1
|
Ming H, Scatolin GN, Ojeda-Rojas OA, Jiang Z. Establishment of bovine extraembryonic endoderm stem cells enables efficient blastoid formation. Cell Rep 2025; 44:115707. [PMID: 40402743 DOI: 10.1016/j.celrep.2025.115707] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2025] [Revised: 03/24/2025] [Accepted: 04/25/2025] [Indexed: 05/24/2025] Open
Abstract
Extraembryonic endoderm stem cells (XENs) resemble the primitive endoderm of blastocyst and provide valuable alternatives for understanding hypoblast development and building stem cell-based embryo models. Here, we report that a chemical cocktail (FGF4, BMP4, IL-6, XAV939, and A83-01) enables the de novo derivation and long-term culture of bovine XENs (bXENs) from blastocysts. Transcriptomic and epigenomic analyses confirm the identity of bXENs and reveal that bXENs resemble the hypoblast lineages of early bovine peri-implantation embryos. Furthermore, we show that bXENs maintain the stemness of expanded embryonic stem cells (EPSCs) and prevent them from undergoing differentiation. The chemical cocktail sustaining bXENs also facilitates the growth of epiblasts in developing pre-implantation embryos. Finally, 3D assembly of bXENs with bovine EPSCs and trophoblast stem cells enables efficient generation of blastoids that resemble blastocysts. bXENs and blastoids established here represent accessible in vitro models for understanding embryogenesis and improving reproductive efficiency in domestic livestock.
Collapse
Affiliation(s)
- Hao Ming
- Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL 32610, USA
| | | | - Oscar Alejandro Ojeda-Rojas
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32610, USA
| | - Zongliang Jiang
- Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL 32610, USA.
| |
Collapse
|
2
|
Feltrin IR, Melo GD, Freitas PP, Morelli KG, Binelli M, Membrive CMB, Pugliesi G. Conceptus signaling markers in immune cells enhance pregnancy prediction in beef cattle. Sci Rep 2025; 15:17548. [PMID: 40394099 PMCID: PMC12092828 DOI: 10.1038/s41598-025-01996-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2024] [Accepted: 05/09/2025] [Indexed: 05/22/2025] Open
Abstract
In beef cattle, estrous synchronization aiming a second artificial insemination (AI) requires a reliable estimation of the pregnancy status 20 days (D20) after the first AI. The hypothesis is that the expression of interferon-stimulated genes (ISGs; ISG15, OAS1, RSAD2, and IFI44) and cytokines (IL1β and IL10) in mononuclear (PBMC) and polymorphonuclear (PMN) cells is regulated by interferon-τ (IFN-τ) and predicts the pregnancy status. PBMC and PMN were isolated from non-pregnant beef cows (N = 9), 10-12 days post-ovulation (D0), and stimulated with 100 ng/mL recombinant ovine (ro) IFN-τ or with pooled uterine flush (UF) from D18 pregnant cows. Both roIFNT and UF stimulated the expression of ISG15, RSAD2, and IFI44 in PBMC and PMN. Expression of IL1β was reduced by UF in both PBMC and PMN. On another experiment, PMN were isolated, and luteal blood perfusion was measured on D20 post-timed-AI in beef females. The accuracy of ISG expression and luteal blood perfusion to predict the pregnancy outcome was determined by ROC curve analysis. All gene combinations were tested, and the best association for increased accuracy (92.7%) and reduction of false-negative results (0.9%, 2/233) was obtained through the combination of the four ISGs (ISG15, OAS1, RSAD2, and IFI44). The criterion was that if the expression levels of at least one of the four genes were greater than the predefined cutoffs, the animal would be considered pregnant. In conclusion, the expression of ISGs and IL1β was upregulated by roIFNT and UF from pregnancy cows. The combined PMN expression of classical (ISG15 and OAS1) and unusual (RSAD2 and IFI44) ISGs provided the greatest predictive accuracy of the pregnancy status on D20 in females with active CL by Doppler and is a potential tool to be used in reproductive programs for beef cattle.
Collapse
Affiliation(s)
- Isabella Rio Feltrin
- Department of Pharmacology and Biotechnology, São Paulo State University (UNESP), Botucatu, SP, Brazil
| | - Gabriela Dalmaso Melo
- Department of Animal Reproduction, University of São Paulo (USP), Avenida Duque de Caxias Norte 225, Jardim Elite, Pirassununga, SP, Brazil
| | - Pedro Pisani Freitas
- Department of Animal Reproduction, University of São Paulo (USP), Avenida Duque de Caxias Norte 225, Jardim Elite, Pirassununga, SP, Brazil
| | - Karine Galhego Morelli
- Department of Animal Reproduction, University of São Paulo (USP), Avenida Duque de Caxias Norte 225, Jardim Elite, Pirassununga, SP, Brazil
| | - Mario Binelli
- Department of Animal Sciences , University of Florida (UF) , Gainesville, FL, USA
| | - Claudia Maria Bertan Membrive
- Department of Pharmacology and Biotechnology, São Paulo State University (UNESP), Botucatu, SP, Brazil
- Department of Animal Sciences , São Paulo State University (UNESP) , Dracena, SP, Brazil
| | - Guilherme Pugliesi
- Department of Animal Reproduction, University of São Paulo (USP), Avenida Duque de Caxias Norte 225, Jardim Elite, Pirassununga, SP, Brazil.
| |
Collapse
|
3
|
Yousef MS, Imakawa K. Epithelial-Mesenchymal Transitions Leading to Conceptus Adhesion in Ruminants: Early Pregnancy Events in Cattle. Int J Mol Sci 2025; 26:3772. [PMID: 40332451 PMCID: PMC12028242 DOI: 10.3390/ijms26083772] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2025] [Revised: 04/11/2025] [Accepted: 04/13/2025] [Indexed: 05/08/2025] Open
Abstract
Trophoblast-endometrium interactions play a critical role in the processes of conceptus elongation, attachment, and adhesion, followed by placental development during early pregnancy in ruminants. The attachment between uterine epithelium and trophoblast cells, which is epithelial in nature, requires epithelial to mesenchymal transition (EMT), where the fetal trophoblasts come into contact with maternal epithelial cells without fully invading the maternal tissues. Understanding the early developmental period driving EMT processes in utero in ruminants is fundamental to improving fertility through the prevention of early pregnancy failure and enhancing overall reproductive efficiency in livestock. This review highlights the key events necessary for the early conceptus to progress properly towards firm adhesion with the endometrium, focusing on trophoblast-endometrium interactions. This field holds the potential to elucidate molecular and cellular mechanisms associated with trophoblast and endometrium attachment and adhesion, leading to reduced early embryonic losses and enhanced economic sustainability by developing effective reproductive management strategies.
Collapse
Affiliation(s)
- Mohamed Samy Yousef
- Research Institute of Agriculture, Tokai University, Kumamoto 862-8652, Japan
- Department of Theriogenology, Faculty of Veterinary Medicine, Assiut University, Assiut 71515, Egypt
| | - Kazuhiko Imakawa
- Research Institute of Agriculture, Tokai University, Kumamoto 862-8652, Japan
| |
Collapse
|
4
|
Zhou C, Huang S, Zheng S, Pius L, Liu M, Xu D. Genome-wide analysis reveals porcine LIFR regulated by DNA methylation promotes the implantation process via the STAT3 signaling. Int J Biol Macromol 2025; 295:139450. [PMID: 39756743 DOI: 10.1016/j.ijbiomac.2024.139450] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2024] [Revised: 12/15/2024] [Accepted: 12/31/2024] [Indexed: 01/07/2025]
Abstract
Embryo-uterine interaction during embryo implantation depends on the coordinated expression of numerous genes in the receptive endometrium. While DNA methylation is known to play a significant role in controlling gene expression, specific molecular mechanisms underlying this regulatory event remain elusive in early porcine pregnancy. Here, we investigated the genome-wide DNA methylation landscape in the Yorkshire and Meishan pig's endometrium. The results revealed a higher degree of DNA methylation modifications on gene promoter regions on day 32 of pregnancy compared to that on day 18 of pregnancy. By integrating the mRNA and methylation profiles, leukemia inhibitory factor receptor (LIFR) was identified as a differentially methylated and expressed gene, crucial in early pregnancy. LIFR expression is epigenetically silenced via promoter hypermethylation from days 18 to 32 of pregnancy. Moreover, functional assays demonstrated that LIFR knockdown inhibited the proliferation, adhesion, and migration of endometrial epithelial cells (EECs) and downregulated the expression of STAT3 signaling and pregnancy-related genes. In vivo studies further revealed a reduction of implanted mouse embryos upon loss of function of LIFR. Furthermore, RUNX1 up-regulates LIFR expression by binding to the differentially methylated region (DMR) of the LIFR promoter. High levels of RUNX1T1, in turn, recruit RUNX1/HDAC1/DNMTs to assemble a regulatory complex that silences LIFR expression through the same locus. Collectively, our findings shed light on the role of dynamic DNA methylation and the epigenetic regulation of LIFR on embryo implantation in early swine pregnancy.
Collapse
Affiliation(s)
- Changfan Zhou
- Colleges of Animal Science & Technology, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Animal Embryo Engineering and Molecular Breeding of Hubei Province, Institute of Animal Sciences and Veterinary Medicine, Hubei Academy of Agricultural Sciences, Wuhan 430070, China
| | - Shuntao Huang
- Colleges of Animal Science & Technology, Huazhong Agricultural University, Wuhan 430070, China
| | - Shuailong Zheng
- Colleges of Animal Science & Technology, Huazhong Agricultural University, Wuhan 430070, China
| | - Lenox Pius
- Colleges of Animal Science & Technology, Huazhong Agricultural University, Wuhan 430070, China
| | - Min Liu
- Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
| | - Dequan Xu
- Colleges of Animal Science & Technology, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China.
| |
Collapse
|
5
|
Elsafadi S, Hankele AK, Giesbertz P, Ulbrich SE. Roe deer uterine fluid metabolome reveals elevated glycolysis, fatty acid breakdown, and spermidine synthesis upon reactivation from diapause†. Biol Reprod 2025; 112:70-85. [PMID: 39673258 PMCID: PMC11736431 DOI: 10.1093/biolre/ioae161] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2024] [Revised: 10/25/2024] [Accepted: 11/28/2024] [Indexed: 12/16/2024] Open
Abstract
The blastocyst of the European roe deer (Capreolus capreolus) undergoes a period of decelerated growth and limited metabolism. During this period known as embryonic diapause, it floats freely in the uterus encircled by the histotroph. Prior to implantation, reactivation is marked by rapid embryonic growth and conceptus elongation. We hypothesized that the uterine fluid, which is known to undergo changes in its composition to support early embryonic development, contributes to controlling embryonic growth during diapause and elongation. We therefore characterized the pre-implantation uterine fluid metabolome during diapause and at elongation by mass spectrometry and particularly assessed nonpolar lipids, polar metabolites, acylcarnitines, and polyamines. Our results show that triglycerides and diglycerides levels decreased at elongation, likely serving as a source for membrane synthesis rather than for energy production. A functional analysis identified glycolysis as a key pathway during elongation, which may compensate for the energy requirements during this phase. We also observed an increase of sphingomyelin; prostaglandin precursors; and the amino acids asparagine, glutamine, and methionine upon elongation. The sphingolipid and glycerophospholipid metabolism pathways were implicated during elongation. Particularly, spermidine, and to some extent spermine but not putrescine-levels significantly increased in the uterine fluid during elongation, indicating their significance for reactivation and/or proliferation at embryo elongation. We conclude that the roe deer uterine fluid sustained dynamic compositional changes necessary to support the energy- and resource-intensive conceptus elongation. However, it remains to be determined whether these changes are the cause or a consequence of embryo elongation. Studying the metabolic changes and molecular interactions in the roe deer during diapause and elongation not only reveals insights into aspects of its reproductive strategy, but also deepens our knowledge of embryo metabolic demands and developmental velocities across species.
Collapse
Affiliation(s)
- Sara Elsafadi
- ETH Zürich, Animal Physiology, Institute of Agricultural Sciences, Universtitätstr. 2, CH-8092 Zurich, Switzerland
| | - Anna-Katharina Hankele
- ETH Zürich, Animal Physiology, Institute of Agricultural Sciences, Universtitätstr. 2, CH-8092 Zurich, Switzerland
| | - Pieter Giesbertz
- Else Kröner-Fresenius-Center of Nutritional Medicine, TUM School of Life Sciences, Technical University of Munich, Freising, Germany
| | - Susanne E Ulbrich
- ETH Zürich, Animal Physiology, Institute of Agricultural Sciences, Universtitätstr. 2, CH-8092 Zurich, Switzerland
| |
Collapse
|
6
|
Martinelle L, Saegerman C. A Review on Reliable and Standardized Animal Models to Study the Pathogenesis of Schmallenberg Virus in Ruminant Natural Host Species. Methods Mol Biol 2025; 2893:207-222. [PMID: 39671040 DOI: 10.1007/978-1-0716-4338-9_16] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2024]
Abstract
In the late summer of 2011, the Netherlands reported a cluster of reduced milk yield, fever, and diarrhea in dairy cattle. In March 2012, congenital malformations appeared, and Schmallenberg virus (SBV) was identified, becoming one of the few orthobunyaviruses distributed in Europe. Initially, little was known about the pathogenesis and epidemiology of these viruses in the European context, so assumptions were largely extrapolated from related viruses and other regions worldwide. To study SBV's pathogenesis and its ability to cross the placental barrier, standardized and repeatable models that mimic clinical signs observed in the field are essential. This review discusses some of the latest experimental designs for infectious disease challenges involving SBV, covering infectious doses, routes of infection, inoculum preparation, and origin. Special attention is given to the placental crossing associated with SBV.
Collapse
Affiliation(s)
- Ludovic Martinelle
- Faculty of Veterinary Medicine, CARE-FEPEX experimental Station, Fundamental and Applied Research for Animal and Health (FARAH) Center, University of Liege, Liege, Belgium
| | - Claude Saegerman
- Faculty of Veterinary Medicine, Research Unit of Epidemiology and Risk analysis applied to Veterinary sciences (UREAR-ULiège), Fundamental and Applied Research for Animal and Health (FARAH) Center, University of Liege, Liege, Belgium.
| |
Collapse
|
7
|
Liu B, Ren S, An H, Liang Y, Sheng X, Qi X, Xiao L, Wang X. Establishment of functional trophoblast organoids from trophoblast cells of bovine placenta. Cells Dev 2024; 180:203970. [PMID: 39243977 DOI: 10.1016/j.cdev.2024.203970] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2024] [Revised: 08/23/2024] [Accepted: 09/03/2024] [Indexed: 09/09/2024]
Abstract
The placenta is an organ that plays a vital role in successful pregnancies, and the failure of early placentation is a significant factor leading to abortion in ruminant species. However, the mechanisms involved in the development and differentiation of bovine placenta remain elusive due to the lack of suitable in vitro placental models. This study aimed to develop an effective method for generating the bovine functional trophoblast organoids by assembling bovine primary trophoblast cells (PBTCs) from the placenta or immortalized bovine placental trophoblast (BTCs) in a 3D culture system in vitro. PBTCs isolated from the 3-month-gestation placenta and BTCs rapidly proliferated and exhibited typical epithelioid morphology in the modified trophoblast organoid medium (TOM) for bovine. Furthermore, PBTCs and BTCs proliferating in the modified TOM were both CK7- and E-cadherin-positive. Both PBTCs or BTCs embedded into Matrigel droplets overlaid with modified TOM proliferated and formed trophoblast organoids after 15 days of culture. Moreover, the expression of syntrophoblast marker genes, including CD71, CD46, and chorionic somatomammotropin hormone 1 (CSH1), was detectable in both organoids derived from different types of trophoblast cells. Notably, the protein expression levels of various genes implicated in the establishment of early pregnancy in endometrial epithelium cells (EECs) was increased following coculture with bovine trophoblast organoids. Collectively, the bovine trophoblast organoids established in our study could serve as robust models for elucidating the essential physical functions of the placenta and the causes of pregnancy failures related to the placenta developmental disorders during early bovine pregnancy.
Collapse
Affiliation(s)
- Bingying Liu
- College of Bioscience and Resources Environment, Beijing University of Agriculture, Beijing 102206, China
| | - Siqi Ren
- Animal Science and Technology College, Beijing University of Agriculture, Beijing 102206, China
| | - Hong An
- Animal Science and Technology College, Beijing University of Agriculture, Beijing 102206, China
| | - Yixuan Liang
- Animal Science and Technology College, Beijing University of Agriculture, Beijing 102206, China
| | - Xihui Sheng
- Animal Science and Technology College, Beijing University of Agriculture, Beijing 102206, China
| | - Xiaolong Qi
- Animal Science and Technology College, Beijing University of Agriculture, Beijing 102206, China
| | - Longfei Xiao
- Animal Science and Technology College, Beijing University of Agriculture, Beijing 102206, China.
| | - Xiangguo Wang
- Animal Science and Technology College, Beijing University of Agriculture, Beijing 102206, China.
| |
Collapse
|
8
|
Pérez-Gómez A, González-Brusi L, Flores-Borobia I, Martínez De Los Reyes N, Toledano-Díaz A, López-Sebastián A, Santiago Moreno J, Ramos-Ibeas P, Bermejo-Álvarez P. PPARG is dispensable for bovine embryo development up to tubular stages†. Biol Reprod 2024; 111:557-566. [PMID: 38832705 PMCID: PMC11402522 DOI: 10.1093/biolre/ioae083] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2024] [Revised: 04/25/2024] [Accepted: 05/31/2024] [Indexed: 06/05/2024] Open
Abstract
Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss, and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors mediate the signaling actions of prostaglandins and other lipids, and, between them, PPARG has been pointed out to play a relevant role in conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG knock-out embryos in vitro using two independent gene ablation strategies and assessed their developmental ability. In vitro development to Day 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass, and trophectoderm cell numbers were similar between wild-type and knock-out D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, embryonic disk formation, and hypoblast migration rates were unaffected by the ablation. The development of tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disk was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages.
Collapse
Affiliation(s)
- Alba Pérez-Gómez
- Animal Reproduction Department, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas, Madrid, Spain
| | - Leopoldo González-Brusi
- Animal Reproduction Department, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas, Madrid, Spain
| | - Inés Flores-Borobia
- Animal Reproduction Department, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas, Madrid, Spain
| | - Nuria Martínez De Los Reyes
- Animal Reproduction Department, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas, Madrid, Spain
| | - Adolfo Toledano-Díaz
- Animal Reproduction Department, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas, Madrid, Spain
| | - Antonio López-Sebastián
- Animal Reproduction Department, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas, Madrid, Spain
| | - Julián Santiago Moreno
- Animal Reproduction Department, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas, Madrid, Spain
| | - Priscila Ramos-Ibeas
- Animal Reproduction Department, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas, Madrid, Spain
| | - Pablo Bermejo-Álvarez
- Animal Reproduction Department, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Consejo Superior de Investigaciones Científicas, Madrid, Spain
| |
Collapse
|
9
|
Mazzarella R, Cajas YN, Gonzalez Martínez ME, Rizos D. Extracellular vesicles: emerging paradigms in bovine embryo-maternal communication. Anim Reprod 2024; 21:e20240065. [PMID: 39286362 PMCID: PMC11404873 DOI: 10.1590/1984-3143-ar2024-0065] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2024] [Accepted: 06/25/2024] [Indexed: 09/19/2024] Open
Abstract
The oviduct and uterus provide an optimal environment for early embryo development, where effective communication between the embryo and the maternal reproductive tract is crucial for establishing and maintaining pregnancy. Oviductal and uterine-derived EVs play pivotal roles in this maternal-embryonic communication and in facilitating early embryo development. However, despite the ability of in vitro culture methods to produce viable embryos, the lack of exchange between the embryo and the mother often results in lower-quality embryos than those derived in vivo. Therefore, there is a pressing need to increase our understanding of the physiological mechanisms underlying embryo interaction with the oviduct and endometrium through EVs and to develop models capable of mimicking the in vivo environment. This review aims to provide up-to-date insights into the communication between the mother and pre-implantation bovine embryo, exploring their applications and perspectives in the field.
Collapse
Affiliation(s)
- Rosane Mazzarella
- Department of Animal Reproduction, National Institute for Agricultural and Food Research and Technology, Spanish National Research Council - INIA-CSIC, Madrid, Spain
| | - Yulia Nathaly Cajas
- Department Agrarian Production, Technical University of Madrid -UPM, Madrid, Spain
- Departamento de Ciencias Biológicas, Universidad Técnica Particular de Loja - UTPL, Loja, Ecuador
| | - Maria Encina Gonzalez Martínez
- Department of Anatomy and Embryology, Veterinary Faculty of the Complutense University of Madrid - FV-UCM, Madrid, Spain
| | - Dimitrios Rizos
- Department of Animal Reproduction, National Institute for Agricultural and Food Research and Technology, Spanish National Research Council - INIA-CSIC, Madrid, Spain
| |
Collapse
|
10
|
Strangstalien A, Braz CU, Miyamoto A, Marey M, Khatib H. Early transcriptomic changes in peripheral blood 7 days after embryo transfer in dairy cattle. J Dairy Sci 2024; 107:3080-3089. [PMID: 38101734 DOI: 10.3168/jds.2023-24199] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2023] [Accepted: 11/12/2023] [Indexed: 12/17/2023]
Abstract
A common goal of the dairy industry is to shorten the calving interval to reap several benefits associated with improved fertility. Early pregnancy detection is crucial to shorten this interval, allowing for prompt re-insemination of cows that failed to conceive after the first service. Currently, the industry lacks a method to accurately predict pregnancy within the first 3 wk. The polypeptide cytokine IFN-tau (IFNT) is the primary signal for maternal recognition of pregnancy in ruminants. As IFNT is released from the early conceptus, it initiates a cascade of effects, including upregulation of IFN-stimulated genes (ISG). Expression of ISG can be detected in the peripheral blood. The present study aimed to characterize peripheral transcriptomic changes, including the ISG, as early as d 7 after embryo transfer. A total of 170 Holstein heifers received in vitro-produced embryos. Whole blood was collected from these heifers within 24 h of the embryo transfer (d 0), d 7, and d 14 after embryo transfer. The heifers were divided into 2 groups, pregnant and nonpregnant, based on pregnancy diagnosis on d 28 via ultrasound. Total RNA was extracted from the peripheral blood of pregnant and nonpregnant heifers, pooled and sequenced. Expression analysis on d 7 heifers resulted in 13 significantly differentially expressed genes mostly related to innate immunity. Differential expression analysis comparing pregnant heifers on d 0 to the same heifers on d 14 showed 51 significantly differentially expressed genes. Eight genes were further quantified through reverse-transcription quantitative real-time PCR for biological validation. On d 7 after embryo transfer, mRNA transcriptions of EDN1, CXCL3, CCL4, and IL1A were significantly upregulated in pregnant heifers (n = 14) compared with nonpregnant heifers (n = 14), with respective fold changes of 8.10, 18.12, 29.60, and 29.97. Although on d 14 after embryo transfer, mRNA transcriptions of ISG15, MX2, OASY1, and IFI6 were significantly upregulated in the blood of pregnant heifers (n = 14) compared with the same heifers on d 0, with respective fold changes of 5.09, 2.59, 3.89, and 3.08. These findings demonstrate that several immune-related genes and ISG are activated during the first 2 wk after embryo transfer, which may explain how the maternal immune system accommodates the allogenic conceptus. To further investigate the diagnostic potentials of these genes, future studies are warranted to analyze the specificity and sensitivity of these biomarkers to predict early pregnancy.
Collapse
Affiliation(s)
- A Strangstalien
- Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, WI 53706
| | - C U Braz
- Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801
| | - A Miyamoto
- Global Agromedicine Research Center, Obihiro University of Agriculture & Veterinary Medicine, Obihiro 080-8555, Japan
| | - M Marey
- Global Agromedicine Research Center, Obihiro University of Agriculture & Veterinary Medicine, Obihiro 080-8555, Japan; Department of Theriogenology, Faculty of Veterinary Medicine, Damanhour University, Behera, 22511, Egypt
| | - H Khatib
- Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, WI 53706.
| |
Collapse
|
11
|
Scatolin GN, Ming H, Wang Y, Iyyappan R, Gutierrez-Castillo E, Zhu L, Sagheer M, Song C, Bondioli K, Jiang Z. Single-cell transcriptional landscapes of bovine peri-implantation development. iScience 2024; 27:109605. [PMID: 38633001 PMCID: PMC11022056 DOI: 10.1016/j.isci.2024.109605] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Revised: 03/14/2024] [Accepted: 03/25/2024] [Indexed: 04/19/2024] Open
Abstract
Supporting healthy pregnancy outcomes requires a comprehensive understanding of the molecular and cellular programs of peri-implantation development, when most pregnancy failure occurs. Here, we present single-cell transcriptomes of bovine peri-implantation embryo development at day 12, 14, 16, and 18 post-fertilization. We defined the cellular composition and gene expression of embryonic disc, hypoblast, and trophoblast lineages in bovine peri-implantation embryos, and identified markers and pathway signaling that represent distinct stages of bovine peri-implantation lineages; the expression of selected markers was validated in peri-implantation embryos. Using detailed time-course transcriptomic analyses, we revealed a previously unrecognized primitive trophoblast cell lineage. We also characterized conserved and divergence peri-implantation lineage programs between bovine and other mammalian species. Finally, we established cell-cell communication signaling underlies embryonic and extraembryonic cell interaction to ensure proper early development. These data provide foundational information to discover essential biological signaling underpinning bovine peri-implantation development.
Collapse
Affiliation(s)
| | - Hao Ming
- Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL 32610, USA
| | - Yinjuan Wang
- School of Animal Sciences, AgCenter, Louisiana State University, Baton Rouge, LA 70803, USA
| | - Rajan Iyyappan
- Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL 32610, USA
| | | | - Linkai Zhu
- Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL 32610, USA
| | - Masroor Sagheer
- Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL 32610, USA
| | - Chao Song
- Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL 32610, USA
| | - Kenneth Bondioli
- School of Animal Sciences, AgCenter, Louisiana State University, Baton Rouge, LA 70803, USA
| | - Zongliang Jiang
- Department of Animal Sciences, Genetics Institute, University of Florida, Gainesville, FL 32610, USA
| |
Collapse
|
12
|
Hao K, Liu X, Chen Y, Zeng W, Chen L, Wang J, Hu G. PPARγ regulates lipid metabolism and viability of sheep trophoblast cells. Reprod Domest Anim 2023; 58:1559-1568. [PMID: 37712626 DOI: 10.1111/rda.14471] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2023] [Revised: 08/15/2023] [Accepted: 08/31/2023] [Indexed: 09/16/2023]
Abstract
Peroxisome proliferator-activated receptor γ (PPARγ) is highly expressed in trophoblast tissues in pregnancy during which the protein participates in diverse events, including embryo implantation and placental formation. However, little is known about the role of PPARγ in embryonic development. This study investigated the function of PPARγ in sheep trophoblast cells. The coding sequence of sheep PPARγ encoded 475 amino acids and included one synonymou mutation compared with the sheep reference sequence for PPARγ. The PPARγ protein was localized in the nucleus and cytoplasm of sheep trophoblasts. The relative expression of PPARγ was elevated in cells treated with rosiglitazone and reduced following administration of GW9662. Activation of PPARγ promoted cell proliferation and mobility, but inhibited apoptosis. In addition, stimulation of PPARγ promoted the expression of lipid metabolism-related genes FABP4 and PLIN2. The expression of prostaglandin metabolism-related genes PLA2G4A, PTGS2 and PTGES also was upregulated significantly in trophoblast cells when PPARγ was activated. In contrast, activation of PPARγ did not impact expression of the prostaglandin-related genes PGFS and SLCO2A1. At the same time, activation of PPARγ activity increased the ratio of PGE2 to PGF2α. Furthermore, fluorescence labelling showed that the numbers of cell lipid droplets increased after stimulation of PPARγ activity, but decreased when PPARγ was inhibited. In conclusion, PPARγ is critical for the regulation of lipid metabolism and prostaglandin synthesis and secretion in sheep trophoblast cells and also has a potent effect on cell proliferation and viability.
Collapse
Affiliation(s)
- Kexing Hao
- College of Animal Science and Technology, Shihezi University, Shihezi, China
| | - Xiuxia Liu
- Shihezi University School of Medicine, Shihezi University, Shihezi, China
| | - Yan Chen
- College of Animal Science and Technology, Shihezi University, Shihezi, China
| | - Weibin Zeng
- College of Animal Science and Technology, Shihezi University, Shihezi, China
| | - Lei Chen
- College of Animal Science and Technology, Shihezi University, Shihezi, China
| | - Jing Wang
- College of Animal Science and Technology, Shihezi University, Shihezi, China
| | - Guangdong Hu
- College of Animal Science and Technology, Shihezi University, Shihezi, China
| |
Collapse
|
13
|
Li X, Yao X, Li K, Guo J, Deng K, Liu Z, Yang F, Fan Y, Yang Y, Zhu H, Wang F. CREB1 Is Involved in miR-134-5p-Mediated Endometrial Stromal Cell Proliferation, Apoptosis, and Autophagy. Cells 2023; 12:2554. [PMID: 37947633 PMCID: PMC10649013 DOI: 10.3390/cells12212554] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2023] [Revised: 09/17/2023] [Accepted: 10/13/2023] [Indexed: 11/12/2023] Open
Abstract
The successful establishment of endometrial receptivity is a key factor in ensuring the fertility of ewes and their economic benefits. Hu sheep have attracted attention due to their high fecundity and year-round estrus. In this study, we found that in the luteal phase, the uterine gland density, uterine coefficient, and number of uterine caruncles of high-fertility Hu sheep were higher than those of low-fertility Hu sheep. Thousands of differentially expressed genes were identified in the endometrium of Hu sheep with different fertility potential using RNA sequencing (RNA-Seq). Several genes involved in endometrial receptivity were screened using bioinformatics analysis. The qRT-PCR analysis further revealed the differential expression of cAMP reactive element binding protein-1 (CREB1) in the Hu sheep endometrium during the estrous cycle. Functionally, our results suggested that CREB1 significantly affected the expression level of endometrial receptivity marker genes, promoted cell proliferation by facilitating the transition from the G1 phase to the S phase, and inhibited cell apoptosis and autophagy. Moreover, we observed a negative linear correlation between miR-134-5p and CREB1 in the endometrium. In addition, CREB1 overexpression prevented the negative effect of miR-134-5p on endometrial stromal cell (ESC) growth. Taken together, these data indicated that CREB1 was regulated by miR-134-5p and may promote the establishment of uterine receptivity by regulating the function of ESCs. Moreover, this study provides new theoretical references for identifying candidate genes associated with fertility.
Collapse
Affiliation(s)
- Xiaodan Li
- Hu Sheep Academy, Nanjing Agricultural University, Nanjing 210095, China; (X.L.)
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Xiaolei Yao
- Hu Sheep Academy, Nanjing Agricultural University, Nanjing 210095, China; (X.L.)
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Kang Li
- Hu Sheep Academy, Nanjing Agricultural University, Nanjing 210095, China; (X.L.)
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Jiahe Guo
- Hu Sheep Academy, Nanjing Agricultural University, Nanjing 210095, China; (X.L.)
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Kaiping Deng
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Zhipeng Liu
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Fan Yang
- Hu Sheep Academy, Nanjing Agricultural University, Nanjing 210095, China; (X.L.)
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Yixuan Fan
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Yingnan Yang
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| | - Huabin Zhu
- Embryo Biotechnology and Reproduction Laboratory, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
| | - Feng Wang
- Jiangsu Livestock Embryo Engineering Laboratory, Nanjing Agricultural University, Nanjing 210095, China
| |
Collapse
|
14
|
Husnain A, Arshad U, Zimpel R, Schmitt E, Dickson MJ, Perdomo MC, Marinho MN, Ashrafi N, Graham SF, Bishop JV, Hansen TR, Jeong KC, Gonella-Diaza AM, Chebel RC, Sheldon IM, Bromfield JJ, Santos JEP. Induced endometrial inflammation compromises conceptus development in dairy cattle†. Biol Reprod 2023; 109:415-431. [PMID: 37540198 PMCID: PMC10577276 DOI: 10.1093/biolre/ioad088] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2022] [Revised: 04/24/2023] [Accepted: 07/28/2023] [Indexed: 08/05/2023] Open
Abstract
Endometrial inflammation is associated with reduced pregnancy per artificial insemination (AI) and increased pregnancy loss in cows. It was hypothesized that induced endometritis alters histotroph composition and induces inflammatory signatures on conceptus that compromise development. In Experiment 1, lactating cows were assigned to control (CON; n = 23) or to an intrauterine infusion of Escherichia coli and Trueperella pyogenes (ENDO; n = 34) to induce endometritis. Cows received AI 26 days after treatment, and the uterine fluid and conceptuses were collected on day 16 after AI. In Experiment 2, Holstein heifers were assigned to CON (n = 14) or ENDO (n = 14). An embryo was transferred on day 7 of the estrous cycle, and uterine fluid and conceptuses were recovered on day 16. Composition of histotroph and trophoblast and embryonic disc gene expression were assessed. Bacterial-induced endometritis in lactating cows altered histotroph composition and pathways linked to phospholipid synthesis, cellular energy production, and the Warburg effect. Also, ENDO reduced conceptus length in cows and altered expression of genes involved in pathogen recognition, nutrient uptake, cell growth, choline metabolism, and conceptus signaling needed for maternal recognition of pregnancy. The impact of ENDO was lesser on conceptuses from heifers receiving embryo transfer; however, the affected genes and associated pathways involved restricted growth and increased immune response similar to the observed responses to ENDO in conceptuses from lactating cows. Bacterial-induced endometrial inflammation altered histotroph composition, reduced conceptus growth, and caused embryonic cells to activate survival rather than anabolic pathways that could compromise development.
Collapse
Affiliation(s)
- Ali Husnain
- Department of Animal Sciences, DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL, USA
| | - Usman Arshad
- Department of Animal Sciences, DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL, USA
| | - Roney Zimpel
- Department of Animal Sciences, DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL, USA
| | - Eduardo Schmitt
- Department of Animal Sciences, DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL, USA
| | - Mackenzie J Dickson
- Department of Animal Sciences, DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL, USA
| | - Milerky C Perdomo
- Department of Animal Sciences, DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL, USA
| | - Mariana N Marinho
- Department of Animal Sciences, DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL, USA
| | - Nadia Ashrafi
- Metabolomics Department, Beaumont Health, Royal Oak, MI, USA
| | - Stewart F Graham
- Metabolomics Department, Beaumont Health, Royal Oak, MI, USA
- Oakland University-William Beaumont School of Medicine, Rochester, MI, USA
| | - Jeanette V Bishop
- Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
| | - Thomas R Hansen
- Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA
| | - Kwang C Jeong
- Department of Animal Sciences, DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL, USA
| | | | - Ricardo C Chebel
- Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
| | - I Martin Sheldon
- Swansea University Medical School, Swansea University, Swansea, UK
| | - John J Bromfield
- Department of Animal Sciences, DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL, USA
| | - José E P Santos
- Department of Animal Sciences, DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, FL, USA
| |
Collapse
|
15
|
Jia GX, Ma WJ, Wu ZB, Li S, Zhang XQ, He Z, Wu SX, Tao HP, Fang Y, Song YW, Xu SR, Wang XQ, Yang QE. Single-cell transcriptomic characterization of sheep conceptus elongation and implantation. Cell Rep 2023; 42:112860. [PMID: 37494181 DOI: 10.1016/j.celrep.2023.112860] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2023] [Revised: 06/19/2023] [Accepted: 07/10/2023] [Indexed: 07/28/2023] Open
Abstract
Bidirectional communication between the developing conceptus and endometrium is essential for pregnancy recognition and establishment in ruminants. We dissect the transcriptomic dynamics of sheep conceptus and corresponding endometrium at pre- and peri-implantation stages using single-cell RNA sequencing. Spherical blastocysts contain five cell types, with 68.62% trophectoderm cells. Strikingly, elongated conceptuses differentiate into 17 cell types, indicating dramatic cell fate specifications. Cell-type-specific gene expression delineates the features of distinctive trophectoderm lineages and indicates that the transition from polar trophectoderm to trophoblast increases interferon-tau expression and likely drives elongation initiation. We identify 13 endometrium-derived cell types and elucidate their molecular responses to conceptus development. Integrated analyses uncover multiple paired transcripts mediating the dialogues between extraembryonic membrane and endometrium, including IGF2-IGF1R, FGF19-FGFR1, NPY-NPY1R, PROS1-AXL, and ADGRE5-CD55. These data provide insight into the molecular regulation of conceptus elongation and represent a valuable resource for functional investigations of pre- and peri-implantation ruminant development.
Collapse
Affiliation(s)
- Gong-Xue Jia
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China; University of Chinese Academy of Sciences, Beijing 100049, China; Qinghai Key Laboratory of Animal Ecological Genomics, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China
| | - Wen-Ji Ma
- State Key Laboratory of Brain and Cognitive Science, CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Center for Single-Cell Omics, School of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Zhao-Bo Wu
- State Key Laboratory of Brain and Cognitive Science, CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
| | - Shuang Li
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China
| | - Xiao-Qian Zhang
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China
| | - Zhen He
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Shi-Xin Wu
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Hai-Ping Tao
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yi Fang
- University of Chinese Academy of Sciences, Beijing 100049, China; Jilin Provincial Key Laboratory of Grassland Farming, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, China
| | - Yong-Wu Song
- Animal Husbandry and Veterinary Station of Gangcha, Haibei 812300, China
| | - Shang-Rong Xu
- Qinghai Academy of Animal Science and Veterinary Medicine, Qinghai University, Xining 810016, China
| | - Xiao-Qun Wang
- State Key Laboratory of Brain and Cognitive Science, CAS Center for Excellence in Brain Science and Intelligence Technology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
| | - Qi-En Yang
- Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China; University of Chinese Academy of Sciences, Beijing 100049, China; Qinghai Key Laboratory of Animal Ecological Genomics, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, China.
| |
Collapse
|
16
|
Wang Y, Ming H, Yu L, Li J, Zhu L, Sun HX, Pinzon-Arteaga CA, Wu J, Jiang Z. Establishment of bovine trophoblast stem cells. Cell Rep 2023; 42:112439. [PMID: 37146606 PMCID: PMC10950030 DOI: 10.1016/j.celrep.2023.112439] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2023] [Revised: 02/17/2023] [Accepted: 04/11/2023] [Indexed: 05/07/2023] Open
Abstract
Here, we report that a chemical cocktail (LCDM: leukemia inhibitory factor [LIF], CHIR99021, dimethinedene maleate [DiM], minocycline hydrochloride), previously developed for extended pluripotent stem cells (EPSCs) in mice and humans, enables de novo derivation and long-term culture of bovine trophoblast stem cells (TSCs). Bovine TSCs retain developmental potency to differentiate into mature trophoblast cells and exhibit transcriptomic and epigenetic (chromatin accessibility and DNA methylome) features characteristic of trophectoderm cells from early bovine embryos. The bovine TSCs established in this study will provide a model to study bovine placentation and early pregnancy failure.
Collapse
Affiliation(s)
- Yinjuan Wang
- School of Animal Sciences, AgCenter, Louisiana State University, Baton Rouge, LA 70803, USA
| | - Hao Ming
- School of Animal Sciences, AgCenter, Louisiana State University, Baton Rouge, LA 70803, USA
| | - Leqian Yu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jie Li
- BGI-Shenzhen, Shenzhen, Guangdong 518083, China; BGI-Beijing, Beijing 102601, China; College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Linkai Zhu
- Department of Animal Sciences, University of Florida, Gainesville, FL 32608, USA
| | - Hai-Xi Sun
- BGI-Shenzhen, Shenzhen, Guangdong 518083, China; BGI-Beijing, Beijing 102601, China; College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
| | - Carlos A Pinzon-Arteaga
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Jun Wu
- Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
| | - Zongliang Jiang
- School of Animal Sciences, AgCenter, Louisiana State University, Baton Rouge, LA 70803, USA; Department of Animal Sciences, University of Florida, Gainesville, FL 32608, USA; Genetics Institute, University of Florida, Gainesville, FL 32610, USA.
| |
Collapse
|
17
|
Mion B, Madureira G, Spricigo JFW, King K, Van Winters B, LaMarre J, LeBlanc SJ, Steele MA, Ribeiro ES. Effects of source of supplementary trace minerals in pre- and postpartum diets on reproductive biology and performance in dairy cows. J Dairy Sci 2023:S0022-0302(23)00216-3. [PMID: 37164845 DOI: 10.3168/jds.2022-22784] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2022] [Accepted: 01/03/2023] [Indexed: 05/12/2023]
Abstract
Our objectives were to evaluate the effects of complete replacement of inorganic salts of trace minerals (STM) with organic trace minerals (OTM) in both pre- and postpartum diets on ovarian dynamics, estrous behavior measured by sensors, preimplantation conceptus development, and reproductive performance in dairy cows. Pregnant cows and heifers (n = 273) were blocked by parity and body condition score and randomly assigned to either STM or OTM diets at 45 ± 3 d before their expected calving. Pre- and postpartum diets were formulated to meet 100% of recommended levels of each trace mineral in both treatments, taking into consideration both basal and supplemental levels. The final target concentrations of Co, Cu, Mn, Se, and Zn were, respectively, 0.25, 13.7, 40.0, 0.3, and 40.0 mg/kg in the prepartum diet, and 0.25, 15.7, 40.0, 0.3, and 63.0 mg/kg in the postpartum diet. The STM group was supplemented with Co, Cu, Mn, and Zn sulfates and sodium selenite, while the OTM group was supplemented with Co, Cu, Mn, and Zn proteinates and selenized yeast. Treatments continued until 156 d in milk (DIM) and were assigned to individual cows using automatic feeding gates. Starting at 21 DIM, ultrasonography examinations of the ovaries were performed weekly to determine the presence of a corpus luteum and postpartum resumption of ovarian cyclicity. Cows were presynchronized with 2 injections of PGF2α at 42 and 56 DIM. Estrous behavior was monitored using electronic activity tags that indirectly measured walking activity. Cows detected in estrus after the second PGF2α were inseminated, and those not detected in estrus by 67 DIM were enrolled in a synchronization program. Cows that returned to estrus after artificial insemination (AI) were reinseminated. Pregnancy diagnosis was performed 33 d after AI, and nonpregnant cows were resynchronized. Transcript expression of interferon-stimulated genes in peripheral blood leukocytes was performed in a subgroup of cows (STM, n = 67; OTM, n = 73) on d 19 after AI. A different subgroup of cows (28 STM, 29 OTM) received uterine flushing 15 d after AI for recovery of conceptuses and uterine fluid for analyses of transcriptomics and metabolomics, respectively. In addition, dominant follicle diameter, luteal size and blood flow, and concentration of progesterone in plasma were measured on d 0, 7, and 15 relative to AI. After flushing, PGF2α was given and the dominant follicle was aspirated 2 d later to measure the concentration of trace minerals by mass spectrometry. Estrous behavior, size of the dominant follicle and corpus luteum, concentration of progesterone, time to pregnancy, and proportion of cows pregnant by 100 d of the breeding period did not differ between treatments. A greater proportion of cows supplemented with OTM had a corpus luteum detected before presynchronization (64.3 vs. 75.2%), and primiparous cows supplemented with OTM tended to resume cyclicity earlier than their STM counterparts. Cows supplemented with OTM had a greater concentration of Cu in follicular fluid than cows supplemented with STM (0.89 vs. 0.77 µg/mL, respectively). In pregnant multiparous cows, expression of receptor transporter protein 4 in peripheral blood leukocytes was 42% greater in the OTM group. Conceptuses of the 2 treatments had 589 differentially expressed transcripts, with many indicating advanced conceptus elongation and greater transcript expression of selenoproteins in the OTM group. In pregnant cows, 24 metabolites were more abundant in the uterine fluid of OTM, including spermidine, sucrose, and cholesterol. In conclusion, replacing STM with OTM caused modest improvements to resumption of ovarian cyclicity and important changes in preimplantation conceptus development, but it did not alter conception risk and pregnancy rate.
Collapse
Affiliation(s)
- B Mion
- Department of Animal Biosciences, University of Guelph, Guelph, ON, N1G 2W1, Canada
| | - G Madureira
- Department of Animal Biosciences, University of Guelph, Guelph, ON, N1G 2W1, Canada
| | - J F W Spricigo
- Department of Animal Biosciences, University of Guelph, Guelph, ON, N1G 2W1, Canada
| | - K King
- Department of Animal Biosciences, University of Guelph, Guelph, ON, N1G 2W1, Canada
| | - B Van Winters
- Department of Animal Biosciences, University of Guelph, Guelph, ON, N1G 2W1, Canada
| | - J LaMarre
- Department of Biomedical Sciences, University of Guelph, Guelph, ON, N1G 2W1, Canada
| | - S J LeBlanc
- Department of Population Medicine, University of Guelph, Guelph, ON, N1G 2W1, Canada
| | - M A Steele
- Department of Animal Biosciences, University of Guelph, Guelph, ON, N1G 2W1, Canada
| | - E S Ribeiro
- Department of Animal Biosciences, University of Guelph, Guelph, ON, N1G 2W1, Canada.
| |
Collapse
|
18
|
Davenport KM, Ortega MS, Johnson GA, Seo H, Spencer TE. Review: Implantation and placentation in ruminants. Animal 2023; 17 Suppl 1:100796. [PMID: 37567669 DOI: 10.1016/j.animal.2023.100796] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2023] [Revised: 03/26/2023] [Accepted: 03/27/2023] [Indexed: 08/13/2023] Open
Abstract
Ruminants have a unique placenta in comparison to other mammalian species. Initially, they possess a non-invasive epitheliochorial type of placenta during conceptus elongation. As the conceptus trophectoderm begins to attach to the luminal epithelium (LE) of the endometrium, binucleate cells (BNCs) develop within the trophoblast of the chorion. The BNCs migrate and fuse with the uterine LE to form multinucleate syncytial plaques in sheep and hybrid trinucleate cells in cattle. This area of the ruminant placenta is semi-invasive synepitheliochorial. The BNCs form the foundation of the placental cotyledons and express unique placenta-specific genes including pregnancy-associated glycoproteins and chorionic somatomammotropin hormone 2 or placental lactogen. Attachment and interdigitation of cotyledons into endometrial caruncles form placentomes that are subsequently vascularized to provide essential nutrients for growth of the fetus. This chapter review will discuss historical and current aspects of conceptus implantation and placenta development in ruminant ungulates with a focus on cattle and sheep. Single-cell analysis promises to provide a much more detailed understanding of the different cell populations and insights into pathways mediating trophoblast and placenta. This fundamental is required to understand pregnancy loss and develop strategies to improve pregnancy outcomes in ruminants.
Collapse
Affiliation(s)
- K M Davenport
- Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA
| | - M S Ortega
- Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, WI 53715, USA
| | - G A Johnson
- Department of Veterinary Integrative Biosciences and Department of Animal Science, Texas A&M University, College Station, TX 7784, USA
| | - H Seo
- Department of Veterinary Integrative Biosciences and Department of Animal Science, Texas A&M University, College Station, TX 7784, USA
| | - T E Spencer
- Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA; Division of Obstetrics, Gynecology, and Women's Health, University of Missouri, Columbia, MO 65211, USA.
| |
Collapse
|
19
|
Hao K, Wang J, Li Z, Chen H, Jia B, Hu G. PPARγ/mTOR Regulates the Synthesis and Release of Prostaglandins in Ovine Trophoblast Cells in Early Pregnancy. Vet Sci 2022; 9:649. [PMID: 36423098 PMCID: PMC9694237 DOI: 10.3390/vetsci9110649] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2022] [Revised: 11/04/2022] [Accepted: 11/17/2022] [Indexed: 09/16/2023] Open
Abstract
Trophoblast cells synthesize and secrete prostaglandins (PGs), which are essential for ruminants in early gestation to recognize pregnancy. Hormones in the intrauterine environment play an important role in regulating PGs synthesis during implantation, but the underlying mechanism remains unclear. In this study, co-treatment of sheep trophoblast cells (STCs) with progesterone (P4), estradiol (E2), and interferon-tau (IFN-τ) increased the ratio of prostaglandin E2 (PGE2) to prostaglandin F2α (PGF2α) and upregulated peroxisome proliferator-activated receptor γ (PPARγ) expression, while inhibiting the mechanistic target of rapamycin (mTOR) pathway and activating cellular autophagy. Under hormone treatment, inhibition of PPARγ activity decreased the ratio of PGE2/PGF2α and cellular activity, while activating expression of the mTOR downstream marker-the phosphorylation of p70S6K (p-p70S6K). We also found that the PPARγ/mTOR pathway played an important role in regulating trophoblast cell function. Inhibition of the mTOR pathway by rapamycin increased the ratio of PGE2/PGF2α and decreased the expression of apoptosis-related proteins after inhibiting PPARγ activity. In conclusion, our findings provide new insights into the molecular mechanism of prostaglandin regulation of trophoblast cells in sheep during early pregnancy, indicating that the PPARγ/mTOR pathway plays an important role in PGs secretion and cell viability.
Collapse
Affiliation(s)
| | | | | | | | - Bin Jia
- College of Animal Science and Technology, Shihezi University, Shihezi 832000, China
| | - Guangdong Hu
- College of Animal Science and Technology, Shihezi University, Shihezi 832000, China
| |
Collapse
|
20
|
Middleton E, Minela T, Ahearne M, Arnold H, Santos A, Pursley J. Dairy heifers have an earlier increase in serum pregnancy-specific protein B compared with lactating dairy cows. Is this an indicator of earlier conceptus attachment? JDS COMMUNICATIONS 2022; 3:291-295. [PMID: 36338019 PMCID: PMC9623710 DOI: 10.3168/jdsc.2021-0198] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 12/02/2021] [Accepted: 03/27/2022] [Indexed: 06/16/2023]
Abstract
This study was designed to use within-individual daily increases in circulating pregnancy-specific protein B (PSPB) to determine time to increase in serum PSPB in nulliparous heifers and multiparous dairy cows following artificial insemination (AI). Weekly cohorts of lactating Holstein cows (n = 56) averaging 122 ± 7 d in milk at AI and nulliparous Holstein heifers (n = 23) averaging 16 ± 0.24 mo old at AI were used in this study. Lactating cows received AI to either Ovsynch (n = 38) or at ~12 h following observed standing estrus (n = 18). All heifers were inseminated ~12 h after observed standing estrus. Blood samples for measurement of PSPB were collected daily from d 15 through d 35 postovulation. Nulliparous heifers had increases in serum PSPB earlier compared with both primiparous and multiparous cows. Day of increase in serum PSPB was defined as the day serum PSPB optical density levels initially increased ≥10% from baseline and continued to increase from baseline of ≥10% the following 2 d. Average PSPB were greater in pregnant heifers compared pregnant cows from d 23 through 29. Early lactation nonpregnant cows maintained greater average optical density of serum PSPB from 15 to 35 d postovulation compared with nonpregnant heifers (n = 38). In summary, fertility differences in heifers versus lactating cows may be due to the differences in timing of increases in serum PSPB. This appears to be one of the first publications that used daily PSPB sampling to investigate possible differences in fertility in heifers versus lactating cows.
Collapse
|
21
|
Pei J, Zhao S, Yin M, Wu F, Li J, Zhang G, Wu X, Bao P, Xiong L, Song W, Ba Y, Yan P, Song R, Guo X. Differential proteomics of placentas reveals metabolic disturbance and oxidative damage participate yak spontaneous miscarriage during late pregnancy. BMC Vet Res 2022; 18:248. [PMID: 35761325 PMCID: PMC9235108 DOI: 10.1186/s12917-022-03354-w] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2022] [Accepted: 06/20/2022] [Indexed: 12/03/2022] Open
Abstract
Background High spontaneous miscarriage rate in yak, especially during late pregnancy, have caused a great economic loss to herdsmen living in the Qinghai-Tibet plateau. However, the mechanism underlying spontaneous miscarriage is still poorly understood. In the present study, placenta protein markers were identified to elucidate the pathological reasons for yak spontaneous miscarriage through isobaric tags for relative and absolute quantification (iTRAQ) proteomic technology and bioinformatic approaches. Results Subsequently, a total of 415 differentially expressed proteins (DEPs) were identified between aborted and normal placentas. The up-regulated DEPs in the aborted placentas were significantly associated with “spinocerebellar ataxia”, “sphingolipid signalling”, “relaxin signalling”, “protein export”, “protein digestion and absorption” and “aldosterone synthesis and secretion” pathway. While the down-regulated DEPs in the aborted placentas mainly participated in “valine, leucine and isoleucine degradation”, “PPAR signalling”, “peroxisome”, “oxidative phosphorylation”, “galactose metabolism”, “fatty acid degradation”, “cysteine and methionine metabolism” and “citrate cycle” pathway. Conclusions The results implied that the identified DEPs could be considered as placental protein markers for yak miscarriage during late pregnancy, and biomacromolecule metabolic abnormality and oxidative damage might be responsible for the high spontaneous miscarriage rate in yak. These findings provide an important theoretical basis for deciphering the pathologic mechanism of late spontaneous miscarriage in yak. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-022-03354-w.
Collapse
|
22
|
Ortega MS, Rizo JA, Drum JN, O'Neil EV, Pohler KG, Kerns K, Schmelze A, Green J, Spencer TE. Development of an Improved in vitro Model of Bovine Trophectoderm Differentiation. FRONTIERS IN ANIMAL SCIENCE 2022. [DOI: 10.3389/fanim.2022.898808] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
The mechanisms regulating early stages of placentation and trophectoderm differentiation in the ruminant conceptus remain poorly understood. Here we present a model of trophectoderm (TE) differentiation in vitro from outgrowths of individual in vitro derived embryos. Cell outgrowths expressed markers of mononucleate (MNC) and binucleate (BNC) TE cells. The percentage of BNC ranged from 14 to 39% in individual outgrowths as determined by flow cytometry. Pregnancy-associated glycoproteins (PAGs), produced by BNC, were measured in culture media on days 35 to 54. Continuous secretion of PAGs was observed and indicative of BNC functionality. Gene expression was evaluated in 20 embryo cell outgrowths derived from two different sires. Expression of HAND1, which is involved in TE differentiation, and CSH2, a BNC-specific gene, was altered in cell outgrowths between the two sires tested. Single-cell RNA-seq analysis of day 40 TE cell outgrowths revealed 11 distinct cell populations, with specific clusters genes involved in TE lineage specification, proliferation, and differentiation. In addition, whole -RNAseq analysis was performed in day 35 and 40 TE cell outgrowths and confirmed sustained expression of genes expressed by BNC, such as CSH2 and some PAGs. The developed in vitro bovine embryo outgrowth culture found evidence for MNC and BNC differentiation and continuous production of PAGs, recapitulating key features of early bovine placenta development. This model can be used to understand the developmental biology of TE cells, provide insights into paternal influences on TE differentiation, and impact our understanding of early pregnancy loss in cattle.
Collapse
|
23
|
Constraint-Based, Score-Based and Hybrid Algorithms to Construct Bayesian Gene Networks in the Bovine Transcriptome. Animals (Basel) 2022; 12:ani12101305. [PMID: 35625151 PMCID: PMC9138007 DOI: 10.3390/ani12101305] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2022] [Revised: 05/10/2022] [Accepted: 05/10/2022] [Indexed: 11/20/2022] Open
Abstract
Simple Summary In this study, we investigated and compared six different Bayesian network algorithms from three different categories to identify hub genes critical to gene expression networks activated in response to progesterone in the bovine uterus. We observed many common hub genes identified between constraint-based algorithms (CBAs) and hybrid algorithms (HAs), while it appeared that score-based algorithm (SBA) methods led to more accurate and relevant predictions of core genes. The results revealed that the identification of hub genes was affected by the type of network reconstruction and by the subsequently used topological parameters. Two identified genes known to have roles during pregnancy are ISG15 and DGAT2. The identified hub genes are associated with biological processes such as amino acid metabolism, hormonal signaling pathways and the immune system. Our analysis revealed a role for miRNAs in the regulation of this system. The biological and physiological roles (enzymatic and hormonal effects) of unannotated identified hub genes should be functionally validated by further studies. Abstract Bayesian gene networks are powerful for modelling causal relationships and incorporating prior knowledge for making inferences about relationships. We used three algorithms to construct Bayesian gene networks around genes expressed in the bovine uterus and compared the efficacies of the algorithms. Dataset GSE33030 from the Gene Expression Omnibus (GEO) repository was analyzed using different algorithms for hub gene expression due to the effect of progesterone on bovine endometrial tissue following conception. Six different algorithms (grow-shrink, max-min parent children, tabu search, hill-climbing, max-min hill-climbing and restricted maximum) were compared in three higher categories, including constraint-based, score-based and hybrid algorithms. Gene network parameters were estimated using the bnlearn bundle, which is a Bayesian network structure learning toolbox implemented in R. The results obtained indicated the tabu search algorithm identified the highest degree between genes (390), Markov blankets (25.64), neighborhood sizes (8.76) and branching factors (4.38). The results showed that the highest number of shared hub genes (e.g., proline dehydrogenase 1 (PRODH), Sam-pointed domain containing Ets transcription factor (SPDEF), monocyte-to-macrophage differentiation associated 2 (MMD2), semaphorin 3E (SEMA3E), solute carrier family 27 member 6 (SLC27A6) and actin gamma 2 (ACTG2)) was seen between the hybrid and the constraint-based algorithms, and these genes could be recommended as central to the GSE33030 data series. Functional annotation of the hub genes in uterine tissue during progesterone treatment in the pregnancy period showed that the predicted hub genes were involved in extracellular pathways, lipid and protein metabolism, protein structure and post-translational processes. The identified hub genes obtained by the score-based algorithms had a role in 2-arachidonoylglycerol and enzyme modulation. In conclusion, different algorithms and subsequent topological parameters were used to identify hub genes to better illuminate pathways acting in response to progesterone treatment in the bovine uterus, which should help with our understanding of gene regulatory networks in complex trait expression.
Collapse
|
24
|
Establishment and characterization of a sheep endometrial epithelial cell line. Biochem Biophys Res Commun 2022; 603:63-68. [DOI: 10.1016/j.bbrc.2022.03.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2022] [Accepted: 03/02/2022] [Indexed: 11/17/2022]
|
25
|
Dirandeh E, Ansari-Pirsaraei Z, Thatcher W. Melatonin as a Smart Protector of Pregnancy in Dairy Cows. Antioxidants (Basel) 2022; 11:292. [PMID: 35204175 PMCID: PMC8868556 DOI: 10.3390/antiox11020292] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Revised: 01/23/2022] [Accepted: 01/26/2022] [Indexed: 12/10/2022] Open
Abstract
The experimental objective was to examine the role of melatonin and its pathways in the maintenance of pregnancy in lactating dairy cows. Blood samples were collected at days 0, 16 and 32 after timed AI from cows (n = 200) in order to consider plasma melatonin concentrations and to conduct AOPP (advanced oxidation products of proteins) and TBARS (thiobarbituric acid reactive substances) tests. Luminal endometrial cells were collected at day 16 using a Cytobrush in all cows to determine mRNA expressions of melatonin receptor 1 (MT1), mouse double minute 2 (MDM2), MDM2 binding protein (MTBP), BCL2-associated X, apoptosis Regulator (BAX), p53 upregulated modulator of apoptosis (PUMA, gene symbol BBC3), mucin 1 (MUC1) and leukemia inhibitory factor (LIF). Plasma concentrations of melatonin were significantly greater in pregnant cows diagnosed pregnant at day 16 who sustained pregnancy to day 32 compared to nonpregnant cows at day 16, or pregnant at day 16 and who lost embryos by days 32. Concentrations of AOPP and TBARS were greater in nonpregnant cows at day 16 or pregnant at day 16 and who lost embryos by days 32 compared to those diagnosed pregnant at day 16 and who sustained pregnancy to day 32. In pregnant cows, endometrial mRNA expressions of MDM2, MTBP, MTR1 and LIF were higher compared to pregnant-embryo-loss cows (p < 0.05). In contrast, mRNA expressions of BBC3 and MUC1 were greater at day 16 in pregnant-embryo-loss cows compared to pregnant cows (p < 0.05). In conclusion, melatonin status is a modulator of embryo well-being and maintenance of pregnancy in lactating dairy cows.
Collapse
Affiliation(s)
- Essa Dirandeh
- Department of Animal Science, Sari Agricultural Sciences and Natural Resources University, Sari, Mazandaran 578, Iran;
| | - Zarbakht Ansari-Pirsaraei
- Department of Animal Science, Sari Agricultural Sciences and Natural Resources University, Sari, Mazandaran 578, Iran;
| | - William Thatcher
- Department of Animal Sciences, University of Florida, Gainesville, FL 32611, USA;
| |
Collapse
|
26
|
SUGINO Y, SATO T, YAMAMOTO Y, KIMURA K. Evaluation of bovine uterine gland functions in 2D and 3D culture system. J Reprod Dev 2022; 68:254-261. [PMID: 35644574 PMCID: PMC9334319 DOI: 10.1262/jrd.2022-029] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
In ruminants, uterine glands play key roles in the establishment of pregnancy by secreting various factors into the uterine lumen. Although a three-dimensional (3D) culture system has been
used for investigating cellular functions in vitro, the detailed functions of uterine gland have not been fully elucidated. In this study, we examined the benefits of 3D
culture system to examine the innate functions of bovine uterine glands. Isolated bovine uterine glands were cultured on Matrigel (2D) or in Matrigel (3D), respectively, and the mRNA levels
of secreted proteins (SERPINA14, MEP1B, APOA1, ARSA, CTGF, and SPP1) were measured in
isolated and cultured uterine glands. The protein expression of estrogen receptor β (ERβ) and progesterone receptor (PR) and the establishment of apico-basal polarity were examined. In
isolated uterine glands, the mRNA levels of secreted proteins changed during the estrous cycle. Although uterine glands cultured in both 2D and 3D expressed ERβ and PR, progesterone did not
affect SERPINA14 mRNA expression. The expression of APOA1 mRNA in 2D cultured uterine glands did not respond to estrogen and progesterone. Additionally, the
mRNA levels of secreted proteins in the 3D culture system were significantly higher than those in the 2D culture system, which might be attributed to the different cellular morphology
between them. The locations of ZO-1 and β-catenin in 2D cultured uterine glands were disordered compared with 3D cultured uterine glands. These results showed that the hormonal
responsiveness of secreted factor expression and cellular morphology were different between 2D and 3D cultured bovine uterine glands.
Collapse
Affiliation(s)
- Yosuke SUGINO
- Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan
| | - Taiki SATO
- Laboratory of Reproductive Physiology, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
| | - Yuki YAMAMOTO
- Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan
| | - Koji KIMURA
- Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan
| |
Collapse
|
27
|
Moses RM, Kramer AC, Seo H, Wu G, Johnson GA, Bazer FW. A Role for Fructose Metabolism in Development of Sheep and Pig Conceptuses. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2021; 1354:49-62. [PMID: 34807436 DOI: 10.1007/978-3-030-85686-1_3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
The period of conceptus (embryo and extraembryonic membrane) development between fertilization and implantation in mammalian species is critical as it sets the stage for placental and fetal development. The trophectoderm and endoderm of pre-implantation ovine and porcine conceptuses undergo elongation, which requires rapid proliferation, migration, and morphological modification of the trophectoderm cells. These complex events occur in a hypoxic intrauterine environment and are supported through the transport of secretions from maternal endometrial glands to the conceptus required for the biochemical processes of cell proliferation, migration, and differentiation. The conceptus utilizes glucose provided by the mother to initiate metabolic pathways that provide energy and substrates for other metabolic pathways. Fructose, however, is in much greater abundance than glucose in amniotic and allantoic fluids, and fetal blood during pregnancy. Despite this, the role(s) of fructose is largely unknown even though a switch to fructosedriven metabolism in subterranean rodents and some cancers are key to their adaptation to hypoxic environments.
Collapse
Affiliation(s)
- Robyn M Moses
- Departments of Animal Science and Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, 77843, USA
| | - Avery C Kramer
- Departments of Animal Science and Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, 77843, USA
| | - Heewon Seo
- Departments of Animal Science and Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, 77843, USA
| | - Guoyao Wu
- Departments of Animal Science and Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, 77843, USA
| | - Gregory A Johnson
- Departments of Animal Science and Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, 77843, USA
| | - Fuller W Bazer
- Departments of Animal Science and Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, 77843, USA.
| |
Collapse
|
28
|
Aranciaga N, Morton JD, Maes E, Gathercole JL, Berg DK. Proteomic determinants of uterine receptivity for pregnancy in early and mid-postpartum dairy cows†. Biol Reprod 2021; 105:1458-1473. [PMID: 34647570 DOI: 10.1093/biolre/ioab190] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2021] [Revised: 08/03/2021] [Accepted: 10/13/2021] [Indexed: 11/14/2022] Open
Abstract
Dairy cow subfertility is a worldwide issue arising from multiple factors. It manifests in >30% early pregnancy losses in seasonal pasture-grazed herds, especially when cows are inseminated in the early post-partum period. Most losses occur before implantation, when embryo growth depends on factors present in maternal tract fluids. Here we examined the proteomic composition of early and mid-postpartum uterine luminal fluid in crossbred lactating dairy cows to identify molecular determinants of fertility. We also explored changes in uterine luminal fluid from first to third estrus cycles postpartum in individual cows, linking those changes with divergent embryo development. For this, we flushed uteri of 87 cows at day 7 of pregnancy at first and third estrus postpartum, recovering and grading their embryos. Out of 1563 proteins detected, 472 had not been previously reported in this fluid, and 408 were predicted to be actively secreted by bioinformatic analysis. The abundance of 18 proteins with roles in immune regulation and metabolic function (e.g. cystatin B, pyruvate kinase M2) was associated with contrasting embryo quality. Matched-paired pathway analysis indicated that, from first to third estrus postpartum, upregulation of metabolic (e.g. creatine and carbohydrate) and immune (e.g. complement regulation, antiviral defense) processes were related to poorer quality embryos in the third estrus cycle postpartum. Conversely, upregulated signal transduction and protein trafficking appeared related to improved embryo quality in third estrus. These results advance the characterization of the molecular environment of bovine uterine luminal fluid and may aid understanding fertility issues in other mammals, including humans.
Collapse
Affiliation(s)
- Nicolas Aranciaga
- Proteins and Metabolites Team, Agresearch, Christchurch, New Zealand.,Faculty of Agriculture and Life Sciences, Lincoln University, Christchurch, New Zealand.,Animal Biotechnology Team, Agresearch, Hamilton, New Zealand
| | - James D Morton
- Faculty of Agriculture and Life Sciences, Lincoln University, Christchurch, New Zealand
| | - Evelyne Maes
- Proteins and Metabolites Team, Agresearch, Christchurch, New Zealand
| | | | - Debra K Berg
- Animal Biotechnology Team, Agresearch, Hamilton, New Zealand
| |
Collapse
|
29
|
mRNA Expressions of Candidate Genes in Gestational Day 16 Conceptus and Corresponding Endometrium in Repeat Breeder Dairy Cows with Suboptimal Uterine Environment Following Transfer of Different Quality Day 7 Embryos. Animals (Basel) 2021; 11:ani11041092. [PMID: 33920430 PMCID: PMC8070175 DOI: 10.3390/ani11041092] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2021] [Revised: 04/01/2021] [Accepted: 04/06/2021] [Indexed: 11/23/2022] Open
Abstract
Simple Summary The mRNA expression of Interferon-τ (IFNT), IFN stimulated genes (ISG15, CTSL1, RSAD2, SLC2A1, CXCL10, and SLC27A6), Peroxisome proliferator-activated receptors (PPARA, D, and G), and Retinoid X receptors (RXRA, B, and G) genes and proteins (IFNT, ISG15, CXCL10, PPARG, RXRG, SLC2A1, and SLC27A6) were lower and MUC1 at mRNA and protein levels, was greater in gestation day (GD) 16 embryo and corresponding endometrium of subclinical endometritis cows, and in cows following transfer of poor quality embryo (Grade 3). All genes and proteins but MUC1 expression was lower in GD16 tubular conceptus and corresponding endometrium vs. GD16 filamentous conceptus and matching endometrium in cows with SCE and in cows following the transfer of Grade 3 embryo. Disrupted embryo-uterine communication by altered expression of candidate genes in SCE cows, and in cows following the transfer of poor GD7 embryo negatively programs the conceptus development and plausibly affects the conceptus survival. Abstract Effect of the gestational day (GD) 7 embryo quality grade (QG) and subclinical endometritis (SCE) on mRNA and protein expressions of candidate genes [Interferon-τ (IFNT), IFN stimulated genes (ISG15, CTSL1, RSAD2, SLC2A1, CXCL10, and SLC27A6), Peroxisome proliferator activated receptors (PPARA, D, and G), Retinoid X receptors (RXRA, B, and G), and Mucin-1 (MUC1)] in GD16 conceptus and corresponding endometrium were evaluated. After screening of performance records (n = 2389) and selection of repeat breeders (n = 681), cows with SCE (≥6% polymorphonuclear neutrophils—PMN; n = 180) and no-SCE (<6%PMN; n = 180) received GD7 embryos of different QGs. Based on GD16 conceptus recovery, cows with SCE (n = 30) and No- SCE (n = 30) that received GD7 embryos QG1 (good, n = 20), 2 (fair, n = 20), and 3 (poor, n = 20) were included for gene analysis. mRNA and protein expressions (IFNT, ISG15, CXCL10, PPARG, RXRG, SLC2A1, and SLC27A6) differed between SCE and embryo QG groups. All genes but MUC1 and all proteins but MUC1 expression was greater in filamentous conceptus and corresponding endometrium vs. tubular conceptus and matching endometrium in SCE and embryo QG groups. In conclusion, disrupted embryo-uterine communication by altered expression of candidate genes in SCE cows, and in cows following the transfer of poor embryo negatively programs the conceptus development and plausibly affects conceptus survival.
Collapse
|
30
|
Madoz LV, Rabaglino MB, Migliorisi AL, Jaureguiberry M, Perez Wallace S, Lorenti N, Domínguez G, Giuliodori MJ, de la Sota RL. Association between progesterone concentration and endometrial gene expression in dairy cows. Domest Anim Endocrinol 2021; 74:106481. [PMID: 32622107 DOI: 10.1016/j.domaniend.2020.106481] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Revised: 03/01/2020] [Accepted: 03/26/2020] [Indexed: 01/10/2023]
Abstract
The objective of this study was to evaluate the association between progesterone concentration on Days 4 and 9 of the estrus cycle and endometrial transcriptome at Day 9 in lactating grazing dairy cows. Blood samples were obtained on Days 0, 4, and 9 for progesterone measurement by chemiluminescence. Cows were assigned to one of the following groups (n = 3 per group): cows with low physiological progesterone on Day 4, cows in anestrous, cows with high physiological progesterone on Day 4, and superovulated cows. Endometrial biopsy samples were obtained on Day 9 for RNA sequencing. Quality control and determination of differentially expressed genes (false discovery rate <0.05) were determined using the edgeR package for R software. We identified 3,042 differentially expressed genes among the 4 groups. Cows having high physiological progesterone and superovulated cows showed high similarities and clustered apart from those in anestrus or having low physiological progesterone. Functional analysis using Database for Annotation, Visualization, and Integrated Discovery revealed that endometrial genes upregulated by low progesterone concentration are enriched genes involved in the immune system and inflammatory response. Conversely, cows with high physiological progesterone concentration presented an endometrial transcriptome with similarities to cows with good genetic merit for fertility, showing upregulation of genes related to uterine relaxation-contraction, focal adhesion, GnRH signaling pathway, and epidermal growth factor-like related terms, suggesting a favorable embryo environment. In conclusion, our results support the concept that there is a threshold of progesterone concentration at the beginning of the luteal phase associated with endometrial expression of critical genes involved in the preparation of the uterine environment for embryo implantation.
Collapse
Affiliation(s)
- L V Madoz
- Instituto de Investigaciones en Reproducción Animal (INIRA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Argentina; National Research Council, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | - M B Rabaglino
- National Research Council, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina; Instituto de Investigación en Ciencias de la Salud, Consejo Nacional de Investigaciones Científicas y Técnicas, Córdoba, Argentina
| | - A L Migliorisi
- Instituto de Investigaciones en Reproducción Animal (INIRA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Argentina
| | - M Jaureguiberry
- Instituto de Investigaciones en Reproducción Animal (INIRA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Argentina; National Research Council, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
| | | | - N Lorenti
- Práctica Privada, Brandsen, BA, Argentina
| | - G Domínguez
- Práctica Privada, Venado Tuerto, SF, Argentina
| | - M J Giuliodori
- Cátedra de Fisiología, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Argentina
| | - R L de la Sota
- Instituto de Investigaciones en Reproducción Animal (INIRA), Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata, La Plata, Argentina; National Research Council, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina.
| |
Collapse
|
31
|
Flinn T, Kleemann DO, Swinbourne AM, Kelly JM, Weaver AC, Walker SK, Gatford KL, Kind KL, van Wettere WHEJ. Neonatal lamb mortality: major risk factors and the potential ameliorative role of melatonin. J Anim Sci Biotechnol 2020; 11:107. [PMID: 33292527 PMCID: PMC7643391 DOI: 10.1186/s40104-020-00510-w] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2020] [Accepted: 09/07/2020] [Indexed: 12/19/2022] Open
Abstract
High incidences of pre-weaning mortality continue to limit global sheep production, constituting a major economic and welfare concern. Despite significant advances in genetics, nutrition, and management, the proportion of lamb deaths has remained stable at 15–20% over the past four decades. There is mounting evidence that melatonin can improve outcomes in compromised ovine pregnancies via enhanced uterine bloodflow and neonatal neuroprotection. This review provides an overview of the major risk factors and underlying mechanisms involved in perinatal lamb mortality and discusses the potential of melatonin treatment as a remedial strategy. Supplementing pregnant ewes with melatonin enhances uterine bloodflow and fetal oxygenation, and potentially birthweight and neonatal thermogenic capacity. Melatonin freely crosses the ovine placenta and blood-brain barrier and provides neuroprotection to the fetal lamb during periods of chronic and acute hypoxia throughout gestation, with improved behavioural outcomes in hypoxic neonates. The current literature provides strong evidence that maternal melatonin treatment improves outcomes for lambs which experience compromised in utero development or prolonged parturition, though to date this has not been investigated in livestock production systems. As such there is a clear basis for continued research into the effects of maternal melatonin supplementation during gestation on pre-weaning survival under extensive production conditions.
Collapse
Affiliation(s)
- Tom Flinn
- Davies Livestock Research Centre, School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA, Australia.
| | - David O Kleemann
- Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, SA, Australia
| | - Alyce M Swinbourne
- Davies Livestock Research Centre, School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA, Australia
| | - Jennifer M Kelly
- Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, SA, Australia
| | - Alice C Weaver
- Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, SA, Australia
| | - Simon K Walker
- Turretfield Research Centre, South Australian Research and Development Institute, Rosedale, SA, Australia
| | - Kathryn L Gatford
- Robinson Research Institute, Adelaide Medical School, The University of Adelaide, Adelaide, SA, Australia
| | - Karen L Kind
- Davies Livestock Research Centre, School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA, Australia
| | - William H E J van Wettere
- Davies Livestock Research Centre, School of Animal and Veterinary Sciences, The University of Adelaide, Roseworthy, SA, Australia
| |
Collapse
|
32
|
Machine learning approach to integrated endometrial transcriptomic datasets reveals biomarkers predicting uterine receptivity in cattle at seven days after estrous. Sci Rep 2020; 10:16981. [PMID: 33046742 PMCID: PMC7550564 DOI: 10.1038/s41598-020-72988-3] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2020] [Accepted: 09/07/2020] [Indexed: 12/12/2022] Open
Abstract
The main goal was to apply machine learning (ML) methods on integrated multi-transcriptomic data, to identify endometrial genes capable of predicting uterine receptivity according to their expression patterns in the cow. Public data from five studies were re-analyzed. In all of them, endometrial samples were obtained at day 6–7 of the estrous cycle, from cows or heifers of four different European breeds, classified as pregnant (n = 26) or not (n = 26). First, gene selection was performed through supervised and unsupervised ML algorithms. Then, the predictive ability of potential key genes was evaluated through support vector machine as classifier, using the expression levels of the samples from all the breeds but one, to train the model, and the samples from that one breed, to test it. Finally, the biological meaning of the key genes was explored. Fifty genes were identified, and they could predict uterine receptivity with an overall 96.1% accuracy, despite the animal’s breed and category. Genes with higher expression in the pregnant cows were related to circadian rhythm, Wnt receptor signaling pathway, and embryonic development. This novel and robust combination of computational tools allowed the identification of a group of biologically relevant endometrial genes that could support pregnancy in the cattle.
Collapse
|
33
|
Simintiras CA, Sánchez JM, McDonald M, Lonergan P. The biochemistry surrounding bovine conceptus elongation†. Biol Reprod 2020; 101:328-337. [PMID: 31181571 DOI: 10.1093/biolre/ioz101] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2019] [Revised: 05/10/2019] [Accepted: 06/07/2019] [Indexed: 12/29/2022] Open
Abstract
Conceptus elongation is a fundamental developmental event coinciding with a period of significant pregnancy loss in cattle. The process has yet to be recapitulated in vitro, whereas in vivo it is directly driven by uterine secretions and indirectly influenced by systemic progesterone. To better understand the environment facilitating this critical reproductive phenomenon, we interrogated the biochemical composition of uterine luminal fluid from heifers with high vs physiological circulating progesterone on days 12-14 of the estrous cycle-the window of conceptus elongation-initiation-by high-throughput untargeted ultrahigh-performance liquid chromatography tandem mass spectroscopy. A total of 233 biochemicals were identified, clustering within 8 superpathways [amino acids (33.9%), lipids (32.2%), carbohydrates (8.6%), nucleotides (8.2%), xenobiotics (6.4%), cofactors and vitamins (5.2%), energy substrates (4.7%), and peptides (0.9%)] and spanning 66 metabolic subpathways. Lipids dominated total progesterone (39.1%) and day (57.1%) effects; however, amino acids (48.5%) and nucleotides (14.8%) accounted for most day by progesterone interactions. Corresponding pathways over-represented in response to day and progesterone include (i) methionine, cysteine, s-adenosylmethionine, and taurine (9.3%); (ii) phospholipid (7.4%); and (iii) (hypo)xanthine and inosine purine metabolism (5.6%). Moreover, under physiological conditions, the uterine lumen undergoes a metabolic shift after day 12, and progesterone supplementation increases total uterine luminal biochemical abundance at a linear rate of 0.41-fold day-1-resulting in a difference (P ≤ 0.0001) by day 14. This global metabolic analysis of uterine fluid during the initiation of conceptus elongation offers new insights into the biochemistry of maternal-embryo communication, with implications for improving ruminant fertility.
Collapse
Affiliation(s)
| | - José M Sánchez
- School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland
| | - Michael McDonald
- School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland
| | - Patrick Lonergan
- School of Agriculture and Food Science, University College Dublin, Belfield, Dublin, Ireland
| |
Collapse
|
34
|
Rocha CC, da Silva Andrade SC, de Melo GD, Motta IG, Coutinho LL, Gonella-Diaza AM, Binelli M, Pugliesi G. Early pregnancy-induced transcripts in peripheral blood immune cells in Bos indicus heifers. Sci Rep 2020; 10:13733. [PMID: 32792605 PMCID: PMC7426272 DOI: 10.1038/s41598-020-70616-8] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2020] [Accepted: 06/08/2020] [Indexed: 01/05/2023] Open
Abstract
Immune cells play a central role in early pregnancy establishment in cattle. We aimed to: (1) discover novel early-pregnancy-induced genes in peripheral blood mononuclear cells (PBMC); and (2) characterize the temporal pattern of early-pregnancy-induced transcription of select genes in PBMC and peripheral blood polymorphonuclear cells (PMN). Beef heifers were artificially inseminated on D0 and pregnancies were diagnosed on D28. On D10, 14, 16, 18, and 20, blood was collected for isolation of PBMC and PMN from heifers that were retrospectively classified as pregnant (P) or non-pregnant (NP). PBMC samples from D18 were submitted to RNAseq and 220 genes were differentially expressed between pregnant (P) and non-pregnant (NP) heifers. The temporal abundance of 20 transcripts was compared between P and NP, both in PBMC and PMN. In PBMC, pregnancy stimulated transcription of IFI6, RSAD2, IFI44, IFITM2, CLEC3B, OAS2, TNFSF13B, DMKN and LGALS3BP as early as D18. Expression of IFI44, RSAD2, OAS2, LGALS3BP, IFI6 and C1R in PMN was stimulated in the P group from D18. The novel early-pregnancy induced genes discovered in beef heifers will allow both the understanding of the role of immune cells during the pre-attachment period and the development of technologies to detect early pregnancies in beef cattle.
Collapse
Affiliation(s)
- Cecilia Constantino Rocha
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil
| | | | - Gabriela Dalmaso de Melo
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil
| | - Igor Garcia Motta
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil
| | - Luiz Lehmann Coutinho
- Laboratory of Animal Biotechnology, School of Agriculture Luiz de Queiroz, University of São Paulo, Piracicaba, São Paulo, Brazil
| | - Angela Maria Gonella-Diaza
- North Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Marianna, FL, USA
| | - Mario Binelli
- Department of Animal Sciences, University of Florida, Gainesville, FL, USA
| | - Guilherme Pugliesi
- Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil.
| |
Collapse
|
35
|
O'Neil EV, Burns GW, Spencer TE. Extracellular vesicles: Novel regulators of conceptus-uterine interactions? Theriogenology 2020; 150:106-112. [PMID: 32164992 PMCID: PMC8559595 DOI: 10.1016/j.theriogenology.2020.01.083] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2020] [Accepted: 01/31/2020] [Indexed: 12/13/2022]
Abstract
This review focuses on extracellular vesicles (EV) in the uterus and their potential biological roles as mediators of conceptus-uterine interactions essential for implantation and pregnancy establishment. Growing evidence supports the idea that EV are produced by both the endometrium and conceptus during pregnancy. Exosomes and microvesicles, collectively termed EV, mediate cell-cell communication in other tissues and organs. EV have distinct cargo, including lipids, proteins, RNAs, and DNA, that vary depending on the cell of origin and regulate processes including angiogenesis, adhesion, proliferation, cell survival, inflammation, and immune response in recipient cells. Molecular crosstalk between the endometrial epithelium and the blastocyst/conceptus, particularly the trophectoderm, regulates early pregnancy events and is a prerequisite for successful implantation. Trafficking of EV between the conceptus and endometrium may represent a key form of communication important for pregnancy establishment. Increased understanding of EV in the uterine environment and their physiological roles in endometrial-conceptus interactions is expected to provide opportunities to improve pregnancy success.
Collapse
Affiliation(s)
- Eleanore V O'Neil
- Division of Animal Sciences, University of Missouri, Columbia, MO, 65203, USA
| | - Gregory W Burns
- Division of Animal Sciences, University of Missouri, Columbia, MO, 65203, USA
| | - Thomas E Spencer
- Division of Animal Sciences, University of Missouri, Columbia, MO, 65203, USA.
| |
Collapse
|
36
|
Simintiras CA, Sánchez JM, McDonald M, Martins T, Binelli M, Lonergan P. Biochemical characterization of progesterone-induced alterations in bovine uterine fluid amino acid and carbohydrate composition during the conceptus elongation window†. Biol Reprod 2020; 100:672-685. [PMID: 30388203 DOI: 10.1093/biolre/ioy234] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2018] [Revised: 10/17/2018] [Accepted: 10/30/2018] [Indexed: 12/15/2022] Open
Abstract
Pregnancy establishment in cattle is contingent on conceptus elongation-a fundamental developmental event coinciding with the time during which most pregnancies fail. Elongation in vivo is directly driven by uterine secretions, indirectly influenced by systemic progesterone concentrations, and has yet to be recapitulated in vitro. To better understand the microenvironment evolved to facilitate this phenomenon, the amino acid and carbohydrate composition of uterine fluid was interrogated using high-throughput metabolomics on days 12, 13, and 14 of the estrous cycle from heifers with normal and high circulating progesterone. A total of 99 biochemicals (79 amino acids and 20 carbohydrates) were consistently identified, of which 31 showed a day by progesterone interaction. Fructose and mannitol/sorbitol did not exhibit a day by progesterone interaction, but displayed the greatest individual fluctuations (P ≤ 0.05) with respective fold increases of 18.39 and 28.53 in high vs normal progesterone heifers on day 12, and increases by 10.70-fold and 14.85-fold in the uterine fluid of normal progesterone animals on day 14 vs day 12. Moreover, enrichment analyses revealed that the phenylalanine, glutathione, polyamine, and arginine metabolic pathways were among the most affected by day and progesterone. In conclusion, progesterone had a largely stabilizing effect on amino acid flux, and identified biochemicals of likely importance to conceptus elongation initiation include arginine, fructose, glutamate, and mannitol/sorbitol.
Collapse
Affiliation(s)
| | - José M Sánchez
- School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland
| | - Michael McDonald
- School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland
| | - Thiago Martins
- Department of Animal Sciences, University of Florida, Gainesville, Florida, USA.,Department of Animal Reproduction, University of São Paulo, Pirassununga, São Paulo, Brazil
| | - Mario Binelli
- Department of Animal Sciences, University of Florida, Gainesville, Florida, USA
| | - Pat Lonergan
- School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland
| |
Collapse
|
37
|
Núñez-Olivera R, Cuadro F, Bosolasco D, de Brun V, de la Mata J, Brochado C, Meikle A, Bó GA, Menchaca A. Effect of equine chorionic gonadotropin (eCG) administration and proestrus length on ovarian response, uterine functionality and pregnancy rate in beef heifers inseminated at a fixed-time. Theriogenology 2020; 151:16-27. [PMID: 32251936 DOI: 10.1016/j.theriogenology.2020.03.031] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2019] [Revised: 03/22/2020] [Accepted: 03/24/2020] [Indexed: 01/08/2023]
Abstract
The objective of the present study was to evaluate the effect of equine chorionic gonadotropin (eCG) administration associated to different proestrus lengths for Fixed-time AI (FTAI) in beef heifers. In Experiment 1, pre-pubertal heifers (n = 46) received a 6-day estradiol/progesterone-based treatment (J-Synch protocol), and were then allocated into four experimental groups in a 2 × 2 factorial design, to receive or not receive eCG (300 IU) at the time of intravaginal progesterone device removal, and to receive GnRH at 48 h or 72 h after device removal (to induce shortened and prolonged proestrus length, respectively). Endometrial samples were obtained 6 d after ovulation from the cranial portion of the uterine horn. The eCG administration induced greater serum estradiol-17β concentrations before ovulation (P < 0.05) and greater proportion of heifers bearing a competent corpus luteum after ovulation (P = 0.054). Delaying GnRH administration from 48 h to 72 h induced a longer interval from device removal to ovulation (i.e., prolonged proestrus; P < 0.05), larger diameter of the ovulatory follicle, and greater progesterone concentrations on Day 10-11 after ovulation. Heifers in eCG + GnRH72h group had more uterine receptors in luminal epithelium than those in eCG + GnRH48h group (PR and ERα), and than those in No eCG + GnRH72h group (PR) (P < 0.05). No effect of eCG or GnRH treatments was found in endometrial gene expression of progesterone and estrogen receptors. In Experiment 2, a total of 2,598 heifers received the J-Synch protocol associated or not with eCG administration at device removal, followed by FTAI/GnRH at 60 or 72 h after device removal (i.e., prolonged proestrus protocol). Heifers that received eCG had greater P/AI than those not receiving eCG (P < 0.05) and there was an interaction between eCG treatment and time of FTAI. The lowest P/AI was found in those heifers that received FTAI/GnRH at 72 h without eCG treatment at device removal (P < 0.05), and no differences were found between the other experimental groups. In conclusion, prolonging the length of proestrus in J-Synch protocol improves ovulatory follicular diameter and luteal function; and the administration of eCG at device removal improves preovulatory estradiol concentrations and luteal function. Finally, P/AI was enhanced by eCG treatment and the improvement was more evident when FTAI/GnRH was performed at 72 h after device removal.
Collapse
Affiliation(s)
- R Núñez-Olivera
- Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Montevideo, Uruguay; Programa de Posgrado de la Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay
| | - F Cuadro
- Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Montevideo, Uruguay; Programa de Posgrado de la Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay
| | - D Bosolasco
- Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Montevideo, Uruguay; Programa de Posgrado de la Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay
| | - V de Brun
- Programa de Posgrado de la Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay; Laboratorio de Endocrinología y Metabolismo Animal, Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay
| | - J de la Mata
- Instituto de Reproducción Animal Córdoba, Córdoba, Argentina; Facultad de Agronomía, Universidad Nacional de La Pampa, Santa Rosa, La Pampa, Argentina
| | - C Brochado
- Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Montevideo, Uruguay; Programa de Posgrado de la Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay
| | - A Meikle
- Laboratorio de Endocrinología y Metabolismo Animal, Facultad de Veterinaria, Universidad de la República, Montevideo, Uruguay
| | - G A Bó
- Instituto de Reproducción Animal Córdoba, Córdoba, Argentina; Instituto A.P. de Ciencias Básicas y Aplicadas, Medicina Veterinaria, Universidad Nacional de Villa María, Villa del Rosario, Córdoba, Argentina
| | - A Menchaca
- Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Montevideo, Uruguay.
| |
Collapse
|
38
|
Moraes JGN, Behura SK, Bishop JV, Hansen TR, Geary TW, Spencer TE. Analysis of the uterine lumen in fertility-classified heifers: II. Proteins and metabolites†. Biol Reprod 2020; 102:571-587. [PMID: 31616912 PMCID: PMC7331878 DOI: 10.1093/biolre/ioz197] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2019] [Revised: 09/09/2019] [Accepted: 10/02/2019] [Indexed: 02/06/2023] Open
Abstract
Survival and growth of the bovine conceptus is dependent on endometrial secretions or histotroph. Previously, serial blastocyst transfer was used to classify heifers as high fertile (HF), subfertile (SF), or infertile (IF). Here, we investigated specific histotroph components (proteins and metabolites) in the uterine lumen of day 17 fertility-classified heifers. Interferon tau (IFNT) was more abundant in uterine lumenal fluid (ULF) of pregnant HF than SF animals as the conceptus was longer in HF heifers. However, no differences in endometrial expression of selected classical and nonclassical interferon-stimulated genes (ISGs) were observed, suggesting that IFNT signaling in the endometrium of pregnant HF and SF heifers was similar. Pregnancy significantly increased the abundance of several proteins in ULF. Based on functional annotation, the abundance of a number of proteins involved in energy metabolism, oxidative stress, amino acid metabolism, and cell proliferation and differentiation were greater in the ULF of pregnant HF than SF heifers. Metabolomics analysis found that pregnancy only changed the metabolome composition of ULF from HF heifers. The majority of the metabolites that increased in the ULF of pregnant HF as compared to SF heifers were associated with energy and amino acid metabolism. The observed differences in ULF proteome and metabolome are hypothesized to influence uterine receptivity with consequences on conceptus development and survival in fertility-classified heifers.
Collapse
Affiliation(s)
- Joao G N Moraes
- Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA
| | - Susanta K Behura
- Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA
| | - Jeanette V Bishop
- Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA and
| | - Thomas R Hansen
- Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA and
| | - Thomas W Geary
- USDA-ARS, Fort Keogh Livestock and Range Research Laboratory, Miles City, Montana, USA
| | - Thomas E Spencer
- Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA
| |
Collapse
|
39
|
Eozenou C, Lesage-Padilla A, Mauffré V, Healey GD, Camous S, Bolifraud P, Giraud-Delville C, Vaiman D, Shimizu T, Miyamoto A, Sheldon IM, Constant F, Pannetier M, Sandra O. FOXL2 is a Progesterone Target Gene in the Endometrium of Ruminants. Int J Mol Sci 2020; 21:ijms21041478. [PMID: 32098259 PMCID: PMC7073057 DOI: 10.3390/ijms21041478] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2020] [Revised: 02/13/2020] [Accepted: 02/19/2020] [Indexed: 02/08/2023] Open
Abstract
Forkhead Box L2 (FOXL2) is a member of the FOXL class of transcription factors, which are essential for ovarian differentiation and function. In the endometrium, FOXL2 is also thought to be important in cattle; however, it is not clear how its expression is regulated. The maternal recognition of pregnancy signal in cattle, interferon-Tau, does not regulate FOXL2 expression. Therefore, in the present study, we examined whether the ovarian steroid hormones that orchestrate implantation regulate FOXL2 gene expression in ruminants. In sheep, we confirmed that FOXL2 mRNA and protein was expressed in the endometrium across the oestrous cycle (day 4 to day 15 post-oestrus). Similar to the bovine endometrium, ovine FOXL2 endometrial expression was low during the luteal phase of the oestrous cycle (4 to 12 days post-oestrus) and at implantation (15 days post-oestrus) while mRNA and protein expression significantly increased during the luteolytic phase (day 15 post-oestrus in cycle). In pregnant ewes, inhibition of progesterone production by trilostane during the day 5 to 16 period prevented the rise in progesterone concentrations and led to a significant increase of FOXL2 expression in caruncles compared with the control group (1.4-fold, p < 0.05). Ovariectomized ewes or cows that were supplemented with exogenous progesterone for 12 days or 6 days, respectively, had lower endometrial FOXL2 expression compared with control ovariectomized females (sheep, mRNA, 1.8-fold; protein, 2.4-fold; cattle; mRNA, 2.2-fold; p < 0.05). Exogenous oestradiol treatments for 12 days in sheep or 2 days in cattle did not affect FOXL2 endometrial expression compared with control ovariectomized females, except at the protein level in both endometrial areas in the sheep. Moreover, treating bovine endometrial explants with exogenous progesterone for 48h reduced FOXL2 expression. Using in vitro assays with COS7 cells we also demonstrated that progesterone regulates the FOXL2 promoter activity through the progesterone receptor. Collectively, our findings imply that endometrial FOXL2 is, as a direct target of progesterone, involved in early pregnancy and implantation.
Collapse
Affiliation(s)
- Caroline Eozenou
- Université Paris-Saclay, INRAE, ENVA, UVSQ, BREED, 78350 Jouy-en-Josas, France; (A.L.-P.); (V.M.); (S.C.); (P.B.); (C.G.-D.); (F.C.); (M.P.)
- Institut Pasteur, UMR 3738, Biologie du Développement et Cellules Souches, Laboratoire de Génétique du Développement Humain, 25 rue du docteur roux, F75015 Paris, France
- Correspondence: (C.E.); (O.S.); Tel.: +33-144389136 (C.E.); +33-134642343 (O.S.)
| | - Audrey Lesage-Padilla
- Université Paris-Saclay, INRAE, ENVA, UVSQ, BREED, 78350 Jouy-en-Josas, France; (A.L.-P.); (V.M.); (S.C.); (P.B.); (C.G.-D.); (F.C.); (M.P.)
| | - Vincent Mauffré
- Université Paris-Saclay, INRAE, ENVA, UVSQ, BREED, 78350 Jouy-en-Josas, France; (A.L.-P.); (V.M.); (S.C.); (P.B.); (C.G.-D.); (F.C.); (M.P.)
| | - Gareth D. Healey
- Swansea University Medical School, Swansea University, Singleton Park, Swansea SA2 8PP, UK; (G.D.H.); (I.M.S.)
| | - Sylvaine Camous
- Université Paris-Saclay, INRAE, ENVA, UVSQ, BREED, 78350 Jouy-en-Josas, France; (A.L.-P.); (V.M.); (S.C.); (P.B.); (C.G.-D.); (F.C.); (M.P.)
| | - Philippe Bolifraud
- Université Paris-Saclay, INRAE, ENVA, UVSQ, BREED, 78350 Jouy-en-Josas, France; (A.L.-P.); (V.M.); (S.C.); (P.B.); (C.G.-D.); (F.C.); (M.P.)
| | - Corinne Giraud-Delville
- Université Paris-Saclay, INRAE, ENVA, UVSQ, BREED, 78350 Jouy-en-Josas, France; (A.L.-P.); (V.M.); (S.C.); (P.B.); (C.G.-D.); (F.C.); (M.P.)
| | - Daniel Vaiman
- Institut Cochin, INSERM U1016, UMR 8104 CNRS, Faculté René Descartes, 24 rue du Faubourg St Jacques, 75014 Paris, France;
| | - Takashi Shimizu
- Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan; (T.S.); (A.M.)
| | - Akio Miyamoto
- Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan; (T.S.); (A.M.)
| | - Iain Martin Sheldon
- Swansea University Medical School, Swansea University, Singleton Park, Swansea SA2 8PP, UK; (G.D.H.); (I.M.S.)
| | - Fabienne Constant
- Université Paris-Saclay, INRAE, ENVA, UVSQ, BREED, 78350 Jouy-en-Josas, France; (A.L.-P.); (V.M.); (S.C.); (P.B.); (C.G.-D.); (F.C.); (M.P.)
| | - Maëlle Pannetier
- Université Paris-Saclay, INRAE, ENVA, UVSQ, BREED, 78350 Jouy-en-Josas, France; (A.L.-P.); (V.M.); (S.C.); (P.B.); (C.G.-D.); (F.C.); (M.P.)
| | - Olivier Sandra
- Université Paris-Saclay, INRAE, ENVA, UVSQ, BREED, 78350 Jouy-en-Josas, France; (A.L.-P.); (V.M.); (S.C.); (P.B.); (C.G.-D.); (F.C.); (M.P.)
- Correspondence: (C.E.); (O.S.); Tel.: +33-144389136 (C.E.); +33-134642343 (O.S.)
| |
Collapse
|
40
|
Challenges in studying preimplantation embryo-maternal interaction in cattle. Theriogenology 2020; 150:139-149. [PMID: 31973965 DOI: 10.1016/j.theriogenology.2020.01.019] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2020] [Accepted: 01/11/2020] [Indexed: 01/10/2023]
Abstract
A comprehensive understanding of the complex embryo-maternal interactions during the preimplantation period requires the analysis of the very early stages of pregnancy encompassing early embryonic development, maternal recognition and the events leading to implantation. Despite the fact that embryo development until blastocyst stage is somewhat autonomous (i.e., does not require contact with the maternal reproductive tract and can be successfully recapitulated in vitro), many studies on ruminant embryo production have focused on the fundamental question of why: (i) only 30%-40% of immature oocytes develop to the blastocyst stage and (ii) the quality of such blastocysts continually lags behind that of blastocysts produced in vivo. Clear evidence indicates that in vitro culture conditions are far from optimal with deficiencies being manifested in short- and long-term effects on the embryo. Thus, enhanced knowledge of mechanisms controlling embryo-maternal interactions would allow the design of novel strategies to improve in vitro embryo conditions and reproductive outcomes in cattle.
Collapse
|
41
|
Scaravaggi I, Borel N, Romer R, Imboden I, Ulbrich SE, Zeng S, Bollwein H, Bauersachs S. Cell type-specific endometrial transcriptome changes during initial recognition of pregnancy in the mare. Reprod Fertil Dev 2019; 31:496-508. [PMID: 30253121 DOI: 10.1071/rd18144] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2018] [Accepted: 08/27/2018] [Indexed: 12/11/2022] Open
Abstract
Previous endometrial gene expression studies during the time of conceptus migration did not provide final conclusions on the mechanisms of maternal recognition of pregnancy (MRP) in the mare. This called for a cell type-specific endometrial gene expression analysis in response to embryo signals to improve the understanding of gene expression regulation in the context of MRP. Laser capture microdissection was used to collect luminal epithelium (LE), glandular epithelium and stroma from endometrial biopsies from Day 12 of pregnancy and Day 12 of the oestrous cycle. RNA sequencing (RNA-Seq) showed greater expression differences between cell types than between pregnant and cyclic states; differences between the pregnant and cyclic states were mainly found in LE. Comparison with a previous RNA-Seq dataset for whole biopsy samples revealed the specific origin of gene expression differences. Furthermore, genes specifically differentially expressed (DE) in one cell type were found that were not detectable as DE in biopsies. Overall, this study revealed spatial information about endometrial gene expression during the phase of initial MRP. The conceptus induced changes in the expression of genes involved in blood vessel development, specific spatial regulation of the immune system, growth factors, regulation of prostaglandin synthesis, transport prostaglandin receptors, specifically prostaglandin F receptor (PTGFR) in the context of prevention of luteolysis.
Collapse
Affiliation(s)
- Iside Scaravaggi
- Clinic of Reproductive Medicine, Department for Farm Animals, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland
| | - Nicole Borel
- Institute of Veterinary Pathology, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland
| | - Rebekka Romer
- Clinic of Reproductive Medicine, Department for Farm Animals, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland
| | - Isabel Imboden
- Clinic of Reproductive Medicine, Department for Farm Animals, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland
| | - Susanne E Ulbrich
- Animal Physiology, Institute of Agricultural Sciences, ETH Zurich, Universitätstrasse 2, 8092 Zurich, Switzerland
| | - Shuqin Zeng
- Clinic of Reproductive Medicine, Department for Farm Animals, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland
| | - Heinrich Bollwein
- Clinic of Reproductive Medicine, Department for Farm Animals, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland
| | - Stefan Bauersachs
- Clinic of Reproductive Medicine, Department for Farm Animals, University of Zurich, Winterthurerstrasse 260, 8057 Zurich, Switzerland
| |
Collapse
|
42
|
Sánchez JM, Simintiras CA, Lonergan P. Aspects of embryo-maternal communication in establishment of pregnancy in cattle. Anim Reprod 2019; 16:376-385. [PMID: 32435281 PMCID: PMC7234086 DOI: 10.21451/1984-3143-ar2019-0075] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Establishment of pregnancy in mammals requires reciprocal molecular communication between the conceptus and endometrium that modifies the endometrial transcriptome and uterine luminal milieu to support pregnancy. Due to the small size of the early embryo and elongating conceptus relative to the volume of the uterine lumen, collection of endometrium adjacent to the developing conceptus is difficult following conventional uterine flushing methods in cattle. Use of endometrial explants in culture can overcome this challenge and reveal information about the dialogue between the developing embryo and the uterus. The aim of this short review is to summarize some of our recent findings in relation to embryo maternal interaction during bovine pregnancy establishment and to put them in the wider context of fertility in cattle.
Collapse
Affiliation(s)
- José M Sánchez
- School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland
| | | | - Patrick Lonergan
- School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland
| |
Collapse
|
43
|
Ealy AD, Wooldridge LK, McCoski SR. BOARD INVITED REVIEW: Post-transfer consequences of in vitro-produced embryos in cattle. J Anim Sci 2019; 97:2555-2568. [PMID: 30968113 DOI: 10.1093/jas/skz116] [Citation(s) in RCA: 92] [Impact Index Per Article: 15.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2019] [Accepted: 04/08/2019] [Indexed: 01/09/2023] Open
Abstract
In vitro embryo production (IVP) in cattle has gained worldwide interest in recent years, but the efficiency of using IVP embryos for calf production is far from optimal. This review will examine the pregnancy retention rates of IVP embryos and explore causes for pregnancy failures. Based on work completed over the past 25 yr, only 27% of cattle receiving IVP embryos will produce a live calf. Approximately 60% of these pregnancies fail during the first 6 wk of gestation. When compared with embryos generated by superovulation, pregnancy rates are 10% to 40% lower for cattle carrying IVP embryos, exemplifying that IVP embryos are consistently less competent than in vivo-generated embryos. Several abnormalities have been observed in the morphology of IVP conceptuses. After transfer, IVP embryos are less likely to undergo conceptus elongation, have reduced embryonic disk diameter, and have compromised yolk sac development. Marginal binucleate cell development, cotyledon development, and placental vascularization have also been documented, and these abnormalities are associated with altered fetal growth trajectories. Additionally, in vitro culture conditions increase the risk of large offspring syndrome. Further work is needed to decipher how the embryo culture environment alters post-transfer embryo development and survival. The risk of these neonatal disorders has been reduced by the use of serum-free synthetic oviductal fluid media formations and culture in low oxygen tension. However, alterations are still evident in IVP oocyte and embryo transcript abundances, timing of embryonic cleavage events and blastulation, incidence of aneuploidy, and embryonic methylation status. The inclusion of oviductal and uterine-derived embryokines in culture media is being examined as one way to improve the competency of IVP embryos. To conclude, the evidence presented herein clearly shows that bovine IVP systems still must be refined to make it an economical technology in cattle production systems. However, the current shortcomings do not negate its current value for certain embryo production needs and for investigating early embryonic development in cattle.
Collapse
Affiliation(s)
- Alan D Ealy
- Department of Animal & Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA
| | - Lydia K Wooldridge
- Department of Animal & Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA
| | - Sarah R McCoski
- Department of Animal & Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA
| |
Collapse
|
44
|
Reliable and Standardized Animal Models to Study the Pathogenesis of Bluetongue and Schmallenberg Viruses in Ruminant Natural Host Species with Special Emphasis on Placental Crossing. Viruses 2019; 11:v11080753. [PMID: 31443153 PMCID: PMC6722754 DOI: 10.3390/v11080753] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2019] [Revised: 07/19/2019] [Accepted: 08/13/2019] [Indexed: 01/03/2023] Open
Abstract
Starting in 2006, bluetongue virus serotype 8 (BTV8) was responsible for a major epizootic in Western and Northern Europe. The magnitude and spread of the disease were surprisingly high and the control of BTV improved significantly with the marketing of BTV8 inactivated vaccines in 2008. During late summer of 2011, a first cluster of reduced milk yield, fever, and diarrhoea was reported in the Netherlands. Congenital malformations appeared in March 2012 and Schmallenberg virus (SBV) was identified, becoming one of the very few orthobunyaviruses distributed in Europe. At the start of both epizootics, little was known about the pathogenesis and epidemiology of these viruses in the European context and most assumptions were extrapolated based on other related viruses and/or other regions of the World. Standardized and repeatable models potentially mimicking clinical signs observed in the field are required to study the pathogenesis of these infections, and to clarify their ability to cross the placental barrier. This review presents some of the latest experimental designs for infectious disease challenges with BTV or SBV. Infectious doses, routes of infection, inoculum preparation, and origin are discussed. Particular emphasis is given to the placental crossing associated with these two viruses.
Collapse
|
45
|
Drews B, Rudolf Vegas A, van der Weijden VA, Milojevic V, Hankele AK, Schuler G, Ulbrich SE. Do ovarian steroid hormones control the resumption of embryonic growth following the period of diapause in roe deer (Capreolus capreolus)? Reprod Biol 2019; 19:149-157. [PMID: 31147267 DOI: 10.1016/j.repbio.2019.04.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2018] [Revised: 04/05/2019] [Accepted: 04/27/2019] [Indexed: 01/01/2023]
Abstract
Embryonic diapause in the European roe deer includes a period of five months from August to December in which embryonic development is extremely decelerated. Following exit from diapause, the embryo rapidly elongates and subsequently implants. In diapausing carnivores and marsupials, resumption of embryonic growth is regulated by ovarian steroid hormones. In the roe deer, the role of steroid hormones is not known to date. In the present study, progesterone (P4), estradiol-17β (E2) and total estrogens (Etot) were determined in blood plasma and endometrium of roe deer shot in the course of regular huntings between September and December. Steroid hormone concentrations were correlated to the corresponding size of the embryo derived from ex vivo uterine flushing and to the date of sampling. The mean plasma concentrations of P4 (5.4 ± 0.2 ng/ml, mean ± SE, N = 87), E2 (24.3 ± 2.6 pg/ml, N = 86) and Etot (21.7 ± 2.6 pg/ml, N = 78) remained constant over the sampling period and were not correlated to embryonic size. Likewise, endometrial concentrations of P4 (66.1 ± 6.5 ng/g), E2 (284.0 ± 24.43 pg/g) and, Etot (440.9 ± 24.43 pg/g) showed no changes over time [corrected]. Therefore, it was concluded that ovarian steroid hormones do not play a determining role in resumption of embryonic growth following the period of diapause in the roe deer.
Collapse
Affiliation(s)
- Barbara Drews
- ETH Zurich, Animal Physiology, Institute of Agricultural Sciences, Switzerland
| | - Alba Rudolf Vegas
- ETH Zurich, Animal Physiology, Institute of Agricultural Sciences, Switzerland
| | | | - Vladimir Milojevic
- ETH Zurich, Animal Physiology, Institute of Agricultural Sciences, Switzerland
| | | | - Gerhard Schuler
- Clinic for Veterinary Obstetrics, Gynecology and Andrology of Large and Small Animals, Justus-Liebig-University, Giessen, Germany
| | | |
Collapse
|
46
|
Biase FH, Hue I, Dickinson SE, Jaffrezic F, Laloe D, Lewin HA, Sandra O. Fine-tuned adaptation of embryo-endometrium pairs at implantation revealed by transcriptome analyses in Bos taurus. PLoS Biol 2019; 17:e3000046. [PMID: 30978203 PMCID: PMC6481875 DOI: 10.1371/journal.pbio.3000046] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2018] [Revised: 04/24/2019] [Accepted: 03/29/2019] [Indexed: 12/14/2022] Open
Abstract
Interactions between embryo and endometrium at implantation are critical for the progression of pregnancy. These reciprocal actions involve exchange of paracrine signals that govern implantation and placentation. However, it remains unknown how these interactions between the conceptus and the endometrium are coordinated at the level of an individual pregnancy. Under the hypothesis that gene expression in endometrium is dependent on gene expression of extraembryonic tissues and genes expressed in extraembryonic tissues are dependent of genes expressed in the endometrium, we performed an integrative analysis of transcriptome profiles of paired extraembryonic tissue and endometria obtained from cattle (Bos taurus) pregnancies initiated by artificial insemination. We quantified strong dependence (|r| > 0.95, empirical false discovery rate [eFDR] < 0.01) in transcript abundance of genes expressed in the extraembryonic tissues and genes expressed in the endometrium. The profiles of connectivity revealed distinct coexpression patterns of extraembryonic tissues with caruncular and intercaruncular areas of the endometrium. Notably, a subset of highly coexpressed genes between extraembryonic tissue (n = 229) and caruncular areas of the endometrium (n = 218, r > 0.9999, eFDR < 0.001) revealed a blueprint of gene expression specific to each pregnancy. Gene ontology analyses of genes coexpressed between extraembryonic tissue and endometrium revealed significantly enriched modules with critical contribution for implantation and placentation, including “in utero embryonic development,” “placenta development,” and “regulation of transcription.” Coexpressing modules were remarkably specific to caruncular or intercaruncular areas of the endometrium. The quantitative association between genes expressed in extraembryonic tissue and endometrium emphasize a coordinated communication between these two entities in mammals. We provide evidence that implantation in mammalian pregnancy relies on the ability of the extraembryonic tissue and the endometrium to develop a fine-tuned adaptive response characteristic of each pregnancy. An integrative analysis of interactions between conceptus and endometrium, in cattle, reveals complex regulatory networks operating at the time of implantation, highlighting the unique transcriptional blueprint of conceptus-maternal communication Implantation in mammals requires a complex crosstalk between the conceptus (the embryo and associated membranes) and the uterus. An imbalanced regulation of the factors contributing to these interactions has negative impacts on the attachment of the fetus, the progression of the pregnancy, and the progeny. Focusing on paired conceptus–endometrium analyses of individual pregnancies in cows, we have determined that communication at implantation encompasses synchronized genome-wide coregulation of genes. Gene regulatory interactions between one conceptus and the surrounding maternal tissue vary between endometrial regions containing or lacking glands. Our data reveal new insights, to our knowledge, on the coordination of molecular mechanisms that contribute to implantation and pregnancy establishment in mammals. We conclude that the biological response of the endometrium is embryo-specific, a phenomenon that deserves further investigation in the context of assisted reproductive technologies.
Collapse
Affiliation(s)
- Fernando H. Biase
- Department of Animal Sciences, Auburn University, Auburn, Alabama, United States of America
- * E-mail: (FB); (OS)
| | - Isabelle Hue
- UMR BDR, INRA, ENVA, Université Paris Saclay, Jouy-en-Josas, France
| | - Sarah E. Dickinson
- Department of Animal Sciences, Auburn University, Auburn, Alabama, United States of America
| | - Florence Jaffrezic
- UMR1313 GABI, INRA, AgroParisTech, Université Paris Saclay, Jouy-en-Josas, France
| | - Denis Laloe
- UMR1313 GABI, INRA, AgroParisTech, Université Paris Saclay, Jouy-en-Josas, France
| | - Harris A. Lewin
- Department of Evolution and Ecology, University of California, Davis, California, United States of America
| | - Olivier Sandra
- UMR BDR, INRA, ENVA, Université Paris Saclay, Jouy-en-Josas, France
- * E-mail: (FB); (OS)
| |
Collapse
|
47
|
Lonergan P, Sánchez JM, Mathew DJ, Passaro C, Fair T. Embryo development in cattle and interactions with the reproductive tract. Reprod Fertil Dev 2019; 31:118-125. [PMID: 32188547 DOI: 10.1071/rd18409] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Embryo mortality is a major contributor to poor reproductive efficiency and profitability in cattle production systems. Coordinated interaction between the developing embryo or conceptus and the maternal reproductive tract is essential for pregnancy establishment in mammals. Up to the blastocyst stage, the embryo can grow in the absence of contact with the oviduct or uterus; however, conceptus elongation after hatching and before implantation, a characteristic of ruminant early development, is entirely maternally driven and is essential to ensure that sufficient quantities of interferon-τ (IFNT) are secreted by the developing conceptus to abrogate the mechanisms that bring about luteolysis. Surprisingly, many questions, such as the threshold level of IFNT required for pregnancy maintenance, remain unanswered. Failure of the conceptus to elongate undoubtedly results in embryonic loss and is thus believed to contribute greatly to reproductive failure in cattle.
Collapse
Affiliation(s)
- P Lonergan
- School of Agriculture and Food Science, University College Dublin, Belfield, D04 N2E, Dublin 4, Ireland
| | - José María Sánchez
- School of Agriculture and Food Science, University College Dublin, Belfield, D04 N2E, Dublin 4, Ireland
| | - Daniel J Mathew
- School of Agriculture and Food Science, University College Dublin, Belfield, D04 N2E, Dublin 4, Ireland
| | - Claudia Passaro
- School of Agriculture and Food Science, University College Dublin, Belfield, D04 N2E, Dublin 4, Ireland
| | - Trudee Fair
- School of Agriculture and Food Science, University College Dublin, Belfield, D04 N2E, Dublin 4, Ireland
| |
Collapse
|
48
|
Sánchez JM, Mathew DJ, Passaro C, Fair T, Lonergan P. Embryonic maternal interaction in cattle and its relationship with fertility. Reprod Domest Anim 2018; 53 Suppl 2:20-27. [PMID: 30238655 DOI: 10.1111/rda.13297] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2018] [Revised: 07/19/2018] [Accepted: 07/23/2018] [Indexed: 11/30/2022]
Abstract
Embryo mortality is a major contributor to poor reproductive efficiency and profitability in cattle production systems. While conception is achieved (i.e., the oocyte is fertilized) in the vast majority of cases if insemination is carried out correctly, a significant proportion of the resulting embryos fail to develop to term. Appropriate communication between the developing conceptus and the maternal endometrium is essential for the establishment and maintenance of pregnancy in all mammals. Up to the blastocyst stage, around Days 7-9, contact worth the female reproductive system is not required. However, the process of conceptus elongation after hatching and prior to implantation is entirely maternally driven and is essential to ensure that sufficient quantities of interferon-tau (IFNT) are secreted by the developing conceptus to abrogate the mechanisms that bring about luteolysis. While the importance of conceptus-derived IFNT in maternal recognition of pregnancy and prevention of luteolysis in cattle is unequivocal, many questions, such as the threshold level of IFNT required for pregnancy maintenance, remain unanswered. Furthermore, the precise role of IFNT-independent mechanisms in pregnancy establishment remains to be elucidated. Irrespective of this, failure of the conceptus to elongate undoubtedly results in embryonic loss and is thus believed to contribute greatly to reproductive failure in cattle. This review will address some of these answered questions and try to shed some light on those gaps in knowledge that could potentially contribute to improved embryo survival and reproductive efficiency.
Collapse
Affiliation(s)
- José María Sánchez
- School of Agriculture and Food Science, University College Dublin, Dublin, Ireland
| | - Daniel J Mathew
- School of Agriculture and Food Science, University College Dublin, Dublin, Ireland
| | - Claudia Passaro
- School of Agriculture and Food Science, University College Dublin, Dublin, Ireland
| | - Trudee Fair
- School of Agriculture and Food Science, University College Dublin, Dublin, Ireland
| | - Pat Lonergan
- School of Agriculture and Food Science, University College Dublin, Dublin, Ireland
| |
Collapse
|
49
|
McCoski SR, Vailes MT, Owens CE, Cockrum RR, Ealy AD. Exposure to maternal obesity alters gene expression in the preimplantation ovine conceptus. BMC Genomics 2018; 19:737. [PMID: 30305020 PMCID: PMC6180665 DOI: 10.1186/s12864-018-5120-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2018] [Accepted: 09/26/2018] [Indexed: 02/07/2023] Open
Abstract
Background Embryonic and fetal exposure to maternal obesity causes several maladaptive morphological and epigenetic changes in exposed offspring. The timing of these events is unclear, but changes can be observed even after a short exposure to maternal obesity around the time of conception. The hypothesis of this work is that maternal obesity influences the ovine preimplantation conceptus early in pregnancy, and this exposure will affect gene expression in embryonic and extraembryonic tissues. Results Obese and lean ewe groups were established by overfeeding or normal feeding, respectively. Ewes were then bred to genetically similar rams. Conceptuses were collected at day 14 of gestation. Morphological assessments were made, conceptuses were sexed by genomic PCR analysis, and samples underwent RNA-sequencing analysis. While no obvious morphological differences existed between conceptuses, differentially expressed genes (≥ 2-fold; ≥ 0.2 RPKM; ≤ 0.05 FDR) were detected based on maternal obesity exposure (n = 21). Also, differential effects of maternal obesity were noted on each conceptus sex (n = 347). A large portion of differentially expressed genes were associated with embryogenesis and placental development. Conclusions Findings reveal that the preimplantation ovine conceptus genome responds to maternal obesity in a sex-dependent manner. The sexual dimorphism in response to the maternal environment coupled with changes in placental gene expression may explain aberrations in phenotype observed in offspring derived from obese females. Electronic supplementary material The online version of this article (10.1186/s12864-018-5120-0) contains supplementary material, which is available to authorized users.
Collapse
Affiliation(s)
- Sarah R McCoski
- Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, 3430 Litton-Reaves Hall (0306), Virginia, Blacksburg, VA, 24061, USA
| | - McCauley T Vailes
- Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, 3430 Litton-Reaves Hall (0306), Virginia, Blacksburg, VA, 24061, USA
| | - Connor E Owens
- Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA
| | - Rebecca R Cockrum
- Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA
| | - Alan D Ealy
- Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, 3430 Litton-Reaves Hall (0306), Virginia, Blacksburg, VA, 24061, USA.
| |
Collapse
|
50
|
Vailes MT, McCoski SR, Wooldridge LK, Reese ST, Pohler KG, Roper DA, Mercadante VR, Ealy AD. Post-transfer outcomes in cultured bovine embryos supplemented with epidermal growth factor, fibroblast growth factor 2, and insulin-like growth factor 1. Theriogenology 2018; 124:1-8. [PMID: 30317067 DOI: 10.1016/j.theriogenology.2018.09.023] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2018] [Revised: 07/28/2018] [Accepted: 09/23/2018] [Indexed: 01/24/2023]
Abstract
This work examined the downstream fetal and placental outcomes of introducing a cocktail of uterine-derived growth factors during bovine embryo culture. Abattoir-derived bovine oocytes were matured and fertilized in vitro. On day 4 post-fertilization, ≥ 8-cell embryos were harvested, pooled and exposed to an embryokine mix, termed EFI, which contained recombinant human epidermal growth factor (10 ng/ml), bovine fibroblast growth factor-2 (10 ng/ml) and human insulin-like growth factor 1 (50 ng/ml) or to a carrier-only control treatment (CON). On day 7, individual, transfer-quality embryos were transferred to recipients. Timed ovulation was completed in mature, non-suckled commercial beef cows. Cows either were artificial inseminated (AI) or received an embryo (ET) on day 7 post-estrus (n = 23-31 cows/treatment over 4 replicate studies). The percentage of grade 1 and 2 morulae and blastocysts was greater (P < 0.05) for EFI-treated embryos than CON. The percentage of pregnant cows diagnosed by transrectal ultrasonography did not differ among the AI and ET groups on days 28, 42 and 56 post-estrus. There also were no differences in the ratio of male to female fetuses determined on day 60 post-estrus by transrectal ultrasonography. On day 21 post-estrus, the relative abundance of three interferon-stimulated gene (ISG) transcripts in peripheral leukocytes were not different based on AI/ET group or the sex of the conceptus. Circulating pregnancy-associated glycoprotein (PAG) concentrations differed (P < 0.05) among days. Also, a difference in PAG concentrations (P < 0.05) were detected between male and female pregnancies in the CON-ET group but not in the AI or EFI-ET groups. Crown-rump length was not affected by AI/ET group on day 42 but were less (P < 0.05) in the CON and EFI-ET groups than the AI group on day 56. These findings implicate EFI supplementation as a means for improving transferable embryo production in a bovine IVP system, but it is not clear if this treatment improves embryo competency after ET.
Collapse
Affiliation(s)
- McCauley T Vailes
- Department of Animal & Poultry Sciences, Virginia Tech, Blacksburg, VA, USA
| | - Sarah R McCoski
- Department of Animal & Poultry Sciences, Virginia Tech, Blacksburg, VA, USA
| | - Lydia K Wooldridge
- Department of Animal & Poultry Sciences, Virginia Tech, Blacksburg, VA, USA
| | - Sydney T Reese
- Department of Animal Sciences, Texas A&M University, College Station, TX, USA
| | - Ky G Pohler
- Department of Animal Sciences, Texas A&M University, College Station, TX, USA
| | - David A Roper
- Department of Animal Sciences & Veterinary Technology, Tarleton State University, Stephenville, TX, USA
| | - Vitor R Mercadante
- Department of Animal & Poultry Sciences, Virginia Tech, Blacksburg, VA, USA
| | - Alan D Ealy
- Department of Animal & Poultry Sciences, Virginia Tech, Blacksburg, VA, USA.
| |
Collapse
|