Profiling equine endometrial gene expression during maternal recognition of pregnancy

2013 Summer.Includes bibliographical references.In order to maintain a pregnancy in the mare the presence of a conceptus in the uterus must be recognized by the endometrium. This is known as maternal recognition of pregnancy (MRP) and is required to prevent the secretion of prostaglandin F2α (PGF), starting on day 14 post-ovulation, from the endometrium into circulation. The secretion of PGF initiates luteolysis of the corpus luteum, which is secreting progesterone, the hormone needed to maintain a pregnancy. However, little is known about maternal recognition of pregnancy in the mare. It is critical that the embryo is mobile throughout the entire uterine lumen to signal maternal recognition of pregnancy between days 12-14. The embryo ceases mobility on day 16 by fixing at the base of one of the uterine horns, independent of the side of ovulation. Previously, an equine specific microarray analysis was performed on days 14, 16, and 18 post-ovulation comparing endometrial gene expression between pregnant and non-pregnant mares. From this analysis, ten genes: juxtaposed with another zinc finger protein 1-like (JAZF1), secretory phospholipase A2 (sPLA2), S100 calcium binding protein G (S100G), estrogen receptor 1 (ESR1), solute carrier family 36 (proton/amino acid symporter), member 2 (SLC36A2), methyltransferase-like protein 7A-like (METTL7A), retinaldehyde dehydrogenase 1-like (RALDH1), eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3), dickkopf 1 homolog (DKK1), and adrenomedullin (ADM), were identified as having consistently higher or lower expression levels in the endometrium of pregnant mares at all three time points. The goal of this study was to confirm and expand upon the results of the microarray on days 14, 16, and 18, by real time PCR (RT-PCR), and to evaluate differential gene expression on day 12. We hypothesized that the expression of the aforementioned ten genes will be the same on day 12 endometrium from pregnant mares as days 14, 16, and 18 endometrium from pregnant mares because day 12 is the start of maternal recognition of pregnancy. To test this hypothesis, 12 normally cycling mares were utilized in a crossover design. Each mare was assigned to a random collection day (day 12, 14, 16, or 18 post-ovulation) and provided endometrial samples from a pregnant cycle and then a non-pregnant (non-mated) cycle (n=3 per day). Endometrial biopsy samples were snap frozen and stored until total RNA was isolated for RT-PCR. This analysis was consistent with the microarray results for days 14, 16, and 18. On day 12, 6 of the 10 differentially expressed genes had the same pattern of expression as day 14, but 4 of the genes had opposite expression levels on day 12. Endometrial samples were then collected on day 13 post-ovulation (n=3) and processed for protein isolation and immunohistochemical analysis. The specificity of rabbit polyclonal antibodies for sPLA2 and DKK1 for equine endometrium were confirmed by Western Blot analysis. Upon conformation of antibody specificity, immunohistochemistry was used to determine the localization of sPLA2, DKK1, and ESR1. sPLA2 was localized to the endometrial epithelium and glandular epithelium in the endometrium from both pregnant and non-pregnant mares. DKK1 showed a localization difference between endometrial samples from pregnant and non-pregnant mares. In the endometrium from the pregnant mare, DKK1 was localized to the endometrial epithelium and the glandular cells, and in the endometrium from the non-pregnant mare DKK1 was localized throughout the glandular region, but not in the endometrial epithelium. ESR1 also showed differential localization based upon pregnancy status. In the endometrium from the pregnant mare, ESR1 was located in the basal glandular region and not close to the lumen or in the endometrial epithelium. In endometrium from non-pregnant mares it was located throughout the entire glandular region and in the endometrial epithelium. This experiment identified the expression patterns of ten genes, previously identified from a microarray analysis, on days 12, 14, 16, and 18 post-ovulation in the endometrium from pregnant and non-pregnant mares. The expression patterns on days 14, 16, and 18 were consistent across each day. Day 12 revealed mixed results for the expression patterns of these genes, indicating that they were undergoing transcriptional regulation based upon the presence or absence of a mobile conceptus. By determining what signal causes these genes to be higher or lower expressed in the endometrium of pregnant mares, it may lead to the identity of the signal for maternal recognition of pregnancy in the mare

Non-Coding RNA Sequencing of Equine Endometrium During Maternal Recognition of Pregnancy.

Maternal recognition of pregnancy (MRP) in the mare is not well defined. In a non-pregnant mare, prostaglandin F2α (PGF) is released on day 14 post-ovulation (PO) to cause luteal regression, resulting in loss of progesterone production. Equine MRP occurs prior to day 14 to halt PGF production. Studies have failed to identify a gene candidate for MRP, so attention has turned to small, non-coding RNAs. The objective of this study was to evaluate small RNA (<200 nucleotides) content in endometrium during MRP. Mares were used in a cross-over design with each having a pregnant and non-mated cycle. Each mare was randomly assigned to collection day 11 or 13 PO (n = 3/day) and endometrial biopsies were obtained. Total RNA was isolated and sequencing libraries were prepared using a small RNA library preparation kit and sequenced on a HiSeq 2000. EquCab3 was used as the reference genome and DESeq2 was used for statistical analysis. On day 11, 419 ncRNAs, representing miRNA, snRNA, snoRNA, scaRNA, and vaultRNA, were different between pregnancy statuses, but none on day 13. Equine endometrial ncRNAs with unknown structure and function were also identified. This study is the first to describe ncRNA transcriptome in equine endometrium. Identifying targets of these ncRNAs could lead to determining MRP

Effects of immunization against bone morphogenetic protein-15 and growth differentiation factor-9 on ovarian function in mares

Currently there is no contraceptive vaccine that can cause permanent sterility in mares. This study investigates the effect of vaccination against oocyte-specific growth factors, Bone Morphogenetic Protein 15 (BMP-15) and Growth Differentiation Factor 9 (GDF-9), on ovarian function of mares. It was hypothesized that immunization against these growth factors would prevent ovulation and/or accelerate depletion of the oocyte reserve. For this study, 30 mares were randomly assigned to three groups (n=10/group) and vaccinated with BMP-15 or GDF-9 peptides conjugated to KLH and adjuvant, or a control of phosphate buffered saline and adjuvant. Horses received vaccinations at weeks 0, 6, 12, and 18. Ovarian activity and estrous behavior were evaluated 3 days a week via ultrasonography and interaction with a stallion. The study was initiated on March1, 2016. Upon evaluation of ovulation rate, the GDF-9 group did not have a difference (P=0.66) in ovulation rate when compared to controls (10.8 and 10.0 ovulations, respectively), but the number of ovulations in the BMP-15 group was less (P=0.02; 4.9 ovulations). Average follicle size prior to ovulation was less (P \u3c 0.0001) in both treatment groups compared to controls. Estrous behavior was altered in both the BMP-15 and GDF-9 groups compared to controls after the second vaccination (P=0.05 and 0.03, respectively). Although further research is required to determine the continued effects of vaccination against GDF-9 on ovulation rates, these results indicate that vaccination against BMP-15 and GDF-9 could serve as a contraceptive in wild horse populations

Coding RNA Sequencing of Equine Endometrium during Maternal Recognition of Pregnancy

Equine maternal recognition of pregnancy (MRP) is a process whose signal remains unknown. During MRP the conceptus and endometrium communicate to attenuate prostaglandin F2α (PGF) secretion, sparing the corpus luteum and maintaining progesterone production. Recognition of a mobile conceptus by the endometrium is critical by days 14–16 post-ovulation (PO), when endometrium produces PGF, initiating luteolysis. The objective of this study was to evaluate endometrial gene expression changes based upon pregnancy status via RNA sequencing. This experiment utilized a cross-over design with each mare serving as both a pregnant and non-mated control on days nine, 11, and 13 PO (n = 3/status/day). Mares were randomly assigned to collection day and pregnancy confirmed by terminal uterine lavage at the time of endometrial biopsy. Total RNA was isolated and libraries prepared using Illumina TruSeq RNA sample preparation kit. Reads were mapped and annotated using HISAT2 and Stringtie. Expression values were evaluated with DESEQ2 (P ≤ 0.05 indicated significance). On day nine, 11, and 13 there were 1435, 1435 and 916 significant transcripts, respectively. Multiple genes with splice variants had different expression patterns within the same day. These are the first data to evaluate the endometrial transcriptome during MRP on days nine, 11, and 13

Effects of immunization against bone morphogenetic protein-15 and growth differentiation factor-9 on ovarian function in mares

Currently there is no contraceptive vaccine that can cause permanent sterility in mares. This study investigates the effect of vaccination against oocyte-specific growth factors, Bone Morphogenetic Protein 15 (BMP-15) and Growth Differentiation Factor 9 (GDF-9), on ovarian function of mares. It was hypothesized that immunization against these growth factors would prevent ovulation and/or accelerate depletion of the oocyte reserve. For this study, 30 mares were randomly assigned to three groups (n=10/group) and vaccinated with BMP-15 or GDF-9 peptides conjugated to KLH and adjuvant, or a control of phosphate buffered saline and adjuvant. Horses received vaccinations at weeks 0, 6, 12, and 18. Ovarian activity and estrous behavior were evaluated 3 days a week via ultrasonography and interaction with a stallion. The study was initiated on March1, 2016. Upon evaluation of ovulation rate, the GDF-9 group did not have a difference (P=0.66) in ovulation rate when compared to controls (10.8 and 10.0 ovulations, respectively), but the number of ovulations in the BMP-15 group was less (P=0.02; 4.9 ovulations). Average follicle size prior to ovulation was less (P \u3c 0.0001) in both treatment groups compared to controls. Estrous behavior was altered in both the BMP-15 and GDF-9 groups compared to controls after the second vaccination (P=0.05 and 0.03, respectively). Although further research is required to determine the continued effects of vaccination against GDF-9 on ovulation rates, these results indicate that vaccination against BMP-15 and GDF-9 could serve as a contraceptive in wild horse populations

Coding RNA Sequencing of Equine Endometrium during Maternal Recognition of Pregnancy.

Equine maternal recognition of pregnancy (MRP) is a process whose signal remains unknown. During MRP the conceptus and endometrium communicate to attenuate prostaglandin F2α (PGF) secretion, sparing the corpus luteum and maintaining progesterone production. Recognition of a mobile conceptus by the endometrium is critical by days 14-16 post-ovulation (PO), when endometrium produces PGF, initiating luteolysis. The objective of this study was to evaluate endometrial gene expression changes based upon pregnancy status via RNA sequencing. This experiment utilized a cross-over design with each mare serving as both a pregnant and non-mated control on days nine, 11, and 13 PO (n = 3/status/day). Mares were randomly assigned to collection day and pregnancy confirmed by terminal uterine lavage at the time of endometrial biopsy. Total RNA was isolated and libraries prepared using Illumina TruSeq RNA sample preparation kit. Reads were mapped and annotated using HISAT2 and Stringtie. Expression values were evaluated with DESEQ2 (P ≤ 0.05 indicated significance). On day nine, 11, and 13 there were 1435, 1435 and 916 significant transcripts, respectively. Multiple genes with splice variants had different expression patterns within the same day. These are the first data to evaluate the endometrial transcriptome during MRP on days nine, 11, and 13

Non-Coding RNA Sequencing of Equine Endometrium During Maternal Recognition of Pregnancy.

Maternal recognition of pregnancy (MRP) in the mare is not well defined. In a non-pregnant mare, prostaglandin F2α (PGF) is released on day 14 post-ovulation (PO) to cause luteal regression, resulting in loss of progesterone production. Equine MRP occurs prior to day 14 to halt PGF production. Studies have failed to identify a gene candidate for MRP, so attention has turned to small, non-coding RNAs. The objective of this study was to evaluate small RNA (<200 nucleotides) content in endometrium during MRP. Mares were used in a cross-over design with each having a pregnant and non-mated cycle. Each mare was randomly assigned to collection day 11 or 13 PO (n = 3/day) and endometrial biopsies were obtained. Total RNA was isolated and sequencing libraries were prepared using a small RNA library preparation kit and sequenced on a HiSeq 2000. EquCab3 was used as the reference genome and DESeq2 was used for statistical analysis. On day 11, 419 ncRNAs, representing miRNA, snRNA, snoRNA, scaRNA, and vaultRNA, were different between pregnancy statuses, but none on day 13. Equine endometrial ncRNAs with unknown structure and function were also identified. This study is the first to describe ncRNA transcriptome in equine endometrium. Identifying targets of these ncRNAs could lead to determining MRP

Non-Coding RNA Sequencing of Equine Endometrium During Maternal Recognition of Pregnancy

Maternal recognition of pregnancy (MRP) in the mare is not well defined. In a non-pregnant mare, prostaglandin F2α (PGF) is released on day 14 post-ovulation (PO) to cause luteal regression, resulting in loss of progesterone production. Equine MRP occurs prior to day 14 to halt PGF production. Studies have failed to identify a gene candidate for MRP, so attention has turned to small, non-coding RNAs. The objective of this study was to evaluate small RNA (<200 nucleotides) content in endometrium during MRP. Mares were used in a cross-over design with each having a pregnant and non-mated cycle. Each mare was randomly assigned to collection day 11 or 13 PO (n = 3/day) and endometrial biopsies were obtained. Total RNA was isolated and sequencing libraries were prepared using a small RNA library preparation kit and sequenced on a HiSeq 2000. EquCab3 was used as the reference genome and DESeq2 was used for statistical analysis. On day 11, 419 ncRNAs, representing miRNA, snRNA, snoRNA, scaRNA, and vaultRNA, were different between pregnancy statuses, but none on day 13. Equine endometrial ncRNAs with unknown structure and function were also identified. This study is the first to describe ncRNA transcriptome in equine endometrium. Identifying targets of these ncRNAs could lead to determining MRP