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The equine placenta is critical in maintaining pregnancy. Not only does it provide essential nutrients and gasses, but it also functions as an endocrine, paracrine, and autocrine organ. For example, it is one of the main sources of estrogen and progesterone in the latter stages of pregnancy. In addition to supplying hormones required for development and pregnancy maintenance, hormones supplied by both the mare and fetus act on the placenta to influence its development and function. The equine placenta possesses both estrogen and progesterone receptors, and a great deal is known about these hormones' effects on placental development. However, very little is known about how, or if, androgens interact with the placenta to affect placental development and/or maintenance. Androgen receptor (AR) has been identified in the placenta of humans, cows, sheep, and rats; however, little is known about its exact functions related to the placenta. The focus of this study was to confirm the presence of AR in the equine placenta. Three term equine placentas from normal pregnancies were utilized in this study. Reverse transcription polymerase chain reaction was used to identify mRNA encoding AR, and Western blotting confirmed the presence of AR protein within the equine placenta. The results of this study provide new insight into the presence of AR, similar to other species including the human, bovine, ovine, and murine placenta.

After spermatogenesis, spermatozoa are not mature and unable to fertilize an oocyte. During transition through the epididymis, spermatozoa mature, and gain fertility and motility. Although epididymal functions are relatively well known, the mechanisms behind maturation are not yet fully understood. Studies indicate proteins are delivered to the spermatozoa during epididymal transit through microvesicles. These microvesicles, called epididymosomes, carry microRNAs (miRNAs). These miRNAs are ∼22 nucleotides in length and regulate mRNA translation. The purpose of this study was to investigate which miRNAs are located within the various regions of stallion epididymal tissue. Three mature stallion epididymides were divided into four sections for miRNA isolation: the caput, proximal corpus, distal corpus and cauda. The contents of the lumen were removed and epididymal tissue was homogenized. Quantitative real-time polymerase chain reaction analysis was used to determine miRNA content. A total of 328 miRNAs were noted in epididymal tissue. A total of 186 of the 346 known equine miRNA transcripts used were found in each section, all four sections also contained miRNAs distinct to specific regions. The cauda contained the greatest number of exclusive miRNA (19) and expressed the greatest number of miRNA (282). This study shows there is indeed a difference in the miRNA profile between regions of the epididymis, and their targeted pathways may effect spermatozoal maturation.

Equine maternal recognition of pregnancy (MRP) is a process whose signal remains unknown. During MRP the conceptus and endometrium communicate to attenuate prostaglandin F2&alpha, (PGF) secretion, sparing the corpus luteum and maintaining progesterone production. Recognition of a mobile conceptus by the endometrium is critical by days 14&ndash, 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 &le, 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.

All epididymal regions are lined with multiple epithelial cell types, each with different functions to provide the luminal environment for spermatozoal maturation. Epithelial cells also create apical blebs, which are released from the apical surface via apocrine secretion and disintegrate in the lumen, thereby releasing epididymosomes. Epididymosomes transport proteins to spermatozoa and contain microRNAs. We hypothesized that epididymosomes also transfer miRNA from epididymal epithelium to spermatozoa. Quantitative real-time polymerase chain reaction was used to determine miRNA profiles of epididymal tissue from caput and cauda, epididymal spermatozoa from caput and cauda, and epididymosomes and from caput, proximal corpus, distal corpus, and cauda. Pathway analysis was performed using DIANA tools on the miRNA unique to caudal spermatozoa. We found 66 newly acquired miRNAs in spermatozoa located in the caudal epididymis. Predicted pathways targeted by these miRNAs suggest a role in cell motility and viability and factors in oocyte and embryo maturation and development. These findings suggest that miRNAs are transported to spermatozoa from epididymal epithelium via epididymosomes.

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

During early pregnancy, the conceptus and mare communicate to establish pregnancy. Cell-secreted vesicles (e.g., exosomes) have been reported in serum. Exosomes contain bioactive materials, such as miRNA, that can mediate cell responses. We hypothesized that a) exosomes are present in mare circulation and quantity varies with pregnancy status, b) exosomes contain miRNAs unique to pregnancy status, and c) miRNAs target pathways in endometrium based upon pregnancy status of the mare. First, serum samples were obtained from mares in a crossover design, with each mare providing samples from a pregnant and nonmated control cycle (n = 3/sample day) on Days 12, 14, 16, and 18 postovulation. Flow cytometry revealed the presence of serum microvesicles in mares in two different-sized populations (greater than or less than 100 nm), validated by transmission electron microscopy. Second, serum was collected on Days 9, 11, and 13 (n = 4/day), and endometrial biopsies were collected on Days 11 and 13 (n = 3/day) from pregnant and nonmated mares. Total RNA from serum exosomes was evaluated with quantitative RT-PCR using equine-specific miRNA sequences. A total of 12 miRNAs were found in different quantities on the specified days. Pathway analysis suggested that miRNAs targeted focal adhesion molecules (FAMs). Transcripts corresponding to FAMs were evaluated in endometrial biopsies. Protein levels and localization for PAK6 and RAF1 were further evaluated. Our data suggest that serum exosomes contain miRNA that differ based upon pregnancy status, and may affect mRNA expression related to focal adhesion pathway in the endometrium, with a potential role in maternal recognition of pregnancy.