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Progesterone is a steroid hormone secreted from the corpus luteum (CL), which is responsible for establishment and maintenance of pregnancy. Early embryonic mortality often occurs due to inadequate regulation of uterine prostaglandin (PG) F2αsecretion, leading to a decrease in progesterone and loss of pregnancy. The objective of the current study was to determine the effects of fish meal supplementation on luteal sensitivity to intrauterine infusions of PGF2α. Nonlactating beef cows received corn gluten meal or fish meal supplementation for 60 days. Cows were administered four intrauterine infusions of 0.25 mL saline at 6-h intervals (n = 6 corn gluten meal; n = 5 fish meal) or two doses of 0.5 mg PGF2αin 0.25 mL saline at 12-h intervals (n = 11 corn gluten meal; n = 11 fish meal) commencing on days 10 to 12 of the estrous cycle. At time of each infusion, luteal biopsies were collected to determine the effects of supplementation on expression of immediate early and steroidogenic genes involved in cholesterol transport and progesterone biosynthesis. Transrectal ultrasonography was performed to measure diameter of CL, and blood samples were collected to determine serum progesterone. Intrauterine infusion of PGF2αresulted in upregulation or no change in FOS, NR4A1, and 3BHSD and downregulation in LDLR, STARD1, and CYP11A1. Although CL diameter decreased, infusion of PGF2αresulted in functional regression in 91% of cows supplemented with corn gluten meal, and only 46% for fish meal supplemented animals. Results demonstrate that fish meal supplementation alters luteal sensitivity to PGF2α, which may affect fertility.Fish meal supplementation alters luteal sensitivity to prostaglandin F2αin the bovine.

Zonadhesin is a rapidly evolving protein in the sperm acrosome that confers species specificity to sperm-zona pellucida adhesion. Though structural variation in zonadhesin likely contributes to its species-specific function, the protein has not previously been characterized in organisms capable of interbreeding. Here we compared properties of zonadhesin in several animals, including the horse (Equus caballus), donkey (E. asinus), and Grevy's zebra (E. grevyi) to determine if variation in zonadhesin correlates with ability of gametes to cross-fertilize. Zonadhesin localized to the apical acrosomes of spermatozoa from all three Equusspecies, similar to its localization in other animals. Likewise, in horse and donkey testis, zonadhesin was detected only in germ cells, first in the acrosomal granule of round spermatids and then in the developing acrosomes of elongating spermatids. Among non-Equusspecies, D3-domain polypeptides of mature, processed zonadhesin varied markedly in size and detergent solubility. However, zonadhesin D3-domain polypeptides in horse, donkey, and zebra spermatozoa exhibited identical electrophoretic mobility and detergent solubility. Equuszonadhesin D3-polypeptides (p110/p80 doublet) were most similar in size to porcine and bovine zonadhesin D3-polypeptides (p105). Sequence comparisons revealed that the horse zonadhesin precursor's domain content and arrangement are similar to those of zonadhesin from other large animals. Partial sequences of horse and donkey zonadhesin were much more similar to each other (>99% identity) than they were to orthologous sequences of human, pig, rabbit, and mouse zonadhesin (52%–72% identity). We conclude that conservation of zonadhesin D3-polypeptide properties correlates with ability of Equusspecies to interbreed.

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.

The placenta is essential for fetal development and nutrient exchange in mammalian pregnancy, and placental malformation can lead to grave complications such as preeclampsia (PE) and intrauterine growth restriction (IUGR) of the fetus. PE occurs in approximately 7% of pregnancies in the United States and can lead to maternal and fetal mortality. Studies have identified increased serum testosterone in PE patients, increased placental expression of androgen receptor, and decreased placental aromatase activity. Treatment of pregnant ewes with testosterone propionate during gestational days 60 to 90 (a well-established model of prenatal androgenization) leads to abnormal developmental programming, including IUGR, polycystic ovarian syndrome in female offspring, and decreased fertility in male offspring. While many of these could be contributed to direct effects on the developing fetus, we hypothesize that prenatal androgenization leads to epigenetic changes and perturbed placental development and differentiation, which in turn leads to abnormal fetal programming. Starting on gestational day 60, pregnant ewes were injected IM biweekly with 100 mg testosterone propionate (treated) or 2 mL cottonseed oil (control). On gestational day 90, 5 placentomes closest to the umbilicus were collected and characterized for each fetus. For all control pregnancies, only type A placentomes were present closest to the umbilicus while a range from type A to D, with increasing fetal cotyledon overgrowth, were present in treated pregnancies. Results demonstrate a decrease in global DNA methylation in placentomes in treated (n=6) compared to control ewes (n=4) according to ELISA (p=0.023). In accordance, real time qPCR analysis revealed a 3.3 fold increase in DNMT3amRNA levels in type D placentomes in treated compared to control placentomes (p=0.032). DNMT3bmRNA levels also were increased (1.7 fold) in caruncles from type A treated compared to controls (p=0.023). There was a 7.3 fold increase in ESR2mRNA levels (p=0.047) in type D placentomes (composed primarily of cotyledon tissues) from treated compared to cotyledons in controls, and a 6.2 fold increase in the imprinted gene H19in type A cotyledons from treated compared to controls (p=0.026). In summary, results indicate that prenatal androgenization leads to changes in placentome morphology and a decrease in global DNA methylation. Furthermore, accompanying changes in a number of transcripts were observed, including the DNA methyltransferases DNMT3aand DNMT3b. The observed increase in ESR2mRNA levels suggests a mechanism for the increased proliferation in cotyledon tissue in the prenatal androgenized ewes. Future work will confirm gene specific changes in methylation using chromatin immunoprecipitation, as well as changes at the protein level. Finally, these results indicate that prenatal androgenization leads to epigenetic changes that result in perturbed placental development and function, and can provide new insight into other placental defects. This project is supported by USDA-NIFA-National Institute of Food and Agriculture Grant #2010-38420-20397 National Needs Graduate Fellowship Program.