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Mares have an atypical hormone profile during pregnancy. Systemic progesterone (P4) levels approach zero by day 220 of gestation. Other reduced pregnanes such as 5��-pregnane-3, 20-dione (5��), 5��-pregnane-3��, 20��-diol (����), 3��-hydroxy-5��-pregnan-3-one (3��) and 20��-hydroxy-5��-prenan-3-one (20��), increase to near ��g/mL levels in the peripheral system of the mare until directly before parturition when they decrease. This unusual hormone profile during gestation indicates the possibility that other pregnanes, not P4, are responsible for uterine quiescence and gonadotropin inhibition during pregnancy. Three experiments were conducted to determine if these steroids have biologic activity. Experiment 1 consisted of jugular vein blood samples taken from mares from ten days pre-partum until the foal heat ovulation, approximately 15 days postpartum. Samples were analyzed for luteinizing hormone (LH), follicle stimulating hormone (FSH), and pregnane content. Concentrations of these hormones were analyzed for serial correlations. There was a serial negative correlation with pregnanes and FSH (p=0.0138), which were analyzed on a same day basis, day -5 to day of foaling. There also was a positive correlation with pregnanes and FSH analyzed from day of foaling to 10 days post-foaling (p<0.00l). There was also a significant negative correlation (p=0.0196) between pregnanes and LH, analyzed on a lag basis, day -5 to day of foaling for pregnanes, and day -5 to day of ovulation for LH. There was also a significant negative correlation when pregnanes were analyzed from day of foaling to 10 days post foaling, and LH was analyzed from 10 days before ovulation to day of ovulation (p=0.004). Maximum pre-partum pregnane levels did not affect time to ovulation (p=0.34). In experiment 2 equine anterior pituitary glands were harvested and the cells plated to begin a primary cell culture. After attachment, the cells were divided into treatment groups: P4, 5��, ����, 20��, 3�� or a control and each group subjected to a 1.0nM Gonadotropin Hormone Releasing Hormone (GnRH) challenge. Subsequently cells and medium were collected and analyzed for LH and FSH content using radioimmunoassay (RIA). The cells did exhibit a response to GnRH (p=0.015 between positive and negative controls) and there was a treatment effect for FSH (p=0.0058); only 3�� resulted in significantly more FSH release than the positive control (p=0.043) after stimulation with GnRH. There was no treatment effect on LH (p=0.56). Experiment 3 analyzed the response of equine uterine myometrial tissue to pregnane treatment. Myometrial tissue was harvested and placed in a 37��C Krebs buffered saline bath, connected to a physiograph and repetitive spontaneous smooth muscle contraction was induced with oxytocin. Tissue was then treated with P4, 5��, ����, 20��, 3�� or a control (ethanol). The amplitude and frequency of the spontaneous contractions were measured and compared to the control. There were no differences between post treatment responses of the control and pregnane treated samples in either frequency (p=0.78) or amplitude (p=0.63) of myometrial contractions. From these data we conclude that in vivo there is a significant and differential physiologic relationship between pre-partum pregnanes and gonadotropins. Due to lack of response it is unlikely that pregnanes inhibit pituitary secretion, and thus may exert their effects elsewhere, such as at the hypothalamus. The involvement of pregnanes in modulating myometrial contractions remains unclear. It is likely that P4 does play a role in decreasing myometrial responses to OT, however, that result was inconsistent in this study. / Graduation date: 2003

Progesterone is unequivocally required for the establishment and maintenance of pregnancy in all mammals studied. Its known functions are complex and encompass global changes in gene expression. Therefore, studies were conducted to characterize the effects of progesterone on expression of genes for endometrial factors having roles in conceptus growth, implantation and establishment of pregnancy. The first study characterized the effect of an artificially induced early increase in circulating progesterone on conceptus growth and development and regulation of expression of galectin-15 (LGALS15), a recently identified protein secreted by the ovine uterine luminal epithelium (LE). Exogenous progesterone beginning on Day 1.5 post-mating accelerated conceptus development on Days 9 and 12. On Day 12 the conceptus was functionally and morphologically advanced to produce greater quantities of interferon tau (IFNT) than blastocysts from control ewes. Further, the endometrium responded to early progesterone and IFNT with early expression of cathepsin L (CTSL), radical S-adenosyl methionine domain containing 2 (RSAD2), and LGALS15 within the endometrium. The second study identifed structural changes within the luminal epithelium which could alter the flux of factors into and out of the uterine lumen to maintain appropriate fetal/maternal communication. In this study, progesterone reduced quantities of proteins associated with both tight and adherens junctions during the elongation period. IFNT subsequently increased these proteins after conceptus elongation. The third and fourth studies identified progesterone-regulated genes which have been implicated as having importance to implantation in sheep, mouse, and human. WNT signaling was transiently downregulated by progesterone, while members of several growth factor families are upregulated including insulin-like growth factor binding proteins (IGFBPs) 1 and 3, hepatocyte growth factor (HGF) and fibroblast growth factor 7 (FGF7), which may enhance conceptus growth. Collectively, these studies assess the role of progesterone in altering gene uterine expression to establish a favorable environment for conceptus development. The long-term goals of these studies are to establish biomarkers of receptivity to conceptus development and implantation, enhance our understanding of gene and pathway regulation in early pregnancy loss, and identify genes which may be targeted in therapeutic strategies to improve reproductive success in humans and animals.

Two experiments were conducted to examine the effect of a cyclopropenoid fatty acid on luteal cell function. In Exp. 1, 12 mature ewes were mated to a fertile ram, assigned to two groups (n = 6/group) and laparotomized on day 18 of gestation. Ewes with corpora lutea (CL) in both ovaries were unilaterally ovariectomized while ewes with a CL in one ovary only were allowed to remain intact. An extract of Sterculia foetida seeds (1.09 mg), consisting of a mixture of fatty acid methyl esters including 750 ug of sterculic acid (SA), or 1.09 mg oleic acid methyl ester (OA) was injected into the artery supplying the ovary bearing CL. Jugular blood was collected on day 18 before surgery and daily thereafter until day 30 of gestation or until detected estrus, whichever occurred first. Sera were assayed for progesterone (P₄) by radioimmunoassay. In Exp. 2, 12 mature ewes were laparotomized on day 10 of the estrous cycle and CL were removed, weighed and sliced for incubation. Corpora lutea from two ewes were pooled for each incubation. Slices of CL were preincubated in medium containing 145 ng/ml of S. foetida extract (100 ng/ml sterculic acid methyl ester) or 145 ng/ml oleic acid methyl ester (control) for 90 min. Then, slices of tissue were washed and reincubated in fresh medium containing 25 ug 22(R)- hydroxycholesterol/ml (0.079 nM final concentration) or 25 ug 5-pregnen-3βol-20-one/m1 (0.084 nM final concentration) for 120 min. Tissue plus medium were analyzed for P₄. Injection of SA or OA on day 18 of gestation caused a reduction in serum concentrations of P4 within 24 h, after which concentrations of steroid remained low and relatively constant in control and those SA-treated ewes that remained pregnant until day 30 of gestation. Three of six ewes that were injected with SA exhibited estrus within 3 to 5 days after treatment. Serum concentrations of P₄ of SA-treated ewes differed from those of OA-injected control ewes (P<0.01). Luteal tissue subjected to SA or OA in vitro did not differ in ability to synthesize P₄ during subsequent incubation in the absence of precursor substrate (incubated controls). Relative to respective incubated controls, P₄ synthesis by tissue previously exposed to SA or OA was not altered by incubation in the presence of 22(R)-hydroxycholesterol. Presence of 5-pregnen-3βol-20-one (pregnenolone) in the medium significantly increased P₄ synthesis by luteal tissue preincubated with SA or OA compared with that of controls. However, response of SA-treated tissue was markedly less than that of tissue exposed to OA (P<0.05). Results of this study suggest that $A can cause regression of CL in 50% of pregnant ewes. Apparently, the luteolytic effect of SA may be caused by its ability to interfere in the conversion of pregnenolone to P₄ by 3β- hydroxysteroid dehydrogenase. / Graduation date: 1991