The biologic activity of 5α-reduced pregnanes in the late gestation mare Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/fj236662f

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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 5α-pregnane-3, 20-dione (5α), 5α-pregnane-3β, 20α-diol (βα), 3β-hydroxy-5α-pregnan-3-one (3β) and 20α-hydroxy-5α-prenan-3-one (20α), increase to near μg/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, 5α, βα, 20α, 3β 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 3β 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, 5α, βα, 20α, 3β 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.
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