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The first ovulation induced by male effect in sheep during seasonal anoestrus usually results in the development of a short cycle that can be avoided by progesterone priming before ram introduction. In elucidating the involvement of the hypothalamic-pituitary-gonadal axis in the occurrence of short cycles, the effects of progesterone and the time of anoestrus on the development of male-induced preovulatory follicles were investigated in anoestrous ewes using morphological, endocrine and molecular approaches. Ewes were primed with progesterone for 2 (CIDR2) or 12 days (CIDR12) and untreated ewes used as controls during early (April) and late (June) anoestrus. The duration of follicular growth and the lifespan of the male-induced preovulatory follicles were prolonged by ∼1.6 days in CIDR12 ewes compared with the controls. These changes were accompanied by a delay in the preovulatory LH and FSH surges and ovulation. Intra-follicular oestradiol concentration and mRNA levels of LHCGR and STAR in the granulosa and theca cells of the preovulatory follicles were higher in CIDR12 ewes than the control ewes. The expression of mRNA levels of CYP11A1 and CYP17A1 also increased in theca cells of CIDR12 ewes. CIDR2 ewes gave intermediate results. Moreover, ewes ovulated earlier in June than in April, without changes in the duration of follicular growth, but these effects were unrelated to the lifespan of corpus luteum. Our results give the first evidence supporting the positive effect of progesterone priming on the completion of growth and maturation of preovulatory follicles induced by male effect in seasonal anoestrous ewes, thereby preventing short cycles., (© 2014 Society for Reproduction and Fertility.)

Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (βA, βB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin βB mRNA levels decreased as follicle diameter increased from 1-3 to >6  mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin βA mRNA at 3-4  mm, and TCs more inhibin α mRNA at >4  mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1-3  mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin βA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s).

Seasonal anoestrus in the ewe results from enhanced oestrogen negative feedback. Recent data have implicated the ventromedial preoptic area (vmPOA) as an important site of oestrogen action. This study addressed whether NO acts within the vmPOA to inhibit LH during seasonal anoestrus. In Experiment 1, microimplants containing Nomega-nitro-l-arginine methyl ester (l-NAME, NOS inhibitor), S-methyl thiocitrulline (SMTC, neural NOS (nNOS) inhibitor) or empty implants (control) were administered during mid-anoestrus to the vmPOA. l-NAME, but not SMTC, significantly increased LH pulse frequency. For Experiment 2, ewes in late anoestrus were administered 7-nitroindazole (7NI; nNOS inhibitor), l-NAME, SMTC, or empty implants. 7NI, but not l-NAME or SMTC, increased LH pulse frequency. In Experiment 3, the effects of microimplants and microinjections of l-NAME were compared in mid-anoestrus. Microinjections of l-NAME (300 nl at 10 microg/microl) increased LH pulse frequency, but microimplants did not. In late anoestrus, similar microinjections were ineffective. Taken together, the results of Experiments 1-3 suggested that NO inhibition may be stronger during the middle than at the end of seasonal anoestrus. To test this hypothesis, ewes in Experiment 4 received microinjection of l-NAME or vehicle thrice during the non-breeding season; none of the treatments increased LH pulse frequency. These results indicate that NO plays a role in the vmPOA in suppressing LH secretion during seasonal anoestrus because NOS inhibitors were consistently stimulatory when LH pulse frequency was low. However, the inconsistent and modest effects of these inhibitors suggest that NO actions in this area cannot completely account for the effects of inhibitory photoperiod.

This study describes progesterone receptor (PR) location within uterine cells and associated morphological changes to the uterine luminal and glandular epithelium in seasonally anoestrous brushtail possums treated with oestradiol and/or progesterone. Twenty-four adult female possums (n = 6/group) were treated with oestradiol, progesterone, oestradiol followed by progesterone or with the oil vehicle alone for 6-day periods. Uterine tissue was recovered, weighed and processed for light and transmission electron microscopy and for immunohistochemistry for PRs. Stereological techniques were used to quantify epithelial cell and constituent volumes for both luminal and glandular tissues. Plasma concentrations of oestradiol and progesterone were determined by radioimmunoassay. Mean uterine wet weights were significantly heavier (P < 0.001) following oestradiol/progesterone treatment and maximum gland dilation, cellular and stromal growth, maximum cell height, and cell and constituent volumes were recorded after this treatment regimen. Cell nuclei and debris were commonly observed in gland lumina, and nuclear PRs were found predominantly in stromal cells following oestradiol-only treatment. Sequential treatment with oestradiol and then progesterone caused a decline in the number of positively stained cells. Epithelial cells contained extensive secretory organelles and degenerating cells were common within the glands. Oestradiol treatment induced cell and cell constituent growth and promoted PR formation in anoestrous possum uterine tissue. Subsequent exposure to progesterone stimulated uterine tissues to reach maximum wet weights and led to the cellular maturation necessary to remodel the uterus in preparation for pregnancy.

Follicular waves and associated circulating hormone concentrations were studied during the anovulatory season in pony mares (n=8). Follicles were monitored by ultrasonography and a blood sample was taken daily from 29 January until ovulation (mean, 28 April). A mid-anovulatory period (largest follicle, 16.0+/-0.5 mm in diameter) and transitional period (largest follicle, 22.4+/-0.5 mm) were distinctive in each mare. The two periods were delineated by an increase in the diameter of the largest follicle to >/=21.0 mm. Follicular waves, identified by significant increases in the mean diameter of the second to sixth largest follicles, were detected during both the mid-anovulatory and transitional periods. The mean number of follicles >/=15.0 mm in diameter and the diameter of the second to sixth largest follicles increased in association with statistically identified FSH surges. The pattern of the FSH concentration changes during surges did not change during the mid-anovulatory and transitional periods. During the declining portion of the FSH surge, follicle growth continued and circulating total inhibin increased, indicating suppression of FSH by inhibin from the growing follicles. Circulating oestradiol or LH did not change relative to wave emergence. Results indicated that follicular waves occurred during the second-half of the anovulatory season, even during the period of lowest follicular activity. On a temporal basis, follicular wave emergence was stimulated by surges in circulating FSH. However, the increase in follicle growth to >/=21.0 mm in diameter for the wave at the beginning of the transitional period and for the subsequent waves was not attributable to a change in the characteristics of the associated FSH surges.