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Jennifer L Juengel, Derek A Heath, Laurel D Quirke and Kenneth P McNatty

A first step to elucidating the roles that steroids may play in the processes of ovarian development and early follicular growth is to identify the cell types that are likely to be receptive to steroids. Thus, cell types expressing receptors for oestrogen (α and β form; ERα and ERβ respectively), androgen (AR) and progesterone (PR) were determined by in situ hybridisation and immunohistochemistry in ovine ovarian tissues collected during ovarian development and follicular formation (days 26–75 of fetal life) as well as during the early stages of follicular growth. Expression of ERβ was observed early during ovarian development and continued to be expressed throughout follicular formation and also during the early stages of follicular growth. ERβ was identified in germ cells as well as in the granulosa cells. At the large preantral stage of follicular growth, expression of ERα was also consistently observed in granulosa cells. AR was first consistently observed at day 55 of fetal life in stroma cells throughout the ovary. Within the follicle, expression was observed in granulosa and thecal cells from the type-2 to -3 stage of follicular growth. PR mRNA did not appear to be expressed during ovarian development (days 26–75 of gestation). However, PR (mRNA and protein) was observed in the theca of type-3 (small preantral) and larger follicles, with mRNA – but not protein – observed in granulosa cells of some type-4 and 5 follicles. Expression of ERβ, ERα and AR, as well as PR, was also observed in the surface epithelium and ovarian stroma of the fetal, neonatal and adult ovary. Thus, in sheep, steroid hormones have the potential to regulate the function of a number of different ovarian cell types during development, follicular formation and early follicular growth.

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Peter Smith, Jo-Ann L Stanton, Laurel Quirke and Jennifer L Juengel

The aim of this study was to examine the relationships between gestational nutrition, fetal ovarian development and offspring fertility in female sheep and to highlight the potential mechanisms underlying these relationships. Adult sheep (n = 79) were fed either a maintenance or 0.6 of maintenance plane of nutrition for the first 55 days of gestation and thereafter fed ad libitum. Fetuses were collected for analysis at days 55 and 75 of gestation. Female offspring were monitored from birth until 19 months of age. Effects of restricted nutrition were observed on maternal plasma concentrations of progesterone, creatinine, albumin and Ca2+ at day 55 and creatinine at day 75. Concentrations of metabolic factors and steroid hormones in day 75 fetal plasma were not affected by the restricted maternal plane of nutrition. At day 55 of gestation, fetal ovarian germ cell development was not affected by maternal plane of nutrition. At day 75 of gestation ovaries from fetuses whose dams were exposed to restricted nutrition contained more germ cells but had lower germ cell proliferation rates than controls. For female offspring at 8 months of age, the dams gestational plane of nutrition did not affect the onset of puberty, ovulation rate (OR) and antral follicle counts (AFC). At 19 months of age, ewes from dams exposed to the restricted plane of gestational nutrition had higher OR, AFC and progesterone concentrations while concentrations of FSH were lower. In conclusion, while effects on fertility per se are yet to be determined, a reduced maternal plane of gestational nutrition can improve indicators of fertility in female offspring.

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Kenneth P McNatty, Derek A Heath, Norma L Hudson, Karen L Reader, Laurel Quirke, Stan Lun and Jenny L Juengel

In mammals with a low ovulation rate phenotype, ovarian follicular development is thought to be hierarchical with few, if any, antral follicles at similar stages of development. The hypothesis being tested herein was that if most follicles are in a functionally different state, then the application of exogenous hormones to increase ovulation rate will not overcome the hierarchical nature of follicular development. Using sheep as the experimental model, the functional states of all non-atretic antral follicles ≥2 mm diameter were assessed in individual ewes (N=10/group) during anoestrus with or without pregnant mare's serum gonadotrophin (PMSG) treatment, or after a standard superovulation regimen, or during the follicular phase of the oestrous cycle. The functional states of these follicles were assessed by measuring the FSH- or human chorionic gonadotrophin (hCG)-induced cAMP responses of granulosa cells in vitro. There were significant overall effects across the treatment groups on the responses of granulosa cells to either FSH or LH (both P<0.001). It was concluded that for anoestrous ewes with or without PMSG treatment, and ewes during the follicular phase, granulosa cell populations of many follicles (≥2 mm diameter) did not share a similar cAMP response to FSH (∼50% of follicles) or hCG (>90% of follicles) either on a per cell or total cell basis. After superovulation, ≤30 and 10% respectively of the granulosa cell populations shared similar responses to FSH and LH with regard to follicular diameter and cAMP output. Thus, exogenous hormone treatments used routinely for increasing oocyte yield do not effectively override the hierarchical pattern of ovarian follicular development during the follicular phase.

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Jennifer L Juengel, Laurel D Quirke, Stan Lun, Derek A Heath, Peter D Johnstone and Kenneth P McNatty

Sheep with a heterozygous inactivating mutation in the bone morphogenetic protein 15 (BMP15) gene experience an increased ovulation rate during either a natural oestrous cycle or a cycle in which exogenous FSH and eCG (gonadotrophins) are given to induce multiple ovulations. The primary aim of these studies was to determine whether ewes immunised against BMP15 would also show an improved superovulation rate following exogenous gonadotrophin treatment. A secondary aim was to determine the effects of BMP15 immunisation on ovarian follicular characteristics. In most ewes (i.e. >75%) immunised with a BMP15-keyhole limpet haemocyanin peptide in an oil-based adjuvant in order to completely neutralise BMP15 bioactivity, there was no superovulation response to exogenous gonadotrophins. In ewes treated with exogenous gonadotrophins following a BMP15-BSA peptide immunisation in a water-based adjuvant to partially neutralise BMP15 bioactivity, the ovulation rate response was similar to the control superovulation treatment groups. Characterisation of follicular function revealed that the water-based BMP15-immunised animals had fewer non-atretic follicles 2.5–3.5 or >4.5 mm in diameter compared with controls. Basal concentrations of cAMP were higher in granulosa cells from animals immunised against BMP15 than control animals. There were no significant differences in the concentrations of cAMP between granulosa cells from BMP15- and control-immunised animals when given FSH or hCG, although there were differences in the proportions of follicles in different size classes that responded to FSH or hCG. Thus, immunisation against BMP15 may have been causing premature luteinisation and thereby limiting the numbers of follicles recruited for ovulation following treatment with exogenous gonadotrophins.

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Jennifer L Juengel, Michelle C French, Laurel D Quirke, Alexia Kauff, George W Smith and Peter D Johnstone

We hypothesised that cocaine- and amphetamine-regulated transcript (CARTPT) would be differentially expressed in ewes with differing ovulation rates. Expression of mRNA for CARTPT, as well as LHCGR, FSHR, CYP19A1 and CYP17A1 was determined in antral follicles ≥1 mm in diameter collected during the follicular phase in ewes heterozygous for the Booroola and Inverdale genes (I+B+; average ovulation rate 4) and ++ contemporaries (++; average ovulation rate 1.8). In ++ ewes (n = 6), CARTPT was expressed in small follicles (1 to <3 mm diameter), where 18.8 ± 2.5% follicles expressed CARTPT. CART peptide was also detected in follicular fluid of some follicles of ++ ewes. In I+B+ ewes, 5/6 ewes did not have any follicles that expressed CARTPT, and no CART peptide was detected in any follicle examined. Expression pattern of CYP19A1 differed between I+B+ and ++ ewes with an increased percentage of small and medium follicles (3 to <4.5 mm diameter) but decreased percentage of large follicles (≥4.5 mm diameter) expressing CYP19A1 in the I+B+ ewes. Many of the large follicles from the I+B+ ewes appeared non-functional and expression of LHCGR, FSHR, CYP17A1 and CYP19A1 was less than that observed in ++ ewes. Expression of FSHR and CYP17A1 was not different between groups in small and medium follicles, but LHCGR expression was approximately double in I+B+ ewes compared to that in ++ ewes. Thus, ewes with high ovulation rates had a distinct pattern of expression of CARTPT mRNA and protein compared to ewes with normal ovulation rates, providing evidence for CART being important in the regulation of ovulation rate.

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Janet L Crawford, Derek A Heath, Karen L Reader, Laurel D Quirke, Norma L Hudson, Jennifer L Juengel and Kenneth P McNatty

The aim of this study was to test the hypothesis that the high ovulation rate in ewes (BB) homozygous for a mutation in the bone morphogenetic protein receptor type 1B (BMPR1B) gene is linked to lower BMP15 and/or GDF9 mRNA in oocytes compared with those in wild-type (++) ewes. Cumulus cell–oocyte complexes (COC) and granulosa cells (GC) were recovered from ≥1 mm diameter follicles of BB and ++ ewes during a prostaglandin-induced follicular phase. Expression levels of GDF9 and BMP15 were measured by multiplex qPCR from individual COC. The gonadotropin-induced cAMP responses of the GC from each non-atretic follicle were measured following treatment with FSH or human chorionic gonadotropin. In a separate validation experiment, GDF9 and BMP15 expression was present only in oocytes and not in cumulus cells. There was no effect of follicular diameter on oocyte-derived GDF9 or BMP15 mRNA levels. The mean expression levels of BMP15, but not GDF9, were significantly lower in all non-atretic follicles, including the subsets containing either FSH- or LH-responsive GC in BB, compared with ++, ewes. No genotype effects were noted for FSH-induced cAMP production by GC either with respect to dose of, or number of follicles responding to, FSH. However, ovaries from BB ewes contained significantly more follicles responsive to LH, with respect to cAMP production in GC. We propose that these findings are consistent with the hypothesis that the higher ovulation rate in BB sheep is due, at least in part, to lower oocyte-derived BMP15 mRNA levels together with the earlier onset of LH-responsiveness in GC.