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K. P. McNatty, S. Lun, N. L. Hudson, and S. Forbes

Summary. The cAMP outputs by granulosa cells from 3–4·5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from ≥5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin. Likewise, the cAMP outputs by cells from 1–2·5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin. At FSH, cholera toxin or forskolin doses of 1 μg/ml, 0·5 μg/ml and 10 −4 m respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P < 0·05) higher than in the respective small FF and medium ++ follicles. The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P < 0·05) observed between follicle sizes but not genotype. No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output.

For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation. For all treatments the rate of cAMP production tended to slow down after 40–60 min.

Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P < 0·05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size. However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small–medium ++ follicles respectively.

These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1–2·5 to 3–4·5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3–4·5 to ≥ 5 mm in diameter. No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size. It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system.

Keywords: FSH; cholera toxin; pertussis toxin; forskolin; cAMP; granulosa cells; Booroola ewes; F gene

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MW Fisher, BJ McLeod, DA Heath, S Lun, and PR Hurst

Physiological and endocrine factors associated with reproductive senescence were assessed in a group of 19 ageing red deer hinds. Reproductive success, defined as the percentage of hinds weaning a calf successfully, decreased gradually from 89% at 6-7 years of age to 50% at 17 years, and subsequently decreased markedly; only one hind reared a calf at 19-20 years of age. When the 12 surviving hinds were approaching 21 years of age, they were compared with ten mature 7-year-old females over the onset of the breeding season. All hinds were subsequently killed, the reproductive tracts were recovered and antral (>/= 2 mm in diameter) and preantral follicle populations were determined by dissection (n = 7 hinds per age group) or stereological analysis (n = 2 ovaries per age group), respectively. Cyclical ovarian activity (plasma progesterone) was evident in fewer aged hinds compared with mature hinds (3/12 versus 10/10, P < 0.001) and mean plasma LH concentrations were higher in aged animals than in mature animals (0.57 +/- 0.05 and 0.20 +/- 0.05 ng ml(-1), P < 0.001). Mean uterine (44.2 +/- 4.5 and 75.4 +/- 4.2 g; P < 0.001) and ovarian masses (0.88 +/- 0.11 and 1.52 +/- 0.12 g; P < 0.001) were lower in the aged hinds, which also had fewer antral follicles than did mature hinds (0.89 +/- 0.35 and 23.5 +/- 4.5 follicles per hind, respectively; P < 0.001). Only one primordial follicle was observed in one of the ovaries of the aged hinds, compared with 7000-21 000 in the ovaries of mature hinds. The high gonadotrophin concentrations, paucity of primordial and antral follicles and failure of ovulation indicate collectively that waning reproductive performance after 17 years of age is primarily due to ovarian failure.

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D. J. Phillips, L. G. Moore, N. L. Hudson, S. Lun, and K. P. McNatty

This study investigated the effects of short-term (20 days) ovariectomy, the effects of FSH assay (radioimmunoassay, receptor assay or in vitro bioassay) and of FecBB genotype on the characteristics of pituitary FSH from Booroola ewes. Pituitary extracts were obtained from ovariectomized homozygous carriers (BB) and non-carriers (++, n = 8 per genotype) and ovary-intact controls (n = 4 per genotype). The extracts (n = 4 per genotype per treatment) were subjected to agarose suspension electrophoresis and the eluates were assayed by the three FSH methods. There were significant effects of ovariectomy (P < 0.01) and assay system (P < 0.05) but not of genotype on the median charge of FSH isoforms. The mean ± sem migration rates for FSH in intact and ovariectomized ewes were 0.469 ± 0.006 and 0.439 ± 0.006 albumin mobility units, respectively (P < 0.01), indicating a shift to more basic isoforms after short-term ovariectomy. When the pituitary extracts were subjected to anion-exchange HPLC, there was a significant (P < 0.01) shift to more basic isoforms in the ovariectomized ewes as shown using agarose electrophoresis, and no gene effects were noted. When the pituitary extracts (n = 4–8 per group) were injected into mature female mice, there were no significant effects of ovariectomy or genotype on the circulating half-lifes of the pituitary FSH isoforms. These results indicate that after short-term ovariectomy, the increase in plasma FSH concentrations is accompanied by a shift in the median charge of pituitary FSH isoforms without any observable change in their metabolic clearance rates. Moreover, the FecBB gene has little effect on the median charge or half-life of pituitary FSH.

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S. Lun, P. Smith, T. Lundy, A. O'Connell, N. Hudson, and K. P. McNatty

The aim of the present study was to establish whether the steroids, progesterone, androstenedione, testosterone and oestradiol, were present in the mesonephric–gonadal complex of female and male sheep fetuses around sexual differentiation (that is, from day 28 to day 45 of gestation, with sexual differentiation occurring at approximately day 32). A second aim was to test whether the mesonephric–gonadal complex, mesonephros (days 35–45 only) and gonad (days 35–45 only) were capable of steroid synthesis in vitro. The steroid contents in the mesonephric–gonadal complex were not detectable before sexual differentiation. However, from day 35 of gestation onwards, the mesonephric–ovarian complex contained mainly oestradiol and the mesonephric–testicular complex contained mainly testosterone: from day 35 until day 45 the increase in content of these two steroids exceeded the increase in the mass of tissue by more than fivefold. From day 40 to day 45 of gestation, the contents of the other steroids in the pathways to oestradiol increased progressively in both sexes but more in parallel with the increase in tissue mass. In contrast to the steroid contents in the tissue at recovery, the mesonephric–gonadal tissue from both sexes in tissue culture was able to synthesize most steroids before and after sexual differentiation and also to metabolise supplementary androstenedione to oestradiol. These findings suggest that many, if not all, of the steroidogenic enzymes in the pathway from cholesterol to oestradiol are present before sexual differentiation. Most of the aforementioned steroids were present in detectable amounts in isolated mesonephros and gonad of both sexes after sexual differentiation. Moreover, for both the isolated mesonephros and gonad, there were increases in the mean contents of most steroids after culture relative to the contents in the tissues at recovery. These data suggest that the mesonephros, as well as the gonad, in both sexes is capable of synthesizing steroid. It is concluded that, in the sheep fetus, the female and male gonads are steroidogenically active after sexual differentiation, that the steroidogenic enzymes develop before sexual differentiation, and that the mesonephros is a site of steroid synthesis.

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K. M. Henderson, K. P. McNatty, R. L. Wards, D. A. Heath, and S. Lun

Summary. The production of inhibin by granulosa cells was studied in vitro using cells from follicles of various sizes and health. Follicles were recovered on Days 10–13 of the oestrous cycle, from Booroola × Romney ewes which were homozygous (FF) carriers or non-carriers (++) of the fecundity (F) gene. Inhibin was measured using a bioassay based on the suppression of follicle-stimulating hormone (FSH) output by cultured pituitary cells from ovariectomized Romney ewes and, in some instances, for comparative purposes, by radioimmunoassay also. Geometric mean inhibin production by granulosa cells from nonatretic follicles increased with increasing follicle diameter, during the first 24 h of culture, for both genotypes. The geometric mean production of inhibin by cells from nonatretic 3–4·5 mm diameter FF follicles (the largest follicles found in FF ewes), was significantly higher (P < 0·05) than that by cells from non-atretic 3–4·5 mm diameter ++ follicles, but similar to that of cells from non-atretic ⩾5 mm diameter ++ follicles. The production of oestradiol-17β by cells cultured in the presence of testosterone (1 μg/ml followed a pattern similar to cellular inhibin production. There was a positive linear correlation between inhibin and oestradiol-17β production during the first 24 h of culture, for both genotypes. In addition to acting as a substrate for oestradiol-17β synthesis, testosterone generally had a slight, stimulatory effect on inhibin production. Irrespective of follicle size, or genotype, no detectable amounts of inhibin were produced by granulosa cells from atretic follicles during the first 24 h of culture, or by cells from nonatretic or atretic follicles during the second 24 h of culture.

These studies show that the highest mean amounts of inhibin produced by granulosa cells in vitro are similar for both genotypes. Moreover, they are achieved with cells from the largest nonatretic follicles in both ++ (i.e. ⩾5 mm diameter) and FF (i.e. 3–4·5 mm diameter) ewes.

Keywords: Booroola ewes; inhibin; granulosa cells; oestradiol-17β

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K. P. McNatty, S. Lun, D. A. Heath, and L. E. O'Keeffe

Summary. The ovaries of 3-month-old Booroola lambs which were heterozygous carriers of a major gene (F) influencing the ovulation rate in mature ewes (i.e. F + lambs) were compared to those ofsimilarly-aged Booroola lambs which were non-carriers of the F-gene (i.e. ++ lambs). The ovaries of the F+ Booroola lambs were significantly lighter (P < 0·01) than those of ++ lambs even though the mean ± s.e.m. number of follicles (≥ 1 mm diam.) in the F+ lambs was greater than that in the ++ lambs (i.e. F+ lambs, 30·2 ± 2·5 follicles; ++ lambs, 18·4±1·2 follicles; P < 0·01).

In granulosa cells from non-atretic follicles (≥1 mm diam.) from F+ and ++ Booroola lambs, FSH (NIAMDD-FSH-S16) doses of 100 and 1000 ng/ml caused significant stepwise increases (P < 0·05) in cyclic adenosine 3′,5′-monophosphate (cAMP) production compared to that achieved at FSH doses of 0 and 1 ng/ml or at any FSH dose in cells from atretic follicles. However, no significant differences in FSH-induced cAMP production were noted with regard to Booroola genotype or follicular diameter.

None of the granulosa cell preparations from non-atretic follicles of 1–2·5 mm diameter from F+ lambs (N = 13) or from non-atretic follicles of 1–4·5 mm diameter from ++ lambs (N = 16) responded to LH (NIAMDD-LH-S24; 10 or 1000 ng/ml) to produce significantly more cAMP than did the controls. In contrast, the granulosa cell preparations from non-atretic follicles of 3–4·5 mm diameter from F+ lambs (N = 4) and from non-atretic follicles of ≥5 mm diameter of ++ lambs (N = 4) produced significantly more cAMP (P < 0·05) in response to LH (1000 and/or 10 ng/ml) relative to that in the controls. The theca interna from follicles of lambs of both genotypes had functional LH receptors as judged by the androstenedione responses to exogenous LH although no genotypic differences were noted.

In F+ lambs, the follicular fluid concentrations of testosterone but not oestradiol (i.e. in 1–4·5 mm diam. follicles) and granulosa cell aromatase activity (i.e. in 3–3·5 mm diam. follicles) were significantly higher (both P < 0·05) than in corresponding follicles or cells from ++ lambs.

Collectively the results suggest that the Booroola F-gene influences the composition and function of sheep ovaries before puberty.

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K. P. McNatty, L. E. O'Keeffe, K. M. Henderson, D. A. Heath, and S. Lun

Summary. Specific receptors for 125I-labelled hCG in ovarian follicle wall were located in the theca interna. No specific binding of 125I-labelled hCG was found in theca externa and/or stromal tissue. The kinetics of 125I-labelled hCG binding to theca interna followed second order kinetics with calculated association rate constants (k a ± s.d.) of 1·57 ± 0·16 × 106 and 0·57 ± 0·02 × 106 litres mol− 1 sec−1 at 37°Cand 22°C respectively. Dissociation of specifically bound 125I-labelled hCG from theca interna was minimal at 37°C and 22°C. The binding of 125I-labelled hCG to theca interna could be displaced with PMSG, FSH-P and sheep LH but other sheep pituitary hormones and LH-releasing hormone showed little or no cross-reaction.

The calculated binding capacities (Bmax) and equilibrium dissociation constants (K d) for 125I-labelled hCG binding to theca interna did not differ between Romney ewes and Booroola × Romney ewes with and without the fecundity (F) gene on Day 10 of the oestrous cycle, during anoestrus or at 36 h after an injection of cloprostenol on Day 10 of the oestrous cycle. When the data for Day 10 and anoestrus were pooled, the median (range) Bmax and K d values in non-atretic follicles ( ≥ 3 mm diameter) were 12·0 (5·1–23·5) fmol/mg protein and 0·10 (0·05–0·16) nM respectively. At 36 h after cloprostenol injection the respective median (range) Bmax and K d values in non-atretic follicles ≥ mm diam.) increased to 46·9 (28·4–70·3) fmol/mg protein and 0·23 (0·13–0·65) nm respectively. In corpora lutea the hCG binding characteristics were similar in all the above breeds/genotypes. On Day 10 of the cycle, the mean Bmax but not the mean K d value was significantly higher (P < 0·01) than the corresponding value at 36 h after cloprostenol injection. In granulosa cells, from follicles of ≥ 5 mm diameter of Romney and Booroola × Romney ( + + ) ewes and from follicles of ≥ mm diameter of Booroola × Romney (F + ) ewes, the hCG binding characteristics were similar. In granulosa cells from smaller sized follicles from the above breeds/ genotypes, no specific hCG binding was noted.

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K. M. Henderson, L. E. Kieboom, K. P. McNatty, S. Lun, and D. Heath

Summary. The influence of follicular size and health on FSH and LH stimulation of cAMP production by granulosa cells in vitro was studied in cells from Booroola × Romney ewes, with (F + ) and without (+ +) a fecundity gene. The granulosa cells were obtained 0–48 h after the initiation of luteolysis on Day 10 of the oestrous cycle by cloprostenol. The highest mean amounts of cAMP produced by granulosa cells challenged with FSH or LH were not significantly different between the genotypes. However, they were achieved using granulosa cells from follicles > 3–4 mm in diameter in F + ewes but from follicles > 4 mm in diameter in + + ewes. Follicles may thus attain ovulatory maturity at a smaller diameter in F+ ewes than in + + ewes. Granulosa cells from most atretic follicles gave a poor cAMP response to FSH or LH, compared to cells from non-atretic follicles. Granulosa cell responsiveness to FSH was independent of the time the cells were recovered after cloprostenol treatment in F + ewes, but not in + + ewes. Cellular responsiveness to LH was independent of time for sheep of both genotypes. There was a significant positive relationship for sheep of both genotypes between the level of aromatase activity in granulosa cells and cellular responsiveness to FSH and LH.

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K. P. McNatty, L. E. Kieboom, J. McDiarmid, D. A. Heath, and S. Lun

Summary. The tissue contents of adenosine cyclic 3′,5′-monophosphate (cAMP) in freshly dissected follicles (0·13–1·00 mm diam.) were significantly higher in Booroola ewes containing a major fecundity gene (FF and F+ ewes) compared to those values in Booroolas with no copy of the gene ( + + animals; P < 0·025). After a 1 h incubation with LH + FSH, the respective proportions of follicles with a diameter of 0·13–0·52 mm (n = 288) and 0·53–1·00 mm (n = 271) that had synthesized ≥0·6 pmol cAMP and ≥1·0 pmol cAMP were significantly influenced by genotype (Booroola ewes homozygous for the F-gene, FF > heterozygous, F+ > ++; P <0·01 for both follicle size ranges).

The contents of progesterone, androstenedione, testosterone and oestradiol-17β in minced ethanolic extracts of freshly dissected follicles (n = 188) were undetectable regardless of Booroola genotype. However, when follicles of 0·53–1·00 mm but not 0·13–0·52 mm diameter were cultured for 48 h with LH + FSH under 70 kPa of a 50% O2, 45% N2 and 5% CO2 gas mixture, the proportions that synthesized high levels of progesterone (≥4·0 ng), androstenedione (≥3 ng), and oestradiol (≥0·8 ng) were significantly influenced by genotype (FF > F+ ≥ ++; P <0·05 for each steroid). No significant genotypic differences were noted for testosterone synthesis.

Collectively, these results show that the Booroola F-gene has an influence on the maturation of ovarian follicles from an early stage of growth.

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J. S. Fleming, P. J. Greenwood, D. A. Heath, N. L. Hudson, S. Lun, L. Shaw, and K. P. McNatty

Homozygous carriers (BB) of the Booroola fecundity gene FecB are characterized by high plasma concentrations of immunoreactive or biologically active FSH and, to a lesser extent, of immunoreactive LH, relative to non-carriers (++). Bovine cDNA probes for the α gonadotrophin, FSHβ and LHβ genes were used to investigate FecB -specific differences in the mRNA species for the gonadotrophin subunits in pituitaries obtained from ++ and BB mid-luteal phase ovary-intact ewes, ovariectomized ewes and ovary-intact or ovariectomized ewes with hypothalamic–pituitary disconnection (HPD) given the same regimen of pulsatile GnRH. No FecB -specific differences in the number or size of mRNA transcripts detected by northern blotting were noted for any of these genes. Densitometry of the northern blots revealed no significant FecB -specific differences in the relative amounts of mRNA encoding α gonadotrophin, FSHβ or LHβ in the pituitaries from any of the experimental groups of ++ and BB sheep. Furthermore, there were no significant FecB -specific differences in the pituitary content of FSH or LH in these animals, despite significantly higher plasma concentrations of FSH in the ovary-intact and ovariectomized HPD groups. These data show that whereas the FecB gene causes increased plasma concentrations of FSH, no consistent effects can be demonstrated on pituitary gonadotrophin content or on gonadotrophin subunit gene transcription, using northern analysis. We suggest that the increased FSH secretion observed in FecB -carriers does not arise from an effect of the FecB gene on the size or stability of the gonadotrophin subunit mRNA, but is more likely to arise from differences in post-translational modification or secretion of the FSH protein in FecB -carriers.