Summary. The ability of bovine granulosa cells to produce inhibin and to synthesize oestradiol-17β increased with increasing follicle size in healthy but not atretic follicles. Granulosa cells from small (≤5 mm diam.) healthy follicles were indistinguishable from cells of atretic follicles in terms of their ability to produce inhibin and to aromatize androgen. However, granulosa cells from healthy and atretic follicles, irrespective of size, differed markedly in their morphological appearance after culture for 24 h. Testosterone (1 μg/ml) stimulated inhibin production by granulosa cells from healthy and atretic follicles while FSH (100 ng/ml) stimulated inhibin production by granulosa cells from healthy follicles only. The relative ability of granulosa cells from different sizes of healthy and atretic follicles to produce inhibin in vitro was reflected in inhibin concentrations in follicular fluid. There was a significant positive correlation between inhibin concentration in follicular fluid and the number of granulosa cells per follicle. There was also a significant positive correlation between follicular diameter and inhibin concentration in follicular fluid, but only in healthy follicles. These findings show that both follicular size and atresia influence follicular inhibin production.

Summary. The presence of a fecundity gene (F) in Booroola Merino ewes increases the ovulation rate. To test how F gene expression affects the gonadotrophin-releasing hormone (GnRH) concentration in hypothalamic or extrahypothalamic regions of the brain, GnRH was measured by radioimmunoassay in acetic acid extracts of various brain tissues from Booroola ewes which were homozygous (FF), heterozygous (F+) or non-carriers (++) of the F gene. The GnRH concentration in brain tissues from FF, F+ and ++ animals which had been ovariectomized 5 months previously was also evaluated.

No significant F gene-specific differences were noted in any of the brain areas tested, in intact or ovariectomized animals. However, in ovariectomized ewes, the concentrations of GnRH increased about 2-fold in the median eminence of the hypothalamus, remained unchanged in the medial basal hypothalamus and dropped to <10% of the values in intact ++ animals in the preoptic area.

These studies suggest that the changed pituitary sensitivity and increased gonadotrophin release in Booroolas carrying the F gene(s) is not attributable to increased hypothalamic GnRH concentrations in these animals.

Keywords: GnRH; sheep; brain; Booroola; F-gene

M.R.C. Unit of Reproductive Biology, 39 Chalmers Street, and * Hormone Laboratory, Department of Obstetrics and Gynaecology, University of Edinburgh, Edinburgh, EH3 9ER, U.K.

Prolactin constitutes part of the luteotrophic complex necessary for the maintenance and secretory activity of the CL in the rat (Evans, Simpson, Lyons & Turpeinen, 1941; Astwood, 1941), mouse (Kovacic, 1964), rabbit (Spies, Hilliard & Sawyer, 1968), hamster (Greenwald & Rothchild, 1968), ferret (Donovan, 1963), pig (du Mesnil du Buisson, 1973), and sheep (Denamur, Martinet & Short, 1973). There is also evidence from in-vitro studies that low concentrations of prolactin are essential for the production of progesterone by preovulatory human granulosa cells (McNatty, Sawers & McNeilly, 1975; McNatty, Bennie, Hunter & McNeilly, 1976) while high concentrations are inhibitory (McNatty et al., 1976). In the present study the effect of various concentrations of prolactin on the production of progesterone by mouse ovaries in organ culture with or without

Hormone Laboratory, Department of Obstetrics and Gynaecology, University of Edinburgh, and * M.R.C. Unit of Reproductive Biology, 39 Chalmers Street, Edinburgh, EH3 9ER, U.K.

Numerous studies have shown that prostaglandin (PG) E-2 can stimulate ovarian steroidogenesis in vitro and can mimic many of the actions of LH on the Graafian follicle in vivo and in vitro (see Neal, Baker, McNatty & Scaramuzzi, 1975). Paradoxically, however, PGF-2α can stimulate or inhibit steroidogenesis by ovarian follicles or isolated cells in vitro, the response varying with the dose employed.

Many of these diverse actions of PGs may be the result of using pharmacological rather than physiological doses of the drugs (see McNatty, Henderson & Sawers, 1975; Neal et al., 1975). In the present investigation, therefore, the effects of various doses of PGE-2 and PGF-2α, alone and in combination, on the secretion of progesterone by mouse ovaries in vitro, with or without added gonadotrophins, were

The aim of the present study in Booroola ewes, either homozygous (BB) or non-carriers (++) of the Fec^B gene, was to test the specificity of the pituitary responses to exogenous hypothalamic releasing hormones by examining the plasma concentrations of FSH, LH, thyroid-stimulating hormone (TSH) and growth hormone (GH) after injecting the animals with different doses of GnRH, thyroid-releasing hormone (TRH) or growth-hormone-releasing hormone, (GHRH) which were administered on separate occasions. The animals (n = 8 per dose) received 0, 3.1 or 12.5 μg of thyroid-releasing hormone and GnRH (i.v.), whereas they (n = 9–13 per dose) received 0, 6.0 or 16.0 μg GHRH (i.v.). For each experiment there were no differences between the genotypes in bodymass or age. Gene-specific differences in the mean pretreatment concentrations of plasma FSH (BB > ++; P < 0.05) but not of LH, TSH or GH were noted. After treatment with GnRH, TRH or GHRH, significant effects of dose were noted for all the hormones; however, a gene-specific effect was observed only for FSH in response to GnRH (BB > ++; P < 0.01) with no genotype × dose interaction (anova). For LH, the effects of genotype and the genotype × dose interaction almost reached significance at the 5% level (genotype, P = 0.055; genotype × dose, P = 0.067). For TSH and GH the respective genotype × dose interactions were not significant. These results support the hypothesis that the Fec^B gene in Booroola ewes influences the release of pituitary hormones in response to hypothalamic releasing hormones only in the case of GnRH, which results mainly in different plasma concentrations of FSH.

Summary. Before castration, the mean plasma concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) did not differ between FF and ++ Booroola rams. After castration, mean LH and FSH concentrations increased after 8 h, and for the next 14 days the rate of increase in FSH, but not LH, secretion was significantly faster in FF than in ++ rams (P < 0·05). Mean FSH concentrations over this period were significantly higher in FF than in ++ rams (P < 0·05). In both genotypes, the ranked FSH values did not significantly change their order over time, i.e. a significant within-ram effect was noted (P < 0·05). Repeated-measures analysis of variance indicated a significant effect of genotype on mean FSH secretion (P < 0·05) and a significant effect of sire in the FF (P < 0·05), but not the ++ (P = 0·76), genotype. From Day 28 to Day 58 after castration, FSH and LH concentrations were variable and no overall increases in concentrations were observed. The mean concentrations of both hormones over this period were not related to genotype.

There were no gene-specific differences in pulsatile LH secretion 14 weeks after castration. However, the mean LH, but not FSH, response to a bolus injection of 25μg of gonadotrophin-releasing hormone (GnRH) was significantly higher in FF than in ++ rams (P < 0·05) and this was not significantly affected by sire.

These studies support the hypothesis that the F gene is expressed in adult rams, in terms of pituitary responsiveness to an injection of GnRH and to the removal of the testes, but it is not clear from this study whether the influence of sire is related to or independent of the apparent gene-specific differences.

Keywords: Booroola; rams; LH; FSH; castration; fecundity

Summary. No gene-specific differences were found with respect to LH or testosterone pulsatile secretion (over 12 h), or in 12 hourly mean FSH concentrations in adult Booroola FF and ++ rams. Also, no differences between genotypes in the LH response to an injection of testosterone propionate, the FSH response to an infusion of bovine follicular fluid, or the testosterone response to injections of PMSG were noted. However, during the phase of seasonal testicular development, mean testosterone pulse amplitude (over 12 h) and the FSH response to 25 μg GnRH were higher in FF than in ++ rams (P < 0·05); there were also significant effects of sire (P < 0·05 in FF genotype only) and litter size (P < 0·05) on testosterone pulse amplitude and GnRH-stimulated FSH release, respectively. During the breeding season, mean LH, but not FSH, concentrations were higher in FF than in ++ rams, after an injection of 0·5 μg GnRH; LH release was not affected by sire or litter size (P > 0·05).

Long-term studies revealed that the FF rams were born in significantly larger litters, they weighed significantly less than ++ rams (P < 0·05), and that bodyweight was significantly correlated (P < 0·05) with litter size. There were no differences in testis size, and testis size was not significantly correlated with bodyweight. There was a strong tendency (P = 0·056) for overall mean FSH concentrations, measured weekly for 9 months, to be highest more often in FF than in ++ rams.

Collectively, these results suggest that the F gene is expressed at the level of the pituitary gland in adult males, although in intact animals gene-specific differences are suppressed by the negative effect of hormones from the testis.

Keywords: Booroola rams; GnRH; LH; FSH; fecundity

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

Summary. Castrated adult Fec^B Fec^B and Fec ⁺ Fec ⁺ Booroola rams were injected with charcoal-treated bovine follicular fluid (bFF) (a source of inhibin-like activity) or given testosterone implants to examine whether the fecundity gene (Fec^B ) influences sensitivity to negative feedback hormones in males. Mean concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) did not differ between genotypes before treatment. In Expt 1, injections of 5 ml bFF, but not of 1 ml (each given four times at intervals of 8 h), significantly (P < 0·05) depressed concentrations of LH and FSH, but there was no effect of genotype. After treatment, gonadotrophin concentrations returned to pretreatment values and for 2–2·5 days scaled (divided by pretreatment mean) LH values (235 ± 49 for Fec^B Fec^B and 96 ± 26% for Fec ⁺ Fec ⁺ rams; P < 0·05) and scaled FSH values (106 ± 5 for Fec^B Fec^B and 85 ± 5% for Fec ⁺ Fec ⁺ rams; P < 0·05) were significantly higher in Fec^B Fec^B than in Fec ⁺ Fec ⁺ rams in the group that received 5 ml bFF. Irrespective of genotype, treatment with 5 ml bFF did not reduce mean FSH to concentrations observed in testis-intact rams.

In Expt 2, Silastic envelopes were implanted subdermally to give physiological or supraphysiological circulating concentrations of testosterone. Both doses significantly reduced scaled LH values in a biphasic manner, such that there was an initial suppression followed by a short-lived increase. During the initial period of suppression in the lower dose group, mean scaled LH values were significantly higher in Fec^B Fec^B than in Fec ⁺ Fec ⁺ rams (48·3 ± 7·5 versus 23·1 ± 5·5%; P < 0·05). Low doses of testosterone decreased LH pulse frequency in both genotypes but decreased (P < 0·05) pulse amplitude and mean concentrations in the Fec ⁺ Fec ⁺ animals only. In nonimplanted control rams, mean LH concentrations (in samples taken every 10 min for 12 h) were significantly lower in Fec^B Fec^B than in Fec ⁺ Fec ⁺ rams (0·6 ± 0·2 versus 1·3 ± 0·1 ng ml⁻¹; P < 0·05). The mean FSH response to testosterone was not related to genotype.

These data suggest that expression of the Fec^B gene results in an altered sensitivity of the pituitary gland to changes in negative feedback from testicular hormones and that, irrespective of genotype, neither testosterone nor inhibin-like activity alone can fully control FSH secretion in castrated rams.

Keywords: Booroola; follicular fluid; testosterone; LH; FSH; ram

Blood samples were collected for 13 days before and 20 days after ovariectomy from carrier (BB) and non-carrier (+ +) ewes of the Booroola Fec^B gene (n = 12 per genotype), at known stages of the oestrous cycle, after which the pituitary glands from these ewes were recovered. Pituitary glands were also collected from cyclic ewes (about day 12; n = 5 per genotype) to compare the effects of ovariectomy on pituitary gonadotrophins. Plasma samples and pituitary extracts were assayed for bioactive (B) FSH, immunoreactive (I) FSH and I-LH. Overall, BB ewes had significantly (P < 0.05) higher plasma I-FSH concentrations than did + + ewes before ovariectomy; the mean value was higher on 16 of the 17 days of the oestrous cycle (P < 0.01). For B-FSH, there were no overall genotypic differences, although the mean for the BB ewes was significantly higher on 13 of the 17 days of the oestrous cycle (P < 0.05), and significantly (P < 0.05) higher between days 13 and 16. No genotypic differences were noted for the plasma bioactive:immunoreactive (B:I) ratio for FSH before ovariectomy. After ovariectomy, there were significant (P < 0.001) increases in plasma for B-FSH, I-FSH and I-LH and a significant (P < 0.05) decrease in the B:I ratio for FSH, irrespective of genotype. Furthermore, BB ewes had significantly (P < 0.05) higher overall concentrations of B-FSH and plasma B:I ratios after ovariectomy than did + + ewes; overall I-FSH concentrations were not significantly different between genotypes but the BB ewes had a higher mean value on 17 of the 20 days after ovariectomy (P < 0.001). With respect to pituitary FSH, there were no significant effects of genotype or ovariectomy on B-FSH or I-FSH contents or concentrations. No genotypic differences were noted in either plasma or pituitary I-LH, except for a higher pituitary I-LH content in BB ewes after ovariectomy. These data show that both ovariectomy and the Fec^B gene cause qualitative and quantitative changes in plasma FSH, but have little effect on pituitary FSH.