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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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K. P. McNatty, N. Hudson, M. Gibb, K. Ball, K. M. Henderson, D. A. Heath, S. Lun, and L. E. Kieboom

Summary. Injection of steroid-free bovine follicular fluid (bFF; 2 × 5 ml s.c. 12h apart) into anoestrous ewes lowered plasma FSH concentrations by 70% and after 24 h had significantly (P < 0·01) reduced the number of non-atretic follicles (≥ 1 mm diam.) without influencing the total number of follicles (≥ 1 mm diam.) compared to untreated controls. Hourly injections of FSH (10 μg i.v. NIH-FSH-S12) for 24 h did not influence the number of non-atretic follicles but did negate the inhibitory effects of bFF on follicular viability. Hourly injections of FSH (50 μg i.v., NIH-FSH-S12) + bFF treatment for 24 h significantly increased the total number of non-atretic follicles, and particularly the number of medium to large non-atretic follicles (≥ 3 mm diam.) compared to the untreated controls (both P < 0·01). The 10μg FSH regimen (without bFF) significantly increased aromatase activity in granulosa cells from large ( ≥ 5 mm diam.; P < 0·01) but not medium (3–4·5 mm diam.) or small (1–2·5 mm diam.) follicles compared to controls. The 10 μg FSH + bFF regimen had no effect on granulosa-cell aromatase activity compared to the controls. However, the 50 μg FSH plus bFF regimen increased the aromatase activity of granulosa cells from large, medium and small non-atretic follicles 2·6-, 8·3- and ≥ 11-fold respectively compared to that in the control cells.

Ewes (N = 11) that ovulated 2 follicles had significantly higher plasma FSH concentrations from 48 to 24 h and 24 to 0 h before the onset of a cloprostenol-induced follicular phase (both P < 0·01) than in the ewes (N = 12) that subsequently ovulated one follicle. Hourly FSH treatment (1·6 μg i.v., NIAMDD-FSH-S15) for 24 h but not for any 6 h intervals between 48 and 24 h or 24 and 0 h before a cloprostenol-induced luteolysis also resulted in significant increases (P < 0·05) in the number of ewes with 2 ovulations.

We conclude that (1) the number of non-atretic antral follicles in sheep ovaries is influenced by plasma FSH concentrations; (2) the level of follicular oestradiol biosynthesis can be enhanced by FSH treatment; and (3) sustained elevations of plasma FSH concentrations for 24 h but not 6 h within 48 h of the onset of luteolysis significantly enhances the ovulation rate in Romney ewes.

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K. M. Henderson, M. D. Prisk, N. Hudson, K. Ball, K. P. McNatty, S. Lun, D. Heath, L. E. Kieboom, and J. McDiarmid

Summary. Romney ewes were injected intramuscularly once or twice daily for 3 days with 0, 0·1, 0·5, 1 or 5 ml of bovine follicular fluid (bFF) treated with dextran-coated charcoal, starting immediately after injection of cloprostenol to initiate luteolysis on Day 10 of the oestrous cycle. There was a dose-related suppression of plasma concentrations of FSH, but not LH, during the treatment period. On stopping the bFF treatment, plasma FSH concentrations 'rebounded' to levels up to 3-fold higher than pretreatment values. The mean time to the onset of oestrus was also increased in a dose-related manner by up to 11 days. The mean ovulation rates of ewes receiving 1·0 ml bFF twice daily (1·9 ± 0·2 ovulations/ewe, mean ± s.e.m. for N = 34) or 5·0 ml once daily (2·0 ± 0·2 ovulations/ewe, N = 25) were significantly higher than that of control ewes (1·4 ± 0·1 ovulations/ewe, N = 35). Comparison of the ovaries of ewes treated with bFF for 24 or 48 h with the ovaries of control ewes revealed no differences in the number or size distribution of antral follicles. However, the large follicles (≥ 5 mm diam.) of bFF-treated ewes had lower concentrations of oestradiol-17β in follicular fluid, contained fewer granulosa cells and the granulosa cells had a reduced capacity to aromatize testosterone to oestradiol-17β and produce cyclic AMP when challenged with FSH or LH. No significant effects of bFF treatment were observed in small (1–2·5 mm diam.) or medium (3–4·5 mm diam.) sized follicles. Ewes receiving 5 ml bFF once daily for 27 days, from the onset of luteolysis, were rendered infertile during this treatment period. Oestrus was not observed and ovulation did not occur. Median concentrations of plasma FSH fell to 20% of pretreatment values within 2 days. Thereafter they gradually rose over the next 8 days to reach 60% of pretreatment values where they remained for the rest of the 27-day treatment period. Median concentrations of plasma LH increased during the treatment period to levels up to 6-fold higher than pretreatment values. When bFF treatment was stopped, plasma concentrations of FSH and LH quickly returned to control levels, and oestrus was observed within 2 weeks. The ewes were mated at this first oestrus and each subsequently delivered a single lamb.

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K. P. McNatty, K. M. Henderson, S. Lun, D. A. Heath, K. Ball, N. L. Hudson, J. Fannin, M. Gibb, L. E. Kieboom, and P. Smith

Summary. A marked difference in both the function and composition of individual ovarian follicles was noted in Booroola × Romney ewes (6–7 years of age) which had previously been segregated on at least one ovulation rate record of 3–4 (F+ ewes, N = 21) or <3 (++ ewes, N = 21).

Follicles in F+ ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In F+ ewes (N = 3), the presumptive preovulatory follicles were 4·4 ± 0·5 (s.e.m.) mm in diameter and contained 2·1 ± 0·3 × 106 (s.e.m.) granulosa cells, whereas in ++ ewes (N = 3), such follicles were 7·3 ± 0·3 mm in diameter and contained 6·5 ± 0·8 × 106 cells. During a prostaglandin (PG)-induced follicular phase, the secretion rate of oestradiol from ovaries containing 3 presumptive preovulatory follicles in F + ewes was similar to that from ovaries with only one such follicle in ++ ewes.

We suggest that the putative 'gene effect' in F+ ewes is manifested during early follicular development and that it may be mediated via an enhanced sensitivity of granulosa cells to pituitary hormones. As a consequence, the development of 3 preovulatory follicles in F+ ewes may be necessary to provide a cell mass capable of producing the same quantity of oestradiol as that from one preovulatory follicle in ++ ewes.