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K. P. McNatty, N. L. Hudson, M. Gibb, and F. L. Collins

Summary. A significant increase in the plasma concentrations of FSH (P < 0·05) and LH (P < 0·001) was observed during the luteal (Days 9–11) phase but not during the subsequent cloprostenol-induced follicular phase in androstenedione-immunized ewes compared to those in control ewes. Over the same time period the geometric mean (and 95% confidence limits) androstenedione antibody titres in the immunized ewes was 1/305 (1/158, 1/590) whereas they were not detectable in the controls. In the subsequent cycle, the ovulation rates were 1·6 ± 0·2 for the immunized ewes and 1·1 ± 0·1 for the control ewes (P < 0·05) and the luteal progesterone concentrations were significantly higher in the immunized ewes compared to the controls (P < 0·01). Collectively, these results suggest that active immunization against androstenedione leads to an increase in the plasma concentrations of both FSH and LH. The results are consistent with the hypothesis that FSH plays a central role in determining the ovulation rate in sheep.

Keywords: FSH; androstenedione immunization; sheep; ovulation rate

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R. W. Kelly, K. P. McNatty, G. H. Moore, D. Ross, and M. Gibb

Summary. Peripheral blood samples were collected throughout pregnancy from 11 red deer hinds. During the same period, 6 other hinds which mated but failed to produce calves were also sampled. Pretreatment of some of these hinds included synchronization of oestrus alone (N = 3) or with injection of 1000 i.u. PMSG (N = 9).

During early and mid-pregnancy, LH and prolactin were frequently undetectable. Prolactin concentrations in pregnant and non-pregnant hinds were high (>250 ng/ml) in December—January. The results of the hormone analyses suggested that the amount of progesterone in plasma correlates with the number of corpora lutea (CL) present. The concentrations of oestradiol and progesterone were low from mid-winter onwards in the non-pregnant hinds, suggesting a reduction in ovarian activity at this time. In pregnant animals, progesterone concentrations were high for the first 200 days of gestation. Oestradiol rose to peak values concomitant with declining progesterone concentrations just before parturition.

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

Summary. In 24-h cultures, steroid production by cells from non-atretic follicles increased with increasing follicular diameter. Cells from atretic follicles, of all sizes, produced low amounts of oestradiol-17β, but very high amounts of progesterone, relative to cells from non-atretic follicles. Increasing the culture period to 72 h caused little change in daily progesterone and oestradiol-17β production by granulosa cells from atretic follicles. In contrast, in cells from non-atretic follicles, daily progesterone production increased and daily oestradiol-17β production decreased to the levels observed with cells from atretic follicles. Dibutyryl cyclic AMP (1·0 mm) significantly stimulated progesterone production by cells from atretic, but not from non-atretic, follicles. Testosterone (1 μg/ml) had no effect on progesterone production by cells from atretic follicles, while oestradiol-17β, oestrone, testosterone, androstenedione and 5α-dihydrotestosterone (0–1000 ng/ml) each significantly suppressed progesterone production by cells from non-atretic follicles in a dose-dependent manner.

Morphometric analysis revealed few subcellular differences between cells from non-atretic and atretic follicles. Mean cell volume was significantly higher for cells from atretic compared to non-atretic follicles, but the mean volumes of the major subcellular components were not influenced by follicle health. The mean surface area of the plasma and nuclear membrane, and granular endoplasmic reticulum was also significantly higher in cells from atretic compared to non-atretic follicles.

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K. P. McNatty, N. L. Hudson, K. M. Henderson, S. Lun, D. A. Heath, M. Gibb, K. Ball, J. M. McDiarmid, and D. C. Thurley

Summary. Overall, significantly more antral follicles ≥ 1 mm diameter were present in Romney ewes during anoestrus than in the breeding season (anoestrus, 35 ± 3 (mean ± s.e.m.) follicles per ewe, 23 sheep; Day 9–10 of oestrous cycle, 24±1 follicles per ewe, 22 sheep; P < 0·01), although the mean numbers of preovulatory-sized follicles (≥5 mm diam.) were similar (anoestrus, 1·3 ± 0·2 per ewe; oestrous cycle, 1·0 ± 0·1 per ewe). The ability of ovarian follicles to synthesize oestradiol did not differ between anoestrus and the breeding season as assessed from the levels of extant aromatase enzyme activity in granulosa cells and steroid concentrations in follicular fluid. Although the mean plasma concentration of LH did not differ between anoestrus and the luteal phase of the breeding season, the pattern of LH secretion differed markedly; on Day 9–10 of the oestrous cycle there were significantly more (P < 0·001) high-amplitude LH peaks (i.e. ≥ 1 ng/ml) in plasma and significantly fewer (P < 0·001) low amplitude peaks (<1 ng/ml) than in anoestrous ewes. Moreover, the mean concentrations of FSH and prolactin were significantly lower during the luteal phase of the cycle than during anoestrus (FSH, P < 0·05, prolactin, P < 0·001).

It is concluded that, in Romney ewes, the levels of antral follicular activity change throughout the year in synchrony with the circannual patterns of prolactin and day-length. Also, these data support the notion that anovulation during seasonal anoestrus is due to a reduced frequency of high-amplitude LH discharges from the pituitary gland.

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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. P. McNatty, N. Hudson, K. M. Henderson, M. Gibb, L. Morrison, K. Ball, and P. Smith

Summary. The mean plasma concentrations of FSH and LH were significantly higher in FF ewes than in ++ ewes with those for F+ animals being consistently in between. These gene-specific differences were found during anoestrus, the luteal phase and during a cloprostenol-induced follicular phase, suggesting that the ovaries of ewes with the F-gene are more often exposed to elevated concentrations of FSH and LH than are the ovaries of ewes without the gene.

The gene-specific differences in LH secretion arose because the mean LH amplitudes were 2–3 times greater in FF compared to ++ ewes with the LH amplitudes for F + ewes being in between. The LH pulse frequencies were similar. In these studies the pulsatile nature of FSH secretion was not defined.

The pituitary contents of LH during the luteal phase, were similar in all genotypes whereas for FSH they were significantly higher in the F-gene carriers compared to ++ ewes. The pituitary sensitivity to exogenous GnRH (0·1, 0·5, 5·0 and 25 μg i.v.) was related to genotype. Overall the LH responses to GnRH were lower in FF ewes than in ++ ewes with the results for the F+ ewes being in between. The FSH responses to all GnRH doses in the FF genotype were minimal (i.e. < 2-fold). In the other genotypes a > 2-fold response was noted only at the highest GnRH dose (i.e. 25 μg). Treatment of FF and F+ but not ++ ewes with GnRH eventually led to a reduced FSH output, suggesting that the pituitary responses to endogenous GnRH were being down-regulated in the F-gene carriers whereas this was not the case in the non-carriers.

Collectively these data confirm that peripheral plasma and the pituitary together with the ovary are compartments in which F-gene differences can be observed. In conclusion, these findings raise the possibility that F-gene-specific differences may also extend to the hypothalamus and/or other regions of the brain.

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

Summary. Differences in the function and composition of individual ovarian follicles were noted in Booroola Merino ewes which had previously been segregated on at least one ovulation rate record of ≥5 (FF ewes, N = 15), 3–4 (F+ ewes, N = 18) or <3 (++ ewes, N = 18).

Follicles in FF and F+ ewes produced oestradiol and reached maturity at a smaller diameter than in ++ ewes. In FF (N = 3), F+ (N = 3) and ++ (N = 3) ewes, the respective mean ± s.e.m. diameters for the presumptive preovulatory follicles were 3·4 ± 0·3, 4·1 ± 0·2 and 6·8 ± 0·3 mm and in each of these follicles the respective mean ± s.e.m. numbers of granulosa cells (× 106) were 1·8 ± 0·3, 2·2 ± 0·3 and 6·6 × 0·3. During a cloprostenol-induced follicular phase, the oestradiol secretion rates from FF ewes with 4·8 ± 0·4 'oestrogenic' follicles, F+ ewes with 3·2 ± 0·2 'oestrogenic' follicles and ++ ewes with 1·5 ± 0·02 'oestrogenic' follicles were not significantly different from one another. Moreover, the mean total numbers of granulosa cells from the 'oestrogenic' follicles from each genotype were identical, namely 5·4 × 106 cells. Irrespective of genotype the mean weight of each corpus luteum was inversely correlated to the ovulation rate (R = 0·91, P <0.001).

Collectively, these findings support the notion that the maturation of ≥ 5 follicles in FF ewes and 3–4 follicles in F+ ewes may each be necessary to provide a follicularcell mass capable of producing the same quantity of oestradiol as that from 1–2 preovulatory follicles in ++ ewes.

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K. P. McNatty, M. Gibb, C. Dobson, K. Ball, J. Coster, D. Heath, and D. C. Thurley

Summary. The patterns of growth and atresia of antral follicles including that of the presumptive preovulatory follicle were examined in sheep ovaries for a 24–48-h period after the induction of luteolysis with a prostaglandin analogue, cloprostenol or cloprostenol + PMSG. Ewes were ovariectomized at various times after the initiation of the treatments. All follicles ≥1 mm in diameter were dissected from the excised ovaries and the antral fluid and granulosa cells recovered. Individual follicles were classified as healthy or atretic on the basis of the number of granulosa cells recovered and then subclassified as to whether they contained intrafollicular levels of oestradiol that were ≥ or < than 100 ng/ml.

In another series of similarly treated ewes, the ovarian secretion rates of oestradiol and the intrafollicular concentrations of oestradiol in all large antral follicles (≥5 mm diameter) as well as the levels of progesterone in peripheral plasma were measured at different times after induction of luteolysis.

The results showed that a large 'oestrogenic' follicle (≥5 mm diameter and secreting ≥1 ng oestradiol/min) appears around 10 h after the cloprostenol injection and that this presumptive preovulatory follicle emerges before the corpus luteum has ceased to function. Moreover, the presumptive preovulatory ('oestrogenic') follicle appears to develop from the pool of small 'oestrogenic' follicles (1–3 mm diameter) after the onset of luteolysis. The emergence of a large 'oestrogenic' follicle is accompanied by a widespread increase in atresia (> 80%) in all other classes of antral follicles (≥1 mm in diameter).

During the first 10 h of cloprostenol-induced luteolysis, PMSG (a) prevented the normal occurrence of atresia in the large follicle population; (b) enhanced oestrogen secretion in a greater proportion of large antral follicles compared to that in control animals; (c) temporarily 'rescued' and/or prevented small antral follicles (1–4 mm diameter) from undergoing atresia; but (d) had little, or no, effect on the overall population of antral follicles (≥1 mm diameter). After 24 h, the atresia-preventing effects of PMSG were no longer discernible and the only obvious difference noted, compared to the controls, was the number of large oestrogen-secreting follicles.

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Duangporn Jamsai, Deborah M Bianco, Stephanie J Smith, Donna J Merriner, Jennifer D Ly-Huynh, Amy Herlihy, Birunthi Niranjan, Gerard M Gibbs, and Moira K O'Bryan

Cysteine-rich secretory protein 2 (CRISP2) is a testis-enriched protein localized to the sperm acrosome and tail. CRISP2 has been proposed to play a critical role in spermatogenesis and male fertility, although the precise function(s) of CRISP2 remains to be determined. Recent data have shown that the CRISP domain of the mouse CRISP2 has the ability to regulate Ca2+ flow through ryanodine receptors (RyR) and to bind to MAP kinase kinase kinase 11 (MAP3K11). To further define the biochemical pathways within which CRISP2 is involved, we screened an adult mouse testis cDNA library using a yeast two-hybrid assay to identify CRISP2 interacting partners. One of the most frequently identified CRISP2-binding proteins was gametogenetin 1 (GGN1). Interactions occur between the ion channel regulatory region within the CRISP2 CRISP domain and the carboxyl-most 158 amino acids of GGN1. CRISP2 does not bind to the GGN2 or GGN3 isoforms. Furthermore, we showed that Ggn1 is a testis-enriched mRNA and the protein first appeared in late pachytene spermatocytes and was up-regulated in round spermatids before being incorporated into the principal piece of the sperm tail where it co-localized with CRISP2. These data along with data on RyR and MAP3K11 binding define the CRISP2 CRISP domain as a protein interaction motif and suggest a role for the GGN1–CRISP2 complex in sperm tail development and/or motility.

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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.