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D. A. Heath, M. Caldani, and K. P. McNatty

Booroola ewes possess a major gene, FecBB, that influences their ovulation rate (number of ovulations per oestrous cycle). Homozygous (BB) carriers of the FecBB gene have higher plasma concentrations of FSH and sometimes LH relative to the non-carriers (++). The aim of this study was to determine whether the plasma concentration differences in FSH or LH between the genotypes were due to a greater number of FSHβ-or LHβ-immunostaining cells in the anterior pituitary gland of BB ewes during the luteal phase of the oestrous cycle. No differences were found between the BB (n = 7) ewes and ++ (n = 8) ewes in total number of pituitary cells, pituitary volume, numbers or diameters of FSHβ- or LHβ-immunostaining cells, notwithstanding significantly higher concentrations of immunoreactive plasma FSH (P < 0.001) but not LH in BB compared with ++ animals. Significant linear relationships were found within each genotype between plasma FSH and number of FSHβ-immunostaining cells. No such relationship was found for plasma LH and number of LHβ cells. For the FSH relationship, the slopes of the regression lines were the same. It is hypothesized that the differences in plasma concentration of FSH between the genotypes is due to a greater output of FSH per pituitary cell in the BB animals.

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J L Crawford, D A Heath, L J Haydon, B P Thomson, and D C Eckery

In eutherian mammals, the gonadotrophins (LH and FSH) are synthesized and stored in gonadotroph cells under the regulation of multiple mechanisms including GnRH. Very little is known about the regulation of gonadotrophin secretion and storage in pituitary glands of marsupials. This study revealed, using quantitative PCR and heterologous RIA techniques, that LHB mRNA expression levels remained constant over the oestrous cycle, regardless of the presence of a preovulatory LH surge, which is characteristic of a hormone secreted under regulation. Our sampling regime was unable to detect pulses of LH during the follicular phase, although GNRHR mRNA levels had increased at this time. Pulses of LH were, however, detected in the luteal phase of cycling females, in anoestrus females and in males. There was a positive correlation between gene expression of FSHB and plasma levels of FSH at different stages of the oestrous cycle and no pulses of FSH were detected at any time; all characteristics of a hormone secreted via the constitutive pathway. Using in situ hybridisation and immunohistochemistry methods, we determined that mRNA expression of LHB and FSHB, and protein storage of gonadotrophins exhibited a similar pattern of localisation within the pituitary gland. Additionally, sexual dimorphism of gonadotroph populations was evident. In summary, these findings are similar to that reported in eutherians and considering that marsupial evolution diverged from eutherians over 100 million years ago suggests that the regulation of gonadotrophins is highly conserved indeed.

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D. J. Tisdall, A. E. Fidler, P. Smith, L. D. Quirke, V. C. Stent, D. A. Heath, and K. P. McNatty

The aim of this study was to investigate stem cell factor and c-kit gene expression and protein localization in the mesonephros and ovary of sheep fetuses at different days of gestation, using RNA in situ hybridization and immunohistochemical procedures. At days 24 and 26 of gestation, stem cell factor mRNA and protein were present in cells throughout the developing gonad and mesonephros. From day 28 to day 40 of gestation, stem cell factor mRNA and protein became increasingly localized to the cortical region of the ovary, where most germ cells were present as actively proliferating oogonia. From day 40 to day 90 of gestation, stem cell factor mRNA and protein localization were confined mainly to the ovarian cortex. Moreover, within the cortical region, stem cell factor mRNA was low or absent where follicles were first forming and highest in the outer ovarian cortex, where germ cells were undergoing mitosis or the early stages of meiosis. In contrast, stem cell factor protein was present in newly forming follicles, as well as in mitotic and meiotic germ cells, which is consistent with the presence of both membrane-bound and soluble forms of this ligand. However, by day 90 of gestation, both stem cell factor mRNA and protein were observed in the granulosa cells of most (> 90%) primordial follicles. C-kit mRNA and protein were observed from day 24 of gestation in both germ cells and somatic cells but, with increasing gestational age, preferentially in germ cells (for example, pre-meiotic germ cells and both isolated oocytes and follicle-enclosed oocytes). C-kit protein, but not mRNA, was also observed in germ cells that were undergoing meiosis. The results indicate that the cells containing stem cell factor mRNA within the ovary up to day 90 of gestation originated from the gonadal blastema and from cells that migrated from the mesonephros before day 28 of gestation. Since stem cell factor mRNA was absent in both mesonephric cells migrating after day 28 of gestation and in regions where follicles were first forming, it is suggested that these later migrating mesonephric cells are the progenitors of the granulosa cells in the first forming follicles. In conclusion, during follicle formation, c-kit mRNA is localized to germ cells whereas c-kit, together with stem cell factor protein, is localized to both germ cells and somatic cells, consistent with the hypothesis that the presence of this receptor–ligand pair is essential to prevent apoptosis.

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K. P. McNatty, D. A. Heath, N. L. Hudson, K. Ball, and L. Condell

Summary. No gene-specific differences were found during either the luteal or follicular phases of the oestrous cycle in the venous secretion rates of ovaries or in concentrations of immunoreactive inhibin in peripheral plasma between Booroola ewes that were homozygous carriers (BB) or non-carriers (++) of the Fec B gene. In three experiments in which concentrations of plasma inhibin and follicle-stimulating hormone (FSH) were compared, gene-specific differences were noted for FSH (P < 0·05), but no significant correlations were noted between FSH and inhibin for either genotype. Granulosa cells and follicular fluid, but not theca interna, stroma or corpora lutea, were the major intra-ovarian sites of inhibin; no gene-specific differences were noted for inhibin concentrations in follicular fluid or in any of the intra-ovarian tissues. The mean concentrations of inhibin in follicular fluid remained constant irrespective of follicular diameter whereas the mean total contents of inhibin increased significantly with increasing diameter (P < 0·05). Inhibin secretion rates were four times higher in ovaries with oestrogen-enriched follicles (i.e. ≥ 50 ng oestradiol ml−1) than in ovaries with no such follicles (P < 0·01). Moreover, inhibin concentrations were higher in follicular fluid of oestrogen-enriched follicles than in those with low oestrogen (i.e. < 50 ng ml−1; P < 0·05). Ovariectomy resulted in a significant reduction in concentrations of immunoreactive inhibin from plasma (P < 0·01). The residual plasma inhibin in some Booroola ewes was not associated with genotype.

It is concluded that, although antral follicles are a major source of inhibin in Booroola ewes, immunoreactive inhibin is not associated with the Fec B gene and is not responsible for the gene-specific differences in concentrations of FSH in plasma.

Keywords: Booroola ewes; Fec B gene; inhibin; FSH; peripheral plasma; ovarian secretion rates

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E. Heath, N. Schaeffer, D. A. Meritt Jr, and R. S. Jeyendran

Summary. Spermatozoa from the ductus deferens of a naked-tail armadillo, Cabassous unicinctus, were arranged in rouleaux. The sperm heads were wafer-thin, with the acrosome and nucleus flattened together. Dense subacrosomal material in the equatorial segment of the acrosomal region was present on one surface but not on the other.

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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. 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, D. A. Heath, N. Hudson, and I. J. Clarke

Summary. Long-term (i.e. ∼70 days) hypophysectomy led to a significant (P < 0·05) reduction in ovarian weight but no reduction in the total number of antral follicles (>0·1 mm in diameter). In hypophysectomized ++ Booroola ewes (N = 8) follicles were always ⩽ 3 mm and in hypophysectomized FF Booroola ewes (N = 6) follicles were always ⩽ 2 mm in diameter; in ewes of both genotypes follicles reached diameters which were ∼40% of their predicted final size at ovulation. Under in-vitro conditions, follicles from the FF and ++ hypophysectomized ewes produced significant increases in cAMP within 1 h of exposure to gonadotrophins (P < 0·05) although no genotypic differences in cAMP production were noted.

We conclude that ovarian follicles in FF and ++ ewes have absolute requirements for pituitary hormone on reaching diameters of 2 mm and 3 mm respectively and that appreciable numbers of antral follicles in ewes of both genotypes remain responsive to pituitary gonadotrophins despite prolonged deprivation of these hormones.

Keywords: hypophysectomy; ovarian follicles; gonadotrophins; cAMP output; Booroola ewes; F gene

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P. Smith, R. Braw-Tal, K. Corrigan, N. L. Hudson, D. A. Heath, and K. P. McNatty

The aims of this study were to examine the effects of the Booroola Fec B gene on ovarian development and reproductive hormones (FSH, LH and inhibin) at days 90, 100, 120 and 135 of gestation (term = 147 days). The effects of litter size were eliminated by transferring equal numbers of homozygous BB and control (++) embryos to recipient ewes. The ovary, but not the body, pituitary, adrenal, kidney or thymus, was heavier (P < 0.05) in BB compared with ++ fetuses at day 90 but not thereafter. In the ovary, gene-specific differences were observed in the total number of germ cells present at days 90 (P< 0.01) and 135 (P < 0.05) with the same tendency being noted at day 100 (P < 0.07); at all of these ages the mean numbers of germ cells in the BB genotype exceeded those in ++ animals. Gene-specific differences were observed in the numbers of oogonia and isolated oocytes at day 90 (i.e. BB > ++), in the number of primordial follicles at days 100 (BB > ++) and 135 (BB > ++), and also in the number of primary or secondary follicles (++ > BB) at day 135. At each gestational age examined no differences were noted with respect to the plasma concentrations of FSH, LH or inhibin between the BB and ++ fetuses. However, the highest mean plasma concentrations of FSH and LH occurred at days 90 and 100 of gestation, which coincided with the first developing primary follicles. Collectively, the results from this and previous studies show that the different effects of the Fec B gene in germ cell development in early gestation continue throughout fetal development independently of litter size. Moreover, during the growth of the first primary and secondary follicles at days 100 and 120, respectively, there are no differences in the plasma concentrations of FSH, LH and inhibin with respect to Booroola genotype.

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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