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  • Author: K. P. McNatty x
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B R Leeuwenberg, P R Hurst, and K P McNatty

ABSTRACT

IGF-I has been implicated as a local and/or systemic regulator of ovarian function by acting alone or as an amplifier of the actions of gonadotrophins, thereby influencing follicular growth and ovulation. In the sheep it is uncertain as to whether IGF-I can fulfil an autocrine or paracrine role, since mRNA expression and peptide synthesis have not previously been demonstrated.

Using in situ hybridization with ovine and human riboprobes, IGF-I mRNA was found in all major steroidogenic cell types of the sheep ovary, namely the granulosa, theca and luteal cells and, to a lesser extent, the stroma. IGF-I mRNA was found to be expressed in the granulosa and thecal cells of early antral follicles and thereafter in these cell types as the follicles increased in diameter. Evidence for IGF-I mRNA expression in preantral follicles could not be demonstrated. No obvious differences in the levels of expression were observed in ovaries recovered during the luteal phase, follicular phase, anoestrus or after exogenous FSH treatment. IGF-I expression was confirmed by Northern blotting and PCR. These findings are consistent with the notion that IGF-I may have an autocrine or paracrine role in enhancing ovarian function in the sheep.

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D J Tisdall, N Hudson, P Smith, and K P McNatty

ABSTRACT

The sites of follistatin and α and βA inhibin gene expression were examined by in situ hybridization in sheep ovaries during the early and mid-luteal phases (days 3 and 10) of the oestrous cycle and a prostaglandin F (PGF)-induced follicular phase.

Follistatin mRNA was detected in the granulosa cells of preantral, antral and early atretic follicles at all stages of the oestrous cycle, and in the corpora lutea at the early and mid-luteal stages of the cycle. However, only low levels of expression of follistatin were observed in the presumptive preovulatory follicle at 56 h after treatment with PGF.

Both α and βA inhibin were shown to be expressed in ovaries at all stages of the oestrous cycle. In situ hybridization localized α subunit mRNA to the granulosa cells of most, but not all, healthy antral follicles, and to no other ovarian cell type. In contrast, expression of the βA subunit was confined to a few medium-to-large healthy antral follicles. In antral follicles expressing βA inhibin, mRNAs for α inhibin and follistatin were always detected, but the converse was not true. Unlike follistatin, no α and βA inhibin expression was seen in preantral follicles, developing corpora lutea, or follicles undergoing atresia.

These results show that, in the adult sheep ovary, follistatin gene expression is a constitutive event in all growing follicles from the early preantral stage, and also provide indirect evidence of the involvement of follistatin, but not inhibin or activin, in the early stages of ovarian follicle development in sheep.

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D J Tisdall, K Watanabe, N L Hudson, P Smith, and K P McNatty

ABSTRACT

A key question in elucidating the role of FSH in ovarian function is to determine when during follicular growth the FSH receptor first appears. The aim of this study was to examine the site and time of FSH receptor gene expression during early follicular growth. This study was carried out on ovaries of adult sheep during the luteal and prostaglandin-induced follicular phase of the oestrous cycle and also on ovaries of fetal sheep at 90, 100, 120 and 135 days of gestation (term=day 147).

Using reverse transcription-PCR and a set of PCR primers spanning exons 8/9/10, two partial FSH receptor cDNAs (500 and 310 bp) were isolated from adult sheep ovary. It was shown by sequencing that exon 8 was deleted in the 310 bp cDNA, implying that this was part of an alternatively spliced FSH receptor transcript. Using RNA in situ hybridisation on ovaries of adult sheep, FSH receptor mRNA was observed in granulosa cells of early preantral follicles with one to two cell layers and it was seen that gene expression continued throughout folliculogenesis into advanced stages of atresia. Moreover, in the fetus, FSH receptor gene expression was detected in follicles with two or more layers of granulosa cells in ovaries taken at 100, 120 and 135 days of gestation.

These results suggest that the FSH receptor gene is expressed after the granulosa cells of a folllicle have begun to divide but not during the earliest stages of follicle growth, namely the transformation of a primordial follicle to a primary follicle.

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R Braw-Tal, D J Tisdall, N L Hudson, P Smith, and K P McNatty

ABSTRACT

The aim of this study was to investigate the sites of follistatin and α and βA inhibin mRNA expression in the ovaries of female sheep fetuses at 90, 100, 120 and 135 days of gestation (term=day 147). At 90 and 100 days primordial follicles were formed, followed by the appearance of primary follicles at 100 days of gestation. At days 120 and 135, primordial, primary and preantral (i.e. secondary) follicles were present in the ovaries, but antral (i.e. tertiary) follicles were not observed at any of these gestational ages. Two Booroola genotypes were studied: homozygous carriers (BB) and non-carriers (++) of the fecundity gene (FecB). Irrespective of genotype no specific hybridization of the α and βA inhibin riboprobes was detected in any ovarian cells at days 90, 100, 120 or 135 of gestation. In control mature ovaries, on the other hand, strong hybridization in the granulosa cells of antral follicles was observed. In contrast to α and βA inhibin, follistatin antisense (but not sense) riboprobes hybridized specifically to the granulosa cells of preantral follicles with two or more layers of cells at days 120 and 135 of gestation. Moreover, hybridization was also evident in the cells of the ovarian rete at days 120 and 135, but not at 90 or 100 days. No follistatin mRNA expression was observed in the granulosa cells of primordial or primary follicles or in any other ovarian cell type at any of the gestational ages examined. No FecB-specific differences in follistatin expression were noted with respect to stage of preantral follicular development and there were no obvious differences in the intensity of expression.

These results show that follistatin mRNA is expressed specifically in the granulosa cells and intraovarian rete. Expression of follistatin in rete cells was coincident with the increasing numbers of growing follicles within the fetal ovary, indicating that rete cell function may have a role in the ontogeny of early follicular growth. Our results suggest that follistatin and α and βA inhibin may not be important for the initiation of follicle growth in the sheep ovary, since these genes are not expressed during the transformation of a primordial follicle to a primary structure. However, the evidence for follistatin mRNA expression in the ovine fetal ovary implies that this hormone is likely to play a role during the early stages of follicle growth.

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D J Tisdall, L D Quirke, P Smith, and K P McNatty

ABSTRACT

Two ovine stem cell factor (oSCF) cDNAs (822 bp and 738 bp) were generated from ovarian follicle mRNA by RT-PCR. Nucleotide sequencing revealed that the oSCF 822 bp cDNA encodes a precursor protein of 274 amino acids. An amino acid change 109E to 109Q was the only sequence difference from that previously described for this species. The smaller (738 bp) oSCF cDNA was shown by nucleotide sequencing to be an mRNA splice variant, equivalent to that found in other mammals, in which an exon (84 bp) encoding a potential proteolytic cleavage site is removed. Northern analysis revealed a single transcript of approximately 6·5 kb in follicles, corpora lutea and stroma of mid-luteal sheep ovaries. In situ hybridization was used to detect oSCF mRNA within ovaries of fetal sheep on days 90, 100, 120 and 135 of gestation (term=147) and of adult sheep within the breeding season. In fetal and adult ovaries, oSCF mRNA was detected in the granulosa cells of follicles at all stages of follicle growth (primordial through to antral). The SCF gene was also expressed in granulosa cells of atretic follicles but appeared to be down-regulated in the cumulus cells surrounding the oocyte at more advanced stages of atresia. In fetal ovaries at day 90 of gestation (90DG), oSCF was expressed in the subepithelial mesenchymal cells of the ovarian cortex. By 100DG the gene expression in the subepithelial cells became restricted to a narrow region below the epithelium, and areas of expression were observed in groups of cells around isolated oocytes, primordial and primary follicles. oSCF gene expression also occurred in the surface epithelial cells of 90DG ovaries, the expression was absent from these cells by 135DG and in adult ovaries. Localization of oSCF mRNA was observed in the ovarian rete and endothelial cells of blood vessels of fetal ovaries. These results suggest that oSCF may have an important and continuous role in the development and/or maintenance of germ cells during follicle growth and atresia in sheep.

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

ABSTRACT

Ovine cDNA probes for the α and βA inhibin subunits and for follistatin were used to investigate the mRNA species for these hormones in ovaries obtained during the luteal phase of the oestrous cycle, from Booroola ewes which were homozygous carriers (BB) or non-carriers (++) of the FecB gene. BB ewes had significantly higher concentrations of peripheral FSH and LH immunoreactivity than ++ ewes, but the peripheral inhibin immunoreactivity and ovarian inhibin and progesterone secretion rates were not significantly different between genotypes. No gene-specific differences in the number or size of mRNA transcripts detected by Northern blotting were noted for any of these genes. A single α inhibin mRNA species at 1.5 kb was observed in the follicle RNA from ++ and BB ovaries. Low amounts of α inhibin hybridization were discerned occasionally in + + and BB stroma and also in BB, but not in ++, corpora lutea. The βA inhibin gene was expressed only in the follicles from both ++ and BB ovaries. At least three βA inhibin transcripts were observed; one at 7.5kb and at least two between 1.4 and 5.0kb. The follistatin cDNA probe detected two major transcripts at 2.7 and 1.5 kb and a minor band at 0.5 kb in both follicle and corpora lutea RNA. Densitometry of the Northern blots revealed no significant gene-specific differences in the levels of α inhibin and follistatin gene mRNA transcripts. However, significantly greater amounts of total βA inhibin hybridization were detected in follicle RNA from BB compared with ++ ovaries (P<0.001) and this FecB-specific difference appeared to be associated with the 7.5 kb transcript. We conclude that the Booroola FecB gene does not influence the synthesis of the α inhibin subunit or follistatin during the luteal phase of the oestrous cycle, but may affect inhibin or activin synthesis in the ovaries of FecB carriers, by increasing the transcription or stability of the βA inhibin mRNA species.