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ABSTRACT

Clusterin or sulphated glycoprotein-2 is a major component of the rete testis fluid, synthesized by the rete testis epithelial cells and Sertoli cells. Differences in the two-dimensional polyacrylamide gel electrophoresis pattern of clusterin-like proteins have been reported in rete testis fluid from Booroola rams carrying the fecundity gene Fec^B, when compared with that from non-carrier rams. In order to determine whether the Fec^B gene influences the expression of the clusterin gene, we used a rat clusterin cRNA probe to investigate mRNA species in the tissues of homozygous (BB) or non-carrier (+ +) Booroola sheep. Northern blots of polyadenylated RNA showed hybridization to the cRNA probe in the testis, ovarian follicles, corpora lutea and stroma, pituitary and liver. A major mRNA transcript was observed at 2·3 kb and a minor transcript in some tissues at 0·8 kb. Densitometry of the autoradiographs revealed no Fec^B-specific differences in the densities of the hybridization signals from + + and BB testis or ovarian follicle, corpora lutea or stromal RNA. We conclude that the gene for ovine clusterin is expressed widely in the tissues of sheep and that its expression is not affected by the presence of the Fec^B gene.

ABSTRACT

A cDNA probe for the β subunit of bovine FSH (FSH-β) detects multiple restriction fragment length polymorphisms (RFLPs) in sheep genomic DNA consistent with an insertion/deletion polymorphism around the FSH-β locus. The presence of the insertion/deletion was confirmed by screening over 100 individuals with two restriction enzymes detecting RFLPs. All individuals showed the same patterns of fragments with both enzymes. A partial restriction map of the FSH-β gene in sheep suggests that the insertion/deletion is approximately 2 kb in size and located downstream from the third exon. Individual DNA samples were analysed from two flocks where the Booroola F gene is known to be segregating. Individuals that were heterozygous for the F gene were shown to be homozygous for one or other of the two alleles. Genetic recombination between the FSH-β locus and the F gene was observed in four pedigrees and there was no evidence that the insertion/deletion is closely linked genetically to the Booroola F gene. A major gene transcript of 2·2–2·3 kb was detected on Northern blots of sheep RNA. Neither the insertion/deletion polymorphism nor the presence of the F gene appeared to influence the size of the FSH-β gene transcript.

ABSTRACT

We have isolated ovine follistatin cDNA from an ovarian follicle cDNA library and determined its sequence. The deduced amino acid sequence of the ovine follistatin precursor is highly homologous (>97%) to the porcine, human and rat follistatins.

Northern analysis was used to characterize follistatin gene expression in ovaries of adult ewes, collected from days 11 to 13 of the oestrous cycle. Two major (about 2.7kb and 1.5 kb) and one minor (about 0.5 kb) transcripts were detected in polyadenylated RNA extracted from ovarian follicles and corpora lutea. The degree of expression of the transcripts varied in the two ovarian compartments, with the 2.7kb species predominating in the follicles and the 1.5kb species being more abundant in the corpora lutea. No transcripts were detected in stromal tissue containing preantral follicles of <1 mm.

ABSTRACT

Ovine cDNA probes for the α and β_A 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 Fec^B 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 β_A inhibin gene was expressed only in the follicles from both ++ and BB ovaries. At least three β_A 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 β_A inhibin hybridization were detected in follicle RNA from BB compared with ++ ovaries (P<0.001) and this Fec^B-specific difference appeared to be associated with the 7.5 kb transcript. We conclude that the Booroola Fec^B 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 Fec^B carriers, by increasing the transcription or stability of the β_A inhibin mRNA species.