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R. M. Moor and I. M. Crosby

Summary. The pattern, chemical nature and biological role of polypeptides secreted by ovine follicles exposed to gonadotrophins in vivo were studied. Follicles were removed at intervals before ovulation (in-vivo groups), separated into granulosa and cumulus compartments, and incubated for 3 h with radiolabelled amino acids. No differences were detected in the polypeptides (M r 46 000–60 000) secreted by granulosa and cumulus cells before exposure to the preovulatory LH surge. Each class of polypeptides was characterized by different degrees of phosphorylation, sulphation and glycosylation. Within 15 h of the LH surge the secretion of the M r 46 000–60 000 polypeptides had ceased and was replaced by a non-sulphated M r 30 000 secretory product. Significant differences in the secretory pattern of granulosa and cumulus cells were detected after exposure to LH.

Intact follicles and granulosa cells were cultured for 24 h (in-vitro groups) and then incubated with radiolabelled amino acids. The profile of polypeptides secreted by intact follicles cultured in the presence or absence of LH corresponded closely with the profile observed in vivo. By contrast, granulosa cells grown as monolayers switched spontaneously to the secretion of M r 30 000 polypeptides in medium devoid of gonadotrophin. This aberrant secretory switch did not occur in granulosa cells maintained in suspension culture. Inhibition of transcription in follicles exposed to LH prevented both the appearance of the M r 30 000 polypeptide and the disappearance of the M r 46 000–60 000 polypeptides. Although the inhibition of steroidogenesis by a variety of steroid enzyme inhibitors was without effect on secretion, evidence was obtained to suggest that one of the secreted polypeptides binds oestradiol-17β

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R. M. Moor and I.M. Crosby

Summary. The susceptibility of sheep oocytes to temperature changes during maturation in vitro was tested by reducing the incubation temperature to 20°C at various stages of meiosis. Cooling induced chromosomal abnormalities including disorganized metaphase plates and multipolar spindles in 28–54% of oocytes cooled at all stages of meiosis from germinal vesicle breakdown (GVBD) to metaphase II. The time of GVBD (8–11 h after the start of culture) was the most sensitive to cooling, whereas fewest abnormalities were found in oocytes cooled in late metaphase I (16–19 h). In addition to the chromosomal abnormalities, unusual vesicles appeared in the cytoplasm of oocytes cooled at 8–11 h and 12–15 h. No abnormalities in protein synthesis were detected by one-dimensional SDS gel electrophoresis.

The consequences of the abnormalities for the developmental potential of the cooled oocytes were tested by transfer to recipient ewes and fertilization in vivo. After 12 days of development only 6% and 11% oocytes cooled at 12–15 h and 20–23 h respectively had developed to expanded blastocysts, compared with 44% of control oocytes.

The results demonstrated that maturing sheep oocytes are very sensitive to a drop in temperature.

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R. M. Moor, J. C. Osborn, and I. M. Crosby

Summary. The development of preovulatory follicles involves an initial phase of somatic cell differentiation and a final phase, initiated by the LH surge, when both the somatic and germinal compartments alter. Abnormalities in this pattern of compartmental development after superovulation have been identified by examining follicles from control, PMSG- and FSH-treated sheep. The pattern of proteins synthesized by oocytes from untreated sheep did not differ after culture of follicles in hormone-free medium from that of germinal vesicle oocytes in vivo. Similarly, 93·5% of oocytes from sheep injected with a pituitary gonadotrophin (FSH-P) synthesized the unchanged germinal vesicle pattern of proteins during culture in an hormonally neutral culture environment. By contrast, the administration of the placental gonadotrophin, PMSG, induced in 28% of oocytes changes in the pattern of synthesis which are normally associated with maturation. An examination of follicular steroidogenesis showed that both total output and particularly oestrogen secretion was over twice as high in follicles from PMSG-treated as compared with FSH-treated animals (P < 0·01).

We conclude that the compartmental pattern of development and steroidogenesis is grossly perturbed in many follicles from PMSG-treated animals. Premature activation of the germinal compartment results in aged or abnormal oocytes and a hostile reproductive tract.

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I. M. Crosby, F. Gandolfi, and R. M. Moor

Summary. Sheep embryos, radiolabelled with [35S]methionine, were studied during the first four cell cycles after fertilization to determine the stage at which the regulation of protein synthesis switches from the direction by maternal mRNA to mRNA transcribed by the embryonic genome. Total protein synthesis was consistently high during the first 2 cleavage divisions, dropped by 95% in the 3rd cell cycle, remained low in the 4th and increased again in the 5th cycle. A consistent pattern of proteins was synthesized during the first 3 cell cycles after fertilization followed by major changes in subsequent cycles. The inhibition of transcription by α-amanitin, an inhibitor of polymerase II, did not affect cleavage or protein synthesis during the first 3 cell cycles but blocked cleavage and protein synthesis thereafter. The results indicate that the full activation of transcription in sheep embryos occurs in the 4th cell cycle.

Keywords: embryo; proteins; α-amanitine; transcription; sheep

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I. M. Crosby, J. C. Osborn, and R. M. Moor

Summary. The developmental capacity of sheep oocytes cultured outside the follicle was greatly increased by the presence of high concentrations of gonadotrophins (10 μg/ml) in the medium. However, even under these conditions, the developmental capacity of the oocytes was only half that of oocytes cultured within the intact follicle. The presence of the cumulus was essential for development; nearly all denuded oocytes failed to undergo cleavage.

Maturational changes in the oocyte involving increased amino acid uptake increased incorporation and specific changes in protein synthesis were inhibited by the follicle cells; this suppression was alleviated by gonadotrophic hormones. The cumulus cells suppressed amino acid incorporation and, to some extent, the changes in protein synthesis. However, the suppression of amino acid uptake required the presence of the whole follicle.

Patterns of protein synthesis by oocytes cultured outside the follicle differed from those in oocytes cultured within the follicle, irrespective of the presence of the cumulus or gonadotrophins. Analysis of single oocytes cultured outside the follicle showed that the protein profiles varied markedly even under identical culture conditions.