The role of heat shock proteins in shielding organisms from environmental stress is illustrated by the large-scale synthesis of these proteins by the organisms studied to date. However, recent evidence also suggests an important role for heat shock proteins in fertilization and early development of mammalian embryos. We found that the presence of anti-HSP70 antibody significantly reduced tight binding of spermatozoa to the zona pellucida of bovine oocytes and interrupted completion of meiosis II and pronuclear formation. Furthermore, the presence of anti-HSP70 in culture medium from day 3 to day 9 of development increased apoptosis and significantly reduced the number of embryos reaching the blastocyst stage. We further observed that the proportion of apoptotic cells in bovine blastocysts was significantly lower after in-vitro culture with a prior exposure to increased temperature. However, nuclear localization of the p53 protein, which is thought to be essential for the up-regulation of genes involved in apoptosis and cell cycle arrest, was detected in the majority of nuclei in blastocysts exposed to increased temperature, whereas in their untreated (control) counterparts, p53 protein was only detected in the cytoplasm. The decrease in apoptosis after exposure of blastocysts to increased temperature may be attributed to cell cycle arrest resulting from nuclear localization of the p53 protein and/or to an increase in heat shock protein synthesis. We propose that HSP70 plays a critical role in fertilization and early embryonic development.
Heat shock protein 70 (HSP70) is part of a superfamily of molecular chaperones, which protect cells from chemical and heat shock. The objectives of this study were to determine the presence of HSP70 in bovine spermatozoa and its subcellular localization during different stages of spermatogenesis. Analysis of sperm proteins by Western blotting using a monoclonal antibody to the inducible form of HSP70 revealed a single immunoreactive band with an estimated molecular weight of 70 kDa in samples from 18 of 18 bulls. Using immunofluorescence microscopy and the same antibody, HSP70 was localized to the cytoplasm of prophase spermatocytes and elongating spermatids, to cytoplasmic droplets of caput epididymal spermatozoa, and to cytoplasmic droplets, acrosome, post-acrosomal region and middle piece of corpus and cauda epididymal spermatozoa. The pattern of distribution changed in freshly ejaculated spermatozoa as HSP70 was detected on the acrosome only. During capacitation and acrosome reaction, HSP70 was once again redistributed, and was localized to the equatorial segment, post-acrosomal region and middle piece. Thus, HSP70 is present in the spermatozoa of mature bulls and redistribution of the protein occurs during capacitation and the acrosome reaction.
Gonadal cell suspensions were made from bovine fetuses of 35-55-, 56-80-, and 80-130-day age groups corresponding to the periods predominated by primordial germ cells (PGCs), oogonia, and meiotic cells, respectively. Germ cells identified on morphological criteria prior to their isolation from suspensions were compared histochemically and morphologically with cells in cryosections, impression smears, and semithin sections of similar gonads. Oocytes were distinguished by their chromosomal configurations in cell spreads. In suspensions from 35-55-day fetuses, cells considered to be PGCs stood out by their size, large nucleus, intracytoplasmic vesicles, and occasional blebbing. The somatic cells were smaller and contained little cytoplasm and few vesicles. In bovine gonads, in contrast to murine gonads, alkaline phosphatase (AP) activity was not specific enough to identify germ cells once they had entered the gonad. In ovaries from the 56-80-day age group, cells similar to PGCs, but slightly larger and with more cytoplasmic vesicles, were identified as oogonia. The cytoplasmic vesicles stained positively for lipid. In ovaries of 80-130-day fetuses, oogonia, oocytes, degenerating germ cells, and multinucleate germ cells were recognized. Degenerating germ cells exhibited a variety of morphological characteristics and were consistently positive for acid-phosphatase activity. Binucleate germ cells appeared around day 85 of gestation, while multinucleate germ cells were seen from day 95. It was concluded that bovine mitotic germ cells can be isolated from gonadal cell suspensions and that the best time to recover them is between 50 and 70 days of gestation.
Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using interspecies gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.
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