Identification of a nuclear localization signal in activin/inhibin betaA subunit; intranuclear betaA in rat spermatogenic cells.
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Activin is a dimeric glycoprotein hormone that was initially characterized by its ability to stimulate pituitary FSH secretion and was subsequently recognized as a growth factor with diverse biological functions in a large variety of tissues. In the testis, activin has been implicated in the auto/paracrine regulation of spermatogenesis through its cognate cell membrane receptors on Sertoli and germ cells. In this study we provide evidence for intranuclear activin/inhibin betaA subunit and show its distribution in the rat seminiferous epithelium. We have shown by transient expression in HeLa cells of beta-galactosidase fusion proteins that the betaA subunit precursor contains a functional nuclear localization signal within the lysine-rich sequence corresponding to amino acids 231-244. In all stages of the rat seminiferous epithelial cycle, an intense immunohistochemical staining of nuclear betaA was demonstrated in intermediate or type B spermatogonia or primary spermatocytes in their initial stages of the first meiotic prophase, as well as in pachytene spermatocytes and elongating spermatids primarily in stages IX-XII. In some pachytene spermatocytes, the pattern of betaA immunoreactivity was consistent with the characteristic distribution of pachytene chromosomes. In the nuclei of round spermatids, betaA immunoreactivity was less intense, and in late spermatids it was localized in the residual cytoplasm, suggesting disposal of betaA before spermatozoal maturation. Immunoblot analysis of a protein extract from isolated testicular nuclei revealed a nuclear betaA species with a molecular mass of approximately 24 kDa, which is more than 1.5 times that of the mature activin betaA subunit present in activin dimers. These results suggest that activin/inhibin betaA may elicit its biological functions through two parallel signal transduction pathways, one involving the dimeric molecule and cell surface receptors and the other an alternately processed betaA sequence acting directly within the nucleus. According to our immunohistochemical data, betaA may play a significant role in the regulation of nuclear functions during meiosis and spermiogenesis. (+info)
The degenerative fate of germ cells not conforming to stage in the pubertal golden hamster testis.
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In the golden hamster (Mesocricetus auratus), pubertal establishment of spermatogenesis includes a defined period (d 26-30 of life) during which elongation of spermatids is selectively arrested. The resulting appearance of germ cell associations not conforming to stage and the phenomenon of desynchronisation-related germ cell degeneration are analysed both quantitatively and qualitatively by means of light and 'retrospective' electron microscopy. From d 26 onwards, the portion of tubules containing non-stage conforming germ cell associations gradually increases up to 37.5% of sectioned tubules on d 32. Concomitantly, the degree of desynchronisation rises to a maturational gap between spermatids and associated younger germ cells of 7 stages of the seminiferous epithelium cycle, i.e. of fully half a cycle. Beyond d 32, the frequency of desynchronised tubule segments decreases again. Some of the arrested round spermatids and, eventually, all belatedly elongating spermatids degenerate and are lost from the epithelium. Thus a regular maturation of advanced spermatids does not succeed under non-stage conforming conditions. Possibly it is not the desynchronisation between the associated germ cell generations and the spermatids by itself that impedes normal further development of the latter cells. Instead this may be due to the maturational delay of the stage-aberrant cells by several stages compared to the seminiferous epithelium as a whole and, especially, in relation to the stage-conditioned functional state of the neighbouring Sertoli cells. (+info)
Genetic analysis of viable Hsp90 alleles reveals a critical role in Drosophila spermatogenesis.
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The Hsp90 chaperone protein maintains the activities of a remarkable variety of signal transducers, but its most critical functions in the context of the whole organism are unknown. Point mutations of Hsp83 (the Drosophila Hsp90 gene) obtained in two different screens are lethal as homozygotes. We report that eight transheterozygous mutant combinations produce viable adults. All exhibit the same developmental defects: sterile males and sterile or weakly fertile females. We also report that scratch, a previously identified male-sterile mutation, is an allele of Hsp82 with a P-element insertion in the intron that reduces expression. Thus, it is a simple reduction in Hsp90 function, rather than possible altered functions in the point mutants, that leads to male sterility. As shown by light and electron microscopy, all stages of spermatogenesis involving microtubule function are affected, from early mitotic divisions to later stages of sperm maturation, individualization, and motility. Aberrant microtubules are prominent in yeast cells carrying mutations in HSP82 (the yeast Hsp90 gene), confirming that Hsp90 function is connected to microtubule dynamics and that this connection is highly conserved. A small fraction of Hsp90 copurifies with taxol-stabilized microtubule proteins in Drosophila embryo extracts, but Hsp90 does not remain associated with microtubules through repeated temperature-induced assembly and disassembly reactions. If the spermatogenesis phenotypes are due to defects in microtubule dynamics, we suggest these are indirect, reflecting a role for Hsp90 in maintaining critical signal transduction pathways and microtubule effectors, rather than a direct role in the assembly and disassembly of microtubules themselves. (+info)
Fertilization, embryonic development, and offspring from mouse eggs injected with round spermatids combined with Ca2+ oscillation-inducing sperm factor.
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Round spermatids, precursor male gametes, are known to possess the potential to achieve fertilization and embryonic development when injected into eggs. However, injection of spermatids alone seldom activates eggs in the mouse, as spermatids by themselves cannot induce an increase in intracellular Ca2+, a prerequisite for egg activation. We injected a mouse round spermatid into an egg simultaneously with partially purified sperm factor from differentiated hamster spermatozoa. The combined injection produced repetitive Ca2+ increases (Ca2+ oscillations) lasting for at least 4 h as observed at fertilization, and induced activation in 92% of eggs. This method provided 75% fertilization success associated with male and female pronucleus formation and development to 2-cell embryos, while only 7% of eggs were fertilized by injection of a spermatid alone. Of the 2-cell embryos, approximately 50% developed to blastocysts during 5 days of culture in vitro, while no blastocysts were obtained following injection of sperm factor alone. Furthermore, the 2-cell embryos, that were created by spermatids and sperm factor and transplanted into foster mothers, developed into normal offspring, although the percentage was only 22%. All infants grew into healthy adults carrying normal chromosomes. The sperm factor served as a complementary factor for successful fertilization by round spermatid injection. (+info)
Histone ubiquitination and chromatin remodeling in mouse spermatogenesis.
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Male infertility in HR6B knockout mice is associated with impairment of spermatogenesis. The HR6B gene is a mammalian, autosomal homolog of the Saccharomyces cerevisiae gene Rad6 encoding a ubiquitin-conjugating enzyme. In addition, X-chromosomal HR6A has been identified, in human and mouse. RAD6 in yeast is required for a variety of cellular functions, including sporulation, DNA repair, and mutagenesis. Since RAD6 and its mammalian homologs can ubiquitinate histones in vitro, we have investigated the pattern of histone ubiquitination in mouse testis. By immunoblot and immunohistochemical analysis of wild-type mouse testis, a high amount of ubiquitinated H2A (uH2A) was detected in pachytene spermatocytes. This signal became undetectable in round spermatids, but then increased again during a relatively short developmental period, in elongating spermatids. No other ubiquitinated histones were observed. In the HR6B knockout mice, we failed to detect an overt defect in the overall pattern of histone ubiquitination. For somatic cell types, it has been shown that histone ubiquitination is associated with destabilization of nucleosomes, in relation to active gene transcription. Unexpectedly, the most intense uH2A signal in pachytene spermatocytes was detected in the sex body, an inactive nuclear structure that contains the heterochromatic X and Y chromosomes. The postmeiotic uH2A immunoexpression in elongating spermatids indicates that nucleosome destabilization induced by histone ubiquitination may play a facilitating role during histone-to-protamine replacement. (+info)
A novel quantitative morphometry of germ cells for the histopathological evaluation of rat testicular toxicity.
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A view that 14 stages of rat spermatogenic cycle could be arranged into 4 groups, viz., conventional stages I-VI, VII-VIII, IX-XI and XII-XIV, according to the features of elongated spermatids was previously presented. A novel morphometry of seminiferous epithelia based on these 4 groups was also proposed. In the present study, utility of the proposed morphometry in the histopathological evaluations of testicular toxicities was monitored in comparison with the conventional one. After administrating adriamycin, ethylene glycol monomethyl ether or 1,3-dinitrobenzene to rats, the viability of their germ cells was estimated by the proposed morphometry and the conventional one employed stages II-III, V, VII, X and XII. In every case, the evaluating results of the proposed morphometry were similar to those of the conventional one. Thus, it was verified that the proposed morphometry was identical with the conventional one in respect of reliable detection of the testicular toxicities. In addition, in situ terminal dUTP nick end labeling indicated that death of spermatogonia, pachytene spermatocytes or round spermatids induced by the above 3 toxic compounds was exclusively apoptotic death. In conclusion, the proposed morphometry would be useful as a practical tool in the evaluation of testicular toxicities. (+info)
Sperm abnormalities and histopathological changes in the testes in Crj:CD(SD)IGS rats.
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In this study, morphological examination and computer-assisted sperm analysis (CASA) of epididymal spermatozoa in non-treated Crj:CD(SD)IGS rats were performed, and the relationship between the data obtained and the retention of step 19 spermatids in Stage IX to XI seminiferous tubules was examined. Retention of step 19 spermatids in Stage IX to XI seminiferous tubules was observed in all 50 untreated males, and the incidence ranged from 3.3% to 100%. Eighteen animals showed a high incidence of retention (74.7 +/- 14.2%, HIR for short), and the others showed a low incidence (24.9 +/- 11.0%, LIR for short). Although the incidence of retention in Stage X and XI seminiferous tubules was very low in LIR males, it was high in HIR males (1.8 +/- 3.0% vs 58.6 +/- 23.2%). Morphological abnormalities of sperms in the caudal region of the epididymis, mainly amorphous head and no head, were more frequently observed in HIR males than in LIR males (36.2 +/- 28.5% vs 1.8 +/- 1.2%). Sperm analysis also revealed some differences between HIR and LIR males: sperm motility in HIR males was severely lower than that in LIR males, and sperm velocity, beat/cross frequency and amplitude of lateral head displacement in HIR males were lower than the corresponding values in LIR males. In summation, retention of step 19 spermatids frequently occurred in the non-treated Crj:CD(SD)IGS males, and a relationship between the retention of these spermatids and sperm abnormalities, such as morphologically abnormal sperms, low motility and other items revealed by sperm analysis (CASA), was suggested. (+info)
Truncated RanGAP encoded by the Segregation Distorter locus of Drosophila.
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Segregation Distorter (SD) in Drosophila melanogaster is a naturally occurring meiotic drive system in which the SD chromosome is transmitted from SD/SD+ males in vast excess over its homolog owing to the induced dysfunction of SD+-bearing spermatids. The Sd locus is the key distorting gene responsible for this phenotype. A genomic fragment from the Sd region conferred full distorting activity when introduced into the appropriate genetic background by germline transformation. The only functional product encoded by this fragment is a truncated version of the RanGAP nuclear transport protein. These results demonstrate that this mutant RanGAP is the functional Sd product. (+info)