Identification of a nuclear localization signal in activin/inhibin betaA subunit; intranuclear betaA in rat spermatogenic cells.
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)
Role of class B scavenger receptor type I in phagocytosis of apoptotic rat spermatogenic cells by Sertoli cells.
Rat Sertoli cells phagocytose apoptotic spermatogenic cells, which consist mostly of spermatocytes, in primary culture by recognizing phosphatidylserine (PS) exposed on the surface of degenerating spermatogenic cells. We compared the mode of phagocytosis using spermatogenic cells at different stages of spermatogenesis. Spermatogenic cells were separated into several groups based on their ploidy, with purities of 60-90%. When the fractionated spermatogenic cell populations were subjected to a phagocytosis assay, cells with ploidies of 1n, 2n, and 4n were almost equally phagocytosed by Sertoli cells. All the cell populations exposed PS on the cell surface, and phagocytosis of all cell populations was similarly inhibited by the addition of PS-containing liposomes. Class B scavenger receptor type I (SR-BI), a candidate for the PS receptor, was detected in Sertoli cells. Overexpression of the rat SR-BI cDNA increased the PS-mediated phagocytic activity of Sertoli cell-derived cell lines. Moreover, phagocytosis of spermatogenic cells by Sertoli cells was inhibited in the presence of an anti-SR-BI antibody. Finally, the addition of high density lipoprotein, a ligand specific for SR-BI, decreased both phagocytosis of spermatogenic cells and incorporation of PS-containing liposomes by Sertoli cells. In conclusion, SR-BI functions at least partly as a PS receptor, enabling Sertoli cells to recognize and phagocytose apoptotic spermatogenic cells at all stages of differentiation. (+info)
Genetic analysis of viable Hsp90 alleles reveals a critical role in Drosophila spermatogenesis.
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)
Histone ubiquitination and chromatin remodeling in mouse spermatogenesis.
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)
Possible carcinogenic effects of X-rays in a transgenerational study with CBA mice.
A lifetime experiment using 4279 CBA/J mice was carried out to investigate whether the pre-conceptual exposure of sperm cells to X-ray radiation or urethane would result in an increased cancer risk in the untreated progeny, and/or increased susceptibility to cancer following exposure to a promoting agent. The study consisted of four main groups, namely a control group (saline), a urethane group (1 mg/g body wt) and two X-ray radiation groups (1 Gy, 2 Gy). At 1, 3 and 9 weeks after treatment, the males of these four parental groups were mated with untreated virgin females. The offspring of each parental group was divided into two subgroups: one received s.c. urethane (0.1 mg/g body wt once) as a promoter, the other saline, at the age of 6 weeks. All animals were evaluated for the occurrence of tumours. K-ras oncogene and p53 tumour suppressor gene mutations were investigated in frozen lung tumour samples. The female offspring of male parents exposed to X-rays 1 week before their mating showed a trend towards a higher tumour incidence of the haematopoietic system than the F1 controls. In addition, a higher percentage of bronchioloalveolar adenocarcinomas in male offspring born to irradiated paternals mated 1 week after X-ray treatment points to a plausible increased sensitivity of post-meiotic germ cell stages towards transgenerational carcinogenic effects. On the other hand, no increased tumour incidence and malignancy were observed in the offspring born to irradiated paternals mated 3 and 9 weeks after X-ray treatment. Paternal urethane treatment 1, 3 and 9 weeks prior to conception did not result in significantly altered incidence or malignancy of tumours of the lung, liver and haematopoietic tissue in the offspring. K-ras mutations increased during tumour progression from bronchioloalveolar hyperplasia to adenoma. Codon 61 K-ras mutations were more frequent in lung tumours of urethane-promoted progeny from irradiated parents than from control parents. P53 mutations were absent from these lung alterations. (+info)
Mouse MutS-like protein Msh5 is required for proper chromosome synapsis in male and female meiosis.
Members of the mammalian mismatch repair protein family of MutS and MutL homologs have been implicated in postreplicative mismatch correction and chromosome interactions during meiotic recombination. Here we demonstrate that mice carrying a disruption in MutS homolog Msh5 show a meiotic defect, leading to male and female sterility. Histological and cytological examination of prophase I stages in both sexes revealed an extended zygotene stage, characterized by impaired and aberrant chromosome synapsis, that was followed by apoptotic cell death. Thus, murine Msh5 promotes synapsis of homologous chromosomes in meiotic prophase I. (+info)
Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein.
We have used immunofluorescent localization to examine the distribution of MLH1 (MutL homolog) foci on synaptonemal complexes (SCs) from juvenile male mice. MLH1 is a mismatch repair protein necessary for meiotic recombination in mice, and MLH1 foci have been proposed to mark crossover sites. We present evidence that the number and distribution of MLH1 foci on SCs closely correspond to the number and distribution of chiasmata on diplotene-metaphase I chromosomes. MLH1 foci were typically excluded from SC in centromeric heterochromatin. For SCs with one MLH1 focus, most foci were located near the middle of long SCs, but near the distal end of short SCs. For SCs with two MLH1 foci, the distribution of foci was bimodal regardless of SC length, with most foci located near the proximal and distal ends. The distribution of MLH1 foci indicated interference between foci. We observed a consistent relative distance (percent of SC length in euchromatin) between two foci on SCs of different lengths, suggesting that positive interference between MLH1 foci is a function of relative SC length. The extended length of pachytene SCs, as compared to more condensed diplotene-metaphase I bivalents, makes mapping crossover events and interference distances using MLH1 foci more accurate than using chiasmata. (+info)
Architecture of the nuclear periphery of rat pachytene spermatocytes: distribution of nuclear envelope proteins in relation to synaptonemal complex attachment sites.
The nucleus of spermatocytes provides during the first meiotic prophase an interesting model for investigating relationships of the nuclear envelope (NE) with components of the nuclear interior. During the pachytene stage, meiotic chromosomes are synapsed via synaptonemal complexes (SCs) and attached through both ends to the nuclear periphery. This association is dynamic because chromosomes move during the process of synapsis and desynapsis that takes place during meiotic prophase. The NE of spermatocytes possesses some peculiarities (e.g., lower stability than in somatic cells, expression of short meiosis-specific lamin isoforms called C2 and B3) that could be critically involved in this process. For better understanding of the association of chromosomes with the nuclear periphery, in the present study we have investigated the distribution of NE proteins in relation to SC attachment sites. A major outcome was the finding that lamin C2 is distributed in the form of discontinuous domains at the NE of spermatocytes and that SC attachment sites are embedded in these domains. Lamin C2 appears to form part of larger structures as suggested by cell fractionation experiments. According to these results, we propose that the C2-containing domains represent local reinforcements of the NE that are involved in the proper attachment of SCs. (+info)