Chronic cyclophosphamide treatment alters the expression of stress response genes in rat male germ cells

Increases in the survival rate of men treated with chemotherapeutic drugs and their desire to have children precipitate concerns about the effects of these drugs on germ cells. Azoospermia, oligospermia, and infertility are common outcomes resulting from treatment with cyclophosphamide, an alkylating agent. Exposure of male rats to cyclophosphamide results in dose-dependent and time-specific adverse effects on progeny outcome. Elucidation of the effects of chronic low-dose cyclophosphamide treatment on the expression of stress response genes in male germ cells may provide insight into the mechanisms underlying such adverse effects. Male rats were gavaged with saline or cyclophosphamide (6 mg/kg) for 4-5 wk; pachytene spermatocytes, round spermatids, and elongating spermatids were isolated; RNA was extracted and probed on cDNA arrays containing 216 cDNAs. After saline treatment, 125 stress response genes were expressed in pachytene spermatocytes (57% of genes studied), 122 in round spermatids (56%), and 83 in elongating spermatids (38%). Cyclophosphamide treatment reduced the number of genes detected in all germ cell types. The predominant effect of chronic cyclophosphamide exposure was to decrease the expression level of genes in pachytene spermatocytes (34% of genes studied), round spermatids (29%), and elongating spermatids (4%). In elongating spermatids only, drug treatment increased the expression of 8% of the genes studied. The expression profiles of genes involved in DNA repair, posttranslational modification, and antioxidant defense in male germ cells were altered by chronic cyclophosphamide treatment. We hypothesize that the effects of cyclophosphamide exposure on germ cell gene expression during spermatogenesis may have adverse consequences on male fertility and progeny outcome.     

Ultrastructural changes in the male germ cells of Bulinus truncatus during spermatogenesis

The structure of the spermatogonia, spermatocytes, spermatids and Sertoli cells of the hermaphroditic snail Bulinus truncatus was studied by electron microscopy. The spermatogonia are small, with relatively large nuclei. The acrosome develops from a small proacrosomal granule which is probably derived from the Golgi apparatus in the spermatocyte stage. Condensation and elongation of the nuclei were found in the spermatids. The shape and components of the Sertoli cells did not change during the spermatogonium and spermatocyte stages. Before spermiation the Sertoli cells have the morphological features of steroid-producing cells. The study showed that the Sertoli cells are involved in the nutrition and transportation of the spermatogenic cells, in spermiation and in hormone production.     

Expression of the soluble adenylyl cyclase during rat spermatogenesis: evidence for cytoplasmic sites of cAMP production in germ cells

To gain insight into the mechanisms of cAMP signaling in germ cells, the expression and subcellular localization of the full-length form of the soluble adenylyl cyclase (sAC) was investigated during rat spermatogenesis and in spermatozoa. A full-length sAC-specific antibody was generated by using a glutathione S-transferase (GST)-sAC carboxyl-terminal region (1399aa-1608aa) fusion protein as the antigen. The selectivity of the purified antibody was confirmed by immunoblotting with lysates from HEK293 cells overexpressing full-length sAC or truncated sAC. Western blot analysis demonstrated that full-length sAC protein appeared on day 25 during testis development. The expression levels increased progressively on days 30 and 35 and remained elevated in adult testis. Full-length sAC protein is retained in spermatozoa from the cauda epididymis. Consistent with the timing of the appearance of the Western blot signal, immunohistochemistry with testis sections at different stages of development detected sAC in late pachytene spermatocytes as well as round and elongating spermatids. Further experiments on the subcellular localization of native or recombinant enzymes revealed that full-length sAC is not only recovered in soluble fractions but also in particulate fractions of testis extracts. Immunofluorescence detection showed localization of the protein in the cytoplasm as well as in organelles of pachytene spermatocytes and spermatids. These findings indicate that cAMP production in spermatids and spermatozoa may occur at sites other than the plasma membrane and suggest that full-length sAC may play a role during spermatid differentiation.     

Expression of senescence-enhanced genes in response to oxidative stress

Expression of the LSC54 gene, encoding a metallothionein protein, has been shown previously to increase during leaf senescence and cell death. Evidence is presented in this paper to indicate that the extent of LSC54 expression is related to levels of oxidative stress in the tissues. Treatment of Arabidopsis cotyledon and leaf tissues with the catalase inhibitor, 3-amino-1,2,4-triazole, or with silver nitrate result in the enhanced expression of LSC54. Combined treatments with quenchers of reactive oxygen species (ROS), such as ascorbate, tiron and benzoic acid indicated that this induced expression was due to increased levels of ROS. The expression of many other senescence-enhanced genes was also found to be inducible by the increase in ROS. Treatment of plant tissue with 3-amino-1,2,4-triazole, followed by silver nitrate, resulted in protection from the severe damage caused by the silver nitrate treatment and reduced expression of many of the genes examined. However, one gene, encoding a lipid hydroperoxide-dependent glutathione peroxidase, showed increased expression in the protected tissue, which may indicate a role for this enzyme in the protection of plant tissue from oxidative stress. ROS-enhanced expression of at least one of the genes investigated required the presence of the salicylic acid signalling pathway, which was not required for the expression of LSC54.     

Expression of HSP86 in male germ cells.

A comparison of HSP84 and HSP86 mRNA expression in adult mouse tissues revealed distinct expression patterns for these highly homologous genes. Particularly striking is the germ cell specificity of HSP86 expression in the testis, suggesting distinct roles for HSP84 and HSP86 with respect to testicular function and development.     

Expression of H-Y antigen during spermatogenesis

With the use of mixed-hemadsorption-hybrid-antibody (MHA-HA) test, H-Y antigen was studied on neonatal testicular cells and fractionated testicular cells from young mice (4–6 weeks old). H-Y antigen was undetectable on spermatogonia cells from neonatal testes but became fully expressed on late spermatids. Our data suggested that there was postmeiotic expression of H-Y antigen.     

Immuno-electron microscopic localization of calmodulin and calmodulin-binding proteins in the mouse germ cells during spermatogenesis and maturation

When extracts of mouse testis were Western-blotted against a monoclonal antibody which reacts with calmodulin in the presence of Ca²⁺, all calmodulin was associated with the macromolecules of molecular weight above 50 kDa. Immuno-electron microscopy of testes using this antibody indicated that calmodulin is localized at higher density in the nucleus and cytoplasm of germ cells during the developmental phase between pachytene and round spermatid, showing the highest level just before meiotic divisions. There was no special association of calmodulin to any organelles in these cells. Extremely low levels of calmodulin occurred in spermatogonia and other testicular tissue cells. Calmodulin decreased dramatically as spermatids underwent metamorphosis, becoming detectable only at the perinuclear space of sperm heads. Further relocation to the postacrosomal region occurred during sperm transit to the cauda epididymis. Immunodetection after the calmodulin overlay on ultrathin sections revealed a sharp increase of calmodulin immunogold deposits in the nuclei of spermatids accompanying their condensation. The results indicate that some calmodulin-binding proteins, but not calmodulin itself, accumulate in the nuclei during the final steps of spermiogenesis.     

Expression of apoptotic genes in testicular germ cells and their modulation by luteinizing hormone

Gonadotropins regulate spermatogenesis by promoting survival and differentiation of germ cells. The molecular markers that are modulated by these hormones to ensure survival however have not been described in great detail. Immunoneutralization of LH in particular leads to apoptotic cell death of the spermatocytes and the round spermatids. In the present study, the expression pattern and regulation of apoptotic markers after specific immunoneutralization of LH in germ cells purified from rats has been investigated at the RNA and protein level. Of the several markers tested, Bax, caspases 1 and 2 and Fadd exhibit differential expression, with the round spermatids expressing higher levels of caspases 1 and 2, and the spermatocytes expressing higher levels of Bax and Fadd. The two cell types therefore exhibit differential expression of apoptotic markers. The cell types also differ with respect to their response to LH antiserum treatment. Fas and Bax both are up-regulated in the round spermatids after 24h of antiserum treatment. In the spermatocytes, Fas was up-regulated as early as 12h after antiserum treatment while Bax was up-regulated after 2 days. These results demonstrate that LH regulates survival of germ cells by modulating the levels of pro and anti-apoptotic proteins.     

Stage-dependent changes in localization of a germ cell-specific lamin during mammalian spermatogenesis

We had earlier identified a 110/120-kDa protein specific to nuclear matrix of rat pachytene spermatocytes (Behal, A., Prakash, K., and Rao, M.R.S. (1987) J. Biol. Chem. 262, 10898-10902). This protein is now shown to be a disulfide-linked homodimer of a 60-kDa polypeptide. Indirect immunofluorescence and Western blot analyses using anti-120-kDa polyclonal antibodies have shown that this protein is a component of the pore-complex lamina structure of spermatogonia. As germ cells enter meiotic prophase and the lamina structure disassembles, this polypeptide is redistributed in the nucleus and can be isolated as a component of synaptonemal complexes. Following meiotic division, this 60-kDa protein is relocalized in the lamina, then representing the sole major component of the lamina structure of round spermatids. The identity of the 60-kDa protein in the pore-complex lamina fraction and synaptonemal complexes was further confirmed by two-dimensional analysis of iodinated tryptic peptides. Such an analysis has also shown that the germ cell-specific 60-kDa protein is related but not identical to somatic lamin B.     

Expression and phosphorylation of TOPK during spermatogenesis

Among normal organs and tissues, the MAPKK-like mitotic protein kinase TOPK is expressed exclusively in the testis. We analyzed the expression and phosphorylation of TOPK to address the functional role of this kinase during spermatogenesis. TOPK protein is expressed mainly in the cytosol of spermatocytes and spermatids, but not in spermatids and spermatogonia in situ. TOPK-Thr-9, a cdk1/cyclin B target residue, was specifically phosphorylated during mitotic and meiotic phases, while TOPK-Thr-198, a key amino acid for the ATP pocket, was constantly phosphorylated irrespective of the cell cycle. These data indicate that spermatogenic germ cells with vital proliferation activity express TOPK. As TOPK-Thr-9 was phosphorylated during both mitosis and meiosis, TOPK was indicted to play a role in cytokinesis and/or chromosomal segregation but not in DNA replication.     

Expression of ethylene response genes during persimmon fruit astringency removal

Thirteen ethylene signaling related genes were isolated and studied during ripening of non-astringent ‘Yangfeng’ and astringent ‘Mopan’ persimmon fruit. Some of these genes were characterized as ethylene responsive. Treatments, including ethylene and CO₂, had different effects on persimmon ripening, but overlapping roles in astringency removal, such as increasing the reduction in levels of soluble tannins. DkERS1, DkETR2, and DkERF8, may participate in persimmon fruit ripening and softening. The expression patterns of DkETR2, DkERF4, and DkERF5 had significant correlations with decreases in soluble tannins in ‘Mopan’ persimmon fruit, suggesting that these genes might be key components in persimmon fruit astringency removal and be the linkage between different treatments, while DkERF1 and DkERF6 may be specifically involved in CO₂ induced astringency removal. The possible roles of ethylene signaling genes in persimmon fruit astringency removal are discussed.     

Radiation-induced malformations after exposure of murine germ cells in various stages of spermatogenesis

We studied radiation effects in day 19 fetuses of the mouse strain 'Heiligenberger' after exposure (2.8 Gy, 137 Cs gamma rays, dose rate 0.28 Gy/h) of their fathers. We observed an increased lethality (exclusively due to preimplantation death and early resorptions) after exposure of all stages of spermatogenesis with the exception of early spermatogonia. In addition, there was a significant increase in the frequency of malformed fetuses (gastroschises only); this increase was observed primarily after exposure of the meiotic stages.