Rat Male Germ Cells Research Articles

Regulation of the aromatase gene expression in adult male rat germ cells: effects of TGFß and TNFα

Les fonctions de reproduction chez le mâle sont sous le controle des hormones hypophysaires. Outre cette regulation endocrine, il existe une regulation locale intragonadique dont depend le bon deroulement de la spermatogenese. Cette regulation locale fait notamment intervenir les steroides, les facteurs de croissance et les cytokines. L’aromatase est l’enzyme responsable de la conversion irreversible des androgenes en estrogenes. Dans la gonade mâle de differentes especes de mammiferes, l’aromatase a ete immunolocalisee non seulement dans les cellules de Leydig, mais aussi dans les cellules germinales. L’objectif de notre travail est d’etudier les facteurs susceptibles de controler la production des estrogenes dans les cellules germinales du rat mâle adulte en precisant notamment, les effets du TGFs et du TNFα sur l’expression du gene de l’aromatase. Les spermatocytes pachytenes et les spermatides rondes ont la capacite d’aromatiser les androgenes en estrogenes; cette activite augmente en fonction de stade de developpement des cellules germinales: elle est 5 fois plus elevee dans les spermatides que dans les spermatocytes pachytenes. A l’aide d’amorces specifiques, nous avons pu mettre en evidence par RT-PCR, l’ARNm de l’aromatase dans les spermatocytes pachytenes et les spermatides rondes. Nos resultats preliminaires obtenus par RT-PCR quantitative competitive ont montre que la testosterone (200ng/ml) stimule la synthese de l’ARNm de l’aromatase dans les deux types de cellules germinales etudies. Cette stimulation est fortement inhibee dans les spermatides rondes par l’ajout du TGFs (1ng/ml); l’effet de ce facteur sur les spermatocytes reste a determiner. L’ajout du TNFα (10ng/ml) aux cellules traitees par la testosterone induit une legere stimulation de la synthese des messagers de l’aromatase dans les spermatocytes pachytenes; cependant, cette cytokine exerce un effet inhibiteur dans les spermatides rondes. En somme, dans le testicule des rongeurs, les cellules germinales meiotiques et post-meiotiques representent une nouvelle source d’estrogene; ces cellules possedent en outre des recepteurs (ERs) pour ces hormones. Ces observations permettent de suggerer un controle estrogenique de la spermiogenese chez certains mammiferes.

Stage-specific nuclear antigen is expressed in rat male germ cells during early meiotic prophase.

A germ cell nuclear antigen with approximately 44-kDa molecular weight was identified by a novel monoclonal antibody designated as Mab 2F2 from the library we have accumulated against rat testicular cells. In immature 20-day-old and adult rat testis the recognized antigen was expressed in the nuclei of early meiotic cells from preleptotene to early pachytene spermatocytes exhibiting a stage-specific appearance in the cycle of the seminiferous epithelium. The immunoreactivity was clearly associated with the meiotic chromosomes. The antigen was not detected in the late pachytene spermatocytes and more advanced stages of spermatogenesis. No labeling was observed in spermatogonia and somatic Sertoli and Leydig cells. The pattern of expression of the recognized antigen during early meiotic stages of spermatogenesis but not in mitotically dividing spermatogonia could strengthen its possible role in meiotic division.

MRNAs Encoding a von Ebner's-like Protein and the Huntington Disease Protein Are Induced in Rat Male Germ Cells by Sertoli Cells

The success of spermatogenesis is dependent upon closely coordinated interactions between Sertoli cells and germ cells. To identify specific molecules that mediate interactions between somatic cells and germ cells in the rat testis, Sertoli cell-germ cell co-cultures and mRNA differential display were used. Two cDNAs, clone 1 (660 nucleotides) and clone 2 (390 nucleotides) were up-regulated when Sertoli cells were co-cultured with pachytene spermatocytes or round spermatids. Northern blot analyses confirmed the differential display expression patterns. Sequence analyses indicated that clone 1 was similar to a von Ebner's gland protein (87% at the nucleotide level and 80% at the amino acid level) and clone 2 was identical to a region of the Huntington disease protein. The von Ebner's-like protein mRNA was induced after 4 h of co-culture, while the Huntington disease protein required 18 h of co-culture for expression. The von Ebner's-like protein was induced in germ cells by a secreted Sertoli cell factor(s) smaller than 10 kDa that is sensitive to freezing and thawing or boiling. The Huntington disease protein was induced in germ cells by a Sertoli cell secreted factor(s) larger than 10 kDa which survives freezing and thawing, but is inactivated by boiling.

Stage-specific apoptosis of male germ cells in the rat: mechanisms of cell death studied by supravital squash preparations

Apoptosis has been proposed as a mechanism by which testis germ cells are removed during normal and various pathological conditions. To establish a new rapid way to detect stage-specific apoptosis in male rat germ cells, their supravital morphology was examined from carefully squashed monolayers of living cells, after several established toxic treatments, using a phase contrast microscope. The results were compared with early detection of apoptosis using annexin V and propidium iodide (PI) stainings. The apoptosis of type-A spermatogonia and round spermatids proceeded in a similar way to somatic cells, while intermediate and type-B spermatogonia, and particularly the dividing spermatocytes, possessed characteristics not entirely typical for apoptosis. Death of elongated spermatids was difficult to assess owing to their compacted chromatin. As the first phases of degeneration seemed different in various germ cell classes, the final stage (karyopycnosis) was similar for most cells. Degenerating cells also showed positive reactions for annexin V and PI. The ‘living cell method’ provides rapid and accurate possibilities for analysis of stage-specific apoptosis during spermatogenesis. This method is not influenced by artefacts induced by fixation, embedding and sectioning. It may be developed further for routine analyses of the accurate stage-specific effects of various physical and chemical effects on mammalian and human spermatogenesis.

Alternative splicing events in the coding region of the cytochrome P450 aromatase gene in male rat germ cells.

Expression of cytochrome P450 mRNA in rat germ cells was characterized by reverse transcription PCR with various primers located at the 3'-end of the coding region. At least two unusual isoforms (Ex10-S and INT) of P450 aromatase (P450arom) mRNA were expressed. Analysis of their sequences demonstrated that an alternative splicing event occurred first at the exon-intron boundary of the GT consensus sequence of the last coding exon, and second in the internal 5' donor inside exon 9 used as a minor cryptic splicing site. These isoforms lacked the last coding exon which contained the heme-binding domain; in addition, for the Ex10-S transcript, the catalytic domain was also absent because of a frameshift in the open reading frame. The deduced amino acid sequences led to truncated P450arom polypeptides without the heme-binding domain, which were probably unable to convert androgens into estrogens. Adult rat germ cells are able to express P450arom mRNA, which is then translated into a biologically active enzyme which is involved in estrogen production. Moreover, for the first time, we report the existence of alternative splicing events of P45Oarom mRNA in pachytene spermatocytes and round spermatids, which probably cannot encode functional aromatase molecules.

Elevated expression of lanosterol 14alpha-demethylase (CYP51) and the synthesis of oocyte meiosis-activating sterols in postmeiotic germ cells of male rats.

Mammalian CYP51 encodes lanosterol 14alpha-demethylase (P45014DM) that is involved in the postsqualene part of cholesterol biosynthesis. This enzyme removes the 14alpha-methyl group from lanosterol and 24,25-dihydrolanosterol producing intermediates in cholesterol biosynthesis, the oocyte meiosis-activating sterols FF-MAS and MAS-412. Human and rat CYP51 messenger RNAs (mRNAs) are expressed in all tissues, with highest levels in the testis due to the presence of an additional shorter CYP51 transcript in this tissue. In situ hybridization shows the highest CYP51 mRNA levels in seminiferous tubules, with only background levels in Leydig cells. The rat testis-specific CYP51 mRNA arises from the use of an upstream polyadenylation site and is restricted to germ cells, being most abundant in elongating spermatids in stages VII-XIV, whereas somatic CYP51 transcripts are present in all cells. In contrast, the mRNA levels of squalene synthase are maximal in round spermatids, and no germ cell-specific transcript is observed. The rat male germ cell-specific CYP51 transcript is translated in vitro to two proteins of approximately 55 and 53.5 kDa. CYP51 activity is higher in protein extracts of testes and germ cells of sexually mature rats than in prepubertal animals, in which postmeiotic germ cells are not yet present. This shows increased capacity for the production of MAS sterols by male germ cells that have already completed meiosis, suggesting that they serve a role different from meiosis activation.

Elevated Expression of Lanosterol 14 -Demethylase (CYP51) and the Synthesis of Oocyte Meiosis-Activating Sterols in Postmeiotic Germ Cells of Male Rats

Elevated Expression of Lanosterol 14 -Demethylase (CYP51) and the Synthesis of Oocyte Meiosis-Activating Sterols in Postmeiotic Germ Cells of Male Rats

Apoptotic response of spermatogenic cells to the germ cell mutagens etoposide, adriamycin, and diepoxybutane.

In testis, apoptosis is a way to eliminate damaged germ cells during their development. In this study, we evaluated the ability of three germ cell mutagens to induce apoptosis (or programmed cell death) at specific stages of rat seminiferous epithelial cycle. These chemicals include the cancer chemotherapy drugs etoposide and adriamycin and the butadiene metabolite diepoxybutane. According to our results, etoposide is a very potent inducer of apoptosis in male rat germ cells and the cell types most sensitive to it include all types of spermatogonia, zygotene, and early pachytene spermatocytes and meiotically dividing spermatocytes. Also, adriamycin causes an increase in apoptosis at specific stages of seminiferous epithelial cycle and the most sensitive cell types are type A3-4 spermatogonia, preleptotene, zygotene, and early pachytene spermatocytes. Diepoxybutane does not cause any significant increase in the frequency of apoptosis in rat testis. In addition, we studied whether p53 is taking part in the apoptotic response of spermatogenic cells by studying the levels of p53 protein in testis before and after chemical treatment. No accumulation of p53 in testis was seen after treatment with these three chemicals. The expression of two p53-regulated genes, p21WAF1 and mdm2, was also studied but no increase in the levels of mRNA of these genes was observed after treatment. The results indicate that apoptosis should be taken into consideration when the genotoxic effects of chemicals are evaluated in germ cells.

Evaluation of the mutagenicity of acetochlor to male rat germ cells

Male rat dominant lethal (DL) assays conducted on the herbicide acetochlor are described. Single dose studies conducted at the maximum tolerated dose (MTD, ≤1000 mg/kg) produced no effects on any of the DL assay parameters at any of the ten weekly sampling periods. It is concluded that acetochlor is non-mutagenic to rat germ cells. Due to initial limited knowledge of the MTD of acetochlor it was also evaluated in the DL assay at a dose level of 2000 mg/kg. At this high dose level severe bodyweight loss and some deaths occurred among the treated animals. In addition, reduced implantations and reduced pregnancy rates were observed at the third sampling period (18–25 days post dosing) in the absence of an increase in early post-implantation deaths. These results indicated that the use of supra-MTD doses of acetochlor had reduced the fertility of the treated males leading to the production of a pseudo-DL assay response, as alerted to and defined by Ehling. Although several such pseudo-DL assay responses have been described, none have been explained mechanistically. It was therefore decided to pursue the effects seen in the DL assay when using supra-MTD doses of acetochlor. Ova analysis of female rats mated with male rats exposed to 2000 mg/kg acetochlor revealed unfertilized ova at the critical third sampling time. Normal fertilization of ova was observed at the first and fifth sampling period and, for a dose of 200 mg/kg acetochlor, at the third sampling period. The magnitude and temporal nature of these effects confirmed the induction of a pseudo-DL assay response, and studies were then undertaken to probe its genesis. Rats treated with 2000 mg/kg acetochlor had normal testicular and epididymal pathology and normal sperm numbers and sperm motility at the critical third sampling period. Despite a small reduction in testicular and epididymal glutathione levels 12 h after exposure to 2000 mg/kg acetochlor, testicular LDH and LDH-X enzyme levels were unaffected. Further, no reduction in the level of free sulphydryl groups (-SH) were observed in epididymal caput sperm heads isolated 0.5, 7 or 14 days after treatment of male rats with 2000 mg/kg acetochlor. The only sperm parameter affected by treatment with 2000 mg/kg acetochlor was an increase in epididymal cauda sperm with head abnormalities. The non-specific nature of this effect was considered inadequate to explain fully the high dose fertility effects seen in the DL assays, which therefore remain unexplained. The present data establish that acetochlor is non-mutagenic to rat germ cells. They also confirm the importance of segregating mutagenic and fertility effects in the DL assay, and emphasize the need for appropriate dose-setting studies prior to the conduct of rodent genetic toxicity assays.

Telomerase activity in female and male rat germ cells undergoing meiosis and in early embryos.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA at the ends of eukaryotic chromosomes. It has been hypothesized that telomerase activity is necessary for cellular immortalization and that telomerase activity is present in cells of germline origin. The objective of the present study was to determine the level of telomerase activity in the following rat cells: 1) oocytes from follicles at different stages of development, 2) spermatogenic cells, and 3) early embryos. Telomerase activity was quantitated using a recently developed, sensitive polymerase chain reaction-based assay and a human kidney cell line (293) as a standard. Telomerase activity was found in oocytes from early antral and preovulatory follicles, as well as in ovulated oocytes. The level of enzyme activity in early antral and preovulatory follicles was comparable to that of the 293 cells, while levels in ovulated oocytes were 50-fold lower. Telomerase activity was present in even lower levels in pachytene spermatocytes and round spermatids, and no telomerase activity was detected in spermatozoa from either the caput or the cauda epididymis. After fertilization, telomerase activity was present in 4-cell embryos. Telomerase activity was also detected in several rat somatic tissues. These data demonstrate that telomerase activity is present in germ cells at several stages of differentiation, with the exception of spermatozoa, and suggest that telomerase activity may be important during meiosis. The high levels of telomerase activity in individual oocytes may serve as a marker for monitoring the effects of hormonal agents, aging, and toxins on oocyte quality.

Inhibition by TGF-beta 1 of the in vitro thymulin-stimulated proliferation of gonocytes from fetal rat testes.

The cytokine transforming growth factor beta 1 (TGF-beta 1) inhibits the growth of certain cells and the differentiation of others. A thymus hormone, thymulin, stimulated the proliferation of fetal male germ cells in explants of testes from 13.5 gestation day rat fetuses. The way in which thymulin acts is unknown. Adding TGF-beta 1 to the culture medium blocked the response of the fetal male rat germ cells to thymulin. We suggest that TGF-beta 1 and thymulin may thus influence the same metabolic chain of events.

Micronuclei are induced in rat spermatids in vitro by 1,2,3,4-diepoxybutane but not by 1,2-epoxy-3-butene and 1,2-dihydroxy-3,4-epoxybutane.

The genotoxic effects of three 1,3-butadiene metabolites, 1,2-epoxy-3-butene (monoepoxide, EB), 1,2,3,4-diepoxybutane (diepoxide, DEB) and 1,2-dihydroxy-3,4-epoxybutane (diolepoxide, DiolEB), on male rat germ cells were studied by the meiotic micronucleus method in vitro. Seminiferous tubular segments from stages XII to XIII containing late pachytene-diakinetic spermatocytes were cultivated in the presence of the test chemical for 4 days. During the culture, spermatocytes passed through meiotic divisions and developed into early spermatids in which micronuclei could be scored. DEB was found to be a very potent micronucleus inducer in rat meiosis. All concentrations tested (5-20 microM) were able to cause a statistically significantly higher frequency of micronuclei (P < 0.05) compared with controls and a linear dose-dependent trend for micronucleus induction was seen (P < 0.01). However, EB and DiolEB caused no increase in micronucleus frequencies in spermatids at the concentrations tested (100-1000 microM for EB and 10-100 microM for DiolEB) and at higher concentrations cytotoxic effects were seen upon dividing cells causing a significant reduction in the number of spermatids. According to these results DEB is the most genotoxic butadiene metabolite in rat germ cells during meiosis.

Evidence for a novel splice variant of the α subunit of G o in rat male haploid germ cells

Northern analysis shows that G oα mRNA is highly expressed in immature germ cells from rat. Whereas α o2 mRNA is the major form in pachytene spermatocytes, a message of shorter chain length is present in large amount in haploid germ cells. This mRNA was detected with an oligonucleotide specific for the 3′-coding region of α o2, but did not hybridize to oligonucleotides specific for the 5′-untranslated and 5′-coding regions. The results indicate the presence of a novel splice variant of α o mRNA, which may code for a G oα protein important for germ cell development.

Factors involved in regulation of the RT7 promoter in a male germ cell-derived in vitro transcription system.

We recently cloned and characterized a rat male germ cell-specific gene, RT7. The RT7 promoter contains a TATA box as well as sequences with homology to binding sites for a number of transcription factors. To investigate the regulation of the RT7 promoter we developed an active in vitro transcription system derived from rat seminiferous epithelium, which, in contrast to total testis, consists mostly of male germ cells. Also, DNase I footprinting analysis and gel retardation experiments were performed to analyze RT7 promoter-protein interaction. The experiments demonstrate that nuclear extracts prepared from rat male germ cells support in vitro transcription and that the RT7 promoter is positively regulated by a testis-specific transcription factor, TTF-D, by a factor similar to the transcription factor CREB, and by a nuclear factor that binds immediately upstream of the RT7 transcription start site.

Tissue-specific genotoxic effects of acrylamide and acrylonitrile.

Acrylamide (AA) has been reported to induce dominant lethal mutations in male rat germ cells and tumors in a variety of organs, including the scrotum, thyroid and mammary glands, but not the liver of rats. The structurally similar vinyl monomer acrylonitrile (ACN) does not induce dominant lethal mutations but does induce tumors of the brain, Zymbal gland, forestomach and mammary gland, but not the liver of rats. Several in vitro and/or in vivo unscheduled DNA synthesis (UDS) assays were employed to examine the potential tissue-specific genotoxic activity of these compounds. Neither AA nor ACN induced DNA repair in either the in vitro or in vivo hepatocyte DNA repair assays. Glycidamide (GA), a mutagenic metabolite of AA, induced DNA repair in the in vitro hepatocyte DNA repair assay. Cyanoethylene oxide (CEO), a mutagenic metabolite of ACN, did not yield a DNA repair response in the in vitro hepatocyte DNA repair assay, but was highly toxic and could not be tested at doses equivalent to GA. AA, but not ACN, produced a DNA repair response in the in vivo spermatocyte DNA repair assay. AA produced a slight response in the in vitro human mammary epithelial cell (HMEC) DNA repair assay in normal cells derived from discarded surgical samples from five different women. GA produced a strong UDS response in all cases in the same assay. CEO, but not its parent compound ACN, produced a response in the HMEC DNA repair assay. These results show a highly tissue-specific pattern of genotoxic activity for AA and ACN that correlates, to the extent that it has been examined, with the tissue-specific pattern of carcinogenic and dominant lethal activity. The induction of DNA repair by GA and CEO confirms the genotoxic potential of these metabolites. While the observation of genotoxic activity of AA in the HMEC DNA repair assay suggests that mammary cells might be a target for carcinogenic activity of this compound in humans, other factors such as pharmacokinetics and epidemiology must be evaluated to establish that effect.

Chromosomal aberrations induced by methylnitrosourea on the germ cells of male rats

Chromosomal aberrations induced by methylnitrosourea on the germ cells of male rats

Chromosomal aberrations induced by methylnitrosourea on the germ cells of male rats

Chromosomal aberrations induced by methylnitrosourea on the germ cells of male rats