Effects of alpha-amanitin on RNA synthesis in cultured rat seminiferous tubules.

The effect of the specific RNA polymerase II inhibitor α-amanitin on the cell morphology and RNA synthesis during spermatogenesis was investigated. Light microscopic autoradiography showed that α-amanitin largely decreased the rate of RNA synthesis in the pachytene spermatocytes and in the spermatids. Degenerative changes occurred most rapidly in the mid pachytene spermatocytes and in the intermediate and type B spermatogonia. Already after 14 hr of culture with α-amanitin (10 μg/ml) a part of these cells showed progressive degenerative changes beginning with the clumping together of the chromosomes. This indicates that at least a part of the HnRNA which is synthesized in the lampbrush loops of the pachytene chromosomes is needed to maintain the normal structure and function of the pachytene spermatocytes. Biochemical evidence suggested also that the formation of HnRNA was specifically inhibited in the pachytene spermatocytes by α-amanitin.     

Developmental stage-specific expression of Rbm suggests its involvement in early phases of spermatogenesis

Rbm is a male infertility gene located on the Y chromosome that is expressed in the testis. To investigate the specific events of spermatogenesis in which Rbm plays a role, the precise pattern of expression of Rbm in the mouse testis was determined. An antibody was generated against the Rbm protein and used to detect a single specific band of 43 kDa in size in mouse testicular lysates. In situ hybridization, immunoblot and immunohistochemistry analyses together indicated that Rbm was expressed in spermatogonia, preleptotene spermatocytes, late leptotene to early pachytene spermatocytes but not in mid-pachytene spermatocytes or subsequent stages of differentiation, including haploid germ cells. These observations suggest that Rbm functions in early but not later stages of male germ cell development.     

Destructive effect of DNA topoisomerase II inhibitor vepesid on mouse spermatogenesis

Inhibition of DNA topoisomerase II with vepesid induced structural and functional reorganization of chromatin in meiotically dividing spermatocytes I, which later led to the block of their differentiation and long-lasting disorders in spermatogenesis. Vepesid induced decondensation of spermatocyte I chromatin, block of desynapsis, and elongation of lateral elements of spermatocyte autosome synaptonemal complexes during late pachytene and diplotene of meiosis. This confirms the involvement of type II DNA topoisomerase in chromatin condensation and homologous chromosome desynapsis at the stage of diplotene and the role of this enzyme in structural organization of the synaptonemal complex. Vepesid induced the formation of dichotomy and breaks of the pericentromer regions of subelements of lateral elements of the autosomal synaptonemal complexes; the number of cells with associations of axial elements of sex chromosomes with autosomal synaptonemal complexes increased, univalents of autosomes and sex chromosomes appeared. Mesna, a modifier of toxic effects of antitumor drugs, had no toxic effect on spermatogenic cells. Mesna reduced the lethal effect of vepesid during combined treatment, but did not ensure long-term protection of spermatogenesis.     

A quantitative study of stage-specific spermatocyte damage following administration of ethylene glycol monomethyl ether in the rat

A quantitative study has been carried out to characterize the stage susceptibility of the spermatocyte to ethylene glycol monomethyl ether (EGM) toxicity. EGM was administered as a single oral dose of 250 mg/kg body wt and rats were examined at time periods after dosing. The number of spermatocytes and round spermatids in tubules at each stage of spermatogenesis was counted. A sharp transition in susceptibility was observed between zygotene spermatocytes in stage XIV which showed no effect and pachytene spermatocytes in stage I which showed death or depletion of 70% of its population after 1 day. A similar transition was seen between dividing spermatocytes and step 1 spermatids, the latter being unaffected. There was a gradual reduction in susceptibility toward midpachytene such that cells in stages VII-XI showed no effect. Analysis of later time periods revealed no effect on spermatogonia or prepachytene spermatocytes but did indicate that midpachytene spermatocytes underwent delayed cell death after further progression through the cycle. In a separate sequential morphological study of early changes, the earliest signs of necrosis were seen 12 hr after dosing and were restricted to spermatocytes in stages V, XI, and XII. Cell death then progressed in a wave-like manner through stages XIII and XIV finally reaching stage I, 24 hr after dosing.     

Specific expression of the mRNA for 25 kDA heat-shock protein in the spermatocytes of mouse seminiferous tubules

 The 25 kDa heat-shock protein (Hsp25) is a member of the family of small heat-shock proteins. We investigated the expression and cellular localization of Hsp25 mRNA in the testis of adult and developing mice using Northern blotting and in situ hybridization techniques. In the early postnatal days, i.e., before the onset of spermatogenesis, no Hsp25 mRNA was detected in the testis. At around 10 days postpartum, Hsp25 mRNA began to be expressed in the testis in coincidence with the onset of the first wave of spermatogenesis and increased in amount progressively toward adulthood. Throughout the testis development, the signal for Hsp25 mRNA was localized exclusively to germ cells and was not detected in Sertoli or interstitial cells. The testis of W/Wv mutant mice, which lack the germ cell line, exhibited no Hsp25 mRNA expression. In the testis of normal adult mice, the abundance of Hsp25 mRNA differed among the seminiferous tubules in different stages of spermatogenesis. The most intense signal for Hsp25 mRNA was localized to the spermatocytes at leptotene, zygotene and early pachytene phases, which are present in the tubules of stages I–III and IX–XII. The signal decreased in intensity in the late pachytene and diplotene spermatocytes and was not detected in spermatids. Spermatogonia were also devoid of the signal. These results suggested that Hsp25 plays some specific role in the meiotic prophase of the testicular germ cell. Accepted: 27 Oct 1998     

Identification of autoantigen-expressing cells in rat testis

Spermatogenic cells expressing autoantigens have been identified using autoantibodies induced against homologous testis tissue in combination with a Staphylococcus Cowan I rosetting technique. The autoantigens first appeared in early pachytene spermatocytes and were present in the spermatogenic cells up to late spermiogenesis. The highest levels of autoantigens were seen in the midpachytene spermatocytes and the late spermatids. These autoantigens probably play a role in the vigorous granulomatotic reactions caused by sperm outside the testis and in the production of autoantibodies after vasectomy.     

Use of seminiferous tubule segments to study stage specificity of unscheduled DNA synthesis in rat spermatogenic cells

DNA repair in spermatogenic cells at various stages of maturity was determined by quantitation of unscheduled DNA synthesis (UDS). Male F-344 rats were exposed (i.p.) to methyl methanesulfonate (MMS, 35 mg/kg); 1 hr later, segments of seminiferous tubules corresponding to spermatogenesis stages II, IV-V, VI, VII, VIII, IX-X, XII, and XIV were isolated with the transillumination pattern of the tubules as a guide. Intact tubule segments were cultured 24 hr in the presence of [3H]thymidine, and UDS was quantitated by autoradiography as net grains/nucleus (NG). In primary spermatocytes from treated rats, NG count increased with increasing maturity from leptotene primary spermatocytes (3.5 NG) up through stage VIII and IX-X pachytene spermatocytes (22 NG), after which NG decreased in stage-XII pachytene and diplotene spermatocytes (to 16 NG and 8 NG, respectively). Round spermatids of steps 2-8 of spermiogenesis all exhibited approximately the same UDS response (8 NG). Elongating spermatids as mature as step 14 underwent UDS after exposure to MMS, but step-15 and later-step spermatids did not. The DNA repair response of pachytene spermatocytes cultured within segments of seminiferous tubule corresponding to stages VIII and IX-X was 4 to 25 times greater, depending on the dose of MMS, than pachytene spermatocytes isolated by enzymatic digestion and cultured in suspension [Bentley and Working, Mutat Res 203:135-142, 1988]. Thus, the use of segments of seminiferous tubule both increased the sensitivity of UDS as an indicator of DNA damage in rat germ cells and enabled the study of UDS in spermatogenic cells at different stages of maturity.     

Successive increases in human cyclin A1 promoter activity during spermatogenesis in transgenic mice

The human cyclin A1 gene is highly expressed in pachytene spermatocytes and is essential for spermatogenesis. To analyze mechanisms of cyclin A1 gene expression in vivo, we cloned a 1.3 kb fragment of the promoter upstream of the cDNA of enhanced green fluorescent protein (EGFP). Four lines of transgenic mice were generated that carried the transgene. Cyclin A1 promoter activity in the organs of the transgenic mice was analyzed using fluorescence microscopy and flow cytometry. Expression of EGFP was seen in male germ cells of all four murine lines. Spermatogonia at the basal membrane expressed low levels of EGFP, but bright green fluorescence was present in spermatocytes entering meiosis. Interestingly, a further sharp increase in EGFP expression was found in spermatocytes approximately at the stage of the first meiotic division. EGFP levels stayed high thereafter and EGFP was present in mature spermatozoa. A portion of c-kit expressing cells in the testis also expressed EGFP indicating cyclin A1 promoter activity in a subpopulation of spermatogonia. These data suggest that cyclin A1 is active not only in pachytene spermatocytes but also in earlier phases of spermatogenesis.     

Quantitative aspects of water buffalo (Bubalus bubalis) spermatogenesis.

In the buffalo, seminiferous tubules occupy about 82% of the testis. Spermatogenesis can be divided into 6 stages according to characteristic cellular associations in the seminiferous epithelium. A-spermatogonia have a volume of approximately 1,400 microns3 and the highest absolute mitochondrial volume of all spermatogenic cells. B-spermatogonia display cellular, nuclear and mitochondrial volumes of approximately half the values of A-spermatogonia. From preleptotene (approximately 470 microns3) to late diplotene (approximately 2,300 microns3), the volume* of primary spermatocytes increases nearly five-fold; their nuclear volumes increase by 3.5 times within the same period. During zygotene mitochondrial cristae start to dilate. Grouping of mitochondria by a dense intermitochondrial substance is most prominent during pachytene and diplotene. In pachytene the absolute size of the Golgi apparatus more than doubles, indicating a high secretory activity. Through zygotene only rER is encountered; in pachytene and diplotene a tubular sER makes its first appearance. Secondary spermatocytes are found only in stage 4 of the cycle. Due to partial cell necrosis and autolytic events, late maturation phase spermatids display no more than 25% of the size of cap phase spermatids. There is no morphological evidence for an active uptake and digestion of residual bodies by the Sertoli cells. Also, no lipid cycle is present in the buffalo seminiferous epithelium. Morphometric evaluations reveal that 63% of all theoretically possible germ cells disappear from the seminiferous epithelium during spermatogenesis. Heavy cell loss is observed in stage 4 of the cycle in the spermatogonial fraction as well as during the second meiotic division.     

Protein expression and cell organelle behavior in spermatogenic cells

Spermatogenic cells stage-specifically produce a wide variety of proteins during spermatogenesis, wherein protein expression is coordinated with cell organelle behavior. It has been shown that the Golgi apparatus and the endoplasmic reticulum (ER) are uniquely coordinated with the expression of an immunoglobulin super-family protein, flagellar plasma membrane MC31 (MC31/CE9), and a molecular chaperone, calmegin, respectively. When the Golgi apparatus begins to generate sperm components in the primary spermatocytes, it actively engages in producing proteins for the acrosome in round spermatids and for the flagellum in elongating spermatids. Structurally, the Golgi apparatus is reduced in size during meiotic division, moves from the apical to the basal region (cytoplasmic lobe) when spermatids differentiate from round to elongating phase, and then collapses in the late maturation phase. The ER is distributed uniformly over the entire cytoplasm of spermatocytes and round spermatids, and then moves distally toward the cytoplasmic lobe along the bundles of microtubule, called the manchette, in elongating spermatids. The ER is resorbed into the radial body in late maturation spermatids. MC31/CE9 expresses strong immunostaining twice on the Golgi apparatus during spermatogenesis, first in early pachytene spermatocytes and then in early elongating spermatids. Calmegin expression exactly parallels ER behavior. This mini-review focuses on the unique relationships in spermatogenic cells, particularly those between protein expression and cell organelle behavior.     

Spermatogenesis in the vasectomized monkey: Quantitative analysis

The seminiferous epithelium in mature vasectomized Macaca fascicularis was examined quantitatively to assess spermatogenesis. Monkeys were bilaterally vasectomized and controls were bilaterally sham operated. At postoperative periods of 10 and 18 months, groups of monkeys were castrated and their testes prepared for morphologic analysis. Diameters were measured in 100 cross sections of seminiferous tubules from each animal. Numbers of spermatogonia (Ad and Ap), preleptotene spermatocytes, pachytene spermatocytes, and step 7 spermatids, relative to Sertoli cell nucleoli, were counted in stage VII tubules. Tubule diameter and germ cell numbers per Sertoli cell nucleoli were not altered by vasectomy. Our study demonstrates quantitatively that spermatogenesis in the monkey is not inhibited up to 18 months following vasectomy.     

A constitutional complex chromosome rearrangement involving meiotic arrest in an azoospermic male: case report

Complex chromosome rearrangements are rare aberrations that frequently lead to reproductive failure and that may hinder assisted reproduction. A 25-year-old azoospermic male was studied cytogenetically with synaptonemal complex analysis of spermatocytes from a testicular biopsy and fluorescence in situ hybridization (FISH) of lymphocytes. The spermatocytes showed a pentavalent plus a univalent chromosome. Cell death occurred mainly at advanced pachytene stages. The sex chromosomes were involved in the multiple, as shown by their typical axial excrescences. Two autosomal pairs, including an acrocentric chromosome (15), were also involved in the multiple. FISH allowed the definite identification of all the involved chromosomes. An inverted chromosome 12 is translocated with most of one long arm of chromosome 15, while the centromeric piece of this chromosome 15 is translocated with Yqh, forming a small marker chromosome t(15;Y). The euchromatic part of the Y chromosome is joined to the remaining piece of chromosome 12, forming a neo-Y chromosome. The patient shows azoospermia and a normal phenotype. The disruption of spermatogenesis is hypothetically due to the extent of asynaptic segments and to sex-body association during pachytene. This CCR occurred 'de novo' during paternal spermatogenesis. Meiotic analysis and FISH are valuable diagnostic tools in these cases.     

Retinoic acid receptor alpha is required for synchronization of spermatogenic cycles and its absence results in progressive breakdown of the spermatogenic process.

Targeted mutagenesis of the retinoic acid receptor alpha (RAR alpha) gene has revealed its essential role in spermatogenesis. Although cells in all stages of spermatogenesis were detected in RAR alpha(-/-) testes, there was an increase in degenerating pachytene spermatocytes and a temporary developmental arrest in step 8-9 spermatids in the first wave of spermatogenesis, a delay in the onset of the second wave, and a temporary arrest in preleptotene to leptotene spermatocytes in the first, second, and third waves. A striking aspect of the mutant phenotype was the failure of spermatids to align at the tubular lumen at stage VIII. Furthermore, there were missing or decreased numbers of the predicted cell types in tubules, and they exhibited a profound asynchrony of mixed spermatogenic cell types. In vivo bromodeoxyuridine labeling revealed a significant decrease in germ cell proliferation in both juvenile and adult RAR alpha(-/-) testes and confirmed the arrest at step 8-9 spermatids. Retinoid signaling through RAR alpha, thus, appears to be critical for establishment of synchronous progression of spermatogenesis and the subsequent establishment of correct cellular associations.     

Interactive effects of chronic phencyclidine and delta 9-tetrahydrocannabinol on spermatogenesis in mice

In this study, the combined effects of chronic phencyclidine (PCP) and delta 9-tetrahydrocannabinol on spermatogenesis in mice were examined. Mice were treated with THC (50 mg/kg, PO) and PCP (15 mg/kg, IP) alone or in combination for 16 days and with PCP alone for 35 days. THC had a significant effect on spermatogenesis and decreased the number of all germinal cells. PCP, on the other hand, affected all germinal cells except spermatids after 35 days of treatment. Combination of THC and PCP treatment caused a significant decrease in resting and pachytene spermatocytes. Similarly, combination of these two drugs caused a significant increase in cauda epididymal abnormal sperms. These results suggest that THC and PCP may cause greater disruption in spermatogenesis when they are abused together.     

GENE EXPRESSION OF A REGION OF CHROMOSOME 17 DURING MURINE SPERMATOGENESIS

The presence of H-2-linked gene products on spermatozoa and their time of appearance during spermatogenesis was determined. Thymus leukaemia antigen specificities 1, 2 and 3 could not be detected on spermatozoa by absorption of the antisera. Immunofluorescent studies with anti-Sip sera did not reveal any specific reactivity with target spermatozoa. In contrast, H-2D antigens were present on somatic as well as germ line components in testes so the time of their first appearance during spermatogenesis could not be precisely specified. Cell separation experiments indicate that H-2D antigens are present on pachytene spermatocytes and increased in quantity on spermatids. The sperm-specific isoenzyme of phosphoglycerate kinase, Pgk-2, appears at a later stage of spermatogenesis than do the H-2 region antigens.     

NF-kappa B is developmentally regulated during spermatogenesis in mice.

To analyze NF-kappa B activity in the testis, we used murine transgenic lines carrying a LacZ reporter gene under the control of a NF-kappa B-responsive promoter (Schmidt-Ullrich et al. [1996] Dev 122:2117-2128). We constructed three independent lines containing the promoter of the gene encoding p105, the precursor of the p50 subunit. This promoter contains three NF-kappa B-binding sites in its proximal part. Our results show that in adult mice, the beta-galactosidase activity which reflects nuclear NF-kappa B activity, is first detected in spermatocytes at the pachytene stage and remains activated in the following steps of germ cell differentiation and maturation. Using transgenic mice carrying a p105nlslacZ construct with the 3 NF-kappa B sites mutated in the p105 promoter, we found a significant reduction in the transgene activity, confirming the important role of NF-kappa B in the activation of the transgene. To confirm the stage of induction during spermatogenesis, we analysed the beta-galactosidase activity in the testes from prepuberal mice in which cells synchrouneously enter meiosis. We detected the transgene activity at 18 days after birth, corresponding to the pachytene stage in spermatocytes. In nuclear extracts prepared from prepuberal mice, we found a peak of NF-kappa B DNA-binding activity made of p50 and p65 subunits at day 18 after birth, which remains high in the later stages. Further analysis showed that I kappa B alpha and beta, but not epsilon are expressed in the testes. Altogether, these data suggest that NF-kappa B factors are stage specifically controlled and may play a role during the development of sperm cells.     

Importin alpha mRNAs have distinct expression profiles during spermatogenesis.

Importin proteins control access to the cell nucleus by mediating the nuclear transport of specific cargoes. We hypothesized that developmental regulation of gene expression may be partially effected by changes in the nuclear transport machinery complement, manifested as regulated expression of importin alpha family genes. We first clarified the identity of the five known mouse importin alpha genes relative to those for human and then determined their expression throughout postnatal rodent testis using PCR and in situ hybridization. Distinct expression patterns were observed for each. At 10 dpp, all importin alpha mRNAs were detected in spermatogonia. In the adult mouse testis, importins alpha1 and alpha3 were detected in spermatogonia and early pachytene spermatocytes. Importin alpha4 mRNA was identified in pachytene spermatocytes, alpha6 mRNA in round spermatids, and alpha2 mRNA in both of these. The distinct importin alpha expression patterns are consistent with their having specific roles and transport cargoes during spermatogenesis.