The spermatogenetic process in Barbus longiceps, Capoeta damascina and their natural sterile hybrid (Teleostei, Cyprinidae)

Testicular ultrastructure was studied in Barbus longiceps, Capoeta damascina and their natural hybrid. The testes of these teleosts belong to the unrestricted or lobular type. Germ cell morphology is similar in the parental males. In the hybrid, spermatogenesis does not extend beyond the pachytene of the first meiotic division, probably due to the unsuccessful pairing of the homologous chromosomes. Hybrid testes are occupied mainly by degenerating primary spermatocytes, at the leptotene and pachytene stages. In both parents and the hybrid, Sertoli and Leydig cells are characterized by the presence of granular endoplasmic reticulum and of mitochondria with tubular cristae. Due to the arrest of spermatogenesis, the male germ cell protective barrier is absent in the hybrid. Germ cell nuclear size was measured by a computerized analysis system, using light-microscopy images. In the parents and the hybrid, germ cells attain a uniform inter-individual nuclear size when they reach the first meiotic prophase. The nuclear size of primary spermatocytes is similar among the three groups of fish, possibly reflecting their close genetic relationship.     

Monitoring meiosis in gametogenesis

Meiotic recombination is essential to hold homologous chromosomes together so that they can separate accurately in the formation of gametes, thus preventing fetal loss due to aneuploidy. How do germ cells know when they have finished genetic recombination and that it is time to enter the meiotic division phase, and what are the elements that signal the onset of the division phase? During spermatogenesis there is no arrest at the end of meiotic prophase (as there is in oogenesis) and signals for progress into the meiotic division phase may be closely related to events of chromosome pairing and recombination. Methods for culture of male germ cells have been used to show that spermatocytes become competent for some aspects of the division phase by the early pachytene stage, long before they would normally enter division. Evidence suggests that establishment of homologous chromosome pairing is one aspect of acquiring competence. Activation of the cell cycle regulator MPF also appears to be important, and there is a requirement for activity of topoisomerase II in order for spermatocytes to exit prophase and enter the meiotic division phase. Understanding how these molecular entities tie into monitoring the completion of recombination and meiotic progress will be instructive about important gametic safeguards preventing aberrant chromosome segregation and resultant aneuploidy.     

The effect of retinoic acid on the meiosis in the chick embryo (Gallus gallus domesticus)

In this study it was shown that the injection of retinoic acid (RA) into incubated eggs on day 9 or 14 induced entry the males germ cells into preleptotene stage of prophase I on day 17, which are absent in the control embryos. At the same time the meiosis marker SCP3 was detected in the germ cells. Which was also absent at control embryos. On day 19 in male embryos the number of male germ cells at the stage preleptoteny increased, but there were no germ cells in the following stages of the prophase of meiosis. In 20-day-old chicks meiotic germ cells were absent. Thus, white it is shown that the influence of RA on the developing chicken embryos induces the entry of germ cells into preleptotene stage of prophase I meiosis. However, further meiotic transformations don't occur. Thus RA is only one of many factors providing meiotic cell division.     

Clastogenic effects of hydroquinone: induction of chromosomal aberrations in mouse germ cells

The clastogenic activity of hydroquinone (HQ) in germ cells of male mice was evaluated by analysis of chromosomal aberrations in primary spermatocytes and differentiating spermatogonia. In the first experiment with treated spermatocytes the most sensitive stage of meiotic prophase to aberration induction by HQ was determined. Testicular material was sampled for microscopic analysis of cells in diakinesis-metaphase I at 1, 5, 9, 11, and 12 days after treatment with 80 mg/kg of HQ, corresponding to treated diplotene, pachytene, zygotene, leptotene and preleptotene. The frequencies of cells with structural chromosome aberrations peaked at 12 days after treatment (p less than 0.01). This indicates that the preleptotene when DNA synthesis occurred was the most sensitive stage of meiotic prophase. In the second experiment the dose response was determined 12 days post treatment by applying 2 additional doses of 40 mg/kg and 120 mg/kg. The clastogenic effects induced by 40 and 80 mg/kg were significantly different from the controls (p less than or equal to 0.01) and higher than the results obtained with 120 mg/kg of HQ. A humped dose-effect relationship was observed. In a third experiment the same doses were used to analyse chromosomal aberrations in dividing spermatogonia of mice 24 h after treatment with HQ. All the administered doses gave results statistically different from the control values (p less than or equal to 0.01) and the data were fitted to a linear equation. HQ was found to be clastogenic in male mouse germ cells. It is concluded that the clastogenic effect in male germ cells is of the same order of magnitude as in mouse bone marrow cells.     

Kinetics of meiosis in azoospermic males: a joint histological and cytological approach

We have developed a protocol for the identification of aberrant chromosome behavior during human male meiosis up to metaphase of the secondary spermatocyte. Histological evaluation by the Johnsen score of a testicular biopsy was combined with immunofluorescence of first meiotic prophase spermatocytes, using antibodies against synaptonemal complex protein 3 (SYCP3) and the product of the ataxia telangiectasia and rad3-related gene (ATR). This combination enables accurate meiotic prophase substaging and the identification of pachytene spermatocytes with asynapsis. Furthermore, we also investigated the competence of late pachytene primary spermatocytes to complete the first meiotic division up to metaphase and of secondary spermatocytes to transform into metaphase by an in vitro challenge with okadaic acid (OA). We tested this protocol on five males with normal Johnsen scores that presented with obstructive azoospermia, five males with low Johnsen scores and non-obstructive azoospermia and six vasectomized control males of proven fertility and normal Johnsen scores. In all azoospermics, the profiling of meiotic prophase stages by immunofluorescence increases the resolving power of the Johnsen score. In both obstructive and non-obstructive azoospermic patients, relatively more leptotene meiotic prophase stages were counted compared to the controls. In non-obstructive azoospermics, a marked heterogeneity in spermatogenesis was found, after combining the results of all three approaches, pointing at functional mosaicism of the germinal epithelium. Asynaptic pachytene spermatocytes were rarely encountered. Also, when first meiotic metaphase could be induced by OA, chiasma counts were normal. In none of the non-obstructive azoospermic males did the pattern of spermatogenesis resemble that of knock-out mouse azoospermics. We conclude that this combined histological and cytological approach enables a detailed phenotypic classification of infertile males, at a level comparable to that applied for male-sterile knock-out mice with a meiotic defect. This may facilitate the identification of candidate genes for human male infertility.     

DNA methyltransferase is developmentally expressed in replicating and non-replicating male germ cells.

Genomic methylation patterns are established during maturation of primordial germ cells and during gametogenesis. While methylation is linked to DNA replication in somatic cells, active de novo methylation and demethylation occur in post-replicative spermatocytes during meiotic prophase (1). We have examined differentiating male germ cells for alternative forms of DNA (cytosine-5)-methyltransferase (DNA MTase) and have found a 6.2 kb DNA MTase mRNA that is present in appreciable quantities only in testis; in post-replicative pachytene spermatocytes it is the predominant form of DNA MTase mRNA. The 5.2 kb DNA MTase mRNA, characteristic of all somatic cells, was detected in isolated type A and B spermatogonia and haploid round spermatids. Immunobolt analysis detected a protein in spermatogenic cells with a relative mass of 180,000-200,000, which is close to the known size of the somatic form of mammalian DNA MTase. The demonstration of the differential developmental expression of DNA MTase in male germ cells argues for a role for testicular DNA methylation events, not only during replication in premeiotic cells, but also during meiotic prophase and postmeiotic development.     

Long glucocorticoid-induced leucine zipper (L-GILZ) protein interacts with ras protein pathway and contributes to spermatogenesis control

Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis.