The aim of the present study was to assess whether the whole meiotic process of spermatogenic cells is able to take place in vitro. Fragments of seminiferous tubules from 20-to 22-or 28-day-old rats were seeded in medium containing 0.2% fetal calf serum in bicameral chambers and then cultured for 4 weeks in a chemically defined medium. The differentiation of meiotic germinal cells was followed by four criteria: (i) ultramicroscopic examination of the different types of germ cells present in the cell layer throughout the culture period; (ii) determination of the changes in DNA content per nucleus of the cell population seeded with time in culture; (iii) assessment of the ability of germinal cells to transcribe genes expressed after completion of meiosis; and (iv) monitoring the fate of BrdU-labeled leptotene spermatocytes. The ultrastructural study showed that the overall organization of the cells in the culture well recalls that of the seminiferous epithelium throughout the culture period. Moreover the identification of young round spermatids 21 days after seeding suggested that these spermatids had been formed very recently in culture. Determination of DNA content per nucleus showed that a 1C cell population could be observed after several days of cultures reaching 6 to 10% of total cells. An exponential-like increase in the amounts of the mRNAs encoding for TP1 or TP2 occurred from the time when 1C cells appeared in the culture until the end of the experiment. Finally, BrdU-labeled leptotene spermatocytes differentiated into pachytene spermatocytes and then into secondary spermatocytes, and BdrU-labeled round spermatids were observed from Day 21 of culture onward. Taken together these results indicate that the whole meiotic process from leptotene spermatocyte to round spermatid can indeed occur in vitro under the present culture conditions.
The present study was aimed at examining, by reverse transcription polymerase chain reaction, the expression of germ cell-specific genes in cocultures of Sertoli cells with either pachytene spermatocytes (PS) or round spermatids (RS). In situ hybridization studies showed that the mRNAs encoding phosphoprotein p19 and the testis-specific histone TH2B were specifically expressed in PS whereas those encoding the transition proteins TP1 and TP2 were specific to RS. This resulted in p19:TP1 and TH2B:TP2 ratios that were much higher in PS fractions than in RS fractions prepared by elutriation. When PS or RS were seeded on Sertoli cell monolayers in bicameral chambers, both the number and the viability of the cells decreased during the coculture. However, both parameters were equal to, or higher than, 60% after 2 wk. In PS-Sertoli cell cocultures, the ratios of p19:TP1 and TH2B:TP2 decreased dramatically during the second week of culture; this was due not only to a decrease in the levels of p19 and TH2B mRNAs but also to an enhancement in the relative amounts of TP1 and TP2 as compared to the amounts present on the first day of the coculture. Conversely, both ratios remained low in RS-Sertoli cell cocultures; this was due to a decrease in the levels of the four mRNAs studied during the coculture period. DNA flow cytometry studies showed the occurrence of a haploid cell population (1C) in PS-Sertoli cell cocultures from Day 2 onward, together with a decrease in the tetraploid cell population (4C). No such changes were observed in Sertoli cell-only cultures. By contrast, the haploid population decreased 3-fold during the first week in RS-Sertoli cell cocultures. Immunocytochemical studies demonstrated further that 5-bromo-2'-deoxyuridine-labeled PS of stages V-VIII were able to differentiate into RS under the present coculture conditions. Hence, although clearly imperfect, the present coculture system should help to clarify the local regulations governing spermatogenesis and should allow easier study of spermatogenic cell gene expression.
The aim of the present study was to set up a culture system allowing most of the meiotic phase of rat spermatogenesis to occur in vitro. For that purpose, the differentiation of spermatogenic cells was monitored by three criteria: 1) examination of expression of genes specifically expressed at a high level in pachytene spermatocytes (the phosphoprotein p19 [p19] and the testis-specific histone TH2B) or in round spermatids (transition protein 1 [TP1] and transition protein 2 [TP2]) by reverse transcription-polymerase chain reaction (RT-PCR); 2) ploidy analysis; and 3) cytological and immunocytochemical study of the germ cells. In the first trial, we determined the changes in the ratios of p19:TP1 and TH2B:TP2 mRNA-related PCR products in the whole testis of rats between 18 and 60 days postpartum and related those results to the sequential appearance of the various types of spermatogenic cells during that period. In the second trial, our aim was to reproduce, in a culture system using seminiferous tubules from 23- to 25-day-old rats, the changes observed in vivo. The p19:TP1 and TH2B:TP2 ratios decreased dramatically in testicular extracts of rats between 32 and 40 days postpartum, i.e., at the time period during which round spermatids become more and more numerous in the testis. When seminiferous tubules were seeded in bicameral chambers, cell viability remained close to 70% of total cells throughout the 3-wk culture period. Both p19:TP1 and TH2B:TP2 ratios decreased during the first week of culture. This was attributable to a decrease in the levels of p19 and TH2B mRNAs and also to an enhancement in the relative amounts of TP1 and TP2. These changes were correlated with the appearance of a 1C cell population in the culture. Histological examination of the culture demonstrated that under the conditions of the present study, 5-bromo-2'-deoxyuridine-labeled pachytene spermatocytes of stages IV-VI were able to differentiate into secondary spermatocytes, then into round spermatids.
The two non-allelic forms of as2-casein, occurring in ovine milk, differ by an internal deletion of nine amino acid residues, including both cysteine residues at positions 34 and 42 in the mature chain. Sequencing of several crs2-casein cDNA, isolated from the mammary cDNA library of a single lactating ewe, showed three new types which differed from that previously studied. In addition to the expected deletion of codons + 34 to + 42 affecting 30-40% of mRNA, another structural difference involving an internal stretch of 44 nucleotides in the 5' untranslated region, was found. S1-nuclease protection assays confirmed the existence of several types of the relevant mRNA and sequencing of in-vitro-amplified genomic DNA demonstrated the presence of the 44-nucleotide stretch in the as2-casein transcriptional unit, thus ruling out the possibility of a cloning artefact.The different a,,-casein mRNA, four in terms of deletion and two in terms of nucleotide substitutions for a given ewe, can be readily explained by partial exon skipping and allelic differences, respectively. This assumption is well supported by the following observations: 5' and 3' ends of both deleted DNA fragments are similar to those of exons; sequences neighbouring the 44-nucleotide stretch of the genomic DNA perfectly match consensus sequences described for 3' and 5' ends of introns; the rather simple patterns observed on Southern blots of different enzymatic digests of genomic DNA strongly suggest the occurrence of only 1 copy as2-casein gene/haploid genome. During the course of evolution, the as2-casein-encoding gene has undergone many mutations and some of them might have occurred in regions corresponding to consensus splicing regions of the pre-mRNA. Thus, complete skipping of some exons might be responsible for the shorter sizes of rat and mouse as2-casein mRNA. If so, the overall organization of the as2-casein gene in the different species might be more similar than expected from structural comparisons of the cognate mRNA or caseins.In ruminants, caseins comprise the major fraction of secretory proteins synthesized in mammary epithelial cells. Primary structures of the four bovine caseins (reviewed in [l]) share little similarity, except for the well-conserved multiple phosphorylation sites and signal peptides [2] of the calciumsensitive caseins, asl, as2 and j?. The proposal that they have a common origin [2] was further substantiated by the similarity in organization of the relevant genes in the region spanning the promoter and the 5' end of the transcription unit [3].The striking similarity (40%) between the N-terminal and C-terminal halves of bovine crs2-casein [4] is partially known for the rat [lo] and bovine [l 13 species. It may contain at least nine introns, but sequence data are only available for the region upstream from exon 11.Previous SDS/PAGE studies of ovine as2-casein, synthesized in both cell-free translation systems and mammary gland explants, revealed the occurrence of two polypeptide chains differing by an internal deletion...
The present work aimed to compare some features of the meiotic process which develops in the testis of pubertal rats, in vivo and in vitro, paying special attention to the time-course of the phenomenon. The differentiation of spermatocytes was assessed in testes of 20- to 46-day-old rats and in tubule segments of 20- or 28-day-old rats cultured over a 4-week period. Very similar results were obtained in vivo and in vitro, during the first week of culture, when considering the changes in the cell populations of different ploidy, the gene expression of germ cells, the kinetics of differentiation of BrdU-labeled early or middle pachytene spermatocytes and the levels of apoptosis in the different cell populations. However, during the second week of culture, the decrease in the proportion of the 4C cell population which was only slightly more marked than that observed in vivo between 27 and 34 days, was not associated with an increase in the 1C cell population as large as in vivo. This result could be explained partly by a high proportion of apoptotic 1C cells beyond one week of culture. Concomitantly, the rate of in vitro differentiation of BrdU-labeled spermatocytes slowed down when reaching the stage of middle pachytene spermatocytes and BrdU-labeled round spermatids were observed 6-11 days later than when BrdU-labeled spermatocytes differentiated in vivo. Taken together, our results indicate that the bottleneck for the development of the meiotic cells in vitro is at the transition from middle to late pachytene spermatocytes. Hence, comparing the expression of locally produced regulatory molecules in vivo and in vitro at different days of culture should allow to identify key regulators of the meiotic step of spermatogenesis.
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