The Whole Meiotic Process Can Occur in Vitro in Untransformed Rat Spermatogenic Cells

Abstract

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.

Quantification of stem cell factor mRNA levels in the rat testis: usefulness of clusterin mRNA as a marker of the amount of mRNA of sertoli cell origin in post pubertal rats

“…However, the ratio provides evidence that the proportion of Sertolian tissue in testis samples may vary greatly according to the different experimental/pathological conditions, even when the number of Sertoli cells remains unchanged. Similar variations were observed whether clusterin mRNA level was measured by Northern blot analysis or by competitive RT-PCR with RNA competitors; the latter method is much more sensitive than the former, enabling analysis from small amounts of tissue such as from clinical biopsies or Sertoli/germ cell co-cultures (Weiss et al 1997, Staub et al 2000. The use of clusterin mRNA as a marker of the amount of RNA of Sertoli cell origin is, however, limited to rats of 20 days old or more, since immature Sertoli cells do not express clusterin mRNA levels as high as those of mature Sertoli cells.…”

“…In order to measure SCF and clusterin mRNA from small amounts of tissue, such as can be obtained from co-cultures of Sertoli and germ cells (Weiss et al 1997), seminiferous tubule cultures (Staub et al 2000) or testicular biopsies, we developed a competitive RT-PCR procedure that monitored both the reverse transcription and the PCR steps.…”

The insulin sensitiser metformin regulates chicken Sertoli and germ cell populations

“…Seminiferous tubules were prepared as described previously (Guibert et al 2013) and seeded in static 24-well cell culture polyethylene terephthalate inserts (pore 0.4 µm, WWR, Fontenay sous Bois, France) as described previously (Djakiew & Dym 1988, Staub et al 2000, Legendre et al 2010. This was carried out in order to preserve germ cells on polarised Sertoli cells in a dynamic culture using the Quasi-Vivo system (Kirkstall Ltd, Rotherham, UK).…”

Ectopic porcine spermatogenesis in murine subcutis: tissue grafting versus cell-injection methods

“…To elucidate the mechanism of spermatogenesis and the conditions required for this process, researchers have long attempted to recapitulate the process in vitro [2,3]. Several reports have claimed to have achieved the completion of meiosis leading to the production of spermatids in vitro [4,5]. Those studies, however, have used the testicular cells of immature animals as experimental material, which include not only spermatogonial stem cells but also spermatocytes from the meiotic stage.…”