Development of the meiotic step in testes of pubertal rats: Comparison between the in vivo situation and under in vitro conditions

Abstract

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.

The effects of FSH and of testosterone on the completion of meiosis and the very early steps of spermiogenesis of the rat: an in vitro study

“…This property of Sertoli cells might also explain, at least partly, the lower percentages of apoptotic germinal cells measured by TUNEL assay than by annexin V binding. These results fit quite well with the proportions of apoptotic germ cells determined by measuring the level of activated caspase 3 in freshly isolated germ cell preparations or in cultured seminiferous tubules (Perrard et al 2003). Many in vivo studies have shown that both FSH and testosterone decrease apoptosis of both PS and RS of the rat (Billig et al 1995, Brinckworth et al 1995 , Marathe et al 1995, El Shennawy et al 1998, but some of them did not produce statistically significant results (Saito et al 2000).…”

“…Besides, several teams have now demonstrated that meiosis can proceed in vitro when mammalian spermatogenic cells are cocultured with Sertoli cells (Parvinen et al 1983, Weiss et al 1997, Hue et al 1998, Staub et al 2000, Lee et al 2001, Sousa et al 2002, Godet et al 2004. In rodents, the kinetics of the meiotic process is similar in vivo and during the first week of culture (Parvinen et al 1983, Perrard et al 2003, but the efficiency of the meiosis events is somewhat lower than in vivo; this can be explained partly by a higher proportion of apoptotic 1C cells in culture and a bottleneck at the transition from middle to late PS which would lead to the phagocytosis of PS by the Sertoli cells (Perrard et al 2003). Nevertheless, it has been shown recently that RS developed in vitro can produce normal offspring in the mouse (Marh et al 2003).…”

“…In the first experiment, the large and the small diameters of the nuclei of the PS were measured at every stage of the seminiferous epithelium on stained histologic sections of testes from adult rats; then, the value obtained after addition of the two diameters was expressed as a percentage of the values obtained for diplotene spermatocytes (stage XIII), which is the largest type of spermatocyte. Thus, three classes of PS were defined: early PS (stages XIV-IV), the sum of the two nucleus diameters (14·0-15·7 µm), which ranged from 59% to 66% of the value obtained for diplotene spermatocytes; middle PS (stages V-IX) (16·5-20·9 µm) (69-88%) and late spermatocytes (stages X-XIII) (21·6-23·8 µm) (91-100%) (Perrard et al 2003). In the second experiment, total germ cell preparations from adult rats were cytospun on glass slides and stained with hematoxylin/eosin.…”

Glial cell-line-derived neurotropic factor and its receptors are expressed by germinal and somatic cells of the rat testis

“…Culture of seminiferous tubule segments was performed as previously described for 21-dayold rat seminiferous tubules (Hue et al 1998, Staub et al 2000, Perrard et al 2003, but omitting testosterone in the culture medium. Indeed, Boitani and co-workers (1993) have shown that, as opposed to FSH, testosterone has no effect on the preservation of the cellular morphology of testicular explants from immature rats.…”

Analyse de la spermatogenèseex vivo

“…Les systèmes développés au laboratoire ont été validés sur de nombreux points de la physiologie. Nous avons ainsi montré que l'ensemble du processus méiotique d'un mammifère peut se dérouler ex vivo [13] et que les caractéristiques de la méiose qui se déroule ex vivo sont très proches de ce qui se produit dans les testicules de rat à la puberté [14]. Par ailleurs, nous avons montré que les pourcentages de spermatocytes aux différents stades de la prophase I-l eptotène, zygotène, pachytène et diplotène 3 précoces qui synthétisent le TGF-1 en plus grande abondance [27], celles-là mêmes qui synthétisent le NGF [24].…”