Retinoids induced t-PA synthesis by C6 glioma cells--role in tumoral haemorrhagic necrosis.

Treatment of rat C6 glioma with high doses of 13 cis-retinoic acid (cRA) was responsible for death related to haemorrhagic necrosis localized to the tumor. Our aim was to explore this adverse effect of retinoid treatment. We show that cRA-treated C6 glioma at 25 mg/kg/day for 18 days exhibits in vivo an increase T-PA activity, which is responsible for a localized tumor fibrinolytic activity. Production of t-PA is supported by specific enhancement of gene expression, as was shown by the increase in t-PA mRNA (x 2.3). This production is a direct effect of cRA when treating the tumor, since tumor cells themselves do not produce enough t-PA and treatment of control rats does not increase the t-PA level. T-PA production by rat C6 glioma is in vivo related to the specific synthesis of t-PA by the C6 cell-line. The stimulation of C6 cell-line by cRA in vitro is dose-dependent and reached a maximum for 3 and 30 microM at the 72nd h. So cRA-treated C6 glioma cells produce t-PA which appears to be the major species associated with the fibrinolytic activity-induced intra-tumoral haemorrhage after exposure to retinoid treatment.

Study in vitro of the phagocytic function of Sertoli cells in the rat.

Aspects of the interaction between residual bodies/cytoplasts from elongated spermatids (RB/CES) and Sertoli cells were studied in vitro. Highly enriched Sertoli cells (91%: experiment A), very highly enriched Sertoli cells (greater than 96%: experiment B), as well as peritubular cells were isolated from testes of 20-day-old rats by means of hypotonic treatment. Isolated Sertoli cells and peritubular cells were also prepared from 45-day-old rats (experiment C). RB/CES were isolated by centrifugal elutriation from testes of rats aged 90-120 days. The kinetics of adhesion of RB/CES to Sertoli cells were similar in all experiments. FSH accelerated binding of RB/CES but markedly reduced the number of RB/CES phagocytosed. Co-culture of the highly enriched Sertoli cells from experiments A and C with isolated peritubular cells did not change the kinetics of adhesion of RB/CES. However, when the contamination of Sertoli cells by peritubular cells was at a minimum (experiment B), addition of peritubular cells induced a slight but significant stimulation of the binding of RB/CES. Transmission electron microscopy revealed the following events within 24 h of co-culture: adhesion of the RB/CES to microvilli of Sertoli cells; internalization of RB/CES; lysis of the membrane of RB/CES; total digestion. Therefore, FSH and peritubular cells modulate the interaction in vitro between Sertoli cells and RB/CES, and the different steps of residual body disposal can be reproduced in co-culture. The co-culture model described in this study provides a useful system for the study of phagocytic activity by Sertoli cells.

[Germ cells and post-natal development of testicular function: in vitro studies]. / Cellules germinales et développement post-natal de la fonction testiculaire: etudes in vitro.

Studies in recent years have clearly established that, in addition to the well known endocrine regulation by gonadotrophin hormones, spermatogenesis is under the modulatory control of a complex set of paracrine regulators. Whereas the role of Leydig cells (testosterone) and of Sertoli cells (nurce cells of germ cells) in spermatogenesis has focused most of the attention, until recently little was known about the contribution of germ cells in the spermatogenetic process. This was the aim of the present experiments. We have used, in vitro, 3 complementary approaches; 1) we measured the influence of the removal of germ cells contaminating Sertoli cell cultures by a hypotonic treatment; 2) in coculture, we examined to what extend isolated germ cells could affect Sertoli cell function; 3) we investigated the effects of germ cell conditioned media on Sertoli cell cultures. Our results indicate that germ cells are able to modulate Sertoli cell function in vitro. This germ cell influence varies according to: 1) the germ cell fraction tested (pachytene spermatocytes, early spermatids or cytoplast from elongated spermatids/residual bodies); 2) the parameter of Sertoli cell function studied (inhibition of oestradiol; stimulation of androgen-binding protein, transferrin...); 3) the age of the Sertoli cell donors; 4) the hormonal environment (+/- FSH). Furthermore we wave demonstrated that germ cell effects were partly at least mediated via proteinaceous factor(s) detected in germ cell spent media. Taking into account previous in vivo studies and these in vitro results, we have hypothesized that germ cells, in conjunction with hormones (LH, FSH, testosterone) play an important role in the ontogenesis of Sertoli cells and therefore in spermatogenesis.

Influence of germ cells upon transferrin secretion by rat Sertoli cells in vitro.

Indirect approach (hypotonic treatment) and direct approaches (co-cultures and conditioned media) were used in order to investigate the effects of germ cells from adult rats upon transferrin secretion by Sertoli cell cultures prepared from 20-day-old rats. Removal of germ cells contaminating the Sertoli cell cultures resulted in a significant decrease in transferrin secretion whereas the addition of crude germ cell preparations or of enriched preparations of pachytene spermatocytes, early spermatids and of liver epithelial cells (LEC) markedly stimulated this parameter. Furthermore, spent media of pachytene spermatocytes and of early spermatids, but not of LEC, also stimulated transferrin production. It is concluded that germ cells normally located within the adluminal compartment of the seminiferous tubules may be capable of controlling their own supply of iron via their influence upon transferrin secretion by the Sertoli cells.

Paracrine control of immature Sertoli cells by adult germ cells, in the rat (an in vitro study). Cell-cell interactions within the testis.

Enriched populations of germ cells prepared from adult rats were found to influence 20-day-old rat Sertoli cell secretory activity by stimulating androgen-binding protein (ABP) and inhibiting oestradiol-17 beta production in the presence of follicle-stimulating hormone (FSH) as well as of dibutyryl cyclic AMP (dbcAMP). Among the different populations tested in coculture, pachytene spermatocytes were the most effective at stimulating ABP and inhibiting oestradiol production, whereas early spermatids had relatively less effects. Cytoplasts from elongated spermatids only slightly stimulated ABP secretion. The influence of germ cells upon Sertoli cells may be mediated via paracrine component(s) detected in nonconcentrated conditioned culture media. The stimulatory (ABP) and inhibitory (oestradiol) effects of pachytene spermatocyte and early spermatid-spent media were reversible (change of media), dose related, specific (no effect of cytoplast, peritubular cell, rat liver epithelial cell or 3T3 cell-conditioned media) and strictly proportional to the cell viability estimated at the end of the incubation periods. Furthermore, the nature of the germ cell factor(s) influencing Sertoli cell secretory function is likely to be proteinaceous since both germ cell-spent media effects were trypsin and heat (100 degrees C; 3 min) sensitive and retained by molecular weight (MW) greater than 10,000 cut-off dialysis membranes. It is hypothesized that germ cells, in particular pachytene spermatocytes and early spermatids, may influence Sertoli cell function during sexual development in the rat.

In vitro effects of germ cells on the secretory activity of Sertoli cells recovered from rats of different ages.

Indirect (hypotonically treated culture) and direct (coculture) approaches were used to study the influence of germ cells on androgen-binding protein (ABP) and 17 beta-estradiol secretion by Sertoli cells prepared from 10-, 15-, 20-, and 45-day-old rats. Using these approaches, the mechanisms that govern Sertoli cell-germ cell membrane recognition were shown to be unaltered by the hypotonic treatment, to vary with the age of the Sertoli cell donors, and to be FSH/(Bu)2cAMP dependent in the younger animals (10-20 days old). Hypotonic treatment had no effect on estradiol levels at all ages studied, but resulted in a marked fall of ABP production by Sertoli cells from 20 days onward. However, the relative stimulation of ABP induced by FSH/(Bu)2cAMP (ABP levels in FSH/(Bu)2cAMP/hypotonically treated Sertoli cell cultures vs. ABP in hypotonically treated cultures) was markedly increased at 20 and 45 days, indicating that germ cells influence Sertoli cell responsiveness to FSH/(Bu)2cAMP. The addition of crude germ cell preparation to the Sertoli cell cultures stimulated ABP and inhibited estradiol levels at all ages studied. Moreover, the addition of germ cells to hypotonically treated cultures induced a decrease in the relative ABP response to FSH and (Bu)2cAMP, thus confirming (see above) that germ cells may be involved in the mechanism of Sertoli cell refractoriness to FSH that occurs with advancing age in the rat. Germ cell-stimulated ABP production was higher in adult Sertoli cells than in immature cells. That Sertoli cell responsiveness to germ cells varies with age is further supported by the data showing that whereas spermatocytes stimulated ABP and inhibited estradiol productions at all ages studied, early spermatids only influenced these parameters from 20 days onward. It is concluded that germ cell control over Sertoli cell function in vitro may be of important physiological significance during Sertoli cell maturation in mammals.

Possible involvement of germ cells in the regulation of oestradiol-17 beta and ABP secretion by immature rat Sertoli cells (in vitro studies).

The effects of germ cells prepared from adult rats and of media conditioned by some of these germ cells have been studied in vitro on both ABP and oestradiol-17 beta secretion by immature rat Sertoli cells. Addition of the germ cells to the Sertoli cell cultures resulted in both a dose-dependent increase of ABP secretion and a dose-dependent inhibition of oestradiol production. These effects were suppressed after removal of germ cells by hypotonic treatment. Furthermore, spent media of highly viable germ cells (SMGC), but not spent media of an epithelial cell line, mimicked the effects of germ cells themselves on ABP and oestradiol levels after FSH or dbcAMP stimulation. These effects were reversible when SMGC were replaced by fresh media and did not result from a change in the conversion of oestradiol to oestrone. SMGC effects were unaltered by heating at 60 degrees C for 30 min, by freezing and thawing and non dialysable (MW greater than 10,000). However, heating at 100 degrees C for 3 min and treatment by trypsin, suppressed the SMGC effects. This indicates that the stimulation of ABP and inhibition of oestradiol levels by germ cells, in vitro, could be mediated by factor(s) of proteinaceous nature.

Stimulation of rat Sertoli cell secretory activity in vitro by germ cells and residual bodies.

The direct influence of germ cells and residual bodies on Sertoli cell basal and FSH-stimulated secretion of androgen-binding protein (ABP) was studied using Sertoli cells, recovered from 20-day-old rats, cultured alone or cocultured with a crude germ cell preparation from adult rats or with pachytene spermatocytes, round spermatids or populations of residual bodies enriched by centrifugal elutriation. The effect of a rat liver epithelial cell line (LEC) on Sertoli cell function was also tested. Addition of a crude germ cell preparation increased basal and FSH-stimulated ABP secretion. Pachytene spermatocytes and residual bodies adhered to the Sertoli cell monolayer to a much greater extent than did round spermatids. Addition of pachytene spermatocytes markedly enhanced basal and FSH-stimulated ABP secretion over 12 days of culture. Round spermatids and residual bodies stimulated ABP secretion although to a lesser extent than did spermatocytes. Furthermore, the increase of FSH-stimulated ABP levels was not maintained after 4 or 8 days of culture. LEC also enhanced basal and FSH-induced ABP levels but the increase of FSH-induced ABP production was only observed until Day 8 of culture. The influence of LEC on Sertoli cell secretion could be mediated through the production of an extracellular matrix. It is concluded that germ cells, particularly pachytene spermatocytes, can directly stimulate Sertoli cell secretory activity in vitro.