Expression of fibronectin and laminin in fetal male gonads in vivo and in vitro with and without testicular morphogenesis.

Sertoli cell differentiation occurs in vitro, even when testicular morphogenesis is inhibited by addition of serum to the culture medium (Magre, S. and A. Jost: Proc. Natl. Acad. Sci. USA 81, 7831-7834 (1984]. Using indirect immunohistochemical technique, we have studied the expression of fibronectin and laminin in gonads lacking testicular morphogenesis, as compared to in vivo controls and gonads cultured in synthetic medium. In undifferentiated gonads in vivo, fibronectin and laminin are distributed uniformly in the blastema. If testicular differentiation occurs in vivo, laminin is detected only in the basement membranes; when it occurs in vitro, laminin is found both in the basement membranes and among the stromal tissue. In gonads without seminiferous cords (cultured in serum-supplemented medium), fibronectin and laminin are both present, they are uniformly distributed among the gonadal cells.

[Effect of an analog of proline (L-azetidine-2-carboxylic acid) on in vitro differentiation of the rat fetal testis]. / Action d'un analogue de la proline (acide L-azétidine-2-carboxylique) sur la différenciation in vitro du testicule foetal de rat.

The initial stages of the development of the seminiferous cords involve the differentiation and the aggregation of primordial Sertoli cells opposite to cells which acquire a mesenchymal-like aspect. The hypothesis that the development of the seminiferous cords depends on epithelial-mesenchymal relations between the two cell types was submitted to experimental test. Male gonadal primordia of rat fetuses were cultured in vitro in a synthetic medium containing the proline competitor, L-Azetidine-2-Carboxylic Acid. This drug is known to disturb the synthesis and secretion of collagen and proline-containing proteins. It prevents testicular organogenesis or destroys it if it has begun. It suppresses the expression of laminin and fibronectin in the gonadal primordium. These observations are taken as evidence that cellular correlations of the epithelial-mesenchymal type play a role in the development of the testis as they do in that of other organs.

Initial phases of the rat testis differentiation in vitro.

Rat gonadal primordia with their supporting mesonephroi were explanted in vitro at the undifferentiated stage (12 days 16 h after fertilization), at the outset of testicular differentiation (13 days 9 h) or when already containing seminiferous cords. The younger foetuses were sexed with the sex chromatin test in the amniotic membrane. The basal medium was CMRL 1066 and the culture period, 1 to 4 days. Testicular differentiation resulted from the appearance of large clear cells, the primordial Sertoli cells, and from their aggregation into seminiferous cords. Addition of 15% foetal calf serum to the medium prevented the differentiation of seminiferous cords, but large clear cells appeared. In testes from 14- or 15-day-old foetuses, the seminiferous cords disintegrated under the influence of serum. The serum did not prevent the differentiation of Sertoli cells, but impaired organogenesis or maintenance of the early seminiferous cords. The results support previous histological observations on the initial stages of testicular differentiation.

Early stages of testicular differentiation in the rat.

The initial phases of the development of the seminiferous cords (future seminiferous tubules) were studied with histological techniques and with electron microscopy. On day 14 after fertilization, seminiferous cords are well differentiated in the anterior part of the testis near the mesonephric tubules. They comprise Sertoli cells which encompass the primordial germ cells. The Sertoli cells shows an expanded clear cytoplasm and microfilaments beneath the outer surface; they differentiate complex contact zones. On day 13 a few cells localized near the mesonephric tubules display the characteristics of the Sertoli cells. These cells become more and more numerous. They aggregate and they form the seminiferous cords. The primordia of male gonads explanted in vitro on the mesonephros, realize testicular organogenesis in a synthetic medium. Adding 15% fetal calf serum to the medium prevents the morphogenesis of the testicular cords, although the Sertoli cells seem to differentiate morphologically and physiologically. In these gonads differentiation of the Sertoli cells was obtained but their aggregation and the morphogenesis of the seminiferous cords were prevented. This gives new insights into testicular morphogenesis and probably provides an experimental model for a new type of gonadal anomaly.

[Effect of fetal calf serum on the in vitro differentiation or maintenance of the seminiferous cords of the testis of the fetal rat]. / Action du sérum de foetus de veau sur la différenciation in vitro ou le maintien des cordons séminifères du testicule du foetus de rat.

The differentiation of seminiferous cords can be obtained in in vitro cultures, in a synthetic medium, of undifferentiated fetal Rat gonads. In older testes the seminiferous cords are maintained under these conditions. On the contrary, adding 15% fetal Calf serum to the medium prevents the differentiation of the seminiferous cords or provokes the disruption of the already formed cords (days 14 and 15). These actions are exerted upon the fetal Sertoli cells.

[Sertoli cells and organogenesis of the fetal testis (author's transl)]. / Cellules de Sertoli et organogenèse du testicule foetal.

In previous papers (1, 2), it was shown that in the rat fetus, testicular differentiation begins 13 days after fertilization, in the depth of the primordium near to mesonephric tubules. A few cells swell and differentiate into Sertoli cells which encompass the germ cells. In the meantime they delineate locally one part of the contour of the developing seminiferous cord, which is completed by the recruitment of new cells during the next hours. Under the electron microscope the differentiation of the Sertoli cells involves cytoplasmic and organelles changes, typical junctions and microfilaments toward the external surface of the seminiferous cord. Testicular differentiation can be obtained in vitro in cultures made in a synthetic medium without or with addition of chick embryo extract. Fetal calf serum prevents the organogenesis of the sex cords or produces the disintegration of those already differentiation (on day 14). These data clearly underline some of the cellular processes involved in the differentiation of the seminiferous cords.