A mouse Sertoli cell line expressing anti-Müllerian hormone and its type II receptor
Introduction
Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance, is a glycoprotein belonging to the transforming growth factor-β (TGF-β) family of growth factors (Cate et al., 1986) which signal through two distantly related serine/threonine kinases, the type I and II receptors (Massagué, 1996). The genes for human (Cate et al., 1986) and mouse (Münsterberg and Lovell-Badge, 1991) AMH have been cloned as well as those for the corresponding type II receptors (AMHR-II) (Imbeaud et al., 1995, Mishina et al., 1996).
AMH is best known through its action on the foetal reproductive tract. In the male foetus, the testis directs the masculinisation of the reproductive tract by secreting two distinct hormones (Jost, 1953): AMH induces the regression of the Müllerian ducts, which give rise to the uterus and upper vagina in females, while testosterone, produced by Leydig cells, virilises the external genitalia and urogenital sinus. Both AMH (Josso et al., 1993) and AMHR-II (Baarends et al., 1994, di Clemente et al., 1994) are expressed by Sertoli cells before and after birth.
Sertoli cells first appear in the gonadal ridge, heralding seminiferous tubule differentiation (Magre and Jost, 1980). They remain immature up to puberty, when they undergo biochemical and morphological changes which allow them to support spermatogenesis. Most genes specifically expressed in Sertoli cells become activated at that time (Gondos and Berndston, 1993). AMH is one of the few that are active mainly during the foetal and early postnatal period and repressed at pubertal maturation (Josso et al., 1990, Münsterberg and Lovell-Badge, 1991). In contrast, developmental expression of AMHR-II is subject to species differences: in the rabbit, puberty leads to its extinction, whereas in the rat, its expression actually increases at adulthood (Baarends et al., 1994, Baarends et al., 1995). The analysis of Sertoli cell function has, for many years, been carried out in primary cultures (Steinberger and Jakubowiak, 1993). However, primary cultures are laborious to establish, have a short life span and lose many of their functional properties after explantation.
Targeted oncogenesis, in which an oncogene is driven by a tissue-specific promoter and cell lines are derived from the resulting tumors, is an attractive method for developing differentiated cell lines from various tissues (Hanahan, 1985, Cory and Adams, 1988, Mellon et al., 1994, Courjault-Gautier et al., 1997). Granulosa cell lines have been obtained from ovarian tumours in mice transgenic for the Simian virus 40 (SV40) early gene placed under the control of the inhibin-α promoter, which in the ovary is specifically expressed by granulosa cells. As planned, the immortalised cell lines expressed the inhibin-α subunit (Kananen et al., 1995). For AMH, a similar strategy has not met with success (Peschon et al., 1992). Nevertheless, using targeted oncogenesis but taking into account the developmental status of the transgenic mouse, we have obtained a clonal cell line with a profile characteristic of prepubertal Sertoli cells and expressing both AMH mRNA and protein.
To the best of our knowledge, this is the first Sertoli cell line stably expressing AMH and its receptor.
Section snippets
Construction of AMH-SV40 oncogene fusion genes
A section of 3.6 kb of the 5′ flanking region of the human AMH gene (position −3573/+9) relative to the major transcriptional initiation site (Guerrier et al., 1990) were placed upstream of the AvrII/BamHI oncogenic fragment of the SV40 virus genomic DNA, either wild-type (encoding both the large T and small t antigens: AT+t construct) or with a deletion (Volckaert et al., 1979) abolishing small t antigen expression (AT−t construct). We checked the sequence of both constructs at the junction
Heritable Sertoli cell tumours in AMH-SV40 oncogene transgenic mice
We constructed fusion genes containing 3.6 kb of the 5′ flanking region of the human AMH gene placed upstream of the SV40 oncogene encoding both large T and small t antigens (AT+t construct) or only the large T antigen (AT−t construct). Two AT+t and 13 AT−t transgenic mice were generated. Integrity of the constructs was checked by Southern hybridisation of genomic DNA for those animals used for further studies.
By dot blot hybridisation, we detected the large T antigen mRNA in testes of most
Discussion
The analysis of AMH regulation in Sertoli cells has suffered for many years from the lack of an adequate in vitro cellular model. AMH production is not maintained for more than a few days by immature Sertoli cells in cell or tissue-culture (Vigier et al., 1985La Quaglia et al., 1986). Capel et al. (1996)derived a cell line composed of several cell types from urogenital ridges of 11-day-old mouse embryos and detected faint AMH expression by RT-PCR after 3 months but no longer than 6 months in
Acknowledgements
We wish to thank Drs Axel Kahn and Alain Vandewalle for counselling and helpful discussion and Jean Feuntun for providing us with wild type and mutant SV40 oncogenes. The WT-1 probe was kindly provided by Dr Philippe Berta, SF-1 by Dr Keith Parker and transferrin, FSHR, SGP-1 and SGP-2 by Dr Michael Griswold. This work was supported by a doctoral fellowship of the Association pour la Recherche sur le Cancer, France, to M.D., a postdoctoral fellowship of the National Research Council (CONICET,
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