Developmental changes in the expression of 18 Leydig cell-specific mRNA species were measured by real-time polymerase chain reaction to partially characterize the developmental phenotype of the cells in the mouse and to identify markers of adult Leydig cell differentiation. Testicular interstitial webs were isolated from mice between birth and adulthood. Five developmental patterns of gene expression were observed. Group 1 contained mRNA species encoding P450 side chain cleavage (P450(scc)), P450(c17), relaxin-like factor (RLF), glutathione S-transferase 5-5 (GST5-5), StAR protein, LH receptor, and epoxide hydrolase (EH); group 2 contained 3beta-hydroxysteroid dehydrogenase (3beta-HSD) VI, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) III, vascular cell adhesion molecule 1, estrogen sulfotransferase, and prostaglandin D (PGD)-synthetase; group 3 contained patched and thrombospondin 2 (TSP2); group 4 contained 5alpha-reductase 1 and 3alpha-hydroxysteroid dehydrogenase; group 5 contained sulfonylurea receptor 2 and 3beta-HSD I. Group 1 contained genes that were expressed in fetal and adult Leydig cells and which increased in expression around puberty toward a maximum in the adult. Group 2 contained genes expressed only in the adult Leydig cell population. Group 3 contained genes with predominant fetal/neonatal expression in the interstitial tissue. Group 4 contained genes with a peak of expression around puberty, whereas genes in group 5 show little developmental change in expression. Highest mRNA levels in descending order were RLF, P450(c17), EH, 17beta-HSD III, PGD-synthetase, GST5-5, and P450(scc). Results identify five genes expressed in the mouse adult Leydig cell population, but not in the fetal population, and one gene (TSP2) that may be expressed only in the fetal Leydig cell population. The developmental pattern of gene expression suggests that three distinct phases of adult Leydig cell differentiation occur.

Proliferation and cord formation by embryonic Sertoli cells are pivotal events involved in testis morphogenesis. A number of growth factors have been implicated in mediating these events. However, the exact level of involvement and importance of each as yet remains elusive. We have adopted an in vitro approach to assess developing mouse Sertoli cells, whereby they are cultured in the presence or absence of fibroblast growth factor (FGF9) and/or extracellular matrix (ECM) gel, since previous studies have shown that ECM gel aids Sertoli cell differentiation. The present findings corroborate this effect, but in addition demonstrate that in the presence of FGF9 (10 ng/ml), cells undergo greater proliferation than those cultured on gel alone. They also display a differentiated epithelial phenotype, with appositional contact of cell membranes in cord-like aggregations. In addition we have shown that cultured Sertoli cells generally express a smaller truncated, nuclear form of the FGFr3, although in the presence of FGF9 and absence of gel, the larger, cytoplasmic form of the receptor is also expressed. Immunolocalisation of FGFr3 in Sertoli cells of whole testes revealed a temporal expression pattern profile, with high levels being abundant in the embryonic testicular cords and at puberty, but an absence in adult Sertoli cells. Our findings suggest that FGF9 plays an important role in proliferation and organisation of embryonic Sertoli cells during testis morphogenesis.

During testicular development, fetal and adult populations of Leydig cells arise sequentially. Previous studies have shown that androgen action is required for normal steroidogenic activity in the mouse testis. Therefore, to determine the role of androgens in regulating fetal and adult Leydig cell differentiation and function, Leydig development has been measured in mice lacking functional androgen receptors (AR-null). The Leydig cell number was normal on day 5 after birth in AR-null mice but failed to increase normally thereafter and was about 30% of the control level on day 20 and about 60% of control level in adult animals. Levels of 15 different mRNA species expressed specifically in Leydig cells were measured by real-time PCR in AR-null and control animals. Expression levels of all mRNA species were normal on day 5 when only fetal Leydig cells are present. In older animals, which contain predominantly adult Leydig cells, five of the mRNA species(3β-hydroxysteroid dehydrogenase (3βHSD) type 1, cytochrome P450scc,renin, StAR protein and luteinising hormone receptor) were expressed at normal or increased levels in AR-null mice. All other mRNA species measured showed significantly reduced expression in older animals, and three of these mRNA species (17β-hydroxysteroid dehydrogenase type III, prostaglandin D(PGD)-synthetase and 3βHSD type VI), which are only expressed in the adult population of Leydig cells, were barely detectable in the adult AR-null mouse. The results show that in the absence of androgen receptors, fetal Leydig cell function is normal, but there is a developmental failure of adult Leydig cell maturation, with cells only aquiring partial characteristics of the adult population.

Previous work in our laboratory showed that pre-Sertoli cells adopt an epithelial phenotype when cultured in the presence of reconstituted basement membrane (RBM), and so cultures were established with and without this substrate. Biological activity of isolated developing mouse Sertoli cells maintained in vitro was assessed in the current study by utilising a co-culture approach, to determine whether the cells were capable of affecting ovarian differentiation. Developing Sertoli cells isolated at embryonic day (E) 12.5 exerted a deleterious effect on E12.5 ovaries in co-culture, inducing a loss of germ cells. However, when cells were isolated a day later and co-cultured with E13.5 ovaries (after entry to meiosis has begun), germ cells survived and showed evidence of meiosis, although ovigerous cords in co-cultures were masculinised compared to those of control cultured ovaries. Thus, both stages examined showed biological effects; cultured pre-Sertoli cells explanted at E12.5 showed a negative effect on female germ cells, whereas those explanted at E13.5 masculinised ovigerous cords. The functional status of isolated developing mouse Sertoli cells in vitro was further assessed by immunocytochemistry to investigate the expression of anti-Müllerian hormone, an early product of pre-Sertoli cells. Positive immunostaining was seen in developing Sertoli cells in vitro, particularly where cells had been explanted to an RBM substrate, demonstrating that good epithelial morphology is associated with improved function. Our culture system is therefore well suited for investigating factors produced by developing Sertoli cells, their role in influencing testicular morphogenesis and their potential to perturb ovarian differentiation. We believe that this in vitro approach provides a more physiological assessment compared with the knockout mouse model, where global effects of genes with housekeeping functions can compromise overall development.