The role of the gonadotrophins in regulating numbers of Leydig and Sertoli cells during fetal and postnatal development was examined using normal mice and hypogonadal (hpg) mice, which lack circulating gonadotrophins. The disector method was used to determine the number of cells from day 16 of gestation until adulthood. The numbers of Leydig cells did not change significantly between day 16 of gestation and day 5 after parturition in normal mice and were not significantly different from numbers in hpg mice at any age up to day 5 after parturition. There was a 16-fold increase in the number of Leydig cells in normal mice between day 5 and day 20 after parturition, followed by a further doubling of number of cells between day 20 and adulthood. The number of Leydig cells in hpg testes did not change between day 5 and day 20 after parturition but doubled between day 20 and adulthood so that the number of cells was about 10% of normal values from day 20 onwards. Leydig cell volume was constant in normal animals from birth up to day 20 and then showed a 2.5-fold increase in adult animals. Leydig cell volume was normal in hpg testes at birth but decreased thereafter and was about 20% of normal volume in adult mice. The number of Sertoli cells increased continuously from day 16 of gestation to day 20 after gestation in normal mice and then remained static until adulthood. The number of Sertoli cells in hpg testes was normal throughout fetal life but was reduced by about 30% on day 1 (day of parturition). Thereafter, Sertoli cells proliferated at a slower rate but over a longer period in the hpg testis so that on day 20 after parturition the number of Sertoli cells was about 50% of normal values, whereas in adult mice the number was 65% of normal. The number of gonocytes did not change between day 16 of gestation and day 1 and did not differ between normal and hpg testes. The number of gonocytes increased nine-fold in normal testes but only three-fold in hpg testes between day 1 and day 5 after parturition. Gonocytes differentiated into spermatogonia in both normal and hpg testes between day 5 and day 20 after parturition. These results show: (i) that fetal development of both Sertoli and Leydig cells is independent of gonadotrophins; (ii) that normal differentiation and proliferation of the adult Leydig cell population (starting about day 10 after parturition) is dependent on the presence of gonadotrophins; and (iii) that the number of Sertoli cells after birth is regulated by gonadotrophins, although proliferation will continue, at a lower rate and for longer, in the absence of gonadotrophins.

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Context:Normal fetal testis development is essential for masculinization and subsequent adult fertility. The second trimester is a critical period of human testicular development and masculinization, but there is a paucity of reliable developmental data.Objective: The objective of the study was to analyze second-trimester human testicular morphology and function.Design: This was an observational study of second-trimester testis development. Setting:The study was conducted at the Universities of Glasgow and Aberdeen.Patients/Participants: Testes were collected from 57 morphologically normal fetuses of women undergoing elective termination of normally progressing pregnancies (11-19 wk gestation).Main Outcome Measure(s): Testicular morphology, cell numbers, and quantitative expression of 22 key testicular genes were determined.

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Prostaglandin D synthetase is expressed relatively highly in the testis and reproductive tract of a number of species, including the mouse. In adult mouse testis, expression is confined largely to the Leydig cells and in this study changes in the expression and localization of prostaglandin D synthetase mRNA during testis development were examined. Initial studies using RT-PCR and isolated testicular compartments indicated that prostaglandin D synthetase expression in the neonatal testis was predominantly within the seminiferous tubules. In situ hybridization studies confirmed that prostaglandin D synthetase mRNA appears to be expressed only in the tubules of neonatal mouse testes and only in the interstitial tissue of the adult testis. TaqMan real-time PCR was used to quantify prostaglandin D synthetase mRNA content during development using an exogenous mRNA as a control standard. Expression per testis decreased after birth to < 10% at day 15 before recovering again by days 25-30. After day 30, expression per testis increased 40-fold during final development to adulthood. Studies using RT-PCR showed that early expression before day 15 was restricted to the tubular compartment, whereas the subsequent increase in expression after day 30 was restricted to the interstitial compartment. Database analysis showed that the 3' end of the prostaglandin D synthetase transcript was subject to alternate splicing. Both splice isoforms were shown by RT-PCR to be present throughout development and without a major change in expression pattern. These results indicate that expression of prostaglandin D synthetase mRNA shifts during development from the tubular compartment of the fetal or neonatal testis to the developing adult Leydig cells, with expression in the Leydig cells increasing markedly after puberty. These changes are similar to those observed for 17beta-hydroxysteroid dehydrogenase type III and may indicate that this developmental process is not uncommon in the testis.

The morphological and functional characteristics of cultured bovine luteal cells were examined. Dispersed luteal cells were cultured in either Ham's F12 medium or Dulbecco's Modified Eagle's Medium (DMEM), plus 10% adult bovine serum. After 18 h of culture, fibronectin was observed to be associated with some cells showing histochemical 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. In DMEM, cells adopted a fibroblast-like appearance with a doubling time of 34 h while in Ham's F12, cells retained an epithelial-like morphology with a doubling time of 91 h. Dibutyryl cyclic AMP (dbcAMP) but not LH slowed proliferation and altered cell morphology in DMEM. Addition of insulin and dbcAMP was required to maintain 3 beta-HSD activity in cells cultured for more than 3 days. Progesterone secretion was significantly greater in Ham's F12 than in DMEM although there was a rapid decline in basal secretion during culture in either medium. Both LH and dbcAMP stimulated progesterone secretion although the effects of dbcAMP were significantly greater than those of LH after 18 h of culture. Addition of insulin significantly enhanced basal, LH-stimulated and dbcAMP-stimulated progesterone secretion after 3 days of culture whereas several antioxidants were without effect. These studies indicate that the morphological and functional characteristics of cultured luteal cells may be best maintained in Ham's F12 plus dbcAMP and insulin.

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