Studies on the Onset of Leydig Precursor Cell Differentiation in the Prepubertal Rat Testis1

Abstract

Leydig cells of the adult rat testis differentiate postnatally from spindle-shaped cells in the testis interstitium during the neonatal-prepubertal period. Which spindle-shaped cell types are the precursor for Leydig cells and the stimulus for initiation of their differentiation are, however, two unresolved issues. In the present study, our objectives were to identify unequivocally which spindle-shaped cells are the precursors to Leydig cells and to test whether the initiation of their differentiation into Leydig cells depends on LH. Testes from fifteen groups of Sprague-Dawley rats (n = 4 per group) from 7-21 days of age were fixed in Bouin solution and embedded in paraffin. Immunoexpression of 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome P450 side-chain cleavage (P450(scc)), 17alpha-hydroxylase cytochrome P450 (P450(c17)), and LH receptors (LHR) in interstitial cells (other than fetal Leydig cells) was observed using the avidin biotin method. Of all spindle-shaped cell types in the testis interstitium, only the peritubular mesenchymal cells showed positive immunolabeling for all three steroidogenic enzymes, beginning from the 11th postnatal day. All three enzymes were expressed simultaneously in these cells, and their numbers increased significantly thereafter. Immunoexpression of LHR in a few of these cells was just evident for the first time on postnatal Day 12 (i.e., after acquiring the steroidogenic enzyme activity). Their numbers gradually increased with time. The number of immunolabeled cells per 1000 interstitial cells (excluding fetal Leydig cells and capillary endothelial cells) was not significantly different for the three steroidogenic enzymes tested at all ages; however, a lower value was observed for LHR at each time-point. Based on these observations, we suggest that 1) the precursor cell type for the adult generation of Leydig cells in the postnatal rat testis is the peritubular mesenchymal cells, 2) precursor cells acquire 3beta-HSD, P450(scc), and P450(c17) enzyme activity simultaneously during Leydig cell differentiation, and 3) onset of precursor cell differentiation during Leydig cell development does not depend on LH.

Comparison of cell types in the rat Leydig cell lineage after ethane dimethanesulfonate treatment

“…We speculate that stem Leydig cells self-renew to keep the homeostasis of adult population of Leydig cells. At postnatal day 11-14 stem Leydig cells commit to spindle-shaped progenitor Leydig cells (Ariyaratne et al 2000), which express some androgen biosynthetic enzymes including cytochrome P450 side-chain cleavage enzyme (CYP11A1, encoded by Cyp11a1), 3bHSD1, and 17a-hydroxylase/20-lyase (CYP17A1, encoded by Cyp17a1) without expression of 17b-hydroxysteroid dehydrogenase 3 (17bHSD3, encoded by Hsd17b3) (Ge & Hardy 1998). Progenitor Leydig cells also contain high levels of 5a-reductase 1 (SRD5A1, encoded by Srd5a1) and 3a-hydroxysteroid dehydrogenase (3aHSD, encoded by Akr1c14) (Ge & Hardy 1998), thus the androgen precursor androstenedione formed by androgen biosynthetic enzymes is further metabolized into androsterone (Ge & Hardy 1998).…”

“…Immature Leydig cells begin to express 17bHSD3 but still have higher levels of SRD5A1 and 3aHSD. Therefore, they primarily produce 5a-androstane-17b,3a,-diol (DIOL; Ariyaratne et al 2000). SRD5A1 disappears in Leydig cells when immature Leydig cells differentiate into ALCs, leading to testosterone becoming the primary androgen (Ge & Hardy 1998).…”

Expression of insulin-like peptide 3 in the postnatal rat Leydig cell lineage: timing and effects of triiodothyronine-treatment

“…newly formed ALC) and the timing of first expression of INSL3 peptide in the ALC lineage in any mammalian species studied to date, and specifically in the rat. It is also interesting to note that there is a time lag of 5 days between the first detection of this cell type in the postnatal rat testis interstium, which occurs on postnatal day 10 (Mendis-Handagama et al 1987, Ariyaratne et al 2000aAriyaratne et al , 2000bAriyaratne et al , 2000c, and the first expression of INSL3 peptide in these cells. The unequivocal identification of the newly formed ALC in the testis interstitium of the prepubertal rat is relatively straightforward because of the characteristic morphology of these cells, as documented in previous studies by basement membrane components, due to their smaller size and little or no cytoplasmic lipid.…”

“…The onset of the differentiation of ALC in the rat and mouse testes is independent of LH (Ariyaratne et al 2000a, 2000b, 2000c, Baker et al 2003. However, LH is required for the steps beyond the differentiation of Leydig stem cells to progenitor cells for further development and maturation of the progenitors to become mature ALC (Mendis-Handagama and Ariyaratne 2001).…”

Role of androgen and gonadotrophins in the development and function of the Sertoli cells and Leydig cells: Data from mutant and genetically modified mice

“…Surprisingly, the immediate progenitors of the adult Leydig cells also remain in some doubt. Earlier studies indicated that the adult Leydig cells differentiate from peritubular mesenchymal cells (Haider et al, 1995;Haider and Servos, 1998;Russell et al, 1995;Ariyaratne et al, 2000). A more recent study has reported, however, that Leydig cells develop from perivascular smooth muscle cells and pericytes during normal development or during Leydig cell regeneration following ablation with the cytotoxic drug ethane dimethane sulphonate (EDS) (Davidoff et al, 2004).…”