165 BIOLOGY OF REPRODUCTION 63, 165–171 (2000) Studies on the Onset of Leydig Precursor Cell Differentiation in the Prepubertal Rat Testis 1 H.B. Siril Ariyaratne, 3 S.M.L. Chamindrani Mendis-Handagama, 2,3 Dale Buchanan Hales, 4 and J. Ian Mason 5 Department of Animal Science, 3 College ofVeterinary Medicine, University of Tennessee, Knoxville, Tennessee 37996 Department of Physiology and Biophysics, 4 University of Illinois at Chicago, Chicago, Illinois 60612 University Department of Reproductive and Developmental Sciences, 5 University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh EH397W, United Kingdom 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-Daw- ley rats (n  4 per group) from 7–21 days of age were fixed in Bouin solution and embedded in paraffin. Immunoexpression of 3-hydroxysteroid dehydrogenase (3HSD), cytochrome P450 side-chain cleavage (P450 scc ), 17-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 intersti- tium, 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 sig- nificantly 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 num- bers gradually increased with time. The number of immunola- beled 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 3-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. development biology, interstitial cells, Leydig cells, testis INTRODUCTION Androgens are important during the neonatal period in the male mammal for activation of the hypothalamo-hy- 1 Supported by grants from COE R180101-08 and UT Minkel to C.M.H., HD27571 to D.B.H., and HD35544 to J.I.M. 2 Correspondence: C. Mendis-Handagama, Department of Animal Sci- ence, College of Veterinary Medicine, University of Tennessee, 2407 River Drive, Knoxville, TN 37996. FAX: 423 974 2215; e-mail: mendisc@utk.edu Received: 2 December 1999. First decision: January 14 2000. Accepted: 24 February 2000.  2000 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org pophyseal-testicular axis, which is essential for completion of the testicular descent, masculinization of the brain, con- trol of Sertoli cell number, and initiation of spermatogenesis and sexual behavior [1]. In the adult male mammal, andro- gens are required for spermatogenesis, maintenance of the accessory sex gland function, sexual behavior, and suc- cessful reproductive performance. Androgens are primarily produced by the Leydig cells in the mammalian testis; therefore, the presence of functional Leydig cells is critical to the adult mammalian male. In general, two morphologically and functionally distinct populations of Leydig cells (i.e., fetal Leydig cells, and adult Leydig cells) are described in the mammalian testes. Fetal Leydig cells differentiate from their mesenchymal precursors around Day 14 of gestation in the rat testis [2] and continue to be present at birth [3, 4] and up to sexual maturity [5, 6]. However, most Leydig cells in the sexually mature rat testis (i.e., the adult population of Leydig cells) differentiate postnatally from spindle-shaped cells in the testis interstitium [3, 4, 7–11] as early as postnatal Day 10 in minute numbers [3]. During Leydig cell development in the postnatal testis, the nonsteroidogenic precursor cells in the testis interstitium should transform into Leydig cells, which have steroidogenic potential, through a series of mor- phological and functional changes. To date, one unresolved issue in this process is which spindle-shaped cells are the precursors to Leydig cells. The testis interstitium has sev- eral types of spindle-shaped cells, namely endothelial cells, pericytes, myoid cells, and fibroblasts. The latter cell type (i.e., fibroblasts) is identified as mesenchymal cells in the present study and is found in the peritubular region as well as scattered randomly throughout the testis interstitium. Another uncertain issue regarding the Leydig cell dif- ferentiation process is what triggers differentiation of the precursor cells to transform into progenitor Leydig cells to begin the process. Several studies have suggested LH as the triggering hormone for Leydig cell differentiation [12– 14]; however, other evidence suggests that LH is not critical to initiate this process. For example, when adult Leydig cells first appear in the neonatal rat testis, circulating LH is at a very low level [15]. After transient neonatal hypo- thyroidism, when circulating LH is also at a very low level [16] Leydig cells still differentiate [17–19]. Therefore, in the present study, we hypothesized that although LH is an important hormone in Leydig cell differentiation, it is not required to initiate the first step, that is, differentiation of precursor cells (i.e., nonsteroidogenic cells) into Leydig cell progenitors (i.e., steroidogenic cells). Although the Leydig cell progenitors are morphologi- cally indistinguishable from the undifferentiated precursor cells, they are definitely committed toward the Leydig cell