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a Department of Animal Science, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37996
b Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612
c University Department of Reproductive and Developmental Sciences, University of Edinburgh, Royal Infirmary of Edinburgh, Edinburgh EH397W, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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-hydroxylase cytochrome P450 (P450c17), 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 3ß-HSD, P450scc, and P450c17 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
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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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 morphological 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 several 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 differentiation 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 hypothyroidism, 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 morphologically indistinguishable from the undifferentiated precursor cells, they are definitely committed toward the Leydig cell lineage, because these cells express steroidogenic enzymes and can produce androgens [20]. Therefore, if our hypothesis is correct, differentiating precursor cells should attain the steroidogenic potential before acquiring LH receptors. To our knowledge, no information is available regarding the timing of these two events (i.e., acquisition of steroidogenic enzymes and LH receptors in precursor cells during Leydig cell differentiation) for any mammalian species in general, and for the rat in particular. Therefore, we designed the present study to determine the timing for the acquisition of LH receptors and the steroidogenic enzymes 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450 side-chain cleavage (P450scc), and 17-hydroxylase cytochrome P450 (P450c17) in postnatal rat testes of Sprague-Dawley rats through 7 to 21 days of age using immunohistochemical techniques. We also examined whether precursor cells acquire these three steroidogenic enzymes simultaneously or at different stages of the process.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Female Sprague-Dawley rats in midpregnancy were purchased from Harlan Industries (Madison, WI). The rats were housed in the animal facility of the University of Tennessee, College of Veterinary Medicine, one rat per cage, under conditions of controlled temperature (25°C) and lighting (14L:10D). Rats were provided food (Agway Prolab rat formula, Syracuse, NY) and water ad libitum, and they were examined for litters twice daily (morning and evening). The day of birth of pups was considered to be Day 1 of age.
Tissue Preparation
Fifteen groups of rat pups (n = 4 per group) from 7 to 21 days of age were used. The animals were killed daily during postnatal Days 7 through 21 by CO2 inhalation. Both testes from each rat were collected and fixed by immersion in Bouin fluid for 5–6 h. Tissue was then washed in 70% ethyl alcohol for several days until the picric acid was removed, processed, and embedded in low-melting Paraplast (Oxford Labware, St. Louis, MO). Serial sections (thickness, 5 µm) were cut from these tissue blocks and adhered on ProbeOn Plus (Fisher Scientific, Pittsburgh, PA) glass slides to use in immunocytochemistry.
Antibodies
The polyclonal antibody against 3ß-HSD was a rabbit immunoglobulin (Ig) G antibody against purified human placental 3ß-HSD [20] and has been used in many previous study for immunolocalization of 3ß-HSD antigen in rat testis, including studies of Leydig cell differentiation in rats during fetal ages [2, 22]. The antibodies used to detect P450scc and P450c17 proteins were raised against bovine adrenal P450scc [23] and porcine testicular P450c17 [24], respectively. These two antibodies have previously been employed in immunoblot [25] and immunohistochemical [26] studies of testicular tissue. A biotinylated goat antirabbit IgG (StrAviGen super sensitive; BioGenex, San Raman, CA) was used as the second antibody.
The antibody directed against purified rat LH receptor was a monoclonal antibody (P1B4) raised in mice and has been described in detail by Indrapichate et al. [27]. It has been used in other studies to immunolocalize LH receptors in the rat testis [2, 28]. Because this is an antibody of the IgM category, a biotinylated goat-antimouse IgM antibody (Sigma, St. Louis, MO) was used as the second antibody.
Immunocytochemistry
For immunocytochemistry, tissue sections were deparaffinized in xylene, rehydrated in a series of graded ethanol, and brought into distilled water. To detect P450scc immunoreactivity, the sections were washed in PBS (pH 7.6) and incubated in 3% H2O2 in absolute methanol for 30 min at room temperature to block endogenous peroxidase activity. The slides were then protein blocked by immersion in a solution of 10% normal goat serum plus 1% bovine serum albumin (BSA fraction-V; Sigma) for 6 h at 4°C. The primary antibody against P450scc was diluted 1:1000 in protein-blocking solution, applied to sections, and incubated overnight at 4°C. On the following day, the slides were washed in PBS, and the bound antibody was detected using the biotin-streptavidin method with a commercially available supersensitive detection kit (BioGenex, San Raman, CA) according to the manufacturer's instructions. This kit uses 3'3-diaminobenzidine hydrochloride as a chromogen. The slides were counterstained with Mayer Haematoxylin (Sigma, St. Louis, MO), dehydrated in a series of increasing concentrations of ethanol, and coverslipped under Permount (Fisher Scientific, Fair Lawn, NJ). A similar procedure was adopted to immunolabel 3ß-HSD and P450c17 antigens, except for the first antibody-dilution step. For the detection of both these antigens, an antibody dilution of 1:2000 was used (i.e., after testing dilutions ranging from 1:200 to 1:3000).
For immunodetection of LH receptors, the rehydrated sections were washed in Tris-buffered saline (TBS; 0.05 M Tris-HCl, 0.85% NaCl [pH 7.4]), and tissue peroxidase activity was inactivated by incubating the sections in 3% H2O2 in water at room temperature for 30 min. Other steps of the procedure were as same as those described for immunolabeling for P450scc, except that instead of PBS, TBS was used as the washing, diluting, and incubating medium. After testing dilutions ranging from 1:200 to 1:3000, a 1:2000 dilution of the primary antibody was employed.
To determine whether each differentiating precursor cell gained all three steroidogenic enzymes at the same time, we employed the following method: After the first immunodetection of the three steroidogenic enzymes in differentiating precursor cells at Day 11 in separate tissue sections, adjacent tissue sections of the previously immunolabeled sections were used to immunolabel the other two steroidogenic enzymes and LH receptors. To compare an area of one section immunolabeled for a particular antigen with the corresponding area of the adjacent section immunolabeled for a different antigen, the microscopic images of the two areas were brought onto two separate color video monitors (Panasonic, Model 1384Y), placed side by side, with the help of a CCD-IRIS color video camera (model DXC-107A; Sony Corporation, Tokyo, Japan) attached to an Olympus BH-2 microscope (Olympus, Tokyo, Japan). For each antibody, control slides were incubated in preimmune serum in place of the primary antibody. Immunolabeling experiments were repeated three times for each antibody to establish the reproducibility of these results.
Cell Counts
To quantify the number of testicular interstitial cells (i.e., progenitors and adult Leydig cells) immunolabeled for each antibody on Days 11, 12, 16, and 21, testicular interstitial cells (excluding fetal Leydig cells and vascular endothelial cells) were counted in single sections using the described Olympus BH-2 microscope under a x40 objective lens and then noting whether they were positive or negative for the antigen tested. Estimates for number of positive cells per 1000 total cells counted were used for comparisons. Approximately 3000 cells per each slide were scored, with eight slides in each age group.
Statistical Analysis
Significant differences (P < 0.05) among the mean numbers of cells stained positively for different antibodies at each age were determined by ANOVA, which was followed by the Duncan multiple range test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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At Day 11, immunolabeling for all three steroidogenic enzymes was detected for the first time in cells other than fetal Leydig cells in the postnatal testis interstitium. These cells were mesenchymal cells, exclusively in the peritubular area and few in number. Myoid cells, endothelial cells, pericytes, and nonperitubular mesenchymal cells were negative at this time (Figs. 1, C–H, and 2B). A few of these differentiating cells appeared to be in the process of rounding up and leaving the peritubular region in some tissue sections, even at postnatal Day 11 (Figs. 1, C, F, and H, and 2B). Moreover, as revealed by immunlabeling of adjacent tissue sections from 11-day-postnatal testes for different enzymes (P450scc, 3ß-HSD, and P450c17), the same progenitor cell contained all three of these enzymes concurrently at Day 11 after birth (Fig. 1, C and D, for P450scc and p450c17; Fig. 1, E and F, for 3ß-HSD and P450scc; Fig. 1, G and H, for 3ß-HSD and P450c17). Immunolabeling of P450scc was also evident in Sertoli cells at this age (Fig. 1, C and F).
From Days 7 to 11, positive labeling for LH receptors was not observed in any cell type in the postnatal testis interstitium other than fetal Leydig cells. When two adjacent sections were surveyed for localization of LH receptors (Fig. 2A) and 3ß-HSD enzyme (Fig. 2B), the cells that expressed 3ß-HSD enzyme at Day 11 did not contain LH receptors. On Day 12, immunolabeling for LH receptors was barely evident in a few Leydig cell progenitors (Fig. 2C). These LH receptor-positive cells were not elongated anymore, and all appeared to be located more toward the intertubular area, somewhat away from the peritubular region. The intensity of the staining in individual cells and the number of cells immunolabeled for each antigen increased with advancing age of the rat (Table 1). Immunolabeling for any of the three steroidogenic enzymes tested and for LH receptors was not detected in myoid cells, endothelial cells, mesenchymal cells in the central interstitium, and pericytes in any age group examined.
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Table 1 shows the results from quantification of immunolabeled testicular interstitial cells (excluding fetal Leydig cells) in the testis interstitium from Day 11 through Day 21. On Day 11, immunoexpression of the three steroidogenic enzymes was observed for the first time in a few peritubular mesenchymal cells. The number of cells that were positive for each antigen increased with age; however, the number of immunolabeled cells that were immunoreactive for different steroidogenic enzymes per 1000 interstitial cells counted at a given age (excluding fetal Leydig cells and vascular endothelial cells) was not different. In contrast, the number of immunolabeled cells for LH receptors (excluding fetal Leydig cells) per 1000 interstitial cells counted (excluding fetal Leydig cells and vascular endothelial cells) was significantly lower than the values for all three steroidogenic enzymes tested at that age.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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The present study also revealed that at postnatal Day 11, spindle-shaped cells immunolabeled for the three steroidogenic enzymes tested are exclusively found in the peritubular region, confirming the peritubular origin of Leydig cell progenitors as suggested by early investigators such as de Kretser [8], Christensen [10], and Van Straaten and Wensing [34]. This observation is also in agreement with more recent studies by Russell et al. [35], who suggested that early recruitment of Leydig cells in the postnatal rat testis is from undifferentiated peritubular fibroblast-like cells. The myoid cells, pericytes, and vascular endothelial cells were not positive for any of these steroidogenic enzymes at any postnatal age. Moreover, at this age, the testicular interstitial space is not filled with lymph but is occupied by a fine network of fibers [3]. Therefore, cells that are identified as lymphatic endothelial cells in the adult rat testis are not yet observed in the testis interstitium; instead, primitive mesenchymal cells are found. Furthermore, we never detected any spindle-shaped cell in the central part of the testis interstitium (i.e., mesenchymal cells other than peritubular type) immunolabeled for the three steroidogenic enzymes tested at any developing age in this study. The detection of P450scc in immature Sertoli cells is in agreement with the fact that these cells are steroidogenically active to some extent at early postnatal ages.
In the present study, vascular entothelial cells positive for 3ß-HSD in the developing testis interstitium were not detected at any age. Although Haider and Servo [31] detected a few 3ß-HSD-positive vascular endothelial cells, which were found only adjacent to some heavily stained Leydig cells in the developing rat testes, they attributed this observation to a paracrine effect of these Leydig cells on the vascular endothelial cells rather than to vascular endothelial cells being potential precursors to Leydig cells. Therefore, it may be possible to exclude myoid cells, pericytes, vascular endothelial cells, and mesenchymal cells in the central interstitium as potential precursors to Leydig cells at the onset of Leydig cell differentiation in the prepubertal rat testis.
The change in shape from elongated to a rounder configuration among the Leydig cell precursors during testicular development is expected after acquiring the steroidogenic potential (i.e., latter stages of the Leydig cell lineage) and indicates of a more advanced stage of differentiation. Therefore, the few rounder cells that were positive for the steroidogenic enzymes found at the peritubular region as well a short distance away from the peritubular region could be considered as being those cells that were initiated earlier, at postnatal Day 11, compared with those that were still elongated in shape but were positive for the steroidogenic enzymes. If the few rounder cells that were steroidogenically positive and found partially away from the peritubular region originated from the mesenchymal cells in the central interstitium, we should have detected elongated cells that were positive for the steroidogenic enzymes in the central interstitium at postnatal Day 10 or earlier, but we did not. These observations exclude the possibility that these steroidogenically positive rounder cells, which were partially away from the peritubular region, originated from the mesenchymal cells in the central interstitial region. Instead, our findings agree with the observation of Mancini et al. [36] regarding differentiation of Leydig cells in the human cryptorchid testis. Those investigators reported that interstitial cells, suggested to be Leydig cell precursors, were first noted in the peritubular region and subsequently left the lamina propria to gain access to the surrounding interstitial tissue. Similar findings have been observed in the adult rat testes after transient neonatal hypothyroidism [17]. From this supportive information and our present findings, we conclude that the peritubular mesenchymal cells are the stem cells of Leydig cells in the adult rat testis. In contrast, Hardy et al. [11] reported that the fusiform cells in the central region of the interstitial space are the precursors for the adult Leydig cells in the rat testis. Our findings are not in agreement with this statement.
The observation that the mesenchymal cells acquire the steroidogenic potential before gaining LH receptors is an important discovery, because this reveals that the initiation of mesenchymal cells to begin Leydig cell differentiation in the prepubertal rat testis does not depend on LH. Moreover, other elongated, spindle-shaped cells in the testis interstitium, such as the endothelial cells, pericytes, and myoid cells, did not show positive labeling for LH receptors at any age tested. Therefore, the present findings do not support the report of Misrahi et al. [37] that vascular endothelial cells express LH receptors. It is also interesting to note that when progenitor Leydig cells acquire LH receptors, they are located partially away from the peritubular region of the testis interstitium, in contrast to their original location at the peritubular region. In addition, they are definitely rounder in shape, in contrast to their original spindle-shape configuration. These findings suggest that in cells committed to the Leydig cell lineage, acquisition of LH receptors occurs during a stage of development later than that at which they first gain the steroidogenic enzymes. Accordingly, these findings suggest that the function of LH in Leydig cell differentiation occurs subsequent to formation of the progenitor cells (i.e., transformation of progenitors to immature and then mature Leydig cells). Other experimental evidence also supports this view. In prepubertal rats [15] and in adult rats after transient neonatal hypothyroidism [38], Leydig cells differentiate [17–19] under very low levels of circulating LH [16], but they do not attain their normal size (i.e., remain as small cells) [17–19], which most probably results from the deficiency of LH in these rats [16]. It has also been reported that during Leydig cell regeneration in the ethane-dimethane-sulphonate (EDS)-treated adult rats, precursor cell proliferation and transformation into progenitor cells occurs without LH, as demonstrated by hypophysectomy or testosterone implants before EDS treatment [39]. Moreover, in fetal rats [2, 40] and mouse [41] testis, Leydig cell differentiation has been observed to be independent of LH.
Demonstration of 3ß-HSD, P450scc, and P450c17 simultaneously in the same progenitor cell reveals that these cells can synthesize androgens using cholesterol as the substrate. Although we have not shown the fourth enzyme in the steroidogenic pathway, 17-ketosteroid reductase (17KSR, also known as 17ß-hydroxy steroid dehydrogenase), the activity of this enzyme is reported to be very low in Leydig cell progenitors [42]. This implies that steroidogenesis in Leydig cell progenitors can proceed only to the formation of androstenedione, and that little or no testosterone is produced by these cells. This is in agreement with the finding that rat testis is capable of increased secretion of androstenedione during this stage of development [43, 44].
In the present study, we have demonstrated that initiation of Leydig cell differentiation in the prepubertal rat testis is independent of LH and similar to what occurs in the prenatal rat [2] and mouse [42] testes. This finding is in contrast to the view that LH stimulates mesenchymal cell differentiation into progenitor cells in the postnatal rat testis [12–14]. Therefore, it is important to investigate what triggers the peritubular mesenchymal cells to initiate Leydig cell differentiation in the postnatal rat testis. Among other factors suggested as being possible candidates, accumulating evidence suggests that the thyroid hormone has an important role in the initiation of precursor cell differentiation in Leydig cell development, because hypothyroidism inhibits [4, 44] and hyperthyroidism advances [44] the differentiation of adult Leydig cell population in the prepubertal rat testis. Therefore, to further understand this phenomenon, it is appropriate to extend these studies to test the effects of thyroid hormone on the acquisition of LH receptors and steroidogenic enzymes among mesenchymal cells in the prepubertal rat testis.
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NOTE ADDED IN PROOF |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 Science, College of Veterinary Medicine, University of Tennessee, 2407 River Drive, Knoxville, TN 37996. FAX: 423 974 2215; mendisc{at}utk.edu
Accepted: February 24, 2000.
Received: December 2, 1999.
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H.B. Siril Ariyaratne, J. Ian Mason, and S.M.L. C. Mendis-Handagama Effects of Thyroid and Luteinizing Hormones on the Onset of Precursor Cell Differentiation into Leydig Progenitor Cells in the Prepubertal Rat Testis Biol Reprod, September 1, 2000; 63(3): 898 - 904. [Abstract] [Full Text]
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