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a Department of Animal Science, College of Veterinary Medicine, The University of Tennessee, Knoxville, Tennessee 37996
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fetal Leydig cells (FLC) and adult Leydig cells (ALC) are two distinct populations. However, in previous quantitative studies except ours [4, 8], differential quantification of FLC and ALC was not attempted [1–3, 5, 7]; and even in our investigations, data for the period between the ages of 21 and 90 days are lacking.
It is known that FLC are present in the rat testis at birth [1–4]. In many species such as the rat [1, 2] and human [9], the fate of the FLC in the postnatal testis is almost universally accepted to be degeneration. However, studies from our laboratory have revealed that FLC numbers do not decline in the postnatal rat testis up to the third postnatal week [4, 8], and cells morphologically similar to FLC have been identified in testes of Sprague Dawley rats throughout development up to sexual maturity (90 days) by Kerr and Knell [6]. Therefore, to ascertain the fate of FLC in the postnatal rat testis, unequivocal identification of this cell type is crucial.
ALC differentiate from their precursor cells during the second week of postnatal age in the rat [4, 8]. It is generally accepted that undifferentiated fibroblast-like cells (referred to as mesenchymal cells in the present study) in the testicular interstitium are the source of ALC [1, 2, 4]. Previously, it was reported that a simple transformation of mesenchymal cells into Leydig cells occurs between Day 14 and Day 28; subsequently one division of these Leydig cells replenishes the population of ALC [7]. However, data are now accumulating to suggest that the kinetics of Leydig cell differentiation is more complex than previously described [10].
To our knowledge, detailed quantitative information on all cell types in the testis interstitium combined with data on changes in the steroidogenic function of the testis from birth to sexual maturity is not available in the rat or any other mammalian species. In addition, the fate and significance of FLC in the postnatal rat testis are unclear at present. Finally, it is important to establish the relationships between Leydig cells and other interstitial cell types in the testis interstitium on the basis of absolute numbers of cells instead of percentages of cell numbers in order to understand the kinetics of Leydig cell differentiation; this has not been demonstrated previously. Therefore, we designed the present study to investigate in detail the changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Sprague Dawley rats in mid pregnancy (to obtain one-day-old and 7-day-old male pups), mothers with a litter (to obtain 14- and 21-day-old males), and male rats of 28, 40, 60, and 90 days of age were purchased from Harlan Industries (Madison, WI). These animals were housed individually in cages in the animal facility of The University of Tennessee College of Veterinary Medicine (Knoxville, TN), with controlled temperature and lighting (14L:10D). Animals had access to rat chow (Agway Prolab, Syracuse, NY) and water ad libitum.
Fixation and Processing of Testicular Tissue
Eight groups of rats of the following ages were used (n = 5 per group): 1, 7, 14, 21, 28, 40, 60, and 90 days of age. Both testes from 1- and 7-day-old rats were fixed by immersing them in a solution of 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Testes from other age groups were fixed by the whole-body perfusion technique as described previously [11]. Briefly, the rats received i.p. injections of heparin (heparin solution for injection, USP, 10 000 U/ml; Steris Laboratory Inc., Phoenix, AZ) at a dose of 10 U/g BW, 15–20 min before deep-inhalation anesthesia (Metofane; Mallincroft Veterinary Inc., Mundelein, IL). One testis from each animal was removed, freed from epididymis, and weighed (fresh testicular weight). The specific gravity of each of these testes was determined by flotation in a series of sucrose solutions of known specific gravity as described previously [12, 13]. Then the testis was immersion-fixed in Bouin's fixative for 4 h and stored in 70% ethyl alcohol until it was embedded in Paraplast for further processing (Oxford Labware, St. Louis, MO). The ipsilateral testis was fixed in situ by vascular perfusion with 2.5% glutaraldehyde solution in 0.1 M cacodylate buffer (pH 7.4). This was accomplished by introducing a cannula into the ascending aorta through a slit made on the left ventricular wall and allowing the fixative to flow under gravity. Initially, a solution of 0.9% NaCl was perfused through the testis for about 30 sec in order to remove blood from the testicular vessels. When the testis was cleared of blood, perfusion of the fixative was begun. After completion of testis fixation within 45–60 min, the fixed testis was weighed (fixed weight), cut into approximately 1-mm cubes, postfixed in a 1:1 mixture of 2% aqueous osmium tetroxide and 3% potassium ferrocyanide, dehydrated in graded alcohol, and embedded in epon araldite. Shrinkage was measured as described previously by Mendis-Handagama and Ewing [13] using whole testes in 1- and 7-day-old rats and fixed testis tissue blocks (approximately 2 x 3 mm in size, 10 blocks per rat) in all other age groups of rats.
Light Microscopic Histology
From polymerized blocks, 1-µm-thick sections were cut using an LKB ultramicrotome V microtome (Pharmacia LKB, Piscataway, NJ) and glass knives. Sections were picked up onto precleaned glass slides, stained with a solution of methylene blue azure II [14], and coverslipped under Permount (Fisher Scientific Company, Fair Lawn, NJ). Various components of testicular parenchyma and different cell types in the interstitium were identified under an Olympus BH-2 laboratory microscope (Tokyo, Japan) according to the method of Mendis-Handagama et al. [4]. FLC and ALC in 1- to 21-day testes were easily distinguished by their morphological characteristics. FLC had an abundance of cytoplasmic lipid. The newly formed ALC (Days 14 and 21) had little cytoplasm, which stained darker than that of mesenchymal cells, but they contained a prominent round nucleus with a very fine rim of heterochromatin. These newly formed ALC in 14- and 21-day-old testes had little or no cytoplasmic lipid droplets. ALC from 28- and 40-day-old rats (immature ALC) had abundant cytoplasmic lipid. The size of individual lipid droplets as well as the number of lipid droplets per cell appeared to be smaller than those of FLC. By contrast, the majority of Leydig cells at Days 60 and 90 contained little or no cytoplasmic lipid in them. FLC clusters in testis of 28- to 90-day-old rats were also surrounded by basement membrane components similar to those of 1- to 21-day FLC. (These were confirmed as FLC by immunocytochemistry for 11ß hydroxysteroid dehydrogenase 1 [11ß-HSD1].) The cytoplasm of macrophages was granular, often contained vacuoles of variable sizes, and stained much lighter than in Leydig cells. The cells touching the seminiferous cords were regarded as myoid cells. Other spindle-shaped cells with lentiform nuclei and little cytoplasm, which were scattered in the interstitial tissue and also formed a concentric arrangement around the seminiferous cords, were identified as mesenchymal cells.
Endothelial cells lined the blood vessels and the lymphatic spaces. Pericytes were observed as crescent-shaped profiles closely associated with blood vessels at all ages. They were easily distinguished from other types of interstitial cells by their location and shape. Each cell contained an intensely stained nucleus surrounded by a thin area of cytoplasm.
Light Microscopic Stereology
Volume of components The volume density of testicular components (defined as the volume of a component per unit volume of testis tissue) was obtained by the point-counting method [15] using an ocular grid with 121 test points fitted to a x10 ocular lens of the microscope. The objective lens used was x40. Four corners of each section were scored (n = 20 blocks/group for Day 1; n = 40 blocks/group for Day 7; n = 50 blocks/group for all other age groups). The absolute volume (mm3) occupied by each testicular component was calculated by multiplying the volume density of each component by the volume (mm3) of the fresh testis; the volume of fresh testis (mm3) was obtained by multiplying the fresh testis weight (g) by density (density = specific gravity in metric units) of the testis [4, 8, 12, 13]. To obtain differential values for the absolute volumes (mm3) of fetal and adult Leydig cells per testis at 28, 40, 60, and 90 days of age, the total Leydig cell volume (mm3) per testis was obtained by multiplying the testis volume (mm3) by the volume density of total Leydig cells. The absolute volumes (mm3) of fetal Leydig cells per testis at 28, 40, 60, and 90 days of age were determined by multiplying the number of fetal Leydig cells per testis by the average volume (µm3) of a fetal Leydig cell. The absolute volume (mm3) of fetal Leydig cells per testis at each of these ages was subtracted from the total Leydig cell volume (mm3) per testis at each age to obtain the absolute volume (mm3) of adult Leydig cells per testis at each age.
Cell number The numerical density (defined as the number of cells per unit volume of the testis tissue, Nv) of each interstitial cell type (Leydig cells, mesenchymal cells, myoid cells, endothelial cells, pericytes, and macrophages) was quantified by the disector method [16] with modifications to the method described by Mendis-Handagama and Ewing [13]. Two 1-µm tissue sections ("reference" section and "look-up" section) that were four sections apart (first and fifth section) were cut from each tissue block and mounted on a glass slide adjacent to each other, stained with methylene blue azure II [14], and coverslipped. The reference and the look-up sections were viewed side by side on two color video monitors. First, an area of the first tissue section (reference section) was viewed by the microscope; this image was captured on the color screen/monitor by a camera adapter (CMA-D7; Sony Corporation, Tokyo, Japan) and a color video camera (DXC-107A; Sony Corporation), and was recorded onto high-quality videotape using a video recorder (PV-7401; Panasonic, Tokyo, Japan). This image was frozen, and the image of the corresponding area of the second tissue section (look-up section) was brought similarly onto the second color monitor so that the two areas could be viewed side by side. For each cell type, the number of unique nuclear profiles (those appearing only in the first area but not in the second) was determined according to the unbiased counting rule of Sterio ([16]; the nuclear profiles touching the upper or left margin of the panel were excluded). Twenty to 30 areas per block and 10 blocks per animal were scored for age groups 14 days and above. For 1- and 7-day groups, 4 and 8 blocks per rat, respectively, were used. The area of the tissue section representing the image on the video monitor was determined by the product of the length and the width of the test area, which were measured by using a slide micrometer. Using the formula Nv = (Q/A x T)(1 - St), Nv was determined, where Q = number of unique nuclear profiles in the test area, A x T = volume of the dissector, and St = total shrinkage of testicular tissue from fresh unfixed state to the final embedded stage. St was determined by use of the equation St = S1 + S2(1 - S1), as published by Mendis-Handagama and Ewing [13]. The total number of each cell type per testis was calculated by multiplying the numerical density of the cell type and testis volume [11].
When Leydig cells were quantified, they were subdivided into FLC and ALC. In 1- to 21-day-old rats, procedures described above were used for this purpose. The nucleator method [17] was used to determine the average cell volume of an FLC in 28- to 90-day-old rats. This procedure is described in the next section (Average Cell Volume). To quantify the FLC number per testis in 28- to 90-day-old rats, we used a modified technique described below. These alternative procedures were necessary to overcome the limitations of reduced sample size for these cells.
First, we quantified the total Leydig cell number per testis (i.e., FLC + ALC) by the dissector method, using the epon-araldite sections stained with methylene blue azure II stain. To subdivide this measurement into FLC and ALC, we performed immunocytochemistry for 11ß-HSD1 in testes of 28- to 90-day-old testes using the sections of Paraplast-embedded tissue as described below. 11ß-HSD1 is a marker for ALC, especially after postnatal Day 26 [18]. This antibody was prepared by the late Dr. Carl Monder [18] in rabbits against rat 11ß-HSD1 antigen and has been used for specific immunolocalization of 11ß-HSD antigen in previous studies [8, 18]. It has been used to differentially identify FLC and ALC in the postnatal rat testis in previous studies [8, 18]. The number ratio of FLC:ALC was determined by the number ratio of 11ß-HSD1-negative:11ß-HSD1-positive cells (f:a) in these testis tissues. Using this ratio (f:a) and the total number of Leydig cells per testis (n) obtained by the dissector method, the numbers of FLC and ALC per testis in these older rats were determined as shown below.
Average cell volume
Except for FLC in 28- to 90-day rats, the average volume of an individual cell was determined by dividing the volume density by the numerical density of each cell type. To determine the average volume of an FLC in 28-, 40-, 60-, and 90-day-old rats, we used the nucleator method [17]. Clusters of FLC were identified in the methylene blue-stained sections of tissue of these age groups using an Olympus BH-2 microscope (Tokyo, Japan) fitted with a color video camera (DXC-107A; Sony Corporation), and the image was brought onto a color video monitor that had a transparent test overlay containing a lattice grid. The objective lens used was x40. Each intersection of the test grid that was on an FLC was taken as a "fixed point," and the distance from the fixed point to the cell boundary in a predetermined direction (l) was measured along the grid line. A slide micrometer was used to determine the length of l in actual units. The average volume (v) of FLC was calculated by the equation v = 4
/3 · -ln [17], where -ln is the average distance from the fixed points to the cell margins.
11ß-HSD1 Immunohistochemistry
Testes of 28- to 90-day-old rats fixed in Bouin's fixative were used to mark ALC with 11ß-HSD1. Fixed testes were decolorized by repeated changes in 70% alcohol over several days, dehydrated in graded alcohol, and embedded in Paraplast. Sections (5-µm thick) were cut with a Leitz rotary microtome (Model 1512; Ernst Leitz, Ontario, Canada) and mounted on Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Deparaffinized and rehydrated sections were incubated with a solution of 3% H2O2 in 100% methyl alcohol for 20 min at room temperature in order to block endogenous peroxidase activity. Then the sections were protein-blocked by incubation in a solution of 10% normal goat serum, 1% BSA, and 0.05% Tween 20 in PBS (pH 7.6) for 6 h at room temperature. Test and control tissue sections were incubated overnight at 4°C in primary antibody or in normal rabbit serum, respectively, diluted in protein block solution. Dilutions ranging from 1:200 to 1:800 were used for the primary antibody. The bound antibody was detected by the peroxidase-antiperoxidase method employing diaminobenzidine tetrahydro-chloride (DAB; BioGenex, San Ramon, CA) as the chromogen according to the manufacturer's instructions. The sections were counterstained with Mayer's hematoxylin, dehydrated in graded alcohol, and coverslipped under Permount (Fisher Scientific Company, Fair Lawn, NJ).
The sections were viewed under an Olympus BH-2 microscope. The ratios of FLC:ALC in 28-, 40-, 60-, and 90-day rat testes were determined by systematically testing entire sections for 11ß-HSD1 negative : positive cells.
Incubation of Testis Tissue In Vitro
Rats of all age groups (n = 8 rats per group, experiment repeated 3 times) were killed by inhalation of CO2 gas. The testes were removed and weighed. One testis from each rat was decapsulated and incubated in basal medium (2 ml Krebs-Ringer bicarbonate solution pH 7.4 supplemented with 0.004 g/ml glucose as previously described [8, 19]). The other testis was incubated in a similar medium containing a maximum stimulatory dose of LH (ovine LH 100 ng/ml) [8, 19–21]. LH was obtained from the NIDDK Hormone Distribution Program, Torrance, CA. Before addition of glucose, the incubation medium was bubbled with air for 10 min as described previously [8]. Incubations were performed in 20-ml scintillation vials at 34°C in an oscillating water bath (90 oscillations/min). At the end of 3 h, the incubation medium was collected and centrifuged at 3000 x g for 10 min, and the supernatant was separated and stored at -70°C until further analysis.
RIA for Testosterone and Androstenedione
Testosterone and androstenedione concentrations in the basic medium and the stimulatory medium were quantified by using commercially available RIA kits (Coat-A-Count; DPC, Los Angeles, CA). The sensitivity of these assays was 0.14 nM for both hormones. The intraassay coefficients of variation for both assays were less than 9%. The cross-reactivity of the antibody used in the testosterone RIA kit was 2.8% for dehydrotestosterone, 0.5% for androstenedione, and less than 0.02% for other steroids. The cross-reactivity of antibody employed in the androstenedione RIA kit was 1.5% for testosterone, 0.21% for dehydrotestosterone, and 0.14% for dehydroepiandrosterone. Samples from all rats (8 from each group) were included in each assay. This part of the experiment was repeated three times to assure reproducibility.
Statistical Analysis
The mean values of various parameters from different age groups were compared using General Linear Models of the Statistical Analysis Systems (SAS) program (SAS Corporation, 1998). When a significant difference was observed among groups, Duncan's New Multiple-Range Test was employed to separate the means. P values of 0.05 or lower were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The average testis weights and body weights in rats increased concomitantly from birth to 90 days of age (Table 1). In testis of 1-day old-rats, the seminiferous cords and the interstitium were quite distinct, and each seminiferous cord was surrounded by several layers of concentrically arranged spindle-shaped cells (Fig. 1a). With advancing age, the number of concentric layers of mesenchymal cells surrounding the seminiferous cords/tubules gradually declined.
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FLC were easily identified in neonatal testes of 1–21 days by their morphological characteristics (Fig. 1). Macrophages were seen dispersed in the interstitial tissue. ALC were first observed at 14 days in small numbers (Fig. 1b) but were abundant from Day 21 onwards.
11ß-HSD1 Immunohistochemistry
In testes of 28-, 40-, 60-, and 90-day-old rats, 11ß-HSD1-positive and -negative Leydig cells were evident in the testis interstitium and were identified as ALC and FLC, respectively, on the basis of the findings of Phillips et al. [18]. Unlabeled FLC in clusters were readily recognized as FLC (Fig. 2, a–d).
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Morphometry
Volume density The volume density of testicular components of postnatal rats from birth to sexual maturity (i.e., 90 days of age) is given in Table 1 and will not be discussed further. Table 1 also shows testis weights and body weights of rats from birth to 90 days of age.
Absolute volume The absolute volumes of testicular components of postnatal rats from birth to sexual maturity (i.e., 90 days of age) are given in Table 2. The absolute volume of the seminiferous cords/tubules per testis increased gradually but significantly with age. This increase was evident in both the cellular and the luminal components. In parallel with the volume changes in seminiferous cords/tubules, a significant increase in the absolute volume of the testicular interstitium was also detected. The absolute volume of the mesenchymal cells per testis did not change significantly from Day 1 to Day 14 but increased significantly from Day 14 to Day 28 and from Day 40 to Day 90. The value at Day 40 was similar to that at Day 21 but significantly lower than those at Days 28, 60, and 90. The absolute volume of macrophages, endothelial cells, pericytes, and myoid cells per testis increased gradually with age; blood vessels and lymphatic spaces followed a similar pattern. The absolute volumes of FLC per testis were not significantly different at 1, 7, 14, 28, 40, 60, and 90 days; however, a significant reduction was observed at Day 21. The absolute volume of ALC was lowest at Day 14 but significantly increased at Days 21, 28, 40, 60, and 90. The ratios of absolute volume per testis of macrophages to adult Leydig cells (macrophage : Leydig cells) in 14-, 21-, 28-, 40-, 60-, and 90-day testes were estimated as 1:2.6, 1:10.8, 1:8.6, 1:2.4, 1:3.3, and 1:3.2, respectively.
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Cell number The number of FLC per testis did not change from 1 to 90 days of age; by contrast, the number of mesenchymal cells, ALC, macrophages, endothelial cells, pericytes, and myoid cells per testis increased gradually with age (Table 3). In addition, as shown in Figure 3A, the increases in mesenchymal and ALC number per testis were linear, although the slopes (i.e., mmes and mALC in the formula Y = mX + c, where Y = cell number, m = slope, X = time, and c = intercept of plot for each cell type) of these graphs were different. The slopes give the rate of change in numbers of each cell type per unit time. mALC = 27.7 x 106, mmes = 15 x 106, mALC/mmes = 27.7 x 106/15 x 106. The ratio mALC:mmes is the comparison of the production rates of ALC and mesenchymal cells. mALC:mmes is approximately 2. This result indicates that the number of ALC increases to twice the number of mesenchymal cells with advancing age.
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Figure 3B shows numbers of different interstitial cell types as a percentage of total interstitial number per testis. The number percentage of mesenchymal cells decreased almost linearly from Day 14 to Day 40 and remained unchanged thereafter. In contrast, the number percentage of ALC increased linearly from Day 14 to Day 40 and remained unchanged thereafter. The number percentages of myoid cells followed a pattern similar to that of mesenchymal cells. The number percentage of endothelial cells, macrophages, and pericytes remained low and unchanged with advancing age. A mirror-image-like relationship was seen between number percentages of mesenchymal cells and ALC and also between myoid cells and ALC.
The number ratios of macrophages to ALC (macrophage : ALC) in 14-, 21-, 28-, 40-, 60-, and 90-day testes were estimated as 1:3.8, 1:10.6, 1:9.1, 1:6.7, 1:6.8, and 1:6.4, respectively.
Average volume of mesenchymal cells, FLCs, ALCs, and macrophages The average volume of different interstitial cell types is given in Table 4. The average volume of a mesenchymal cell decreased significantly from Day 1 to Day 14, increased gradually up to Day 28, and declined significantly thereafter; the highest value was at Day 28, and the lowest values were detected at Days 14 and 90. The average volume of FLC did not change significantly from birth to Day 14, but a significant reduction was observed at Day 21. The average volume of ALC on Days 14 and 21 was not significantly different; a gradual and continued increase was observed thereafter, reaching a 3-fold increase at 60 and 90 days. No significant change was observed in the average volume of a macrophage from birth to 90 days of age.
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Testicular Testosterone and Androstenedione Production In Vitro
Figure 4A shows LH-stimulated testosterone production per testis in vitro. Total testosterone production per testis in vitro was not significantly different at Day 1 compared to Days 7, 14, 21, and 28. Significant increases were observed at Days 40 and 60. Values at Days 60 and 90 were not significantly different. Testosterone production per testis in vitro at the basal level (no LH) followed the same trend, with considerably lower numerical values at each age (results not shown).
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Figure 4B shows LH-stimulated androstenedione production per testis in vitro. Total androstenedione production per testis in vitro gradually increased with age from birth to 90 days. Androstenedione production per testis in vitro at the basal level (no LH) followed the same trend, with considerably lower numerical values at each age (results not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The significance of the reduction in the average volume of an FLC on Day 21 was not addressed in the present study and is still not clear at present. This observation agrees with our previous findings [4, 8], but other studies are not available for comparison with this observation. Low levels of circulating LH at this age [23] may be a factor contributing to the hypotrophy of FLC at Day 21. In addition, hypothetically it is possible that the factors that stimulate transformation of mesenchymal cells into ALC may cause transient hypotrophy of FLC. This view could be supported by several previously published observations. It was observed that hypotrophy of FLC is inhibited with the arrest of mesenchymal cell differentiation into ALC in hypothyroid neonatal rats [8]. Moreover, when the FLC population was extended by hCG administration to the neonatal rats, the differentiation of ALC was delayed [24]. By contrast, premature removal of FLC by ethane dimethane sulphonate treatment can induce an early proliferation and differentiation of mesenchymal into Leydig cells [25]. These observations suggest a possible reciprocal inhibitory relationship between FLC and differentiating ALC.
The significance of the presence of FLC in the postnatal rat testis throughout development is also an issue worthy to be addressed. Although we do not know the exact answer, the results of the present study disclose some important aspects of FLC in the postnatal rat testis. Our study confirms our previous observations [8] that FLC are the only steroid secretory cell type in the testis interstitium from Days 1 to Day 7 and the primary source of testosterone and androstenedione. Except for the reduced cell size on Day 21, FLC do not show any regressive changes beyond Day 7. Moreover, as newly formed ALC mainly secrete androstenedione and 5
-reduced androgens because of their lack or reduced levels of 17 ketosteroid reductase enzyme and the presence of high levels of testosterone-metabolizing enzymes such as 5-reductase [26–28], it is logical to suggest that FLC have a contribution to the testicular testosterone secretion beyond postnatal Day 7 through Day 35 (ALC begin to secrete significant amounts of testosterone only after Day 35 [23, 29]). On the basis of such information, it appears that all important functions that require testosterone during the neonatal-prepubertal period, such as the activation of the hypothalamo-hypophyseal-testicular axis (reviewed in [9]) are heavily dependent on FLC. It is also suggested that this activation is essential for the completion of the testicular descent, masculinization of the brain, control of Sertoli cell number, initiation of spermatogenesis, and sexual behavior [9]. This view is also supported by the observation that the inhibition of the neonatal rise in testosterone is known to delay puberty in male marmoset monkeys [30].
Several previously published reports have shown that testosterone-producing capacity per FLC at neonatal ages is significantly greater than that of ALC at Day 90 [3, 31]. From the results of the present study, LH-stimulated testosterone secretory capacity per FLC in vitro at postnatal Days 1 and 7 is calculated to be 70 and 87 pg per cell, respectively (total testosterone secretion per testis ÷ number of FLC per testis). These values are very much greater than those obtained previously with isolated rat Leydig cells of 90 days of age (0.18 pg [32] and 0.1 [33] pg per Leydig cell). This difference in the steroidogenic capacities between FLC and ALC is thought to be due to the absence of many negative influences of hormonal and paracrine factors on FLC in contrast to ALC [3, 31]. If no contribution from the FLC at Day 90 is assumed, LH-stimulated testosterone secretory capacity per ALC in situ obtained by the testicular incubation method (calculated by total testosterone secretion per testis ÷ number of ALC per testis at Day 90) is estimated as 1.43 pg per ALC. This value is about 8- to 14-fold greater than those values published previously with isolated rat Leydig cells of 90-day-old rats [32, 33]. This finding can be explained by one of the following suggestions: the isolated Leydig cells may not represent the population of Leydig cells in the intact testis, or the FLC are still functionally active at 90 days of age, and therefore there is an additional contribution from the FLC for this testicular function.
Emergence of the ALC population in the rat testis has been shown to be as early as 10 days after birth [4]. In the present study and in a previous study published recently [8], we detected adult Leydig cells from postnatal Day 14 onwards. They significantly increased in number and volume per testis at Day 21 [4, 8]. The present investigation extended beyond Day 21 up to 90 days of age and showed clearly that changes occur in this cell population in number and average cell volume. To our knowledge, the present study is the first to document such changes in adult Leydig cells from the time of emergence in the postnatal testis up to sexual maturity in a mammalian species in general, and the rat in particular.
It is known that macrophages and Leydig cells have a functional relationship [34]. Although macrophages are thought to stimulate Leydig cell differentiation in the prepubertal testes [35], it is reported that they exert an inhibitory effect on Leydig cell steroidogenesis [34]. These findings suggest that the role of macrophages varies in postnatal rat testes at different stages of postnatal life. In the present study, we observed that in testes of 14-, 40-, 60-, and 90-day rats, 1 macrophage was associated with 3 Leydig cells; and in testes of 21- and 28-day rats, each macrophage was associated with 9–11 Leydig cells. The ratio obtained in the present study for a 90-day-old rat compares favorably with what we have published previously [11]; however, there are no other reports with which we can compare our present findings on all other ages. The volume ratios of macrophage : Leydig cells of the present study also showed that at Days 21 and 28 the macrophage : Leydig cell ratio is widened. Because macrophage-Leydig cell interactions are complex [34], at this juncture it is difficult to explain the observations of the present investigation.
Testosterone secretory capacity per testis was not different from Day 1 to Day 14 and can be explained by the absence of change in the absolute volume of FLC per testis from birth to 14 days of age. In spite of a significant reduction in the absolute volume of FLC per testis at Day 21, testosterone production per testis was unchanged, which can be explained by the contribution by the ALC population, as has been seen before [8]. With ages beyond Day 21 (i.e., 28, 40, 60, and 90 days), continued hypertrophy and hyperplasia of ALC explain the increase in testosterone production per testis in vitro. However, despite the increase in the ALC volume per testis from Day 21 to Day 40, levels of androstenedione production per testis of 21-, 28-, and 40-day old-rats was maintained at a constant level. This information suggests a decline in androstenedione secretory capacity per ALC when age advances from 21 days to 40 days. Therefore, it appears that the increased androstenedione-producing capacities of 60- and 90-day testes are most probably due to the increase in ALC number and not to an increase in the capacity per ALC with age. This is not surprising because newly formed adult Leydig cells change into primarily testosterone-secreting cells with the progression of development.
It is accepted that elongated spindle-shaped cells in the testis interstitium are the precursors for Leydig cells in the postnatal rat testis [2, 4, 36–38]. However, there are many types of these cells, namely, peritubular myoid cells, fibroblasts (identified as mesenchymal cells in the present study), endothelial cells, and pericytes. Differences in opinion exist on which spindle-shaped cell type(s) is the precursors to Leydig cells [7, 38]. The present study demonstrates that the numbers of all spindle-shaped cell types in the rat testis interstitium continuously increased with age. These observations are reasonable because increases in numbers and volumes of spindle-shaped cells are necessary to accommodate the increase in the testis volume and expansion of the testis interstitium with advancing age. The changes in numbers of elongated spindle-shaped cells in the testis interstitium with age agree with the findings of Hardy et al. [7] for many cell types except for mesenchymal cells. In contrast to our present findings on continuous increase in mesenchymal cell number per testis from birth to 90 days, Hardy et al. [7] reported a 50% reduction in the mesenchymal cell number per testis from Day 28 to Day 56. We cannot explain this discrepancy. However, it is difficult for us to comprehend how a 485-mm3 testis [7] could have a 2-fold increase in the number of mesenchymal cells compared to a 1225-mm3 testis [7]. Moreover, on the basis of percentages of Leydig and mesenchymal cell numbers, which are relative measures, Hardy et al. [7] proposed that mesenchymal cell numbers decrease and Leydig cell numbers increase during testicular development [7]. By contrast, on the basis of the absolute cell numbers per testis, the present study demonstrates that both mesenchymal and Leydig cells increase linearly with age. The slopes of these graphs further revealed that the rate of increase in cell numbers from birth to 90 days is 2-fold greater in Leydig cells compared to mesenchymal cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was supported by Grants R180101 (Center of Excellence, The University of Tennessee), IBN-94-0928 (National Science Foundation), and Minkel (The University of Tennessee). 
2 Correspondence: S.M.L.C. Mendis-Handagama, Department of Animal Science, College of Veterinary Medicine, The University of Tennessee, 2407 River Drive, Knoxville, TN 37996. FAX: 423 974 2215; mendisc{at}utk.edu
Accepted: October 21, 1999.
Received: August 9, 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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