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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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-dihydrotestosterone are vital for normal postnatal development and functioning of the male reproductive system. Moreover, testosterone aromatized to estrogen is critical for imprinting of the central nervous system during development so that the male pattern of gonadotropin secretion and masculine sexual behavior occur in the adult life [7]. Therefore, it is clear that the presence of fetal Leydig cells at birth and the early neonatal period is a requirement for the proper postnatal development of the male reproductive function. However, it has been shown that when the adult population of Leydig cells appears in significant numbers in the third postnatal week in the rat testis, fetal Leydig cells show cell atrophy [4]. Although the significance of this change in fetal Leydig cells is still not known, these observations suggest that the emergence of the adult Leydig cell population and the cell atrophy in the fetal Leydig cell population occur at the same time. Whether these events are interdependent remains to be determined. More importantly, what triggers these events in the neonatal rat testis is still poorly understood. Establishing the adult Leydig cell population is of utmost importance to the mammalian male for many functions associated with reproduction. It has been suggested that the adult population of Leydig cells differentiates from mesenchymal cell precursors in the neonatal rat testis [2–4, 8]. However, the primary stimulus that initiates this differentiation process is still unknown. Although LH, FSH, and testosterone have been suggested as possible candidates in triggering this differentiation process [9–12], the following observations suggest that none of these could be the primary trigger in this process. First, serum testosterone and LH levels have been observed to be at the lowest levels around Day 10 [13, 14], when adult Leydig cells first appear in the neonatal rat testis [4]. Second, Leydig cells still differentiate under conditions of suppressed LH, FSH, and testosterone levels, i.e., after transient neonatal hypothyroidism [15–17].
Previous studies have shown that transient neonatal thyroid hormone deficiency results in an increase in the number of Leydig cells per testis at adulthood [15–17]. This finding suggests that thyroid hormone has a regulatory role on cell populations of the testis interstitium. However, the mechanism underlying the 2-fold increase in Leydig cell numbers in transiently hypothyroid rats during the neonatal period is still uncertain. Leydig cell numbers could increase as a result of proliferation of the precursor cells and/or of the existing Leydig cells. To address this issue, details of the changes in the interstitial cell populations under hypothyroid conditions during the neonatal period are crucial. Therefore, the present study was designed to examine the regulatory effects of thyroid hormones on the testicular interstitial cell populations (namely, fetal and adult Leydig cells, connective tissue/mesenchymal cells) and the steroidogenic capacity of the neonatal Sprague Dawley rat.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovine LH-25 was a gift from the National Hormone and Pituitary Program, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases (Rockville, MD).
Testicular Tissue Preparation for Light Microscopic Morphometry
Both testes of each control and PTU rat pup of 1 and 7 days (n = 5 rats per group) were removed under Metafane (Mallincroft Veterinary Inc., Mundelein, IL) inhalation anesthesia and weighed. The specific gravity of the fresh testis was determined by the flotation technique [18,19], and the fresh testis volume (V1) was calculated. This step was followed by fixing these testes by immersion in 2.5% glutaraldehyde in 1 M cacodylate buffer. In 14- and 21-day-old rats (n = 5 rats per group), one testis was removed under inhalation anesthesia (Metafane, Mallincroft), and the fresh testis volume was determined as described above. The other testis of each rat was fixed by whole-body perfusion followed by immersion fixation, with 2.5% glutaraldehyde in 0.1 M cacodylate buffer. Fixed testes of 1- and 7-day rats were processed without cutting into blocks. In 14- and 21-day-old rats, fixed testes were cut into 1- to 2-mm cubes. All tissue blocks were postfixed and further processed to be embedded in epon-araldite as described [19]. Volume changes that occurred in testis tissue from the fresh to the processed state were estimated as described previously [16].
Microscopy and Morphometry
Two tissue sections, 1 µm in thickness and 3 µm apart (i.e., the first and fourth sections), were cut from each of the testis tissue blocks and prepared for microscopy using an LKB IV ultramicrotome (Pharmacia LKB, Piscataway, NJ) and glass knives. These pairs of sections were obtained to perform the Disector technique [20], the first section, taken randomly, serving as a "reference section," and the fourth section serving as a "look-up section." Blocks were accumulated as follows: 1- and 7-day-old) 2 blocks per rat as each testis provided 1 block, thus 10 blocks per group; 14 days) 8 blocks per rat, thus 40 blocks per group; 21 days) 10 blocks per rat, thus 50 blocks per group. For 1- and 7-day-old rats, both testes were used. In these rats, in addition to the pair of sections obtained for the Disector method, a second pair of sections was cut 40 µm or more deeper than the previous pair. Therefore, total pairs of reference and look-up sections for the Disector technique were 20 in these two age groups; a total of 40 and 50 such pairs were used for 14- and 21-day-old rats, respectively. These sections were stained with methylene blue azure stain and viewed and photographed using an Olympus BH-2 (Tokyo, Japan), photomicroscope. The numerical density of cells (Nv, defined as the number of cells per unit volume of testis), namely, fetal Leydig cells, adult Leydig cells, and mesenchymal cells, in fresh unfixed testis tissue was determined by the Disector method [20] as described previously by Mendis-Handagama and Ewing [19] using the equation Nv = [
Q-/(n x a x 3t)] x (1 - ST)]. Q- was the total number of unique nuclei of each cell type counted in all reference sections per rat, n was the number of disectors per rat, 3t was the height of a disector (i.e., number of sections between the reference and look-up section x section thickness or 3 x 1 µm), and ST was the total shrinkage of testicular tissue from the fresh to the final processed state [19]. During counting, the fields were selected randomly without overlap (area of the test frame = 4230 µm2). A total of 123, 101, 97, and 89 fetal Leydig cells were counted for 1-, 7-, 14-, and 21-day-old control rats, respectively, and a total of 129, 111, and 108 fetal Leydig cells were counted for PTU rats of 7, 14, and 21 days of age, respectively. The total numbers of adult Leydig cells counted for 14- and 21-day controls were 138 and 672, respectively. Summation average graphs were used to test the sampling adequacy [21]. The number of each cell type per testis was obtained from multiplying the fresh testis volume by the numerical density of each cell type.
To determine the average volume of a Leydig cell, we first determined the volume density (Vv) of Leydig cells in both treatment groups using the point counting method [22] as described by us [23]. The first tissue section of each Disector pair was used for this procedure (8–10 sections per rat ). Vv was divided by Nv to obtain the average volume of a Leydig cell as published previously [23].
These cell types were identified by their characteristic morphology as described previously [4]. In brief, fetal Leydig cells in sections appeared as circular or polygonal. Lipid droplets were abundant in their cytoplasm, and their nuclei showed abundant euchromatin and a thin peripheral rim of heterochromatin. Adult Leydig cells had little or no cytoplasmic lipid droplets, had a relatively thicker peripheral rim of heterochromatin in their nuclei, and were smaller than fetal Leydig cells (except at Day 21). All elongated spindle-shaped cells in the testis interstitium, namely fibroblasts, endothelial cells, pericytes and peritubular myoid cells, were collectively categorized as mesenchymal cells.
11ß Hydroxy Steroid Dehydrogenase 1 (11ß-HSD1) Immunocytochemistry
11ß-HSD1 has been shown to be present in adult Leydig cells of neonatal rats but not in fetal Leydig cells [24]. With immunofluorescent techniques, this enzyme was first detected at Day 26 of postnatal life [24]. However, in a separate study on testicular development in the rat we noted that 11ß-HSD1-positive cells were present in the rat testis interstitium at Day 21 (unpublished results), although fewer in number than at the older ages used in that study. Therefore, we performed immunocytochemistry on testes of 21-day-old control and PTU rats to compare the presence of 11ß-HSD1-positive cells in the testis interstitium under euthyroid and hypothyroid conditions at this age (i.e., 21 days). Twenty-one-day-old control and PTU rat testes (n = 4 per group) were fixed in Bouin's solution for 4 h and washed in 70% ethanol for several days until all picric acid was removed from the tissue. Tissue was left in 70% ethanol at 4°C until processed and embedded in paraffin. Sections were made from these paraffin-embedded tissue blocks at 5-µm thickness and adhered on ProbeOn Plus slides (Fisher Scientific, Pittsburgh, PA). These sections were protein blocked using a PBS solution containing 1% BSA and 5% normal goat serum. Anti-11ß-HSD1 antibody was diluted (1:800) in the blocking solution and was applied to the sections and incubated overnight at 4°C. Control sections were incubated with preimmune serum. Binding of anti-11ß-HSD1 was detected using the protocol described for the peroxidase-anti peroxidase kit (Biogenex, San Ramon, CA).
LH-Stimulated Steroidogenesis In Vitro
Testes (n = 8 per group) were removed, cleaned of fat, decapsulated, and incubated in Krebs-Ringer bicarbonate buffer (aerated for 10 min) containing 2% glucose and 100 ng per ml LH (maximum stimulatory dose [16, 25, 26]), at 34°C for 3 h in a shaking water bath at 90 oscillations per minute as described previously [27]. The incubation chamber was aerated continuously throughout the incubation period. Testosterone and androstenedione levels in the supernatant of the incubation medium were determined by RIA using diagnostic kits (ICN, Irvine, CA) as published previously [28–30].
Statistical Analysis
Significant differences (p < 0.05) between the means of different age groups were determined by ANOVA followed by Duncan's Multiple Range Test. Significant differences between the means at a particular age in control and PTU rats were determined by t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In both control and PTU rats, body weights increased with age (Table 1). Comparison between the two treatment groups revealed that the body weights were lower in PTU rats at all ages studied, but a statistically significant difference was observed only at Day 21 (Table 1).
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Testis Weights
Testis weights of both control and PTU rats gradually increased with age, although the values were lower in PTU rats at all ages; significant reductions were observed at Days 14 and 21 (Table 1).
Qualitative Morphology
Fetal Leydig cells were observed in testes of both control and PTU rats at all ages; a few mitotic figures were also observed in these cells (Fig. 1). In 1- through 14-day-old control rats and 7- through 21-day PTU rats, fetal Leydig cells showed their characteristic morphology. However, fetal Leydig cells with irregular nuclear profiles were more frequently observed at Day 21, compared to all other treatment groups. Cytoplasm of all fetal Leydig cells was loaded with lipid droplets, which often appeared as empty spaces because of the extraction of lipid during tissue processing. In Day 21 controls, these profiles were smaller but otherwise identical in their morphological characteristics to those at other ages. Adult Leydig cell profiles were observed only in controls; they were abundant at Day 21 and fewer in number at 14 days of age. These approximated fetal Leydig cells in size at Day 21, but they contained hardly any cytoplasmic lipid (Fig. 2).
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11ß-HSD1 Immunocytochemistry
11ß-HSD1-positive cells (i.e., adult Leydig cells) were absent in testis interstitium of 21-day-old PTU rats but were present in controls of the same age in small numbers (Fig. 3).
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Morphometry
Morphometric studies revealed that the number of fetal Leydig cells per testis was not significantly different between controls and PTU rats at any age tested (Table 2).
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Adult Leydig cells were observed only in 14- and 21-day-old controls (Table 2); they were significantly greater in number at Day 21 than at Day 14. Mesenchymal cell number per testis concomitantly increased with age in both control and PTU rats. In addition, there were significantly greater numbers of mesenchymal cells in PTU testes at 7, 14, and 21 days than in controls (Table 2).
The average volume of a fetal Leydig cell in controls and in PTU rats was not significantly different up to 14 days of age. A significant reduction was observed at Day 21 in controls (Table 3). The average volume of an adult Leydig cell in controls was not significantly different at 14 and 21 days (Table 3).
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Steroid Hormone Secretory Capacity In Vitro
LH-stimulated (LH 100 ng/ml) testosterone secretory capacity per testis was not significantly different from Days 1 through 21 within each treatment group. Additionally, on Days 7 and 14, there were no significant differences between age-matched controls and PTU rats. However, a significantly greater value was observed in PTU rats compared to controls at Day 21 (Fig. 4). LH-stimulated (LH 100 ng/ml) androstenedione secretory capacity per testis was not significantly different at Days 7 and 14 between controls and PTU rats. At Day 21, a sharp rise (asterisk) was observed in controls. By contrast, this increase was absent in PTU rats, and this value at Day 21 was not significantly different from those at younger ages (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present investigation, we demonstrate that adult Leydig cells do not appear in the neonatal rat testis up to 21 days under hypothyroid conditions. These findings are in agreement with Teerds et al. [31], although they used a different strain of rats (Wistar strain). Direct comparisons between the two strains are possible to some extent as testicular development of the two strains has been well studied [1–6, 8]. Because Leydig cells are differentiated from the mesenchymal cells in the neonatal testis, it appears that the differentiation of mesenchymal cells into adult Leydig cells is delayed or prevented under hypothyroidism. In agreement with this view, we also observed that the mesenchymal cell number per testis in PTU rats was significantly greater than the corresponding number in controls at every postnatal age tested. We suggest that this is due to the continued proliferation of mesenchymal cells in the absence of differentiation as discussed in the next paragraph. Teerds et al. [31] could not show a statistically significant increase in the percentage of labeled mesenchymal cells in their investigation; however, absolute numbers are not available in their study to compare with those in the present paper.
The concomitant increase in mesenchymal cell numbers per testis observed with advancing age in the present investigation can be explained by the expansion of the testis interstitium due to the growth of the seminiferous tubules and the testis itself. These results are consistent with our previous findings on the increase in the volume of mesenchymal cells per testis with advancing age from 1 through 21 days [4]. Additionally, these findings compare favorably with those of Hardy et al. [8] at similar ages. The finding that PTU testes contain a greater number of mesenchymal cells per testis than their age-matched controls is also in agreement with the study of Amin and El-Shiekh [32]; they showed that hypothyroidism causes proliferation of connective tissue cells in the testis interstitium in neonatal rats. This increase is intriguing, because it reveals that there are significantly greater numbers of Leydig cell precursors per testis in these hypothyroid rats than in the controls.
Findings in the present study of LH-stimulated testicular steroid secretory capacity in vitro are consistent with these observed changes in the testicular interstitial cells. However, no other study is available to compare our results on the LH-stimulated steroid hormone secretory pattern in vitro of the developing rat testis during the neonatal period under either normal or hypothyroid conditions. Testosterone is the primary androgen secreted by the fetal Leydig cells. The fact that testosterone secretory capacity per testis in PTU rats is maintained up to Day 21 reveals that the functional characteristics of the fetal Leydig cell population are unaffected by the PTU treatment up to 21 days. Moreover, the latter finding can be explained by the unaltered fetal Leydig cell size and number per testis up to 21 days in the PTU-treated rats. Although fetal Leydig cell atrophy was observed on Day 21 in controls, there was no change in the testosterone secretory capacity per testis. Therefore, it appears that an additional contribution is made by the newly formed adult Leydig cells at Day 21 to help the testis maintain its testosterone secretory capacity at a level similar to that at younger ages. However, even with this additional source of testosterone, the amount of this hormone produced by the 21-day-old control testis was significantly lower than the amount produced by the 21-day-old PTU testis. The sharp rise in androstenedione secretion in control testis at Day 21 could be explained by the increase in the adult Leydig cell number per testis at that age, because one of the primary androgens secreted by the adult-type Leydig cells in the prepubertal testis is androstenedione [33–36]. Its absence in the age-matched PTU rats is clearly due to the absence of newly formed adult Leydig cells and is consistent with our morphometric and immunocytochemical investigations. Low levels of androstenedione secretion in 1- to 14-day controls and 7- to 21-day PTU rats can be attributed to the secretion by the fetal Leydig cells.
The findings of the present study suggest that transient neonatal hypothyroidism produces an increase in the number of mesenchymal cells, which are precursors to Leydig cells, in 7-, 14-, and 21-day-old testes. Therefore, it is logical to suggest that, upon withdrawal of the PTU treatment, the inhibition is removed and an increased number of Leydig cell precursors are available to be differentiated. As a result, an increase in the number of Leydig cells per testis is established at adulthood in rats subjected to the treatment of transient neonatal hypothyroidism [15–17]. This view does not agree with the conclusion of Hardy et al. [37] that proliferation of Leydig cells rather than increased proliferation of their mesenchymal precursors is the principal mechanism responsible for the increase in the Leydig cell number in adult rat testis after neonatal hypothyroidism.
According to our present study and the study of Teerds et al. [31], adult Leydig cells do not differentiate under hypothyroid conditions. Therefore, if the labeling index of Leydig cells is high in PTU rats during this neonatal period, as shown by Hardy et al. [37], it implies that fetal Leydig cells, but not immature adult Leydig cells, are undergoing mitosis under hypothyroid conditions at a higher rate than controls. If this is true, an increase in the number of fetal Leydig cells per testis would be expected in PTU rats at least by Day 21. However, fetal Leydig cell number per testis did not change up to 21 days. Moreover, Teerds et al. [31] failed to observe an increase in the labeling index for Leydig cells in PTU rats compared to controls and do not agree with the observation of Hardy et al. [37] on this issue. In addition, it is difficult to comprehend why Leydig cell proliferation was absent in controls in the latter study of Hardy et al. [37] from birth to 21 days of age but was present in their previous study [8].
In the present study, fetal Leydig cells in mitosis in the testis interstitium were observed in both controls and in PTU rats. It appears that similar observations have been made by Hardy et al. [33] in a recent study. The Leydig cell in mitosis shown in that paper [33] is identified as an immature Leydig cell (adult type) and not as a fetal Leydig cell, and it is presented as evidence for the occurrence of cell division in immature Leydig cells in the neonatal PTU testis. It is true that both these Leydig cell types (i.e., fetal and immature adult types) contain a considerable amount of cytoplasmic lipid. However, we believe that this feature alone is inadequate to differentially identify these two cell types. The demonstrated Leydig cell in mitosis in a PTU rat, which is identified as an "immature Leydig cell" by these authors [33], fits well into the category of fetal Leydig cells in the neonatal PTU rat on the basis of its ultrastructural characteristics and size (which do not change with PTU treatment). This view is also supported by the presence of surrounding basement membrane components, which are unique to fetal Leydig cells [6]. When these observations are taken together, it is difficult for us to accept that the proliferation of Leydig cells rather than increased proliferation of their mesenchymal cell precursors in the hypothyroid testis is the principal mechanism responsible for the increase in Leydig cell number in adult rats subjected to transient neonatal hypothyroidism.
In summary, the present study demonstrates that mesenchymal cell differentiation into adult Leydig cells in the neonatal rat testis interstitium is prevented by PTU-induced hypothyroidism. On the basis of these findings, it is possible to conclude that thyroid hormones may have an important role in triggering the differentiation of mesenchymal cells into adult Leydig cells in the neonatal rat testis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: (423) 974–8222; mendisc{at}utk.edu
Accepted: March 23, 1998.
Received: October 20, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxylase,C17,20 lyase and the receptor for luteinizing hormone (LH) in the fetal rat testis suggests that the onset of Leydig cell steroid production is independent of LH action. Biol Reprod 1998; 58:520–525.-reduced androgen secretion by rabbit testes-epididymides perfused in vitro. Endocrinology 1975; 96:610–617.[Abstract]This article has been cited by other articles:
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  T. F. M. Mikkila, J. Toppari, and J. Paranko Effects of Neonatal Exposure to 4-Tert-Octylphenol, Diethylstilbestrol, and Flutamide on Steroidogenesis in Infantile Rat Testis Toxicol. Sci., June 1, 2006; 91(2): 456 - 466. [Abstract] [Full Text] [PDF]
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 I.-s. Kim, H.B. Siril Ariyaratne, and S.M.L. C. Mendis-Handagama Changes in the Testis Interstitium of Brown Norway Rats with Aging and Effects of Luteinizing and Thyroid Hormones on the Aged Testes in Enhancing the Steroidogenic Potential Biol Reprod, May 1, 2002; 66(5): 1359 - 1366. [Abstract] [Full Text] [PDF]
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 S. L. P. Matta, D. A. R. Vilela, H. P. Godinho, and L. R. Franca The Goitrogen 6-n-Propyl-2-Thiouracil (PTU) Given during Testis Development Increases Sertoli and Germ Cell Numbers per Cyst in Fish: The Tilapia (Oreochromis niloticus) Model Endocrinology, March 1, 2002; 143(3): 970 - 978. [Abstract] [Full Text] [PDF]
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S.M.L. Chamindrani Mendis-Handagama and H.B. Siril Ariyaratne Differentiation of the Adult Leydig Cell Population in the Postnatal Testis Biol Reprod, September 1, 2001; 65(3): 660 - 671. [Abstract] [Full Text] [PDF]
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H.B. S. Ariyaratne, N. Mills, J. I. Mason, and S.M.L. C. Mendis-Handagama Effects of Thyroid Hormone on Leydig Cell Regeneration in the Adult Rat Following Ethane Dimethane Sulphonate Treatment Biol Reprod, October 1, 2000; 63(4): 1115 - 1123. [Abstract] [Full Text]
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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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H.B.S. Ariyaratne, S.M.L.C. Mendis-Handagama, and J.I. Mason Effects of Tri-Iodothyronine on Testicular Interstitial Cells and Androgen Secretory Capacity of the Prepubertal Rat Biol Reprod, August 1, 2000; 63(2): 493 - 502. [Abstract] [Full Text]
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H.B. Siril Ariyaratne, S.M.L. Chamindrani Mendis-Handagama, D. Buchanan Hales, and J. Ian Mason Studies on the Onset of Leydig Precursor Cell Differentiation in the Prepubertal Rat Testis Biol Reprod, July 1, 2000; 63(1): 165 - 171. [Abstract] [Full Text]
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J. J. Buzzard, J. R. Morrison, M. K. O'Bryan, Q. Song, and N. G. Wreford Developmental Expression of Thyroid Hormone Receptors in the Rat Testis Biol Reprod, March 1, 2000; 62(3): 664 - 669. [Abstract] [Full Text]
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H.B. S. Ariyaratne and S.M.L. Chamindrani Mendis-Handagama Changes in the Testis Interstitium of Sprague Dawley Rats from Birth to Sexual Maturity Biol Reprod, March 1, 2000; 62(3): 680 - 690. [Abstract] [Full Text]
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K. J. Teerds, D. G. de Rooij, F. H. de Jong, and L. H. van Haaster Development of the Adult-Type Leydig Cell Population in the Rat Is Affected by Neonatal Thyroid Hormone Levels Biol Reprod, August 1, 1998; 59(2): 344 - 350. [Abstract] [Full Text]
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