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Regular Article |
a Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7
ABSTRACT
Effective interactions among the various compartments of the testis are necessary to sustain efficiency of the spermatogenic process. To study the intercellular communication between the Sertoli and Leydig cells in the complete absence of FSH receptor signaling, we have examined several indices of Leydig cell function in FSH receptor knockout (FORKO) mice. The serum testosterone levels were reduced in the 3- to 4-mo-old adult FORKO males compared to wild-type mice despite no significant alteration in circulating LH levels. Treatment with ovine LH resulted in a dose-dependent increase in serum testosterone levels in all three genotypes (+/+, +/-, and -/-). However, the response in FORKO males was significantly reduced. Similarly, the total intratesticular testosterone per testis was also lower, but the intratesticular testosterone per milligram of testis was significantly elevated in the FORKO males. Western blot analysis revealed an apparent higher expression of the enzyme 3ß-hydroxysteroid dehydrogenase (3ß-HSD) as well as LH-receptor density in the testis of FORKO males. Immunohistochemistry also showed an increase in the intensity of 3ß-HSD staining in the testicular sections of FORKO males. Although LH receptor binding increased per unit weight in FORKO mice, the total LH binding remained the same in all genotypes. Taken together, the results of the present study suggest that, in the absence of FSH receptor signaling, the testicular milieu is altered to affect Leydig cell response to LH such that circulating testosterone is reduced in the adult mutant. Studies are currently under way to understand the mechanisms underlying this phenomenon.
follicle-stimulating hormone receptor, Leydig cells, luteinizing hormone, testis, testosterone
INTRODUCTION
Sertoli and Leydig cells represent two major somatic cells that are characteristic of testicular architecture and function. Sertoli cells, which provide the anchor for germ cell development, are restricted to the tubular compartment of the testis, whereas Leydig cells are present in the intertubular/interstitial space. The Sertoli cells express functional receptors for FSH, whereas the Leydig cells express LH receptors. Thus, these two receptors are mutually exclusive. The LH-stimulated Leydig cell androgen production is essential for the development and maintenance of the male reproductive tract and spermatogenesis [1]. Although LH is the primary tropic hormone that regulates the steroidogenic activity of Leydig cells, several studies have suggested that FSH may also exert a stimulatory effect on Leydig cells [2–4]. However, LH contamination during the preparation of FSH is considered to be among the possible reasons for the stimulation of androgen by this hormone [5], because these studies were performed before the availability of recombinant FSH. The binding of FSH is specific to Sertoli cells [6], and receptors for this hormone are absent in Leydig cells [7]. However, FSH administration in hypophysectomized animals has been shown to increase both Leydig cell steroidogenesis and LH receptors [3, 8].
We have recently produced the FSH receptor knockout (FORKO) mouse to understand the regulatory role of FSH receptor signaling in reproductive functions of the male and female. The FORKO females are infertile, whereas the males exhibit reduced fertility, with drastic reduction in testis size and circulating testosterone levels [9]. In the mammalian testis, Leydig cells are responsible for testosterone production. The aim of the present study was to understand the changes that might have occurred in intercellular communication between Sertoli and Leydig cells in the absence of FSH receptor signaling. Our hypothesis was that Leydig cell function might be compromised in the complete absence of FSH receptor signaling. Accordingly, we have examined key markers of Leydig cell activity in adult FORKO males and assessed their functional capacity to tropic hormone challenge. Our data demonstrate that, even though LH and LH receptor expressions are not significantly perturbed, the output of the Leydig cell is compromised in these mutants.
MATERIALS AND METHODS
Reagents
The LH receptor peptide (amino acids 21–41) antibody used in this study was a gift from Dr. N.R. Moudgal (Indian Institute of Science, Bangalore, India). Antibody to recombinant mouse 3ß-HSD I (hydroxy steroid dehydrogenase) protein was kindly provided by Dr. Anita Payne (Stanford University, Stanford, CA). The actin antibody (A 2066) was procured from Sigma (St. Louis, MO). Testosterone antibody, kindly supplied by Dr. A.J. Rao (Indian Institute of Science), was used for the RIA. Ovine LH (oLH) was purified from the sheep pituitary glands according to the standard protocol of our laboratory [10]. Recombinant human FSH as well as the RIA kit for rat LH were obtained from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) through Dr. A.F. Parlow (University of California at Los Angeles, Los Angeles, CA). Carrier-free NaI125 was supplied by Amersham, Inc. (Chicago, IL). Polyvinylidene fluoride (PVDF) membranes were obtained from NEN Life Science Products (Boston, MA). The Western blot detection system ECL+plus was procured from Amersham (Buckinghamshire, UK). For immunohistochemistry, we used the ImmunoCruz staining system from Santa Cruz Biotechnology (Santa Cruz, CA). Actin antibody was used to compare protein loading in Western blots. A Carl Zeiss microscope (Thornwood, NY) and was used for histochemical analysis.
Animals
This investigation was approved by the ethics committee of the Clinical Research Institute of Montreal and was conducted according to accepted standards of animal experimentation. The generation of mice with targeted disruption of the FSH receptor has been recently described [9]. This alteration results in elimination of the entire repertoire of FSH receptor forms, producing a complete loss of hormone signaling. Breeding F2 heterozygous males and females produced mice of all three genotypes in the SV129 background. The animals were maintained under well-controlled conditions of temperature (22°C), light (12L:12D), and humidity, with food and water provided ad libitum. The primers and amplification conditions used for the multiplex polymerase chain reaction (PCR) to identify the genotypes have been described in detail elsewhere [11]. Thus, a single PCR performed on each sample allowed unambiguous identification of +/+, +/-, and -/- mice. Adult wild-type and FORKO males (n = 5 per group, unless indicated otherwise) were used in the experiments.
In Vivo and In Vitro Testicular Response of Testis to oLH
Experiment 1 To understand how LH might stimulate testosterone production in the absence of FSH receptor signaling, we examined the in vivo testicular response of adult wild-type and FORKO mice following s.c. injection of a high dose of oLH (preparation 1, 10 µg). Control animals received an equal volume of saline. Testosterone was measured in the serum 2 h after hormone administration.
The method followed to study the in vitro testicular response was essentially that described by Chandrashekar and Bartke [12]. Briefly, each testis from 3- to 4-mo-old wild-type and FORKO mice (n = 6 per group) was dissected out, cut into pieces, and incubated in a 24-well culture plate, with the wells containing 2 ml of Dulbecco modified Eagle medium (Gibco BRL, Burlington, ON, Canada). Thus, each well contained fragments from one testis. The tissues were treated with different doses of oLH (preparation 2) or recombinant human FSH for 2 h at 37°C in a shaking (100 rpm) water bath. After 2 h, the contents were centrifuged, and the medium was frozen at -20°C until assayed directly for testosterone by RIA.
Experiment 2 Three- to four-month-old adult animals of all three genotypes were treated with saline or with 1 or 10 µg of oLH (preparation 2), and the mice were killed after 2 h by an overdose of anesthetic. Blood was collected by cardiac puncture and transferred to tubes precoated with EDTA. The testes were removed, weighed, and frozen at -80°C. One testis from each animal was homogenized in phosphate-buffered saline, and intratesticular testosterone was measured in the cytosol [13]. The remaining testis was processed individually for Western blot analysis (see below).
Hormone Measurements by RIA
Serum LH was measured with kits supplied by the National Hormone and Peptide Program (NHHP) (NIDDK) using rat LH for iodination and the corresponding antibody. The suggested protocol was adopted for the RIA, and the serum LH values are expressed in terms of the mouse LH reference preparation AFP-5306A. Serum testosterone was measured by RIA following solvent extraction using procedures standardized in the laboratory [14]. Intratesticular testosterone was measured in the aliquots of testicular cytosol [13]. The sensitivity of the assay was 20 pg/tube. The inter- and intraassay variations were 6% and 10%, respectively.
Western Blot Analysis and LH Receptor Binding
One testis of all three genotypes in experiment 2 was extracted as described using a lysis buffer [15] for examining the solubilized LH receptors and the 3ß-HSD enzyme. Heart and skeletal muscles from the same animal were also extracted to serve as control tissues. Equivalent amounts of solubilized protein (25 µg) were run on SDS-PAGE and transferred onto PVDF membrane for reaction with antibodies. Following reaction with a 1:10 000 (v/v) dilution rabbit antibody of LH receptor corresponding to the 21–41 amino acid peptide sequence or a 1:500 (v/v) dilution of the 3ß-HSD antibody, detection was performed by chemiluminescence using the ECL+plus kit. Molecular weight markers were used to estimate the mass of the detected bands. After tests with LH-receptor antibodies, each blot was stripped for reprobing with an actin antibody to test for equivalent loading and transfer. Similarly, another blot was probed with the 3ß-HSD antibody.
The level of LH receptor binding in mouse testicular membrane preparations was determined using purified, radiolabeled oLH (preparation 2) according to a slight modification of published procedures adopted in our laboratory [16]. Thus, in a separate experiment, the testes from each animal (n = 3 of each genotype) were dissected and homogenized in 25 mM Tris-HCl buffer (pH 7.5) containing 0.25 M sucrose and centrifuged at 1000 x g for 15 min to remove debris. The supernatant was then centrifuged at high speed (15 000 x g) for 20 min to collect the particulate material. The pellet was then dispersed in buffer containing 1 mg/ml of bovine serum albumin and 10 mM MgCl2. Tests were performed in duplicate tubes calculated to contain the membrane equivalent of 15 mg wet weight of each testis. The reaction was performed in a total volume of 0.5 ml, containing 50 000 cpm of labeled oLH, and the tubes were incubated overnight at room temperature. Specific binding was calculated by subtracting the binding to the membranes determined in the presence of excess oLH (1 µg/tube) from the binding seen in the absence of unlabeled oLH. The results were calculated to express the binding as femtomoles of LH bound per milligram of testis as well as per pair of testis for each animal.
Immunohistochemistry
After processing, testes were embedded in paraffin wax and sectioned at 5-µm thickness before detection of 3ß-HSD antigen. Antigen was immunolocalized using prediluted rabbit antibody and employing a biotin-streptavidin-immunoperoxidase method. Before incubation of the sections with primary antibody, all sites were blocked with normal rabbit serum for 20 min. Primary antibody was applied to the sections and allowed to incubate overnight at 4°C. At this point, the sections were treated exactly as described in the manufacturer's instructions supplied with the immunostaining kit for 3',5'-diaminobenzidine as a chromogen. Hematoxylin counterstaining of the sections was performed briefly (30–60 sec), and sections were mounted with Permount. As controls, additional sections were handled with primary antibody omitted and normal rabbit sera treated in place of primary antibody.
Statistical Analysis
When necessary, statistical significance was calculated by performing one-way ANOVA (P < 0.05).
RESULTS
Circulating LH in FORKO Males
Because pituitary LH is the primary regulator of testicular androgen production by the Leydig cells and the circulating level of this steroid is reduced in adult FORKO males [9, 17], there might be a reduction of this tropic hormone in the circulation of FORKO males as well. This possibility was verified by comparing serum LH levels in 3- to 4-mo-old wild-type and FORKO males. The hormone levels as measured by RIA for the two groups remained essentially similar (Fig. 1); the slight increase in the mutants was not statistically significant. This suggests that the perturbations leading to reduced androgen might be at the target site, the Leydig cells.
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Testicular Response to Hormone Challenge
The primary objective of the present study was to understand the intercellular communication between the Sertoli and Leydig cells in the absence of FSH receptor signaling. To determine the testicular response to LH in the mutants, we injected adult wild-type and FORKO males with oLH. As previously reported, the basal serum testosterone level in adult FORKO males was significantly lower than that in +/+ littermates [9]. In experiment 1, the serum testosterone levels increased in response to oLH in both wild-type and FORKO males after 2 h (Fig. 2A). However, the response to exogenous LH was significantly lower in the FORKO males.
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During the in vitro experiment, we treated the testicular fragments with recombinant human FSH and pituitary oLH. Testosterone secretion was unaltered following a 2-h treatment of testicular fragments placed in the culture wells with different doses of recombinant human FSH in both +/+ and -/- mice (Fig. 2B). The only difference noted was a higher release of testosterone into the medium under basal conditions in -/- males. This pattern remained unaltered following recombinant human FSH treatment (Fig. 2B). The response of testis to oLH was clearly different from that during the FSH treatments. The following differences were evident between the +/+ and -/- testis. At 1 ng/well, the lowest concentration used in this series of experiments, steroidogenesis in the +/+ testis was already reaching maximal levels, because with the next 10-fold higher dose of oLH, the response was significantly decreased. In contrast, the response of the FORKO testis was significantly lower compared to that of the wild-type mouse testis (Fig. 2C). Maximal steroidogenesis did appear to attain the same level as in +/+ testis with a higher concentration of LH. In both groups, testosterone output was reduced at 100 and 1000 ng/ml of LH.
In experiment 2, which compared the testicular response patterns for all three genotypes, circulating testosterone levels were significantly reduced in the saline-treated FORKO males compared to the wild-type males (Fig. 3A). Treatment with oLH (preparation 2) resulted in a dose-dependent increase in the circulating testosterone levels in all three genotypes. However, the stimulation was significantly lower in the FORKO males compared to wild-type mice of the respective treatment groups (Fig. 3A). The total intratesticular testosterone content per testis was also significantly lower in the FORKO males compared to wild-type males (Fig. 3B, see saline). On treatment with oLH (1 or 10 µg), intratesticular testosterone was also elevated in FORKO males, but not to the same extent as in +/+ mice, even at the higher LH concentration. Interestingly, the intratesticular testosterone produced per milligram of testis was significantly high in the FORKO males compared to wild-type males (Fig. 3C).
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Status of Leydig Cell Markers
As markers of Leydig cell status, we studied two well-established molecular entities using their respective specific antibodies. The 3ß-HSD antibody reacted with a protein having an estimated molecular weight of 40 kDa, whereas the LH receptor antibody revealed a 77-kDa protein equivalent to the mass of the full-length glycosylated LH receptor (Fig. 4A). Extracts of skeletal muscle used as control showed no reaction at either the 40- or 77-kDa region in the respective blots. A weaker band observed for another control tissue, the heart, at 77 kDa is attributed to some minor contamination or to a nonspecific reaction. Differences for these two markers in the testicular extracts of the three genotypes were readily apparent. To appreciate the relative differences, we normalized the values for protein loading in respective lanes using the ß-actin antibody (Fig. 4A, bottom row). The intensity of the ß-actin band was most pronounced in the skeletal muscle. On normalization to ß-actin, the testis of both wild-type and heterozygous animals showed lower contents of 3ß-HSD and LH receptor proteins compared to the FORKO males (compare the saline group in Fig. 4, A–C). Although we have depicted the results typical of a representative mouse from each genotype including the treatments, this pattern was consistent for all three animals in the different groups. Following oLH treatment in vivo (1 or 10 µg) for 2 h, the intensity of the bands corresponding to 3ß-HSD and LH receptor increased in the +/+ and +/- testis, but not in the FORKO males. These differences following treatment were greatest for the +/+ males (LH treated in Fig. 4, A–C), and the observations were reproducible.
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We also determined radiolabeled hormone binding to the testicular membranes as an extension of the Western blot analysis of LH receptor protein. These experiments, which were performed with a different set of animals from 3 to 4 mo of age, revealed differences among the three genotypes (Fig. 4, D and E). The LH receptor binding was approximately 2.5-fold higher in FORKO mice for an equivalent wet weight of testis (Fig. 4D). This difference was highly significant and consistent for all mutants. However, the total LH receptor content per animal was not much different (Fig. 4E), because testicular weight is reduced to approximately half in adult FORKO males compared to the wild type [17].
When we stained the testicular sections to localize the 3ß-HSD enzyme, the cytoplasmic staining was confined to the Leydig cells (Fig. 5) in both wild-type and FORKO males. Comparison of the immunohistochemistry results showed an apparent, dramatic increase in 3ß-HSD-positive Leydig cells in the FORKO males compared to the wild-type males (Fig. 5). The increase in interstitial space created by the reduction in volume of the seminiferous tubules in FORKO mice appeared to be occupied by the resident structures, which included the Leydig cells. In testicular sections from FORKO mice (Fig. 5, C and E), an increase in the groups of intensely stained Leydig cells was observed. It may be noted that omission of the primary antibody did not produce any color (Fig. 5A), assuring the specificity of the tests.
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DISCUSSION
Although LH is the primary regulator of Leydig cell function, several studies have indicated that FSH may also exert a stimulatory effect, either directly or indirectly, on the steroidogenic activity of Leydig cells. The FORKO mouse, in which we have ablated all FSH receptor signaling, provides an interesting model for examining and extending some of these issues relevant to male reproduction. In the present study, we have examined the functional status of Leydig cells in the complete absence of FSH receptor signaling in adult male mice. First, we have established that the lower level of circulating testosterone in mutants is not due to a reduced secretion of pituitary LH. From these experiments, we can also infer that the hypothalamic-pituitary-gonadal axis is not seriously perturbed as far as LH is concerned. Despite this, a significant reduction is found in testicular weight from the prepubertal stage [18], and this important change persists in the adult mutant [17]. We have shown that the 50% reduction in testicular weight in the adult mutant is due to a reduction in the number of Sertoli cells and tubular shrinkage [18]. These observations clearly indicate that FSH receptor signaling is essential for maintaining testicular volume in the normal animal.
The significant decrease in basal as well as LH-stimulated testosterone production in FORKO males as found during experiment 1 (Fig. 2) is in conformity with our initial observation [9]. This decrease in testosterone production may be due to the 50% decline in testicular size, Sertoli cell numbers, and reduced activity of the Leydig cells in the mutants. It is important to note that in vitro treatment of the testicular fragments with different doses of recombinant human FSH did not alter testosterone production in either wild-type or FORKO testis, showing that this hormone, at the doses tested, has no direct effect on the Leydig cells of both genotypes. Because of other cell types present in the testicular fragments used, we can also exclude their possible participation in FSH action. Because this same human FSH preparation was highly active in FSH receptor-transfected cells investigated during another study [15], there could be no doubt regarding its biological activity. Thus, our observations noting a lack of direct effect on the mouse testis (Leydig cells) is contradictory to those of several earlier studies in which investigators noted that FSH stimulated testosterone production both in vivo [2, 19] and in vitro [3, 20]. However, such effects may be due to the LH contamination in the FSH preparations, because these studies predate the availability of recombinant FSH [5]. Other previous findings involving the administration of recombinant FSH to neonatal rats [13] or GnRH-immunized adult rats [21] failed to show effects on the levels of circulating and testicular androgens, which supports our data (Fig. 2). In the same manner, recombinant FSH in vivo has no effect on intratesticular testosterone in the hpg mouse model [22], a system that lacks both gonadotropins due to a genetic deficiency of GnRH. Thus, taken together, these data indicate that a direct effect of FSH on Leydig cells is highly unlikely. These findings also imply that the effect of FSH on Leydig cell function must be mediated indirectly through its receptor via its maintenance of the integrity of the Sertoli cells.
In our experiments, the use of highly purified oLH as a positive control significantly stimulated testosterone production in the testis of both +/+ and -/- genotypes, but in a different manner. This shows that the Leydig cells can remain functional in the complete absence of FSH receptor signaling. However, their capacity to produce testosterone appears to be reduced, which is a conclusion supported by several lines of evidence in the present investigation. First, the in vivo study, in which exogenous LH was used in the testis, indicated significant reduction in the overall response (Figs. 2A and 3, A and B), suggesting that Leydig cells deprived of their normal environment may be unable to reach their maximum steroidogenic functional capability. Because we examined only the adult mutants in this study, we cannot be certain when these changes might have occurred. Second, in the in vitro incubations, Leydig cells from the FORKO testis showed a tendency to be less sensitive, because a low LH concentration, which produced maximal steroidogenesis in the +/+ genotype, caused only approximately half the amount of testosterone to be produced under identical conditions. In addition, the elevated testosterone production induced in the FORKO testis at higher LH concentrations might suggest alterations in the metabolic behavior of Leydig cells. However, further investigation is necessary to understand the mechanisms underlying these differences. In analyzing these issues, one must consider the alterations in the Leydig cell-to-germ cell ratio in the knockout males and/or the possibility of Leydig cell hyperplasia (by at least 28%) in the absence of FSH receptor signaling. This can be deduced from the following evidence. In a recent study using flow cytometric analysis, we have shown a 28% increase in the 2C cells in FORKO testis. This population, which consists of Sertoli and Leydig cells as well as spermatogonia in adult FORKO males, is enhanced compared to that in wild-type males [17]. Because we have estimated a 50% decrease in Sertoli cells in this 2C population [18], the ratio of Leydig to other somatic and germ cells becomes higher. This might account for the increases that are seen in the various parameters of Leydig cells, including testosterone secretion per milligram of testis. However, the level of testosterone produced by each Leydig cell could be lower, explaining the lower levels in the serum and in the total testis.
Pending an understanding of the molecular mechanisms underlying changes in Leydig cell structure and function in FORKO mice, we have presently explored two important markers that form part of the androgenic compartment in the testis. Surprisingly, the results of the Western blot analysis of 3ß-HSD and LH receptor in the FORKO testis lysates showed apparent increases compared to +/+ or +/- littermates (Fig. 4, A–C). Because these two key participants that lead to testosterone production already seem to be higher in the FORKO testis, they do not appear to be further influenced by the action of exogenous LH, at least according to the present analytical data. This may, indeed, account for the higher intratesticular testosterone concentration per milligram of testis. The simplest explanation for these findings is that the relative contribution of Leydig cells in the FORKO testis becomes higher due to tubular shrinkage and reduction in testicular weight. Similar arguments may explain the stronger staining patterns for 3ß-HSD in the FORKO testis.
Evidence gathered from the estimation of LH receptor binding in FORKO testis suggests that, whereas hormone binding per unit weight of testis is enhanced, the total binding per animal remains unaltered. From this, we can infer that, in the adult FORKO testis, full expression of LH receptor in the Leydig cells is independent of FSH receptor signaling. Because LH receptor expression appears to be fully preserved, it is reasonable to conclude that any compromise in steroidogenic efficiency of the FORKO testis might be due to perturbation of post-LH receptor events.
In conclusion, the present study reveals, to our knowledge for the first time, that Leydig cells appear to undergo changes sufficient to reduce their steroidogenic capacity in the absence of complete FSH receptor signaling. One or more of the regulatory molecules secreted from the healthy Sertoli cell [23] under the influence of FSH receptor signaling is required to maintain Leydig cell function. Further studies using FORKO males might be helpful in the identification and characterization of these substances.
ACKNOWLEDGMENTS
We thank Maria Gerdes and Yinzi Yang for their helpful management of the mouse colony, Karin Cote for assistance in some aspects of this investigation, and Odile Royer for her help in preparation of the manuscript. We are grateful to Drs. N.R. Moudgal, A.J. Rao, A.H. Payne, and A.F. Parlow for providing the various reagents used in this investigation.
FOOTNOTES
1 Supported by the Medical Research Council of Canada and the Canadian Institutes of Health Research (CIHR). N.D. is a recipient of a doctoral award from the CIHR. 
2 Correspondence: M. Ram Sairam, Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, PQ, Canada H2W 1R7. FAX: 514 987 5585; sairamm{at}ircm.qc.ca
Accepted: May 30, 2001.
Received: March 2, 2001.
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