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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-2072
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The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 3 1062-1068
Copyright © 2006 by The Endocrine Society

Follicle-Stimulating Hormone-Independent Functions of Primate Sertoli Cells: Potential Implications in the Diagnosis and Management of Male Infertility

Yendrembam Sangeeta Devi1, Kanchan Sarda1, Benjamin Stephen, P. Nagarajan and Subeer S. Majumdar

Division of Cellular Endocrinology, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India

Address all correspondence and requests for reprints to: Subeer S. Majumdar, Division of Cellular Endocrinology, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India. E-mail: subeer{at}nii.res.in.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: FSH is known to augment the production of essential germ cell (Gc) survival factors, lactate and estradiol, by Sertoli cells (Sc) of 18-d-old pubertal rats. However, the failure of gonadotropin and androgen treatment to initiate spermatogenesis in testis of some infertile men bearing Sc and Gc is intriguing. The role of FSH in regulation of lactate and estradiol production by primate Sc is currently unknown.

Objective: The objective of the study was to determine the role of FSH in regulating lactate and estradiol production by primate Sc.

Methods: Gc differentiation was initiated in male juvenile rhesus monkeys by pulsatile administration of GnRH for 4–5 wk. Sc from these pseudopubertal monkeys and pubertal rats were cultured. Production of lactate and estradiol in response to FSH and 8-bromoadenosine-cAMP was evaluated. Inhibin-ßB expression, cAMP production, and cell proliferation were also assayed.

Results: Unlike Sc from pubertal rats, Sc from pseudopubertal monkeys constitutively aromatized testosterone to estradiol and produced large amounts of lactate without FSH stimulation. Increasing doses of recombinant monkey FSH or 8-bromoadenosine-cAMP failed to augment lactate production, although they significantly augmented proliferation of Sc. Production of cAMP and expression of inhibin-ßB mRNA were also remarkably augmented by recombinant monkey FSH.

Conclusions: These results suggest that lactate and estradiol production by monkey Sc is not governed by FSH, as previously thought based on studies of rat Sc. Thus, in a clinical situation, assessment of such gonadotropin-independent functions of Sc may be obligatory for the diagnosis and management of certain forms of idiopathic male infertility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MORE THAN 60 countries are facing the threat of infertility at or below population replacement levels (1). Up to 50% of the cases of male infertility are idiopathic in nature. A clear understanding of testicular function and related defects may help in counteracting this situation. In the seminiferous tubule, germ cell (Gc) development and differentiation are solely dependent on the milieu generated by the Sertoli cells (Sc). Several functions of the Sc are known to be regulated by FSH and testosterone (T) through their receptors expressed in these cells (2). However, in certain cases of hypogonadotropic hypogonadism, treatment with FSH and T fails to initiate spermatogenesis (3, 4). This may occur due to defective signal transduction of hormones in the testis. Alternately, it can be hypothesized that primate Sc secrete certain factors essential for the Gc development whose production is not governed by FSH and T and that constitutive defect(s) in production of these factors may be responsible for the failure of initiation of spermatogenesis despite FSH and T supplementation to some infertile individuals. Infertile patients suffering from varying degrees of damage to testicular Sc are currently untreatable due to lack of knowledge regarding hormonal regulation of primate Sc functions (5, 6).

The Sc metabolites estradiol (E2) and lactate are absolutely essential for the development and differentiation of Gc. Lactate produced by Sc is known to be the principal source of energy for Gc (7, 8), and pharmacological deprivation of lactate in Sc leads to a decline in Gc viability (9). Lactate also prevents Gc apoptosis in men (10). The necessity of E2 for male fertility is also well established. The estrogen receptor-{alpha}-null male mouse is infertile (11). The inability of P450 aromatase-null mice to convert T to E2 is associated with a drastic rise in Gc apoptosis (12, 13). The role of E2 as a Gc survival factor has also been established in men (14, 15). Treatment with an aromatase inhibitor leads to arrest of spermatid maturation in monkeys (16). Also, men with aromatase deficiency consecutive to a P450 aromatase gene mutation are found to be sterile (17, 18). Hence, it is reasonable to hypothesize a major role for lactate and E2 in the development and differentiation of Gc into sperm.

It is believed that FSH plays an important role in the regulation of Sc functions responsible for the initiation of spermatogonial differentiation during puberty (19, 20). Hence, it is necessary to evaluate the hormonal regulation of Sc function during puberty (21). Most information in this regard has been generated using Sc from 18-d-old pubertal rat testes, which have spermatogonia-B and spermatocytes (19). During this phase of pubertal development, lactate production as well as aromatizing ability of rat Sc was shown to be significantly augmented in response to FSH treatment (7, 22, 23, 24). However, the scarcity of primate testes and lack of established procedures for isolation and culture of primate Sc have limited the knowledge of primate Sc functions (25, 26).

It is difficult to identify the exact age and time of the onset of puberty in primates because it exhibits a great deal of individual variation. Hence, the present study used juvenile rhesus monkeys in which precocious puberty was induced by pulsatile infusion of GnRH, resulting in the initiation of Gc differentiation and the appearance of spermatogonia-B as well as spermatocytes, similar to the situation in the 18-d-old rat testis (27). Sc from such pseudopubertal monkeys were used to determine their dependency on FSH for the production of lactate and E2, which are necessary for Gc development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

All animals were kept and used as per the national guidelines provided by the Committee for the Purpose of Control and Supervision of the Experiments on Animals. Protocols for the experiments were approved by the Institutional Animal Ethics Committee and the Committee for the Purpose of Control and Supervision of the Experiments on Animals. Wistar rats (Rattus rattus, 18 d old) were obtained from the Small Animal Facility of the National Institute of Immunology (New Delhi, India). Twelve juvenile (18–22 months old) rhesus monkeys (Macaca mulatta) from the Primate Research Center of National Institute of Immunology (New Delhi, India) were used.

Reagents

Recombinant monkey FSH, ovine (o) FSH, GnRH, and anti-cAMP antibody were obtained from National Hormone and Pituitary Program (NHPP), National Institutes of Health (NIH; Torrance, CA). All other reagents, unless stated otherwise, were procured from Sigma Chemical Co. (St. Louis, MO).

Preparation of pseudopubertal monkeys

For inducing precocious puberty, the dormant pituitary-testicular axis was activated by treatment with pulsatile GnRH in 10 juvenile rhesus monkeys. For this purpose, juvenile rhesus monkeys were surgically implanted with chronic indwelling catheters via femoral or internal jugular vein under sterile conditions (28). The catheter was exteriorized in the midscapular region. The exteriorized catheter was protected by a nylon jacket and a flexible stainless steel tether (36 in. long, 0.5 in. inner diameter) attached to a swivel device on top of the cage. This system allowed normal free movement of the monkeys without affecting continuous access to venous circulation via the catheter. Monkeys were treated with intermittent pulsatile GnRH (0.3 µg GnRH per 2 ml saline per 2 min per 3 h) for 4–5 wk, until serum T levels reached the adult range. Weekly blood samples were collected via catheter to measure the circulating levels of T, before and after the GnRH pulse.

Isolation and culture of Sc

Rat. For each experiment, 12 testes from six rats were pooled for the isolation of cells. Sc were isolated from testis of 18-d-old Wistar rats following the procedure of Welsh and Wiebe (29) with minor modifications (30). The cells were cultured in DMEM/nutrient mixture F-12 Ham (DMEM/F12 HAM) containing 1% fetal calf serum for first 24 h in a humidified CO2 (5%) incubator at 34 C. Equal numbers of cell clusters (0.5 x 104 clusters/well) were plated in each well on d 1 of culture. One day later, cells were washed and cultured in medium containing serum replacement factors (5 µg/ml sodium selenite, 10 µg/ml insulin, 5 µg/ml transferrin, and 2.5 ng/ml epidermal growth factor). Medium was replaced every 24 h.

Monkey. Testes from the pseudopubertal monkeys were surgically removed under general anesthesia and Sc were isolated following the procedure described for juvenile rhesus monkeys (31) with minor modifications. Briefly, the testes were washed twice in Hanks’ balanced salt solution (HBSS), decapsulated, and minced into five to six pieces using a sterile blade. The tissue was digested in prewarmed collagenase solution (6.6 mg collagenase per 25 ml HBSS) in presence of DNase (500 kU) at 33 C for 10 min in a shaking (120 oscillations/min) water bath. Small fragments of tubules were removed along with the supernatant, and the remaining pieces of tissue at the bottom of flask were digested further under similar conditions. This digestion was repeated thrice. All supernatants containing small tubular fractions were pelleted at 75 x g, and pellets were resuspended in HBSS and washed twice (at 75 x g) to remove interstitial cells in the supernatant. All pellets were resuspended in 10 ml HBSS containing pancreatin (2.5 mg) and DNase (250 kU). The digestion was carried out (8–10 min) at room temperature until a large cell aggregate consisting mainly of peritubular cells appeared, which was discarded. The remaining cells were pelleted (75 x g), washed three times, and resuspended in medium containing 1% fetal calf serum. They were plated (1 ml containing 0.5 x 104 cell clusters/well) in 24-well plate(s) and maintained as described for the rat Sc.

In vitro treatments

For each set of treatments, cells from at least three different GnRH-treated monkeys were used. On d 4 of rat as well as monkey Sc culture, the few residual Gc were removed by hypotonic shock (32). On d 5 of culture, rat Sc were treated with either media alone or oFSH (1 µg/ml) or T (10–7 M) or a combination of both oFSH and T in the presence of isobutylmethylxanthine (10–4 M). Sc from rhesus monkeys were treated with either media alone or recombinant monkey (rm) FSH (5, 10, 20 ng/ml), T (10–7 M), a combination of both rmFSH and T or 8-bromo- adenosine-cAMP (8-Br-cAMP; 150, 300, 600 µM) in the presence of isobutylmethylxanthine (10–4 M). The dose of rmFSH was based on previous reports that showed that the circulating level of FSH in adult monkeys is approximately 5 ng/ml when a heterologous RIA is used using rhesus monkey FSH as the standard (33) and less than 1 ng/ml when homologous RIA is used (34). After 24 h of treatment, culture supernatants were collected for measuring lactate and E2. Sc were resuspended using trypsin-EDTA, counted, washed, pelleted, and treated with Trizol for RNA isolation (35). In additional experiments, monkey Sc were treated with 5 ng/ml of rmFSH for 30 min after which media were collected for estimation of cAMP. To evaluate the bioactivity of rmFSH and 8-Br-cAMP, Sc from 18-d-old rats were treated with rmFSH (5 ng/ml) or 8-Br-cAMP (150 µM) in addition to oFSH (1 µg/ml) treatment for 24 h. Culture supernatants were collected after 24 h and cells in each well were counted.

Cytochemical evaluation of the cultured cells

On d 6 of culture, cell viability was determined by trypan blue exclusion. Leydig cell contamination was evaluated by cytochemical staining specific for the 3ß-hydroxysteroid dehydrogenase activity (36). Peritubular cells were identified by staining for alkaline phosphatase activity, and lipid droplets in Sc were stained using Oil Red-O (37).

Tissue histology

After castration, tissue samples from testes of two untreated juvenile and three GnRH-treated juvenile rhesus monkeys were fixed in Bouin’s solution at room temperature for 24 h. Dehydration of tissues was done in a series of ascending concentrations of ethanol for 1 h in each grade of ethanol. The tissues were embedded in paraffin and 4-µm sections were cut. Sections were stained with hematoxylin and eosin for evaluating the status of spermatogenesis.

Lactate estimation

Lactate was measured enzymatically in the Sc culture supernatants. For this purpose, a lactate assay kit (Sigma) was used.

Hormone assays

E2 was measured in the 24-h culture supernatants by RIA to evaluate the aromatase activity in the cultured Sc. The intra- and interassay coefficients of variation were less than 5 and less than 9%, respectively.

Plasma levels of T were measured by RIA. The intra- and interassay coefficients of variation were less than 7 and less than 10%, respectively.

cAMP assay

cAMP concentrations in culture medium were analyzed by RIA by using 2–0' monosuccinyl cAMP tyrosine methyl ester and anti-cAMP in accordance with the instructions provided by the NHPP (National Institute of Diabetes and Digestive and Kidney Diseases, NIH). Antiserum to cAMP (lot CV-27) was procured from NHPP.

RT-PCR analyses

Total RNA was isolated from Trizol-treated samples. The eluted RNA was treated with DNase I to remove trace DNA contamination. At the end of the treatment, DNase I was removed from the RNA using a DNase I inactivation solution (Ambion Inc., Austin, TX). Reverse transcription of 1 µg total RNA was carried out using a reverse transcription system (Promega Corp., Madison, WI) with AMV reverse transcriptase and oligo (dT)15 for the single-strand cDNA synthesis. Subsequent PCRs (10 µl reaction volume) were carried out using 0.5 µl of the reverse transcription reaction for the inhibin-ßB gene (forward, 5'-AGCCTCCAGGATACCAGCAAA-3'; reverse, 5'-TCAAACGGTCATTGCCCCT-3'). Expression of inhibin-ßB in the control and FSH-treated Sc wells was normalized using cyclophilin A (forward, 5'-TCACCATTTCCGACTGTGGAC-3'; reverse, 5'-ACAGGACATTGCGAGCAGATG-3') as the housekeeping gene.

Presentation of data and data analysis

Treatments within each set of experiment (a set of culture from one monkey or six rats) were carried out in triplicate wells. Data from at least three monkeys for each set of experiments are presented. Results from lactate, E2, cAMP, and cell proliferation assays for each set of experiments were expressed as the mean ± SEM of three or more wells. The statistical analysis to determine significant/nonsignificant responses to various treatments was limited within experimental group, and results for each monkey are reported separately.

Each bar represents the mean of three or more culture wells from a treatment group of one set of culture. The statistical significance was determined by multiple measures one-way ANOVA followed by Dunnett’s posttest using the InStat statistical program (version 3.0, GraphPad Software, Inc., San Diego, CA). The P values for significance levels are specified in the figure legends.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of GnRH treatment of the juvenile rhesus monkeys

The plasma T levels in GnRH-treated animals reached peak values of 6–10 ng/ml from 0.27–0.72 ng/ml, similar to that found during puberty, by 4–5 wk of treatment (see supplemental data published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). The testicular weight increased approximately 4-fold in pseudopubertal animals, compared with that in control juvenile animals. Histological analysis of the testis of control juvenile monkeys showed only spermatogonial populations (mainly spermatogonia A; Fig. 1AGo), whereas testicular sections of GnRH-treated juvenile monkeys showed enlargement of seminiferous tubules and initiation of Gc differentiation associated with the presence of spermatogonia B and primary spermatocytes (Fig. 1BGo).


Figure 1
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  		FIG. 1. A, Cross-section of the testis of a juvenile rhesus monkey. The testis contains mainly spermatogonia A (arrow). B, Testicular cross-section of a GnRH-treated juvenile rhesus monkey; the increase in the size of the seminiferous tubules and the presence of spermatogonia B (bold arrow) and spermatocytes (arrow) can be noted. C, Confluent monolayer of Sc (nuclei stained with hematoxylin) isolated and cultured from testis of GnRH-treated rhesus monkey.

 
Viability and purity of Sc cultures

More than 98% of cells were found to be viable on d 6 of all the cultures (from 18-d-old rats and GnRH-treated rhesus monkeys). There was no Leydig cell contamination, judged by the absence of 3ß-hydroxysteroid dehydrogenase staining in the cultures. Peritubular cell contamination was found to be less than 2% in all cultures. In all Sc cultures, more than 95% of the cells were positively stained by Oil Red-O (see supplemental data published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). A confluent monolayer of pseudopubertal monkey Sc in culture is shown in Fig. 1CGo.

Lactate and E2 production by 18-d-old rat Sc

Lactate production by rat Sc was augmented significantly (P < 0.05) due to treatment with oFSH (1 µg/ml), whereas T did not enhance lactate production by Sc (Fig. 2AGo). Rat Sc cultured in the presence of T alone did not show any increase in E2 above the basal levels. However, conversion of T to E2 by rat Sc was significantly (P < 0.05) increased by FSH treatment (Fig. 2BGo).


Figure 2
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  		FIG. 2. Lactate (A) and E2 (B) production by Sc cultured from 18-d-old rats. C, Control; F, oFSH (1 µg/ml); T, 10–7 M; F+T, wells treated with combination of FSH and T. Each bar represents mean ± SEM of three or four wells from one culture group. Each panel represents one set of experiments. *, P < 0.05 vs. control; multiple-measures one-way ANOVA followed by Dunnett’s posttest. The bar corresponding to T treatment is hatched in column B to highlight the aromatizing ability of the Sc in the absence of FSH.

 
Lactate and E2 production by pseudopubertal monkey Sc

Untreated monkey Sc produced large amounts of lactate. No increase in lactate production was observed when Sc were treated with rmFSH or the combination of T and rmFSH (Fig. 3AGo). These Sc also constitutively converted T to E2 in the absence of rmFSH (Fig. 3BGo). Recombinant monkey FSH failed to increase the aromatizing ability of these Sc any further. Consistent results for lactate as well as E2 production were obtained in the Sc cultured from five pseudopubertal rhesus monkeys. Higher doses of rmFSH and increasing doses of 8-Br-cAMP also failed to stimulate lactate production by these Sc (Fig. 4Go). The effect of higher doses of FSH or 8-Br-cAMP on E2 production was not evaluated because E2 production by juvenile monkey Sc was found to be constitutively enhanced (approximately 7-fold, compared with basal) when exposed to T alone.


Figure 3
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  		FIG. 3. Production of lactate (A) and E2 (B) by Sc cultured from pseudopubertal rhesus monkeys. C, Control; F, rmFSH (5 ng/ml); T, 10–7 M; F+T, wells treated with combination of FSH and T. The constitutively high (approximately 7-fold) E2 production by pseudopubertal monkey Sc treated with T alone is notable. Each bar represents mean ± SEM of three or four wells from one culture group. Each panel represents one set of experiments. *, P < 0.05 vs. control; multiple-measures one-way ANOVA followed by Dunnett’s posttest. The bar corresponding to T treatment is hatched in column B to highlight the aromatizing ability of the Sc in the absence of FSH.

 

Figure 4
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  		FIG. 4. Lactate production by Sc of pseudopubertal monkeys in response to increasing doses of rmFSH and 8-Br-cAMP. F5, rmFSH (5 ng/ml); F10, rmFSH (10 ng/ml); F20, rmFSH (20 ng/ml); 150, 150 µM of 8-Br-cAMP; 300, 300 µM of 8-Br-cAMP; 600, 600 µM of 8-Br-cAMP. Each panel represents one set of experiments. Each bar represents mean ± SEM of three or four wells of cultured Sc. *, P < 0.05 vs. control; multiple-measures one-way ANOVA followed by Dunnett’s posttest.

 
Effect of rmFSH and 8-Br-cAMP on rat Sc

Sc from 18-d-old rats were used as a test system to evaluate the bioactivity of rmFSH used in the study. In response to treatment with 5 ng/ml rmFSH or 150 µM 8-Br-cAMP, lactate production by rat Sc was significantly (P < 0.05) augmented, indicating their efficacy at these doses (Fig. 5Go).


Figure 5
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  		FIG. 5. Determination of bioactivity of FSH preparations and the cAMP analog in Sc from 18-d-old rats. oFSH, 1 µg/ml; rmFSH, 5 ng/ml; 8-Br-cAMP, 150 µM. Each bar represents mean ± SEM from three sets of experiments. *, P < 0.05 vs. control; one-way ANOVA followed by Dunnett’s posttest.

 
Bioactivity of rmFSH and 8-Br-cAMP in monkey Sc

Treatment with rmFSH (5 ng/ml) led to a significant (P < 0.05) rise in cAMP production by monkey Sc (Fig. 6AGo). The expression of inhibin-ßB mRNA in the monkey Sc was also significantly (P < 0.05) augmented in response to treatment with rmFSH (Fig. 6Go, B and C). The numbers of pseudo- pubertal monkey Sc increased significantly (P < 0.05) in response to treatment with rmFSH as well as 8-Br-cAMP (Table 1Go).


Figure 6
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  		FIG. 6. Determination of bioactivity of rmFSH in Sc from pseudopubertal monkeys. A, Production of cAMP by monkey Sc in response to rmFSH. B, upper panel, Inhibin-ßB expression in untreated (lane 1) Sc and rmFSH-treated (5 ng/ml; lane 2) Sc. Lower panel, Expression of the housekeeping gene, cyclophilin A, in untreated (lane 1) and rmFSH-treated (lane 2) Sc. C, Densitometric analysis of the inhibin-ßB expression in untreated vs. rmFSH-treated monkey Sc; cyclophilin A expression was used to normalize data. C, Control; F5, rmFSH (5 ng/ml). Each bar represents mean ± SEM of three or four wells from one group of culture.*, P < 0.01 vs. control; multiple-measures one-way ANOVA followed by Dunnett’s posttest.

 

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  		TABLE 1. Effect of recombinant monkey FSH or 8-Br-cAMP on proliferation of Sc cultured from pseudopubertal juvenile rhesus monkeys

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study was designed to evaluate the need of FSH for the production of two spermatogenically important Sc metabolites, lactate and E2, in primates. It is known that lack of FSH or FSH receptor in men does not necessarily result into complete blockade of spermatogenesis (38). However, lack of E2 or estrogen receptor activity is associated with infertility (17, 18), and injection of lactate in cryptorchid testes of adult rats significantly augments sperm production (39), lending support to the notion that lactate and E2 are essential for spermatogenesis. In a recent study, E2 has been demonstrated to induce Gc differentiation in 88% of seminiferous tubules of irradiated rats (40).

In the present study, lactate and E2 production by Sc cultured from 18-d-old rats was significantly augmented on treatment with FSH. Interestingly, Sc from pseudopubertal rhesus monkeys produced large amounts of lactate basally, and high doses of rmFSH failed to augment lactate production. In rat Sc, cAMP is known to augment the production of lactate to the same extent as that by FSH (41). The failure of 8-Br-cAMP to augment lactate production by monkey Sc is consistent with the inability of FSH to augment lactate production by these cells. In concurrence with previous reports, oFSH significantly stimulated aromatization of T to E2 by the 18-d-old rat Sc (15, 16, 42). In contrast, monkey Sc constitutively aromatized T to E2 without the aid of FSH. Whereas there was a 7-fold increase in the aromatizing ability of rat Sc due to FSH treatment, monkey Sc displayed similar high levels of aromatization of T to E2 even in the absence of FSH. These observations suggest that production of lactate and aromatization of T (E2 production) by monkey Sc are FSH-independent functions.

The bioefficacy of rmFSH and 8-Br-cAMP was validated by their ability to significantly (P < 0.05) augment lactate production by 18-d-old rat Sc. Pulsatile administration of recombinant human FSH to juvenile rhesus monkeys for 11 d is known to result in nearly doubling Sc numbers in vivo, indicating successful signaling by FSH to the Sc of juvenile monkeys (20). A significant (P < 0.05) increase in cAMP production by monkey Sc in response to rmFSH and an increase in cell numbers in response to rmFSH or 8-Br-cAMP treatment of monkey Sc confirmed that the dose of rmFSH (5 ng/ml) and 8-Br-cAMP (150 µM) used in this study was sufficient to trigger efficient signal transduction in these cells in vitro. Hence, the lack of FSH-mediated increase in lactate and E2 production cannot be attributed to defect(s) in FSH-receptor or signal transduction.

It cannot be ruled out that constitutive production of lactate and E2 by monkey Sc may be an artifact of the in vitro culture system due to altered environment or removal and/or dilution of factors that inhibit their production in vivo. However, this is unlikely because rmFSH as well as 8-Br-cAMP did induce Sc proliferation in vitro. Moreover, the increase in inhibin-ßB mRNA expression by rmFSH treatment also effectively demonstrates the ability of monkey Sc to respond to FSH in vitro. The reason for this species difference between the hormonal regulation of rat and monkey Sc functions is unknown and is worth exploring. Although increases in sperm production by FSH treatment are well documented, it is noteworthy that some men with inactivating mutations of the FSH receptor are reported to be fertile (38). This could also suggest that the production of every factor essential for Gc survival and formation of sperm may not be necessarily dependent on FSH in humans.

Our study does not question the importance of FSH therapy for the treatment of male infertility (27) but emphasizes that certain spermatogenically relevant functions of Sc (such as lactate and E2 production) are independent of FSH in primates. Hence, defects in such Sc functions may not necessarily be corrected by treatment with FSH and/or T. The inability of primate Sc to produce lactate and/or E2 may contribute to defective Gc differentiation in certain forms of male infertility. Treatment leading to elevation of E2 and lactate in the testicular milieu may complement the hormonal (FSH and T) therapy for obtaining qualitatively and quantitatively normal sperm in such individuals. Because live births have been reported using in vitro matured spermatids from male Gc (43, 44), a fraction of seminiferous tubules or Gc from such infertile individuals could be cultured in FSH-, T-, lactate-, and E2-rich medium for obtaining mature spermatids for use in assisted reproduction. Our study thus widens the scope for the diagnosis of certain forms of idiopathic male infertility and treatment of individuals who fail to respond to hormone therapy in a clinical situation.


   Acknowledgments
 
We are thankful to the director of the National Institute of Immunology for supporting this study. We are thankful to the staff of the Primate Research Center and Small Animal Facility. Thanks are due to Mr. Ram Singh and Mr. Birendra Roy for the technical assistance. We thank Dr. A. F. Parlow (NHPP, NIH) for providing the hormones used in this study. We thank Dr. Tony M. Plant for a critical review of the manuscript.

All experimental animals were maintained and handled according to the guidelines provided by Institutional Animal Ethics Committee, National Institute of Immunology, New Delhi, India.

None of the material submitted in this manuscript has been published or is under consideration for publication.


   Footnotes
 
First Published Online December 20, 2005

1 Y.S.D. and K.S. contributed equally to this work Back

Abbreviations: 8-Br-cAMP, 8-Bromoadenosine-cAMP; E2, estradiol; Gc, germ cell; HBSS, Hanks’ balanced salt solution; o, ovine; rm, recombinant monkey; Sc, Sertoli cell; T, testosterone.

This work was supported by grants to the National Institute of Immunology from the Department of Biotechnology and Indo-US Contraceptive and Reproductive Health Research Program.

Present address for Y.S.D.: Department of Physiology and Biophysics, University of Illinois, Chicago, Illinois 60612-7432.

Present address for B.S.: Biocon Biopharmaceuticals and Enzymes, 20th KM Hosur Road, Electronics City, Bangalore, India.

The authors have nothing to declare as a conflict of interest related to the material being published in this manuscript.

Received September 19, 2005.

Accepted December 9, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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