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Recombinant Human Follicle-Stimulating Hormone Administration Increases Testosterone Production in Men, Possibly by a Sertoli Cell-Secreted Nonsteroid Factor

División Endocrinología, Hospital Carlos G. Durand (O.L., S.A., C.T., C.A.), and Laboratorio de Estudios Hormonales (C.Z.), Buenos Aires; and Instituto de Analisis Biologicos Endocrinologies (D.A., M.C., H.S.), La Plata, Argentina

Address all correspondence and requests for reprints to: Dr. Oscar A. Levalle, División Endocrinología, Hospital Carlos G. Durand, Díaz Velez 5044, 1405 Buenos Aires, Argentina.

	Abstract

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We previously showed that recombinant human FSH (R-FSH) in males increased the testosterone (T) concentration in spermatic venous blood (SB). To investigate the effect of R-FSH on spermatic steroid levels and the action of steroid- and LH-free SB on isolated Leydig cells, nine normospermic males were studied during spermatic cord surgery. Peripheral blood and SB samples were collected before and 30 min after iv administration of 150 U R-FSH to measure LH, FSH, T, estradiol, 17-hydroxyprogesterone, and sex hormone-binding globulin, and in SB, androstenedione (⁴) and dehydroepiandrosterone (DHEA) were also measured. LH bioactivity was assessed by in vitro production of T in isolated Leydig cells. The actions of R-FSH and SB (steroid and LH free) were analyzed in the bioassay. Data are expressed as the mean ± SE.

FSH in peripheral blood and SB increased by 411% and 477% after R-FSH administration. R-FSH induced a significant increase in spermatic T (basal vs. 30 min, 326.4 ± 98.5 vs. 732.4 ± 152.8 ng/mL; P < 0.047) and in spermatic estradiol (289.5 ± 66.9 vs. 535.6 ± 83.4 pg/mL; P < 0.036). The T/⁴ ratio (36.9 ± 9.2 vs. 74.5 ± 13.3; P < 0.019) and the T/DHEA ratio (10.8 ± 1.1 vs. 22.4 ± 4.9; P < 0.024) increased significantly. In isolated Leydig cells, R-FSH did not change T production, but the SB (steroid and LH free) after R-FSH administration induced an increase in T production (3.3 ± 0.6 vs. 4.9 ± 0.6 ng/tube; P < 0.04). LH-like activity was found in a more than 50,000-Da fraction after centrifugation in Amicon filters, even in the presence of anti-LH.

These results suggest that R-FSH increases the production of T by Leydig cells through a Sertoli cell-released nonsteroid factor with a molecular mass greater than 50 kDa. The increase in the T/⁴ and T/DHEA ratios indicates that this factor would act by amplifying the LH response through the ⁵ pathway and the 17ß-hydroxysteroid dehydrogenase enzyme.

	Introduction

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FSH INDUCES, by acting on its specific receptor in the Sertoli cell (1), the synthesis and secretion of multiple proteins, including inhibin, androgen-binding protein, insulin-like growth factor I (IGF-I), transferrin, ceruloplasmin, plasminogen activator, and aromatase inhibitor, among many other factors (2), acting as a paracrine regulator of spermatogenesis and Leydig cell steroidogenesis by mechanisms that have not been completely elucidated to date. In particular, a protein secreted by cultured Sertoli cells has been recently reported to stimulate biosynthesis in the Leydig cell (3), although this action has not yet been confirmed in vivo.

However, there is also evidence that FSH-stimulated rat Sertoli cells synthesize estradiol (E) from testosterone (T) (4) and that FSH induces aromatase activity in cultured immature rat Sertoli cells (5). In a previous study we inferred that infertile idiopathic males with selective elevation of FSH had high concentrations of intratesticular E, which suggests that endogenous FSH could stimulate its production in Sertoli cells (6).

The effects of FSH on testicular steroidogenic function in men have been difficult to confirm to date, firstly because most of the in vivo or in vitro studies were performed in animals, and secondly because the FSH preparations used were contaminated with different degrees of LH, and therefore the possibility that small amounts of LH act directly on Leydig cells (7) cannot be excluded. Furthermore, it is known that under certain conditions, peripheral blood (PB), unlike spermatic venous blood (SB), may not be useful for the detection of variations in the production of testicular hormones due to a phenomenon of dilution in blood and extracellular fluid. Recently, we investigated T concentrations in SB in men before and after iv administration of recombinant human FSH (R-FSH) with no LH contamination, observing an increase in T in all cases (8).

The aims of the present study were 1) to investigate the effect of R-FSH on several sexual steroid levels in SB in patients undergoing inguinal canal surgery for pathologies unrelated to gonadal dysfunction, trying to establish its sites or mechanisms of action; and 2) to evaluate the effect of steroid- and LH-free SB on isolated Leydig cells, trying to confirm the presence of a nonsteroid factor capable of modifying testicular steroidogenesis in men. The understanding of these mechanisms might represent a further advance toward the rational treatment of male fertility disorders.

	Subjects and Methods

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Subjects

Nine patients, aged 21–43 yr, who had to undergo inguinal canal surgery for cysts in the spermatic cord, hydrocele, varicocele, or inguinal hernia, with no other demonstrable pathology were included in the study. All showed previous fertility and had no other history of congenital or acquired pathologies affecting fertility.

Testicular volume was normal (15 mL or more, assessed with Prader’s orchidometer). Neither endocrine nor any other chronic diseases were detected, and none of the patients was taking medication. Sperm counts (after 3 days of sexual abstinence) were made three times in six patients and twice in two patients over a period of 1–3 months. Semen parameters were within the normal range according to WHO guidelines (9).

Methods

Patients were administered rachidial anesthesia. During surgery, after locating the spermatic cord, 10 mL PB and 10 mL SB were collected. Immediately afterward, 150 U R-FSH were injected into the antecubital vein. Blood samples were withdrawn again 30 min after the R-FSH injection, by the end of the surgical procedure. Four men into whom saline instead of R-FSH was injected were used as controls. Recombinant human FSH for this clinical trial was supplied by Serono Laboratory (Braintree, MA).

LH, FSH, T, E, 17-hydroxyprogesterone (17OHP), and sex hormone-binding globulin (SHBG) were measured in PB and SB. Androstenedione (⁴), dehydroepiandrosterone (DHEA), and LH biological activity of SB were also measured. The biological activity of LH was determined in a bioassay with isolated Leydig cells by in vitro production of T. In the same bioassay, R-FSH was analyzed to rule out its possible direct effect on Leydig cells as well as the action of the SB itself on T production, with the aim of establishing a possible LH-like effect of SB on Leydig cells.

Hormonal measurements. For the determination of LH bioactivity, samples were previously extracted by ethyl ether to eliminate endogenous steroids. Samples were incubated for 6 h with Leydig cells from Siberian hamsters, which were isolated from testes by decapsulation, collagenization, and separation of seminiferous tubules by filtration through a 50-µm pore nylon mesh (10). A dilution of about 1,000,000 viable cells/tube was used. The incubation was performed in a Dubnoff metabolic incubator in an air environment containing 5% CO₂. Cell-induced T production was determined by RIA in the supernatant obtained from incubation. The method for T measurement is described below. The LH supplied with the Delfia LH Spec kit (Wallac, Turku, Finland) for measuring circulating LH was used as the standard. The sensitivity was 1 IU/L.

Additionally, to establish whether the SB samples exhibited LH-like activity, anti-LH antibody (National Pituitary Agency, NIH, Bethesda, MD) was added to the incubation medium to absorb endogenous LH from the sample. Preliminary experiments were performed under the bioassay conditions described above to determine the optimal anti-LH concentration to inhibit LH-induced T production by Leydig cells. As a result, a 1:40,000 final dilution was chosen, which inhibited the T production induced by the highest LH concentration used in the bioassay (75 IU/L).

LH and FSH were measured by a Delfia kit. The sensitivity for LH was 0.02 IU/L.

T was measured by a competitive RIA using a polyclonal antibody developed in our laboratory; T-H3 was used as tracer, and the free fraction was separated from the antibody-bound fraction with dextran carbon, as previously described (8). The method intra- and interassay errors were 5.5% and 7.5%, respectively. Quality controls, including sensitivity and specificity, have been previously described (8).

E was determined by a competitive RIA kit using antibody-coated tubes (Diagnostic Products Corp., Cleveland, OH). Intra- and interassay errors were 6.3% and 9.7%, respectively. The assay sensitivity and specificity were those reported by the manufacturer.

17OHP was determined by a competitive RIA kit using antibody-coated tubes from Immunotech (Marseille, Cedex, France). Intra- and interassay errors were 4.5% and 8.5%, respectively. The method sensitivity and specificity were those reported by the manufacturer. DHEA was measured by a competitive RIA kit from Immunotech, with antibody-coated tubes and ¹²⁵I-labeled DHEA as a tracer. The RIA was performed on ether-extracted serum or plasma samples. The antibody specificity was determined by assaying the cross-reactivity of 27 steroids, both natural and synthetic. The antibody showed 104% cross-reaction with DHEA sulfate and 68% cross-reaction with DHEA glucuronidate, which cannot be extracted with ether. The assay sensitivity was 0.3 ng/mL. Intra- and interassay coefficients of variation were 6% and 10%, respectively.

⁴ was determined by a competitive RIA kit from Immunotech, using antibody-coated tubes. Intra- and interassay errors were 4.5% and 8.2%, respectively. The assay sensitivity and specificity were those reported by the manufacturer.

The SHBG binding capacity was determined by Scatchard analysis with a solid phase method using concanavalin A-Sepharose, as described by Dunn et al. (11).

Statistical analysis. The t test for paired samples was used. When the data did not show a normal distribution, the Mann-Whitney test was used. Results are expressed as the mean ± SE. Statistical significance was considered at P < 0.05.

Patient’s consent. Written consent was obtained from all patients for their participation in the clinical trial, following a complete explanation of its characteristics. This study was approved by the ethics committee of the Durand Hospital of Buenos Aires.

	Results

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The iv administration of 150 U R-FSH induced a significant increase in circulating FSH levels in both PB and SB (411% and 477%, respectively), confirming that the dose administered was enough to induce pharmacological effects. There were no significant changes in LH, bioactive LH, or SHBG concentrations in PB or SB (Table 1). There was a significant increase in T (basal, 326.4 ± 84.9 ng/mL; 30 min, 732.4 ± 144.1 ng/mL; P < 0.039) and E (basal, 289.5 ± 66.9 pg/mL; 30 min, 535.6 ± 83.4 pg/mL; P < 0.019) in SB, with no changes in the remaining steroids. T levels in SB from control men were 322.5 ± 143.9 and 399.25 ± 201.04 ng/mL before and after surgery for inguinal hernia (no statistically significant differences were observed).

The bioassay of R-FSH in isolated Leydig cells did not show an increase in T, which confirms that this pharmacological preparation has no direct effect on Leydig cell steroidogenesis.

The Leydig cell bioassay of steroid-free SB collected 30 min post-R-FSH treatment incubated in the presence of anti-LH induced a significant increase in T production compared to that in basal SB (basal, 3.3 ± 0.6 ng/tube; 30 min, 4.9 ± 0.6 ng/tube; P < 0.009; Fig. 1).

View larger version (52K): [in this window] [in a new window]   Figure 1. T production induced by steroid-free SB added to the bioassay with isolated Leydig cells and anti-LH antibody in the nine patients and in the entire group before and after R-FSH administration. Dotted and ruled bars indicate before and after R-FSH administration, respectively. *, P < 0.05; **, P < 0.001; ***, P = < 0.009. Statistics were obtained by comparison of T values from triplicate Leydig cell incubations.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Multiple experimental and clinical studies have made it possible to establish with certainty the physiological action of LH in the human testis. This gonadotropin not only induces the synthesis and release of T, but also acts on the Leydig cell aromatase system, causing a significant increase in the intratesticular release and concentration of E (5, 12). Conversely, the difficulties for isolating Sertoli cells in culture to be used in bioassays as well as the contamination with LH of conventional human menopausal gonadotropin (HMG) preparations for treatments or clinical studies have made it impossible to determine with accuracy the testicular events induced by FSH.

Based on a previous study in which we observed that the iv administration of a R-FSH preparation increased the T concentration in SB in men (8), we designed the present work to investigate the mechanism of action of this phenomenon. To this end, firstly we determined several precursors and metabolites of T in SB before and after R-FSH administration. Secondly and to rule out the possible direct action of R-FSH on Leydig cells, we studied the production of T in vitro, adding R-FSH to the assay. Finally, the free steroid spermatic serum was added to a similar assay with anti-LH antibody, with the aim of establishing whether a nonsteroid factor was responsible for the action on steroidogenesis. It is known that the determination of testicular steroids in PB, because of their dilution in the bloodstream and extracellular fluid, may not reflect exactly the drug-induced physiological testicular changes. For example, T levels in SB are 50- to 100-fold higher than those in PB. Despite the possible variations resulting from dilution with arterial blood by anastomosis, this is a safer way of analyzing intratesticular events (13). With the iv administration of R-FSH, a significant increase in circulating FSH was obtained in both plasmatic and SB; therefore, it is assumed that the dose was enough to induce pharmacological effects, the most noticeable being the significant increases in T and E in the SB of the patients evaluated. Addition of the R-FSH to the in vitro assay of isolated Leydig cells did not induce T production, which rules out a direct effect of the hormone on such cells. In the bioassay with anti-LH, the greater in vitro production of T induced by steroid-free SB after R-FSH treatment suggests that the effect is produced by a Sertoli cell-secreted nonsteroid factor with a molecular mass greater than 50 kDa.

Our results appear to confirm in vivo the action of a Sertoli cell-secreted protein able to stimulate Leydig cell steroidogenesis (3), which is in agreement with previous findings in hypophysectomized immature rats, in which FSH not only stimulated the seminiferous tubule, but also induced hypertrophy, hyperplasia, and an increase in the number of Leydig cell LH receptors. The validity of these studies was questioned because of the possible LH contamination of the FSH preparations used (14, 15). However, recent studies using highly purified pituitary FSH or recombinant human FSH preparations indicate that FSH increases the number of LH receptors and the Leydig cell steroidogenic response in hypophysectomized rats (16, 17).

R-FSH did not change SHBG or LH levels or the bioactivity of LH in circulating blood; therefore, the changes observed do not seem to be related to a pituitary or steroid-binding effect.

Even though no R-FSH-induced changes were detected in concentrations of 17OHP, ⁴, and DHEA in SB, the significant increase in T/⁴ and T/DHEA ratios, but not in the T/17OHP ratio, and the decrease in the ⁴/17OHP ratio indicate that the Sertoli cell-secreted factor would act by amplifying the LH response through the ⁵ pathway and through the 17ß-hydroxysteroid dehydrogenase enzyme, an enzyme that catalyzes the conversion of ⁴ to T and that of DHEA to androstenediol (18, 19).

The increase in T observed after the R-FSH injection was a true effect, because the levels of T in four men treated with saline showed no statistically significant differences. The rise in SB E observed in the present study could be due to the T increment observed. However, a direct stimulation of Sertoli cell aromatase by R-FSH or an indirect stimulation through a Sertoli cell-secreted factor that would act on the 17ß-hydroxysteroid dehydrogenase enzyme cannot be discarded, because it is known that this enzyme not only catalyzes the conversion of T from its precursors ⁴ and DHEA, but also acts in the conversion of estrone to E (18, 19).

In conclusion, the results of this study show that the iv administration of pharmacological doses of R-FSH induces significant changes in testicular steroidogenesis, increasing the release of T and E. In vitro studies rule out a direct action of the hormone on Leydig cell and support the presence of a Sertoli cell-secreted nonsteroid factor that would act on testicular androgenic production. A number of factors (>40) have been found to modulate testicular function through autocrine, intracrine, and paracrine mechanisms (20). About 50% of those factors (or its messenger ribonucleic acids) were found in the human testis. However, only a few were shown to have stimulatory effects on animal steroidogenesis in vivo and/or in vitro. Among those detected in the human testis are inhibin, epidermal growth factor, IGF-I, TRH, substance P, endothelin I, and transforming growth factor-ß. The latter was shown to enhance the expression of a 50-kDa protein related to 2',5'-oligoadenylate synthetase in human sperm cells (21). IGF-I is a 7.5-kDa peptide; however, it is associated with six binding proteins, rendering complexes of 50 kDa and higher (22). Although we have mentioned only stimulatory factors described in the human, some substances found in other species should not be discarded. For example, a 70-kDa protein complex able to stimulate Leydig cell steroidogenesis secreted from rat Sertoli cells was reported (23). Further studies will allow identification of this factor to clarify the precise mechanisms involved in the in vivo stimulatory effect of R-FSH. These findings could be a step toward a rational use of physiological doses of FSH in therapy for reproductive disorders.

Received December 10, 1997.

Revised June 24, 1998.

Accepted July 6, 1998.

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