This Article Abstract Full Text (PDF) All Versions of this Article: 91/10/3962    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Matthiesson, K. L. Articles by Meachem, S. J. Search for Related Content PubMed PubMed Citation Articles by Matthiesson, K. L. Articles by Meachem, S. J. Related Collections Male Endocrinology Neuroendocrinology and Pituitary

The Relative Roles of Follicle-Stimulating Hormone and Luteinizing Hormone in Maintaining Spermatogonial Maturation and Spermiation in Normal Men

Prince Henrys Institute (K.L.M., R.I.M., L.O., D.M.R., P.G.S., S.J.M.) and Departments of Obstetrics and Gynaecology (K.L.M., R.I.M., D.M.R.) and Urology (M.F.), Monash University, Monash Medical Centre, Clayton, Victoria 3168, Australia

Address all correspondence and requests for reprints to: Kati Matthiesson, MBBS, FRACP, Prince Henrys Institute, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: kati.matthiesson{at}princehenrys.org.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context:. Male hormonal contraception via gonadotropin and intratesticular androgen withdrawal disrupts spermatogenesis at two principal sites: 1) spermatogonial maturation, and 2) spermiation.

Objective: The objective of this study was to explore the relative dependence of each stage of germ cell development on FSH and LH/intratesticular androgen action.

Design, Setting, and Participants: Eighteen men enrolled in this prospective, randomized 14-wk study at Prince Henrys Institute.

Interventions: Subjects (n = 6/group) were assigned to 6 wk of 1) testosterone (T) implant (4 x 200 mg sc once)+depot medroxy progesterone acetate (DMPA; 150 mg im once); 2) T implant+DMPA+FSH (300 IU sc twice weekly); and 3) T implant+DMPA+human chorionic gonadotropin (hCG; 1000 IU sc twice weekly as an LH substitute). Men then underwent a vasectomy and testicular biopsy with previously reported control data used for comparison.

Main Outcome Measures: Germ cell number (assessed by the optical disector stereological approach) and intratesticular androgen levels were determined.

Results: T+DMPA alone significantly suppressed type B spermatogonia, preleptotene through to pachytene spermatocytes, and round spermatids from control (P < 0.05). All germ cell subtypes were maintained at control levels by either FSH or LH activity, except pachytene spermatocytes, which were found to be lower in the hCG vs. FSH (P < 0.01) and control groups (P < 0.05).

Conclusions: FSH and LH maintained spermatogenesis independently in this gonadotropin-suppressed model. Compared with LH, FSH showed better maintenance of pachytene spermatocyte number, whereas improved conversion to round spermatids was suggested with hCG treatment. Future contraceptive treatment strategies must consider independent regulation of spermatogenesis by both FSH and LH/intratesticular androgens for maximum efficacy.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
IN MAN, BOTH gonadotropins are required to reinitiate and maintain quantitatively normal sperm production after experimentally induced oligoazoospermia (1, 2, 3, 4). However, the relative roles of FSH and LH/intratesticular androgens on germ cell development remain unclear. Male hormonal contraceptive regimens using androgens, alone or combined with a progestin, achieve similar conception rates to typical first-year usage of the female oral contraceptive pill (5, 6, 7, 8, 9). However, inadequate suppression of sperm density, delayed onset of action, and the potential for reappearance of sperm in the ejaculate (10, 11) remain significant issues for patient acceptability and efficacy. Previous work has reported predominant effects of male hormonal contraception on spermatogonial maturation and spermiation in testicular tissue from normal men (12, 13). A better understanding of the roles of FSH and LH/intratesticular androgens in germ cell development may allow for more effective approaches that hasten the onset of azoospermia and prevent reappearance of sperm in the ejaculate.

It is recognized that both FSH and LH are necessary for the initiation of spermatogenesis (14) and the establishment of fertility in men with hypogonadotropic hypogonadism (15). However, once initiated, human chorionic gonadotropin (hCG; as an LH substitute) alone can maintain spermatogenesis, albeit at a submaximal rate over the longer term (16). In contrast, the administration of FSH and testosterone (T) to men with hypogonadotropic hypogonadism is unable to induce or maintain spermatogenesis, with sperm concentrations falling to a mean of 0.3 ± 0.1 million/ml at 3 months and azoospermia by 6 months (15).

Additional data on the relative roles of FSH and LH in spermatogenesis come from a series of studies using gonadotropin withdrawal followed by selective reinitiation in normal young men. After treatment with T, 200 mg im weekly, and the induction of oligoazoospermia, hCG (5000 IU im three times weekly) (2, 4) or pituitary-derived LH (1100 IU sc daily) (17) was found to restore sperm concentration partially. In a similar study, suppression of serum LH and selective FSH replacement (100 IU sc daily) also partially restored sperm concentration (1). In such studies, the complete elimination of one or other gonadotropin cannot be assured, but such data support the contention that both FSH and LH/intratesticular androgens are needed for quantitatively normal spermatogenesis to occur.

In vivo animal models suggest that spermatogonial maturation is largely FSH dependent (18, 19, 20, 21); spermiogenesis appears to be reliant on LH/intratesticular T (22, 23), whereas spermiation requires both FSH and LH/intratesticular T (24). Mice solely lacking FSH action [receptor (25) or ß-subunit (26)] are able to complete spermatogenesis, albeit at a reduced level. In contrast, the loss of both FSH and LH action in the GnRH-deficient mouse (27) or of LH action alone [receptor (28, 29) or ß-subunit (30)] results in germ cell arrest at the pachytene spermatocyte or round spermatid stage, respectively. The late onset of limited spermatogenesis in the LH receptor knockout mouse appears to be due to LH-independent androgen action (31). Complete androgen receptor knockout mice (32) also do not progress past the pachytene spermatocyte stage, with the Sertoli cell-specific androgen receptor knockout model showing only occasional round spermatids (33, 34). Such data illustrate that germ cell development in the mouse can be completed in the absence of FSH but not androgen action.

In contrast, men with LH ß-subunit mutations (35, 36) may produce low numbers of sperm, but there are no data on germ cell development nor intratesticular androgen levels, leaving open the possibility of LH-independent androgen secretion/action as observed in the LH receptor knockout mouse. Data from the few reported men with FSH ß-subunit (37) or receptor (38) mutations indicate that most patients have severe oligospermia but others have higher sperm densities, suggesting that, although FSH may not be a prerequisite for completion of spermatogenesis, it may be of relatively greater importance in human compared with murine spermatogenesis.

The aim of this study was to establish whether exogenous FSH and LH are independently able to support spermatogenesis and specifically whether they differ in their support of germ cell progression. To this end, we performed a prospective, randomized, three-arm, 6-wk study using a "suppress and replace" model. Endogenous gonadotropins were suppressed using a combined androgen/progestin male hormonal contraceptive regimen together with their selective maintenance via administration of recombinant human (rh) FSH or hCG as an LH substitute. End points included reproductive hormone levels, sperm concentration, stereological evaluation of germ cell number, and testicular steroid measurement.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Eighteen men were recruited through media advertisement to participate in this study. The study protocol was approved by the Southern Health Human Research Ethics Committee, and all subjects gave written informed consent before screening. Men underwent medical interview, physical examination, and biochemical investigations. Subjects were required to fulfill each of the following criteria: 1) age 21–45 yr; 2) normal physical findings and normal testicular volumes; 3) two normal semen analyses according to World Health Organization criteria (39); 4) normal serum FSH, LH, T, SHBG, and estradiol (E2); and 5) normal liver and renal function and complete blood count. Men with a past history of hypertension; significant cardiovascular, thromboembolic, renal, hepatic, prostatic, and testicular disease; or infertility were excluded. Men with a past history of drug abuse or who were taking significant prescribed medications were also excluded, as were men who were subject to testing for steroid usage in competitive sports.

Study design

This research study was divided into three phases: a 4-wk screening phase, a 6-wk treatment phase, and a 4-wk recovery period. After screening, men were randomly assigned to one of the following three treatments groups (n = 6/group): 1) T implant, 800 mg sc once (4x 200 mg; Organon, Lane Cove, Australia) + depot medroxyprogesterone acetate (DMPA), 150 mg im once (2 ml @ 150 mg/ml; Pfizer, West Ryde, Australia); 2) T implant, 800 mg sc once, + DMPA, 150 mg Im once, + FSH, 300 IU sc twice weekly (Puregon; Organon); 3) T implant, 800 mg sc once, + DMPA, 150 mg im once, + hCG, 1000 IU sc twice weekly (Pregnyl; Organon). Blood for reproductive hormone analysis was drawn weekly at screening and before gonadotropin administration and vasectomy. Gonadotropin administration was performed by the investigators on d 0 of each week, with men in groups 2 and 3 educated to self administer either FSH (300 IU) or hCG (1000 IU sc) on d 4. Men were required to provide weekly semen samples with a minimum of 2 d abstinence before collection.

Assays

Serum FSH and LH were measured using sensitive immunofluorometric assays (40) (Delfia, hFSH kit no. A0710201; and hLH spec kit no. A031-101, Perkin-Elmer, Wallac Inc., Turku, Finland). The sensitivities of the FSH and LH assays were 0.01 and 0.015 IU/liter with an interassay variation of 13.4% and 15.3%, respectively. The dose ranges of the standards used in the respective assays were 0.015–10 IU/liter for FSH and 0.01–10 IU/liter for LH.

Serum hCG was measured in an automated immunoassay (Beckman Coulter Inc., Chaska, MN) with an assay sensitivity of 1.0 IU/liter and interassay variation across the working range of 2.3–9.5%.

Serum T was measured by an automated chemiluminescent immunoassay (Chiron Diagnostics, East Walpole, MA) with a sensitivity of 0.3 nmol/liter and interassay variation across the working range of 5.0–11.4%.

Serum SHBG was measured by an automated chemiluminescent immunoassay (catalog no. LKSH5; Diagnostic Products Corp., Los Angeles, CA) with a sensitivity of 2 nmol/liter and an interassay variation across the working range of 7.9–14.7%.

Serum E2 was measured by double-antibody competitive immunoassay (catalog no. KE2D5; Diagnostic Products Corp.) with a sensitivity of less than 20 pmol/liter and interassay variation across the working range of 7.3–14.7%.

Testicular biopsies and stereological assessment

The testicular biopsies were performed under general anesthesia using a previously described technique (41). Tissue for stereological analysis was fixed in Bouin’s fluid and embedded in methacrylate, and 25-µm sections were cut and stained with hematoxylin and eosin and germ cell number (type A dark spermatogonia, type A pale spermatogonia, all type A spermatogonia, type B spermatogonia, preleptotene spermatocytes, leptotene to zygotene spermatocytes, pachytene spermatocytes, steps 1–2 round spermatids, steps 3–6 elongating spermatids, and steps 7–8 elongated spermatids) was assessed using the optical disector approach as previously described (13).

Testicular steroids

Testicular steroids including T, dihydrotestosterone (DHT), 3Adiol, and 3ßAdiol were quantitated as previously described (13, 41). For 3Adiol and 3ßAdiol, all samples were assayed in the one assay, with intraassay variations (n = 3–4) from parallel fragments from the same testis sample of 10.0 and 18.1%, respectively. The intraassay variations for T and DHT were 15.4 and 8.0%, respectively, whereas interassay variations (n = 2) were 10.4–14.1% for T and 6.5–13.9% for DHT.

Control data

Previously reported control data on intratesticular steroid levels from five subjects were used for comparative analysis of intratesticular T, DHT, and 3Adiol levels (13). Testis biopsies from the same five control subjects (13) were processed for stereology and were counted along with biopsies from the current study, providing control data for comparison of germ cell numbers. Baseline characteristics for the control group (13) and three treatment groups were assessed with no significant differences found.

Statistical analyses

Data are shown as mean ± SEM, n = 5–6/group for all parameters. Statistical comparisons were performed using Sigmastat (Systat, Point Richmond, CA). All data were log transformed before analysis, and the variances were analyzed for homogeneity. Nonparametric statistics were used when equal variance testing failed. Serum hormone and sperm concentration data were analyzed by repeated-measures ANOVA followed by Tukey test to examine differences across time within treatment groups. Differences between control and treatment groups were analyzed by one-way ANOVA followed by Tukey test or, when normality testing failed, by Kruskal-Wallis one-way ANOVA on ranks followed by Dunn’s method for post hoc comparison.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Baseline characteristics

As outlined in Table 1, no baseline differences were detected between groups.

Serum FSH levels declined significantly at d 7–42 in the T+DMPA and T+DMPA+hCG groups, falling to nadir of 0.09 IU/liter (1.5% of baseline, d 35, P < 0.001) and 0.03 IU/liter (0.8% of baseline, d 28, P < 0.001), respectively (Fig. 1A). Although FSH levels appeared to be further suppressed in the T+DMPA+hCG group over T+DMPA alone, no significant differences were determined. Administration of FSH maintained levels at baseline and higher than that in the T+DMPA and T+DMPA+hCG treatment groups (d 7–42, P < 0.05). Serum LH levels declined significantly from baseline in all groups at d 7–42, with no differences in LH levels between groups detected (Fig. 1B).

View larger version (13K): [in this window] [in a new window]   FIG. 1. Changes in serum FSH (A) and LH (B) over the 6-wk treatment phase in 18 men (n = 6/group) randomized to receive: 1) T implant, 800 mg sc, + DMPA, 150 mg im once; 2) T implant+DMPA+FSH, 300 IU sc twice weekly; or 3) T implant plus DMPA plus hCG, 1000 IU sc twice weekly. Serum FSH and LH levels are shown at screening and then weekly thereafter. Serum FSH levels declined from baseline, d 7–42, P < 0.01 in the T+DMPA and T+DMPA+hCG groups. In the T+DMPA+FSH group, FSH levels were maintained at baseline and were higher than that of the other two treatment groups, d 7–42, P < 0.05. Serum LH levels declined from baseline in all groups, d 7–42, P < 0.001, with no differences in LH levels between groups detected. Data are graphed logarithmically and shown as mean ± SEM, n = 6/group.

In the T+DMPA group, sperm concentration fell from baseline from d 28 onward (P < 0.001) (Fig. 2). In the T+DMPA+FSH group, sperm concentration was decreased (mean concentration of 44 ± 27 million/ml) at d 42 (P = 0.003). No fall in sperm concentration was seen in the T+DMPA+hCG group during the treatment period (mean sperm concentration at d 42, 49 ± 11 million/ml, P = 0.627). Sperm concentration was found to be higher in the FSH and hCG groups compared with the T+DMPA group, d 28 and 35 (P < 0.05) and at d 42 (hCG group alone, P < 0.05). No differences were found between the T+DMPA+FSH and T+DMPA+hCG treatment groups at d 42. In the T+DMPA+FSH group, one subject had a d-42 sperm concentration of 152 million/ml, whereas the other four available subject sperm concentrations were less than 30 million/ml. In the T+DMPA+hCG group, four of the five available subject sperm concentrations were greater than 30 million/ml at d 42.

View larger version (16K): [in this window] [in a new window]   FIG. 2. Changes in sperm concentration after 6-week treatment phase with: 1) T implant, 800 mg sc, + DMPA, 150 mg im once; 2) T implant+DMPA+FSH, 300 IU sc twice weekly; or 3) T implant+DMPA+hCG, 1000 IU sc twice weekly. In the T+DMPA group, sperm concentration fell from baseline, d 28 onward (P < 0.001). Sperm concentration was higher in the FSH and hCG groups compared with the T+DMPA group, d 28 and 35 (P < 0.05) and at d 42 (hCG group alone, P < 0.05). Data were graphed logarithmically and shown as mean ± SEM, n = 5–6/group.

No differences in T levels from baseline were detected in the T+DMPA treatment group, whereas there were higher levels found in the T+DMPA+FSH group at some time points (d 14–28 and 42, P < 0.05) (Fig. 3A). In the T+DMPA+hCG group, T levels were higher than baseline, except on d 35 (198–263% of baseline, P < 0.05) throughout the study treatment period (Fig. 3A). Serum SHBG levels remained in the normal reference interval (13–71 nmol/liter), with no differences detected except a 77% of baseline decrease at d 21 (P = 0.021) in the T+DMPA+FSH group (data not shown). Serum E2 levels in the T+DMPA group and T+DMPA+FSH group remained in the normal range (25–130 pmol/liter). In the T+DMPA+hCG group, E2 levels were higher than baseline and the other two treatment groups (P < 0.05) throughout the treatment phase of the study (Fig. 3B).

View larger version (25K): [in this window] [in a new window]   FIG. 3. Changes in serum T (A) and E2 (B) levels in 18 men (n = 6/group) randomized to receive: 1) T implant, 800 mg sc, + DMPA, 150 mg im once; 2) T implant+DMPA+FSH, 300 IU sc twice weekly; or 3) T implant+DMPA+hCG, 1000 IU sc twice weekly. Serum levels are shown at screening and then weekly thereafter. Data are shown as mean ± SEM, n = 6/group. Shaded areas represent normal reference intervals. No differences in T levels from baseline were determined in the T+DMPA group. Higher levels from baseline were found in the T+DMPA+FSH group, d 14–28 and 42 (P < 0.05), and the T+DMPA+hCG group, d 7–42 except on d 35 (P < 0.05). Serum E2 levels were elevated in the T+DMPA+hCG group alone, d 7–42 (P < 0.01).

Germ cell data for control and treated men are expressed as number per Sertoli cell [a constant denominator with contraceptive treatment (42)] (Table 2) and as percent control (Fig. 4). Relative to controls, T+DMPA treatment resulted in suppression of type B spermatogonia through to round spermatids (P < 0.05). No decrease in any germ cell subtype was seen in the T+DMPA+FSH group, with only pachytene spermatocytes being reduced in the T+DMPA+hCG group compared with control (P < 0.05).

View larger version (23K): [in this window] [in a new window]   FIG. 4. Changes in germ cell number over the 6-wk treatment phase in men (n = 6/group) randomized to receive: 1) T implant, 800 mg sc, + DMPA, 150 mg im once; 2) T implant+DMPA+FSH, 300 IU sc twice weekly; or 3) T implant+DMPA+hCG, 1000 IU sc twice weekly. Data are expressed as a percentage of that seen in control subjects (n = 5). Germ cell subtypes are described along the x-axis as follows: type A dark spermatogonia (Ad); type A pale spermatogonia (Ap); all type A spermatogonia (All A); type B spermatogonia (B); preleptotene spermatocytes (Pl); leptotene to zygotene spermatocytes (L-Z); pachytene spermatocytes (PS); steps 1–2 round spermatids (rST); steps 3–6 elongating spermatids (3–6 ST); steps 7–8 elongated spermatids (7–8 ST); sperm concentration in the ejaculate on the day of testicular biopsy (sperm). T+DMPA treatment suppressed type B spermatogonia through to rSTs (P < 0.05). All germ cell subtypes were maintained similar to control in the FSH- and hCG-treated groups except PS in the T+DMPA+hCG group (P < 0.05). PS were significantly lower in the T+DMPA+hCG compared with T+DMPA+FSH group (P < 0.01). Logarithmic second y-axis depicting sperm concentration in the ejaculate on the day of testicular biopsy (sperm) with sperm concentrations of 0 million/ml assigned a value of 0.001. No differences between treatment group 7–8 ST were determined. However, significantly higher sperm concentrations in the T+DMPA+hCG group compared with T+DMPA group (P < 0.05) were noted, with a similar, although not significant, trend also found in the T+DMPA+FSH group.

In regard to the process of spermiation, examination of steps 7–8 elongated spermatid numbers revealed no differences among treatment groups or compared with the control group. However, mean sperm concentrations derived from semen analyses collected on the day of testicular biopsy were significantly higher in the T+DMPA+hCG group compared with T+DMPA alone (P < 0.05), with a similar, although not significant, trend also noted in the T+DMPA+FSH group.

Testicular steroid levels

T+DMPA and T+DMPA+FSH treatments resulted in falls in intratesticular T (0.5–2% of control, P < 0.001), with the administration of hCG resulting in increased intratesticular T levels (262% of control, P = 0.006) (Table 3). T+DMPA and T+DMPA+hCG administration did not result in significant changes in intratesticular DHT levels (55% of control, P = 0.156; and 223% of control, P = 0.086, respectively), whereas levels in the T+DMPA+FSH group significantly decreased (27% of control, P < 0.01). Levels of 3Adiol differed significantly from control in only the T+DMPA+FSH treatment group (13% of control, P = 0.011). A positive correlation was found between intratesticular T levels and preleptotene (R = 0.817, P = 0.047), pachytene (R = 0.936, P = 0.006) and round spermatid number (R = 0.847, P = 0.033) in the hCG treatment group alone (data not shown).

Recovery and adverse events

Gonadotropin and T levels were followed until recovery. All treatments were well tolerated, with no serious adverse events.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
This study has shown that FSH and LH independently support spermatogenesis in a gonadotropin-suppressed human model. Using stereological approaches to assess germ cell development, the effect of selective gonadotropin maintenance in men receiving an androgen-progestin hormonal contraceptive regimen was examined. In the absence of gonadotropin supplementation, a similar reduction in sperm concentration and germ cell number (from type B spermatogonia through to round spermatids) to that previously reported (13, 41) was noted. FSH treatment delayed a fall in sperm concentration until d 42, whereas hCG treatment prevented any decline. All germ cell subtypes were maintained by either gonadotropin, except for pachytene spermatocytes, which were lower in the hCG-treated group. The data suggest that FSH is more efficient that hCG in supporting pachytene spermatocyte populations in this experimental paradigm. Also, the conversion of pachytene spermatocytes to round spermatids, a process associated with completion of the second meiotic division, appeared to be more efficient in the hCG-treated group despite reduced numbers of pachytene spermatocytes.

In rodents and monkeys, spermatogonial maturation is largely reliant on FSH, whereas the later stages of spermatogenesis, including completion of meiosis, spermiogenesis, and spermiation, are thought to be more dependent on intratesticular androgens (12, 43). To the extent possible, this study of limited subject number has now shown that FSH or LH/intratesticular androgens can similarly maintain germ cell development. Low levels of serum FSH (1–2% control) and LH (<0.4% control), and intratesticular T (<2% control), persist after combined androgen-progestin treatment, but it is unclear whether such levels are biologically active. LH-independent Leydig cell androgen secretion (31) may exist in man and may thereby lie beyond the reach of existing male hormonal contraceptive regimens. One could speculate whether synergistic effects may occur between an observed residual intratesticular T of 26 nmol in the FSH-treated group, or between serum FSH levels of 0.04 IU/liter in the intratesticular T-replete hCG group. Also of consideration is the role of other nonsteroidal hCG-stimulated Leydig cell products, such as INSL3, which may provide spermatogenic support via reduced germ cell apoptosis (44).

In the present study, spermatogonial and preleptotene through to zygotene spermatocyte numbers in the FSH- and hCG-treated groups were not different. Data in rats have shown the withdrawal of FSH via passive immunoneutralization results in decreased spermatogonial number (75% of control) (45, 46). Similarly, Bonnet monkeys (Macaca radiata) immunized against the FSH receptor demonstrate reduced spermatogonial to spermatocyte conversion (47). LH suppression has not been shown to affect spermatogonial maturation in rats significantly (48), with data in monkeys lacking. Despite previous data suggesting a relatively greater role for FSH than LH/intratesticular androgens in the process of spermatogonial maturation in rodents and monkeys, the current study findings suggest that, at least in the short term, LH/intratesticular androgens can maintain spermatogonia and their progression into spermatocytes.

The present study supports that FSH and/or LH/intratesticular androgens are able to support spermatocyte development. However, FSH appears potentially more effective than LH/intratesticular androgens in supporting pachytene spermatocytes, given the reduction in their number in the hCG-treated group. This is consistent with animal data that has shown that FSH can partially maintain spermatocyte number in hypophysectomized (49, 50, 51) and GnRH antagonist-treated rats (52) and monkeys (21). It is generally accepted that both FSH and intratesticular androgens can support spermatocyte survival in rodents (53). However, FSH appears more effective than intratesticular androgens in the current experimental paradigm.

Either gonadotropin was able to support spermiogenesis to a similar degree, although analysis was limited due to the 6-wk study timeframe. Interestingly, round spermatid numbers were not different in the FSH- and hCG-treated groups, despite the latter having reduced pachytene spermatocyte numbers, possibly indicating increased efficiency of conversion between these two germ cell subtypes in the presence of maintained intratesticular androgens. This proposition is supported by studies in mice with Sertoli cell-specific ablation of androgen receptors (SCARKO mice) in which androgen has been shown to be essential for the completion of meiosis and the production of haploid spermatids (34).

Spermiation was supported by either gonadotropin in a similar way to that seen in rats (24). However, hCG may be more effective in supporting this process, given that only the hCG-treated group maintained sperm concentration at baseline, despite similar stage 7–8 elongated spermatid numbers across both treatment groups. Furthermore, although FSH treatment was able to maintain sperm concentrations similarly to hCG, only one man in the FSH-treated group maintained a sperm concentration above 30 million/ml, compared with four in the hCG-treated group.

As with previous studies, the combination of T+DMPA resulted in a profound fall in intratesticular T levels to 1–2% baseline (13, 41, 54). In this study, a dose of hCG 1000 IU sc twice weekly was chosen based on recently reported testicular needle aspiration data (55), with the aim of maintaining high-normal intratesticular T levels. However, substantially higher intratesticular T levels (262% of control) were achieved than those seen in that study in which a minimum total weekly dose of 750-1000 IU hCG was required to maintain baseline intratesticular T and a dose of 1500–2000 IU weekly resulted in a 26% increase over baseline (55). Our data suggest that lower doses of hCG may be necessary to better mimic physiological intratesticular T levels. However, based on rodent data (43), it is unlikely that there would be any difference in spermatogenic outcome between normal and the modestly elevated intratesticular T levels seen in this study.

Intratesticular 5 reduced androgen levels did not fall to the same extent as intratesticular T, with T+DMPA supporting the up-regulation of 5 reductase in the face of reduced intratesticular T (41, 56). Although FSH did not maintain intratesticular DHT and intratesticular 3Adiol levels similar to that of control, there were no significant differences between the T+DMPA and T+DMPA+FSH groups or ratios of intratesticular T:intratesticular DHT. Previous work in rodents has shown a positive role for FSH in the regulation of type 1, 5 reductase (57), a finding not supported by the current study data, which suggested reduced 5 reductase activity in the FSH-treated group.

In conclusion, the stereological assessment of germ cell number after short-term selective maintenance of FSH or LH/intratesticular T showed that either gonadotropin was able to support all stages of the spermatogenic process including spermatogonial maturation, meiosis, spermiogenesis, and spermiation. Both FSH and hCG were able to maintain germ cell numbers at that of control with pachytene spermatocytes, the only germ cell subtype to differ significantly between treatment groups (lower with hCG), underscoring the role of FSH in maintenance of spermatocyte number. In contrast, no differences in round spermatid number were found, potentially highlighting the importance of intratesticular androgens in the completion of meiosis. This study also demonstrates that relatively small doses of hCG are required to maintain physiological intratesticular T and may have implications for dose adjustment in the treatment of men with secondary testicular failure. These data characterize the dual gonadotropic dependence of human spermatogenesis and accentuate the need for male hormonal contraceptive strategies to withdraw FSH and LH/intratesticular androgens maximally to initiate and maintain spermatogenic suppression.

	Acknowledgments

 
The authors acknowledge the excellent assistance of Fiona McLean for intratesticular steroid assays, G. A. Balourdos for germ cell counting, Enid Pruysers for serum hormone measurements, and Clinical Research Nurses Ms. Joanne McKenzie and Ms. Elise Forbes.

	Footnotes

 
This study was funded by the National Health and Medical Research Council (NH&MRC) of Australia, Program Grant no. 241000, NH&MRC Post Graduate Scholarship Grant ID 241031 (to K.L.M.), and Research Fellowships 169020 (to R.I.M.) and 169201 (to D.M.R.).

The authors have nothing to disclose.

First Published Online August 8, 2006

Abbreviations: DHT, Dihydrotestosterone; DMPA, depot medroxyprogesterone acetate; E2, estradiol; hCG, human chorionic gonadotropin; rh, recombinant human; T, testosterone.

Received May 26, 2006.

Accepted July 31, 2006.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Matsumoto AM, Karpas AE, Paulsen CA, Bremner WJ 1983 Reinitiation of sperm production in gonadotropin-suppressed normal men by administration of follicle-stimulating hormone. J Clin Invest 72:1005–1015[Medline]
2. Matsumoto AM, Bremner WJ 1985 Stimulation of sperm production by human chorionic gonadotropin after prolonged gonadotropin suppression in normal men. J Androl 6:137–143[Abstract/Free Full Text]
3. Matsumoto AM, Karpas AE, Bremner WJ 1986 Chronic human chorionic gonadotropin administration in normal men: evidence that follicle-stimulating hormone is necessary for the maintenance of quantitatively normal spermatogenesis in man. J Clin Endocrinol Metab 62:1184–1192[Abstract]
4. Bremner WJ, Matsumoto AM, Sussman AM, Paulsen CA 1981 Follicle-stimulating hormone and human spermatogenesis. J Clin Invest 68:1044–1052[Medline]
5. WHO Task Force on Methods for the Regulation of Male Fertility 1990 Contraceptive efficacy of testosterone-induced azoospermia in normal men. Lancet 336:955–959[CrossRef][Medline]
6. WHO Task Force on Methods for the Regulation of Male Fertility 1996 Contraceptive efficacy of testosterone-induced azoospermia and oligozoospermia in normal men. Fertil Steril 65:821–829[Medline]
7. Gu YQ, Wang XH, Xu D, Peng L, Cheng LF, Huang MK, Huang ZJ, Zhang GY 2003 A multicenter contraceptive efficacy study of injectable testosterone undecanoate in healthy Chinese men. J Clin Endocrinol Metab 88:562–568[Abstract/Free Full Text]
8. Turner L, Conway AJ, Jimenez M, Liu PY, Forbes E, McLachlan RI, Handelsman DJ 2003 Contraceptive efficacy of a depot progestin and androgen combination in men. J Clin Endocrinol Metab 88:4659–4667[Abstract/Free Full Text]
9. Trussell J, Kost K 1987 Contraceptive failure in the United States: a critical review of the literature. Stud Fam Plann 18:237–283[CrossRef][Medline]
10. Gui YL, He CH, Amory JK, Bremner WJ, Zheng EX, Yang J, Yang PJ, Gao ES 2004 Male hormonal contraception: suppression of spermatogenesis by injectable testosterone undecanoate alone or with levonorgestrel implants in chinese men. J Androl 25:720–727[Abstract/Free Full Text]
11. Gu YQ, Tong JS, Ma DZ, Wang XH, Yuan D, Tang WH, Bremner WJ 2004 Male hormonal contraception: effects of injections of testosterone undecanoate and depot medroxyprogesterone acetate at eight-week intervals in chinese men. J Clin Endocrinol Metab 89:2254–2262[Abstract/Free Full Text]
12. Matthiesson KL, McLachlan RI 2006 Male hormonal contraception: concept proven, product in sight? Hum Reprod Update 12:463–482[Abstract/Free Full Text]
13. McLachlan RI, O’Donnell L, Stanton PG, Balourdos G, Frydenberg M, de Kretser DM, Robertson DM 2002 Effects of testosterone plus medroxyprogesterone acetate on semen quality, reproductive hormones, and germ cell populations in normal young men. J Clin Endocrinol Metab 87:546–556[Abstract/Free Full Text]
14. Matsumoto AM, Bremner WJ 1987 Endocrinology of the hypothalamic-pituitary-testicular axis with particular reference to the hormonal control of spermatogenesis. Baillieres Clin Endocrinol Metab 1:71–87[CrossRef][Medline]
15. Schaison G, Young J, Pholsena M, Nahoul K, Couzinet B 1993 Failure of combined follicle-stimulating hormone-testosterone administration to initiate and/or maintain spermatogenesis in men with hypogonadotropic hypogonadism. J Clin Endocrinol Metab 77:1545–1549[Abstract]
16. Depenbusch M, von Eckardstein S, Simoni M, Nieschlag E 2002 Maintenance of spermatogenesis in hypogonadotropic hypogonadal men with human chorionic gonadotropin alone. Eur J Endocrinol 147:617–624[Abstract]
17. Matsumoto AM, Paulsen CA, Bremner WJ 1984 Stimulation of sperm production by human luteinizing hormone in gonadotropin-suppressed normal men. J Clin Endocrinol Metab 59:882–887[Abstract]
18. Haywood M, Spaliviero J, Jimemez M, King NJ, Handelsman DJ, Allan CM 2003 Sertoli and germ cell development in hypogonadal (hpg) mice expressing transgenic follicle-stimulating hormone alone or in combination with testosterone. Endocrinology 144:509–517[Abstract/Free Full Text]
19. Meachem SJ, Stanton PG, Schlatt S 2005 Follicle-stimulating hormone regulates both Sertoli cell and spermatogonial populations in the adult photoinhibited djungarian hamster testis. Biol Reprod 72:1187–1193[Abstract/Free Full Text]
20. Meachem SJ, Wreford NG, Robertson DM, McLachlan RI 1997 Androgen action on the restoration of spermatogenesis in adult rats: effects of human chorionic gonadotrophin, testosterone and flutamide administration on germ cell number. Int J Androl 20:70–79[CrossRef][Medline]
21. Weinbauer GF, Behre HM, Fingscheidt U, Nieschlag E 1991 Human follicle-stimulating hormone exerts a stimulatory effect on spermatogenesis, testicular size, and serum inhibin levels in the gonadotropin-releasing hormone antagonist-treated nonhuman primate (Macaca fascicularis). Endocrinology 129:1831–1839[Abstract]
22. O’Donnell L, McLachlan RI, Wreford NG, de Kretser DM, Robertson DM 1996 Testosterone withdrawal promotes stage-specific detachment of round spermatids from the rat seminiferous epithelium. Biol Reprod 55:895–901[Abstract]
23. O’Donnell L, McLachlan RI, Wreford NG, Robertson DM 1994 Testosterone promotes the conversion of round spermatids between stages VII and VIII of the rat spermatogenic cycle. Endocrinology 135:2608–2614[Abstract]
24. Saito K, O’Donnell L, McLachlan RI, Robertson DM 2000 Spermiation failure is a major contributor to early spermatogenic suppression caused by hormone withdrawal in adult rats. Endocrinology 141:2779–2785[Abstract/Free Full Text]
25. Dierich A, Sairam MR, Monaco L, Fimia GM, Gansmuller A, LeMeur M, Sassone-Corsi P 1998 Impairing follicle-stimulating hormone (FSH) signaling in vivo: targeted disruption of the FSH receptor leads to aberrant gametogenesis and hormonal imbalance. Proc Natl Acad Sci USA 95:13612–13617[Abstract/Free Full Text]
26. Kumar TR, Wang Y, Lu N, Matzuk MM 1997 Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat Genet 15:201–204[CrossRef][Medline]
27. Singh J, O’Neill C, Handelsman DJ 1995 Induction of spermatogenesis by androgens in gonadotropin-deficient (hpg) mice. Endocrinology 136:5311–5321[Abstract]
28. Zhang FP, Poutanen M, Wilbertz J, Huhtaniemi I 2001 Normal prenatal but arrested postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice. Mol Endocrinol 15:172–183[Abstract/Free Full Text]
29. Lei ZM, Mishra S, Zou W, Xu B, Foltz M, Li X, Rao CV 2001 Targeted disruption of luteinizing hormone/human chorionic gonadotropin receptor gene. Mol Endocrinol 15:184–200[Abstract/Free Full Text]
30. Ma X, Dong Y, Matzuk MM, Kumar TR 2004 Targeted disruption of luteinizing hormone beta-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility. Proc Natl Acad Sci USA 101:17294–17299[Abstract/Free Full Text]
31. Zhang FP, Pakarainen T, Poutanen M, Toppari J, Huhtaniemi I 2003 The low gonadotropin-independent constitutive production of testicular testosterone is sufficient to maintain spermatogenesis. Proc Natl Acad Sci USA 100:13692–13697[Abstract/Free Full Text]
32. Yeh S, Tsai MY, Xu Q, Mu XM, Lardy H, Huang KE, Lin H, Yeh SD, Altuwaijri S, Zhou X, Xing L, Boyce BF, Hung MC, Zhang S, Gan L, Chang C 2002 Generation and characterization of androgen receptor knockout (ARKO) mice: an in vivo model for the study of androgen functions in selective tissues. Proc Natl Acad Sci USA 99:13498–13503[Abstract/Free Full Text]
33. Chang C, Chen YT, Yeh SD, Xu Q, Wang RS, Guillou F, Lardy H, Yeh S 2004 Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells. Proc Natl Acad Sci USA 101:6876–6881[Abstract/Free Full Text]
34. De Gendt K, Swinnen JV, Saunders PT, Schoonjans L, Dewerchin M, Devos A, Tan K, Atanassova N, Claessens F, Lecureuil C, Heyns W, Carmeliet P, Guillou F, Sharpe RM, Verhoeven G 2004 A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proc Natl Acad Sci USA 101:1327–1332[Abstract/Free Full Text]
35. Valdes-Socin H, Salvi R, Daly AF, Gaillard RC, Quatresooz P, Tebeu PM, Pralong FP, Beckers A 2004 Hypogonadism in a patient with a mutation in the luteinizing hormone ß-subunit gene. N Engl J Med 351:2619–2625[Abstract/Free Full Text]
36. Weiss J, Axelrod L, Whitcomb RW, Harris PE, Crowley WF, Jameson JL 1992 Hypogonadism caused by a single amino acid substitution in the ß subunit of luteinizing hormone. N Engl J Med 326:179–183[Medline]
37. Phillip M, Arbelle JE, Segev Y, Parvari R 1998 Male hypogonadism due to a mutation in the gene for the ß-subunit of follicle-stimulating hormone. N Engl J Med 338:1729–1732[Free Full Text]
38. Tapanainen JS, Aittomaki K, Min J, Vaskivuo T, Huhtaniemi IT 1997 Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility. Nat Genet 15:205–206[CrossRef][Medline]
39. World Health Organization 1999 WHO Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge, UK: Cambridge University Press
40. Robertson DM, Pruysers E, Stephenson T, Pettersson K, Morton S, McLachlan RI 2001 Sensitive LH and FSH assays for monitoring low serum levels in men undergoing steroidal contraception. Clin Endocrinol (Oxf) 55:331–339[CrossRef][Medline]
41. Matthiesson KL, Stanton PG, O’Donnell L, Meachem SJ, Amory JK, Berger R, Bremner WJ, McLachlan RI 2005 Effects of testosterone and levonorgestrel combined with a 5-reductase inhibitor or gonadotropin-releasing hormone antagonist on spermatogenesis and intratesticular steroid levels in normal men. J Clin Endocrinol Metab 90:5647–5655[Abstract/Free Full Text]
42. Zhengwei Y, Wreford NG, Royce P, de Kretser DM, McLachlan RI 1998 Stereological evaluation of human spermatogenesis after suppression by testosterone treatment: heterogeneous pattern of spermatogenic impairment. J Clin Endocrinol Metab 83:1284–1291[Abstract/Free Full Text]
43. McLachlan RI, O’Donnell L, Meachem SJ, Stanton PG, de Kretser DM, Pratis K, Robertson DM 2002 Identification of specific sites of hormonal regulation in spermatogenesis in rats, monkeys, and man. Recent Prog Horm Res 57:149–179[Abstract/Free Full Text]
44. Kawamura K, Kumagai J, Sudo S, Chun SY, Pisarska M, Morita H, Toppari J, Fu P, Wade JD, Bathgate RA, Hsueh AJ 2004 Paracrine regulation of mammalian oocyte maturation and male germ cell survival. Proc Natl Acad Sci USA 101:7323–7328[Abstract/Free Full Text]
45. Shetty J, Marathe GK, Dighe RR 1996 Specific immunoneutralization of FSH leads to apoptotic cell death of the pachytene spermatocytes and spermatogonial cells in the rat. Endocrinology 137:2179–2182[Abstract]
46. Meachem SJ, McLachlan RI, Stanton PG, Robertson DM, Wreford NG 1999 FSH immunoneutralization acutely impairs spermatogonial development in normal adult rats. J Androl 20:756–762; discussion 755[Abstract/Free Full Text]
47. Moudgal NR, Sairam MR, Krishnamurthy HN, Sridhar S, Krishnamurthy H, Khan H 1997 Immunization of male bonnet monkeys (M. radiata) with a recombinant FSH receptor preparation affects testicular function and fertility. Endocrinology 138:3065–3068[Abstract/Free Full Text]
48. Marathe GK, Shetty J, Dighe RR 1995 Selective immunoneutralization of luteinizing hormone results in the apoptotic cell death of pachytene spermatocytes and spermatids in the rat testis. Endocrine 3:705–709
49. Russell LD, Corbin TJ, Borg KE, De Franca LR, Grasso P, Bartke A 1993 Recombinant human follicle-stimulating hormone is capable of exerting a biological effect in the adult hypophysectomized rat by reducing the numbers of degenerating germ cells. Endocrinology 133:2062–2070[Abstract]
50. Kerr JB, Maddocks S, Sharpe RM 1992 Testosterone and FSH have independent, synergistic and stage-dependent effects upon spermatogenesis in the rat testis. Cell Tissue Res 268:179–189[CrossRef][Medline]
51. Bartlett JM, Weinbauer GF, Nieschlag E 1989 Differential effects of FSH and testosterone on the maintenance of spermatogenesis in the adult hypophysectomized rat. J Endocrinol 121:49–58[Abstract]
52. Chandolia RK, Weinbauer GF, Behre HM, Nieschlag E 1991 Evaluation of a peripherally selective antiandrogen (Casodex) as a tool for studying the relationship between testosterone and spermatogenesis in the rat. J Steroid Biochem Mol Biol 38:367–375[CrossRef][Medline]
53. O’Donnell L, Meachem S, Stanton PG, McLachlan RI 2006 The endocrine regulation of spermatogenesis. In: Neill JD, ed. Knobil and Neill’s physiology of reproduction. 3rd ed. San Diego: Elsevier; 1017–1069
54. Coviello AD, Bremner WJ, Matsumoto AM, Herbst KL, Amory JK, Anawalt BD, Yan X, Brown TR, Wright WW, Zirkin BR, Jarow JP 2004 Intratesticular testosterone concentrations comparable with serum levels are not sufficient to maintain normal sperm production in men receiving a hormonal contraceptive regimen. J Androl 25:931–938[Abstract/Free Full Text]
55. Coviello AD, Matsumoto AM, Bremner WJ, Herbst KL, Amory JK, Anawalt BD, Sutton PR, Wright WW, Brown TR, Yan X, Zirkin BR, Jarow JP 2005 Low-dose human chorionic gonadotropin maintains intratesticular testosterone in normal men with testosterone-induced gonadotropin suppression. J Clin Endocrinol Metab 90:2595–2602[Abstract/Free Full Text]
56. Anderson RA, Wallace AM, Wu FC 1996 Comparison between testosterone enanthate-induced azoospermia and oligozoospermia in a male contraceptive study. III. Higher 5 alpha-reductase activity in oligozoospermic men administered supraphysiological doses of testosterone. J Clin Endocrinol Metab 81:902–908[Abstract]
57. Pratis K, O’Donnell L, Ooi GT, Stanton PG, McLachlan RI, Robertson DM 2003 Differential regulation of rat testicular 5alpha-reductase type 1 and 2 isoforms by testosterone and FSH. J Endocrinol 176:393–403[Abstract]

This article has been cited by other articles:

				S. T. Page, J. K. Amory, and W. J. Bremner Advances in Male Contraception Endocr. Rev., June 1, 2008; 29(4): 465 - 493. [Abstract] [Full Text] [PDF]

				S. M. Ruwanpura, R. I. McLachlan, K. L. Matthiesson, and S. J. Meachem Gonadotrophins regulate germ cell survival, not proliferation, in normal adult men Hum. Reprod., February 1, 2008; 23(2): 403 - 411. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 91/10/3962    most recent Author Manuscript (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Matthiesson, K. L. Articles by Meachem, S. J. Search for Related Content PubMed PubMed Citation Articles by Matthiesson, K. L. Articles by Meachem, S. J. Related Collections Male Endocrinology Neuroendocrinology and Pituitary