Biol Reprod
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print February 1, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.048413
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
74/5/938    most recent
biolreprod.105.048413v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Allan, C. M.
Right arrow Articles by Handelsman, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Allan, C. M.
Right arrow Articles by Handelsman, D. J.
Agricola
Right arrow Articles by Allan, C. M.
Right arrow Articles by Handelsman, D. J.
BIOLOGY OF REPRODUCTION 74, 938–944 (2006)
DOI: 10.1095/biolreprod.105.048413
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Maintenance of Spermatogenesis by the Activated Human (Asp567Gly) FSH Receptor During Testicular Regression Due to Hormonal Withdrawal1

Charles M. Allan 2 , Alvaro Garcia , Jenny Spaliviero , Mark Jimenez , and David J. Handelsman 

Andrology Laboratory, University of Sydney, ANZAC Research Institute, Concord Hospital, Sydney, New South Wales 2139, Australia

ABSTRACT

The first activating mutation of the FSH receptor (FSHR*D567G) was identified in a gonadotropin-deficient hypophysectomized man who exhibited persistent spermatogenesis and fertility with only androgen replacement. We have determined the ability of FSHR* activity to maintain spermatogenesis and/or steroidogenesis during gonadotropin and androgen deprivation in mature transgenic FSHR* mice (Tg(Abpa-FSHR*D567G)1Cmal), hereafter referred to as Tg-FSHR* mice. Testes of untreated adult Tg-FSHR* males were equivalent in weight to nontransgenic controls but exhibited increased total Sertoli cell (24%) and spermatogonia (34%) numbers and nonsignificantly elevated spermatocyte-spermatid numbers (13%–17%). During sustained GNRH1 agonist treatment that markedly reduced (96%–98%) serum LH and testosterone (T) and decreased serum FSH (68%–72%), the testes of GNRH1 agonist-treated Tg-FSHR* mice remained significantly larger than treated nontransgenic controls. After 4 wk of gonadotropin suppression, Sertoli cell numbers were reduced in Tg-FSHR* testes to levels comparable with nontransgenic testes, whereas spermatogonia numbers were maintained at higher levels relative to nontransgenic testes. However, after 8 wk of GNRH1 agonist treatment, the total spermatogonia, spermatocyte, or postmeiotic spermatid numbers were reduced to equivalent levels in Tg-FSHR* and nontransgenic mice. FSHR* effects were further examined in gonadotropin-deficient hypogonadal Gnrh1hpg/Gnrh1hpg (Gnrh1–/–) mice during testicular regression following withdrawal of T after maximal T-stimulated spermatogenesis. After 6 wk of T withdrawal, spermatogonia, spermatocyte, and postmeiotic spermatid numbers in Tg-FSHR* Gnrh1–/– testes decreased to levels found in untreated Tg-FSHR* Gnrh1–/– testes. Basal serum T levels in untreated Tg-FSHR* Gnrh1–/– males were 2-fold higher than Gnrh1–/– controls, but following T treatment/withdrawal, serum T and epididymal weights declined to basal levels found in nontransgenic Gnrh1–/– mice. Therefore, FSHR* was unable to sustain circulating T or androgen-dependent epididymal size or postmeiotic spermatogenic development. We conclude that FSHR* activity enhances Sertoli and spermatogenic development in normal testes but has limited ability to maintain spermatogenesis during gonadotropin deficiency, in which the testicular response provided by the FSHR*D567G mutation resembled typical FSH-mediated but not steroidogenic activity.

follicle-stimulating hormone receptor, Sertoli cells, spermatogenesis, testis, testosterone

INTRODUCTION

The gonadal actions of FSH are mediated via a specific G-protein coupled FSH receptor (FSHR) located in the testicular Sertoli or ovarian granulosa cells [1]. An inactivating mutation in the human FSHR permits men to remain fertile despite having smaller testes [2], whereas affected females have complete ovarian failure [2–6]. Similarly, targeted disruption of mouse Fshr or Fshb genes preserved male fertility despite reducing testis size, whereas all females remained sterile [7–9]. In contrast, infertility occurs in men lacking normal circulating FSH [10–15], which suggests that FSH is necessary for human male fertility (reviewed in [16]). The requirement for FSH activity to support human spermatogenesis was further highlighted by the identification of a mutated FSHR (FSHR*D567G, denoted FSHR*) in a hypophysectomized man who retained spermatogenesis and fertility despite receiving testosterone (T) but with uncorrected complete gonadotropin deficiency [17]. Because the single amino acid substitution (Asp567Gly) occurred in the third cytoplasmic loop that resembled known sites for activating mutations in the related LH [18] and TSH receptors [19], it was proposed that FSHR* provided the necessary constitutive FSH activity in the absence of circulating ligand. In vitro analysis verified that this mutant FSHR* exhibits increased ligand-independent constitutive activity (elevating basal intracellular cAMP levels) relative to wild-type (wt) FSHR [1, 20–22], although in vivo verification of the activating status of this FSHR mutation has been difficult to demonstrate [23].

We previously showed that transgenic FSHR* stimulated autonomous FSH-like activity in hypogonadal (Gnrh1–/–, also known as hpg) mouse testes [24], which normally fail to develop postnatally because of a major deletion in the Gnrh1 gene producing complete functional gonadotropin deficiency [25]. Whereas FSHR* promoted early germ cell development, the incomplete postmeiotic germ cell development in Tg-FSHR* Gnrh1–/– testes contrasted with the full spermatogenesis and fertility in the original hypophysectomized man with the mutant FSHR* [17]. However, this transgenic Gnrh1–/– model evaluated the ability of FSHR* to initiate spermatogenesis on a background of congenital gonadotropin deficiency, but not the capacity of FSHR* to maintain spermatogenesis following gonadotropin loss after completion of testicular development at maturity. We previously showed different hormonal requirements for initiation versus maintenance of spermatogenesis in Gnrh1–/– mice, whereby maintenance required at least an order of magnitude lower T than initiation [26]. Therefore, it is possible that the arrested spermatogenic phenotype of Tg-FSHR* Gnrh1–/– mice was due to a different capacity of FSHR* to initiate, rather than maintain, complete germ cell maturation. The Tg-FSHR* Gnrh1–/– model also suggested that paracrine actions of constitutive FSHR signalling elevated T production [24]. Despite pituitary ablation, the original FSHR* case also maintained higher than expected circulating T levels (≥4.9 nmol/L) and normal semen sperm parameters during 4 mo withdrawal from T replacement therapy. These observations raise the possibility that the mutated FSHR* maintained spermatogenesis and/or stimulated androgen production in the absence of gonadotropins. Therefore, our current work has evaluated the capacity of transgenic FSHR* to maintain spermatogenesis and/or androgen levels following gonadotropin or androgen deprivation in Tg-FSHR* mice.

MATERIALS AND METHODS

Animals

The transgenic RR.3 and RR.4 lines expressing the mutant (Asp567Gly) human FSHR* under control of the rat Abpa promoter (Tg(Abpa-FSHR*D567G)1Cmal) have previously been described [24], and are herein referred to as Tg-FSHR* mice. Males expressing transgenic FSHR* on a gonadotropin-deficient hypogonadal Gnrh1hpg/Gnrh1hpg (Gnrh1–/–) background were obtained by crossbreeding animals heterozygous for the Gnrh1 gene deletion, determined by detection of wt or disrupted Gnrh1 gene or Tg-FSHR* PCR products as described [24, 27]. Animals were raised and housed at the ANZAC Research Institute under controlled conditions (12L:12D, 19–22°C) with ad libitum access to food and water. All animal procedures were approved by the Central Area Health Services Animal Welfare Committee and performed in accordance to the National Health and Medical Research Council code of practice for the care and use of animals and the NSW Animal Research Act (1985). Littermates or age-matched control Gnrh1–/– or wt (Gnrh1+/+ or Gnrh1+/–) males were used for comparison with transgenic males.

Animal Treatments, Serum, and Tissue Collection

GNRH1 agonist (Zoladex) treatment. Adult Tg-FSHR* males and age-matched nontransgenic controls were injected with Zoladex (a gift from AstraZeneca) for 4 (s.c. 3.5 mg pellet) or 8 wk (consecutive 3.5 mg pellets on Day 1, then Day 28). All untreated and GNRH1 agonist-treated animals were killed at 26 wk of age.

T treatment. Tg-FSHR*, nontransgenic littermates, or age-matched Gnrh1–/– males received a subdermal 1-cm Silastic implant (Dow Corning) containing crystalline T at 21 days old as described [28]. After 6 wk of T treatment, the implants were removed under anesthesia, and animals were examined after 2 or 6 wk of T withdrawal. Under terminal Rompun-ketamine anesthesia, blood was collected via cardiac puncture and serum stored at –20°C; then, animals were perfused with Bouin fixative and tissue was collected, weighed, processed, and embedded in hydroxymethylmethacrylate resin (Technovit 7100; Kulzer and Co.) as described [28]. Thin (3-µm) or thick (18- to 20-µm) testis sections were cut using a Polycut S microtome (Reichert Jung) and stained with 0.5% toluidine blue or periodic acid-Schiff respectively; the latter sections were used for stereological evaluation (n = 4–8 mice/group).

Hormone Assays

Serum FSH levels were determined using a two-site immunofluorometric assay as described [29], using mouse FSH standard (AFP-5308D; NIDDKD). Serum LH levels were measured by radioimmunoassay as described [30], using iodination grade rat LH (rLH I-9) and mouse LH standard (AFP-5306A; NIDDKD). Serum samples were assayed in duplicate and assay detection was 200 pg/ml FSH and 70 pg/ml LH. Serum T levels were measured in duplicate by radioimmunoassay as previously described [31].

Stereological Analysis

Testicular Sertoli and germ cell populations were quantified using the optical-disector technique as described [28]. Uniform random sampling of fixed tissue sections (three blocks representing upper, middle, and lower poles of testis) was performed by light microscopy (x100/1.35 objective) using unbiased sample frames created by Olympus CAST grid software (Olympus Corp.). Total cell numbers per testis = numerical cell densities x testicular volume (testis weights/specific gravity d = 1.04 g/ml). Germ cell estimates were broadly grouped into spermatogonia, primary spermatocytes (preleptotene-pachytene), round spermatids, and elongated spermatids.

Data Analysis

All statistical analysis was performed using SPSS, version 11.0 (SPSS, Inc.), or NCSS (NCSS). For GNRH1 agonist or T treatment studies, comparisons of transgenic and treatment effects were determined by two-way ANOVA. Data for transgenic versus nontransgenic samples at equal time points used unpaired t-tests, and comparison of transgenic or nontransgenic samples at different time points used one-way ANOVA. Differences were regarded as significant when P < 0.05. All data and graph plots are presented as mean ± SEM.

RESULTS

FSHR* Activity in Normal or GNRH1 Agonist-Treated Mice

Untreated mice. The in vivo effects of transgenic FSHR* expression on normal spermatogenesis and serum hormone levels were examined in untreated mature males. Serum LH, FSH, and T levels were not significantly different in age-matched transgenic and nontransgenic males (Fig. 1). In addition, Tg-FSHR* testes weights were similar to nontransgenic testes (Fig. 2); however, stereological analysis revealed differences in the testicular cell populations. The absolute number of Sertoli cells was significantly higher (24%, P < 0.01) in untreated Tg-FSHR* testes compared to nontransgenic controls (Fig. 2). Likewise, spermatogonia numbers were significantly greater (34%, P < 0.05) in Tg-FSHR* compared to nontransgenic testes (Fig. 3). Postmitotic germ cell populations were not significantly different, although primary spermatocytes (13%, P = 0.171) and postmeiotic spermatid numbers (17%, P = 0.086) were nonsignificantly higher in Tg-FSHR* relative to nontransgenic testes (Fig. 3).


Figure 1
View larger version (14K):
[in this window]
[in a new window]
  		FIG. 1. Serum FSH, LH, and T levels in mature 26-wk-old Tg-FSHR* (black) or nontransgenic (white) males either untreated (0 wk) or administered GNRH1 agonist (Zoladex) for 4 or 8 wk to suppress gonadotropin secretion. All hormone levels were significantly reduced after 4 and 8 wk GNRH1 agonist treatment. Values are mean ± SEM.


Figure 2
View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2. Testis weights and total Sertoli cell numbers in mature 26-wk-old Tg-FSHR* (black) or nontransgenic (white) males either untreated (0 wk) or administered GNRH1 agonist (Zoladex) for 4 or 8 wk. Testis weights were significantly reduced by GNRH1 agonist-induced gonadotropin suppression, but Tg-FSHR* testes were significantly heavier (two-way ANOVA), and at 4 wk GNRH1 agonist treatment, weights were maintained, compared to significantly reduced (shown by asterisk) nontransgenic testes. GNRH1 agonist-induced suppression of serum gonadotropins and T significantly reduced the above-normal (shown by asterisk) Sertoli cell numbers in Tg-FSHR* testes to the level found in treated nontransgenic testes. Values are mean ± SEM.


Figure 3
View larger version (29K):
[in this window]
[in a new window]
  		FIG. 3. Total testicular spermatogonia, primary spermatocyte (preleptotene-pachytene), round spermatid, and elongated spermatid numbers (x 106) in mature untreated (wk 0) or GNRH1 agonist-treated Tg-FSHR* (black) or nontransgenic (white) mice were quantified as described in Materials and Methods. All germ cell populations were significantly reduced by gonadotropin suppression. Significant changes are detailed in Results, noting that total spermatogonia were significantly higher in untreated and 4-wk GNRH1 agonist-treated transgenic compared to nontransgenic mice, as indicated by asterisks. Values are mean ± SEM.

Gonadotropin-deprived mature mice. Effects of transgenic FSHR* on spermatogenic maintenance during gonadotropin suppression were examined in mature mice receiving sustained GNRH1 agonist (Zoladex) delivery to reduce gonadotropin secretion. Body weights of GNRH1 agonist-treated or untreated age-matched Tg-FSHR* and nontransgenic males were not significantly different (data not shown). Four and eight weeks of sustained GNRH1 agonist administration significantly reduced serum FSH (two-way ANOVA, P < 0.001), LH, and T (two-way ANOVA, P < 0.002) levels relative to untreated age-matched controls (Fig. 1). Eight weeks of Zoladex suppressed serum FSH levels (68%–72%) to equivalent concentrations and markedly decreased serum LH and T (96%–98%) to similar levels in Zoladex-treated Tg-FSHR* and nontransgenic males (Fig. 1).

Gonadal effects of transgenic FSHR* during GNRH1 agonist treatment were first examined by comparison of testis weights between Tg-FSHR* and nontransgenic groups. GNRH1 agonist treatment significantly reduced testis weights of Tg-FSHR* and nontransgenic mice (two-way ANOVA, P < 0.001); however, testis weights of Tg-FSHR* mice were significantly higher (two-way ANOVA, P < 0.05) than those of nontransgenic mice. After 4 wk of GNRH1 agonist, testes weights of Tg-FSHR* mice were not significantly reduced compared to those of untreated transgenic mice (one-way ANOVA, P = 0.12), whereas testes of nontransgenic mice were significantly reduced (one-way ANOVA, P < 0.05) by 18% (Fig. 2). After 8 wk of prolonged GNRH1 agonist administration, the testes weights of transgenic and nontransgenic mice were significantly reduced to 54% and 41%, respectively, of untreated age-matched animals (Fig. 2).

Stereological assessment of testis cell types revealed that GNRH1 agonist administration significantly reduced the total Sertoli cell population in Tg-FSHR* (two-way ANOVA, P < 0.01) but not in nontransgenic mice (Fig. 2). After 8 wk of Zoladex treatment, Sertoli cell numbers declined 35% in GNRH1 agonist-treated Tg-FSHR* testes to the levels found in treated nontransgenic testes. Administration of GNRH1 agonist significantly reduced (two-way ANOVA, P < 0.0001) all four germ cell populations examined in transgenic and nontransgenic mice (Fig. 3). However, Tg-FSHR* testes contained higher total spermatogonia (two-way ANOVA, P < 0.01) and primary spermatocyte (two-way ANOVA, P < 0.05) numbers compared to nontransgenic testes, whereas total round (two-way ANOVA, P = 0.055) and elongated (two-way ANOVA, P = 0.062) spermatid numbers were nonsignificantly higher in Tg-FSHR* relative to nontransgenic testes. In particular, after 4 wk of GNRH1 agonist, total spermatogonia numbers, although reduced, were 37% higher (one-way ANOVA, P < 0.01) in treated Tg-FSHR* compared to treated nontransgenic males, and were equivalent to numbers in untreated nontransgenic mice (Fig. 3). Stained sections from Tg-FSHR* and nontransgenic testis demonstrate the comparison of Sertoli and spermatogonia cells before and after 4 wk of GNRH1 agonist treatment (Fig. 4). After 8 wk of GNRH1 agonist treatment, the reduced total spermatogonia and different postmitotic germ cell populations were not significantly higher (e.g., P = 0.26 or P = 0.38 for round or elongated spermatids respectively) in Tg-FSHR* relative to nontransgenic testes (Fig. 3).


Figure 4
View larger version (142K):
[in this window]
[in a new window]
  		FIG. 4. Testicular histology of untreated or GNRH1 agonist treated Tg-FSHR* or nontransgenic 26-wk-old mice. Toluidine-blue stained 3-µm sections of testes from untreated nontransgenic (A) or Tg-FSHR* (B) mice in comparison to treated nontransgenic (C) or Tg-FSHR* (D) after 4 wk of sustained GNRH1 agonist administration, all shown at same magnification. Distinct Sertoli (SC) or spermatogonia (Sg) cells are indicated by triangle or narrow arrows, respectively. Original magnification x40.

FSHR* activity in Gnrh1–/– mice

To evaluate transgenic FSHR* activity in the functional absence of circulating FSH ligand, qualitatively complete spermatogenesis in gonadotropin-deficient hypogonadal Gnrh1–/– mice was first induced by T treatment, and then spermatogenic maintenance was examined following T withdrawal. Testes of T-treated Tg-FSHR* Gnrh1–/– mice were 1.7-fold (P < 0.001) larger than treated nontransgenic Gnrh1–/– testes before T withdrawal (Fig. 5). T withdrawal significantly decreased testes weights of Tg-FSHR* and nontransgenic Gnrh1–/– mice (two-way ANOVA, P < 0.001); however, Tg-FSHR* Gnrh1–/– testes remained significantly larger (two-way ANOVA, P < 0.001) than treated nontransgenic Gnrh1–/– testes. Two and six weeks of T withdrawal significantly reduced Tg-FSHR* Gnrh1–/– testis weight by 60% and 78%, respectively (Fig. 5). Likewise, nontransgenic Gnrh1–/– testis weight significantly declined 51% and 74% after 2 and 6 wk of T withdrawal, consistent with our previous regression data [26]. After 6 wk of androgen withdrawal, Tg-FSHR* Gnrh1–/– testes remained 1.4-fold greater (P < 0.05) than nontransgenic Gnrh1–/– controls. Stained sections of Tg-FSHR* and nontransgenic Gnrh1–/– testes after T treatment and following T withdrawal highlighted changes in germ cell populations, such as higher spermatogonia numbers in Tg-FSHR* males and overall loss of germ cells after T withdrawal (Fig. 6).


Figure 5
View larger version (16K):
[in this window]
[in a new window]
  		FIG. 5. Testis weights and total Sertoli cell numbers in Tg-FSHR* (black) and nontransgenic (white) Gnrh1–/– mice during the T withdrawal regime described in Materials and Methods. Three-week-old gonadotropin-deficient hypogonadal Gnrh1–/– mice received T implants for 6 wk to induce spermatogenesis (wk 0), followed by T withdrawal for 2 or 6 wk as indicated. Significant differences are detailed in Results, noting that T withdrawal significantly reduced testes weights but that Tg-FSHR* Gnrh1–/– testes remained significantly higher than nontransgenic Gnrh1–/– controls. Sertoli cell numbers in transgenic or nontransgenic Gnrh1–/– mice were unchanged by T withdrawal. Values are mean ± SEM.


Figure 6
View larger version (142K):
[in this window]
[in a new window]
  		FIG. 6. Testicular histology of nontransgenic or Tg-FSHR* Gnrh1–/– mice after 6 wk T treatment (A and B, respectively) and then after 6 wk T withdrawal (C and D, respectively) as described in Materials and Methods. Shown are toluidine blue-stained 3-µm testis sections, all at the same magnification. Representative Sertoli (SC) or spermatogonia (Sg) cells are indicated by triangles or narrow arrows respectively. Original magnification x40.

Stereological estimation was used to compare total Sertoli and germ cell numbers in Tg-FSHR* and nontransgenic Gnrh1–/– testes after the T implant treatment/withdrawal regime. Total Sertoli cell numbers were unchanged in Tg-FSHR* and nontransgenic Gnrh1–/– testes after T treatment/withdrawal (Fig. 5). Total spermatogonia numbers were 2.7-fold higher (P < 0.05) in T-treated Tg-FSHR* relative to those of treated nontransgenic Gnrh1–/– testes, and were significantly decreased by T withdrawal (two-way ANOVA, P < 0.001) in transgenic but not in nontransgenic Gnrh1–/– testes. After 6 wk of T withdrawal, spermatogonia remained significantly higher (2-fold, P < 0.02) than corresponding numbers in nontransgenic Gnrh1–/– testes (Fig. 7). Likewise, T withdrawal significantly reduced total primary spermatocyte numbers by 32%–42% (P < 0.001) in transgenic and nontransgenic Gnrh1–/– testes, but spermatocyte numbers remained significantly higher (two-way ANOVA, P < 0.001) in these Tg-FSHR* compared to nontransgenic Gnrh1–/– testes. Round and elongated spermatid numbers were significantly reduced (two-way ANOVA, P < 0.001) after T withdrawal but remained higher (two-way ANOVA, P < 0.005) in Tg-FSHR* compared to nontransgenic Gnrh1–/– testes (Fig. 7). After 6 wk of T removal, total primary spermatocytes were significantly higher (1.7-fold, P < 0.02) and total spermatids nonsignificantly higher (3-fold, P = 0.11) in Tg-FSHR* compared to nontransgenic Gnrh1–/– testes. However, compared to untreated Tg-FSHR* Gnrh1–/– testes, there was no significant difference found between the total numbers of spermatogonia (P = 0.25), spermatocyte (P = 0.84), or round (P = 0.19) or elongated (P = 0.14) spermatid populations in Tg-FSHR* Gnrh1–/– testes after T treatment/withdrawal (Fig. 7).


Figure 7
View larger version (29K):
[in this window]
[in a new window]
  		FIG. 7. Total testicular germ cell populations (x 106) in Tg-FSHR* (black circles) and nontransgenic (white circles) Gnrh1–/– mice during the T withdrawal regime were quantified as described in Materials and Methods. Three-week-old mice received T treatment for 6 wk to induce spermatogenesis (shown as wk 0), followed by T withdrawal for 2 or 6 wk as indicated. All four germ cell populations were significantly higher in Tg-FSHR* compared to nontransgenic Gnrh1–/– testes; however, after 6 wk T withdrawal there were no significant differences in germ cell numbers, as detailed in Results, in treated Tg-FSHR* compared to untreated mature Tg-FSHR* Gnrh1–/– testes (shown by black triangles for comparison). Values are mean ± SEM.

To determine whether FSHR* stimulated steroidogenesis independently of gonadotropins, serum T levels were compared in Tg-FSHR* and nontransgenic Gnrh1–/– males. Serum T concentrations in untreated Tg-FSHR* Gnrh1–/– males were 2-fold higher (P < 0.02) than those in nontransgenic Gnrh1–/– males (5.0 ± 2.7 vs. 2.9 ± 1.6 nM; n = 17 per group). T treatment via silastic implants maintained elevated serum T levels of approximately 40 nM in transgenic and nontransgenic Gnrh1–/– mice [24]. Serum T declined to equivalent basal levels in Tg-FSHR* and nontransgenic Gnrh1–/– mice after 6 wk of T withdrawal (1.2 ± 0.4 vs. 0.9 ± 0.09 nM; n = 9 transgenic and 7 nontransgenic). Epididymal weights were also similar in transgenic and nontransgenic Gnrh1–/– males after 6 wk of T withdrawal (5.01 ± 0.39 vs. 4.79 ± 0.48 mg; n = 5 transgenic and 6 nontransgenic).

DISCUSSION

We have evaluated the ability of the activating FSHR* mutation to maintain spermatogenesis during gonadotropin deficiency, which was highlighted by its actions in the original hypophysectomized FSHR* man who retained spermatogenesis and fertility in the absence of both gonadotropins [17]. Our past analysis of in vivo FSHR* actions focused on the initiation of spermatogenesis in Tg-FSHR* gonadotropin-deficient hypogonadal Gnrh1–/– mice [24]. To extend this research, we present two approaches to explore the in vivo consequences of FSHR* activity upon the maintenance of preexisting spermatogenesis, as existed in the first FSHR* case described, rather than the initiation of germ cell maturation in Tg-FSHR* mice. Pharmacological suppression of gonadotropins in mature Tg-FSHR* mice allowed investigation of FSHR* maintenance of full spermatogenesis, whereas examination of Tg-FSHR* Gnrh1–/– mice by using a T treatment-withdrawal regime permitted analysis of potential spermatogenic and steroidogenic maintenance independently of circulating FSH ligand.

Expression of transgenic FSHR* in normal mouse testes produced cellular changes indicative of enhanced FSH activity. Total Sertoli cell and spermatogonia populations were increased in Tg-FSHR* testes, suggesting that this mutant receptor increased overall FSH-mediated activity during the crucial postnatal window for FSH-induced Sertoli cell proliferation. We previously showed that transgenic FSHR* expression increased the capacity of postnatal testis to bind FSH [24]. Therefore, higher numbers of FSH-responsive Sertoli and spermatogonial cells in mature Tg-FSHR* males may reflect increased Sertoli cell-surface FSHR expression and upregulated mitogenic signaling. An increased Sertoli cell population in Tg-FSHR* mice is consistent with studies that showed that postnatal administration of high doses of exogenous FSH elevated total Sertoli cell numbers above normal in adult rats [32, 33]. However, mice heterozygous for a disrupted wt Fshr have normal Sertoli and germ cell numbers [34], demonstrating that 2-fold changes to functional wild-type Fshr presence has no apparent impact upon testicular development. Therefore, it is more likely that elevated Sertoli and early germ cell development reflect increased constitutive activity provided by the FSHR* mutation than simply FSHR overexpression per se. Despite significant changes to the mitotic cells within the seminiferous tubules, testis size was not significantly altered and there was no observed difference in the fertility of Tg-FSHR* males compared to normal nontransgenic males [24].

In our present study, the higher than normal total Sertoli cell population in mature Tg-FSHR* mice was unexpectedly reduced by pharmacological suppression of serum FSH, LH, and T, whereas Sertoli cell numbers remained unchanged in nontransgenic controls. It is possible that the maintenance of higher Sertoli cell numbers in Tg-FSHR* testes was dependent upon circulating FSH levels, but a precise mechanism for the GNRH1 agonist-induced Sertoli cell loss remains unknown. Despite reduced hormone levels and Sertoli cell loss, the testicular weights of treated Tg-FSHR* mice were initially maintained, in contrast to the significantly regressed weights of nontransgenic testes. The reduced testicular regression of Tg-FSHR* relative to nontransgenic males was consistent with more germ cells maintained in Tg-FSHR* testes. In particular, transgenic FSHR* expression preferentially sustained the FSH-responsive spermatogonia population after 4 wk of sustained GNRH1 agonist treatment. However, the presence of transgenic FSHR* did not provide longer-term protection for spermatogenesis, because testicular regression from prolonged GNRH1 agonist treatment reached equivalent levels in transgenic and nontransgenic animals. Therefore, despite delaying regression of spermatogenic function, transgenic FSHR* was unable to significantly maintain any additional germ cell development in the gonadotropin-deprived adult mouse testis.

Circulating FSH levels were not as completely reduced as serum LH and T during prolonged GNRH1 agonist treatment, highlighting the well-known distinct regulation of FSH and LH pituitary secretion. Because residual FSH activity may influence the pattern of testicular regression observed in this pharmacological model, we further examined the spermatogenic and steroidogenic effects of FSHR* during testicular regression in the Gnrh1–/– mouse, which has complete postnatal functional FSH/LH deficiency [16, 35, 36]. Using an established T treatment to stimulate maximal androgen-specific spermatogenic completion in Gnrh1–/– testes [26–28], subsequent withdrawal of T for 6 wk was predicted to produce testicular regression, manifest as loss of testis weight and, in particular, loss of androgen-dependent meiotic and postmeiotic germ cells [26].

Total Sertoli numbers were not reduced following T withdrawal in either Tg-FSHR* or nontransgenic Gnrh1–/– testes. Maintenance of Sertoli cells in FSH-deficient Tg-FSHR* Gnrh1–/– testes may indicate that the loss of Sertoli cell numbers in GNRH1 agonist-treated normal (Gnrh1+/+ or Gnrh1+/–) Tg-FSHR* testes was due to suppression of FSH but not T. Overall germ cell numbers were elevated in treated Tg-FSHR* Gnrh1–/– testes, consistent with recent work showing that FSHR* activity increased early germ cell development in Gnrh1–/– males [31]. After T withdrawal, higher spermatogonia numbers in Tg-FSHR* testes were significantly reduced, whereas lower spermatogonia numbers in nontransgenic Gnrh1–/– testes did not change during testicular regression, which suggests that FSHR* and T activity combined in Gnrh1–/– testes to enhance spermatogonial development. Meiotic and postmeiotic germ cells were all significantly reduced in Tg-FSHR* and nontransgenic Gnrh1–/– testes following androgen withdrawal. Although germ cell populations were higher in transgenic relative to nontransgenic Gnrh1–/– testes, 6 wk of androgen deprivation reduced the spermatogonia and meiotic and postmeiotic germ cell populations in regressed Tg-FSHR* Gnrh1–/– testes to the numbers found in untreated Tg-FSHR* Gnrh1–/– testes. Thus, complete enumeration of cell populations revealed that FSHR* maintained FSH-like spermatogenic development in gonadotropin-deficient Gnrh1–/– testes following withdrawal of T after maximal T-induced spermatogenesis. However, FSHR* did not maintain any significant androgen-dependent germ cell development in these Gnrh1–/– males. The in vivo FSH-like activity provided by FSHR* in the absence of circulating FSH ligand is more likely due to increased constitutive activity provided by the mutation, although some contribution from potential FSHR overexpression has not been fully excluded. However, previous studies showed that higher expression (increased transfection DNA) of wt FSHR did not increase constitutive signaling (basal cAMP levels) in cultured Sertoli [1] or CHO cell lines [37], which suggests that mutation-specific effects and not receptor overexpression per se may be necessary for FSH-like activity in Gnrh1–/– mice.

The above findings indicate that transgenic FSHR* has only a limited capacity to maintain spermatogenesis during testicular regression following suppression of gonadotropins in the mouse. Although basal serum T levels were 2-fold higher in gonadotropin-deficient Gnrh1–/– males expressing transgenic FSHR*, supporting our previous proposal that constitutive FSHR signaling may promote steroidogenesis [24], these higher serum T concentrations were not sustained in Tg-FSHR* Gnrh1–/– mice following the T hormone withdrawal regime to induce spermatogenic regression. In addition, the androgen-responsive epididymis was reduced to an equivalent size in Tg-FSHR* and nontransgenic Gnrh1–/– males following T withdrawal. Furthermore, the GNRH1 agonist-treated model demonstrated that despite the presence of functional mature Leydig cells in adult Gnrh1+/+ or Gnrh1+/– males, FSHR* expression alone was not sufficient to maintain serum T levels following pharmacological suppression of LH. Therefore, our overall findings are not able to explain the higher than castrate levels of circulating T reported in the original hypophysectomized FSHR* man after androgen withdrawal [17]. It is possible that persistent low serum T in this FSHR* individual resulted from residual levels of administered androgen, or from an incomplete LH deficiency that sustained low-level steroidogenesis. Our current findings demonstrate that the presence of transgenic FSHR* in mice has little capacity to stimulate functional steroidogenesis or provide significant androgen-mediated germ cell development in the absence of circulating LH or FSH.

In summary, our transgenic FSHR* paradigm showed that the mutant activated human FSHR* can increase the FSH-responsive Sertoli and germ cell populations in the adult mouse testis. However, analysis of two testicular regression strategies has clearly demonstrated that this mutant activated FSH receptor maintains only limited support of germ cell development during the suppression or withdrawal of circulating androgen and gonadotropins, in which the testicular response provided by this mutant FSHR* resembled typical FSH-mediated but not steroidogenic-like activity.

FOOTNOTES

1 Supported in part by a National Health & Medical Research Council project grant. Back

2 Correspondence: Charles M. Allan, Andrology Laboratory, ANZAC Research Institute, Concord Hospital, Sydney NSW 2139, Australia. FAX: 61 2 9767 9101; charles{at}anzac.edu.au Back

Received: 10 October 2005.

First decision: 17 November 2005.

Accepted: 1 February 2006.

REFERENCES

  1. Simoni M, Gromoll J, Nieschlag E, The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr Rev 1997 18:739-773[Abstract/Free Full Text]
  2. Tapanainen JS, Aittomaki K, Min J, Vaskivuo T, Huhtaniemi IT, Men homozygous for an inactivating mutation of the follicle-stimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility. Nat Genet 1997 15:205-206[CrossRef][Medline]
  3. Aittomaki K, Lucena JL, Pakarinen P, Sistonen P, Tapanainen J, Gromoll J, Kaskikari R, Sankila EM, Lehvaslaiho H, Reyes Engel A, Nieschlag E, Huhtaniemi I, et al Mutation in the follicle-stimulating hormone receptor gene causes hereditary hypergonadotropic ovarian failure. Cell 1995 82:959-968[CrossRef][Medline]
  4. Meduri G, Touraine P, Beau I, Lahuna O, Desroches A, Vacher-Lavenu MC, Kuttenn F, Misrahi M, Delayed puberty and primary amenorrhea associated with a novel mutation of the human follicle-stimulating hormone receptor: clinical, histological, and molecular studies. J Clin Endocrinol Metab 2003 88:3491-3498[Abstract/Free Full Text]
  5. Allen LA, Achermann JC, Pakarinen P, Kotlar TJ, Huhtaniemi IT, Jameson JL, Cheetham TD, Ball SG, A novel loss of function mutation in exon 10 of the FSH receptor gene causing hypergonadotrophic hypogonadism: clinical and molecular characteristics. Hum Reprod 2003 18:251-256[Abstract/Free Full Text]
  6. Touraine P, Beau I, Gougeon A, Meduri G, Desroches A, Pichard C, Detoeuf M, Paniel B, Prieur M, Zorn JR, Milgrom E, Kuttenn F, et al New natural inactivating mutations of the follicle-stimulating hormone receptor: correlations between receptor function and phenotype. Mol Endocrinol 1999 13:1844-1854[Abstract/Free Full Text]
  7. Abel MH, Wootton AN, Wilkins V, Huhtaniemi I, Knight PG, Charlton HM, The effect of a null mutation in the follicle-stimulating hormone receptor gene on mouse reproduction. Endocrinology 2000 141:1795-1803[Abstract/Free Full Text]
  8. Dierich A, Sairam MR, Monaco L, Fimia GM, Gansmuller A, LeMeur M, Sassone-Corsi P, 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 U S A 1998 95:13612-13617[Abstract/Free Full Text]
  9. Kumar TR, Wang Y, Lu N, Matzuk MM, Follicle stimulating hormone is required for ovarian follicle maturation but not male fertility. Nat Genet 1997 15:201-204[CrossRef][Medline]
  10. Phillip M, Arbelle JE, Segev Y, Parvari R, Male hypogonadism due to a mutation in the gene for the beta-subunit of follicle-stimulating hormone. N Engl J Med 1998 338:1729-1732[Free Full Text]
  11. Lindstedt G, Nystrom E, Matthews C, Ernest I, Janson PO, Chatterjee K, Follitropin (FSH) deficiency in an infertile male due to FSHbeta gene mutation. A syndrome of normal puberty and virilization but underdeveloped testicles with azoospermia, low FSH but high lutropin and normal serum testosterone concentrations. Clin Chem Lab Med 1998 36:663-665[CrossRef][Medline]
  12. Mantovani G, Borgato S, Beck-Peccoz P, Romoli R, Borretta G, Persani L, Isolated follicle-stimulating hormone (FSH) deficiency in a young man with normal virilization who did not have mutations in the FSHbeta gene. Fertil Steril 2003 79:434-436[CrossRef][Medline]
  13. Layman LC, Lee EJ, Peak DB, Namnoum AB, Vu KV, van Lingen BL, Gray MR, McDonough PG, Reindollar RH, Jameson JL, Delayed puberty and hypogonadism caused by mutations in the follicle-stimulating hormone beta-subunit gene. N Engl J Med 1997 337:607-611[Free Full Text]
  14. Layman LC, Porto AL, Xie J, da Motta LA, da Motta LD, Weiser W, Sluss PM, FSH beta gene mutations in a female with partial breast development and a male sibling with normal puberty and azoospermia. J Clin Endocrinol Metab 2002 87:3702-3707[Abstract/Free Full Text]
  15. Giltay JC, Deege M, Blankenstein RA, Kastrop PM, Wijmenga C, Lock TT, Apparent primary follicle-stimulating hormone deficiency is a rare cause of treatable male infertility. Fertil Steril 2004 81:693-696[CrossRef][Medline]
  16. Allan CM, Handelsman DJ, In vivo FSH actions. In: Skinner MK, Griswold MD (eds.) Sertoli Cell Biology San Diego: Elsevier Academic Press 2005 171-197
  17. Gromoll J, Simoni M, Nieschlag E, An activating mutation of the follicle-stimulating hormone receptor autonomously sustains spermatogenesis in a hypophysectomized man. J Clin Endocrinol Metab 1996 81:1367-1370[Abstract]
  18. Laue L, Chan WY, Hsueh AJ, Kudo M, Hsu SY, Wu SM, Blomberg L, Cutler GB, Jr, Genetic heterogeneity of constitutively activating mutations of the human luteinizing hormone receptor in familial male-limited precocious puberty. Proc Natl Acad Sci U S A 1995 92:1906-1910[Abstract/Free Full Text]
  19. Parma J, Duprez L, Van Sande J, Cochaux P, Gervy C, Mockel J, Dumont J, Vassart G, Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature 1993 365:649-651[CrossRef][Medline]
  20. De Leener A, Montanelli L, Van Durme J, Chae H, Smits G, Vassart G, Costagliola S, Presence and absence of FSH receptor mutations provide some insights to spontaneous ovarian hyperstimulation syndrome physiopathology. J Clin Endocrinol Metab 2006 91:555-562 Published 8 November 2005, doi: 10.1210/jc.2005-1580 [Abstract/Free Full Text]
  21. Montanelli L, Van Durme JJ, Smits G, Bonomi M, Rodien P, Devor EJ, Moffat-Wilson K, Pardo L, Vassart G, Costagliola S, Modulation of ligand selectivity associated with activation of the transmembrane region of the human follitropin receptor. Mol Endocrinol 2004 18:2061-2073[Abstract/Free Full Text]
  22. Smits G, Olatunbosun O, Delbaere A, Pierson R, Vassart G, Costagliola S, Ovarian hyperstimulation syndrome due to a mutation in the follicle-stimulating hormone receptor. N Engl J Med 2003 349:760-766[Free Full Text]
  23. Nordhoff V, Gromoll J, Foppiani L, Luetjens CM, Schlatt S, Kostova E, Huhtaniemi I, Nieschlag E, Simoni M, Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of human FSH receptor promoter. Asian J Androl 2003 5:267-275[Medline]
  24. Haywood M, Tymchenko N, Spaliviero J, Koch A, Jimenez M, Gromoll J, Simoni M, Nordhoff V, Handelsman DJ, Allan CM, An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice. Mol Endocrinol 2002 16:2582-2591[Abstract/Free Full Text]
  25. Mason AJ, Hayflick JS, Zoeller RT, Young WS III, Phillips HS, Nikolics K, Seeburg PH, A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse. Science 1986 234:1366-1371[Abstract/Free Full Text]
  26. Handelsman DJ, Spaliviero JA, Simpson JM, Allan CM, Singh J, Spermatogenesis without gonadotropins: maintenance has a lower testosterone threshold than initiation. Endocrinology 1999 140:3938-3946[Abstract/Free Full Text]
  27. Singh J, O'Neill C, Handelsman DJ, Induction of spermatogenesis by androgens in gonadotropin-deficient (hpg) mice. Endocrinology 1995 136:5311-5321[Abstract]
  28. Haywood M, Spaliviero J, Jimenez M, King NJC, Handelsman DJ, Allan CM, Sertoli and germ cell development in hypogonadal (hpg) mice expressing transgenic follicle-stimulating hormone alone or in combination with testosterone. Endocrinology 2003 144:509-517[Abstract/Free Full Text]
  29. Jimenez M, Spaliviero JA, Grootenhuis AJ, Verhagen J, Allan CM, Handelsman DJ, Validation of an ultrasensitive and specific immunofluorometric assay for mouse follicle-stimulating hormone. Biol Reprod 2005 72:78-85[Abstract/Free Full Text]
  30. Dong Q, Li B, Rintala H, Blair S, Spaliviero J, Handelsman DJ, LH pulsatility, biopotency, and clearance during undernutrition in orchidectomized mature rats. Am J Physiol 1993 265:304-313
  31. Allan CM, Garcia A, Spaliviero J, Zhang FP, Jimenez M, Huhtaniemi I, Handelsman DJ, Complete Sertoli cell proliferation induced by follicle-stimulating hormone (FSH) independently of luteinizing hormone activity: evidence from genetic models of isolated FSH action. Endocrinology 2004 145:1587-1593[Abstract/Free Full Text]
  32. Atanassova N, McKinnell C, Walker M, Turner KJ, Fisher JS, Morley M, Millar MR, Groome NP, Sharpe RM, Permanent effects of neonatal estrogen exposure in rats on reproductive hormone levels, Sertoli cell number, and the efficiency of spermatogenesis in adulthood. Endocrinology 1999 140:5364-5373[Abstract/Free Full Text]
  33. Meachem SJ, McLachlan RI, de Kretser DM, Robertson DM, Wreford NG, Neonatal exposure of rats to recombinant follicle stimulating hormone increases adult Sertoli and spermatogenic cell numbers. Biol Reprod 1996 54:36-44[Abstract]
  34. Krishnamurthy H, Babu PS, Morales CR, Sairam MR, Delay in sexual maturity of the follicle-stimulating hormone receptor knockout male mouse. Biol Reprod 2001 65:522-531[Abstract/Free Full Text]
  35. Cattanach BM, Iddon CA, Charlton HM, Chiappa SA, Fink G, Gonadotrophin-releasing hormone deficiency in a mutant mouse with hypogonadism. Nature 1977 269:338-340[CrossRef][Medline]
  36. Allan CM, Haywood M, Swaraj S, Spaliviero J, Koch A, Jimenez M, Poutanen M, Levallet J, Huhtaniemi I, Illingworth P, Handelsman DJ, A novel transgenic model to characterize the specific effects of follicle-stimulating hormone on gonadal physiology in the absence of luteinizing hormone actions. Endocrinology 2001 142:2213-2220[Abstract/Free Full Text]
  37. Kudo M, Osuga Y, Kobilka BK, Hsueh AJ, Transmembrane regions V and VI of the human luteinizing hormone receptor are required for constitutive activation by a mutation in the third intracellular loop. J Biol Chem 1996 271:22470-22478[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
74/5/938    most recent
biolreprod.105.048413v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Allan, C. M.
Right arrow Articles by Handelsman, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Allan, C. M.
Right arrow Articles by Handelsman, D. J.
Agricola
Right arrow Articles by Allan, C. M.
Right arrow Articles by Handelsman, D. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS