Gonadotrophins regulate germ cell survival, not proliferation, in normal adult men
1 Prince Henry's Institute, Clayton, Level 4, 43-51 Kanooka Grove, Victoria 3168, Australia 2 Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria 3168, Australia 3 Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria 3000, Australia
4 Correspondence address. Tel: +61-3-9594-7913; Fax: +61-3-9594-7909; E-mail: sarah.meachem{at}princehenrys.org
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
BACKGROUND: Gonadotrophins support spermatogenesis via poorly understood mechanisms. We aimed to determine the effect of FSH/LH suppression in regulating germ cell apoptosis and proliferation in normal fertile men.
METHODS: Testicular tissues were obtained after gonadotrophin suppression induced by testosterone alone or combined with depot medroxyprogesterone acetate for 2 or 6 weeks and an untreated group of men (referred to as ‘normal men’) served as controls (n = 5 or 10 men per group). Apoptosis and proliferation were identified by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and proliferating cell nuclear antigen (PCNA) labelling methods, respectively. Intrinsic and extrinsic apoptotic pathways were identified by immunohistochemistry using the pathway-specific proteins: activated caspase (aCaspase) 9 and 8 and quantified using stereological techniques.
RESULTS: By 2 and 6 weeks, the proportion of TUNEL-labelled spermatogonia increased to 354% and 268% respectively, compared with normal men (P < 0.001), with increased caspase 9 [223 and 166% compared with normal men (P < 0.001)], but no increase in caspase 8, immunoreactivity. At 6 weeks, the proportions of TUNEL-labelled spermatocytes and round spermatids tended to increase (303 and 180% compared with normal men, NS), as did caspase 9 (199 and 147% compared with normal men, NS) and caspase 8 immunoreactivities (286 and 243% compared with normal men, NS and P = 0.06), respectively. The proportion of TUNEL-labelled elongating/elongated spermatids tended to increase (144 and 138% compared with normal men, NS) at 2 and 6 weeks, respectively, with no change in either caspase immunoreactivities. Even though the number of PCNA-labelled cells did not change with gonadotrophin suppression, the balance between proliferation and apoptosis was lower in spermatogonia (P = 0.01) and spermatocytes (P = 0.3) between treated and untreated normal men.
CONCLUSIONS: We demonstrated that gonadotrophins act as spermatogonial survival factors via the regulation of intrinsic apoptotic pathway, whereas having no effect of cellular proliferation in normal men.
Key words: spermatogonia/caspase/apoptosis/intrinsic pathway/extrinsic pathway
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
Spermatogenesis is a dynamic process occurring in three phases: mitosis (spermatogonia), meiosis (spermatocytes) and spermiogenesis (the morphological transformation of spermatids) (McLachlan et al., 2002a
; Saunders 2003). Sperm output relies upon the coordinated proliferation, differentiation and survival of each progressively maturing germ cell type, as regulated by a complex network of signals, notably the pituitary gonadotrophins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), via their regulation of intratesticular androgen levels.
In men, gonadotrophins are required to reinitiate and maintain quantitatively normal sperm production after experimentally induced oligoazoospermia (Matsumoto et al., 1983, 1986; Matsumoto and Bremner, 1985). To understand the sites of gonadotrophin action within the spermatogenic process, we used the experimental model of exogenous testosterone administration, alone or in combination with a progestin, given to normal men in order to suppress FSH and LH and, thereby, sperm production (Zhengwei et al., 1998a, McLachlan et al., 2002b; Matthiesson et al., 2005). We have demonstrated the striking inhibition of type A pale and B spermatogonia maturation as determined by a 60–90% reduction in type B spermatogonia, in addition to the retention of mature spermatids in the epithelium strongly suggesting a failure of sperm release (spermiation). These observations held true in later androgen-based contraceptive studies involving the addition of a gonadotrophin releasing hormone antagonist or a 5 alpha reductase inhibitor to further withdraw gonadotropic/androgenic support for spermatogenesis (Matthiesson et al., 2005, 2006).
The mechanisms by which gonadotrophins regulate spermatogonial development in men are unknown; however, the regulation of apoptosis is a potentially important factor. There are two described pathways of apoptosis in the testis: the intrinsic and extrinsic pathways (reviewed in Sinha-Hikim et al., 2003a). The intrinsic pathway (or mitochondrial pathway) involves translocation of bax from the cytosol to the mitochondria which results in the release of cytochrome C into the cytosol, where it binds to apoptotic protease activating factor (Apaf)-1 and then activates initiator caspase 9, leading to activation of executioner caspases 3, 6 and 7 that cleave intracellular proteins and effect apoptosis (Adams and Cory, 1998; Green, 2000; Hengartner, 2000). The extrinsic pathway (or death receptor pathway) involves FasL stimulation of Fas on target cells which then activates initiator caspase 8, which subsequently activates executioner caspases, effecting apoptosis (Nagata and Golstein, 1995; Lee et al., 1997). Vera et al. (2006) suggested that spermatocytes and spermatids undergo apoptosis via the intrinsic pathway in human seminiferous tubule cultures under hormonal free conditions, as evidenced by activation of p38 mitogen-activated protein kinase and induction of inducible nitric oxide synthase leading to an activation of caspases. In a similar experimental paradigm, an immuno-localization of fas to spermatocytes and spermatids has been observed, suggesting an involvement of the extrinsic pathway (Pentikainen et al., 1999). However, to our knowledge, there is no evidence suggesting spermatogonial apoptosis is regulated by gonadotrophins in normal men.
Whether gonadotrophins regulate human germ cell proliferation remains unclear. Takagi et al. (2001) demonstrated that increased apoptosis, rather than reduced proliferation, underlie the reduction of spermatogonial number in men with hormonally independent idiopathic infertility. In men with congenital gonadotrophin deficiency, increased germ cell apoptosis was noted but proliferation was not studied (Francavilla et al., 2000). In chronically gonadotrophin-depleted monkeys, spermatogonial mitotic activity [based on bromo-deoxyuridine (BrdU) incorporation] was not altered by gonadotrophin treatment (Marshall et al., 2005).
We aimed to investigate whether gonadotrophin suppression in normal men results in accelerated germ cell apoptosis (via activation of the intrinsic and/or extrinsic pathways) or a change in proliferation. Using testicular tissue from previous hormonal contraceptive studies (McLachlan et al., 2002b; Matthiesson et al., 2005), we aimed to determine the mechanisms (apoptosis or proliferation) and pathway(s) involved in germ cell loss by employing antibody detection systems directed to the specific activated caspase (aCaspase) forms (aCaspase 9, intrinsic; aCaspase 8, extrinsic) in combination with germ cells enumeration using stereological techniques.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
Subjects
Testicular biopsies were obtained from 30 normal men aged 31–46 years who underwent either no treatment or androgen-based male contraceptive treatment for 2 or 6 weeks prior to a previously planned vasectomy and informed consent (McLachlan et al., 2002b
; Matthiesson et al., 2005). These studies were approved by the Southern Health Human Research and Ethics Committee, Melbourne, Australia and the Institutional Review Board of the University of Washington, Seattle, USA. The clinical trial by McLachlan et al. (2002b) was conducted between March 1998 and March 2000 (Melbourne). The trial by Matthiesson et al. (2005) was completed on 27 June 2004 (Seattle).
Design
Groups of 5 men received either a weekly dose of 200 mg of testosterone enanthate (TE: Primotestin depot, Schering AG Berlin, Germany) alone or in combination with a single dose of 300 mg of depot medroxyprogesterone acetate [DMPA (progestin): Upjohn Pharmaceuticals, Rydalmere, Australia] by intramuscular injections for the 2 or 6 weeks before surgery (McLachlan et al., 2002b) and these groups are referred to as ‘gonadotrophin-suppressed’ throughout this study. These sex steroid treatments resulted in feedback inhibition of gonadotrophin release and thereby spermatogenic inhibition. As these two treatments, TE and TE plus DMPA led to similar suppression of serum gonadotrophin levels (mean serum FSH 1.2–1.3% and mean serum LH 0.3–0.5% of baseline), intratesticular T (iT) levels (
2% of controls) and germ cell types (McLachlan et al., 2002b), they were pooled (n = 10 per/group) for the 6-week time point. Despite reported significant differences in gonadotrophin levels (mean serum FSH 11.5% versus 4.2%, mean serum LH 13.8% versus 2.7% of baseline, respectively) and germ cell numbers at the 2-week time point for TE and TE plus DMPA treatments, no differences were observed in iT levels and apoptotic or proliferative end-points using a t-test, and accordingly, data were also pooled in a single group of 10 men. Ten normal men not receiving any treatment served as controls and this group is referred to as ‘normal men’ throughout this study (McLachlan et al., 2002b; Matthiesson et al., 2005).
Testicular biopsy and tissue processing
A fragment of the single testicular biopsy from one testis of each man was immersion fixed in Bouin's solution for 3–5 h and paraffin embedded (Zhengwei et al., 1998a) for either in situ detection of apoptosis or immunohistochemistry. Prior to immunostaining, 5 µm tissue sections were prepared, deparaffinized and hydrated by successive series of ethanol and rinsed in phosphate-buffered saline (PBS; 10 mM, pH 7.4).
Assessment of apoptosis
In situ detection of cells with DNA strand breaks (apoptosis) was performed on tissue sections by the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP nick-end labelling (TUNEL) technique using an ApopTag® Peroxidase In Situ Apoptosis Detection kit (Chemicon International, Temecula, USA). Previously, the ApopTag kit technique has been validated for in-situ detection of germ cell apoptosis in the human (Sinha-Hikim et al., 1998, 2003b; Takagi et al., 2001; Vera et al., 2006). In brief, to eliminate non-specific binding, tissues were subjected to microwave antigen retrieval in EDTA–NaOH buffer (1 mM, pH 8; 90–95°C for 10 min then room temperature for 1 h), not proteases treatment (Billig et al., 1995). Tissues were incubated with a mixture containing digoxigenin-conjugated nucleotide and TdT at 37°C for 1 h, whereas this mixture was substituted with the same volume of milli-Q water on negative control sections. Endogenous peroxidase activity was blocked by immersing tissue sections in 3% hydrogen peroxide in methanol for 15 min followed by two washes with PBS. Tissues were blocked with CAS block (Invitrogen, San Fransico, USA) for 30 min to prevent non-specific binding. Subsequently, tissues were incubated with anti-Dig peroxidase for 30 min to highlight the incorporated digoxigenin-conjugated nucleotides in fragmented DNA. After washing, diaminobenzidine (DAB; DAKO, Carpenteria, USA) was added for 2–3 min to reveal sites of anti-Dig binding with a dark brown reaction product. Sections were counterstained with Mayer's haematoxylin for 3 min and blued in Scott's tap water for 1 min, and finally dehydrated and mounted in DepeX (BDH Laboratory Suppliers, Poole, UK) under coverslips.
Assessment of proliferation
The expression of proliferating cell nuclear antigen (PCNA; to DNA polymerase-delta found during the activation of DNA replication) is used as an index of the proliferative activity in human testicular tissues (Hall and Levison, 1990; Steger et al., 1998; Takagi et al., 2001). Tissue sections were subjected to antigen retrieval in EDTA–NaOH buffer, and then endogenous peroxidase activity was quenched. The sections were treated with 10% normal rabbit serum in PBS (Serotec, Raleigh, USA) and incubated with mouse monoclonal anti-human PCNA antibody (5 µg/ml in PBS; Biosciences, Franklin Lakes, USA) for 1 h. Sections were then incubated with biotinylated rabbit anti-mouse immunoglobulin G (IgG) secondary antibody (10 µg/ml in PBS; Zymed Labs, San Francisco, USA) for 30 min with pre- and post-washes with PBS, followed by incubation with avidin–biotin–peroxidase (ABC) complex (Vectastain Elite, Vector Laboratories, Burlingame, USA) according to the manufacturer's instructions for 30 min. After washing, DAB was added for 2–3 min, and sections were counterstained, blued, dehydrated and mounted in DepeX under coverslips. The negative control sections were performed in a similar manner, except the primary antibody was substituted with the same concentration of mouse IgG antibody (Biosciences) to determine any false positive cross reactivity of secondary antibody.
Assessment of apoptotic pathways
The activation of apoptotic pathways have been identified with previously validated immunohistochemistry procedures by employing antibodies against the activated forms of pathway specific caspases (Bozec et al., 2005). In brief, testicular tissue sections were subjected to antigen retrieval in EDTA–NaOH buffer, endogenous peroxidase activity was quenched, and sections were blocked with 10% normal goat or rabbit sera, then incubated over night with either aCaspase 9 antibody (0.76 µg/ml in PBS, rabbit polyclonal human specific cleaved caspase 9 detecting p17 and p37 of active caspase 9; Cell Signaling Technology, Danvers, USA) or with caspase 8 (in their activated form; aCaspase 8) antibody (2.4 µg/ml in PBS; mouse monoclonal caspase 8 which detects only the N-terminal region of the p18 subunit; Novocastra Laboratories, Newcastle, UK). Subsequently, sections were incubated with biotinylated sheep anti-rabbit IgG (2 µg/ml in PBS; Chemicon International) for 1 h or with biotinylated rabbit anti-mouse IgG secondary antibody (10 µg/ml; Zymed Labs) for 30 min with pre- and post-washes with PBS. Following ABC complex treatment, DAB was added for 2–3 min, and sections were counterstained and blued and mounted. On negative control sections, the primary aCasapse 9 and 8 antibodies were substituted with same concentration of rabbit and mouse IgG antibodies (Biosciences), respectively.
Quantification of labelled cells
Stereological techniques were applied to determine the percentages of TUNEL, PCNA and aCaspase 9- or 8-labelled cells. TUNEL and PCNA-labelled cell types were identified by deep brown nuclear staining and aCaspase-labelled cells were identified by brown nuclear, cytoplasmic or whole cell staining (upon activation, caspases translocate from the cytoplasm to nucleus, therefore localization of aCaspase varies along the apoptotic pathway), based on their location within the seminiferous tubules, their size and the shape of cell nucleus. Cells were classified into four groups: spermatogonia, spermatocytes, round spermatids and elongating/elongated spermatids (Russell et al., 1990). The percentages of labelled cells were assessed using an unbiased counting frame of 2914 µm2 superimposed on video image by CASTGRID V1.60 software package per field (Olympus, Denmark, Germany), where 50–300 cell nuclei for each cell group was counted from one section per testis in a systematic uniform random sampling manner. All slides were masked prior to the analysis. The rate of apoptosis and proliferation and caspase activities were quantified by dividing the number of labelled cells by the total number of labelled and unlabelled cells in each group (PCNA: n = 5 men per group, TUNEL and caspases: n = 10 men per group).
Immunofluorescence and confocal studies
To determine the prevalence of cells undergoing each apoptotic pathway, the co-localization of TUNEL-labelled cells with aCaspase 9 or 8 proteins was detected by confocal microscopy using immunofluorescence dual labelling (Sinha-Hikim et al., 2003b; Vera et al., 2004). In situ detection of cells with DNA fragmentation was performed on tissue sections using an Apoptag® Fluorescein In Situ Apoptosis Detection Kit (Chemicon International), without protease treatment. In brief, tissues were subjected to antigen retrieval in EDTA–NaOH buffer to achieve lower background (Billig et al., 1995), incubated with a mixture containing digoxigenin-conjugated nucleotide and TdT, and were treated with 488 antidigoxigenin-fluorescein for 30 min in the dark. For staining of aCaspases, slides were washed and then incubated with 10% normal goat serum for 20 min and treated with antibodies to aCaspase 9 or 8 (2.4 µg/ml in PBS; rabbit monoclonal cleaved caspase 8 which detects only the cleaved product p18, p41 and p43 of active caspase 8) (Cell Signaling Technology) overnight, followed by goat anti-rabbit Alexa 546 secondary antibody (Molecular Probes, Eugene, USA) for 45 min. Slides were washed and mounted with Fluorsave (Calbiochem, La Jolla, USA). For negative control sections, TdT was omitted and the lack of secondary antibody cross-reactivity was verified with the equivalent concentration of antibody of the same isotype control.
Confocal images were obtained and processed using a Fluoview FV300 computer package and Olympus microscope (Olympus Australia, Mt Waverly, Australia). The proportions of TUNEL-labelled germ cells that were either aCaspase 9 or 8 positive were quantified by counting all the labelled and dual labelled cells per group (n = 10 men per group).
Statistical analysis
One way analysis of variance (ANOVA) test was used to determine the differences between normal and gonadotrophin-suppressed men for 2 and 6 weeks and that data were normally distributed for all histological data. If data did not show normal distribution, then a Kruskal–Wallis one way ANOVA on ranks was performed. A two sample t-test was also used in some cases to determine differences between two groups. All statistical analyses were performed using Sigmastat for Windows version 3.1 (Jandel Corporation, Canada). Data are expressed as mean ± standard error of mean (SEM), n = 10 per group, except for the assessment of proliferation, where n = 5 per group.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
Gonadotrophin suppression induces germ cell apoptosis, rather than proliferation in normal men
In normal men, TUNEL-labelling was observed in all germ cell types; spermatogonia, spermatocytes, round spermatids and elongating/elongated spermatids (1.6 ± 0.2%, 1.6 ± 0.5%, 1.9 ± 0.4% and 3.7 ± 1.0%, respectively; Figs. 1A and 2A). Gonadotrophin suppression for 2 and 6 weeks increased the percentage of TUNEL-labelled spermatogonia to 354 and 268% compared with normal men (1.6 ± 0.2% versus 5.6 ± 0.9%, 4.2 ± 0.8%: P < 0.001), respectively. After 6 weeks treatment, an apparent increase in TUNEL-labelled spermatocytes and round spermatids to 303% (1.6 ± 0.5% versus 4.9 ± 1.8%) and 180% (1.9 ± 0.4% versus 3.4 ± 0.9%) compared with normal men, respectively, did not achieve statistical significance. At 2 and 6 weeks, the apparent increase in the percentage of TUNEL-labelled elongating/elongated spermatids to 144 and 138% compared with normal men (3.6 ± 1.0% versus 5.2 ± 2.0%, 5.0 ± 0.8%), respectively, also did not achieve significance (Figs. 1A and 2B).
|
|
PCNA indices of germ cell proliferation following 2 and 6 weeks of gonadotrophin suppression were similar to those of normal men (Figs. 1B, 2D and 2E).
The ratios of the PCNA-labelled to the TUNEL-labelled cell rate were analysed as an index of the balance between cell proliferation and apoptosis of germ cells (Hall and Levison, 1990; Takagi et al., 2001). The ratios for spermatogonia were significantly lower in gonadotrophin-suppressed men for 2 and 6 weeks compared with normal men (P = 0.01), but not significantly altered in regard to spermatocytes (P = 0.3: Table I).
|
The intrinsic and extrinsic pathways are present in total germ cell apoptosis in normal men
In normal men, percentages of TUNEL-labelled germ cells with aCaspase 9 and aCaspase 8 co-reactivity were 25.4 ± 2.3% and 25.7 ± 2.5%, respectively (Figs. 3A, 3C and 4). At 2 and 6 weeks, gonadotrophin suppression tended to increase the percentage of TUNEL-labelled germ cell that were positive for aCaspase 9 to 30.9 ± 2.6% and 29.5 ± 5.2% (NS) (Figs. 3B and 4) and tended to decrease percentage of TUNEL-labelled germ cell that were positive for aCaspase 8 to 18.5 ± 1.7% and 20.1 ±2.2% (P = 0.07) (Figs. 3D and 4) compared with normal, respectively, but neither achieved statistical significance.
|
|
Gonadotrophin suppression affects spermatogonial apoptosis via the intrinsic apoptotic pathway
In normal men, aCaspase 9-labelling was observed in all germ cell types; spermatogonia, spermatocytes and round spermatids (2.6 ± 0.7%, 0.8 ± 0.2% and 2.3 ± 0.6%, respectively: Figs. 5A and 6A). Gonadotrophin suppression for 2 and 6 weeks increased the percentage of aCaspase 9-labelled spermatogonia to 223 and 166% compared with normal (2.6 ± 0.2% versus 5.8 ± 0.7%, 4.3 ± 0.8%: P < 0.001), respectively. Increases in aCaspase 9-labelled spermatocytes and round spermatids to 199% (0.8 ± 0.2% versus 1.6 ± 0.8%: P = 0.6) and 147% (2.3 ± 0.6% versus 3.4 ± 1.1%: P = 0.1) compared with normal were observed at 6 weeks, respectively, but neither achieved significance (Figs. 5B and 6A).
|
|
Gonadotrophin suppression possibly affects spermatocytes and round spermatid apoptosis via the extrinsic apoptotic pathway
In normal men, no aCaspase 8-labelled spermatogonia were observed (Fig. 6B). In response to gonadotrophin suppression, there were only few or no aCaspase 8-labelled spermatogonia observed after 2 and 6 weeks of gonadotrophin suppression, respectively.
In normal men, aCaspase 8-labelling was observed in spermatocytes and round spermatids (0.9 ± 0.3% and 1.8 ± 0.5%, respectively: Figs. 5D and 6B). Following gonadotrophin suppression, there were trends towards increases in aCaspase 8-labelled spermatocytes to 286% (0.9 ± 0.3% versus 2.5 ± 0.9%: P = 0.4) following 6 weeks and in aCaspase 8-labelled round spermatids to 199 and 243% compared with normal (1.8 ± 0.5% versus 3.7 ± 0.7%, 4.5 ± 1.0%: P = 0.06), after 2 and 6 weeks respectively (Figs. 5E and 6B).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
In this study, we report that gonadotrophins act as survival factors by regulating both the intrinsic and extrinsic pathways, rather than proliferative factors in the human testis. We have previously reported a 60–90% decrease in type B spermatogonial numbers following gonadotrophin suppression induced by androgen-based hormonal contraceptive regimens (Zhengwei et al., 1998a
; McLachlan et al., 2002b; Matthiesson et al., 2005; 2006). We now show that this decrease in spermatogonia is attributable to an increase in apoptosis, principally via the intrinsic pathway. Gonadotrophins possibly act as a survival factor for later germ cell types by regulating both the intrinsic and the extrinsic pathways. This suggests that the mechanisms by which gonadotrophins influence germ cell survival differs between germ cell subtypes.
We investigated the effect of gonadotrophin withdrawal at two time points that were chosen in previous studies of hormonal contraceptive effects on serum gonadotrophin levels and germ cell populations in the short (2 weeks) and medium term (6 weeks). As germ cell development and apoptotic death are dynamic processes, any assessment at a particular time point will encounter cells at different phases of these processes. This fact must be taken into account when interpreting the data. For example, we were unable to investigate the rate of apoptosis that occurs during the early period of gonadotrophin suppression or the most active apoptotic phase nor account in a numerical sense for the fate of individual cells. We suppressed gonadotrophins using TE alone or in combination with DMPA. In this model, we observed profound gonadotrophin suppression and marked reduction in iT. In rodents, FSH has shown to be involved in stimulation of androgen binding proteins and androgen receptor numbers resulting in an increased iT (Verhoeven and Callieau, 1988; Ottenweller et al., 2000). The most likely explanation for the reduction of iT in this model is that reduced FSH following gonadotrophin suppression may result in a reduced production of androgen binding proteins, thus the reduction of T within the testis. Therefore, we envisage that the only mechanism by which this may induce germ cell regression and apoptosis would most likely be through direct or indirect effects of the withdrawal of gonadotropic support.
In terms of spermatogonia, in this study, we were unable to distinguish whether type A and/or B spermatogonial subpopulations were affected. However, since type A numbers remain near normal in these gonadotrophin suppressed men (McLachlan et al., 2002b), we speculate that B spermatogonia are subject to gonadotrophin-dependent apoptosis. We have shown significant increases in apoptotic rates following 2 and 6 weeks of gonadotrophin suppression. Moreover, by comparing the balance between spermatogonial proliferation and apoptosis, decreases in this balance following gonadotrophin suppression were strongly inclined towards apoptosis even after most active apoptotic phase. However, the specific site at which spermatogonial development is being inhibited following gonadotrophin suppression in humans has been found to be different to non human primates, such as old world monkeys. Several studies have indicated that type A spermatogonia, followed by type B spermatogonia, are affected following gonadotrophin withdrawal upon treatment with a GnRH-antagonist (Schlatt and Weinbauer, 1994; Zhengwei et al., 1998b; Marshall et al., 2005). These differences noted between primates and human may be due to different sensitivities of the spermatogonial subtypes to FSH and/or androgen. Additionally, Schlatt and Weinbauer (1994) reported a reduction in spermatogonial mitosis as determined by PCNA labelling after short-term GnRH antagonist-treatment, but apoptosis was not assessed in these monkeys. However, it is possible that GnRH antagonist treatment can cause a more profound suppression of circulating FSH and intra-testicular testosterone in monkeys than T-induced gonadotrophin withdrawal in human.
Our data and that of Takagi et al. (2001) suggest that spermatogonial proliferation in men is gonadotrophin independent. This study shows that 40–50% of spermatogonia were PCNA-labelled in both control and gonadotrophin-suppressed men, whereas Takagi et al. (2001) reported a similar level of PCNA-labelled spermatogonia in idiopathic infertile men with normal levels of gonadotrophins. Therefore, these studies suggest that a significant component of spermatogonial proliferation occurs despite the presence or absence of gonadotrophins in the testis. This is consistent with the observation that incorporation of BrdU by type A spermatogonia was not altered in chronically gonadotrophin-deplete adult rhesus monkeys following gonadotrophin replacement (Marshall et al., 2005). In addition, gonadotrophin independent type A spermatogonial proliferation was observed during juvenile development in rhesus monkeys exhibiting a natural hypogonadotrophic state, compared with that during their infancy with elevated levels of gonadotrophins (Simorangkir et al., 2005). One caveat in our study is that the very small levels of gonadotrophins persist despite sex steroid treatment (range 11–1.3%: FSH and 2–1%: LH of baseline) which may support spermatogonial proliferation.
The relative roles of FSH and or LH (via intratesticular androgens) in regulating spermatogonial survival remains unknown but we speculate that the suppression of FSH underlies the increase in spermatogonial apoptosis. Administration of FSH to GnRH antagonist-treated cynomolgus (Weinbauer et al., 1991), rhesus (van Alphen et al., 1988; Marshall et al., 1995; Marshall et al., 2005) and bonnet (Moudgal et al., 1997) monkeys results in an increase in spermatogonial numbers. Similarly, Weinbauer et al. (2001) reported that the inhibition of spermatogonia following gonadotrophin withdrawal related to suppression of FSH rather than to testicular androgen levels in the cynomolgus monkeys. Similar data in rodent models suggested that FSH plays a key role in promoting spermatogonial survival (Shetty et al., 1996; Meachem et al., 1999, 2005).
The trend towards increases in spermatocytes and round spermatids apoptosis after 6 weeks, and in elongating/elongated spermatids apoptosis after 2 and 6 weeks, suggest that gonadotrophins may also act as survival factors during meiosis and spermiogenesis, rather than as proliferative factors in normal men. Consistent with these data, human seminiferous tubules cultured in a medium without FSH or testosterone for 4–24 h reported significant increases in DNA fragmentation in primary spermatocytes and elongating/elongated spermatids (Tesarik et al., 1998, 2002; Pentikainen et al., 1999; Vera et al., 2006). This also held true for non human primate models, where increases in spermatocytes and spermatid apoptosis were observed in rhesus monkeys following gonadotrophin suppression for up to 4 weeks by testosterone undecanoate (Zhou et al., 2001; Zhang et al., 2003). Our ability to detect significant changes in germ cell apoptosis is limited by the subject numbers but also by the recognized and marked heterogeneity of germ cell response that is not accounted for by phenotypic characteristics or hormonal changes during male hormonal contraceptive treatment (Gu et al., 2003; Matthiesson et al., 2005).
We have demonstrated that gonadotrophins regulate spermatogonial survival via the intrinsic apoptotic pathway, and not the extrinsic apoptotic pathway, as evidenced by increased aCaspase 9 positive cells following 2 and 6 weeks of gonadotrophin suppression in the human testis. In male germ lines, Bcl-2 family proteins, such as bax, are involved in the intrinsic pathway by controlling the release of cytochrome C from mitochondria (Adams and Cory, 1998). In bax knockout mice, increased spermatogonial numbers suggest that intrinsic apoptotic pathways regulate their survival (Knudson et al., 1995; Russell et al., 2002). In addition, when Apaf-1 was removed via gene targeting, male mice were rendered infertile with high levels of degenerating spermatogonia (Honarpour et al., 2000). The mechanisms by which the lack of gonadotrophins is transduced within spermatogonia to direct their death via the intrinsic pathway are unclear. Perhaps, the lack of gonadotrophins changes the mitochondrial permeability transition, allowing factors such as cytochrome C and bax to be transported in and out of the mitochondria, causing apoptosis (Erkkila et al., 1999). The reason spermatogonia were unable to undergo apoptosis via the extrinsic pathway may be due to the fact that the up-stream component of this pathway, fas is not expressed in spermatogonia (Nandi et al., 1999).
Our data suggest that gonadotrophins possibly regulate both the intrinsic and extrinsic apoptotic pathways during meiosis and spermiogenesis in normal men. However, Vera et al. (2006) suggested that spermatocytes and spermatids undergo apoptosis via the intrinsic apoptotic pathway as evidenced by activation of p38 mitogen-activated protein kinase and induction of inducible nitrogen oxide synthase in human seminiferous tubules cultured under hormone free conditions for 4 h. In rodents, such increases in p38 mitogen-activated protein kinase and nitrogen oxide synthase are accompanied by a marked perturbation of the BCL-2 and BAX rheostat, cytochrome C release from mitochondria and caspase 9 activation suggesting an involvement of the intrinsic pathway (Vera et al., 2006). In addition, in a similar experimental paradigm of human seminiferous tubule culture, fas had been found to localize to apoptotic spermatocytes and spermatids by electron microscopy (Pentikainen et al., 1999), suggesting an involvement of the extrinsic apoptotic pathways. It has also been suggested that administration of exogenous testosterone to Rhesus monkeys induces spermatocyte and spermatid apoptosis by increases in Fas/FasL expression (Zhou et al., 2001) and Bcl-2/Bax (Zhang et al., 2003) expression, therefore via extrinsic and intrinsic apoptotic pathways, respectively. However, cross talk between the apoptosis pathways is evidenced by the fact that in some cells, aCaspase 8 leads to a cleavage of the Bcl-2 protein family member, Bid. Bid can then induce Bax-mediated release of cytochrome C from mitochondria, further committing the cell apoptosis via the intrinsic pathway (Sinha-Hikim et al., 2003a). We did not attempt to establish whether caspase 8-mediated cleavage Bid is responsible for stimulation of intrinsic pathway signaling, although this might explain the observation that spermatocytes and spermatids undergo apoptosis via both pathways.
In normal adult men, germ cells die via both the intrinsic and extrinsic apoptotic pathway at similar levels (25% of apoptotic cells for each pathway). This was also found to be true in normal adult rats with similar levels of aCaspase 9 and 8 activities measured using fluorometric protease assays (Eid et al., 2002). We observed that 50% of TUNEL-labelled cells did not register as undergoing either pathway suggesting the involvement of another pathway; i.e. endoplasmic reticulum (ER) pathway. Although this was not studied, there is evidence to suggest that Bcl-2 proteins may also serve to regulate germ cell apoptosis via the ER pathway in rodents, as translocation of Bax to ER were seen in germ cells shortly after heat treatment, compared with controls where only trace amount of Bax was found in ER (Sinha-Hikim et al., 2003b). Perhaps, another explanation for only 50% of TUNEL-labelled cells having a determinable apoptotic pathway may be deferring time courses of positivity between TUNEL and caspase detection methods. TUNEL staining marks the latter phase of apoptosis DNA fragmentation, whereas caspase activation begins during the earlier phase of apoptosis. There can be reduced or lost caspase reactivity in some cells before reaching their latter phases.
In conclusion, this study reveals that gonadotrophins act as survival factor for spermatogonia via the intrinsic pathway of apoptosis, and possibly regulate spermatocyte survival via both the intrinsic and extrinsic pathways, but do not modulate germ cell proliferation in man. Understanding the basic mechanisms in which hormones regulate germ cell progression is a crucial step towards improved understanding of fertility disorders and fertility regulation.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
Supported by the National Health and Medical Research Council of Australia, Program Grant No. 241000 (S.J.M., K.L.M. and R.I.M.).
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingReferences |
|---|
Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science (1998) 281:1322–1326.
van Alphen MM, van de Kant HJ, de Rooij DG. Follicle-stimulating hormone stimulates spermatogenesis in the adult monkey. Endocrinology (1988) 123:1449–1455.
Billig H, Furuta I, Rivier C, Tapanainen J, Parvinen M, Hsueh AJ. Apoptosis in testis germ cells: developmental changes in gonadotropin dependence and localization to selective tubule stages. Endocrinology (1995) 136:5–12.[Abstract]
Bozec A, Ruffion A, Decaussin M, Andre J, Devonec M, Benahmed M, Mauduit C. Activation of caspases-3, -6, and -9 during finasteride treatment of benign prostatic hyperplasia. J Clin Endocrinol Metab (2005) 90:17–25.
Eid NA, Shibata MA, Ito Y, Kusakabe K, Hammad H, Otsuki Y. Involvement of Fas system and active caspases in apoptotic signalling in testicular germ cells of ethanol-treated rats. Int J Androl (2002) 25:159–167.[CrossRef][Web of Science][Medline]
Erkkila K, Pentikainen V, Wikstrom M, Parvinen M, Dunkel L. Partial oxygen pressure and mitochondrial permeability transition affect germ cell apoptosis in the human testis. J Clin Endocrinol Metab (1999) 84:4253–4259.
Francavilla S, D'Abrizio P, Rucci N, Silvano G, Properzi G, Straface E, Cordeschi G, Necozione S, Gnessi L, Arizzi M, et al. Fas and Fas ligand expression in fetal and adult human testis with normal or deranged spermatogenesis. J Clin Endocrinol Metab (2000) 85:2692–2700.
Green D. Apoptotic pathways: paper wraps stone blunts scissors. Cell (2000) 102:1–4.[CrossRef][Web of Science][Medline]
Gu YQ, Wang XH, Xu D, Peng L, Cheng LF, Huang MK, Huang ZJ, Zhang GY. A multicenter contraceptive efficacy study of injectable testosterone undecanoate in healthy Chinese men. J Clin Endocrinol Metab (2003) 88:562–568.
Hall PA, Levison DA. Review: assessment of cell proliferation in histological material. J Clini Pathol (1990) 43:184–192.[CrossRef]
Hengartner M. The biochemistry of apoptosis. Nature (2000) 407:770–776.[CrossRef][Medline]
Honarpour N, Du C, Richardson JA, Hammer RE, Wang X, Herz J. Adult Apaf-1-deficient mice exhibit male infertility. Dev Biol (2000) 218:248–258.[CrossRef][Web of Science][Medline]
Knudson CM, Tung KS, Tourtellotte WG, Brown GA, Korsmeyer SJ. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science (1995) 270:96–99.
Lee J, Richburg JH, Younkin SC, Boekelheide K. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology (1997) 138:2081–2088.
Marshall GR, Zorub DS, Plant TM. Follicle-stimulating hormone amplifies the population of differentiated spermatogonia in the hypophysectomized testosterone-replaced adult rhesus monkey (Macaca mulatta). Endocrinology (1995) 136:3504–3511.[Abstract]
Marshall GR, Ramaswamy S, Plant TM. Gonadotropin-independent proliferation of the pale type A spermatogonia in the adult rhesus monkey (Macaca mulatta). Biol Reprod (2005) 73:222–229.
Matsumoto AM, Bremner WJ. Stimulation of sperm production by human chorionic gonadotropin after prolonged gonadotropin suppression in normal men. J Androl (1985) 6:137–143.
Matsumoto AM, Karpas AE, Paulsen CA, Bremner WJ. Reinitiation of sperm production in gonadotropin-suppressed normal men by administration of follicle-stimulating hormone. J Clin Invest (1983) 72:1005–1015.[Web of Science][Medline]
Matsumoto AM, Karpas AE, Bremner WJ. 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 (1986) 62:1184–1192.
Matthiesson KL, Stanton PG, O'Donnell L, Meachem SJ, Amory JK, Berger R, Bremner WJ, McLachlan RI. Effects of testosterone and levonorgestrel combined with a 5alpha-reductase inhibitor or gonadotropin-releasing hormone antagonist on spermatogenesis and intratesticular steroid levels in normal men. J Clin Endocrinol Metab (2005) 90:5647–5655.
Matthiesson KL, McLachlan RI, O'Donnell L, Frydenberg M, Robertson DM, Stanton PG, Meachem SJ. The relative roles of follicle-stimulating hormone and luteinizing hormone in maintaining spermatogonial maturation and spermiation in normal men. J Clin Endocrinol Metab (2006) 91:3962–3969.
McLachlan RI, O'Donnell L, Meachem SJ, Stanton PG, De Kretser DM, Pratis K, Robertson DM. Identification of specific sites of hormonal regulation in spermatogenesis in rats, monkeys, and man. Recent Prog Horm Res (2002) a57:149–179.
McLachlan RI, O'Donnell L, Stanton PG, Balourdos G, Frydenberg M, de Kretser DM, Robertson DM. Effects of testosterone plus medroxyprogesterone acetate on semen quality, reproductive hormones, and germ cell populations in normal young men. J Clin Endocrinol Metab (2002) b87:546–556.
Meachem SJ, McLachlan RI, Stanton PG, Robertson DM, Wreford NG. FSH immunoneutralization acutely impairs spermatogonial development in normal adult rats. J Androl (1999) 20:756–762.
Meachem SJ, Ruwanpura SM, Ziolkowski J, Ague JM, Skinner MK, Loveland KL. Developmentally distinct in vivo effects of FSH on proliferation and apoptosis during testis maturation. J Endocrinol (2005) 186:429–446.
Moudgal NR, Sairam MR, Krishnamurthy HN, Sridhar S, Krishnamurthy H, Khan H. Immunization of male bonnet monkeys (M. radiata) with a recombinant FSH receptor preparation affects testicular function and fertility. Endocrinology (1997) 138:3065–3068.
Nagata S, Golstein P. The Fas death factor. Science (1995) 267:1449–1456.
Nandi S, Banerjee PP, Zirkin BR. Germ cell apoptosis in the testes of Sprague Dawley rats following testosterone withdrawal by ethane 1,2-dimethanesulfonate administration: relationship to Fas? Biol Reprod (1999) 61:70–75.
Ottenweller JE, Li MT, Giglio W, Anesetti R, Pogach LM, Huang HF. Alteration of follicle-stimulating hormone and testosterone regulation of messenger ribonucleic acid for Sertoli cell proteins in the rat during the acute phase of spinal cord injury. Biol Reprod (2000) 63:730–735.
Pentikainen V, Erkkila K, Dunkel L. Fas regulates germ cell apoptosis in the human testis in vitro. Am J Physiol (1999) 276:E310–E316.[Web of Science][Medline]
Russell LD, Ettlin RA, Sinha-Hikim AP, Clegg ED. Histological and Histopathological Evaluation of the Testis (1990) Clearwater: Cache River Press.
Russell LD, Chiarini-Garcia H, Korsmeyer SJ, Knudson CM. Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis. Biol Reprod (2002) 66:950–958.
Saunders PT. Germ cell-somatic cell interactions during spermatogenesis. Reprod Suppl (2003) 61:91–101.[Medline]
Schlatt S, Weinbauer GF. Immunohistochemical localization of proliferating cell nuclear antigen as a tool to study cell proliferation in rodent and primate testes. Int J Androl (1994) 17:214–222.[Web of Science][Medline]
Shetty J, Marathe GP, Dighe RR. Specific immunoneutralisation of FSH leads to apoptotic cell death of the pachytene spermatocytes and spermatogonial cells in the rat. Endocrinology (1996) 137:2179–2182.[Abstract]
Simorangkir DR, Marshall GR, Ehmcke J, Schlatt S, Plant TM. Prepubertal expansion of dark and pale type A spermatogonia in the rhesus monkey (Macaca mulatta) results from proliferation during infantile and juvenile development in a relatively gonadotropin independent manner. Biol Reprod (2005) 73:1109–1115.
Sinha-Hikim AP, Wang C, Lue Y, Johnson L, Wang XH, Swerdloff RS. Spontaneous germ cell apoptosis in humans: evidence for ethnic differences in the susceptibility of germ cells to programmed cell death. J Clin Endocrinol Metab (1998) 83:152–156.
Sinha-Hikim AP, Lue Y, Diaz-Romero M, Yen PH, Wang C, Swerdloff RS. Deciphering the pathways of germ cell apoptosis in the testis. J Steroid Biochem Mol Biol (2003) a85:175–182.[CrossRef][Web of Science][Medline]
Sinha-Hikim AP, Lue Y, Yamamoto CM, Vera Y, Rodriguez S, Yen PH, Soeng K, Wang C, Swerdloff RS. Key apoptotic pathways for heat-induced programmed germ cell death in the testis. Endocrinology (2003) b144:3167–3175.
Steger K, Aleithe I, Behre H, Bergmann M. The proliferation of spermatogonia in normal and pathological human seminiferous epithelium: an immunohistochemical study using monoclonal antibodies against Ki-67 protein and proliferating cell nuclear antigen. Mol Hum Reprod (1998) 4:227–233.
Takagi S, Itoh N, Kimura M, Sasao T, Tsukamoto T. Spermatogonial proliferation and apoptosis in hypospermatogenesis associated with nonobstructive azoospermia. Fertil Steril (2001) 76:901–907.[CrossRef][Web of Science][Medline]
Tesarik J, Guido M, Mendoza C, Greco E. Human spermatogenesis in vitro: respective effects of follicle-stimulating hormone and testosterone on meiosis, spermiogenesis, and Sertoli cell apoptosis. J Clin Endocrinol Metab (1998) 83:4467–4473.
Tesarik J, Martinez F, Rienzi L, Iacobelli M, Ubaldi F, Mendoza C, Greco E. In-vitro effects of FSH and testosterone withdrawal on caspase activation and DNA fragmentation in different cell types of human seminiferous epithelium. Hum Reprod (2002) 17:1811–1819.
Vera Y, Diaz-Romero M, Rodriguez S, Lue Y, Wang C, Swerdloff RS, Sinha Hikim AP. Mitochondria-dependent pathway is involved in heat-induced male germ cell death: lessons from mutant mice. Biol Reprod (2004) 70:1534–1540.
Vera Y, Erkkila K, Wang C, Nunez C, Kyttanen S, Lue Y, Dunkel L, Swerdloff RS, Sinha-Hikim AP. Involvement of p38 mitogen-activated protein kinase and inducible nitric oxide synthase in apoptotic signaling of murine and human male germ cells after hormone deprivation. Mol Endocrinol (2006) 20:1597–1609.
Verhoeven G, Callieau J. Follicle-stimulating hormone and androgens increase the concentration of the androgen receptor in Sertoli cells. Endocrinology (1988) 122:1514–1550.
Weinbauer GF, Behre HM, Fingscheidt U, Nieschlag E. 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 (1991) 129:1831–1839.
Weinbauer GF, Schlatt S, Walter V, Nieschlag E. Testosterone-induced inhibition of spermatogenesis is more closely related to suppression of FSH than to testicular androgen levels in the cynomolgus monkey model (Macaca fascicularis). J Endocrinol (2001) 168:25–38.[Abstract]
Zhang ZH, Zhou XC, Wei P, Hu ZY, Liu YX. Expression of Bcl-2 and Bax in rhesus monkey testis during germ cell apoptosis induced by testosterone undecanoate. Arch Androl (2003) 49:439–447.[CrossRef][Web of Science][Medline]
Zhengwei Y, Wreford N, Royce P, de Kretser DM, McLachlan RI. Stereological evaluation of human spermatogenesis after suppression by testosterone treatment: heterogeneous pattern of spermatogenic impairment. J Clin Endocrinol Metab (1998) a83:1284–1291.
Zhengwei Y, Wreford NG, Schlatt S, Weinbauer GF, Nieschlag E, McLachlan RI. Acute and specific impairment of spermatogonial development by GnRH antagonist-induced gonadotrophin withdrawal in the adult macaque (Macaca fascicularis). J Reprod Fertil (1998) b112:139–147.
Zhou XC, Wei P, Hu ZY, Gao F, Zhou RJ, Liu YX. Role of Fas/FasL genes in azoospermia or oligozoospermia induced by testosterone undecanoate in rhesus monkey. Acta Pharmacol Sin (2001) 22:1028–1033.[Web of Science][Medline]
Submitted on August 27, 2007; resubmitted on October 12, 2007; accepted on October 23, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Z. He, M. Kokkinaki, J. Jiang, I. Dobrinski, and M. Dym Isolation, Characterization, and Culture of Human Spermatogonia Biol Reprod, February 1, 2010; 82(2): 363 - 372. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D.R. Simorangkir, S. Ramaswamy, G.R. Marshall, C.R. Pohl, and T.M. Plant A selective monotropic elevation of FSH, but not that of LH, amplifies the proliferation and differentiation of spermatogonia in the adult rhesus monkey (Macaca mulatta) Hum. Reprod., July 1, 2009; 24(7): 1584 - 1595. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Lue, C. Wang, Y. Cui, X. Wang, J. Sha, Z. Zhou, J. Xu, C. Wang, A. P. Sinha Hikim, and R. S. Swerdloff Levonorgestrel Enhances Spermatogenesis Suppression by Testosterone with Greater Alteration in Testicular Gene Expression in Men Biol Reprod, March 1, 2009; 80(3): 484 - 492. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Sa, R. Neves, S. Fernandes, C. Alves, F. Carvalho, J. Silva, N. Cremades, I. Malheiro, A. Barros, and M. Sousa Cytological and Expression Studies and Quantitative Analysis of the Temporal and Stage-Specific Effects of Follicle-Stimulating Hormone and Testosterone During Cocultures of the Normal Human Seminiferous Epithelium Biol Reprod, November 1, 2008; 79(5): 962 - 975. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M Ruwanpura, R. I McLachlan, P. G Stanton, K. L Loveland, and S. J Meachem Pathways involved in testicular germ cell apoptosis in immature rats after FSH suppression J. Endocrinol., April 1, 2008; 197(1): 35 - 43. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Ruwanpura, R. I. McLachlan, P. G. Stanton, and S. J. Meachem Follicle-Stimulating Hormone Affects Spermatogonial Survival by Regulating the Intrinsic Apoptotic Pathway in Adult Rats Biol Reprod, April 1, 2008; 78(4): 705 - 713. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||