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BOR - Papers in Press, published online ahead of print December 19, 2007.
Biol Reprod 2007, 10.1095/biolreprod.107.065912
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BIOLOGY OF REPRODUCTION 78, 705–713 (2008)
DOI: 10.1095/biolreprod.107.065912
© 2008 by the Society for the Study of Reproduction, Inc.

Testis; 

Follicle-Stimulating Hormone Affects Spermatogonial Survival by Regulating the Intrinsic Apoptotic Pathway in Adult Rats1

Saleela M. Ruwanpura 3 4, Robert I. McLachlan 3, Peter G. Stanton 3, and Sarah J. Meachem 2 3

Prince Henry's Institute of Medical Research,3 Clayton, Victoria 3168, Australia Department of Obstetrics & Gynaecology,4 Monash University, Clayton, Victoria 3168, Australia

ABSTRACT

Follicle-stimulating hormone plays a key role in spermatogonial development in adult rats via poorly understood mechanisms. We aimed to identify the role of this hormone in the regulation of germ cell apoptosis and proliferation in adult rats by suppression of FSH action following passive immunoneutralization with a rat FSH antibody for 4 and 7 days. Apoptosis and proliferation were identified by TUNEL and proliferating cell nuclear antigen labeling methods, respectively. Intrinsic and extrinsic apoptotic pathways were identified by immunohistochemistry, stereological techniques, and RT-PCR by assessing pathway-specific proteins and genes. Following FSH suppression for 4 and 7 days, we have previously reported a 30% decrease in spermatogonial number, with increased apoptosis in a stage-specific manner. The present study also shows stage-specific increases in apoptosis with no changes in proliferation. This increase in apoptosis was attributable to an increase in spermatogonial apoptosis via the intrinsic rather than extrinsic pathway, as shown by increased activated caspase 9-positive spermatogonia. The concomitant suppression of FSH and LH/testosterone showed that testosterone alone or together with FSH was more important in spermatocyte and spermatid survival by regulating both apoptotic pathways. A reduction in the level of the intrinsic pathway transcript Bcl2l2 (apoptosis suppressor gene) following FSH suppression for 4 days shows that FSH regulates some components of the intrinsic pathway. This study reveals that FSH predominantly acts as a survival factor for spermatogonia by regulating the intrinsic pathway while having no affect on germ cell proliferation in rats in vivo.

apoptosis, caspase, extrinsic pathway, follicle-stimulating hormone, intrinsic pathway, mechanisms of hormone action, proliferation, spermatogenesis, spermatogonia, testis

INTRODUCTION

Spermatogenesis is a dynamic process occurring in three phases: mitosis (spermatogonia), meiosis (spermatocytes), and spermiogenesis (the morphological transformation of spermatids) [1, 2]. Sperm output relies upon coordinated proliferation, differentiation, and survival of each progressively maturing germ cell type, as regulated by a complex network of signals, most notably the pituitary gonadotropins, FSH, and LH/intratesticular androgens. In brief, FSH acts to support spermatogonial populations and partially support spermatocyte maturation in adult rats [3–5]. Luteinizing hormone and testosterone are required at least in part for spermatocyte maturation, but absolutely for spermatid maturation, most importantly round spermatid progression [6].

It is clear that FSH is critical in establishing the size of the Sertoli cell population in early testicular development which, in turn, is a major determinant of sperm output in the adult animal [7]. In adult rats, FSH is required to reinitiate and maintain quantitatively normal sperm production after hormonal manipulation [3–5, 8–10]. To understand the site of FSH action within the spermatogenic process, we have used the experimental model of FSH suppression by passive immunoneutralization via an FSH antibody given to normal adult rats for up to 1 wk [5]. We have previously demonstrated the reduction in spermatogonial number by 30% and slight reduction (nonsignificant) in preleptotene spermatocyte populations by this treatment [5]. These observations held true for FSH restoration studies where gonadotropin-depleted rats were administered with recombinant human FSH, showing complete restoration of spermatogonia and partial restoration of spermatocytes within 7 days [3], whereas testosterone alone failed to restore the initial phase of spermatogenesis [4]. These studies suggest that FSH plays an important role in spermatogonial development.

Follicle-stimulating hormone may potentially regulate spermatogonial development via control of its proliferation and/or apoptosis [3, 5, 10, 11]. To date, the data regarding proliferation in rodents are inconsistent. We have shown that FSH does not acutely stimulate germ cell proliferation in long-term gonadotropin-depleted adult rats [3]. In contrast, FSH has been reported to stimulate DNA synthesis in spermatogonia and spermatocytes in vitro, based on measurement of [3H]thymidine incorporation in 48-h cultures of adult rat seminiferous tubule segments [11]. In contrast, there is agreement that acute suppression of FSH leads to spermatogonial and spermatocyte apoptosis, as evidenced by in situ analysis of DNA strand breakage [10] or stage-specific changes in cell numbers [5].

There are two described pathways of apoptosis in the testis: the intrinsic and extrinsic pathways (reviewed in Sinha-Hikim et al. [12]). 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-1 (APAF1). This in turn activates initiator caspase 9, leading to activation of executioner caspases 3, 6, and 7 that cleave intracellular proteins and effect apoptosis [13–15]. The BCL2 protein family members, such as BCL2L2 (formerly BCL-W), have been shown to be involved in this pathway by the formation of dimers with BAX [13]. The intrinsic pathway is important in short-term heat-induced programmed germ cell death in the testis [16]. The extrinsic pathway (or death receptor pathway) involves Fas ligand (FASL) stimulation of FAS on target cells, which then activates initiator caspase 8 and subsequently activates executioner caspases, effecting apoptosis [17, 18]. The extrinsic pathway is involved in germ cell apoptosis following selective withdrawal of testosterone in the adult rat [19, 20]. However, the contribution of FSH-mediated signals to each of these apoptotic pathways is undefined.

We hypothesized that acute FSH suppression results in accelerated germ cell apoptosis via activation of the intrinsic and/or extrinsic pathways, rather than by a change in proliferation. In this study 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 forms (aCaspase 9: intrinsic; aCaspase 8: extrinsic) in combination with germ cell enumeration using stereological applications. In addition, this model has enabled quantification of candidate genes that contribute to the FSH-mediated pathways of apoptosis.

MATERIALS AND METHODS

Animals

Male outbred 75- to 90-day-old Sprague-Dawley rats were obtained from Monash University Animal Services (Clayton, Australia). They were maintained at 20°C in a fixed 12L:12D cycle with free access to food and water in accordance with the Australian Code of Practice for Care and Use of Animals for Scientific Purposes (National Health and Medical Research Council, Australia, 1997). This study was approved by the Monash Medical Centre Animal Ethics Committee.

Experimental Design

Passive immunization against FSH. Rats (n = 5 per group) were immunized either with a purified in-house polyclonal ovine immunoglobulin fraction derived from antisera raised against rat FSH (FSHAb) [4] or with control sheep immunoglobulin (ConAb). Each animal received a daily dose of 2 mg/kg in saline by subcutaneous injections for 4 and 7 days, a dose of FSHAb capable of neutralizing the serum FSH levels in adult rats by more than 90% (reaching the limit of assay detection) without affecting serum and testicular testosterone levels [4, 5]. Rats were killed 24 h after their final injection. Five rats remained untreated for 7 days and served as another set of controls.

To assess the potential for a synergistic effect of FSH and testosterone, adult rats were administered with the GnRH antagonist Cetrorelix (100 µg/kg/day in saline by subcutaneous injections; Asta Medica, Frankfurt, Germany) [21] for 7 days to suppress gonadotropin levels. They also received the antiandrogen flutamide (20 mg/kg/daily subcutaneously in peanut oil; Sigma-Aldrich, Milwaukee, WI) [22] and FSHAb (2 mg/kg) to suppress residual testicular testosterone and serum FSH, respectively. This group will be referred to as concomitant FSH and LH/testosterone suppression throughout this study.

Positive control tissues. The heat-treated adult rat testis was used as a positive control for identification of intrinsic pathway activation (courtesy of Dr. Amiya Sinha-Hikim), as short-term exposure of the testis to mild heat (43°C for 15 min) results in activation of germ cell apoptosis via the intrinsic pathway within 6 h [16].

Immature (18 days postpartum) rat testis was used as a positive control for identification of extrinsic pathway activation. In normal rats, aCaspase 8 expression has been detected throughout postnatal life [23].

Tissue Collection and Preparation

Prior to whole-body perfusion, right testes were removed and snap frozen in liquid nitrogen and stored at –80°C for molecular analyses. Following perfusion fixation with Bouin solution, the left testes were weighed and then sampled using a systematic sampling scheme from a random starting point [24]. The sampled tissues then were embedded in paraffin 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 PBS (10 mM, pH 7.4).

Apoptosis Analysis

In situ detection of cells with DNA strand breaks (apoptosis) was performed in tissue sections by the TUNEL method [5]. On negative control sections, the TdT enzyme was omitted and substituted with milli-Q water (Millipore, Billerica, MA). Apoptotic cells were visualized as staining deep brown using the chromogen diaminobenzidine (DAB).

Using stereological techniques, three tissue sections of adult rat testis were examined to determine the percentage of tubules containing TUNEL-labeled cells in untreated controls and in rats that received ConAb or FSHAb for 4 or 7 days. Each tubule cross section was classified into one of three stage groupings (XIV–III, IV–VIII, and IX–XIII) [25] based on the reliable stage identification using broad groupings of basal cells, since more precise stage groupings are difficult due to the absence in staining of round spermatid acrosomes with hematoxylin. In order to estimate the frequency of tubules containing TUNEL-labeled cells, 60–150 tubules for each stage grouping were studied per rat using a crosshair superimposed on video image by CASTGRID V1.60 software package (Olympus). All slides were masked prior to the analysis. The extent of apoptosis was calculated by dividing the number of tubules containing one or more TUNEL-labeled cells by the total number of tubules in stage groupings. Previously, this method of quantification has been validated for assessing TUNEL-labeled cells in the same experimental paradigm [5].

Assessment of Proliferation

The expression of proliferating cell nuclear antigen (PCNA) is used as an index of the proliferative activity in rat testicular tissues [26, 27]. Tissue sections from rats that received ConAb or FSHAb for 4 or 7 days were subjected to antigen retrieval in EDTA-NaOH buffer (1 mM, pH 8; 90°C–95°C for 10 min, then room temperature for 1 h), and then endogenous peroxidase activity was quenched. The sections were treated with 10% normal rabbit serum (Serotec, Raleigh, NC) in PBS and incubated with mouse monoclonal anti-human PCNA antibody (5 µg/ml in PBS; Biosciences, Franklin Lakes, NJ) for 1 h. Sections were then incubated with biotinylated rabbit anti-mouse IgG secondary antibody (10 µg/ml in PBS; Zymed Labs, San Francisco, CA) for 30 min with prewashes and postwashes with PBS, followed by incubation with ABC complex (Vectastain Elite; Vector Laboratories, Burlingame, CA) 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 cover slips. The negative control sections were performed in a similar manner, except the primary antibody was substituted with the same concentration of mouse IgG (Biosciences).

Assessment of Apoptotic Pathways

The activation of apoptotic pathways has been identified with previously validated immunohistochemistry procedures by employing antibodies against the activated forms of pathway-specific caspases [28]. Testicular tissue sections were subjected to antigen retrieval in EDTA-NaOH buffer, endogenous peroxidase activity was quenched, blocked with 10% normal goat or rabbit sera, and then incubated overnight with either activated caspase 9 (aCaspase 9) antibody (0.76 µg/ml in PBS; this rabbit polyclonal antibody detects p17 and p37 of active caspase 9 protein; Cell Signaling Technology) or with aCaspase 8 antibody (2.4 µg/ml in PBS; this mouse monoclonal antibody 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) or with biotinylated rabbit anti-mouse IgG secondary antibody for 1 h (10 µg/ml; Zymed Labs) and 30 min, respectively, with prewashes and postwashes with PBS. Following ABC complex (Vector Laboratories) treatment, DAB was added for 2–3 min, and sections were counterstained and blued and mounted. On negative control sections, the primary aCaspase 9 and 8 antibodies were substituted with the same concentration of rabbit and mouse IgG antibodies (Biosciences), respectively.

Quantification of Labeled Cells

Stereological techniques were applied to determine the percentages of PCNA and aCaspase 9- or 8-labeled cells. Proliferating cell nuclear antigen-labeled cell types were identified by deep brown nuclear staining, and aCaspase-labeled cells by brown nuclear, cytoplasmic, and whole-cell staining. (Upon activation, caspases translocate from the cytoplasm to the nucleus; therefore, localization of activated caspase varies along the apoptotic pathway). Germ cell types were identified by their location within the seminiferous tubules, in conjunction with their nuclear size and shape. Cells were classified into groups: spermatogonia (type A, intermediate, and B spermatogonia), preleptotene spermatocytes, leptotene-pachytene spermatocytes (leptotene, zygotene, and pachytene spermatocytes), round spermatids, and elongating/elongated spermatids [25]. The percentages of labeled cells were assessed using an unbiased counting frame of 2914 µm2 per field superimposed on a video image by CASTGRID V1.60 software package (Olympus), where 50–400 cell nuclei for each cell group were counted per rat. All slides were masked prior to the analysis. The extent of proliferation and caspase activities were quantified by dividing the number of labeled cells by the total number of labeled and unlabeled cells in each group. Previously, this method of quantification has been validated for assessing PCNA and caspase-labeled cells in the human testis [29].

Immunofluorescence and Confocal Studies

To determine the prevalence of cells undergoing each apoptotic pathway, the colocalization of TUNEL-labeled cells with aCaspase 9 or 8 proteins was detected by confocal microscopy using immunofluorescence dual labeling [16, 30]. In situ detection of cells with DNA fragmentation was performed on tissue sections using an Apoptag fluorescein in situ apoptosis detection kit (Chemicon). In brief, to eliminate nonspecific binding to meiotic cells, tissues were subjected to microwave antigen retrieval in EDTA-NaOH buffer (1 mM, pH 8; 90°C–95°C for 10 min, then room temperature for 1 h) rather than protease K treatment [31]. Tissues then were 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 (Cell Signaling Technology) or aCaspase 8 (2.4 µg/ml in PBS; this rabbit monoclonal antibody detects only the cleaved product p18, 41, 43 of active caspase 8 protein; Cell Signaling Technology) overnight, followed by goat anti-rabbit Alexa 546 secondary antibody (Molecular Probes, Eugene, OR) for 45 min. Slides were washed and then mounted with Fluorsave (Calbiochem). 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-labeled germ cells that were either aCaspase 9 or 8 positive were quantified by counting all the labeled and dual-labeled cells. This method of quantification has been validated for assessing TUNEL-labeled cells with caspase activity in human testis [29]. TUNEL-labeled cells were observed with varying intensities of aCaspase staining, and therefore we designated them as aCaspase low or aCaspase high. Only aCaspase-high TUNEL cells were included in the quantification for dual labeling.

Total RNA Extraction and Reverse Transcription

Real-time PCR was used to measure the relative levels of five candidate apoptotic pathway-specific genes. Total RNA was extracted from testes treated with ConAb; FSHAb for 4 (n = 5 per group) and 7 (n = 3 per group) days; and gonadotropin antagonist, FSHAb, and flutamide for 7 (n = 5 per group) days using a total RNA extraction kit (Qiagen, Hilden, Germany). Any contaminating residual genomic DNA was removed using the DNAse-free kit (Ambion) according to the manufacturer's instructions. Reverse transcription was performed on 500 ng total RNA/sample using AMV-reverse transcriptase (8 units; Roche, Mannheim, Germany), random hexamer primers (200 ng; Amersham Biosciences), dinucleotide triphosphates (20 nmol each; Roche), RNasin (20 units; Promega, Madison, WI), and 5x reaction buffer (Roche) in a final volume of 20 µl for 90 min at 46°C and 2 min at 95°C, prior to storage at –20°C. The absence of contaminating genomic DNA in total RNA samples was confirmed using reactions in which AMV-reverse transcriptase was omitted.

Real-Time PCR Analysis

Quantitative real-time PCR analysis was performed using the Roche LightCycler and the FastStart DNA Master SYBR-Green 1 system (Roche). Oligonucleotide primer sequences for Gapdh, Bax, and Bcl2l2 were designed using Primer 3 (www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) [32], and caspase 9 (Casp9), caspase 8 (Casp8), and Fas were obtained from published sources (Table 1). For PCR analysis, sample cDNA was diluted 1:4- to 1:80-fold, with a primer concentration of 40 pmol. Polymerase chain reaction conditions, including Mg2+ concentration, anneal temperature, anneal time, and extension time, were optimized for each primer pair and are summarized in Table 1. For all PCR analyses, standard curves were produced using dilutions of an adult control rat testicular cDNA preparation assigned an arbitrary unitage. Polymerase chain reaction of all standards was performed using duplicate reactions, and samples were performed using triplicates for approximately 38–40 cycles, after which a melting curve analysis was performed to monitor PCR product purity (Table 1). Amplification of a single PCR product was confirmed by agarose gel electrophoresis and DNA sequencing (data not shown). To compare the expression levels of the apoptotic pathway-specific genes in groups, we normalized the data collected in our RNA analysis with the housekeeping gene, Gapdh.


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  		TABLE 1. Primer-specific LightCycler conditions used for PCR amplification of candidate genes.

Statistical Analysis

All statistical analyses were performed using Sigmastat for Windows version 3.1 (Jandel Corp.). Normally distributed data for ConAb- and FSHAb-treated samples were analyzed using t-test, and if data did not show normal distribution, a Mann-Whitney test was performed. A one-way ANOVA was used to determine the differences between ConAb, FSHAb, and gonadotropin antagonist, FSHAb, and flutamide-treated rats for 7 days for all normally distributed data. If data did not show normal distribution, then a Kruskal-Wallis one-way ANOVA on ranks was performed. Data are expressed as mean ± SEM (for all histological and testicular weight data) or SD (for all gene expression levels), n = 3–5 rats per group.

RESULTS

Testicular Weights Are Not Affected by Acute FSH Suppression

The testicular weights of rats treated for 4 or 7 days with either FSHAb (1.8 ± 0.1 g and 1.8 ± 0.2 g, respectively) or ConAb (1.9 ± 0.2 g and 1.9 ± 0.2 g, respectively) were not significantly different. In response to 7 days of concomitant FSH and LH/testosterone suppression, testicular weights were reduced to 71% (1.4 ± 0.1 g vs. 1.9 ± 0.2 g; P < 0.001) of those in corresponding ConAb-treated rats.

FSH Suppression Induces Apoptosis in a Stage-Specific Manner

In untreated rats, the frequencies of seminiferous tubules containing TUNEL-labeled cells were highest at stage groupings XIV–III and IX–XIII (29% and 23%, respectively) and lowest at stage grouping IV–VIII (3%; Fig. 1A). There were no differences between the frequency of TUNEL-labeled tubules in any stage grouping between ConAb-treated and untreated rats at either time point.


Figure 01
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  		FIG. 1. The percentage of tubules in adult testes displaying TUNEL-labeled germ cells in stages XIV–III, IV–VIII, and IX–XIII of the seminiferous cycle in untreated (grid patterned bars), in rats that received ConAb (white bars) or FSHAb (black bars) for 4 and 7 days, and gonadotropin antagonist, FSHAb, and flutamide (stippled bar) for 7 days (A). Data are means ± SEM, n = 5 per group. Asterisks denote significant differences between FSH and/or LH/testosterone-suppressed rats and the corresponding ConAb-treated samples (*P < 0.05; **P < 0.01). Photomicrographs represent the TUNEL and PCNA staining in cross sections of the testis from ConAb-treated (B, D) and FSHAb-treated (C, E) rats for 4 days, respectively. Insets are negative control sections. T, testosterone. Bar in B = 100 µm (B-E) and 400 µm (insets in B-E).

In response to 4 days of FSHAb treatment, there was a 5-fold increase in the frequency of stage grouping IV–VIII tubules displaying TUNEL-labeled cells compared with ConAb-treated controls (4.1% vs. 20.8%; P = 0.005); however, no significant differences were observed in stage groupings XIV–III and IX–XIII. Furthermore, in response to 7 days of FSHAb treatment, there were increases in stage groupings XIV–III (1.4-fold of ConAb-treated rats, 29.4% vs. 41.0%; P = 0.011) and IV–VIII (3.1-fold of ConAb-treated rats, 4.3% vs. 13.3%; P = 0.056) compared with ConAb-treated rats, with no differences in stage grouping IX–XIII (Fig. 1, A–C). In concomitant FSH and LH/testosterone-suppressed rats for 7 days, the frequencies of seminiferous tubules containing TUNEL-labeled cells at stage groupings XIV–III, IV–VIII, and IX–XIII (78%, 95%, and 95%, respectively) were significantly increased compared with their corresponding ConAb-treated rats (Fig. 1A).

The number of apoptotic germ cells per tubule did not differ between FSHAb- and ConAb-treated rats at either time point (data not shown). However, concomitant FSH and LH/testosterone suppression for 7 days led to an increase in the number of apoptotic germ cells per tubule compared with that of corresponding ConAb-treated rats (1.9 ± 0.1 vs. 7.9 ± 0.9; P = 0.008).

FSH Suppression Did Not Affect Germ Cell Proliferation

Proliferating cell nuclear antigen indices of germ cell proliferation following 4 and 7 days of FSH suppression were similar to those of corresponding ConAb-treated rats (Fig. 1, D and E, and Table 2).


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  		TABLE 2. The percentage of PCNA-labeled germ cell populations of the seminiferous epithelium in untreated rats and adult rats that received FSHAb for 4 or 7 days.a

FSH Suppression Alters Both the Intrinsic and Extrinsic Pathways

Four days of FSH suppression increased the proportion of TUNEL-labeled germ cells that were positive for aCaspase 9 to 68.9% ± 3.2% vs. 49.1 ± 1.8% (P < 0.001) and decreased aCaspase 8 to 42.4% ± 1.4% vs. 52.0% ± 3.1% (P = 0.020) compared with ConAb-treated rats, respectively (Fig. 2, A, C, D, F, and G).


Figure 02
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  		FIG. 2. A) The proportion of TUNEL-labeled germ cells with caspase 9 or 8 reactivity in adult rats that received ConAb (white bars) or FSHAb (black bars) for 4 or 7 days and rats that received GnRH antagonist, FSHAb, and flutamide for 7 days (patterned bars). Data are means ± SEM, n = 5 per group. Asterisks denote significant differences between FSH and/or LH/testosterone-suppressed rats and the corresponding ConAb-treated samples (*P < 0.05; **P < 0.01). B–G) Photomicrographs illustrate the staining of caspases 9 (B–D) and 8 (E–G), with markers of intrinsic and extrinsic pathways, respectively in red, in rat germ cells undergoing apoptosis (DNA fragmentation using TUNEL in green) in cross sections of rat testis. Dual staining of caspases with TUNEL appears in yellow (arrows). B) Heated adult rat testis (quality control [QC] for the intrinsic pathway is courtesy of Dr. A. Sinha-Hikim). E) 18-day-old rat testis (QC for extrinsic pathway). C, F) Sections are stained for caspases 9 and 8 from rats receiving ConAb for 4 days, respectively. D, G) Sections are stained for caspases 9 and 8 from rats receiving FSHAb for 4 days, respectively. Insets are negative controls (primary antibodies are replaced with equivalent concentrations of nonspecific antibodies with same isotope control). Bar in B = 20 µm (B-G) and 80 µm (insets in B-G).

After 7 days of FSH suppression, there were no significant differences in the proportion of TUNEL-labeled cells that were positive for aCaspase 9 or aCaspase 8 compared with ConAb-treated rats (Fig. 2A). Seven days of concomitant FSH and LH/testosterone suppression showed a marginal change in the proportion of TUNEL-labeled germ cells that were positive for caspase 9 activity to 45.0% ± 2.7% vs. 54.0% ± 3.0% compared with ConAb-treated rats (P = 0.053), whereas aCaspase 8 activity was reduced to 24.9% ± 2.9% compared with 46.1% ± 3.7% of corresponding ConAb-treated rats (P = 0.002; Fig. 2A).

FSH Suppression Affects Spermatogonial Apoptosis via the Intrinsic Apoptotic Pathway

In rats treated with ConAb for 4 and 7 days, low levels of aCaspase 9 labeling was observed in spermatogonia, preleptotene spermatocytes, and leptotene-pachytene spermatocytes (Fig. 3, A and B). FSHAb treatment for 4 and 7 days, respectively, resulted in 2- to 3-fold increases in aCaspase 9-labeled spermatogonia (4 days: 1.2% ± 0.8% vs. 3.8% ± 0.7%; P = 0.040; 7 days: 3.1% ± 0.6% vs. 6.9% ± 1.3%; P = 0.030) compared with the corresponding ConAb-treated rats. A 4.8-fold increase in aCaspase 9-labeled preleptotene spermatocytes at 4 days compared with ConAb-treated rats was also observed; however, this did not achieve significance (0.7% ± 0.4% vs. 3.3% ± 1.8%: P = 0.20; Fig. 3, A and C).


Figure 03
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  		FIG. 3. A) The percentage of aCaspase9-labeled germ cells in germ cell groups—spermatogonia, preleptotene spermatocytes, and leptotene-pachytene spermatocytes—in adult rats that received ConAb (white bars) or FSHAb (black bars) for 4 or 7 days, and GnRH antagonist, FSHAb, and flutamide for 7 days (patterned bars). Data are means ± SEM, n = 5 per group. Asterisks denote significant differences between FSH and/or LH/T suppressed rats and the corresponding ConAb-treated samples (*P < 0.05). Photomicrographs represent the staining of aCaspase 9 (B, C) and aCaspase 8 (D, E), markers of intrinsic and extrinsic pathways, respectively, in cross sections of the testis from ConAb-treated (B, D) and FSHAb-treated (C, E) rats for 4 days, respectively. Insets represent negative controls. Bar in B = 100 µm (B-E) and 400 µm (insets in B-E).

In concomitant FSH and LH/testosterone-suppressed rats for 7 days, there was an apparent 1.5-fold increase in aCaspase 9-labeled spermatogonia (3.1% ± 0.6% vs. 4.8% ± 0.9%; P = 0.05) but no significant change in aCaspase 9-labeled preleptotene spermatocytes (1.3% ± 0.7% vs. 3.1% ± 1.5%; P = 0.50) compared with the corresponding ConAb-treated rats (Fig. 3A). In addition, aCaspase 9 labeling was observed in round spermatids (4.8% ± 1.3%) and elongating/elongated spermatids (6.1% ± 2.4%) in concomitant FSH and LH/testosterone-suppressed rats, even though none were observed in their corresponding ConAb-treated rats.

In ConAb- and FSHAb-treated rats for 4 and 7 days, low levels of aCaspase 8 labeling was observed in spermatogonia, preleptotene spermatocytes, leptotene-pachytene spermatocytes, round spermatids, and elongating/elongated spermatids (Fig. 3, D and E; quantification data not shown). Conversely, concomitant FSH and LH/testosterone suppression for 7 days led to significant increases in the amount of aCaspase 8 labeling of preleptotene spermatocytes (0.8% ± 0.5% vs. 4.9% ± 2.0%; P = 0.020), leptotene-pachytene spermatocytes (0.8% ± 0.2% vs. 5.4% ± 1.0%; P < 0.001), round spermatids (0.3% ± 0.2% vs. 10.2% ± 3.4%; P = 0.008), and elongating/elongated spermatids (0.1% ± 0.1% vs. 7.1% ± 1.0%; P = 0.009) compared with that of the corresponding ConAb-treated rats.

Identification of FSH-Regulated Genes Involved in Apoptosis

Changes in expression of three intrinsic pathway-specific genes, Casp9, Bax, and Bcl2l2, were examined by real-time RT-PCR of whole testis. After 4 days of FSH suppression, both Bax and Bcl2l2 showed 2-fold significant (both P < 0.001) reductions in mRNA levels (Fig. 4, B and C), whereas Casp9 mRNA did not show any significant change, compared with the corresponding ConAb-treated rats (Fig. 4A).


Figure 04
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  		FIG. 4. The mRNA expression of Casp9, Bax, Bcl2l2, Casp8, and Fas levels in adult rats that received ConAb (white bars) or FSHAb (black bars) for 4 or 7 days, and GnRH antagonist, FSHAb, and flutamide for 7 days (patterned bars). Data are means ± SD, n = 3–5 per group. Asterisks denote significant differences between FSH and/or LH/testosterone-suppressed rats and the corresponding ConAb-treated samples (*P < 0.05; **P < 0.01).

After 7 days of FSH suppression, Casp9, Bax, and Bcl2l2 mRNA levels did not show any significant changes compared with ConAb-treated rats (Fig. 4, A–C). In response to 7 days of concomitant FSH and LH/testosterone suppression, there were decreases in mRNA expression of Bax (by 1.6-fold; P < 0.001), Bcl2l2 (by 1.7-fold; P < 0.001), and Casp9 (by 1.6-fold; P = 0.04) compared with the corresponding ConAb-treated rats (Fig. 4, A–C).

The expression of two extrinsic pathway-specific genes, Casp8 and Fas, were also examined in whole testis. After 4 days of FSH suppression, a significant 1.4-fold reduction in Casp8 mRNA levels (P = 0.006; Fig. 4D) was observed; however, there was no change in Fas mRNA levels compared with ConAb-treated rats (Fig. 4E). Following 7 days of concomitant FSH and LH/testosterone suppression, no significant differences in the mRNA expression of Casp8 and Fas compared with the corresponding ConAb-treated rats were observed (Fig. 4, D and E).

DISCUSSION

In this study, we report that FSH acts as a survival factor by regulating the intrinsic pathway in the adult rat testis. We previously reported a 30% decrease in type A/intermediate spermatogonial numbers, with increased apoptosis in a stage-specific manner following acute FSH suppression [5]. We now show that these increases in apoptosis are attributable to an increase in spermatogonial apoptosis via the intrinsic pathway, not the extrinsic pathway. FSH had no effect on germ cell proliferation in this model. In addition, we have shown that testosterone alone or together with FSH is important in spermatocyte and spermatid survival through regulation of both apoptotic pathways. Following FSH suppression for 4 days, the reduction in Bcl2l2 mRNA levels supports the contention that FSH regulates components of the intrinsic pathway.

The present data demonstrate that FSH suppression induces apoptosis in a stage-specific manner in adult rat testis, in agreement with our previous observations in a similar paradigm [5]. Both studies showed a similar increase in the frequency of stage XIV–III tubules with apoptosis after 7 days and, additionally, the present study shows increases in stages IV–VIII after 4 and 7 days following FSH suppression. We were unable to discern what subpopulation of spermatogonia was affected. However, a decrease in type A/intermediate spermatogonial development (associated with stages I–IV) has been suggested by the time course of decline in spermatogonial number corresponding to an inhibition of A3–A4 spermatogonial maturation at stages XIV–I [5]. The mutant mouse models of FSH deficiency, FSH receptor knockout mice, showed that FSH is required for quantitatively normal spermatogenesis and suggested that spermatogonia may undergo increased apoptosis as assessed by flow cytometry [35]. In addition, FSH-beta knockout mice show reduction in spermatogonial numbers; however, apoptosis has not been assessed in these mice [36]. The effect of FSH on midstage germ cell apoptosis (VI–VIII) at 4 days may reflect the disruption of androgen action in this experimental setting, despite testicular testosterone concentration remaining normal [5]. The mechanism of this effect may be due to FSH regulation of androgen receptor number and androgen binding protein [37, 38]. Nonetheless, FSH has been shown to support the restoration [3] and maintenance [9] of pachytene spermatocyte and round spermatid number in gonadotropin-depleted rats, and to enhance spermatocyte survival in vitro [10], suggesting a direct effect of FSH on these germ cell types.

Our data provide no evidence that FSH regulates germ cell proliferation as assessed by PCNA for both control and FSH-suppressed rats. This conclusion is consistent with our previous data, where no change in the proliferative index was observed with acute replacement of FSH in chronically gonadotropin-depleted rats when assessed using bromodeoxyuridine incorporation [3]. These data suggest that germ cell proliferation in normal adult rats is FSH independent. In the context of the rat in vivo studies, it must be noted that low but potentially biologically significant (less than 10%) levels of FSH persisting in FSH-suppressed and gonadotropin-depleted rats may support germ cell proliferation. This potentially low level of FSH within our model may also explain an unchanged number of apoptotic cells per tubule and 5%–25% of tubules not displaying apoptosis following FSH suppression alone or together with LH suppression, respectively. Consistent with the present study, FSH receptor knockout mice showed no change in bromodeoxyuridine-labeled cells by indicating that the kinetics of spermatogenesis have been unaffected by the lack of FSH [35]. In contrast to our data, the hypogonadal (hpg) mice expressing transgenic FSH hormone (tgFSH/hpg) provide compelling evidence that FSH supports spermatogonial proliferation and the stimulation of meiotic and postmeiotic germ cell development [39]. It also has been shown that FSH stimulates early germ cell development in in vitro cultures of adult rat seminiferous tubule segments [11].

This study demonstrates for the first time that FSH regulates spermatogonial survival via the intrinsic apoptotic pathway and not the extrinsic apoptotic pathway in rats, as evidenced by increased aCaspase 9- but not aCaspase 8-positive spermatogonia following 4 and 7 days of FSH suppression. To date, FSH regulation of apoptotic pathways has not been (directly) shown. Intrinsic pathway activation of spermatogonial cell death has been observed in models of estrogen manipulation [40, 41]. Administration of the estrogenic compound, diethylstilbestrol, for up to 7 days activated the intrinsic apoptotic pathway in adult rats observed as the translocation of BAX to mitochondria, resulting in the release of cytochrome c [40]. In vitro culture systems confirmed that these estrogen-induced changes lead to spermatogonial death via intrinsic apoptotic pathway [41]. However, as estrogen inhibits gonadotropin (predominantly FSH) secretion in vivo (Tena-Sempere et al. [42], reviewed in O'Donnell et al. [43]), the failure to assess FSH levels in in vivo models of estrogen exposure raises the possibility that activation of intrinsic pathway may have resulted specifically or partially from FSH withdrawal. Additional evidence for intrinsic pathway regulation of spermatogonial survival was provided by the Bax gene knockout mouse, wherein a rise in spermatogonial cell number suggested the failure of physiological apoptosis [44, 45]. Furthermore, when Apaf1 was removed via gene targeting, high numbers of degenerating spermatogonia were seen, suggesting apoptosis occurs via the intrinsic pathway in male germ line [46].

In our study, the concomitant FSH and LH/testosterone-suppressed group showed a level of increased aCaspase 9-positive spermatogonia similar to the FSH alone-suppressed group. Despite the fact that this latter result was nonsignificant, the similarity in responses suggests increased spermatogonial apoptosis stems from the suppression of FSH rather than testosterone. Similar to our data, Chausiaux and colleagues [47] have reported spermatogonial apoptosis in response to gonadotropin deficiency in hpg mice, at least in part via the intrinsic pathway. In addition, we showed that testosterone, alone or together with FSH, supports survival of meiotic and spermiogenic cells via both the intrinsic and extrinsic pathways in rats, as evidenced in concomitant FSH and LH/testosterone compared with FSH alone-suppressed rats. The extrinsic pathway has been found to be involved in germ cell apoptosis after testosterone withdrawal [19, 20]. Selective testosterone deprivation by EDS (a Leydig cell toxicant) treatment resulted in the apoptosis of pachytene spermatocytes and spermatids via extrinsic pathway, as determined by the colocalization of FASL expression in those cells [20] and increased FAS protein [19] after 8 days of treatment. Increases in FAS and BAX protein and their mRNA levels after GnRH antagonist treatment [48], and increased expression of Casp8 and Casp9 mRNA in hpg mice [47], also suggest a synergistic action of both FSH and testosterone in regulating both the intrinsic and extrinsic pathways.

Intriguingly, decreases in aCaspase 8 activities of apoptotic cells after 4 days of FSH and 7 days of co-joint FSH and LH/testosterone suppression were observed. This decrease may be due to limitations in the dual-labeling techniques or the clearance (reduction) of apoptotic cells expressing caspase activity (as seen in low-aCaspase-labeled cells that were excluded from quantification) at earlier time points. However, caspase activity was assessed using light microscopy, and increases in the number of aCaspase 8-labeled cells were observed in spermatocytes and spermatids, making the technical problem with staining less likely. There may be another possible explanation for a reduction in caspase 8 activity. It may be due to cross talk between apoptotic pathways. Cross talk between apoptotic pathways is evidenced by the fact that in some cells, activated caspase 8 leads to a cleavage of the BCL2 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 [12]. This type of cross talk has been seen in the spermatocyte apoptosis during the first wave of spermatogenesis in normal rats [49]. In this study, we did not assess cross talk between apoptotic pathways in germ cells, and to our knowledge there is no evidence for cross talk in germ cells of adult rats.

The molecular events governing apoptosis in response to FSH suppression are not yet fully understood. We have investigated several candidate genes that have been identified as important regulators of testicular cell apoptosis via intrinsic and extrinsic pathways [13, 19, 33]. We have demonstrated that FSH suppression acutely (within 4 days) reduced testicular Bcl2l2 mRNA levels, suggesting an involvement of the intrinsic pathway. These data are consistent with those from Yan and colleagues [50], who observed upregulation of Bcl2l2 mRNA levels in seminiferous tubules in vitro following FSH exposure. Activation of the intrinsic pathway involves the BCL2 protein family [13], of which BCL2L2 is an important prosurvival member [51, 52] that participates in the regulation of apoptosis by dimerizing with the proapoptotic factor BAX [50]. The competitive action of the prosurvival and proapoptotic BCL2 family proteins regulates the activation of caspases [13]. However, we did not show disturbances in the dynamic equilibrium between Bcl2l2 to Bax (data not shown), but a reduction in Bax was observed at the 4-day time point following FSH suppression. The possible explanation for this reduction in proapoptotic factor may be due to downregulation of Bax by the remaining germ cells as part of a survival mechanism to minimize cell death. Surprisingly, FSH suppression showed no change, whereas the concomitant FSH and LH/testosterone suppression showed reductions in mRNA expression of Bcl2l2 and Bax after 7 days. A possible explanation for this may be that testosterone in FSH-suppressed rats compensates for prolonged FSH suppression and returns apoptotic gene mRNA expression to normal levels in order to limit further induction of cell death and prevent severe damage to the testis. Fas mRNA levels were unchanged after FSH and or LH/testosterone suppression.

Even though caspase activation is often thought of principally at the protein level controlling proteolytic cascades, some studies have reported increases in caspase mRNA due to apoptotic stimuli [33, 53]. We observed no change in Casp9 mRNA after 4 and 7 days but, surprisingly, did find a significant decrease in Casp8 mRNA expression levels after 4 days of FSH suppression. In addition, a decrease in Casp9 mRNA expression level was observed following concomitant FSH and LH/testosterone suppression for 7 days. We cannot explain these data, but it must be noted that genes give only an indication of the transcript levels that may but not necessarily reflect active protein concentration, due to the potential for posttranslational regulation of protein activities; however, active protein levels were not quantified in the present study.

In conclusion, this study reveals that FSH regulates the intrinsic pathway in the testis of adult rats. In the seminiferous tubules, FSH regulates spermatogonial survival via the intrinsic pathway, not the extrinsic pathway. Additionally, FSH suppression leads to reduction of intrinsic pathway-specific Bcl2l2 transcript levels in the whole testis. Testosterone alone or in conjunction with FSH is more important in meiotic and spermiogenic cell survival via both the intrinsic and extrinsic pathways. The FSH suppression model enables us to understand the basic mechanisms in which germ cells develop, as it may have some clinical relevance to infertility.

ACKNOWLEDGMENTS

Thank you to Dr. Amiya Sinha Hikim, Harbor-University of California and Los Angeles Biomedical Research Institute, for providing the heated-testis tissues as the quality control for intrinsic pathway, and Mr. Simon Degen, Prince Henry's Institute for designing primers.

FOOTNOTES

1Supported by the National Health and Medical Research Council of Australia, Program Grant 241000, to S.J.M., P.G.S., and R.I.M. Back

Correspondence: 2FAX: 61 3 9594 7909; e-mail: sarah.meachem{at}princehenrys.org

Received: 3 October 2007.

First decision: 23 October 2007.

Accepted: 13 December 2007.

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