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a The Molecular Reproduction Research Laboratory, Clinical Research Institute of Montreal, Montreal, Quebec, Canada H2W 1R7
b Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada H3A 2B2
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Some recent reports on human mutations are also beginning to draw attention to the role of FSH and/or its receptor in maintaining optimal testicular function conducive to normal spermatogenesis. Phillip et al. [12] have reported that hypogonadism and azoospermic condition in a man may be due to the inactivating mutation in the FSHß subunit gene. Gromoll et al. [11] noted that a man carrying activating type of FSH receptor mutation could autonomously sustain spermatogenesis in the absence of gonadotropins. In Finland, some men exhibit an inactivating mutation of FSH receptor gene that leads to a large but not complete elimination of its function [22]. In that study, one out of the three men tested was infertile, indicating variable degree of spermatogenic failure and fertility. Two other men whose fertility status remains unknown also had low sperm counts.
Gene disruption studies involving both FSH [18] and its receptor [23, 24] have now been reported, providing new animal models for further analysis. Recent reports from our laboratory on targeted disruption of FSH receptor gene [23, 24], as well as another study in which the FSHß subunit gene was deleted in mice [18], have clearly demonstrated that FSH signaling is required for maintaining normal testicular size, seminiferous tubular diameter, sperm number, and motility. However, FSHß mutant males are reportedly fully fertile [18] while FSH receptor mutant males exhibit reduced fertility [23, 24]. FSH receptor knockout males, which we now designate by the term FORKO, show increased levels of FSH and decreased testosterone levels with normal LH values. The FORKO males exhibit low sperm counts, decreased sperm motility, and aberrant sperm morphology [24]. However, no morphological sperm abnormalities were reported in the FSHß mutants that had normal circulating levels of LH and testosterone. Histological examination of the testes of FORKO males, while showing a decrease in tubular diameter, did not reveal visible changes in gross morphology or in the appearance of Sertoli and Leydig cells even though the mice exhibited reduced fertility [23, 24]. In order to find a cellular basis to explain this apparent discordance, we explored other analytical procedures. As flow cytometry provides a rapid and quantitative analysis of cells, we have applied this technique to examine the alterations that could contribute to reduced fertility observed in FORKO males. In the present study we have performed morphometric analysis of the seminiferous tubules and quantified the testicular germ cell percentages, absolute number of elongated spermatids, the percentage of spermatogonial cells undergoing proliferation, and elongated spermatid number as well as spermatid nuclear condensation during spermiogenesis. The thiol status of the cauda epididymal sperm and their susceptibility to acid denaturation in vitro using the sensitive sperm chromatin structure assay were also evaluated to elucidate the cellular basis of aberrant spermatogenesis in the complete absence of FSH receptor signaling in mice. These investigations demonstrate that disruption of FSH receptor signaling in Sertoli cells alters the testicular milieu to impede normal spermatogenesis by destabilizing sperm DNA. Our data may be of relevance in uncovering clues that may be useful in the treatment of infertile patients and in the development of a male contraceptive.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The following reagents were purchased from Sigma (St. Louis, MO): bromodeoxyuridine (BrdU), dithiothreitol (DTT), Nonidet P-40, pepsin, propidium iodide (PI), RNase, and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Anti-BrdU monoclonal antibody was from Dako Diagnostica (Ontario, ON, Canada), anti-mouse c-kit monoclonal antibody from Pharmingen (Ontario, ON, Canada), and acridine orange (AO) from Polysciences (Warrington, PA). Dulbecco's PBS was from Gibco (Grand Island, NY); testosterone RIA kit Coat-A-count from Diagnostic Products (Los Angeles, CA); Tissue Tearor homogenizer, model 985–370, from Biospec Products (Racine, WI). All other chemicals were of the highest grade available from commercial suppliers.
Animals
Groups of 10- to 12-wk-old male mice were used in the current study. As previously described [24], breeding heterozygous males with heterozygous females produced these animals. The animals were maintained under well-controlled conditions of temperature (22°C), light (14L:10D), and humidity with food and water provided ad libitum. Upon weaning, all the littermates were genotyped by polymerase chain reaction (PCR) using DNA from tailpieces and three different primers. These oligonucleotides were designed to amplify segments from the first exon in mouse FSH receptor upstream of the translation start site and a part of the neomycin gene, which had been included in the disruption strategy. In this manner, a single PCR performed on each sample allowed unambiguous identification of +/+, +/-, and -/- mice.
Histological Analysis
Wild-type and FORKO mice were anesthetized with phenobarbital, and the abdominal cavity was opened by sagittal incision. The testes were isolated and injected with 100 µl of Bouin's fixative under the tunica albuginea with a 26-gauge needle. The testicular tissues were then immersed in Bouin's fixative for 48 h at 4°C. Epididymis was also dissected out and fixed directly in Bouin's fluid. The fixative was then extracted with 70% ethanol at room temperature (RT) for 3 days, and the tissues were embedded in paraffin. Mounted sections (5 µm) were deparaffinized, rehydrated, and stained with hematoxylin and eosin.
The seminiferous tubular diameter, luminal diameter, and area of the seminiferous epithelium were measured using a computer-aided Eclipse image analyzer and Carl Zeiss (Thornwood, NY) microscope. Similarly, the tubular diameter and luminal diameter of the caput epididymal duct were also recorded.
Preparation of Testicular Cells and Epididymal Sperm
The animals were weighed and killed by an overdose of anesthetic ether. Blood was collected by heart puncture and allowed to clot. Serum testosterone was analyzed by RIA using commercial kits (sensitivity 4 ng/dl). The complete reproductive tract was dissected and placed in a Petri dish containing calcium- and magnesium-free Dulbecco's PBS. The testis, epididymis, and seminal vesicle weights were recorded. The animals used for BrdU incorporation experiments received intraperitoneal injections of 100 mg/kg body weight of this reagent in saline 1 h before they were killed.
Testes from wild-type, heterozygous, and FORKO males were minced in PBS and gently aspirated to disperse the cells, filtered using 100-µm nylon filter, and washed in PBS. After centrifugation and aspiration, the cells were resuspended in PBS, fixed in 70% chilled ethanol, and stored at 4°C until further analysis. Similarly, sperm from cauda epididymidis were prepared and stored. A drop of cauda sperm suspension was used for phase-contrast microscopy before the samples were fixed in ethanol.
Quantification of Testicular Cells by DNA Flow Cytometry
The procedure followed to stain DNA in testicular cells was essentially similar to that described elsewhere for mice [25]. Briefly, an aliquot of 1–2 x 106 ethanol-fixed testicular cells was washed with PBS and treated with 0.25% pepsin solution for 10 min at 37°C. After centrifugation, cells were stained with PI staining solution (25 µg/ml PI, 40 µg/ml RNase, and 0.3% Nonidet P-40 in PBS) at RT for 20 min. The PI-stained cells were analyzed on Coulter flow cytometer EPICS XL (Miami, FL) equipped with 15-mW argon-ion laser at an excitation wavelength of 488 nm. The fluorescence signals of the PI-stained germ cells were collected at 620 nm.
Quantification of BrdU-Labeled Testicular Cells by Dual-Parameter Flow Cytometry
The quantification of BrdU-positive cells by flow cytometry can be considered a measure of actively dividing spermatogonial cells [26]. Quantification of BrdU-labeled testicular cells was done according to the procedure described by Terry et al. [27]. An aliquot of ethanol-fixed testicular cells (1–2 million) was washed, pepsinized, acid denatured, and incubated with 200 µl of a monoclonal antibody against BrdU at 1:7.5 dilution for 1 h in the dark. The cells were washed and incubated with 200 µl of FITC-conjugated goat anti-mouse IgG at 1:10 dilution for 30 min in the dark, and cells were counterstained with PI. Control cells were processed similarly without the primary antibody incubation step. The intensity of green fluorescence of FITC- and red fluorescence of PI-stained cells was measured at 530 and 620 nm, respectively, on a Coulter flow cytometer.
Quantification of c-kit-Positive Spermatogonia by Immunocytometry
We used the modified procedure of Schonfeldt et al. [28] to quantify the c-kit-positive spermatogonia by immunocytometry. Freshly prepared testicular cells were fixed in 1% buffered paraformaldehyde (pH 7.4) at a concentration of 1 x 106/ml for 30 min at 4°C. An aliquot of 1 x 106 cells was washed twice with 3 ml of staining buffer (PBS containing 1% BSA), and the cells were resuspended in 100 µl of staining buffer containing 1 µg of anti-mouse c-kit monoclonal antibody and incubated at RT for 1 h. The cells were washed with 3 ml of staining buffer and again incubated with 100 µl of staining buffer containing fluorosceinated goat anti-mouse IgG (1:50 dilution) in the dark for 30 min at RT. The cells were again washed with 3 ml of staining buffer before counterstaining with 1 ml of DNase-free PI staining solution containing 40 µg/ml RNase and 25 µg PI. The cells were analyzed on a Coulter flow cytometer using the same filters and excitation as described above for BrdU-labeled cells. Control cells were processed in the same manner excluding the c-kit antibody.
Elongated Spermatid Counts
After decapsulation, testicular parenchyma was homogenized using a Tissue Tearor homogenizer at maximum speed (30 000 rpm) for 1 min in 2 ml of normal saline containing 0.05% Triton X-100 and 3.8 mM sodium azide [29]. Homogenization-resistant elongated spermatids (steps 14–16) were counted using a hemocytometer. The numbers were expressed as per testis.
PI Binding to Sperm DNA Following DTT Treatment
The potential influence of nuclear protein transitions on DNA compaction during spermatogenesis can be measured by examining PI binding to sperm rendered accessible to the chromophore. The procedure followed for obtaining sperm nuclear decondensation by means of exposure to varying concentrations of the reducing agent DTT was similar to that described by Aravindan et al. [30]. After incubation with DTT, the samples were washed, and the sperm pellet was resuspended in 0.5 ml of PI (PI staining solution; 25 µg PI/ml, 40 µg RNase/ml, and 0.3% Nonidet P-40). The addition of RNase in the solution assures that only native or single-stranded DNA is analyzed. The PI-stained samples were analyzed using a Coulter flow cytometer. The PI fluorescence signals were collected at 620 nm. Before the samples were analyzed on the flow cytometer, a drop of sperm suspension was taken on the slide for fluorescence microscopic visualization of sperm heads.
Sperm Chromatin Structure Assay (SCSA)
The SCSA is another sensitive measure of denaturability of sperm DNA in situ by flow cytometry. The AO staining procedure used was similar to that described by Evenson and Jost [31]. About 0.2 x 106 ethanol-fixed cauda sperm were washed twice in TNE buffer (0.01 M Tris, 0.15 M NaCl, and 1 mM EDTA, pH 7.4), and the pellet was resuspended in 200 µl TNE and treated with 0.4 ml of acid/Triton X-100 solution (0.15 M NaCl, 0.1% Triton X-100, and 0.08 N HCl in double-distilled water). After 30 sec, 1.2 ml of AO staining solution (0.15 M NaCl, 0.1 M citric acid, 0.2 M Na2PO4, pH 6, and 6 µg/ml AO) was added, and the stained samples were analyzed using a Coulter flow cytometer equipped with a 15-mW argon-ion laser. The AO-stained samples were excited at 488 nm; the green fluorescence was collected at 530 nm and that of red at 620 nm. SCSA quantitates the shift from double-stranded to single-stranded DNA following acid denaturation. The extent of denaturation is quantitatively denoted by the term alpha t (
t), a value that can range from 0 to 1. The t value is a ratio of red fluorescence to total (green and red) fluorescence, and it was calculated as follows: mean channel of red fluorescence/mean channel of red fluorescence + mean channel of green fluorescence. A higher shift signifies greater DNA denaturability and reduced/loss of fertility [31].
Data Analysis
The statistical significance was assessed by one-way ANOVA and Mann-Whitney t-test. Level of significance was set at 5%.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At 3 mo, the age at which all current studies were done, the FORKO males exhibited about a 50% decline in testis weight, which was significantly low compared to that of wild-type males (Fig. 1). Similarly, around 30% reduction in the epididymis weight was observed in FORKO males compared to wild type. The seminal vesicles were also smaller in FORKO males, exhibiting a 23% reduction (Fig. 1). We surmise that this difference in seminal vesicle weight may be even greater because the values for +/+ or +/- males may be underestimated due to loss of some of the viscous secretions during the dissection; the loss was minimal in FORKO mice. However, there was no difference in the testes, epididymal, and seminal vesicle weight of heterozygous and wild-type males. There was no change in body weight between the genotypes (Fig. 1). The reduction in serum testosterone levels (Table 1) in the FORKO males probably accounts for the lowering of the weight of sex accessories.
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Morphological Analysis of Testis and Epididymis
Histological examination of testis from FORKO males revealed that all seminiferous tubules were normal in terms of histological features and distribution of stages of the cycle. The cellular integrity of the seminiferous epithelium also appeared to be normal (Fig. 2, A–D). No disruption or alteration of any given step of spermatogenesis was noted in the testis of FORKO males. However, it was evident that the seminiferous tubular diameter was smaller in FORKO males. The interstitial space appeared to be increased in FORKO males. To verify this observation a quantitative analysis was conducted. Two stages of spermatogenesis (i.e., stage VII and XI) were selected for comparison. The tubular diameter of the cross sections of seminiferous tubules of FORKO males was significantly reduced compared to that for the same stages of the cycle of wild-type mice (Table 2). The luminal diameter was also significantly smaller in mutant males compared to wild-type mice (Table 2). Measurement of the area (relative volume) of the seminiferous epithelium revealed a significant decrease only at stage VII, not at stage XI, in FORKO males (Table 2). Similar analysis was conducted on the epididymides. Histologically, the epididymis of the FORKO males had normal appearance, but not all the lumina were filled with spermatozoa. Some tubules of the caput epididymidis were devoid of sperm (Fig. 2, E and F). It was also noted that the tubular diameter of cross sections of the epididymis of FORKO males was smaller than that of wild-type males (Fig. 2 and Table 2). Quantitative analysis conducted on the caput epididymidis confirmed this observation (Table 2).
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Testicular Cell Composition as Determined by DNA Flow Cytometry
As the classical histological examination of testicular sections did not reveal major changes in cellular associations between wild-type and FORKO males [24], we resorted to a more rapid and sensitive analysis of testicular germ cells by quantitative DNA flow cytometry. Figure 3 shows the frequency distribution histograms of PI-stained testicular cells as determined by DNA flow cytometry. Based on the DNA content, five quantifiable populations were discernible: elongated spermatids (HC; H, hypostainability of elongated spermatids due to condensation of nuclear DNA during spermiogenesis); round spermatids (1C); spermatogonia and testicular somatic cells (2C); spermatogonial cells synthesizing DNA (S-phase); and primary spermatocytes and G2 spermatogonia (4C). It may be noted that the percentage of testicular somatic cells (Sertoli, Leydig, and peritubular myoid cells) is less than 3% of total testicular cells [32, 33] and falls within the 2C population.
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Analysis of testicular cells revealed that FORKO males at 3 mo of age exhibited a 28% increase in 2C cells compared to the wild type, and this difference was significant (Table 3). Interestingly, the percentage of HC cells was decreased by 25% in FORKO males compared to wild type (Table 3). However, no change in the composition of testicular cells was evident in the heterozygous male compared to wild type (Table 3).
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Analysis of Proliferating Spermatogonia by BrdU Incorporation
To assess the percentage of actively dividing spermatogonial cells, we quantified BrdU-incorporated cells as a measure of DNA synthesis in vivo. The percentage of S-phase cells as determined by univariate analysis did not show any change between the different genotypes studied. The reason may be that the percentage of S-phase cells obtained by univariate analysis of DNA histograms of testicular cells may be subject to possible errors due to the presence of debris as well as the cell aggregates contributing to the background in this region [34, 35]. Therefore, the percentage of S-phase cells was determined by bivariate analysis (Fig. 4). Using this method, the distribution of BrdU-labeled cells has been predicted to be 50% of preleptotene spermatocytes, 25% of B spermatogonia, and 12.5% of intermediate spermatogonia, with different types of A spermatogonia accounting for 12.5% in the population [36]. The percentage of BrdU-positive cells in FORKO males determined by pulse labeling for 1 h was no different from that for wild-type or heterozygous males (Table 4).
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Analysis of c-kit-Positive Cells
The c-kit ligand, also called steel or stem cell factor, is a Sertoli cell product [37] that stimulates DNA synthesis in the A spermatogonial cells in the mouse [38]. The survival and/or proliferation of type A1 to A4 spermatogonia depends on the interaction of c-kit ligand supplied by the Sertoli cell and the receptor located on germ cells [39]. To examine whether or not the observed elevation in the percentage of 2C cells in FORKO males was attributable to increase in the c-kit-positive spermatogonia, in the present study we used an anti-mouse c-kit monoclonal antibody as marker to quantify c-kit-positive spermatogonia by immunocytometry (Fig. 5). Interestingly, FORKO males exhibited a significant (105%, P < 0.03) increase in c-kit-positive 2C cells over the wild-type males. Surprisingly, heterozygous males also showed an increase in c-kit-positive cells compared to wild type; however, this difference was not statistically significant.
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Elongated Spermatid Count
The numbers of homogenization-resistant spermatids are presented in Figure 6. The absolute number of homogenization-resistant spermatids per testis was significantly (P < 0.0001) lower in FORKO males than in wild-type males.
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Nuclear Condensation During Spermiogenesis in Mutants
The differences observed in the fluorochrome-binding pattern of haploid cells (round and elongated spermatids) have been attributed to changes known to occur in nuclear transition proteins during spermiogenesis [40]. Such changes, referred to as condensation, lead to a decreased binding of the fluorochrome by elongated spermatids despite their having the same DNA content as round spermatids [41]. Therefore, this analysis becomes a sensitive indicator of nuclear changes induced in sperm. The nuclear condensation pattern during spermiogenesis was determined by the PI stainability of elongated spermatids. The differences in the PI stainability of elongated spermatids in the three genotypes are shown in Figure 7. The mean channels of fluorescence intensity of PI-bound elongated spermatids from wild-type, heterozygous, and FORKO males are presented in Table 5. The elongated spermatids of FORKO males exhibited a 23% increase, which may be considered quite dramatic, as any increase signifies less DNA condensation. These cells from heterozygous mice also showed a 6% increase in PI bindability over those from wild-type males (Table 5). This small increase was not only consistent but also statistically significant, emphasizing that even a subtle perturbation in FSH receptor signaling, as in the heterozygous mice, could have an effect on the quality of spermiogenesis.
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Microscopic Examination of the Cauda Epididymal Sperm
The cauda epididymal sperm from FORKO males, in addition to showing decreased motility [24], exhibited morphological abnormalities like retention of the cytoplasmic droplet (Fig. 8). More than 80% of the sperm showed the cytoplasmic droplet in the case of the FORKO males. While these were also present in sperm of heterozygous mice, they were absent in the wild-type animals. The retention of the cytoplasmic droplet by ejaculated spermatozoa has been correlated with reduced fertility in animals [42].
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DTT-Induced Sperm Nuclear Decondensation In Vitro
Sperm released from the testis are rich in arginine and cysteine residues [43] containing free sulphydryl groups that are oxidized during epididymal transit to form inter-intraprotein-linked disulfide bridges [44] leading to further compaction of nuclear DNA. Nuclear compaction is essential for production of viable spermatozoa. Evenson et al. [45] have demonstrated using flow cytometry that sperm from the mouse and rat cauda region of the epididymis bind less fluorochrome than those from the caput region. Thus, a measurement of this parameter in the presence of compounds like reducing agents provides an index of chromatin status. Exposure of sperm collected from the cauda region of all the three genotypes to different concentrations of the reductant DTT prior to PI staining led to a dose-dependent increase in the fluorescence intensity (Fig. 9). Microscopic examination of the PI-bound sperm as represented in Figure 10 revealed that sperm of FORKO males exhibited larger head size than sperm of wild-type or heterozygous males. Although we did not measure the magnitude of the increase in size of sperm heads, a great majority in FORKO males showed this property. In flow cytometry, the PI-binding pattern of sperm from FORKO males was significantly higher at all concentrations of the reducing agent DTT than that for wild-type males. Similarly, the sperm from heterozygous males showed a significant increase in PI stainability after treatment with 2.5 and 10 mM DTT (Table 6). Although the sperm from FORKO males showed a tendency toward a higher PI-binding pattern in the absence of DTT compared to wild type, it was not statistically significant (Table 6). This indicates inadequate compaction of the chromatin that may have contributed to the increased head size of sperm in FORKO males (Fig. 10).
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Sperm Chromatin Structure Assay
The dye AO intercalates into double-stranded DNA, producing a green fluorescence signal, while its binding to single-stranded DNA or RNA causes a red fluorescence. The SCSA provides an assessment of the degree of DNA denaturation that the sperm nucleus undergoes upon exposure to stringent acid treatment. The extent of denaturation can be determined by the metachromatic shift from green to red fluorescence of the AO-stained sperm. Figure 11 shows a representative dot plot of AO-bound sperm from the cauda epididymal region of wild-type, heterozygous, and FORKO males. Each dot represents the intensity of green and red fluorescence exhibited by an individual spermatozoon. The sample debris seen at the lower left of each box was eliminated from analysis. The SCSA is so accurate that minute changes can be detected in assessing the quality of sperm following exposure of animals to physical or chemical treatments or genetic mutation. The t values signifying the ratio of red fluorescence to total fluorescence exhibited by sperm from FORKO males were significantly increased compared to those for sperm from wild-type males. There was no difference in the t values of the sperm from heterozygous males and wild-type males (Fig. 11).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The aim of the current study was to understand the reasons for reduced fertility observed in FORKO male mice. The dramatic decrease in testicular weight of FORKO males (54%) compared to wild-type mice is a primary indication that FSH receptor signaling is required for maintenance of normal testicular size. A similar decrease in testicular weight has also been observed in FSHß-deficient mice despite their showing normal circulating testosterone values [18]. Restoration of normal testicular size has also been demonstrated through genetic rescuing of the FSHß-deficient mice with appropriate expression of human FSHß gene [46].
In other studies, concomitant administration of recombinant human FSH to adult rats [47] and monkeys [10] attenuated the GnRH antagonist-induced testicular weight loss. Similar beneficial effects of FSH treatment upon testicular volume in normal monkeys [9] and fertile men [48] also constitute direct evidence for maintaining size. Since Jégou et al. [49] have demonstrated that seminiferous tubular fluid secretion is under the control of FSH, we believe that the dramatic decrease in the seminiferous tubular diameter observed in FORKO males [24] may be due to a combination of low tubular fluid secretion by Sertoli cells as well as reduction in absolute numbers of germ cells.
The normal weight and secretory activity of the seminal vesicle and other accessory sex organs are dependent on the circulatory levels of androgen [50]. The decrease in the levels of testosterone in FORKO males compared to wild type was shown earlier [24] and confirmed in the present study (Table 1). The reduction in the sizes of seminal vesicle and epididymis in FORKO males may be due to the decreased levels of testosterone. The decrease in epididymis and seminal vesicle weight in FORKO males is an effect different from that in the FSHß-deficient mice [18], in which no change in these organs was observed. This latter finding may be attributable to the fact that levels of testosterone in FSHß-deficient males were unaltered [18].
This difference between the two mutant models may have several important functional implications. First, unknown factor(s) may have compensated to maintain some degree of Sertoli cell function by interacting with the FSH receptor system that apparently remains fully intact in the FSHß knockout animals [18]. Secondly, it is unlikely that intercellular communication was perturbed in FSHß knockout males because testosterone was unaffected. Thirdly, a normal androgen level could allow progression of a sufficient number of sperm into their mature state, contributing to full fertility. Induction of these changes in FORKO mice indicates that the effects of receptor signaling disruption are more severe, compromising the normal Sertoli-Leydig cell intercellular communication. Earlier studies in adult mice clearly showed that the intratesticular injection of FSH significantly increased testosterone levels [51]. In immature hypophysectomized rats, FSH induced Leydig cell proliferation and maturation [52]. Furthermore, a stimulatory effect of FSH on basal and LH-induced testosterone secretions by rat Leydig cells in vitro has also been demonstrated [53]. However, these investigations predate the availability of recombinant FSH. Detailed studies are currently under way in our laboratory to clarify the molecular mechanisms underlying these interactions in the mutants.
As no gross changes were perceptible upon cursory histological examination of testicular sections of FORKO males compared to wild type at 3 mo of age [24], despite the large constriction of the tubule including its walls, we performed morphometric analysis. The decrease in the tubular and luminal diameter, as well as area of the seminiferous epithelium, in both stage VII and XI in FORKO males indicates a quantitative reduction in the absolute number of germ cells undergoing spermatogenesis. Thus, in the absence of FSH receptor signaling, cellular components required for association of germ cells may be perturbed. Because of the reduced sperm production in mutant males, the lumen of the caput epididymidis showed few or no sperm (Fig. 2F). With the object of uncovering subtle changes that may have gone unnoticed in FORKO males, we have quantified the testicular germ cells by a sensitive DNA flow cytometric technique. In the present study, the decrease in testicular weight (Fig. 1) and the absolute number of elongated spermatids (Fig. 6) in FORKO males suggests that a quantitatively lower number of spermatogonia are entering the process of spermatogenesis. The lack of change in the percentage of BrdU-positive cells of FORKO males indicates that the kinetics of spermatogenesis is unaffected by the lack of FSH signaling. The lower number of germ cells indicates that the 28% augmentation in the cells of the 2C compartment of FORKO males may be due to the relative increase in testicular somatic cells. It is interesting that a similar pattern of change in the 2C cells was observed in adult male rats [15] and monkeys [54] that had been passively immunized against FSH to disrupt testicular function. Likewise, active immunization of bonnet monkeys with oFSH, as well as recombinant FSH receptor protein producing infertility, also results in a significant increase in the percentage of 2C population cells [20, 21].
It has been shown that the c-kit receptor is localized on type A spermatogonia as well as on the Leydig cells [55]. The c-kit ligand secreted by the nursing Sertoli cells of the testis [37] stimulates DNA synthesis in type A spermatogonia [38]. Studies demonstrating that the survival and/or proliferation of type A1 to A4 spermatogonia depends on effective interaction of c-kit ligand and receptor [39], as well as the strong evidence for FSH regulation of stem cell factor [38], indicate how any perturbation might dislocate the spermatogenic process. Even though we did not quantify the level of c-kit ligand in our previous study, we did observe a perceptible reduction in the gene expression [24]. Simultaneously there was also an increase c-kit gene expression. The enhancement in the percentage of c-kit-positive cells compared to a 28% increase in the overall 2C cells of FORKO males in the current work indicates that the observed increase in the population of cells in the 2C compartment (determined by PI staining) may be due to increase in Leydig cells as well as in type A spermatogonia that are dividing as actively as in the wild type (determined by BrdU labeling). We can therefore speculate that the increase in the percentage of c-kit-positive cells and decrease in the testosterone values of FORKO males may be due to the reduced levels of stem cell factor available for intercellular communication in the absence of FSH receptor signaling. However, we cannot at this point exclude alterations in other factors that may also affect communication.
In the normal testis, although cohorts of spermatogonial stem cells enter spermatogenesis at any given time, not all spermatogonia or their subsequent cell types survive and complete the terminal differentiation process [56], a process that is likely to determine the final outcome. Thus, it has been demonstrated that selective deprivation of endogenous FSH achieved by treating adult male rats with specific FSH antiserum enhances the number of apoptotic cells in the testis, particularly the premeiotic germ cells [17]. The reduced PI stainability of 2C peak in FORKO males (Fig. 7), as well as the significant reduction in mean channel number of 2C peak (Table 5), suggests that the germ cells in the 2C compartment may also be undergoing increased apoptosis. Previous studies using similar techniques have shown that decrease in the mean channel number of 2C peak of mice treated with a toxicant such as methoxyacetic acid [25], as well as of monkeys treated with the synthetic steroid norethisterone [57], is due to the loss of DNA from the apoptotic cells.
The significant reduction in the percentage of the HC population and absolute number of homogenization-resistant elongated spermatids of FORKO males is evidence that lack of FSH receptor signaling quantitatively reduces the production of elongated spermatids. This observation is also consistent with results obtained in FSH-deprived rats [15] and monkeys using bioneutralization techniques [21]. As opposed to the reduction of elongated spermatids in FORKO males, a balanced, active, and continuous proliferation of spermatogonia in wild-type animals would ensure overall adequate turnover of germ cells resulting in the production of abundant sperm number.
During spermiogenesis, the round spermatid nuclei progressively condense, resulting in the production of sperm having a highly compacted nucleus [58]. Spermatids in the early steps of differentiation contain basic proteins like those present in the somatic cell nucleus [59]. However, during mid-spermiogenesis, synthesis of different types of proteins occurs, resulting in the eventual replacement of histones by protamines that are responsible for chromatin condensation and compaction of DNA [40]. These essential molecular changes that occur during normal spermiogenesis are reflected by the decreased accessibility of DNA to intercalating dyes [41]. The increase in the PI stainability of elongated spermatids in the testis of FORKO males (Fig. 7) may be due to the disturbance in the normal replacement of histones by protamines during spermiogenesis leading to poor condensation of spermatid nuclei. Elongated spermatid nuclei of monkeys immunized with oFSH are susceptible to DTT-induced decondensation [60]. Such inadequately processed elongated spermatids may differentiate into sperm lacking fertilizing ability. The increased PI stainability of cauda sperm of FORKO males, indicating either the lack of or inappropriate sperm maturation, is an interesting feature that parallels several observations in humans. Engh et al. [61] have demonstrated that sperm from patients attending infertility clinics exhibit high DNA stainability. In addition, various other reports have shown that the sperm voided by infertile men exhibit abnormal protein complements with an increase in the histone level and/or an altered protamine P1/P2 ratio [62–65]. The FSH-immunized monkeys also show defective sperm in their ejaculate [29]. Studies in vitro on human spermatogenesis reveal that FSH, but not testosterone, could induce rapid morphological changes like spermatid nuclear condensation, elongation, and flagellar growth, indicating a specific role for FSH in spermiogenesis [66].
In the FORKO mouse sperm, defects are not restricted to the nucleus or size of the head (Fig. 10). Aberrations are also observed in the tail portion. Most importantly, in addition to the bent tail, which impedes mobility [24], a great majority of the sperm retain their cytoplasmic droplets. This structure is a small mass of cytoplasm that is present in spermatozoa as they are released into the lumen from anchorage in the seminiferous epithelium. Because it is a transient structure, it is gradually displaced from its original position on the flagellum to be eliminated completely by the time the spermatozoa complete their transit to the cauda epididymidis. This shedding process, which is also an integral part of sperm maturation, is required for fertility [42]. Thus, the lack of FSH receptor signaling in FORKO mice results in release of immature and infertile sperm into the ejaculate.
Abnormal sperm chromatin structure can be revealed by studying susceptibility of DNA to in situ denaturation by SCSA [31]. Thus, the increase in the t values of sperm from FORKO males, measured by a metachromatic shift in the AO-bound sperm from green fluorescence (native DNA) to red (denatured single-stranded DNA), indicates the susceptibility of the sperm nuclear DNA to acid-induced denaturation (Fig. 11). Although several reports show the usefulness of SCSA in evaluating fertility potential as well as in reproductive toxicology studies, this is the first time in rodents we have experimentally demonstrated that abolishing FSH receptor function affects sperm chromatin status by SCSA. Apparently, sperm cells recognized by the DNA denaturation assay are entities that are irreparable and consequently considered reproductively dead [67]. These conclusions are consistent with reports that have shown aberrant sperm chromatin packaging in FSH-deprived adult male monkeys [60, 68]. The t values are very precise, as this ratio, calculated from the SCSA, appears to show minimal variation within a single group over a period of time. Thus, the coefficient of variation is 10% for a group of 45 volunteer men as determined from analysis of eight monthly semen samples [69]. Because the t values have been shown to be useful in assessing the fertility rating of bulls [70, 71], we can justifiably argue that improperly processed sperm are functionally ineffective. Furthermore, Evenson et al. [72] have shown that even though fertilization could occur with sperm having high t value, postfertilization development is terminated prematurely, resulting in early embryonic death. Indeed, this may be the reason we see a reduction in live pups sired by FORKO males, and more detailed studies are under way to investigate this phenomenon. Several studies have also demonstrated that although sperm may appear morphologically normal, hidden defects in chromatin packaging could indeed be present. These can be revealed only [73–77] by more discriminating procedures such as we have applied in the study of FORKO males.
In conclusion, we have shown that genetic disruption of the FSH receptor in the mouse affects critical phases of spermatogenesis in the male. The combined effect of these qualitative and quantitative perturbations produces dysfunctional sperm contributing to reduced fertility. As these parameters are even more dramatically altered in male primates with FSH receptor blockade [60], species differences in testicular susceptibility become readily apparent. This model should be useful for understanding intercellular communication in the testis and for identifying mechanisms of sperm development and maturation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant from Medical Research Council of Canada. 
2 Correspondence: M. Ram Sairam, Clinical Research Institute of Montreal, 110 Pine Avenue West, Montreal, PQ, Canada H2W 1R7. FAX: 514 987 5585; sairamm{at}ircm.qc.ca
Accepted: November 29, 1999.
Received: September 16, 1999.
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