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Department of Andrology (U.S., K.M., D.J.H.), ANZAC Research Institute, University of Sydney, Sydney, New South Wales 2139, Australia; and Department of Medicine (R.A.D., J.D.Z.), Austin Health, University of Melbourne, Melbourne, Victoria 3084, Australia
Address all correspondence and requests for reprints to: Professor D. J., Handelsman, ANZAC Research Institute, Sydney, New South Wales 2139, Australis. E-mail: djh{at}anzac.edu.au.
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Introduction |
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Prostate and seminal vesicles secrete fluids that form the seminal plasma. Seminal plasma serves as a transport vehicle and transient nutritional medium for spermatozoa and, in rodents, formation of a copulatory plug. The development and secretory function of prostate and seminal vesicles are highly androgen dependent (5) with castration producing involution of both organs (6). In rodents, reduced prostate and seminal vesicle secretions and changes in their volume and composition lead to reduced copulatory plug formation after mating (7, 8).
During spermatogenesis, spermatozoa acquire the structural basis for movement and fertilization of oocytes, whereas these functions are acquired during transit through the epididymis (9, 10). The epididymis secretes into its luminal environment specific proteins/glycoproteins that modify spermatozoal surface during their epididymal transit (9). Androgen action is essential for not only spermatogenesis but also the maturation of spermatozoal functions. Androgen receptor (AR) protein is highly expressed in tubular epithelial cells and interstitial stromal cells in the epididymis (11, 12, 13), and androgens regulate epididymal epithelial secretion (14, 15). However, the specific mechanism of epididymal secretory proteins on sperm maturation and its regulation by androgens are not well understood (16, 17, 18, 19).
This study examines the effect of selective abrogation of androgen action in sex accessory organs on male fertility in mice. Previously it has been difficult to distinguish the effects of androgens on the testis from those on sex accessory glands. We recently generated a mouse model with prostate epithelial androgen receptor deficiency (PEARKO) using the Cre/LoxP system involving a prostate epithelium-directed Probasin promoter (20). In these mice, unexpected Cre expression was also detected in seminal vesicles and epididymis leading to reduced AR activity and weight of these organs, although testis development and serum testosterone levels were unaffected (20). The dichotomy of reduced androgen action in prostate, seminal vesicles, and epididymis with preserved androgen action in the testis provides an unique opportunity to analyze the role of androgen action in sex accessory organ function and sperm maturation in male fertility.
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Materials and Methods |
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Tissue collection
At 8 wk of age, male mice were killed by cardiac exsanguination under ketamine/xylazine anesthesia. Serum was stored frozen at –20 C. Male reproductive organs (separate prostate lobes, seminal vesicle, epididymis, and testis) were dissected free of fat and connective tissue and weighed separately. Seminal vesicles were also weighed after extruding secretions by manual pressure with the weight of seminal vesicle secretion defined as the difference between intact and empty seminal vesicle weights. Tissues were either snap frozen with liquid nitrogen and stored in –80 C or fixed in Bouins solution for 4 h at room temperature.
X-gal staining and histology
Functional recombinase activity of transgenic Cre in male reproductive organs was analyzed using ARR2PBi-Cre mice crossed with R26R (ROSA) reporter mice (23). Expression of functional Cre recombinase by lacZ activity in tissues was detected by β-galactosidase staining of dissected male reproductive organs as previously described (20). Whole-mount staining for reproductive organs was performed at 1 wk of age. Additionally, seminal vesicles were stained as whole mount at 5 wk of age, embedded in paraffin, and sectioned (5 µm) for analysis of cell-specific expression. Cell-specific Cre expression in epididymis was analyzed at 8 wk of age by lacZ staining of frozen sections. For histological analysis, sections (5 µm) were cut from fixed, paraffin-embedded tissues and stained with hematoxylin and eosin.
Immunohistochemistry for smooth muscle 
-actin (SMActin) was performed on 5-µm-thick dewaxed paraffin sections (clone 1A 4; Sigma, Sydney, Australia). Signal was visualized with EnVision rabbit/mouse (permanent red) kit (Dako, Carpentaria, CA). Sections were counterstained with Harris hematoxylin.
RNA extraction and RT-PCR
For RT-PCR and real-time RT-PCR, total RNA was extracted using RNeasy Plus mini kit (QIAGEN, Doncaster, Australia) and cDNA synthesized with Omniscript reverse transcriptase (QIAGEN) from 250 ng of total RNA, respectively, using oligo-deoxythymidine (Invitrogen Australia Pty., Mount Waverley, Australia). Final reverse transcription reactions were diluted 1:5 for storage at –20 C. Expression of normal and exon 3 excised AR was performed as previously described (20, 24). Global AR knockout (ARKO) testis was used as a control for expression of exon 3-deleted AR. ARKO mice were generated as previously described (22).
Real-time RT-PCR
Quantitative real-time RT-PCR analyses were performed on cDNA using QuantiTect SYBR Green PCR kit (QIAGEN) and Rotor-Gene 2000 system (Corbett Research, Mortlake, Australia) as previously described (20). Primer sequences, product size, and annealing temperatures for cyclophilin, seminal vesicle secreted 2 (SVS2), Adam7, Lipocalin (Lcn)-5 and Lcn8, and glutathione peroxidase type (Gpx)-5 are previously described (14, 25, 26). Primer sequences for seminal vesicle secretory protein 99 (SVP99) were forward, 5'-GGA AGA CAG CAA GAG AGC AT-'3 and reverse, 5'-GAC ACT GTG TGA CTC CAT CA-'3 (60 C, 147 bp). SVS2 and SVP99 as well as Adam7, Lcn5 and Gpx5 were chosen as androgen-responsive markers expressed in seminal vesicles and epididymis, respectively (14, 27, 28, 29). Lcn8 was analyzed as a control for androgen-independent, testicular factor-dependent gene expression in the epididymis (30). Epididymal gene expression was analyzed in the caput-corpus region of epididymis because selected genes have no or very low expression in the caudal region of the epididymis (14, 31).
Natural matings
Littermate control and PEARKO males were housed singly at 7–8 wk of age with a fertile female (>11 wk of age) for a period of 3 months. The timing of litters and number of offspring born were recorded.
Mating behavior and plug formation
To monitor mating behavior and plug formation, two fertile females were induced to superovulate with 10 IU of pregnant mare’s serum gonadotropin (Folligon; Intervet, Bendigo, Australia) followed 48 h later by 10 IU of human chorionic gonadotropin (Sigma). They were then mated with a single littermate control or PEARKO male (7–8 wk of age) either overnight with the presence of plugs (or any signs of plug such as threads around the vagina) checked in the morning or between 0600 and 0800 h and plug formation recorded 4 h after start of mating trial. Females were killed, pubic bones cut, and the vaginal plugs carefully removed. Plugs were photographed, fixed in 70% ethanol, dried at room temperature, and weighed.
Sperm assessment
Homogenization-resistant sperm head count
Excised testes were stored frozen at –80 C. Thawed testis and cauda, caput, and corpus epididymis were homogenized in 1 ml of PBS without Triton X-100 (32) and diluted as needed to count homogenization-resistant sperm head (stage 14–16 spermatids) in a Neubauer hemocytometer.
Collection of fresh epididymal sperm
Cauda and caput epididymides were quickly excised from mice killed under anesthesia placed in separate 1 ml of prewarmed human tubal fluid medium (hTFM) (33) containing 3 mg/ml of BSA. Tissue was teased and gently cut using a 27-gauge (insulin) needle, and sperm were gently squeezed out. Sperm was further incubated at 37 C for 10 min to allow motile sperm to swim out. Sperm number was analyzed by hemocytometer.
In vitro fertilization assay
Control colony females were superovulated and killed under anesthesia by cervical dislocation 14–16 h after human chorionic gonadotropin injection. Cumulus oocyte complexes (COCs) were collected gently from oviducts into HEPES-buffered hTFM, washed, and divided into 5-ml fertilization tubes containing 1 ml of hTFM containing 3 mg/ml BSA (
20–30 oocytes/tube). Fresh epididymal sperm collected from 8- to 10-wk-old males were allowed to capacitate for 30 min at 37 C, and 3,000–300,000 sperm were added to tubes containing COCs and further incubated at 37 C for 5 h, and fertilization assessed by the number of oocytes containing two pronuclei visualized by Hoechst 33342 (Sigma) staining.
In vivo fertilization assay
Fertile females were superovulated and mated with control or PEARKO males between 0600 and 0800 h. Females that mated were killed in the afternoon and COCs collected and then incubated with hyaluronidase (final 0.3 mg/ml) to remove granulosa cells before analyzing fertilization using Hoechst 33342 staining.
Acrosome reaction
Cauda epididymal sperm acrosomes were stained as described (34). Briefly, sperm were spun down (3500 rpm, 5 min, Eppendorf 5415D), fixed in 4% paraformaldehyde for 10 min, washed with 9.0 M ammonium buffer twice, smeared onto the glass slides, and left to air dry. One million sperm were added into 0.5 ml hTFM containing 3 mg/ml BSA in 5-ml fertilization tubes and incubated at 37 C and sample for acrosome evaluation obtained after 0, 10, 20, 30, or 40 min of incubation. Dry slides were stained with 0.2% Coomassie blue solution [methanol-water- glacial acetic acid 50:10:40 (vol/vol)] for 10 min and washed twice with distilled water. At least 100 acrosome intact or reacted sperm were counted using CASTGRID (version 1.10; Olympus Corp. Albertslund, Denmark) software to generate counting frames.
Sperm morphology
Coomassie blue-stained slides were analyzed using CASTGRID version 1.10 software to generate counting frames. At least 200 sperm were categorized as normal, flagellar bent, or flagellar spiral/hairpin.
Statistics
Statistical analysis was performed using one-way ANOVA or covariance with the least significant difference method as a post hoc test or Fisher’s test as appropriate using SPSS (Chicago, IL), NCSS (Kaysville, UT), or StatXact software (Cytel Statistics, Cambridge, MA). Fractional (percentage) data were subject to an arcsin transformation before statistical analysis. In case of nonhomogenous variances (Levene’s test, P < 0.01), the nonparametric Kruskal-Wallis ANOVA was used followed by the Mann-Whitney U test. Reproductive performance was characterized by Kaplan-Meier survival analysis to provide median time to first and second litters. P < 0.05 were considered statistically significant. Data are expressed as mean and SEM or mean and 95% confidence interval (for fractional data) unless otherwise specified.
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Results |
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). Epididymal Cre expression was localized in the epithelial cells of the epididymis (Fig. 1B). Strong Cre expression in seminal vesicles was detected at 5 wk of age, and the Cre expression was localized on the stromal smooth muscle (Fig. 1, C and D).
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). Both wt and exon 3-deleted AR were detected in each part of PEARKO epididymis (caput, corpus, and cauda), whereas only wt AR was detected in control epididymis (Fig. 1E). Similarly to the epididymis, seminal vesicle, coagulating gland, and vas deferens of PEARKO also expressed both wt and exon 3-deleted AR (Fig. 1E). The presence of exon 3 deleted AR in all prostate lobes, seminal vesicles, and epididymis remained strong until 1 yr of age (data not shown).
Sex accessory organ phenotype
PEARKO seminal vesicles were significantly smaller (55% of littermate control at 8 wk of age; P < 0.001) due to significantly (P < 0.01) reduced weight of secretion but also due to reduced weight of emptied gland (Fig. 2, A and B) (P < 0.01). The epithelial cells of PEARKO seminal vesicles, most frequently in proximal region, appeared to be very low, cuboidal, with very little cytoplasm, and the epithelial layer was less folded when compared with control (Fig. 3, A and B). However, the lumen was filled with a dense homogenous acidophilic mass, similar to that in the normal vesicle (Fig 3). The distal epithelium appeared to have a normal, tall secretory morphology, with occasional small foci of hyperplastic epithelial cells and a decrease in the size of the acini (Fig. 3, C and D). Immunohistochemical detection of SM-Actin demonstrated that the stromal smooth muscle layer in PEARKO seminal vesicles was thinner and disorganized when compared with control (Fig. 3, E and F).
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). However, stromal thickening was detected in the corpus and caudal region of PEARKO epididymis (Fig. 4). Morphology of testis was normal in PEARKO males (data not shown).
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) of PEARKO mice. In addition, expression of Lcn5 and Adam7, androgen-dependent markers of epididymal function, was significantly (P < 0.05) reduced in combined caput and corpus epididymis of PEARKO, whereas no significant difference was detected in expression of Gpx5 or Lcn8 between control and PEARKO caput-corpus region of epididymis (Fig. 5B).
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). Whereas control males had an average of 3.7 ± 0.4 litters over the 90-d breeding trial, however, only five of 15 tested PEARKO males (33%) had a single litter, and only one of 15 had two litters within the 90-d mating period (Fig. 6A). Median time to the first litter was 21 d (20–22; 95% confidence interval) and 42 (39–45) d for second litter for control males, compared with more than 90 d for PEARKO males for both first and second litters. The mean number of pups per litter for control males was 9.6 ± 2.7 (mean ± SD, n = 26 litters), whereas for the minority of PEARKO males that sired a litter, the mean number of pups per litter was 6.2 ± 1.8 (n = 6 litters), significantly (P < 0.01) different from controls.
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In vitro fertilization capacity of PEARKO sperm was significantly reduced at lower sperm concentrations (<50,000 sperm/ml, P < 0.002) but not at higher sperm concentrations (>50,000 sperm/ml, P = 0.59), demonstrating a mild defect in sperm binding to the zona pellucida, which can be overcome by higher sperm numbers (Fig. 6C).
Plug formation
Analysis of the copulatory plugs formed by control and PEARKO males 4 h after coitus or after overnight mating with hormone stimulated females were discrepant. At 4 h after coitus, plugs were detected in equivalent proportions (P = 0.32) of females mating with control or PEARKO males (Fig. 7A). However, when examined after overnight mating, fewer females mated with PEARKO males had significantly fewer (P = 0.023) copulatory plugs (Fig. 7B). Whereas females mated with control males had hard plugs tightly filling the cervical opening, only fibrous residue was detected in cervical opening of some females mated with PEARKO males. Copulatory plugs recovered after 4 h mating were significantly smaller (Fig. 7C) and lighter (P < 0.001) in weight (Fig. 7D). Plugs formed by PEARKO males were soft with fibrous consistency.
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). However, caput sperm were reduced (P < 0.01), corpus sperm were similar, and caudal epididymal sperm numbers were significantly increased (P < 0.05) in PEARKO males when compared with control (Fig. 8).
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, A–D).
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Discussion |
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To further investigate the role of androgens in sex accessory organ function and sperm maturation in male fertility, we generated a mouse model in which the AR is inactivated in the posttesticular accessory organs without effecting androgen-dependent testicular function using the Cre-loxP system. ROSA reporter mice revealed that whereas the Cre activity in ARR2PBi-Cre males is localized in epithelial cells of prostate (20, 21) and epididymis (present study), unexpected Cre activity was also detected in the seminal vesicles localized to the smooth muscle layer surrounding the epithelial cells. Comparably, both exon 3-deficient (functionally inactive) and wt AR expression were detected in all prostate lobes (20), seminal vesicles, epididymis, and vas deferens, indicating only partial deletion of functional wt AR. Despite this partial deletion of the wt AR in these organs, androgen action was significantly reduced as indicated by significantly reduced weight of these androgen-responsive organs as well as mRNA expression of androgen-responsive genes analyzed, except Gpx5 in epididymis. The latter observation may be because Gpx5 expression, although regulated by androgens (14, 15), is less androgen sensitive because castration does not fully inhibit its expression. Only normal, wt AR expression was detected in the testis of PEARKO males.
Despite the normal sperm production in PEARKO testis, reduced androgen action in sex accessory organs significantly reduced fertility in PEARKO males. Only five of 15 PEARKO males (33%) were fertile, with only one of 15 siring a second litter within a 90-d mating trial. PEARKO males had a much delayed median time to first litter (>90 vs. 21 d), compared with control males that all had sired two litters within 69 d. Because androgen action was reduced in epididymis as well as prostate and seminal vesicles, we further analyzed the specific effects of reduced androgen action on these sex accessory organs in trying to determine whether the acquisition of sperm fertilizing capacity or the formation of a copulatory plug were more important factors in the marked subfertility of PEARKO males.
PEARKO males had significantly smaller prostate and seminal vesicles (20) accompanied by reduced epithelial function and abnormal prostate histology (20). The reduction in seminal vesicle weight was due to significant reduction in weight (volume) of seminal vesicle secretion but was also due to reduced weight of tissue. This was also detected in the seminal vesicle histology, in which the size of lumen appeared to be reduced but was still filled with strongly eosinophilic secretions, suggestive of partly preserved epithelial secretory function.
Prostate and seminal vesicles produce seminal plasma that provides energy, protection, and propulsion for spermatozoa in the female reproductive tract and compose a copulatory plug in rodents (1). Reduced function of prostate and seminal vesicles in PEARKO males lead to defects in copulatory plug formation after mating, implying that the malformed and fibrous plug detected in females mated with PEARKO males is not able to properly seal the vagina. This is further supported by finding that after overnight mating, the proportion of plug positive females after mating with PEARKO males was notably lower. By contrast, soon after mating (4 h), PEARKO and control males produced similar numbers of copulatory plugs, indicating successful mating. The reduced copulatory plugs after overnight mating is probably due the malformed and small plugs produced by PEARKO males being more readily dissolved or dropped. The role of copulatory plug in fertility is contradictory, with conflicting and species-specific reports (2, 37, 38), and it is also argued that the role of vaginal plug is to prevent subsequent insemination by rival males (39). In rats presence and normal size of copulatory plug is required for sperm progression through the cervix (8, 38, 40, 41, 42), whereas in mice and guinea pigs, removal of the coagulating gland and prevention of plug formation was not shown to impair fertility (38, 43). Yet the removal of seminal vesicles also significantly reduced fertility in mice (43), demonstrating the role of seminal plasma in mouse fertility.
In mammals, seminal fluid also influence fertility by affecting sperm transport and survival in the female reproductive tract (2, 37, 44). As an example, murine SVS2, whose expression was significantly reduced in PEARKO seminal vesicles, is reported to be major component of the rodent copulatory plug (45) but also function as a decapacitation factor in the female reproductive tract, thereby improving the fertilization rate in the oviduct (46). Therefore, it is concluded that whereas the copulatory plug formation by PEARKO males is disrupted, factors other than a defective copulatory plug may also be important in reducing the in vivo fertility of these males. Because our model still has some secretions, compared with removal of the whole gland, it may be a valuable tool for analyzing specific androgen- dependent contributions of the seminal vesicle and prostate secretory proteins influencing sperm survival.
In addition to reduced seminal vesicle and prostate secretions, defects in sperm epididymal maturation process could also influence sperm survival and fertilizing capacity in the female reproductive tract. Androgens influence the overall rate of protein synthesis in the epididymis (47) but not to the extent observed in prostate and seminal vesicles. It has been suggested that the postcastration involution of the epididymis may reflect androgen regulation of protein degradation more than protein synthesis (48). Androgens are available from luminal fluid as well as the vasculature (49), but other testicular molecules arriving via the ductal lumen also induce epithelial responses (50, 51, 52). Androgens are known to regulate AR expression and, compared with other male reproductive organs, epididymal epithelial cells appear to have greater ability to maintain AR levels during androgen deprivation (53). This implies that androgens play an important role in epididymal epithelial cells.
Epididymal weight was significantly reduced in PEARKO males (20), and there appeared to be gross morphological differences between control and PEARKO epididymis, demonstrating the effect of reduced androgen action in this organ. As anticipated based on normal testicular function, the sperm was still present in lumen of each part of PEARKO epididymis. However, the significant distortion of distribution of sperm in the PEARKO epididymis suggests changes in sperm transit time kinetics in the epididymis. The reduction in caput with an increase in caudal sperm numbers suggests that sperm may transit more rapidly through caput and corpus epididymis, whereas the storage time in cauda epididymis may be increased. The different regions of the epididymis (caput, corpus, cauda) display distinct patterns of gene or protein expression (54, 55) as well as absorptive capacity (15), leading to changes in luminal environment and therefore may have different functions in sperm maturation. The majority of qualitative maturation of epididymal sperm occurs in caput and corpus region (15), and therefore, the reduced transit time through these regions could lead to defects in sperm maturation process.
Whereas androgens are known to regulate expression of various epididymal genes (14, 56), the specific role of androgens in this process is not clear. Our results suggest that reduced androgen activity in epididymal epithelial cells affects sperm epididymal maturation, as shown by a significant increase in PEARKO sperm spontaneous acrosome reaction and could lead to premature capacitation of sperm in female reproductive tract after ejaculation. This was accompanied by an increased angulated and spiral sperm flagellar morphology in PEARKO cauda epididymal sperm. These sperm morphological changes are assumed to be due to sperm failure in volume regulation caused by the time spermatozoa spent in the distal cauda epididymis and shown to prevent spermatozoa to reach the eggs in the oviduct (as reviewed in Refs. 18 and 57) and therefore could be a factor contributing to reduced fertility in PEARKO males. However, there was only a mild defect in quantitative in vitro fertilization ability of PEARKO male sperm, which was overcome by higher sperm numbers. This demonstrates that despite reduced androgen action in epididymis, the sperm retains its ability to bind and penetrate with zona pellucida of eggs. This is not entirely surprising because rare litters were produced by PEARKO males, indicating that the sperm is capable of fertilization. In hamsters, removal of seminal vesicles and coagulating gland also influences embryonic development after fertilization (58). However, our in vivo fertilization findings, in which significantly fewer fertilized oocytes were recovered from females mated with PEARKO males when compared with control males, demonstrate that subfertility in the present model is mainly due to defective prefertilization loss, not postfertilization.
This study is the first to in vivo analyze the role of reduced androgen action in sex accessory organs and its impact on male fertility in the presence of normal spermatogenesis. Our results provide strong evidence to suggest that the PEARKO subfertility is likely to be caused by reduced accessibility of PEARKO sperm to oocytes in female reproductive tract due to defects in seminal plasma production and composition as well as epididymal sperm maturation. These findings and further analysis of specific mechanisms in sperm maturation defects in PEARKO males may have implications for better understanding of mechanisms of idiopathic male infertility and novel tissue targeted hormonal male contraceptives.
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Acknowledgments |
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Footnotes |
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Disclosure Summary: The authors state no conflict of interest.
First Published Online March 20, 2008
Abbreviations: AR, Androgen receptor; ARKO, AR knockout; COC, cumulus oocyte complex; Gpx, glutathione peroxidase; hTFM, human tubal fluid medium; Lcn, Lipocalin; PEARKO, prostate epithelial androgen receptor deficiency; ROSA, R26R; SMActin, smooth muscle -actin; SVP99, seminal vesicle secretory protein 99; SVS2, seminal vesicle secreted 2; wt, wild type.
Received December 31, 2007.
Accepted for publication March 13, 2008.
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-reductase inhibitor. Reprod Toxicol 5:337–346[CrossRef][Medline]-reductase inhibitor, finasteride, is due to deficits in copulatory plug formation. Reprod Toxicol 5:353–362[CrossRef][Medline]
-glutamyl transpeptidase catalytic activity and protein level in the initial segment of the rat epididymis by testicular factors: role of basic fibroblast growth factor. Biol Reprod 58:197–206This article has been cited by other articles:
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  P. Bebas, C. P. Goodall, M. Majewska, A. Neumann, J. M. Giebultowicz, and P. E. Chappell Circadian clock and output genes are rhythmically expressed in extratesticular ducts and accessory organs of mice FASEB J, February 1, 2009; 23(2): 523 - 533. [Abstract] [Full Text] [PDF]
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, 
) and second litter (
, 
). B, In vivo fertility measured by percent oocytes recovered from oviduct fertilized 6 h after copulation [mean ± SE, n = 83 oocytes for control (from six females) and 46 oocytes for PEARKO (five females)]. *, Significantly different from control (P < 0.05). C, In vitro fertilization capacity of control and PEARKO cauda epididymal sperm (percent of oocytes fertilized at different sperm concentration in fertilization dish). Values are mean ± SE, average 123 (range 91–182) and 138 (99–191) oocytes were analyzed for each sperm concentration from average seven (five to 11) separate control and PEARKO males, respectively.
