Department of Physiology, Institute of Biomedicine (P.S., M.M., J.P., I.H., M.P.), and Turku Graduate School of Biomedical Sciences (P.S., J.P.), University of Turku, FIN-20520 Turku, Finland; Institute of Reproductive Medicine of the University (T.G.C., C.-H.Y.), D-48129 Münster, Germany; and Reproduction and Development Research Group (J.D.), Blaise Pascal University, Centre National de la Recherche Scientifique Unité Mixte de Recherche, F-63177 Aubière, France
Address all correspondence and requests for reprints to: Matti Poutanen, Department of Physiology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-20520 Turku, Finland. E-mail: matti.poutanen{at}utu.fi.
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ABSTRACT |
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INTRODUCTION |
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In this study, we have taken the approach of immortalizing and/or disrupting the function of the epididymal epithelium by targeting expression of the Simian Virus 40 large T-antigen (SV40 Tag) into the epididymis of transgenic mice by using the murine GPX5 promoter. Recently, the 5'-flanking sequences of two epididymis-specific proteins have been shown to direct the transgene expression into the epididymis, namely those of murine epididymal retinoic acid-binding protein (mE-RABP; Ref. 5) and murine glutathione peroxidase 5 (GPX5; Ref. 6). GPX5 is a well characterized epididymal secretory protein that belongs to the glutathione peroxidase family (7, 8). The expression of GPX5 in the caput epididymidis is under the control of androgens (7, 9), and we have shown that the 5.0-kb 5' flanking region of the mouse GPX5 gene is suitable for directing transgene expression to the caput epididymidis (6). The function of GPX5 is not totally understood, but it has been shown to bind to epididymal spermatozoa (10, 11) at the acrosomal region, and therefore it has been suggested that GPX5 could protect sperm membranes from oxidative damage (8, 12, 13).
SV40 Tag is able to transform cells (14), and it is widely used in studies on genetically targeted tumorigenesis owing to its capacity of transforming even well differentiated cell types (15, 16, 17). The exact mechanisms for immortalization and transformation by SV40 Tag are not yet known, but several immortalization pathways are likely to be involved (for review, see Ref. 18). It has been suggested that binding of SV40 Tag to two cell cycle regulatory proteins, p53 and retinoblastoma susceptibility protein, is needed for transformation and immortalization (19), but p53-independent pathways have also been reported (18, 19). Furthermore, it has been shown that in addition to tumorigenesis, T-antigen expression in transgenic mice can cause apoptosis, for example in the mammary gland (20).
Epididymal epithelial cells are highly differentiated and possess different characteristic features in different regions of epididymis. As shown in the present study, a high level of expression of SV40 Tag in the epididymis leads to severe dysplasia, whereas a low level of expression was found to cause epididymal dysfunction, resulting in a maturation defect of the sperm leading to angulated sperm flagella and infertility.
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RESULTS |
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GPX5-Tag1 females typically died at the age of 36 months, and the histo-pathological changes found in the females were highly variable. The most constant finding was that they developed adrenal tumors. In addition, tumors were infrequently detected in the pituitary gland and uterus. By contrast, the GPX5-Tag2 female mice had a normal life span, and no tumor formation was found in them.
Expression of the GPX5-Tag mRNA in GPX5-Tag1 and GPX5-Tag2 Male Mice
Northern blot and RT-PCR analyses were used to identify the transgene expression in GPX5-Tag1 and GPX5-Tag2 males at the ages of 50 d and 4 months. In the GPX5-Tag1 males, transgene expression was found in the epididymis, seminal vesicles, vas deferens, and adrenal gland at the age of 50 d (Fig. 1B), and at the age of 4 months the transgene mRNA was further detected in the prostate tumors formed. In contrast, Northern blot analysis of the GPX5-Tag2 males indicated that the transgene was expressed exclusively in the epididymis at both age groups analyzed (Fig. 1C
). In addition to that found by the Northern analysis, RT-PCR analysis also showed a weak expression in the testis in both of the mouse lines and in vas deferens and seminal vesicles in the GPX5-Tag2 mice.
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Terminal Uridine Deoxynucleotidyl Nick End Labeling (TUNEL) Staining
To study whether increased apoptosis rate was the cause of decreased testicular and epididymal size in GPX5-Tag2 males, TUNEL staining was performed. In the GPX5-Tag2 initial segment, the number of apoptotic cells per independent microscopic frame was significantly higher (13.4 ± 1.93; P < 0.001; n = 7; Fig. 6A) as compared with the WT initial segment (1.1 ± 0.51; Fig. 6B
). Also, in the caput region the number of apoptotic cells was higher in GPX5-Tag2 males (3.0 ± 1.0; P < 0.05) than in the WT males (0.7 ± 0.29). By contrast, no difference was found in the number of apoptotic cells between the genotypes in corpus (WT, 0.7 ± 0.42; GPX5-Tag2, 0.7 ± 0.56) and cauda regions (WT, 0; GPX5-Tag2, 0.4 ± 0.42). Furthermore, the number of apoptotic cells in the different spermatogenic stages (IVI, VIIVIII, and IXXII) was equal between WT (0.4 ± 0.20, 0.6 ± 0.41, and 1.1 ± 0.24, respectively) and GPX5-Tag2 males (0.1 ± 0.06, 0.4 ± 0.11, and 0.8 ± 0.27).
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Reproductive Performance of the GPX5-Tag2 Mice
Because of the severely altered testis structure, the lack of sperm, and the drastic structural changes in the epithelium in the epididymis and seminal vesicles, it was not surprising to find that all of the GPX5-Tag1 males were infertile. However, all of the GPX5-Tag2 males analyzed were also infertile, although histological analyses of the reproductive tissues of these male mice did not show any obvious reason for the infertility. The reproductive performance of the GPX5-Tag2 males was therefore examined further. Because the mice possessed normal mating behavior (copulatory plugs in 18 of 18 females studied), and the uterus was full of sperm after mating, anejaculation was obviously not the reason for the infertility observed in the GPX5-Tag2 males. The ability of the sperm to fertilize oocytes in vivo was studied next. After mating the GPX5-Tag2 males with WT females, neither sperm nor fertilized oocytes were found in the oviducts of the females (Table 2). Hence, it was likely that the spermatozoa produced by the GPX5-Tag2 males were unable to reach the oviduct in normal matings. As a control, the oocytes in the females mated with WT males were analyzed, and 96% of the oocytes were fertilized in vivo (Table 2
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Sperm Motility
Spermatozoa from the cauda epididymidis of both GPX5-Tag2 and WT males were equally motile upon release in two test media used, and there was no difference in the maintenance of motility over the 2.5-h follow-up period. Because kinematic parameters were uninfluenced by the substrates, data from the two media were pooled. Although the percentage of motile sperm from the cauda epididymidis was not different between GPX5-Tag2 and WT mice at either time point, there were marked differences in the flagella forms (Fig. 7). The majority of motile WT sperm had straight flagella, whereas most motile sperm from GPX5-Tag2 mice had hairpin bends (Fig. 7
). The slight reductions in mean values of curvilinear velocity (micrometers per second), average path velocity (micrometers per second), straight-line velocity (micrometers per second), linearity (percentage), and amplitude of lateral head displacement (micrometers) of sperm from GPX5-Tag2 mice did not reach statistical significance.
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Osmotic Pressure of Luminal Contents
The osmotic pressure of the cauda epididymal contents from WT and GPX5-Tag2 males was also measured, and it was found to be significantly higher in WT (mean ± SEM, 396 ± 8 mmol/kg; n = 8) than in the GPX5-Tag2 mice (358 ± 10 mmol/kg; n = 6).
Epididymal Gene Expression in the GPX5-Tag2 Mouse Line
To further characterize the epididymal dysfunction in GPX5-Tag2 mice, the expression of various epididymal and ion transporter genes (Table 3) was analyzed by RT-PCR and Northern blot analysis. There were no differences in the expression levels of 12 of 15 genes analyzed (data not shown). However, the mRNAs for two initial segment-specific genes, cystatin-related epididymal spermatogenic (CRES) and mouse epididymal protein 17 (mEP17), were found to be missing from GPX5-Tag2 mice (Fig. 9
, A and B). Also, the mouse homolog for human epididymal 6 (HE6) was not expressed in the initial segment of GPX5-Tag2 mice, and the expression was lower in GPX5-Tag2 mice in distal caput and cauda compared with WT mice (Fig. 9C
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DISCUSSION |
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Several oncogenes have been used in studies aimed at developing animal models for tumor development. Some of these studies indicate that both the properties of the activated oncogene and the tissue context determine the final outcome, such as malignant transformation (23). There are several transgenic mouse models for tumor development in the prostate (15), testis (16), and adrenal gland (17). By contrast, in the epididymis only hyperplasia has been reported (23, 24, 25), and hyperplasia also seems to be a common feature in the seminal vesicles expressing an oncogene (24). These observations are in line with the mouse models generated in the present study. In the GPX5-Tag1 mice, tumors developed in the prostate and adrenal gland, whereas the seminal vesicles and epididymides were only hyperplastic with severe dysplasia. The disrupted spermatogenesis with involuted seminiferous tubules in GPX5-Tag1 mice is suggested to be caused by the occluded epididymal lumen at the corpus and caudal regions. Furthermore, it is also likely that the altered tubular structure in the initial segment and caput do not support the normal fluid flow in the epididymis. The conclusion of epididymal dysfunction leading to disrupted spermatogenesis is also supported by the fact that only a minor SV40 Tag expression was detected in the testis by RT-PCR and that in the earlier studies, no pathology has been observed in mice expressing SV40 Tag in haploid spermatids (26, 27).
The phenotypic changes in GPX5-Tag2 males were less severe than in GPX5-Tag1 males. This is in line with a lower SV40 Tag expression in the GPX5-Tag2 males and the stricter expression of SV40 Tag in the epididymis. In the GPX5-Tag2 mouse line, one macroscopically notable difference was that the initial segment was lacking its normal reddish color, indicating a decrease in the vascularization. Increased vascularization is characteristic of the initial segment (28, 29) and is thought to underlie the high metabolic activity of this region (30). Decreased vascularity characterizes c-ros knockout mice, which lack the initial segment (31, 32). GPX5-Tag2 mice do have an initial segment, although light microscopy revealed stellate profiles in this region of the GPX5-Tag2 mice, which are not typical for the mouse. Such stellate profiles are characteristic of the initial segment of other species and are unlikely as such to contribute to an abnormal epididymal environment. The other difference between the GPX5-Tag2 and WT mice was the lower testis weight in the GPX5-Tag2 mice at older age. An increased rate of apoptosis has been reported in many SV40 Tag mouse models, and some studies indicate that apoptosis has a role in regulating tumorigenesis (33, 34, 35), whereas other studies indicate that the expression of SV40 Tag directly causes apoptosis (20). In our GPX5-Tag2 mouse model, the dysplastic initial segment was found to have increased apoptosis rate, suggesting that the induced proliferative pressure by SV40 Tag expression is counter-balanced by increased rate of apoptosis. The increased apoptosis rate in the epididymides of SV40 Tag-expressing mice might be one of the mechanisms resulting to the fact that no tumor formation was detected in the epididymis. Also, in the caput epididymidis apoptosis rate was increased in GPX5-Tag2 males. The rest of the epididymal regions, i.e. corpus and cauda, had normal apoptotic rate compared with WT males, suggesting a correlation between the observed alterations in the epididymal histology at the initial segment and increased apoptosis rate. However, there was no difference in the number of apoptotic cells between WT and GPX5-Tag2 testes in any of the spermatogenic stages studied, and hence, the reason for the reduced testis weight with qualitatively normal spermatogenesis remains to be explored.
No change in serum testosterone and LH levels and only a mild change in the FSH and inhibin B at the old adult age was detected in the GPX5-Tag2 males, further suggesting that the infertility found in the mice was not due to hormonal factors but rather to a nonhormonal effect in the epididymis.
The most obvious feature of the epididymal sperm in the GPX5-Tag2 mice was the angulated flagellum that was maintained while the sperm were swimming and did not hinder motility significantly. From the fixed epididymal preparations, it was clear that over 80% of the spermatozoa produced by the testes were straight in shape and the bending of the flagellum occurred gradually within the epididymal canal and especially as sperm entered the cauda epididymidis. By comparison, the c-ros knockout mice, whose in vivo infertility is due to failure of sperm in the uterus to negotiate the oviduct because of the hairpin forms of the sperm, exhibit much less extensive tail angulation within the epididymis, although the angulation is exaggerated upon suspension in culture medium (32, 36). Sperm angulation is a response to osmotic swelling, which forces the tail to bend at the enlarged cytoplasmic droplet, affecting an increase in cell volume without unsustainable stretching of the cell membrane. It has been shown that sperm maturation in the epididymis includes changes in properties of cell volume regulation (32), but it is not known whether the process involves modification of sperm membrane transport, sperm osmolytes, or osmolarity of the luminal milieu. The latter is much higher than the osmolarities of fluids in the rete testis or the female tract. The in situ angulation of the epididymal sperm in the GPX5-Tag2 mice might be related to the observed decrease in osmolality of the cauda epididymidal fluid, which could be a result of epididymal malfunction.
Elimination of membrane restraint by a detergent allows angulated sperm from the c-ros knockout mice to straighten out (36). By contrast, such treatment reduced, but did not abolish, hairpin forms from GPX5-Tag2 sperm found in situ. This failure to straighten out may reflect prolonged fixation of the bent tail by thiol oxidation within the epididymis because incubation in a thiol-reducing agent induced a further reduction in hairpin forms in sperm both before and after demembranation.
The tail angulation may well explain the lack of fertilized oocytes in the oviduct and the infertility of the GPX5-Tag2 males in vivo as well as in vitro, because similar changes in sperm morphology have been shown to be the cause of infertility in the c-ros knockout mouse (37). In the c-ros knockout mice, the initial segment is lacking, suggesting that the initial segment in particular is involved in the formation of the phenotype of sperm angulation. Unlike the c-ros knockout mice, the GPX5-Tag2 mice contain the initial segment, but its mere presence may not necessarily denote normal function and critical secretions of the epithelial cells that could be missing from the GPX5-Tag2 transgenic males. The data suggest that the lower osmotic pressure of epididymal fluid could be the cause for the bent sperm phenotype seen in GPX5-Tag2 mice. However, the expression levels of all the tested ion transporters were unchanged, but whether there is a functional defect in some of the ion transporters causing the lower osmolality of caudal fluid in GPX5-Tag2 epididymides remains to be studied.
It has been reported previously that SV40 Tag transformation of prostatic epithelial cells was accompanied by down-regulation of the differentiated function, demonstrated by the loss of differentiation-specific secretory proteins (15). Accordingly, we suggest that the SV40 Tag expression in the GPX5-Tag2 mice results in a defect in differentiation of the initial segment. This is supported by the lack of expression of certain initial segment-specific genes, such as CRES and mEP17. By contrast, all of the tested caput-, corpus-, and cauda-specific genes were normally expressed. The only exception was HE6, for which expression levels were lower than in controls in GPX5-Tag2 mice in all of the epididymal regions.
In addition to being merely markers for the lack of differentiation of initial segment, these proteins could also be partially the cause of bend sperm phenotype seen in GPX5-Tag2 sperm. CRES is a member of the cystatin superfamily of cysteine protease inhibitors, and it is suggested to be involved in the regulation of proprotein processing (38). The target proteins for CRES and its function are still unclear, but the absence of CRES from the initial segment may lead to undesirable protein processing by cysteine proteases either in the epididymal epithelium or sperm plasma membrane. mEP17 is a new member of the lipocalin superfamily suggested to transport retinoids within the epididymis (39), and HE6 belongs to the seven transmembrane-domain receptor superfamily (40). Animal studies have shown the importance of the retinoic acid signaling pathway for the function of epididymal epithelium, and retinoids have been shown to regulate epididymal gene expression (41), whereas HE6 has been suggested to be an orphan membrane receptor. However, the direct role of these proteins in the sperm function remains to be explored further.
In conclusion, the GPX5-Tag2 is a novel mouse line with epididymal defect, and it is useful for analyzing further the role of epididymis in posttesticular sperm maturation, especially to analyze genes involved in the regulation of the posttesticular sperm maturation.
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MATERIALS AND METHODS |
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Establishment of the GPX5-Tag Transgenic Mouse Lines
The GPX5-Tag transgene was cleaved from the pGEM-7Zf vector by XhoI/SacI digestions (Fig. 1A), purified by a Quick-Pick Electroelution Capsule Kit (QIAGEN, Valéncia, CA) and Elutip DEAE-columns (Schleicher & Schüell, Dassel, Germany), and diluted to the final concentration of 2 ng/µml. Transgenic founder mice were generated in the genetic background of the FVB/N strain by microinjecting the DNA into pronuclei of fertilized oocytes using standard techniques. Integration of the transgene was verified by PCR screening using tail DNA isolated using the salting-out method. The PCR consisted of 30 cycles (1 min 97 C, 1.5 min 56 C, 2 min 72 C), and the following primers were used: 5'-primer, 5'-CAGCTAATGGACCTTCTAGG-3'; 3'-primer, 5'-GCAATCGAAGCAGTAGCAATC-3'. The founder mice were mated with WT FVB/N mice to create specific transgenic mouse lines (GPX5-Tag1 and GPX5-Tag2). All mice were handled in accordance with the institutional animal care policies of the University of Turku (Turku, Finland). The mice were specific pathogen-free and were fed with complete pelleted chow and tap water ad libitum in a room with controlled light (12 h light, 12 h darkness) and temperature (21 ± 1 C).
To confirm the transgene integration and analyze the copy numbers of the integrated transgene, Southern blot analysis was performed using previously described protocols (6).
RNA Analysis
Total RNA from various tissues was isolated using the single-step method, and the GPX5-Tag transgene expression in the GPX5-Tag1 and GPX5-Tag2 mouse lines was studied using Northern blot analysis and RT-PCR. In addition, the expression of several epididymal genes in the GPX5-Tag2 epididymides was studied using semiquantitative RT-PCR and Northern blot analysis. For Northern blot analysis, 20 µg denatured total RNA were resolved on a 1% denaturing agarose gel and transferred onto nylon membranes (Hybond-XL). The membranes were hybridized with the [32P]CTP-labeled cDNA for the large T-antigen gene, 308-bp-long RT-PCR product for CRES gene, and 300- and 431-bp-long RT-PCR products for mEP17 and HE6, respectively, using standard techniques. Hybridization signals were detected by autoradiography using x-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan) or a phosphor imager (Fuji Photo Film Co., Ltd.).
In the RT-PCR analysis, 1 µg of DNase I (Life Technologies, Inc., Paisley, Scotland, UK)-treated total RNA was reverse-transcribed (10 min, 50 C) using avian myeloblastosis virus reverse transcriptase (Promega Corp.) and amplified using Dynazyme II-polymerase (Finnzymes, Espoo, Finland) in the same reaction tube. The primers used were identical to those used for genotyping the GPX5-Tag mice.
3'-End labeling of RT-PCR products using digoxigenin-11-dideoxy-uridine triphosphate (Boerhringer Mannheim, Basel, Switzerland) was done as described previously (42). Total RNA was DNase I-treated before RT-PCR. Primers for ß-actin (used as an internal control) and for each tested gene, annealing temperatures, and cycle numbers are represented in Table 3.
Morphological, Histological, and Immunochemical Analyses
Histological evaluation of tissues of WT, GPX5-Tag1, and GPX5-Tag2 mice was made in two age groups, 5060 d and 4 months. The mice were anesthetized by ip injection of 300600 µl of 2.5% Avertin (Sigma, St. Louis, MO), blood was collected by cardiac puncture, and tissues were dissected out for macroscopic analyses and for organ weights. For histological analyses, Bouin- and paraformaldehyde-fixed paraffin sections of tissues were stained with hematoxylin/eosin. For immunohistochemistry, paraffin sections from epididymides, testes, seminal vesicle, prostate and adrenal gland of the WT and GPX5-Tag1 males, and epididymides and testes from the GPX5-Tag2 males were boiled in 1 M urea in a microwave oven for 15 min. Samples were then immunostained with a mouse monoclonal SV40 Tag (Ab-2) antibody (Oncogene Research Products, Boston, MA; 1:100 to 1:300 dilution in PBS). The antigen-antibody complexes were visualized by biotinylated anti-mouse antibody (Vector Laboratories, Inc., Burlingame, CA) combined with avidin-horseradish peroxidase complex (ABC kit, Vector Laboratories, Inc.) and 3,3'-diaminobenzidine tetrahydrochloride (DAB-Plus Substrate Kit, Zymed Laboratories, Inc., San Francisco, CA).
TUNEL Staining
To study whether the increased apoptosis rate was the cause of decreased epididymis and testis size, TUNEL staining was performed on epididymal and testicular sections of WT and GPX5-Tag2 males. The DNA strand breaks present in paraffin-embedded sections were labeled with the In-situ Cell Death Detection Kit (Roche Molecular Biochemicals, Québec, Canada) according to the manufacturers instructions. In different parts of epididymides (initial segment, caput, corpus, cauda), the number of TUNEL-positive cells present in tubular cross-sections on seven individual microscopic frames (20x objective) were then calculated. The cross-sections of testicular seminiferous tubules were divided into three different groups according to the spermatogenic stages: IVI, VIIVIII, and IXXII; the number of apoptotic cells was calculated from each group using 5 testicular cross-sections from 3 mice, or 15 sections altogether.
Hormone Measurements
After blood collection, the separated serum samples were stored at -70 C until assayed for hormones. Serum FSH was measured by an immunofluorometric assay for rat FSH, essentially as described before (43). LH of the sera was measured by a sensitive immunofluorometric assay for rat LH (Delfia; Wallac, Inc. OY, Turku, Finland) as described earlier (44). Testosterone was measured from diethyl ether extracts of the sera using RIA as described earlier (45). Serum inhibin B concentrations were measured by using inhibin-B assay kit (Oxford Bio-Innovation Ltd., Oxford, UK).
Analysis of the Fertility of the GPX5-Tag Male Mice
Continuous matings were performed to analyze the fertility of males at the age of 7 wk to 3 months from both GPX5-Tag1 and GPX5-Tag2 lines, and none of the mice was found to be fertile. For statistical analysis of the infertility, six males from both lines were mated with three WT females each. Copulatory plugs were sought daily, and the plugged females were followed for 34 wk to determine the number of litters and offspring produced by each male.
For GPX5-Tag2 males, additional studies were performed to determine whether the existing spermatozoa were able to fertilize oocytes in vivo. Four GPX5-Tag2 males, at the age of 2 months, and six age-matched WT males were mated with six adult females each, and females were checked daily for copulatory plugs. The plugged females were killed, and oocytes were collected from the oviductal ampulla. The number of fertilized oocytes and the total number of oocytes in the oviducts were counted. The oocytes were furthermore incubated in KSOM Embryo Culture Medium (Specialty Media Inc., Lavallette, NJ) at 37 C overnight to analyze whether the fertilized oocytes divided normally. In vitro fertilization was performed to GPX5-Tag2 males as described previously (46).
Analysis of Sperm Motility
To analyze whether sperm motility was affected by metabolic substrates, we followed a previously described method (47, 48). Sperm from GPX5-Tag2 mice were released from a few loops of tubule of the cauda epididymidis into two different media; medium H contained glucose, pyruvate, and lactate as substrates, and medium G contained glucose only. The sperm were allowed to disperse for 12 min at 37 C in 5% CO2, and sperm suspensions were then further diluted to a concentration appropriate for motility assessment at 37 C, immediately and again after a 2.5-h incubation at 37 C. The sperm suspension was placed on a siliconized slide under an 18 x 18-mm coverslip to provide a depth of 40 µm. Motility was recorded by videotaping using pseudo-darkfield optics created by using a 4x objective with a 40x condenser ring and a 3.3x photo ocular. The percentages of the motile and immotile sperm with straight or bent flagella were measured using phase contrast optics and a 20x objective. The kinematic parameters of 100200 motile sperm per sample were measured by analyzing the videotapes at 25 Hz for 30 frames by the Hamilton-Thorne HTM-C 10.6 system (Beverly, MA).
Analyzing Sperm Shape
The entire epididymis of GPX5-Tag2 mice was immerse-fixed overnight in 5% glutaraldehyde. The organs were divided into seven regions: proximal and distal caput; proximal, middle, and distal corpus; and proximal and distal cauda epididymidis. Tubule fragments were rinsed and minced in PBS before brief sonication to release individual sperm from the fixed, agglutinated clumps. Five-microliter aliquots were examined for categorization of the sperm tail as straight, slightly angulated (obtuse angle), greatly angulated (acute angle), or hairpin bends.
In an additional experiment, spermatozoa were released by cutting a few tubule segments from the caput, corpus, and cauda epididymidis of WT and GPX5-Tag2 mice and transferring the luminal contents into BWW medium (Specialty Media Inc.) containing glucose, pyruvate, and lactate and sufficient NaCl to be of the same osmolality as that of cauda epididymidal fluid from GPX5-Tag2 mice. After dispersion, 20 µl were either fixed in 2.5% glutaraldehyde in PBS or treated with 1% Triton X-100 in PBS for 1 min before fixation to see whether the bending was caused by membrane restraint. Other aliquots of caudal sperm were released into medium containing 1 mM DTT and incubated for 45 min at 37 C, after which time they were fixed as above. For each sample, 100 sperm were categorized for their morphology. The total percentage of angulated forms was calculated from all angulated flagella, including hairpin forms. The percentage of sperm bearing cytoplasmic droplets was calculated by adding the straight sperm bearing visible droplets to those that were angulated at the site of the droplet.
Measurement of Osmotic Pressure
The osmotic pressure of 3 µl of undiluted cauda epididymidal contents was measured by a Vapro vapor pressure osmometer as described by Yeung et al. (36), with the exception that fluid was flushed out with a medium possessing osmolality of 430 mmol NaCl/kg water. Six samples were obtained from different GPX5-Tag2 males and 8 samples from WT males. For each sample, the mean of two measurements (taken after 10 and 15 min equilibration) was taken. In addition to calibration with standards of 100, 290, and 1000 mmol/kg as recommend by the supplier, standards of 400 mmol NaCl/kg water were measured under identical conditions before, during, and after measurements of the epididymal fluids to confirm the absence of instrument drift.
Statistical Analyses
Statistical analyses were performed by SigmaStat-program (version 2.0 for Windows 95, SPSS, Inc., Chicago, IL). For TUNEL results, t test or Mann-Whitney rank sum test was performed for analyzing the statistical significance (P < 0.05). For hormone results, Kruskal-Wallis one-way analysis or one-way ANOVA was performed, and in the case of statistically significant results, Dunns or Tukeys test was performed for pair-wise multiple comparisons. For tissue weights, the same analyses were performed. Two-way ANOVA was used to examine the differences in the percentages of morphological forms of the sperm for each genotype, in each epididymal region separately, in the absence and presence of Triton X-100. One-way ANOVA was used to examine the effects of DTT on tail morphology of caudal GPX5-Tag2 sperm. The t test was used to compare the osmotic pressures of epididymal fluid between genotypes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: CRES, Cystatin-related epididymal spermatogenic; DTT, dithiothreitol; EGFP, enhanced green fluorescent protein; GPX5, glutathione peroxidase 5; HE6, human epididymal 6; mEP17, mouse epididymal protein 17; SV40 Tag, Simian virus 40 large T-antigen; TUNEL, terminal uridine deoxynucleotidyl nick end labeling; WT, wild-type.
Received for publication March 11, 2002. Accepted for publication July 23, 2002.
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REFERENCES |
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