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1 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba Science City, Ibaraki 305-8572, Japan
2 RIKEN, Bioresource Center, Tsukuba Science City, Ibaraki 305-0074, Japan
3 Laboratory of Genome and Biosignal, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresSperm recovery from uterusArtificial inseminationPreparation of uterine fluidsStatistical analysisReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSperm recovery from uterusArtificial inseminationPreparation of uterine fluidsStatistical analysisReferences |
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An acrosomal serine protease, acrosin, has been long believed to participate in limited proteolysis of ZP, thus enabling sperm to penetrate the egg coat. However, our previous work using acrosin-deficient (Acr–/–) mice conclusively showed that this serine protease is not essential both for sperm penetration through the ZP and for fertilization (Baba et al. 1994). The loss of acrosin results in delayed sperm penetration of the ZP at the early stages of fertilization in vitro (IVF), probably owing to the delay in dispersal of acrosomal proteins during the acrosome reaction (Yamagata et al. 1998b). Because p-aminobenzamidine (pAB), a competitive inhibitor for trypsin-like serine proteases, blocks penetration of Acr–/– sperm through the ZP (Yamagata et al. 1998a), pAB-sensitive protease(s) besides acrosin may function in the penetration event. Indeed, a gelatin-hydrolyzing trypsin-like serine protease with a size of 42 kDa is present in the sperm extracts of wild-type and Acr–/– mice (Baba et al. 1994; Yamagata et al. 1998a, 1999). Thus, the 42-kDa protease is a candidate enzyme involved in sperm penetration of the ZP at least in the mouse, although participation of acrosin and ubiquitin–proteasome system (Sutovsky et al. 2004) cannot be ruled out completely.
We (Honda et al. 2002) previously found that the 42-kDa sperm serine protease corresponds to testicular serine protease 5 (Tesp5) identical to esp-1 (Inoue et al. 1998), testisin (Hooper et al. 1999), and tryptase 4 (Wong et al. 2001) belonging to a 21st member of serine protease family Prss21. Prss21/Tesp5 is initially synthesized as a 43-kDa precursor in the testis, and the precursor is converted into the 42- and 41-kDa active enzymes during sperm transport in the epididymis (Honda et al. 2002). The enzymatic property of Prss21 is similar to but distinguishable from those of acrosin and trypsin by the substrate specificity and inhibitory effects of serine protease inhibitors. Importantly, Prss21 is included on lipid rafts of the sperm membrane as a glycosylphosphatidylinositol (GPI)-anchored protein (Honda et al. 2002; Kim et al. 2005). In human, PRSS21 has been suggested to function as a non-classical type II tumor suppressor because of its complete loss in the testicular tumors of germ cell origin (Hooper et al. 1999; Scarman et al. 2001). On the contrary, PRSS21 mRNA is abundantly expressed in ovarian carcinoma cells despite little or no expression in the normal ovary (Shigemasa et al. 2000). The protease activity of PRSS21 is required for cancer transformation, and knockdown of PRSS21 in the tumor cell lines by RNA interference results in increased apoptosis and reduced cell growth (Tang et al. 2005). Thus, PRSS21 may play a role in promoting growth and progression of human ovarian cancer. Despite the importance of PRSS21 in tumor cells, little is known of the PRSS21 function in fertilization.
In this study, we have produced mutant mice lacking Prss21 (Prss21–/–) and shown that despite normal fertility of Prss21–/– male mice, the epididymal sperm are severely defective in the ability to fertilize the eggs in vitro. Moreover, the reduced fertility of Prss21–/– epididymal sperm was rescued by exposure of the sperm to uterine microenvironment and by in vitro treatment of the sperm with uterine fluids. These findings provide a new insight into the mechanisms of sperm capacitation in the uterus, acrosome exocytosis, and sperm penetration through the ZP.
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Results |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSperm recovery from uterusArtificial inseminationPreparation of uterine fluidsStatistical analysisReferences |
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Despite normal fertility of Prss21–/– sperm in vivo, Prss21–/– cauda epididymal sperm revealed a severe defect in fertilizing cumulus-intact, metaphase II-arrested eggs in vitro (Fig. 2A); the IVF rate of Prss21–/– epididymal sperm was approximately 10% of that of wild-type epididymal sperm. The ability of epididymal sperm to bind the cumulus-free egg ZP and to fuse with the ZP-free eggs was also diminished noticeably by the loss of Prss21 (Fig. 2B and C, and Supporting Information Figs S2 and S3).
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4, an enzyme essential for the ZP glycoprotein-induced acrosome reaction (Fukami et al. 2001, 2003), indeed exhibited a negligibly low level of the acrosome reaction. Moreover, the differences in the relative ratio of acrosome-reacting and acrosome-reacted sperm to all ZP-binding sperm were significant between wild-type and Prss21–/– mice (18.1 ± 1.5% and 1.9 ± 0.6%, respectively; Student's t-test, P < 0.001) and between wild-type and Plc4–/– mice (18.1 ± 1.5% and 2.9 ± 0.6%, respectively; P < 0.001). These data suggest that Prss21 as well as Plc4 may be implicated in the acrosome reaction normally induced by ZP glycoproteins.
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4–/– sperm passed through the ZP under the experimental conditions employed.
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Prss21–/– epididymal sperm exhibited the defects in sperm/ZP binding (Fig. 2B) and the acrosome reaction on the ZP surface (Fig. 3). To ascertain whether the presence of other sperm proteins is affected by the loss of Prss21, we carried out immunoblot analysis of protein extracts from TGC and epididymal sperm (Fig. 5C). Four proteins, Adam3 involved in sperm/ZP binding (Shamsadin et al. 1999; Nishimura et al. 2001), Izumo1, Plc4, and proacrosin, were normally present in Prss21–/– TGC and sperm. In addition, the levels of six other proteins, including Crisp1 (Rankin et al. 1992; Nixon et al. 2006) and Pbp (Nixon et al. 2006) known as decapacitation factors secreted from the epididymis and testis, respectively, were similar between Prss21+/+ and Prss21–/– sperm. These data suggest that Prss21–/– epididymal sperm normally contain these ten proteins involved in the known sperm functions.
The discrepancy in the fertility of Prss21–/– sperm between in vivo and in vitro implies that the impaired functions of Prss21–/– epididymal sperm are presumably rescued during transport of ejaculated sperm through the female reproductive tract. To ascertain this possibility, effects of sperm exposure to the uterine microenvironment on their functions were examined (Fig. 6A). Wild-type female mice were artificially inseminated by injection of capacitated epididymal sperm into the uterus. The 2-cell embryos fertilized by Prss21–/– sperm were retrieved from the oviduct at a rate similar to that found in Prss21+/+ sperm, although the IVF rate of the Prss21–/– epididymal sperm was low (Fig. 6B). A control experiment indicated that artificial insemination of Adam1a–/– epididymal sperm incapable of migrating from the uterus into the oviduct (Nishimura et al. 2004) indeed yields no fertilized egg from the oviduct. When sperm were recovered from the uterus 30 min after natural mating, Prss21–/– uterine sperm showed dramatic increases both in fertilizing the cumulus-intact eggs and in fusing with the ZP-free eggs in vitro (Fig. 6C). These results strongly suggest that the reduced fertility of Prss21–/– epididymal sperm is restored by exposure of the sperm to the uterus. No significant difference in the levels of other sperm proteins, including Adam3, Ph-20, Crisp1, Pbp, Plc4, and proacrosin, was found between Prss21+/+ and Prss21–/– uterine sperm (Fig. 6D). We also verified that no proteolytic processing of these proteins occurs in uterine sperm.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSperm recovery from uterusArtificial inseminationPreparation of uterine fluidsStatistical analysisReferences |
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Prss21–/– and Plc4–/– epididymal sperm barely undergo the acrosome reaction on the egg ZP (Fig. 3). The failure of Prss21–/– sperm to undergo the ZP-induced acrosome reaction may be due to the impaired function in binding the ZP (Figs 2 and 3). However, Prss21–/– sperm are indeed capable of penetrating the ZP, albeit at a very low rate (Fig. 4), apparently inconsistent with the general concept that sperm/ZP binding and subsequent acrosome exocytosis are essential for sperm penetration through the ZP (Snell & White 1996; Wassarman 1999, 2005). We previously demonstrated that sperm binding to the ZP is severely reduced by the loss of Adam1a, probably due to the lack of Adam3 on the sperm surface (Nishimura et al. 2004). Despite the impaired ability to bind the ZP, Adam1a–/– sperm are capable of penetrating the ZP and fertilizing the egg normally. Moreover, approximately 35% of matings between Plc4–/– male and female mice has been reported to result in pregnancy (Fukami et al. 2001), although Plc4–/– epididymal sperm are unable to initiate the ZP-induced acrosome reaction, as described above. Thus, while the above general concept seems correct, there are some exceptions and additional complexities.
The mouse ZP structure is composed of three glycoproteins, ZP1, ZP2, and ZP3 (Yanagimachi 1994; Wassarman 2005). The ZP3 glycoprotein has long been thought to serve as a sperm receptor and to induce sperm to undergo the acrosome reaction (Wassarman 1999, 2005). Comparative analysis using mouse egg ZPs lacking any one of these three ZP proteins and containing either of human ZP2 and ZP3 (Rankin et al. 2003; Baibakov et al. 2007) revealed that sperm/ZP binding is not mediated by only a single ZP glycoprotein, and the three-dimensional structure of the ZP, which is determined by the cleavage status of ZP2, may be responsible for formation of the sperm binding site. It is also proposed as a "mechanosensory signal transduction" model (Baibakov et al. 2007) that the continued forward motility of sperm bound onto the ZP surface transduces a mechnosensory signal leading to mobilization of acrosomal Ca2+ stores and induction of the acrosome reaction. However, whether all sperm undergo the mechanosensory signal transduction-induced acrosome reaction to penetrate the ZP remains unclear. Even if the mechanosensory signal transduction model is correct, the acrosome reaction triggered by the ZP glycoproteins appears to be the predominant mechanism governing sperm entry into and penetration through the ZP.
An important question is whether Prss21–/– and Plc4–/– sperm, which have penetrated the egg ZP, undergo the acrosome reaction. In vitro penetration assays using Cd9–/– eggs show that wild-type and Prss21–/– sperm pooled in the perivitelline space are all acrosome-reacted (Supporting Information Fig. S6). On the basis of these results, it is interesting to suppose that the forward motility of Prss21–/– sperm bound onto the ZP surface may enable the sperm to enter into the ZP matrix, which may lead to the acrosome reaction induced by a mechanism possibly similar to that of "mechanosensory signal transduction" (Baibakov et al. 2007) or by an unknown mechanism, albeit at a low frequency. Proacrosin present in the acrosome of Prss21–/– sperm (Figs 5C and 6D) may partially assist the fertilizing sperm to penetrate the ZP. Nevertheless, to further clarify the functional roles of acrosin, Prss21, and other serine protease(s) in sperm penetration through the ZP, we need to characterize mutant mice lacking both acrosin and Prss21.
Noteworthy is that reduced fertility of Prss21–/– epididymal sperm in vitro is rescued by exposure of the sperm to the uterine microenvironment (Fig. 6) and by treatment of the sperm with uterine fluids (Fig. 7). Sperm capacitation naturally takes place within the female reproductive tract under autonomical nervous and hormonal controls (Yanagimachi 1994; Suarez 2006). Although physiological changes of sperm, including elevation of intracellular ions (pH) and hyperpolarization of the sperm plasma membrane, have been reported to occur during capacitation, the molecular mechanism is poorly understood. Serine protease Prss21 is localized on lipid rafts of sperm plasma membrane as a GPI-anchored protein (Honda et al. 2002; Kim et al. 2005), and exhibits a substrate specificity similar to but distinguishable from those of rat acrosin and bovine pancreatic trypsin (Honda et al. 2002). Our data suggest that a Prss21-like protease(s) may be secreted from the uterus and/or oviduct into uterine fluids, and compensate for the loss of Prss21 on the sperm surface, possibly by proteolytic processing of functionally latent proteins and barrier proteins covering the sperm plasma membrane. It is also possible that an unknown molecule(s) in uterine fluids may confer additional functions to the sperm deposited in the uterus. Thus, the uterine molecule(s) involved in the sperm gain-of-function system remains to be identified. At any rate, Prss21–/– male mice are useful for elucidating the molecular mechanism of sperm capacitation in the female reproductive tract. Identification and characterization of the unknown uterine factor(s) will contribute to an improvement in clinical Assisted Reproductive Technology and fertility controls in the future.
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Experimental procedures |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSperm recovery from uterusArtificial inseminationPreparation of uterine fluidsStatistical analysisReferences |
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A targeting vector containing an expression cassette of neo flanked by approximately 5- and 1.7-kbp genomic regions of Prss21/Tesp5 was constructed by using a genomic clone, HGC-14 (Honda et al. 2002). For negative selection, the MC1 promoter-driven herpes simplex virus thymidine kinase gene (tk) was inserted at the 3'-end of the 1.7-kbp Prss21 genomic region. Following electroporation of the targeting vector, which had been linearized by digestion with SalI, into mouse D3 ES cells, homologous recombinants were selected using G418 and gancyclovir, as described previously (Kashiwabara et al. 2002). Thirteen ES cell clones carrying the targeted mutation were selected from 644 clones resistant to G418 and gancyclovir, and injected into C57BL/6 mouse blastocysts. Chimeric male mice were crossed to ICR females (Japan SLC Inc., Shizuoka, Japan) to establish mouse lines heterozygous for the Prss21 mutation. The homozygous mice were obtained by mating of the heterozygous males and females. All animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals in University of Tsukuba. Prss21–/– mice have been also produced by Lexicon Genetics (Woodlands, TX) and are available from Taconic Farms (Germantown, NY), although no phenotype is described in the literature (see http://korepository.taconic.com/).
Blot hybridization
Genomic DNA was prepared from mouse tail, digested by EcoRI, separated by agarose gel electrophoresis, and transferred onto Hybond-N+ nylon membranes (GE Healthcare Amersham Biosciences). Total cellular RNA was prepared from testicular tissues using Isogen (Nippon Gene, Toyama, Japan), as described previously (Kashiwabara et al. 2002). The RNA samples were glyoxylated, separated by agarose gel electrophoresis, and transferred onto nylon membranes. The blots were probed by 32P-labeled DNA fragments and analyzed by an FLA-2000 Bio-Image Analyzer (Fuji Photo Film, Tokyo).
Antibodies
Polyclonal antibodies against Prss21/Tesp5 (Honda et al. 2002), Adam1a (Kim et al. 2003), Adam1b (Kim et al. 2006), Adam3 (Kim et al. 2004), Ph-20 (Baba et al. 2002), Plc4 (Fukami et al. 2001), and proacrosin (Yamagata et al. 1999) were prepared as described previously. Anti-Adam2 (Ikawa et al. 2001) and anti-Izumo1 (Inoue et al. 2005) antibodies were generously provided by Dr M. Okabe. Polyclonal antibodies against β-1,4-galactosyltransferase (GalTase) (Gong et al. 1995) and Crisp1 (Rankin et al. 1992) were kind gifts of Drs. B. D. Shur and R. J. Matusik, respectively. Anti-phosphotyrosine 4G10 monoclonal and anti-phosphatidylethanolamine binding protein 1 (Pbp or Rkip) polyclonal antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Horseradish peroxidase-conjugated goat antibodies against rabbit or mouse IgG (H + L) were purchased from Jackson Immunoresearch Laboratories. Alexa Flour 488-conjugated goat antibody against rabbit IgG and Alexa Flour 568-conjugated goat antibody against mouse or rat IgG were purchased from Invitrogen, Eugene, OR.
Immunoblot analysis
Testicular tissues were minced with a razor blade in PBS, filtered through a nylon mesh (75 µm), and centrifuged at 800 g for 5 min at 4 °C, as described previously (Nishimura et al. 2004). Fresh cauda epididymal sperm were washed with PBS by centrifugation. TGC and epididymal sperm were then suspended in a lysis buffer consisting of 20 mM Tris–HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, and 1% protease inhibitor cocktail (Sigma-Aldrich), gently rotated for 6 h, and centrifuged at 13 400 g for 10 min at 4 °C. Proteins in the supernatant solution were denatured by boiling for 5 min in the presence of 1% SDS and 1% 2-mercaptoethanol, separated by SDS-PAGE, and transferred onto Immobilon-P membranes (Millipore). After blocking with 3% skim milk, the blots were incubated with primary antibodies, and then with horseradish peroxidase-conjugated secondary antibodies. Immunoreactive proteins were detected by an ECL Western blotting detection kit (GE Healthcare). Protein concentration was determined using a Coomassie protein assay reagent kit (Pierce).
Fertilization in vitro (IVF)
Female BDF1 mice (8–10-weeks old, Japan SLC) were superovulated by intraperitoneal injection of pregnant mare's serum gonadotropin (5 units, Teikoku Zoki Co., Tokyo, Japan) followed by human chorionic gonadotropin, hCG (5 units, Teikoku Zoki) 48 h later. Metaphase II-arrested eggs tightly packed with cumulus cells were collected from the oviductal ampulla 14 h after hCG injection, and placed in a 90-µL drop of a modified Krebs-Ringer bicarbonate solution (TYH medium) (Toyoda et al. 1971) covered with mineral oil. Unless otherwise stated, TYH medium contained bovine serum albumin, BSA (4 mg/mL, Sigma-Aldrich, A3311–10G). Fresh cauda epididymal sperm from 3- to 5-month-old mice were capacitated by incubation in a 0.2-mL drop of TYH medium for 2 h at 37 °C under 5% CO2 in air. An aliquot (10 µL) of the capacitated sperm suspension (1.5 x 103 cells/µL) was mixed with the above 90-µL drop of TYH medium containing the eggs. After incubation at 37 °C under 5% CO2 in air, the fertilized eggs were treated with bovine testicular hyaluronidase (350 units/mL, Sigma-Aldrich) for 10 min to remove cumulus cells, fixed with PBS containing 0.25% glutaraldehyde and 0.5% polyvinylpyrrolidone (PVP), and washed with PBS containing 0.5% PVP. The female and male pronuclei in the eggs were stained with Hoechst 33342 (2.5 µg/mL), and then viewed under an IX71 fluorescence microscope (Olympus, Tokyo, Japan).
Sperm-ZP binding
Metaphase II-arrested eggs tightly packed with cumulus cells from BDF1 mice (8–10 weeks old, Japan SLC) were treated with bovine hyaluronidase, washed, and placed in a 90-µL drop of TYH medium covered with mineral oil. Capacitated sperm (1.5 x 104 cells/10 µL) were mixed with the 90-µL TYH drop containing cumulus-free eggs and 2-cell embryos, and the mixture (0.1 mL) was incubated for 30 min at 37 °C under 5% CO2 in air. The eggs were transferred to a 0.1-mL drop of fresh TYH medium, washed by pipetting to remove loosely bound and unbound sperm, and fixed in PBS containing 0.25% glutaraldehyde and 0.5% PVP on ice for 15 min. After washing with PBS containing 0.5% PVP, the number of sperm tightly bound to the egg ZP was counted under an Olympus IX71 microscope equipped with a DP-12 camera (Olympus), as described previously (Yamagata et al. 1998a; Yamashita et al. 2007). The 2-cell embryos were used as an internal negative control for nonspecific and loose sperm binding, and the average number of bound sperm per 2-cell embryo was less than 1.0 under the above conditions.
Sperm-egg fusion
ZP-free eggs were mechanically prepared by using a piezo-driven micromanipulator (Prime Tech Ltd., Ibaraki, Japan) without the use of 
-chymotrypsin or acid Tyrode, as described previously (Yamagata et al. 2002). The ZP-intact eggs were treated with Hoechst 33342 (1 µg/mL) for 10 min, and washed three times with TYH medium. After removal of the ZP, the ZP-free eggs in a 90-µL TYH drop were mixed with capacitated sperm (1.5 x 104 cells/10 µL), and the mixture (0.1 mL) was incubated for 30 min at 37 °C under 5% CO2 in air. After washing with PBS containing 0.5% PVP, the eggs were fixed in PBS containing 0.25% glutaraldehyde and 0.5% PVP, and observed under an Olympus IX71 fluorescence microscope.
Acrosome reaction on ZP surface
Capacitated epididymal sperm (3 x 104 cells/10 µL) were mixed with a 90 µL TYH drop containing cumulus-free, ZP-intact eggs and 2-cell embryos, as described above. After incubation for 30 min at 37 °C under 5% CO2 in air, the eggs were transferred to a 0.1-mL drop of fresh TYH medium, washed by rotary shaking, fixed with PBS containing 4% paraformaldehyde and 0.5% PVP for 15 min, and washed with PBS containing 0.5% PVP. Sperm bound onto the ZP were immuno-reacted with anti-Izumo1 antibody, incubated with Alexa Flour 488-conjugated antibody against rabbit IgG, counterstained with Hoechst 33342, and observed under an Olympus IX71 fluorescence microscope, as described previously (Yamashita et al. 2007).
Sperm penetration assay
Cumulus-intact eggs of Cd9–/– mice in a 90-µL TYH drop were mixed with capacitated epididymal sperm (1.5 x 104 cells/10 µL). After incubation for 6 h at 37 °C under 5% CO2 in air, the eggs were treated with hyaluronidase, fixed with 0.25% glutaraldehyde and 0.5% PVP in PBS, and washed with PBS containing 0.5% PVP. The sperm nuclei within the perivitelline space of egg were stained with Hoechst 33342 (2.5 µg/mL), and then viewed under an Olympus IX71 fluorescence microscope.
Spontaneous acrosome reaction
Cauda epididymal sperm were dispersed in a 0.2-mL drop of TYH medium free from BSA for 10 min, and incubated in BSA (4 mg/mL)-containing TYH medium at 37 °C under 5% CO2 in air. The sperm samples were transferred into a 1.5-mL microtube, washed with PBS by centrifugation, fixed with 4% paraformaldehyde in PBS on ice for 15 min, stained with 0.04% Coomassie brilliant blue at room temperature for 5 min, washed with PBS, and viewed under an Olympus IX71 microscope, as described previously (Yamagata et al. 1998a; Yamashita et al. 2007).
Protein tyrosine phosphorylation
Cauda epididymal sperm were dispersed in a 0.2-mL drop of mKRB medium (Rodeheffer & Shur 2004) free from CaCl2 and BSA for 5 min, washed with the same medium, and re-suspended in mKRB medium containing CaCl2 and BSA (4 mg/mL). After incubation at 37 °C under 5% CO2 in air, sperm were collected by centrifugation, washed with PBS, re-suspended in an SDS sample buffer free from 1% 2-mercaptoethanol, boiled for 5 min, and centrifuged at 13 400 g for 5 min. The supernatant solution was transferred into a 1.5-mL microtube, re-boiled in the presence of 5% 2-mercaptoethanol for 5 min, and then subjected to immunoblot analysis using anti-phosphotyrosine 4G10 monoclonal antibody, as described previously (Visconti et al. 1995). The Immobilon-P membranes were blocked with 5% fish gelatin (Sigma-Aldrich), instead of skim milk.
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Sperm recovery from uterus |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSperm recovery from uterusArtificial inseminationPreparation of uterine fluidsStatistical analysisReferences |
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Artificial insemination |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSperm recovery from uterusArtificial inseminationPreparation of uterine fluidsStatistical analysisReferences |
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Preparation of uterine fluids |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSperm recovery from uterusArtificial inseminationPreparation of uterine fluidsStatistical analysisReferences |
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Statistical analysis |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSperm recovery from uterusArtificial inseminationPreparation of uterine fluidsStatistical analysisReferences |
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3) unless otherwise stated. Student's t-test was used for statistical analysis; significance was assumed for P < 0.05.
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Acknowledgements |
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Footnotes |
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These authors contributed equally to this work. 
* Correspondence: acroman{at}sakura.cc.tsukuba.ac.jp |
References |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresSperm recovery from uterusArtificial inseminationPreparation of uterine fluidsStatistical analysisReferences |
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Received: 4 June 2008
Accepted: 3 July 2008
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