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Division of Reproductive Biology (G.L., F.X., B.J., J.C., E.L., M.C.), Department of Obstetrics and Gynecology, and Department of Comparative Medicine (M.A.G.), Stanford University School of Medicine, Stanford, California 94305; and Department of Pharmacology (D.R.S.), University of Washington, Seattle, Washington 98195
Address all correspondence and requests for reprints to: M. Conti, Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Drive, Room A344, Stanford, California 94305-5317. E-mail: marco.conti{at}stanford.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A possible explanation for the effects of GPCR in spermatozoa is that conventional G protein-coupled transmembrane (TM) ACs coexist with sAC in male germ cells. However, evidence that TM cyclases coupled to G proteins contribute to cAMP accumulation in spermatozoa is marred by conflicting observations. Several publications suggest that forskolin stimulates sperm cyclase (12, 14, 15) and that the zona component, ZP3, stimulates cAMP through a G protein-mediated mechanism (16, 17), whereas other reports have refuted this possibility (18, 19). Very recently, Fraser and Adeoya-Osiguwa (11) provided evidence that agents influencing membrane-associated AC, but not sAC, modulate cAMP production in mouse spermatozoa and that forskolin significantly stimulates cAMP accumulation in these cells. In addition, the presence of AC2, AC3, and AC8 in the acrosomal and flagellar regions, and of AC1 and AC4 at lower levels in the midpiece and acrosomal cap regions, has been reported (15, 20). However, Western blot analysis investigating the presence of these enzymes in sperm extracts has yielded results that are inconsistent with the presence of full-length active enzymes.
In olfactory neurons, AC3 is a 130-kDa glycosylated protein involved in the cascade required for detection of odorants (21). In these cells, this cyclase is activated mainly through the G protein Golf expressed in the olfactory epithelium. In a strikingly similar fashion, it has been reported that the olfactory type cyclase (AC3) and Golf are expressed in the male germ cells of the rodent testis (22, 23). In rat, AC3 mRNA is expressed in rat spermatids with the protein localized in the acrosomal region of spermatids (22, 23). These findings demonstrate that olfactory receptors, G protein, and cyclase involved in olfactory signaling are expressed in spermatids and are possibly retained in spermatozoa, suggesting that an olfactory-like signaling pathway including AC3 may have a key role in spermatogenesis and spermatozoa functions. The involvement of a TM cyclase in chemotaxis has been implied by pharmacological manipulation in human spermatozoa (13).
The AC3 knockout (KO) mouse was developed to investigate olfactory-dependent behaviors (24). These mice display anosmia as suggested by the loss of electroolfactogram responses induced by odorants with consequent high neonatal mortality. Because of this loss of olfaction, AC3 null pups are not able to locate the nipple and nurse. These authors reported that homozygous mating was unproductive even though AC3–/– males exhibited normal anogenital sniffing and mounting behavior with females. Mating is thought to be dependent on the function of the vomeronasal organ, and AC3 is not expressed in this organ (25, 26, 27). Thus, impairment of male fertility due to defects outside the brain may be present in these mice.
With the present report, we have reinvestigated the expression of AC3 in mouse testes and spermatozoa. Furthermore, we investigated the reproductive phenotype of the AC3–/– males and demonstrated that the in vivo and in vitro fertilizing ability of AC3–/– spermatozoa is compromised.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) of this cyclase showed specific products of the expected size (570 bp; Fig. 1A). By using testis RNA from adult rat (data not shown) or from human, the same amplification product could be detected. No PCR products were detected in the minus reverse transcriptase control. Therefore, the AC3 messenger is present in the testis of rodents and humans.
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). These levels of expression were confirmed with two different sets of AC3 primers (see Table 1). Sequencing of the fragment obtained with both sets of primers confirmed that the AC3 sequence was amplified.
In situ hybridization of mouse testes with an AC3 antisense probe detected a specific signal in male germ cells. The signal was minimal at the base of the tubule, intense in the area corresponding to spermatocytes, and was still detected in the inner portion of the tubule where round spermatids are located (Fig. 2). The AC3 sense probe was used as a negative control, and only diffuse background signal was observed throughout the section (data not shown). A probe for sAC used as a positive control showed a similar pattern of expression at the same stages of spermatogenesis (Fig. 2). Thus, sAC and AC3 most likely coexist in spermatocytes and spermatids. Mouse epididymi also were used for in situ hybridization with AC3 but no signal could be detected in this tissue (data not shown).
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, A–C). When immunofluorescence was used, the AC3 protein was more clearly localized in the acrosomal area of these cells (Fig. 3, A and B). In elongated spermatids, AC3 immunofluorescence was detected on the acrosomal cap (Fig. 3B). The signal was retained in elongating spermatids, which sometimes showed a clear localization along the acrosomal membrane (Fig. 3, B and C). As a control of the specificity of the antibody, immunofluorescence was performed on COS7 cells transfected with a construct encoding for rat AC3 (Fig. 3D). Transfected cells showed a strong signal in Golgi and plasma membrane, whereas cells transfected with the antisense construct showed no signal. To further confirm the specificity of the signal observed in testis, the AC3 antibody used for immunohistochemistry was preincubated with a 500-fold excess of the AC3 peptide. The signal was greatly reduced with only faint staining seen in round spermatids (data not shown). The immunolocalization of AC3 in mouse testis is in agreement with previously published data in rat (22, 23).
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Inactivation of the Mouse AC3 Gene and Transcript Expression in Testis
Inactivation of the AC3 gene in mouse has been previously carried out by homologous recombination targeting the first exon that codes for the amino terminus and part of the first set of TM domains (24). Surprisingly, when RT-PCR was used to evaluate the effect of homologous recombination, an increase in AC3 mRNA steady state was observed in the testis. To further determine the nature of these transcripts, pairs of primers were designed to determine the presence of exon 1 and the C1 C2 coding regions of the AC3 open reading frame (ORF) (Fig. 4A). This analysis (Fig. 4B) showed that the transcript detected in testis, brain, and kidney lacked exon 1, thus confirming that the homologous recombination had effectively removed this exon. However, a transcript could be detected when primers that encompass the catalytic domain of AC3 (primers AC3b for exon 4–6 and AC3c for exon 17–20) were used, and their expression was significantly increased in testis from AC3–/– mice when assessed by semiquantitative RT-PCR (Fig. 4, B and C).
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). Thus, we surmise that the increased level of expression of this transcript is probably attributable to the murine phosphoglycerate kinase promoter that drives the Neo resistance gene (24), and that this promoter active in germ cells directs the expression of a truncated AC3 (Tr-AC3).
The expression of this Tr-AC3 in human embryonic kidney (HEK)293 cells caused the accumulation of a protein of approximately 100 kDa detected with either a V5- or AC3-selective antibody (Fig. 5); however, no AC activity was associated with this expressed protein (Fig. 5). Conversely, expression of a full-length AC3 cDNA in the same cells led to a significant increase in both basal as well as forskolin-stimulated activity (Fig. 5). These data strongly indicate that the mRNA originating from the recombined AC3 locus codes for a truncated protein with no detectable AC activity. Thus, the homologous recombination of the AC3 gene indeed produces a catalytically inactivated AC3. However, we cannot exclude the possibility that the truncated protein encoded by the recombined allele has additional unknown functions.
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). The staining was no longer localized only to the acrosome cap but appeared more diffuse as a rim surrounding the acrosome and the nucleus of the spermatozoon. The whole of these data indicate that, although accumulation of AC3 transcript and proteins is not prevented in vivo, AC3 homologous recombination yields a catalytically inactive and mislocalized protein. Attempts to verify the presence of the shorter protein in spermatozoa of the AC3–/– mice by Western blot were unsuccessful.
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). Spermatozoa were isolated from the cauda epididymis, and their contamination was assessed by microscopic examination before incubation with different agents. cAMP production was measured in the absence or presence of forskolin, a known stimulator of the TM-AC, or in the presence of bicarbonate known to stimulate sAC. In AC3+/+ spermatozoa, a small but significant increase in cAMP levels was observed in the presence of forskolin. This stimulation was not always detectable and varied considerably among different sperm preparations from different mice (Fig. 7). Conversely, bicarbonate stimulation was always present (data not shown). When AC3–/– spermatozoa were used, basal cAMP accumulation was not significantly different from that of wild-type controls. Incubation of AC3–/– spermatozoa with forskolin did not produce a significant effect. Conversely, bicarbonate stimulated cAMP accumulation in AC3–/– spermatozoa in a manner similar to wild-type controls (data not shown). These findings suggest that a forskolin-sensitive cyclase activity is present in mouse spermatozoa but is only a minor component of the overall sperm cAMP synthesizing capacity.
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). Both AC3–/– and AC3+/– males were mated with AC3+/+ or AC3+/– females. Whereas AC3+/– males were fully fertile (nine of nine), AC3–/– males produced few or no litters; only three males of the 11 tested had offspring (Table 2). These data are consistent with the reported finding by Wong et al. (24) that AC3–/– mating is unproductive. Furthermore, AC3+/+ or AC3+/– female mice housed overnight with AC3–/– males showed vaginal plugs the following morning. Therefore, infertility of AC3–/– males is not due to a compromised reproductive behavior after disruption of olfaction.
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). Thus, the AC3 inactivation greatly reduced male fecundity.
Morphology of the Testis and Epididymis of the AC3 KO
To understand the cause of this subfertility, we studied the morphology of the reproductive tract of the AC3–/– males in greater detail. No gross anomalies could be seen along the reproductive tract; testis and accessory glands had a normal shape and size. When histological examinations of the AC3–/– reproductive tract were performed, all spermatogenic stages could be detected in the testis with a normal proportion of the different germ cells (Fig. 8A). Similarly, the epididymis showed no gross abnormality, and the lumen was filled with spermatozoa. Lastly, the number of spermatozoa retrieved from the epididymis of AC3+/+ and AC3–/– animals was evaluated. AC3–/– males produced slightly less spermatozoa than AC3+/+ mice, but this decrease was not statistically significant (data not shown). The AC3–/– spermatozoa did not display structural anomalies (head location, flagellar folding) in comparison with the AC3+/+ spermatozoa at light microscopic level (Fig. 8, B and C). Neither could structural defects be detected at the electron microscopic level (Fig. 8, D–H). All the structural components were present and appeared to be organized correctly.
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AC3 Inactivation Perturbs Sperm Motility
Because the infertility of the AC3–/– males is not associated with an overt perturbation of spermatogenesis or of any testicular function, the motility of AC3–/– spermatozoa was assessed. The percentage of motile spermatozoa (showing flagellar beat) was significantly decreased (P < 0.001) in the AC3–/– sperm population (Fig. 9). This decrease in motility was confirmed by computer-assisted spermatozoa analysis (CASA) (data not shown). In addition, the percentage of AC3–/– spermatozoa recovered from the swimming-up procedure was consistently reduced by 80% compared with wild-type controls (P < 0.01).
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). Conversely, AC3+/+ spermatozoa used in the same conditions and in similar numbers showed a high in vitro fertilization (IVF) success rate (Fig. 10A). This finding conclusively demonstrates that the low fecundity of the AC3–/– males comes from a defect at the spermatozoon level. It is important to notice that although decreased fertilization was observed when AC3–/– spermatozoa were incubated with the cumulus-oocyte complex (COC), we noticed binding of many spermatozoa to the oocytes, suggesting that the initial sperm egg interactions are normal. By using a live/dead staining on AC3–/– and AC3+/+ spermatozoa, more than 95% of spermatozoa were alive with no difference between the two genotypes. Therefore, the deficiency in fertilization cannot be attributed to a lower survival rate of the AC3–/– spermatozoa.
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). Using these oocytes with acrosome-reacted AC3+/+ and AC3–/– spermatozoa, we obtained a comparable number of embryos with both sperm populations. Therefore, it is possible to restore a normal fertility rate with AC3–/– spermatozoa. These data indicate that AC3 inactivation produces subtle changes in the spermatozoon, including deficits in motility and zona-induced acrosome reaction.
Finally, we measured the percentage of acrosome-reacted spermatozoa untreated or treated with 1 µM or 10 µM concentrations of the calcium ionophore (Fig. 11). Interestingly, in the untreated AC3–/– spermatozoon group, the percentage of spermatozoa undergoing spontaneous acrosome reaction was significantly higher than in the AC3+/+ spermatozoa. When spermatozoa was treated with calcium ionophore to artificially induce the acrosome reaction, no significant difference could be observed between the AC3+/+ and AC3–/– spermatozoa. Altogether, these data indicate that both the motility and the acrosome reaction may be affected by AC3 inactivation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Expression of AC3 in mouse spermatids is suggested by both the detection of the mRNA in germ cells and the immunocytochemical data. In situ hybridization clearly shows that spermatogenic cells expressing sAC also express AC3 mRNA. In addition, forskolin-sensitive cyclase activity is readily detected in seminiferous tubules and in enriched spermatid preparations, even though somatic cell ACs may contribute to this activity. That AC3 is retained in epididymal spermatozoa has been reported in several studies (15, 20). In agreement with these findings, we show that mouse epididymal spermatozoa are immunoreactive to AC3 antibodies with staining confined to the acrosomal region. A caveat of our studies and those of others is that the localization of AC3 relies on the use of a single antibody. Unlike previous reports, specific staining in the mid or principal piece of the flagellum was minimal. Surprisingly, staining in the head of sperm is still present in spermatozoa from the AC3 null mice. Because this staining is not only in acrosomal cap, and in view of the presence of Tr-AC3 mRNA in the testis, our interpretation of the data is that a protein lacking a critical membrane-anchoring domain is produced in the AC3 null mice and is retained in spermatozoa. On the basis of the expression in a heterologous system, this protein is catalytically inactive. This latter conclusion is consistent with the cAMP measurements even though it has been difficult to detect forskolin-stimulated cyclase activity in spermatozoa. Several previous reports have indicated the presence of forskolin-regulated cyclase in sperm from different species, even though other reports have refuted this possibility (16, 17, 18, 19).
Ablation of AC3 does not produce an overt disruption of spermatogenesis differentiation. Testis weight, histological appearance of seminiferous tubules, expression of key mRNAs, and ultrastructure of spermatozoon AC3 null testis are undistinguishable from control littermates. The number of sperm produced in the AC3 mice is also normal. Thus, ablation of AC3 in spermatids does not appear to have a major impact on qualitative or quantitative differentiation of these cells. Given the fact that sAC is active in production of cAMP in spermatids (29), it is possible that cAMP produced by this cyclase is sufficient to compensate for the loss of AC3 in spermatids. In addition, one should consider the possibility that additional TM cyclases are expressed in spermatids. Indeed mRNAs for AC9 are readily detectable in mouse testes (data not shown).
Despite apparently normal spermatogenesis, AC3–/– males are largely infertile, a finding consistent with the initial report of difficulty in producing offspring with homozygous null mating (24). The results of the homozygous mating are complicated by the fact that females also exhibit greatly reduced fertility. It is unlikely, however, that this infertility is due to the altered behavior of mice with disruption of the olfactory system. Mounting behavior is present, and vaginal plugs can be detected in wild-type females housed with AC3 null males. The finding that sexual behavior is unaffected in these mice is consistent with the notion that the vomeronasal organ, not affected by AC3 ablation, is more relevant for this function (27). By probing the function of sperm produced by the AC3 null mice, we have determined that AC3 spermatozoa display a greatly reduced ability to fertilize an egg in vitro. The yield of embryos using sperm preparations from five different AC3–/– mice was reduced by 90%. Because the fertilizing ability can be restored by dispersion of the cumulus matrix and removal of the zona, we concluded that AC3 null sperm are unable to efficiently penetrate the oocyte vestments. This defect may be caused by a subtle disruption in flagellar movements, by altered or untimely acrosome reaction, or both. Although a defect in motility was uncovered with the swim-up procedure, this may only indirectly cause inefficient fertilization, because the same number of motile spermatozoa was used in the IVF experiment.
In view of the difficulty of detecting the expression of AC3 activity in spermatozoa, we cannot formally exclude that the effect of AC3 disruption on fertility is indirect and caused by a loss of function early during spermatid differentiation. This latter possibility, however, is inconsistent with the normal morphology of the testis and spermatozoa and the normal expression of key genes involved in spermatid differentiation. Although the adcy3 heterozygous mice had no fertility phenotype, we cannot exclude the possibility that the truncated protein derived from the recombined allele has biological effects other than cAMP production in spermatids or spermatozoa, perhaps functioning as a dominant-negative allele.
It is remarkable that many of the components of the transduction pathway used for sensing olfactory cues are expressed in spermatids and spermatozoa. Olfactory receptor mRNAs and, in some instances, proteins have been detected in germ cells from different species (30, 31). The olfactory Golf protein is expressed in spermatids and retained in spermatozoa (22). As shown here, the major olfactory cyclase AC3 is expressed together with the phosphodiesterases PDE1 and PDE4A, all expressed at high levels in the olfactory system. Cyclic nucleotide-gated channels also are shared by spermatozoa and olfactory cells (32). To explain this remarkable similarity, it has been proposed that an olfactory-like signaling pathway may be used by the male gamete to sense the external chemical environment (31, 33). In agreement with this hypothesis, it has been reported recently that an olfactory receptor expressed in human spermatozoa is involved in sperm chemotaxis (13). Given the pharmacological data suggesting that this olfactory receptor is coupled to a cyclase with the properties of a TM-AC (13, 34), it is tempting to speculate that the olfactory cyclase AC3 functions downstream of this receptor. If this hypothesis is correct, AC3 null spermatozoa may carry defects in the chemosensory system required to recognize and penetrate an egg. Although no obvious deficit in sperm binding to the egg was observed during IVF, this possibility needs to be explored further.
In summary, our data indicate that the AC3 TM cyclase is directly or indirectly necessary for sperm function and fertility. Even though the exact biochemical lesion disrupting AC3 fertility remains to be determined, these findings suggest a complex interplay between bicarbonate-sensitive sAC and other ACs and GPCRs. In view of the probable localization of these cyclases in different spermatids and spermatozoa compartments, the site of cAMP production may be an important determinant of the biochemical functions of these different components of cAMP signaling. Thus, spermatozoa provide a unique model in which to study this signaling compartmentalization.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Experimental Animals
Wild-type mice were purchased from Charles River Laboratories, Inc. (Wilmington, MA). All animal procedures were in accordance with accepted standards of humane animal care and were approved by the Animal Care and Use Committee at Stanford University.
Analysis of the Genotype and Mating the AC3-Deficient Mice
AC3+/– mice provided by Daniel Storm from the University of Washington Animal Facility were used to generate AC3–/– mice to study male fertility (24). Due to the poor efficiency of this mating, some AC3+/– males were also mated with AC3–/– females to improve the yield of AC3–/– pups. To reduce litter size and increase the survival rate, pups were transferred to foster mothers as soon as possible after birth. To genotype the mice, PCR was performed on genomic DNA extracted from tails of the offspring. The primers 5'-cctgtgctctagtagcttacgg-3' and 5'-ctgtgaagtaggttcctacctg-3' identified the mutant allele, and 5'-ctggtgaagtggcttgacct-3' and 5'-gttatgaagaaggagaagaca-3' corresponded to the wild-type allele.
AC3+/– and AC3–/– males between 2 and 6 months of age were mated (two females per male) with AC3+/+ or AC3+/– females. No difference was observed in the fertility of AC3+/– or AC3+/+ females. The presence of vaginal plugs was recorded on many occasions for both AC3+/– and AC3–/– males. When pregnancy was detected by an increased weight, females were removed from the mating cage and housed separately until delivery. Males siring at least one litter were defined as fertile, and the number of litters per month obtained by one male was used as the index of fecundity.
Histology and Electron Microscopy
Testes and epididymi of AC3–/–and AC3+/+ mice were fixed in Bouin’s solution for 6 h at room temperature and then dehydrated and stained with eosin. After embedding in Paraplast, 5-µm sections were cut, deparaffinized, rehydrated, and then stained with hematoxylin and eosin. Slides were cleared with xylene, mounted, and analyzed by light microscopy with a Zeiss microscope (Carl Zeiss, Thornwood, NY) fitted with AxioCam (Zeiss, Oberkochen, Germany).
For electron microscopy, spermatozoa suspensions were fixed in 2% glutaraldehyde in PBS on ice. Next, samples were washed several times in PBS, dehydrated through an ethanol series, and embedded. Samples were visualized on a Philips CM12 transmission electron microscope.
Immunohistochemistry and Immunofluorescence
Detection of AC3 protein in mouse testis and spermatozoa was performed as previously described (35). Briefly, testis were fixed in Bouin’s solution or 4% paraformaldehyde (PFA) and then either embedded in Paraplast or frozen in optimal cutting temperature compound (Tissue-Tek, Torrance, CA). Sections (5–10 µm) were cut. For immunohistochemistry, the Vectastain Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA) was used. Paraplast sections were rehydrated and then incubated 1 h at room temperature with 10% goat serum, followed by antibodies overnight incubation with anti-AC3 antibodies (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in a humidified chamber at 4 C. The distribution of the primary antibody was revealed with a biotinylated goat antirabbit secondary antibody, and the avidin-biotin-peroxidase complex was visualized with 3,3'-diaminobenzidine. For immunofluorescence, frozen sections were treated similarly but incubated with AC3 antibodies diluted 1:1000, and the primary antibody was visualized with a Texas Red goat antirabbit secondary antibody (Vector Laboratories).
Fresh spermatozoa were fixed in 4% PFA (30 min at 4 C), washed in PBS, and dried on slides coated with poly-L-lysine. If the slides were not to be used immediately, they were stored at 4 C.
The specificity of staining was monitored by replacing the first antibody with nonimmune IgG or by blocking the signal with an excess of peptide. As a further positive control for the antibody, COS7 cells were transfected (Effectene transfection kit; QIAGEN, Chatsworth, CA) with 2 µg of constructs encoding the rat sense or antisense AC3 cDNA (22). Cells were fixed 24 h later in 4% PFA (15 min at 4 C) and then rinsed in PBS and stored at 4 C before staining.
In Situ Hybridization
In situ hybridization was carried out as previously described (35). Briefly, a partial segment of 387 bp (3301–3687 bp) of rat AC3 cDNA (M55075) subcloned in pcDNA3.1/V5-His-TOPO vector was used as template for the synthesis of radioactive 35S-labeled RNA probes.
Mouse testes were fixed in 4% PFA for 6 h and incubated in 0.5 M sucrose overnight at 4 C. Testes were embedded in OCT (Tissue-Tek), cut into 10-µm sections, and mounted. Slides were postfixed in PFA and treated as previously described. Radioactive hybridized sections were exposed in NTB2 Emulsion (Eastman Kodak, Rochester, NY) for 7 d, developed photographically, counterstained with Gill’s hematoxylin and eosin (0.25% wt/vol in ethanol), cleared with xylene, and mounted with Permount (Fisher Scientific Co., Pittsburgh, PA). Testes were visualized and photographed with AxioCam.
IVF: Preparation of Spermatozoa
Males of ages ranging between 2.5 and 4 months were caged alone at least 5 d before the IVF was performed. Mice were euthanized by CO2 narcosis and hypoxia. The cauda epididymi and a portion of the vas deferens were removed and quickly transferred into a culture dish containing 2 ml of M2 medium and then transferred into another dish with 200 µl of M2 medium. Four incisions were made in the cauda epididymis to allow spermatozoa to swim out. After 10 min of incubation at 37 C, tissue fragments were discarded, and the spermatozoa suspension was collected. Cells were counted and, if necessary, diluted to obtain similar concentrations in AC3+/+ and AC3–/– preparations. Some of the spermatozoa suspension (50 µl) was transferred to the bottom of a 1.5-ml Eppendorf tube containing 200–450 µl of KSOM [potassium simplex optimized medium (Specialty Media, Phillipsburg, NJ), supplemented with 4 mM glucose and 4% BSA (later referred to as modified-KSOM, mKSOM) (36)]. Capacitation was allowed to proceed for 1 h at 37 C, in a 5% CO2 incubator and then 50 µl was collected from the upper fraction of the spermatozoa suspension, which represents a population of highly motile capacitated spermatozoa, and the concentration of the spermatozoa in the fraction was determined and, if necessary, adjusted to obtain similar concentrations of AC3+/+ and AC3–/– sperm. Additionally, live/dead staining was performed with SYBR 14 dye (Live/Dead Spermatozoa Viability Kit; Molecular Probes, Inc., Eugene, OR) according to the recommendations of the manufacturer.
IVF: Preparation of Oocytes
C57BL/6 female mice (25-d old) (Charles Rivers Laboratories) were induced to superovulate by ip injection of 5 IU pregnant mare’s serum gonadotropin (Calbiochem) followed by 5 IU human chorionic gonadotropin (Goldline Laboratories, Fort Lauderdale, FL) 48 h later. Animals were killed by CO2 14 h after chorionic gonadotropin injection. Oviducts were collected in a 35-mm dish containing 2 ml M2 medium. The COCs were recovered by gentle dissection of the oviducts. Each COC was transferred to a 20-µl droplet of capacitated spermatozoa suspension in mKSOM and covered with mineral oil.
IVF Procedure
Highly motile spermatozoa (2–5 x 105/ml) from the swim-up fraction were added to each fertilization droplet with the COCs and placed in an incubator for 4 h. Oocytes then were washed twice in 50 µl mKSOM under oil and finally transferred to droplets of the same medium. The dishes were incubated at 37 C in 5% CO2 in a humidified incubator. Fertilization was assessed by recording the number of two-cell embryos 24 h after fertilization. Some of the dishes were maintained in culture to monitor two-cell embryo progression to later stages of development. Embryos were observed at x400 magnification on the warmed stage (37 C) of an inverted microscope.
In a set of experiments, zona drilling was performed on mouse oocytes, and motile spermatozoa from the swim-up fraction were artificially acrosome reacted. First the oocytes from the COC were denuded by treatment with 0.03% hyaluronidase in M2 medium at 37 C. The denuded oocytes then were treated for 1–2 sec with acidic Tyrode’s solution until almost complete disappearance of the zona and then washed with several rinses of medium before being transferred to the IVF drop. Spermatozoa were treated with calcium ionophore (10 µM A23187) for 30 min to induce a synchronous acrosome reaction. The ionophore was then quenched with a high concentration of BSA (20 mg/ml), and the spermatozoa suspension was diluted 10- to 20-fold before being added to the oocytes for a 2-h incubation. Oocytes were then washed twice as described above.
Measurement of Spermatozoa Motility
Motility of the spermatozoa was assessed subjectively using an inverted microscope. After a brief incubation into mKSOM, spermatozoa were diluted in PBS, and motile and total spermatozoa were counted. For each point, at least 200 spermatozoa were evaluated. Whenever possible, these data were confirmed by CASA (integrated visual optical system from Hamilton Thorne Biosciences, Inc., Beverly, MA).
Measurement of cAMP Production
The spermatozoa were collected from cauda epididymi and allowed to swim out into BWW medium (95 mM NaCl; 4.8 mM KCl; 1.3 mM CaCl2; 1.2 mM MgSO4; 1.2 mM KH2PO4; 20 mM sodium lactate; 5 mM glucose; 0.25 mM sodium pyruvate; 25 mM NaHCO3, pH 7.4) supplemented with 10 mM HEPES (pH 7.4) and 3% BSA (37). Spermatozoa were counted and normalized to 20 million cells per ml and pretreated for 10 min with 0.5 mM isobutylmethylxanthine. Of this suspension, 50 µl was added to 50 µl of the medium with or without 25 mM bicarbonate or 100 µM forskolin and incubated for 15 min at 37 C. To stop the reaction, 1 ml of ice-cold 0.1% trichloroacetic acid in ethanol was added. Samples were then centrifuged at 3000 rpm for 30 min at 4 C, and the supernatant was evaporated while centrifuging under vacuum and reconstituted with 500 µl of PBS. cAMP was measured by the RIA method of Harper and Brooker (38).
Measurement of Acrosome Reaction
After capacitation, spermatozoa preparations were incubated for 30 min with or without various concentrations of calcium ionophore (A23187) and then fixed in 4% PFA for 10 min at room temperature. Spermatozoa suspensions were then centrifuged at 1000 x g for 10 min and resuspended with 500 µl of 0.15 M ammonium acetate (pH 9). Spermatozoa suspensions were centrifuged one more time and resuspended in 50 µl PBS, and then spotted and dried on slides coated with 0.1% poly-L-lysine. G-250 Coomassie blue (4% in 3.5% perchloric acid) was added onto the slide for 2–3 min. The slides then were washed four times, 5 min each, in water. The acrosome-reacted cells were counted with x1000 magnification. Acrosome reaction caused the loss of intense staining on the anterior aspect of the spermatozoa head. Data were expressed in percentage of acrosome-reacted cells compared with the total number of cells. At least 200 cells were counted for each point.
RT-PCR and 5'-RACE
Testes were quickly removed from the animals, washed in PBS, and frozen at –80 C. RNA isolation was performed with 1 ml of Trizol reagent (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s protocol. To avoid contamination with genomic DNA, RNA was treated with deoxyribonuclease. Reverse transcriptase reactions were performed with 5 µg of RNA and oligo-dT primers. PCR with specific primer for AC3, sAC, TP1, and ß-actin was performed. The primers sequences, the size of the amplified bands, and the annealing temperatures are given in Table 1. To ensure that we specifically amplified the type 3 AC, and no other similar AC, the PCR products were sequenced. Semiquantitative RT-PCR also was performed, and the expression of these genes was compared with the ß-actin expression. This protocol assures that all measurements were taken during the exponential phase of the PCR.
5'-RACE was performed on brain and testis RNAs using commercially available kits (5' RACE System for Rapid Amplication of cDNA Ends, Invitrogen; Marathon-Ready cDNA, CLONTECH Laboratories, Inc.) following the manufacturer’s instructions. The products of the reaction were sequenced and sequences were analyzed using the BLAT or Browser utilities of the UCSC Genome Bioinformatics web site (http://genome.ucsc.edu/index.html?org=Mouse).
Expression of the Recombinant Proteins and AC Activity
Full-length AC3 (FL-AC3) and truncated Tr-AC3 (Tr-AC3) tagged with V5 were transfected in HEK293 cells using Mirus transfection reagents following the manufacturer’s directions. Cells were harvested 48 h after transfection and protein was extracted and measured for AC activity as described earlier (29). An aliquot of the extract was separated on 8% SDS-PAGE, transferred on immobilon membrane, and immunoblotted with AC3 or V5 antibodies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online February 10, 2005
Abbreviations: AC, Adenylyl cyclase; CASA, computer-assisted spermatozoa analysis; COC, cumulus-oocyte complex; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; IVF, in vitro fertilization; KO, knockout; mKSOM, modified potassium simplex optimized medium; ORF, open reading frame; PFA, paraformaldehyde; RACE, rapid amplication of cDNA ends; sAC, soluble AC; TM transmembrane; Tr-AC3, truncated AC3.
Received for publication August 6, 2004. Accepted for publication February 1, 2005.
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o, Gi2, and adenylyl cyclase II as major components of a pheromone signaling cascade. J Neurosci 16:909–918This article has been cited by other articles:
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