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J. Biol. Chem., Vol. 279, Issue 37, 38194-38200, September 10, 2004
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**
From the
Mass Spectrometry Resource, Division of Endocrinology, Departments of Medicine and of **Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110, the ¶Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801, and the ||Division of Experimental Diabetes, Mount Sinai School of Medicine, New York, New York 10029
Received for publication, June 10, 2004 , and in revised form, July 1, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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) is the first recognized cytosolic Ca2+-independent PLA2 and has been proposed to participate in arachidonic acid (20:4) incorporation into glycerophosphocholine lipids, cell proliferation, exocytosis, apoptosis, and other processes. To study iPLA2 functions, we disrupted its gene by homologous recombination to generate mice that do not express iPLA2. Heterozygous iPLA2+/– breeding pairs yield a Mendelian 1:2:1 ratio of iPLA2+/+, iPLA2+/–, and iPLA2–/– pups and a 1:1 male:female gender distribution of iPLA2–/– pups. Several tissues of wild-type mice express iPLA2 mRNA, immunoreactive protein, and activity, and testes express the highest levels. Testes or other tissues of iPLA2–/– mice express no iPLA2 mRNA or protein, but iPLA2–/– testes are not deficient in 20:4-containing glycerophosphocholine lipids, indicating that iPLA2 does not play an obligatory role in formation of such lipids in that tissue. Spermatozoa from iPLA2–/– mice have reduced motility and impaired ability to fertilize mouse oocytes in vitro and in vivo, and inhibiting iPLA2 with a bromoenol lactone suicide substrate reduces motility of wild-type spermatozoa in a time- and concentration-dependent manner. Mating iPLA2–/– male mice with iPLA2+/+, iPLA2+/–, or iPLA2–/– female mice yields only about 7% of the number of pups produced by mating pairs with an iPLA2+/+ or iPLA2+/– male, but iPLA2–/– female mice have nearly normal fertility. These findings indicate that iPLA2 plays an important functional role in spermatozoa, suggest a target for developing male contraceptive drugs, and complement reports that disruption of the Group IVA PLA2 (cPLA2
) gene impairs female reproductive ability. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Of mammalian PLA2s so far cloned, secretory PLA2s are low molecular weight enzymes that require millimolar Ca2+ concentrations for catalysis and affect eicosanoid generation, inflammation, and other processes (1). The platelet-activating factor-acetylhydrolase PLA2 family exhibits substrate specificity for platelet-activating factor and oxidized phospholipids. Of Group IV cytosolic PLA2 (cPLA2) family members (1), cPLA2 was the first identified and prefers substrates with sn-2 20:4 residues, catalyzes 20:4 release for subsequent metabolism, associates with its substrates in membranes upon rises in cytosolic [Ca2+] in stimulated cells, and is also regulated by phosphorylation (6).
The Group VI PLA2 (iPLA2) enzymes (1, 2, 7, 8) do not require Ca2+ for catalysis and are inhibited by a bromoenol lactone (BEL) suicide substrate that does not inhibit secretory PLA2 or cPLA2 at similar concentrations (9, 10). The Group VIA PLA2 (iPLA2) resides mainly in the cytoplasm of resting cells, but the Group VIB PLA2 (iPLA2
) contains a peroxisomal targeting sequence and is membrane-associated (11).
Many cells express multiple distinct PLA2s, and this might reflect redundancy or specific functions of an individual PLA2. Physiological roles for PLA2s can be studied with genetic gain- or loss-of-function manipulations. Overexpressing iPLA2 in insulinoma cells, for example, provides evidence for its participation in exocytosis, cell proliferation, and apoptosis (12–14), and cPLA2 gene disruption by homologous recombination has produced cPLA2-null mice that reveal a role for cPLA2 in parturition, allergic responses, and post-ischemic brain injury (15, 16).
We have used homologous recombination to generate iPLA2-null mice. Among various tissues, testes of wild-type mice express the highest iPLA2 levels, and male iPLA2–/– mice produce spermatozoa with reduced motility and impaired ability to fertilize mouse oocytes in vitro and in vivo. Male iPLA2–/– mice are also much less fertile than wild-type males, but female iPLA2–/– mouse fertility is not markedly impaired. Our findings indicate that iPLA2–/– plays an important functional role in spermatozoa.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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gene fragment by screening a 129/SvJ mouse genomic DNA library with rat iPLA2 cDNA (8). The 7.8-kb EcoRV-BglII fragment containing exons 7–10 was subcloned into pBluescript SK–. A single XhoI site mapped to exon 9 near sequence encoding the 463GTSTG467 lipase motif. A pGK-neo-poly(A) cassette with a neomycin resistance gene (neo) was inserted at this site to disrupt iPLA2 coding sequence and provide a positive selection marker. A pGK-thymidine kinase gene was inserted into the BglII site of the genomic fragment as a negative selection marker. This yielded a vector with 4.1 and 3.7 kb of the 5' and 3' sequences, respectively, homologous to the native gene for recombination.
The targeting fragment was excised with EcoRV and BglII and introduced into 129/SvJ mouse embryonic stem cells by electroporation. Clones resistant to G418 and ganciclovir were isolated and screened for homologous recombination by Southern blotting of genomic DNA digested with EcoRV. Six embryonic stem clones contained 6.7-kb fragments characteristic of iPLA2 gene disruption and, as expected, 8.7-kb fragments from the wild-type allele. The clones were injected into C57BL/6 mouse blastocysts, which were implanted for gestation to yield chimeras that were mated with wild-type mice to yield heterozygotes. Mating iPLA2+/– mice with each other gave iPLA2–/–, iPLA2–/+, and iPLA2+/+ pups.
Mice were genotyped with Southern blots of tail clipping genomic DNA digested with EcoRV using a 32P-labeled probe prepared by PCR amplification or restriction endonuclease digestion with EcoRV and BglII to yield an 0.95-kb fragment located downstream of the targeting sequence. This probe hybridizes with an 8.7-kb DNA fragment in iPLA2+/+ mice, with a 6.7-kb fragment in iPLA2–/– mice, and with both in iPLA2+/– mice (see Fig. 1D). The iPLA2 gene-targeting sequence inserts a new EcoRV site in genomic DNA to yield a fragment that hybridizes with the probe that is shorter than that from wild-type genomic DNA.
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cDNA probes labeled by random priming. The iPLA2 cDNA probe was amplified using reverse transcription-PCR (sense primer, 5'-TGTGACGTGGACAGCACTAGC; antisense primer, 5'-CCCCAGAGAAACGACTATGGA), which hybridizes to both short and long isoforms of iPLA2 (2). This region of cDNA represents the sequence that encodes amino acid residues 307–552 of the short isoform of iPLA2. A final stringency wash was followed by autoradiography, and the filters were then stripped and hybridized with cDNA probes to rat glyceraldehyde-3-phosphate dehydrogenase to mark RNA load (8). Western blotting was performed as described with tissue homogenates and iPLA2 antibody that had been raised in rabbits (12). Ca2+-independent Phospholipase A2 Activity Assay—Tissue Ca2+-independent PLA2 specific activity was determined as described (8) in cytosol by monitoring hydrolysis of 1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine in assay buffer (40 mM Tris, pH 7.5, 5 mM EGTA) to [14C]linoleate as measured by TLC and liquid scintillation spectrometry. Specific activity was calculated from released [14C]disintegrations/min and protein content.
Electrospray Ionization Mass Spectrometry of Lipids—Tissue lipids were extracted and infused into the ESI source of a triple stage quadrupole mass spectrometer in CHCl3/CH3OH containing LiOH, as described (17–19). Glycerophosphocholine (GPC) lipids and triacylglycerols (TAGs) were analyzed as [M+Li]+ ions. Seminolipid and glycerol-, inositol-, and ethanolamine-glycerophospholipids and phosphatidic acids were analyzed as [M-H]– ions. Tandem spectra were obtained by accelerating selected ions into a collision cell to induce dissociation, and product ions were analyzed in the third quadrupole. Internal standards, e.g. 14:0/14:0-GPC, were used for quantitation.
Analyses of Motility of Spermatozoa and Effect of the iPLA2 Inhibitor BEL—Male mice 8–10 weeks old were euthanized with pentobarbital in a protocol approved by our Animal Studies Committee. Caudae epididymides, and the vasa deferentia were removed and placed in prewarmed and pregassed human tubal fluid medium (Irvine Scientific, Irvine, CA). Morphology and viability of spermatozoa were assessed as described (20–22), did not differ between iPLA2+/+ and iPLA2–/– mice, and were unaffected by BEL. Sperm suspension was placed in an incubation chamber (37 °C), and motility was quantified using CEROS computer-assisted semen analyses (version 10; Hamilton Thorne Research, Beverly, MA), as discussed (23). Total and progressive motilities were analyzed in about 104 spermatozoa from each genotype. A swim-up motility assay was also used that involved centrifugation, incubation (1 h, 37 °C), and counting spermatozoa that migrated into supernatant, as described (22). Spermatozoa were treated in some cases with various concentrations of BEL (0–20 µM), and motility was analyzed after various periods (0–30 min).
In Vitro Fertilization Assay—Female mice about 30 days old were injected with pregnant mare serum gonadotropin (7.5 IU intraperitoneally) and 48 h later were injected with human chorionic gonadotropin (hCG; 7.5 IU intraperitoneally), as discussed (24). Oviducts were collected 13 h later, and the oocyte cumulus complexes were removed. One or two complexes were placed in a culture dish (100 mm, 50 µl minimum essential medium, 25 mM NaHCO3, 1% fatty acid-free bovine serum albumin; Sigma), and droplets were covered by embryo-tested mineral oil (Sigma). Spermatozoa collected from cauda epididymis were allowed to swim into minimum essential medium (10 min, 37 °C), aspirated, incubated (37 °C, 60 min, 5% CO2, 4 x 106/ml) to permit capacitation, diluted, and added to oocyte droplets to achieve a concentration of 105 or 106 spermatozoa/ml. Spermatozoa and oocytes were co-incubated (5–24 h, 37 °C, 5% CO2). Oocytes and zygotes were passed through a pipette to remove cumulus cells, fixed (1% paraformaldehyde), placed on a microscope slide with affixed coverslip, treated with acetic acid/ethanol (1/3 v/v, 2 min), stained (1% lacmoid/45% acetic acid), destained (45% acetic acid), and examined for oocyte fertilization reflected by a second polar body and two pronuclei with one near a sperm tail (24).
In Vivo Fertilization Assay—Superovulation of 6–8-week-old female mice was induced by injecting pregnant mare serum gonadotropin (10 IU, intraperitoneally; Sigma) and, after 48 h, hCG (10 IU, intraperitoneal, Sigma), as described (21–24). Those mice were then mated with iPLA2+/+ or iPLA2–/– males overnight, and, after 48 h, the mice were euthanized with pentobarbital according to a protocol approved by our Animal Studies Committee. Dissected uterine horns were flushed with human tubal fluid medium containing 0.25% bovine serum albumin (Sigma) to retrieve one- and two-cell structures, which were then counted and cultured (human tubal fluid with 0.25% bovine serum albumin media microdroplets under mineral oil, 37 °C, 5% CO2) for 72 h. The blastocyst embryos were then counted.
Fertility Tests—Male mice were placed with females for 6 weeks and then removed, and the pups were counted, as described (21). Females mated with iPLA2–/– males had a normal frequency of vaginal semen plugs, determined as described (25).
Statistical Analyses—Comparisons between two groups or among three or more groups were performed with Student's t test or with analysis of variance using posthoc analysis (Statview 4.51, Abacus), respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-null Mice—Fig. 1 illustrates our scheme to generate mice with a disrupted iPLA2 gene and to determine their genotypes. An iPLA2 gene-targeting construct was introduced into mouse embryonic stem cells, and those that incorporated it by homologous recombination, which disrupts the iPLA2 gene coding sequence (Fig. 1B), were introduced into mouse blastocysts that were then implanted into pseudo-pregnant female mice. Progeny included chimeras, which were mated with wild-type mice, and litters included iPLA2+/– mice, reflecting iPLA2– allele germ-line transmission. Mating pairs of iPLA2+/– mice yielded iPLA2+/+, iPLA2+/–, and iPLA2–/– pups in a nearly Mendelian 1:2:1 distribution with a 1:1 male/female gender distribution (Table I).
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mRNA content exceeds that of muscle, pancreas, kidney, liver, brain, heart, adipose, and epididymis (Fig. 2A). No iPLA2 mRNA was detected in testes or other tissues of iPLA2–/– mice. Western blots also revealed high iPLA2 protein expression in wild-type testes, but no iPLA2 protein was detected in iPLA2–/– testes or spermatozoa (Fig. 2B).
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(10–12), it is stimulated by ATP and inhibited by a BEL suicide substrate (Fig. 3B). Testes and other tissues of iPLA2–/– mice exhibit much less total Ca2+-independent PLA2 activity than wild-type mouse tissues (Fig. 3A), suggesting that iPLA2 is ordinarily the major tissue Ca2+-independent PLA2 and that there is little up-regulation of other Ca2+-independent PLA2s, such as cPLA2 and iPLA2 (1–2), to compensate for the loss of iPLA2.
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Gene Disruption on Tissue Content of Arachidonic Acid-containing Phosphatidylcholine and Other Lipids—Studies of [3H8]arachidonic acid incorporation into P388D1 tumor cells have been taken to imply that the function of iPLA2 is to generate lysophosphatidylcholine acceptors for arachidonic acid (20:4) incorporation into GPC lipids (26), and it might thus be expected that iPLA2–/– mouse tissues would be deficient in 20:4-containing GPC lipids. The lipids were extracted from wild-type and iPLA2–/– mouse testes and analyzed by ESI/MS in the presence of LiOH. Fig. 4A is the ESI/MS positive total ion current spectrum for a lipid extract from wild-type mouse testes, and it contains ions with even m/z values for Li+ adducts (17) of internal standard (m/z 684) dimyristoyl-GPC (14:0/14:0-GPC) and of endogenous 16:0/18:1-GPC (m/z 766) and the 20:4-containing species 16:0/20:4-GPC (m/z 788) and 18:1/20:4-GPC (m/z 814).
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The ESI/MS spectrum for Li+ adducts of iPLA2–/– mouse testes lipids (Fig. 4B) is nearly identical to that for iPLA2+/+ mice (Fig. 4A), and abundances of ions for 20:4-containing GPC lipids (m/z 788 and m/z 814) relative to the internal standard (m/z 684) are virtually identical in panels A and B of Fig. 4, indicating that iPLA2–/– mouse testes are not deficient in 20:4-containing GPC lipids. Negative ion ESI/MS analyses (19) of testes lipids reveal 20:4-containing glycerophosphoethanolamine and glycerophosphoinositol lipids, e.g. 18:0/20:4-glycerophosphoethanolamine and 18:0/20:4-glycerophosphoinositol, and other lipid species. Negative ion ESI/MS spectra for iPLA2–/– and iPLA2+/+ testes lipids are also nearly identical (not shown). Lack of iPLA2 thus has little effect on testes phospholipid composition or 20:4-content, and iPLA2–/– and iPLA2+/+ testes also do not differ in gross or microscopic anatomy or weight.
Motility of Spermatozoa from Wild-type and iPLA2–/– Mice—Although there is a modest reduction in number of spermatozoa produced by iPLA2–/– mice (1.5 ± 0.4 x 107/mouse) compared with wild-type mice (3.4 ± 0.7 x 107/mouse), there is a marked reduction in motility of iPLA2–/– mouse spermatozoa. Fig. 5A summarizes swim-up motility analyses and illustrates that motility of spermatozoa from iPLA2–/– mice is less than 5% of that of spermatozoa from iPLA2+/+ or iPLA2+/– mice. Fig. 5B illustrates computer-assisted spermatozoa analyses of motility and also demonstrates reduced motility of spermatozoa from iPLA2–/– compared with iPLA2+/+ or iPLA2+/– mice.
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Activity with a BEL Suicide Substrate on Motility of Spermatozoa—To examine further the role of iPLA2 in motility, suspensions of spermatozoa were treated with varied concentrations of the iPLA2 inhibitor BEL (9–10) for various intervals, and motility of spermatozoa was determined. BEL reduced motility of spermatozoa in a concentration- and time-dependent manner at [BEL] as low as 5 µM (Fig. 6A) and at times as early as 5 min (Fig. 6B).
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–/– spermatozoa suggested that they might be impaired functionally. Fig. 7A illustrates that wild-type spermatozoa exhibit a concentration-dependent ability to fertilize oocytes from wild-type female mice in vitro, but spermatozoa from iPLA2–/– mice achieved fertilization only about 5% as often as wild-type spermatozoa at the highest concentration tested, indicating that iPLA2–/– spermatozoa are functionally impaired.
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–/– male mice. Products from oocytes were recovered from the uterus, and it was determined whether fertilization had occurred and whether there had been subsequent development of blastocysts. Fig. 7B illustrates that when female mice were mated with control, reproductively competent male mice, 84 two-cell structures were observed by 48 h after hCG injection, and 86% of them later developed into blastocysts. In contrast, when female mice were mated with iPLA2–/– male mice, only 2 two-cell structures were observed by 48 h after hCG injection, and neither developed into a blastocyst (Fig. 7B). Both in vitro (Fig. 7A) and in vivo (Fig. 7B) fertilization experiments thus demonstrate functional impairment of spermatozoa from iPLA2–/– mice.
Fertility of iPLA2–/– Male Mice—The reduced motility of iPLA2–/– mouse spermatozoa and their impairment in fertilizing oocytes raised the question of whether these mice would exhibit reduced fertility. Male iPLA2+/+, iPLA2+/–, and iPLA2–/– mice were thus mated with female iPLA2+/+, iPLA2+/–, and iPLA2–/– mice. Ten breeding pairs were examined for each possible genotypic pairing. Each pair was allowed to mate for 6 weeks, and the number of pups each pair produced during this period was determined. Table II illustrates that with wild-type iPLA2+/+ male partners, female iPLA2+/+ mice bore 59 pups and female iPLA2–/– mice produced 70 pups, indicating that female iPLA2–/– mice exhibit no severe reproductive defect.
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+/+ males or heterozygous iPLA2+/– males with female mice of all three genotypes. A total of 214 pups were sired by iPLA2+/+ males, and 235 pups were sired by iPLA2+/– males (Table II), indicating that iPLA2+/– males are not reproductively impaired. In contrast, iPLA2–/– male mice sired only 16 pups or about 7% of the number sired by iPLA2+/+ or iPLA2+/– males, reflecting severe reproductive impairment in iPLA2–/– males despite a normal frequency of vaginal semen plugs in females with which they were mated.
Male iPLA2–/– males were not completely infertile and sired more pups with wild-type iPLA2+/+ female partners than with iPLA2+/– or iPLA2–/– female partners (Table II). No mechanistic explanation for that finding is obvious because iPLA2+/– females produced more pups (total of 183) than did iPLA2+/+ females (total of 148 pups) when mated with iPLA2+/+ or iPLA2+/– males. The severe impairment of fertility of male iPLA2–/– mice is reflected by the fact that they sired only a single pup with iPLA2+/– female partners under these conditions. The larger number of pups resulting from matings of iPLA2+/+ females with iPLA2–/– males might reflect chance variation, although an occult mechanistic basis cannot be excluded.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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–/– knockout mice have greatly reduced fertility that would impose a selection bias against that genotype, although female iPLA2–/– mouse reproductive ability is not dramatically impaired. The low iPLA2–/– male fertility is associated with markedly reduced motility of spermatozoa. Inhibition of iPLA2 with BEL also reduces motility of wild-type spermatozoa in a concentration- and time-dependent manner that resembles the effect of eliminating iPLA2 by gene disruption. The iPLA2-null mouse thus joins a group of recently reported mouse models involving gene disruption that produce selective impairment of male (but not female) fertility associated with reduced motility of spermatozoa. Other such models include disruption of the genes for soluble adenylyl cyclase (20), for the voltage-gated cation channels Catsper1 and CatSper2 (27–29), and for plasma membrane Ca2+-ATPase 4 (25).
Signals that regulate motility of spermatozoa include changes in cAMP and intracellular [Ca2+] (27–33), and both parameters are affected by products of PLA2 action, which include a free fatty acid and a 2-lysophospholipid. Mice deficient in soluble adenylyl cyclase activity are infertile because of a severe sperm motility defect (20), and both abnormalities also occur in mice null for the catalytic subunit of cAMP-dependent protein kinase A (33). Products of iPLA2 action affect down-stream effects of cAMP, and iPLA2 overexpression in insulinoma cells amplifies secretion induced by glucose and agents that elevate [cAMP] (12). The iPLA2 reaction products 2-lysophosphatidylcholine (LPC) and 2-lysoplasmenylcholine activate cAMP-dependent protein kinase A and enhance phosphorylation of the cAMP response element-binding protein in cardiac myocytes (34), and LPC generated by iPLA2 also regulates cAMP- and cAMP-dependent protein kinase A-dependent events in macrophages and endothelial cells (35, 36).
The flagellar motion underlying sperm motility is cyclic and is associated with [Ca2+] oscillations at the base of the flagellum that occur at the frequency of the flagellar beat (37). Hyperactivated motility involves Ca2+ release from intracellular sequestration sites, such as the redundant nuclear envelope that surrounds the axoneme at its origin in the flagellar base (30). The fact that motility of spermatozoa is impaired by knockout of channels that mediate Ca2+ entry (27–29) or a plasma membrane pump that extrudes Ca2+ (25) is consistent with a requirement for Ca2+ oscillations for flagellar motion.
Insulin secretion by -cells is oscillatory and associated with [Ca2+] oscillations produced by a cyclic process that involves glucose-induced Ca2+ release from endoplasmic reticulum (ER) and resultant activation of a nonselective plasma membrane cation channels (38). Activation of these store-operated channels (SOC) depolarizes the plasma membrane and causes voltage-operated Ca2+ channels to mediate Ca2+ influx. This results in refilling of ER Ca2+ stores and inactivation of the depolarizing SOC (38). This cycle repeats itself in an oscillatory manner in the continued presence of stimulus.
Recently, iPLA2 has been found to participate in regulating SOC (39). Ca2+ store depletion-induced activation of depolarizing SOC and resultant activation of voltage-operated Ca2+ channels in smooth muscle cells involves production of Ca2+ influx factor by ER. Ca2+ influx factor then interacts with calmodulin so as to release it from and relieve its tonic inhibition of iPLA2, which then catalyzes phospholipid hydrolysis. LPC produced by iPLA2 then activates SOC (39). Pharmacologic and biochemical evidence supports operation of this pathway in vascular smooth muscle cells and -cells (38–41). The operation of a similar pathway in spermatozoa could rationalize the requirement for Ca2+ store release (30), iPLA2 (this report), Ca2+ entry channels (27–29), and a Ca2+ extrusion pump (25) in the oscillatory flagellar motion that underlies the motility of spermatozoa.
The free fatty acid product of iPLA2 action could also participate in regulating [Ca2+] in spermatozoa subcellular compartments. Arachidonic acid (20:4) facilitates Ca2+ entry from the extracellular space and Ca2+ release from ER (42), and 20:4-containing plasmenylethanolamine species are abundant in ER and are excellent iPLA2 substrates (43). Moreover, Ca2+ influx factor is an arachidonate oxygenation product (44), and its production could involve iPLA2 action.
The fact that spermatozoa from iPLA2–/– mice are defective in in vitro fertilization in which a high concentration of spermatozoa are placed in close proximity to oocytes suggests that these spermatozoa might be defective in properties in addition to motility. Ca2+ signaling in the tail of spermatozoa is involved in regulating flagellar motion, and [Ca2+] in the head of spermatozoa is involved in the acrosomal reaction induced in spermatozoa by oocyte zona pellucida (45), a reaction in which iPLA2 could participate. A PLA2 is activated during induction of the acrosomal reaction by zona pellucida and releases arachidonic acid and LPC from spermatozoa membrane phospholipids (46). LPC also induces the acrosome reaction in spermatozoa (30, 32), and one species of LPC (2-lysoplasmanylcholine) is the precursor of the platelet-activating factor, which is produced by spermatozoa and is an autocrine inducer of capacitation (47).
Our finding that homozygous iPLA2 gene disruption impairs male reproductive ability by causing production of spermatozoa with reduced motility complements reports that cPLA2 gene disruption impairs female reproductive ability by preventing parturition (15–16). PLA2 activities are thus involved in multiple steps of the reproductive process, and the reduced motility and fertilization competence of spermatozoa from male iPLA2–/– mice coupled with the reduction in motility of spermatozoa induced by inhibiting iPLA2 with BEL suggest that iPLA2 is a potential target for developing male contraceptive agents.
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FOOTNOTES |
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Both authors contributed equally to this work.
To whom correspondence should be addressed: Box 8127, Washington University School of. Medicine, 660 S. Euclid, St. Louis, MO 63110. E-mail: jturk{at}wustl.edu.
1 The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; iPLA2, Group VI PLA2; BEL, bromoenol lactone; ESI, electrospray ionization; MS, mass spectrometry; GPC, glycerophosphocholine; TAG, triacylglycerol; hCG, human chorionic gonadotropin; LPC, 2-lysophosphatidylcholine; ER, endoplasmic reticulum; SOC, store-operated channel.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, p < 0.01).