Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice
Hiroyasu Sato, … , Ichiro Kudo, Makoto Murakami
Hiroyasu Sato, … , Ichiro Kudo, Makoto Murakami
Published April 26, 2010
Citation Information: J Clin Invest. 2010;120(5):1400-1414. https://doi.org/10.1172/JCI40493.
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Research Article

Group III secreted phospholipase A2 regulates epididymal sperm maturation and fertility in mice

Abstract

Although lipid metabolism is thought to be important for the proper maturation and function of spermatozoa, the molecular mechanisms that underlie this dynamic process in the gonads remains incompletely understood. Here, we show that group III phospholipase A2 (sPLA2-III), a member of the secreted phospholipase A2 (sPLA2) family, is expressed in the mouse proximal epididymal epithelium and that targeted disruption of the gene encoding this protein (Pla2g3) leads to defects in sperm maturation and fertility. Although testicular spermatogenesis in Pla2g3–/– mice was grossly normal, spermatozoa isolated from the cauda epididymidis displayed hypomotility, and their ability to fertilize intact eggs was markedly impaired. Transmission EM further revealed that epididymal spermatozoa in Pla2g3–/– mice had both flagella with abnormal axonemes and aberrant acrosomal structures. During epididymal transit, phosphatidylcholine in the membrane of Pla2g3+/+ sperm underwent a dramatic shift in its acyl groups from oleic, linoleic, and arachidonic acids to docosapentaenoic and docosahexaenoic acids, whereas this membrane lipid remodeling event was compromised in sperm from Pla2g3–/– mice. Moreover, the gonads of Pla2g3–/– mice contained less 12/15-lipoxygenase metabolites than did those of Pla2g3+/+ mice. Together, our results reveal a role for the atypical sPLA2 family member sPLA2-III in epididymal lipid homeostasis and indicate that its perturbation may lead to sperm dysfunction.

Authors

Hiroyasu Sato, Yoshitaka Taketomi, Yuki Isogai, Yoshimi Miki, Kei Yamamoto, Seiko Masuda, Tomohiko Hosono, Satoru Arata, Yukio Ishikawa, Toshiharu Ishii, Tetsuyuki Kobayashi, Hiroki Nakanishi, Kazutaka Ikeda, Ryo Taguchi, Shuntaro Hara, Ichiro Kudo, Makoto Murakami

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Figure 1

Targeted disruption of the Pla2g3 gene.

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Targeted disruption of the Pla2g3 gene. (A) Expression of transcript...
(A) Expression of transcripts for Pla2g3 in various tissues of male C57BL/6 mice was assessed using RT-PCR. After electrophoresis of the PCR products in agarose gels with ethidium bromide (top panel), the intensities of the band for Pla2g3, relative to those for Gapdh, were quantified using a densitometer (CS analyzer 2.0; mean ± SD; n = 3) (bottom panel). (B) RT-PCR of Pla2g3 mRNA expression in male and female reproductive organs from C57BL/6 mice. (C) Immunoblotting of sPLA2-III protein in sperm and fluid from the cauda epididymidis of C57BL/6 mice. Arrow indicates a fully processed sPLA2-III. (D) Schematic representation of the Pla2g3 gene, targeting vector, and mutated Pla2g3 allele. Details of the construction of the targeting vector are given in Methods. Arrows indicate positions of primers for PCR genotyping. Boxes indicate exons of the Pla2g3 gene. Large arrows indicate the 5.5-kb long-arm and 1.7-kb short-arm genomic fragments of the Pla2g3 gene. Small arrows indicate the positions of primers used for PCR genotyping (III-F, WT-R, and MT-R indicate Pla2g3 WT forward primer, WT reverse primer, and mutant reverse primer, respectively). “X” indicates the regions where homologous recombination occurs. A bold bar shows a region used as a probe for Southern blotting. (E) Genotyping using PCR of samples from mouse tails. The WT and mutant alleles gave 375-bp and 627-bp amplified fragments, respectively. (F) Confirmation by RT-PCR of the absence of Pla2g3 mRNA in the testis and epididymis of nullizygous mice. (G) PLA2 enzymatic activity in cauda epididymal fluid of Pla2g3–/– mice and their littermates. (H) PLA2 enzymatic activity in cauda epididymal fluid of Pla2g10–/– mice and their littermates. (G and H) Values are mean ± SD; n = 4. *P < 0.05.