Impaired fertility in mice deficient for the testicular germ-cell protease PC4 - PubMed (original) (raw)

. 1997 Jun 24;94(13):6842-6.

doi: 10.1073/pnas.94.13.6842.

H Tadros, N Ishida, C P Lerner, E De Lamirande, A Chen, M El-Alfy, Y Clermont, N G Seidah, M Chrétien, C Gagnon, E M Simpson

Affiliations

Impaired fertility in mice deficient for the testicular germ-cell protease PC4

M Mbikay et al. Proc Natl Acad Sci U S A. 1997.

Abstract

PC4 is a member of the proprotein convertase family of serine proteases implicated in the processing of a variety of polypeptides including prohormones, proneuropeptides, and cell surface proteins. In rodents, PC4 transcripts have been detected in spermatocytes and round spermatids exclusively, suggesting a reproductive function for this enzyme. In an effort to elucidate this function, we have disrupted its locus (Pcsk4) by homologous recombination in embryonic stem cells and have produced mice carrying the mutation. In intercrosses of heterozygous mutant mice, there was low transmission of the mutant Pcsk4 allele to the progeny, resulting in lower than expected incidence of heterozygosity and null homozygosity. The in vivo fertility of homozygous mutant males was severely impaired in the absence of any evident spermatogenic abnormality. In vitro, the fertilizing ability of Pcsk4 null spermatozoa was also found to be significantly reduced. Moreover, eggs fertilized by these spermatozoa failed to grow to the blastocyst stage. These results suggest that PC4 in the male may be important for achieving fertilization and for supporting early embryonic development in mice.

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Figures

Figure 1

Figure 1

(A) Disruption of the Pcsk4 gene. Vertical boxes represent exons, the hatched area an acceptor splice site, and short horizontal bars DNA probe locations. B, _BstZ_I; X, _Xho_I; S, _Sph_I. (B and C) Southern blot genotyping of ES cell clones (B) and mouse tail tips (C) with a 3′ (B Upper and C) or the 5′ probe (B Lower). Open circles, solid circles, and asterisks identify the wild-type allele, the mutant allele, and recombinant clones, respectively. L stands for 1-kb ladder. (D) Northern blot analysis of testicular RNA of wild-type (+/+), heterozygous mutant (−/+), and homozygous mutant (−/−) mice for PC4 mRNA (Upper) and 18S rRNA (Lower). (E) Western blot analysis of epididymal sperm. The two immunoreactive proteins of about 52 and 23 kDa detectable in wild-type and heterozygote mouse samples are PC4 related. They were not observed after the antiserum had been preabsorbed with purified PC4 (not shown), unlike the two proteins of about 40 and 44 kDa detectable in all three samples, which are likely due to a nonspecific immunoreaction.

Figure 2

Figure 2

(A) Toluidine blue stained semi-thin sections (×1,000) of seminiferous tubes from normal control (+/+) and Pcsk4 null mice (−/−). Arrows point at some pachytene spermatocytes (SP), round (rS) and elongated (eS) spermatids. (B) Staining for β-gal activity in the testis of wild-type (+/+) and mutant (−/−) mice. The reaction dark product is associated with testicular tubules.

Figure 3

Figure 3

In vitro fertilizing ability of spermatozoa and their capacity to support embryo development. Spermatozoa from 4 wild-type and 4 PC4-null mice were studied in 4 separate experiments involving 283 and 250 wild-type B6 oocytes, respectively. Significance was determined by χ2 test. The differences between the two genotypes were significant in all cases (P < 0.0001).

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