Serine protease PRSS55 is crucial for male mouse fertility via affecting sperm migration and sperm-egg binding

Abstract

Testis-specific PRSS55 is a highly conserved chymotrypsin-like serine protease among mammalian species. So far, the physiological function of PRSS55 remains unknown. Here, we show that PRSS55 is a GPI-anchored membrane protein, specifically expressed in adult mouse testis and mainly observed in the luminal side of seminiferous tubules and sperm acrosome. Mice deficient for Prss55 develop male infertile with normal reproduction-related parameters observed. Interestingly, in vivo fertilization rate of Prss55^-/- males is dramatically decreased, possibly due to incapable migration of Prss55^-/- sperm from uterus into oviduct. However, in vitro fertilization rate has no difference between two genotypes although Prss55^-/- sperm presents defective recognition/binding to zona-intact or zona-free oocytes. Further study reveals that mature ADAM3 is almost undetectable in Prss55^-/- sperm, while precursor ADAM3 remains unchanged in the testis. However, it is shown that ADAM3 has no interaction with PRSS55 by immunoprecipitation with anti-PRSS55 antibody. The expression levels of several proteins known to be related to the observed phenotypes remain comparable between wt and Prss55^-/- mice. Moreover, we found that Prss55 deficiency has no effect on PRSS37 or vice versa albeit two mutant males share almost the same phenotypes. Microarray analysis reveals a total of 72 differentially expressed genes in Prss55^-/- testis, most of which are associated with cellular membrane and organelle organization, protein transport and complex assembly, and response to stimulus and signaling. In conclusion, we have demonstrated that PRSS55 plays vital roles in regulating male fertility of mice, including in vivo sperm migration and in vitro sperm-egg interaction, possibly by affecting the maturation of ADAM3 in sperm and the expression of multiple genes in testis.

Keywords: Knockout mouse model; Male infertility; Serine protease PRSS55; Sperm migration; Sperm–egg recognition/binding.

Fig. 1

PRSS55 is a GPI-anchored membrane protein specifically expressed in adult mouse testis. a Tissue expression profile of Prss55 mRNA in adult mice is shown by semi-quantitative and real-time quantitative RT-PCR. Actb serves as an internal control. Bar chart shows mean ± SE values from three replicates. b Prss55 mRNA relative to Actb levels in the testis and epididymis of the mice at the indicated age. Bar chart shows mean ± SE values from three replicates. c PRSS55 protein levels in the testis and epididymis of the mice at the indicated age, and GAPDH as a loading control are shown. d The protein levels in cytoplasmic (C), membrane (M) and nuclear (N) subfractions of adult mouse testicular cells were analyzed by Western blot. GAPDH, LAMIN A/C and NaKATPASE represent cytoplasmic, nuclear and membrane fractions, respectively. e The intact testicular cells from adult mice were treated with the indicated concentrations of PI-PLC, and the supernatants were collected for Western blot analysis. uPAR was detected as a positive control for GPI-anchored protein

Fig. 2

Generation of Prss55-KO mice. a The targeting strategy for disruption of the mouse Prss55 gene. The boxes in black represent the exons with coding region. The targeting vector contains 3192 bp of 5′ and 3043 bp of 3′ homologous fragments. PGK-Neo and HSV-TK were used for positive and negative selections, respectively. P1-P7, the primers for genotyping and their relative positions, are indicated. N, NotI; H, HindIII; B, BamHI. b PCR on genomic DNA from ES cell clones was performed using the primers P1 and P2 for the 5′ arm and P3 and P4 for the 3′ arm. c Triple primer PCR on mouse tail genomic DNA was performed using primers P5-P7 as routine genotyping of mice. The expected size of PCR products (arrowheads) is shown on the right of the images. d RT-PCR for the expression of Prss55 mRNA in the testis of adult mice with three different genotypes. Actb was used as an internal control. e Western blot analysis of PRSS55 protein in the testis and epididymis of adult mice with three different genotypes. ACTB was used as a loading control

Fig. 3

Prss55-deficiency dramatically reduces fertilization rate and the defect in sperm migration into oviduct in vivo. a wt and Prss55^−/− male mice were mated with superovulated wt females and oocytes were recovered from plugged females. The numbers marked in or above each column represent the total 2-cell embryos/total oocytes obtained from several experiments. b Sperm count in the oviduct 3.5 h after coitus. The oviducts of three plugged females each group were dissected and the sperm in the oviducts were flushed out and counted under light microscope. c EGFP-expressing wt (Prss55^+/+EGFP^tg/+) and Prss55^−/− (Prss55^−/−EGFP^tg/+) males were mated with wt females. The uterus and oviduct were visualized under fluorescence microscope 3.5 h after coitus. Boxed areas in the middle images are enlarged as the bottom ones

Fig. 4

Prss55 disruption results in defective sperm–egg binding but normal fertilization rate in vitro. a Zona-intact and zona-free eggs were incubated with capacitated wt or Prss55^−/− sperm in vitro. Prss55^−/− sperm show severe defects in recognition/binding to either zona-intact or zona-free oocytes. One representative experiment out of three is shown. b IVF rate is compared between wt and Prss55^−/− sperm. The numbers indicated in each column are the total oocytes developed to 2-cell stage and the total oocytes incubated with sperm at the beginning of the fertilization assays from several experiments

Fig. 5

Prss55 deficiency impedes ADAM3 maturation with no effects on the related proteins as marked. a The total lysates of testis and sperm were subjected to immunoblot analysis using the indicated antibodies. No significant differences between wt and mutant mice in protein levels are shown. ATP5A1 and GAPDH are used as loading controls. b Western blot analysis of ADAM3 precursor and mature ADAM3 in the testis and sperm of wt, Prss55^−/− and Prss37^−/− mice. GAPDH is used as a loading control. c, d, e Testicular cell extracts from wt and Prss55^−/− mice were immunoprecipitated and probed with antibodies against ADAM3, PRSS55, and tACE. Testicular cells lysates were loaded as the input control. No evidence for the interaction of PRSS55 with ADAM3 and tACE was found

Fig. 6

Differentially expressed genes in the testis of Prss55^−/− mice identified by microarray analysis. a Total RNA extracted from the testes of three pairs of wt and Prss55^−/− mice were used for gene expression analysis using whole mouse genome 4 × 44 k microarray (Agilent Technologies). A total of 72 genes were identified to be differentially expressed significantly (p < 0.05, f_c ≥ 2), which are displayed as the heat map. b GO analysis of the 72 DE genes, which primarily clustered into 7 functional groups with varied numbers. c Fold changes of ten genes (seven up-regulated and three down-regulated genes) were examined by qRT-PCR. The housekeeping gene Actb was used for expression normalization. Bar chart shows mean ± SE values of six mice