Rescue of male infertility by human PRSS55 in transgenic mice establishes a contraceptive research model

Abstract

The development of non-hormonal male contraceptives requires validated preclinical models. This study investigated whether human orthologs of two mouse testis-specific serine proteases, PRSS55 and TMPRSS12, both essential for male fertility in mice, could functionally rescue the infertility phenotypes of their respective knockout mouse lines. We generated transgenic mouse lines expressing human PRSS55 with either an extracellularly or intracellularly positioned C-terminal 3xFLAG tag (RES GPI or RES TM), and a line expressing human TMPRSS12 with a C-terminal 3xFLAG tag (T12 RES), all on their respective mouse null backgrounds. Fertility was assessed through continuous mating trials, and sperm parameters were evaluated. Both hPRSS55 rescue lines sired offspring; RES GPI males exhibited partially restored fertility with significantly fewer pups per litter compared to controls, while RES TM males demonstrated fertility comparable to controls. In contrast, T12 RES males remained infertile, exhibiting severe defects in sperm motility and other parameters, despite confirmed transgene mRNA expression. These findings indicate that human PRSS55, particularly when tagged intracellularly, can functionally replace its mouse counterpart, validating the RES TM line as a promising model for testing human PRSS55-targeted contraceptives. The inability of hTMPRSS12 to rescue fertility highlights challenges potentially due to sequence divergence or unconfirmed protein expression levels.

Fig. 1

Developing hPRSS55 and hTMPRSS12 transgenic mouse line. (a) Graphic of the hPRSS55 with a 3XFlag tag added at the C-terminus of the protein to generate the PRSS55 transgenic mouse lines. The GPI construct has the Flag tag inserted prior to the GPI anchor making the tag extracellular, while the TM construct has the Flag tag following the transmembrane domain so that flag tag would be located intracellularly. Graphic of the hTMPRSS12 with a 3XFlag tag added at the C-terminus of the protein to generate the TMPRSS12 transgenic mouse line. (b) Graphic detailing the genotypes of the transgenic mice. (c) Genotyping of PRSS55 alleles. Primers shown in Fig. 1c amplify specific amplicons for the WT (Fw-WT x Rv-WT), KO (Fw-KO x Rv-KO), TG GPI (Fw-TG GPI x Rv-TG GPI), and TG TM (Fw-TG TM x Rv-TG TM) alleles. This gel image was cropped so only 4 wells of the gel are present, the full gel can be found in the Supplementary Fig. S3. (d) Genotyping of TMPRSS12 alleles. Primers shown in Fig. 1d amplify specific amplicons for the T12 WT (Fw-WT x Rv-WT), T12 KO (Fw-KO x Rv-KO) and T12 TG (Fw-TG x Rv-TG) alleles. This gel image was cropped so only 3 wells of the gel are present (lanes 4–6), the full gel can be found in the Supplementary Fig. S1. (e) Immunoblot analysis to confirm expression of PRSS55 in HET, KO, RES GPI, and RES TM mice in the first blot. With confirmatory blotting of Anti-FLAG in the transgenic mice in the second blot and Anti-GAPDH in all mice in the third blot. These blots were cropped so only bands between the molecular weight of 75 kDa and 35 kDa are shown, the full blots can be found in the Supplementary Fig. S4.

Fig. 2

Waveform Analysis of Prss55 KO mice and Tmprss12 KO compared to WT mice. (a) Comparison of sperm waveform between WT and Prss55 KO mice at 15- and 90-min show that KO mice appear to have a wider range of flagellar movement at 15 min compared to WT mice. (b) Comparison of sperm waveform analysis of WT and Tmprss12 KO mice at 15- and 90-min show that KO mice have what appears to be a more rigid tail movement than the wider loops of the WT mouse.

Fig. 3

CASA for RES GPI and RES TM mice at 90 min. There was no difference in sperm count (a, e), sperm motility (b, f), or progressive cells (c, g) for RES GPI or RES TM mice at 90 min. Differences were observed in hyperactivation (d, h) for RES GPI (P = 0.01) and RES TM (P < 0.0001) compared to WT mice at 90 min.

Fig. 4

CASA for T12 RES at 15 and 90 min. At 15 min differences were noted in motility (P = 0.04) (b), static cells (P = 0.04) (d), hyperactivation (P = 0.008) (e), VAP (P = 0.0007) (f), VCL (P < 0.0001) (g), VSL (P = 0.004) (h), ALH (P = 0.0009) (i), BCF (P < 0.0001) (j), LIN (P = 0.0002) (k), and WOB (P < 0.0001) (m). At 90 min T12 RES males differed from HET males in motility (P = 0.04) (o), static cells (P = 0.04) (q), hyperactivation (P = 0.0003) (r), VAP (P < 0.0001) (s), VCL (P < 0.0001) (t), VSL (P = 0.0001) (u), ALH (P = 0.0003) (v), BCF (P = 0.0017) (w), LIN (P = 0.01) (x), and WOB (P = 0.0006) (z). There were no observed differences in sperm count (a,n), progressive cells (c,p), or STR (l,y), at either time point.

Fig. 5

Prss55 transgenic mice can rescue fertility in Prss55 KO mice. (a) Average pup counts for the number of RES GPI mice differed (P = 0.0019) from the TG_HET GPI mice. Litter size was measured by the number of pups born. (b) Average litter per male per month, from natural mating of RES GPI mice compared to TG_HET GPI mice did not differ. (c) Average pup counts for the number of RES TM mice did not differ from the TG_HET TM mice. Litter size was measured by the number of pups born. (d) Average litter per male per month, from natural mating of RES TM mice compared to TG_HET TM mice did not differ. (e) The average body weight for RES GPI and RES TM mice did not differ between WT mice. (f) The average testis weight for RES TM and RES TM mice did not differ between WT mice.

Fig. 6

The Tmprss12 transgene is not able to rescue fertility in Tmprss12 KO mice. (a) Average litter per male per month, from natural mating of HET, T12 TG, and T12 RES mice. The average pup counts of T12 RES mice was 0, indicating it did not rescue fertility while the average pup counts between HET and T12 TG mice did not differ. (b) Average litter per male per month, from natural mating of HET, T12 TG, and T12 RES mice. Litter size was measured by the number of pups born. T12 RES mice had no litters and were unable to rescue fertility. (c) The average body weight from HET, T12 TG, and T12 RES mice. (d) The average testis weight from HET, T12 TG, and T12 RES mice.