Fig 2: Fertilizing ability and motility analysis of spermatozoa from Tmprss12 del/del mice. (A) Average numbers of ZP binding spermatozoa in vitro. The number of ZP binding spermatozoa in Tmprss12del/del mice (2.49 ± 1.07 spermatozoa), and Adam3−/− mice (9.40 ± 3.23 spermatozoa) were significantly reduced compared to that of WT mice (32.93 ± 10.43 spermatozoa). *P < 0.05. (B) IVF rates in TYH using spermatozoa from control, Adam3−/−, and Tmprss12del/del mice. Average fertilization rates of control, spermatozoa from Adam3−/−, and Tmprss12del/del mice were 93.7 ± 6.0%, 85.9 ± 5.7%, and 0%, respectively. Spermatozoa from WT and Tmprss12del/wt mice were used as controls. *P < 0.05, **P < 0.01. (C) Computer-assisted sperm analysis motility parameters; (left to right) average path velocity (VAP), curvilinear velocity (VCL), straight line velocity (VSL). Spermatozoa were incubated in capacitation medium (TYH) and motility measured at 10 min (uncapacitated) and 120 min (capacitated) to assess motility before and after capacitation. More than 200 spermatozoa were analyzed for each male. Spermatozoa from WT, Tmprss12d8/wt, and Tmprss12del/wt mice were used as controls. Tmprss12del/del KO sperm velocity decreased compared to those of controls and Adam3 KO males. *P < 0.05, **P < 0.01. (D) In vitro fertilization rates spermatozoa from Tmprss12del/wt and Tmprss12del/del mice in CARD medium. Average fertilization rates of spermatozoa from Tmprss12del/wt and Tmprss12del/del mice were 98.8 ± 1.0 and 58.2 ± 19.7, respectively.

Fig 3: 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 4: Intron Retention Is a Key Feature of the Male Meiotic Transcriptome(A) Bar graph showing percentages of events annotated in FAST-DB (dashed, light gray columns) and of those differentially regulated between spermatocytes (sp.cytes) and spermatids (sp.tids) (black columns) within each AS pattern. p Values above the graph indicate a significant enrichment for regulated events within specific AS pattern in our dataset compared with the reference database (modified Fisher's test). Red box highlights the IR pattern.(B) Bar graph representing percentages of up- and downregulated events among exonic and intronic splicing events. Enrichment in upregulated events in IR with respect to all other AS events was statistically significant (p = 4.9 × 10−61; χ2 test).(C) (Top) Visualization of the RNA-seq reads profile of the intron-retaining genes Adam3, Adam18, and Stag3 in sp.cytes (upper graph) and sp.tids (lower graph). Sequence reads (vertical gray lines), exons (blue boxes), and introns (horizontal lines) are shown. (Bottom) Bar graphs showing results of qPCR analyses for the expression of the retained and non-retained introns (red and black dashed boxes, respectively) relative to their flanking exons in sp.cytes and sp.tids (mean ± SD, n = 3; ∗p ≤ 0.05, ns = not significant; one-way ANOVA).(D) Bar graphs showing results of qPCR analyses for the expression of indicated introns relative to spliced product of their flanking exons in sp.cytes, and round and elongated sp.tids (mean ± SD, n = 4; ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; ns, not significant; one-way ANOVA).See also Figures S2 and S3.

Fig 5: SPACA4 is required for efficient fertilization in male mice. (A and B) Overview of the C57BL/6J-Spaca4 knockout alleles generated by CRISPR-Cas9–mediated targeted mutagenesis. One allele contains a 117-nt in-frame deletion after amino acid 42. The other allele contains a 77-nt out-of-frame deletion after amino acid 47. (A) Schematic of the wild-type and knockout alleles. Yellow dashed lines indicate the site of the deletions. Predicted disulfide bridges are indicated in gray. SP, signal peptide; TM, transmembrane region. (B) Genotyping of Spaca4 knockout mice. Spaca4 PCR products are separated based on their sizes on an agarose gel. (C) Spaca4 knockout male mice are subfertile. Litter sizes of C57BL/6J-Spaca4 wild-type (+/+), heterozygous (+/−), and transheterozygous (−/−) males caged with B6129F1 wild-type females or B6129F1 wild-type males caged with transheterozygous (−/−) females. Successful mating was confirmed by plug checks. Data are means ± SD. ***P < 0.0001 (Kruskal–Wallis test with Dunn multiple-comparisons test); n.s., not significant. n = number of litters; m = number of male mice tested. (D) Sperm morphology is normal in the absence of SPACA4 protein. DIC, differential interference contrast image. The sperm heads (boxed areas) are shown below at higher magnification. Immunostaining of sperm detects SPACA4 protein (red) under permeabilizing conditions in the head of sperm from wild-type (+/+) mice but not in sperm from Spaca4 knockout (−/−) mice. DAPI (cyan) staining labels the sperm nucleus. (Scale bars, 10 µm.) (E) Sperm from Spaca4 knockout mice has a severely reduced ability to fertilize wild-type oocytes. IVF of oocytes from C57BL/6J wild-type females using sperm from C57BL/6J-Spaca4 wild-type (+/+), heterozygous (+/−), or transheterozygous (−/−) males. Plotted is the percentage of two-cell stage embryos as a measure of successful fertilization. Data are means ± SD. *P = 0.014 (Kruskal–Wallis test with Dunn multiple-comparisons test); n.s., not significant. n = total number of oocytes; m = number of males tested. (F) IZUMO1 and ADAM3 proteins are expressed and processed normally in sperm and testis from Spaca4 knockout mice. Western blot analysis showing the absence of SPACA4 protein in testes and spermatozoa of Spaca4 knockout mice (Left) and expression and processing of ADAM3 and IZUMO1 proteins similar to wild-type controls. Testes and spermatozoa of wild-type mice, as well as BASIGIN protein, which undergoes proteolytic cleavage during sperm maturation (7), are used as control. (G) IZUMO1 localization is normal in Spaca4 knockout mice. Immunostaining of sperm under permeabilizing conditions shows normal localization of IZUMO1 (green) in acrosome-intact wild-type (+/+) and SPACA4-deficient (−/−) sperm and normal relocalization of IZUMO1 in AR sperm (asterisk) in both genotypes. BF, brightfield image. (Scale bars, 10 µm.) (H) Quantification of the percentage of AR sperm 2 or 4 h after incubation of capacitated sperm. Acrosome reaction was assessed by immunostaining for IZUMO1. Data are means ± SD; n.s., not significant. n = total number of sperm; m = number of males tested.