PRSS55 plays an important role in the structural differentiation and energy metabolism of sperm and is required for male fertility in mice

Abstract

Orderly and stage-specifically expressed proteins are essential for spermatogenesis, and proteases play a key role in protein activation and function. The present study aimed to investigate serine protease 55 (PRSS55), which was reported to play a role in sperm-uterotubal junction (UTJ) migration and sperm-zona pellucida (ZP) binding. We found that PRSS55 was specifically expressed in testicular spermatids and epididymal spermatozoa. By constructing knockout mice targeting all transcripts of Prss55, we demonstrated that deletion of Prss55 resulted in a serious decline of male fertility, with significantly increased sperm malformation and decreased sperm motility. In Prss55^-/- mice, increased structural abnormality, including deficient "9 + 2" microtubules, damaged peripheral dense fibre, and defective mitochondrial cristae, were found in sperm. In addition, sperm showed decreased expression of electron transfer chain molecules and lower ATP contents. These could be the potential causes of the astheno/teratozoospermia phenotype of the Prss55^-/- mice, and provided new evidence for the previously reported impaired sperm-UTJ migration. Moreover, preliminary studies allowed us to speculate that PRSS55 might function by activating type II muscle myosin in the testis, which is involved in many processes requiring motivation and cytoskeleton translocation. Thus, PRSS55 is essential for the structural differentiation and energy metabolism of sperm, and might be a potential pathogenic factor in astheno/teratozoospermia. Our results provide an additional explanation for the male sterility of Prss55^-/- mice, and further reveal the role of PRSS55.

Keywords: PRSS55; energy metabolism; male infertility; sperm motility; spermiogenesis.

Figure 1

Expression and Location of the serine protease PRSS55 in mice. (A) Detection of the expression of Prss55 mRNA in various tissues of mice, showing that it was only expressed in male reproductive organs. (B) The testicular Prss55 mRNA expression profile was tested at the indicated time points after birth, demonstrating that its expression first appeared at approximately the second week. (C) Immunofluorescence localization of PRSS55 in the adult mouse testis showed it was restrictively expressed at steps 12‐16 of spermiogenesis. Each image exhibits a stage of the seminiferous epithelial cycle, denoted by Roman numerals at the top of each image. (D) Immunofluorescence localization of the PRSS55 protein in adult mouse epididymis, showing its expression in sperm but not in epithelial cells

Figure 2

Detection of Prss55 transcripts in mice testes and generation of Prss55 gene knock out mice. (A) Schematic diagram of the possible pre transcriptional levels of Prss55. T1 is the known and confirmed transcript, and T2‐T4 are the predicted transcripts (NCBI). (B) We designed specific primer 1, which was located in exons 2 and 3, to detect all the transcripts of Prss55, primer 2 was designed to detect the T2 transcript, and PCR products were sequenced. (C) Schematic strategies for the generation of Prss55 ^−/− (Homo) mice using CRISPR/Cas9 technology. 53 bp of Prss55 were deleted from Exon 3. (D) The genotype of each Homo mouse was confirmed using PCR. (E) PRSS55 expression in the testes and epididymis of Homo mice was evaluated using immunofluorescence analysis, showing no positive signal in the elongating spermatids on one side of the tubule lumen. (F) PRSS55 expression in the epididymis of Homo mice was evaluated through immunofluorescence analysis, showing no positive signal in sperm. ① Corresponding to transcripts T1 and T2, ②‐1 and ②‐2 corresponding to transcripts T3 and T4, respectively, and ③ corresponding to transcript T2

Figure 3

Evaluation of fertility and testicular structure. (A) Each male was mated with two females. Tests were performed for litter size on wild‐type (WT) and gene knock out mice, showing the serious damage of fertility in Prss55 ^−/− (Homo) males. Data are presented as mean ± SD (n = 6). ***P < .001. (B) Morphology and size of WT and Homo mice testes showing no significant difference and the testis/body weight ratio for WT and Homo mice showing no significant difference (n = 3). (C) Haematoxylin and eosin (H&E)‐stained sections of testes from WT and Homo male mice. All the images showed normal spermatogenesis (n = 3). (D) The number of spermatogenic cells in the spermatogenic tubules of stages Ⅶ, Ⅷ and XI were counted and showed no significant difference between the WT and Homo mice (n = 3). PL: preleptotene, Z: zygotene, P: pachytene, D: diplotene, Rst: round spermatids, Est: elongated spermatids. Scale Bars, 20 μm

Figure 4

Apoptosis detection in the testis. (A) Terminal deoxynulceotidyl transferase nick‐end‐labelling (TUNEL) staining (red) in testes and the column diagram of statistics showing no significant difference (n = 3). Scale bars, 50 µm. (B) Apoptosis‐related molecules were detected in testis samples using Western blotting, the grey intensity analysis showed that their expression levels were not significantly different between the wild‐type (WT) and Prss55 ^−/− (Homo) mice (n = 3)

Figure 5

Evaluation of epididymis structure and sperm parameters. (A) The morphology and size of the epididymis, as well as the epididymis/body weight ratio, showed no significant difference between the wild‐type (WT) and Prss55 ^−/− (Homo) mice (n = 3). (B) Observing the haematoxylin and eosin (H&E)‐stained sections showed similar histological structures of the epididymis in WT and Homo mice, with no significant abnormalities. (C) Sperm count from the cauda epididymis of WT and Homo mice showed no significant differences (n = 6). (D) Percentage of motile and progressively motile sperm from Homo mice decreased significantly (n = 6). ***P < .001

Figure 6

Morphological and ultrastructural observation of sperm. (A) The sperm morphology of the two groups of mice is shown using light microscopy at low magnification (the abnormal sperm is indicated by arrows). (B) Morphological abnormality of sperm in Prss55 ^−/− (Homo) mice revealed at high magnification by light microscopy. Bar = 10 μm. (C) Statistical analysis showing that the sperm malformation rate in Prss55 ^−/− male mice was significantly increased compared with that in wild‐type (WT) mice (n = 5). (D‐E) The normal structure of WT mice sperm tail was observed under a transmission electron microscope (D: longitudinal section; E: transverse section). (F‐I) The abnormal structure of Prss55 ^−/− mice sperm under the transmission electron microscope (*: More than one set of flagellum structures contained in the same cross section; △: Absence of microtubules; ☆: Sperm head bending towards the tail; ▲: Abnormal peripheral dense fibres; ★: Lack of or blurred mitochondrial cristae).***P < .001

Figure 7

Molecular assessments of abnormal sperm structure and motility. (A) Detection of sperm ATP content showed a significant decrease in Prss55 knockout mice (Homo) compared with that in wild‐type (WT) mice (n = 3). (B) The mRNA expression levels of key markers in the mitochondrial electron transfer chain in Homo sperm were obviously lower than those in the WT mice (n = 3). (C) Detection of ODF1 in sperm samples from WT and Homo mice using Western blotting; the grey intensity analysis shows an obvious decrease in Homo mice (n = 3). *P < .05, **P < .01, ***P < .001

Figure 8

Identification of potential target substrates of PRSS55. (A) PRSS55 protein (black arrow) was successfully expressed in HEK293T cells. (B) PRSS55 and proteins that might interact with PRSS55 (red arrow) were eluted. (C) The proteins in the immunoprecipitation eluent were identified using mass spectrometry. Five proteins were found to be absent and four proteins showed decreased levels (>1.5 fold) in the experimental group compared with those in the control group