Hyaluronidase 6 Does Not Affect Cumulus-Oocyte Complex Dispersal and Male Mice Fertility

Abstract

Glycosylphosphatidylinositol-anchored sperm hyaluronidases (HYAL) assist sperm penetration through the cumulus-oocyte complex (COC), but their role in mammalian fertilization remains unclear. Previously, we demonstrated that sperm from HYAL 5 and 7 double-knockout (dKO) mice produced significantly less offspring than sperm from wild-type mice due to defective COC dispersal. However, the HYAL6 gene remained active in the sperm from the dKO mice, indicating that they were not entirely infertile. This study explored the role of HYAL6 in fertilization by analyzing HYAL6-mutant mice. In this mouse model, HYAL5 and HYAL7 were present in the HYAL6-knockout sperm, and they could disperse hyaluronic acid. We found that HYAL6 was present on the surface of sperm. However, male mice lacking the HYAL6 gene had normal fertility, testicular integrity, and sperm characteristics. Furthermore, in vitro fertilization assays demonstrated that HYAL6-deficient epididymal sperm functioned normally. Therefore, HYAL6 is dispensable for fertilization.

Keywords: COC dispersal; HYAL6; fertility; hyaluronidase; mouse model; sperm.

Figure 1

Chromosomal orientation and expression patterns of mouse hyaluronidase (HYAL) genes. (A) Seven genes (HYAL1 through HYAL7) are located on chromosomes six and nine. Arrows (→) indicate the protein-coding direction. TM, telomere; CM, centromere. (B) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. Mouse tissue complementary DNA was amplified by PCR, and the fragments were then stained with ethidium bromide. GAPDH; glycer-aldehyde-6-phosphate dehydrogenase.

Figure 2

Generating hyaluronidase 6 (HYAL6) knockout mice using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9. (A) Mouse HYAL6 contains four exons interrupted by three introns. The ATG translation initiation codon and stop codon are in exons two and four, respectively. F and R indicate primers for genomic PCR analysis. (B) Genomic sequence of the CRISPR target site in HYAL6. Letters inside the dotted lines indicate the target deletion. (C) Asterisks (*) indicate amino acids shared between the two sequences, and the hashtag symbol (#) indicates the stop codon.

Figure 3

Characterizing male HYAL6 knockout (KO) mice. (A) Genomic DNA polymerase chain reaction (PCR) results from wild-type (WT) and KO mice. An asterisk (*) indicates a WT band, and arrows (→) indicate Hyal6 KO. (B) Reverse transcription PCR analysis of HYAL2, HYAL5, HYAL6, HYAL7, and glycerol-3-phosphate dehydrogenase (i.e., GAPDH) using complementary DNA from WT and KO tissues. (C) Sperm-specific hyaluronan-hydrolyzing hyaluronidase activity test. Two asterisks indicate high-molecular-weight hyaluronic acid, while two arrows (→) indicate low-molecular-weight hyaluronic acid digested by sperm hyaluronidase.

Figure 4

HYAL6 KO does not affect spermatogenesis or the fertilization rate. (A) Mean litter size from HYAL6 KO male mice. mWT, male wild-type; fWT, female wild-type; mKO, male knock-out. (B) Indirect immunofluorescent analysis of capacitated epididymal sperm. The HYAL6 molecule was probed by affinity-purified anti-HYAL6 antibody in WT sperm. No significant staining was observed in the KO sperm. Sperm were also stained with 4′-6-diamidino-2-phenylindole (i.e., DAPI). The arrows (→) indicate mouse HYAL6 protein expression in WT sperm. There was no staining in HYAL6 KO sperm. (C) Hematoxylin and eosin staining of the HYAL6 KO mouse testis. Losing HYAL6 does not affect spermatogenesis or the fertilization rate.

Figure 5

Cumulus cell dispersal by HYAL6 KO sperm and sperm extracts. A. The cumulus–oocyte complexes (COCs) were incubated with KO sperm (A) and KO sperm extracts (B).

Figure 6

Zymography and in vitro fertilization assay. (A) Sperm protein fractions were prepared from wild-type mice, HYAL6 KO sperm, and HYAL5/HYAL7 double-KO (dKO) sperm; then, 30 µg of the sperm extracts were separated by 0.1% hyaluronan sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions and analyzed by zymography. (B) Fertilization ratio of intact eggs inseminated with capacitated cauda epididymal sperm from HYAL6 and dKO (open column). (C) Fertilization ratio was determined after inseminating cumulus-free eggs with capacitated cauda epididymal sperm derived from HYAL6 KO (shaded column) and dKO (open column) mice.