Sperm hyaluronidase is critical to mammals' fertilization for its ability to disperse cumulus-oocyte complex layer

Abstract

Glycosylphosphatidylinositol-anchored sperm hyaluronidases have long been believed to assist in sperm penetration through the cumulus-oocyte complex (COC); however, their role in mammalian fertilization remains unclear. Previously, we have shown that hyaluronidase 5 (Hyal5)/Hyal7 double-knockout (dKO) mice produce significantly fewer offspring than their wild-type (WT) counterparts because of defective COC dispersal. Male infertility is mainly caused by a low sperm count. It can be further exacerbated by the deficiency of sperm hyaluronidase, which disperses the cumulus cells of the outer layer of the COC. In the current study, we evaluated the effects of a low count of Hyal-deficient sperm and conditions of ovulated oocytes on the fertilization rate using a mouse model. Our results demonstrated that a low sperm count further decreases the in vitro fertilization (IVF) rate of Hyal-deficient dKO spermatozoa. In addition, the dKO spermatozoa resulted in a fertilization rate of 12.5% upon fertilizing COCs with a thick cumulus layer, whereas the IVF rate was comparable to that of WT spermatozoa when oocytes with a thin or no cumulus layer were fertilized. Finally, we proved that the IVF rate of dKO spermatozoa could be recovered by adding rat spermatozoa as a source of sperm hyal. Our results suggest that a deficiency of proteins involved in fertilization, such as sperm hyal, has a vital role in fertilization.

Keywords: HYAL5; HYAL7; cumulus–oocyte complex; fertilization; infertility; sperm hyaluronidase.

Figure 1

Fertilized oocytes following IVF by different sperm numbers from wild-type (WT) and Hyal5/Hyal7 dKO mice. Cauda epididymidal spermatozoa at different numbers (1.5 × 10⁶, 1.5 × 10⁵, and 1.5 × 10⁴ cells) from WT and Hyal5/Hyal7 dKO mice were incubated with COCs in a 200 μl TYH drop for 6 h. (a) Three types of oocytes were stained with Hoechst 33342 dye. The presence of two pronuclei indicated fertilization, whereas a single pronucleus (haploid) in the oocyte indicated that a spermatozoon did not enter the oocyte. Scale bar = 50 μm. (b) The presence of oocytes with two pronuclei were defined as “fertilized oocytes”. The oocytes with two pronuclei are indicated by asterisks. Pronuclei were stained with Hoechst 33342 dye (blue). Scale bar = 100 μm. (c) Rate of fertilization. Significant (P < 0.05) differences are indicated by an asterisk. The results of IVF were expressed as the mean ± s.e.m. of three separate experiments. Hyal5: hyaluronidase 5; IVF: in vitro fertilization; WT: wild-type; dKO: double-knockout; COCs: cumulus–oocyte complexes; s.e.m.: standard error of mean.

Figure 2

IVF by hyaluronidase-deficient mouse spermatozoa and various types of COCs. (a) Criterion for determining the status of mouse oocytes based on the surrounding cumulus cell mass. The status of the COCs was categorized as follows: type I, the oocytes were tightly packed with cumulus cells; type II, the oocytes had a loosely associated cumulus mass but still had many cumulus cells; type III, the oocytes had a few cumulus cells; and type IV, the oocytes completely lost cumulus cells (naked eggs). Scale bar = 100 μm. (b) Two pronuclei are indicated by asterisks. Pronuclei were stained with Hoechst 33342 (blue). Scale bar = 100 μm. (c) IVF assay of Hyal5/Hyal7 dKO spermatozoa. The fertilization rate of four types of COCs inseminated with capacitated cauda epididymidal spermatozoa from WT (black column) and Hyal5/Hyal7 dKO (shaded column) mice is shown. Significant (P < 0.05) differences are indicated by an asterisk. The results of IVF are expressed as the mean ± s.e.m. of three separate experiments. Hyal5: hyaluronidase 5; IVF: in vitro fertilization; WT: wild-type; dKO: double-knockout; COCs: cumulus–oocyte complexes; s.e.m.: standard error of mean.

Figure 3

IVF using Hyal5/Hyal7 dKO spermatozoa mixed with rat spermatozoa. Caudal epididymidal sperm of WT mice (black column), Hyal5/Hyal7 dKO mice (open column), Hyal5/Hyal7 dKO mice and rat spermatozoa (mosaic column), and only rat spermatozoa (gray) were incubated with mouse oocytes for 6 h. (a) The oocytes containing female and male pronuclei were defined as “fertilized oocytes”. Scale bar = 50 μm. Pronuclei (indicated by an asterisk) were stained with Hoechst 33342 (blue). (b) The fertilization rate. Significant (P < 0.05) differences are indicated with an asterisk. Hyal5: hyaluronidase 5; IVF: in vitro fertilization; WT: wild-type; dKO: double-knockout.

Figure 4

Competitive fertilization of COCs with a mixed population of WT and Hyal5/Hyal7 dKO spermatozoa. PCR analysis of the genomic DNA obtained from the embryos resulting from in vitro fertilization. Metaphase II-arrested mouse oocytes with intact cumulus cells were incubated with an equally mixed suspension of WT and Hyal5/Hyal7 dKO mouse spermatozoa. After initial incubation for 6 h, the fertilized oocytes were further incubated for 96 h. Genomic DNA was prepared from each of the developing embryos and then used as a template for PCR amplification. Two DNA fragments of 523 bp and 212 bp, originating from the WT and Hyal5/Hyal7 KO alleles, respectively, were detected by 1.2% agarose gel electrophoresis. Hyal: hyaluronidase; WT: wild-type; dKO: double-knockout; COCs: cumulus–oocyte complexes.