Function of the acrosomal matrix: zona pellucida 3 receptor (ZP3R/sp56) is not essential for mouse fertilization

Abstract

In mammalian fertilization, sperm-zona pellucida binding is considered to be a critical aspect of gamete interaction. In this study, we examine the mouse sperm acrosomal matrix protein zona pellucida 3 receptor (ZP3R; formerly called sp56) because of our interest in defining the function of the acrosomal matrix, the particulate compartment within the sperm secretory acrosome. Using targeted deletion of the Zp3r gene by homologous recombination, we examined the fertility of nullizygous animals. Our experiments showed that males and females homozygous for the affected gene exhibited no differences in litter sizes compared to wild-type and heterozygous animals. Testis weights of nullizygous males were equivalent to those of wild-type and heterozygous males, and no differences in the number of sperm produced by mice of three genotypes were found. In vitro fertilization rates using cumulus-intact and cumulus-free oocytes were also equivalent. Examination of sperm-binding zonae of unfertilized eggs and the ability of the sperm to undergo acrosomal exocytosis in response to calcium ionophore A23187 displayed no differences between wild-type, heterozygous, and nullizygous mouse sperm. These results provide further evidence that either ZP3R is not involved in sperm-zona pellucida binding or this process might be functionally redundant, involving multiple proteins for gamete interactions.

FIG. 1.

Targeted deletion of Zp3r gene. A) Diagram of the vector construct. B) Genotyping results. C) Expression of Zp3r gene in different tissues. B, brain; H, heart; K, kidney; Li, liver; Lu, lung; O, ovary; S, spleen; T, testis; Th, thyroid gland; U, uterus.

FIG. 2.

Expression of ZP3R in wild-type, heterozygous, and nullizygous male mice. A) Testis weight (n = 8) and sperm count and motility. B) Northern blot analysis of Zp3r mRNA from testis of wild-type and Zp3r-null mice. C) Immunoblotting analysis of ZP3R protein in wild-type and Zp3r-null mice. D) Indirect immunofluorescence analysis of ZP3R protein in wild-type versus targeted-deletion mice. Still frames from Supplemental Movies 1–4 illustrating sperm motility from Zp3r^+/+ and Zp3r^−/− males after the incubation under capacitating conditions for 15 min (E and F, respectively [Movies 1 and 2]) and 120 min (G and H, respectively [Movies 3 and 4]) are shown. Original magnification ×400 (D) and ×40 (E–H).

FIG. 3.

Fertilizing capacity of ZP3R-deficient sperm. Sperm from Zp3r^+/+, Zp3r^+/−, and Zp3r^−/− were used to inseminate cumulus-intact (A) or cumulus-free (B), metaphase II-arrested eggs using in vitro fertilization. Results are expressed as the mean ± SD of four independent experiments. No statistical differences were observed between groups (P > 0.05).

FIG. 4.

Binding of wild-type, heterozygous, and nullizygous mouse sperm to ZP. Mouse sperm from Zp3r^+/+, Zp3r^+/−, and Zp3r^−/− males were incubated with metaphase II-arrested eggs for 30 min to promote adhesion to the ZP. Four independent experiments with more than 200 oocytes were scored per antibody. No statistical differences were observed between groups (P > 0.05).

FIG. 5.

Acrosomal exocytosis in ZP3R-deficient sperm. Mouse sperm from Zp3r^+/+, Zp3r^+/−, and Zp3r^−/− males were incubated under capacitating conditions for 180 min. Then, calcium ionophore A23187 (final concentration, 1 μM) was added, and the cells were incubated for an additional 15 min. Next, cells were washed with PBS and observed under the fluorescence microscope for the presence of soluble green fluorescent protein in their acrosomes. Results are expressed as the mean ± SD of four independent experiments. No statistical differences were observed between groups (P > 0.05).