Human ZP4 is not sufficient for taxon-specific sperm recognition of the zona pellucida in transgenic mice

Abstract

The molecular basis of human fertilization remains enigmatic. Mouse models are often used to study sperm-egg recognition, but the mouse zona pellucida surrounding ovulated eggs contains three proteins (ZP1, ZP2, and ZP3) whereas the human zona contains four (ZP1, ZP2, ZP3, and ZP4). Human sperm are fastidious and recognize human but not mouse eggs. Transgenic mouse lines were established to ascertain whether human ZP4 is the sole determinant of human sperm binding. Human ZP4 expressed in transgenic mice had a molecular mass similar to the range of native protein isoforms and was incorporated into the extracellular zona matrix. Transgenic females were fertile with normal litter sizes. Mouse sperm readily recognized transgenic ovulated eggs, but human sperm did not. We conclude that human ZP4 is not sufficient to support human sperm binding to the zona pellucida in transgenic mice and that other zona proteins may be needed for human gamete recognition.

Figure 1

Transgenic mice expressing human ZP4. (A) Schematic of 11.6 kb human ZP4 gene locus composed of a 2.4 kb promoter, 8.2 kb coding region, and 1.0 kb 3′ of the last exon. Exons are indicated by Arabic numbers and PCR primers by arrowheads. (B) Tissue-specific expression of human ZP4 was determined by RT-PCR. Total RNA was isolated from the brain (Br), muscle (Mus), heart (Hrt), lung (Lun), kidney (Kid), liver (Liv), spleen (Spl), uterus (Ut), testes (Ts), and ovary (Ov) of transgenic mice. Ovarian RNA without reverse transcriptase was a negative control (C). After RT to obtain ssDNA, PCR amplification was performed with exon-specific primers (A) to distinguish ssDNA (485 bp) from genomic DNA (1052 bp). Gapdh expression (572 bp) was used as an internal control for RNA integrity and quantity. Molecular mass markers are indicated at the left. (C) In situ hybridization of normal and human ZP4 transgenic ovarian sections. Specific ³⁵S-labeled sense and anti-sense human ZP4 cRNA probes were hybridized to OCT-embedded ovarian sections from 15-day-old transgenic females. Sections were viewed with bright- and darkfield microscopy. (D) Plastic-embedded ovarian sections from 3- to 4-week-old human ZP4 transgenic and normal female mice. Sections were stained with periodic-acid Schiff’s reagent to highlight the zona pellucida (arrow), surrounding developing oocytes and counterstained with hematoxylin. (E) Immunoblot of human and mouse eggs. Lane 1 (Hu), uninseminated, immature human oocyte (2); lane 2 (Mo), normal mouse eggs (30); lane 3 (Tg), human ZP4 transgenic eggs (30); and lane 4 (Mix), mixture of lanes 1 (Hu) and 3 (Tg). Blot was probed with a MAB specific to human ZP4 and detected with HRP-conjugated secondary antibodies and chemiluminescence. Molecular weights are indicated to the left.

Figure 2

Confocal microscopy of ovulated eggs. Ovulated eggs from normal and human ZP4 transgenic females were stained with MABs specific to mouse ZP1, mouse ZP2, mouse ZP3, or human ZP4 and imaged by confocal microscopy. The zona pellucida surrounding human ZP4 transgenic eggs contained ZP4, which was absent in normal eggs.

Figure 3

Taxon-specific sperm binding to ovulated eggs. (A) Ovulated eggs and two-cell embryos from normal and human ZP4 transgenic females were incubated with capacitated epididymal mouse sperm or capacitated human sperm from fertile donors. Washes to remove nonadherent sperm were performed until three or fewer sperm were bound to control two-cell embryos. Insets are higher magnification of individual eggs from lower magnification field. Eggs, embryos, and sperm were stained with DAPI to detect DNA. (B) Same as (A), but with uninseminated, immature human oocytes.