The mouse zona pellucida: folliculogenesis, fertility and pre-implantation development

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Abstract

Perinatally, oocytes within the mouse ovary become surrounded by a layer of flattened granulosa cells and form primordial follicles. The subsequent accretion of the zona pellucida between the oocytes and granulosa cells provides a biochemical marker of folliculogenesis. In mice, the zona matrix is composed of three proteins (ZP1, ZP2, ZP3). Mouse lines lacking either ZP1 or ZP3 have been established and have abnormal folliculogenesis. Without ZP1, structurally defective zonae are formed resulting in decreased fecundity due to early embryonic loss. More strikingly, without ZP3, the zona matrix is absent, no 2-cell embryos are formed and females are infertile. The structural integrity of the zona matrix can be restored by substituting human homologues for the missing mouse protein and these ‘humanized’ zona matrices should prove useful in investigating the molecular basis of fertilization.

Introduction

At birth the mouse ovary contains 10 000–15 000 oocytes, which by 2 days post-partum have completed the prophase of the first meiotic division and are arrested in the dictyate stage. Perinatally, oocytes within the mouse ovary become surrounded by a layer of flattened granulosa cells and form primordial follicles. At the onset of follicular development, granulosa cells become cuboidal and proliferate to form a stratified epithelium while a morphologically distinct layer of thecal cells eventually differentiates from the gonadal stroma to define the outer boundary of the follicle. Concomitant with the onset of granulosa cell proliferation, the oocyte initiates its own growth during which time the zona pellucida is first observed and becomes a uniform matrix that surrounds the oocyte.

Section snippets

Folliculogenesis and ovulation

During the initial stages of follicular growth, oocytes growing within the follicles accumulate approximately 90 pg of poly(A+) RNA. Some mRNAs are stored in a stable, untranslated form while others are directly translated into proteins during oocyte growth (for review, see Liang and Dean, 1992). Folliculogenesis becomes gonadotropin-dependent at the late pre-antral stage when granulosa cells proliferate in response to FSH and an antral cavity forms among the cells. At approximately the same

Fertilization and early development

Fertilization takes place in the ampulla of the oviduct, usually within 16 h of ovulation. After ejaculation into the female reproductive tract, sperm undergo a poorly understood maturation process (capacitation) before reaching the ovulated egg. Acrosome intact sperm pass through the enveloping cumulus oophorus, composed of the glycosaminoglycan-rich matrix and the cumulus cells. The initial binding of sperm to the zona triggers the acrosome reaction that releases lytic enzymes from the sperm

Zona pellucida: structure and function

In mice, the zona pellucida is a well-organized matrix composed of three, major sulfated glycoproteins: ZP1, ZP2, and ZP3. These proteins are synthesized and secreted by the growing oocyte during folliculogenesis, resulting in a 7 μm thick matrix surrounding the ovulated mouse egg (for review, see Wassarman, 1988). The transcripts encoding the zona proteins accumulate coordinately during oocyte growth, representing about 1.5% of the total mRNA of the oocyte at peak levels (Epifano et al., 1995

Null mutations of the zona genes

Using targeted mutagenesis in embryonic stem cells, it has been possible to establish mouse lines with null mutations in each of the mouse zona pellucida genes. In each case, mice mutated in one zona gene continue to express the other two zona proteins. Mice lacking any one of the zona proteins exhibit abnormal folliculogenesis and varying degrees of infertility. For example, mice without ZP1 have a zona pellucida composed only of ZP2 and ZP3, but the matrix is structurally flawed (Fig. 2B).

‘Humanized’ zonae pellucidae

The absence of a zona matrix in the Zp3 null females precludes the assessment of the role of ZP3 in sperm binding to the zona pellucida. As an alternative approach, transgenesis was used to replace the endogenous mouse ZP3 protein with the conserved, but heterologous, human ZP3 protein. Normally, human sperm will not bind to the mouse zona pellucida (Bedford, 1977) and it was reasoned that if ZP3 was solely responsible for sperm binding, then human sperm would bind to the ‘humanized’ zona

Acknowledgements

We appreciate our constructive discussions with other members of the laboratory. Portions of this review have appeared elsewhere.

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