OBJECTIVE: The mechanisms of sperm binding to the zona pellucida (ZP) of ovulated eggs remain unclear. Mouse models are often used to study sperm-egg recognition, but the mouse zona contains three proteins (ZP1, ZP2, ZP3) whereas the human zona contains four (ZP1, ZP2, ZP3, ZP4). Human sperm are fastidious and bind to human but not mouse eggs. We sought to determine if human ZP4 expressed in transgenic mice would be sufficient to support human sperm binding and provide an explanation of the observed taxon-specificity of fertilization. DESIGN: Transgenic mouse model. MATERIALS AND METHODS: Human ZP4 genomic DNA containing the coding sequence and 5' promoter (12.4kb) was isolated by DNA recombineering. Purified DNA was microinjected into one-cell embryos and transgenic founder lines established. Ovulated eggs and embryos (negative control) from transgenic mice were incubated with human sperm from fertile male donors. Persistent sperm binding was assessed after fixation and imaging with confocal microscopy. The assay was performed in triplicate with three separate human sperm samples. RESULTS: The human ZP4 transgene was integrated into the mouse genome as confirmed by PCR and Southern blot. Ovary-specific expression was confirmed by RT-PCR and human ZP4 was detected in the zona matrix by confocal microscopy. Recombinant ZP4 on western blot had a similar molecular mass (65-70kDa) as native ZP4 from discarded human oocytes. Transgenic females were fertile with normal litter sizes. Mouse sperm bound readily to transgenic ovulated eggs (36.4 +/- 3.5 SEM/egg, n=27), but human sperm bound little or not at all (1.5 +/- 0.34 SEM/egg, n=36). CONCLUSIONS: Human ZP4 alone is not sufficient to support human sperm binding in transgenic mice. Previous research in transgenic mice demonstrated similar results with human ZP2 and ZP3. Thus, the presence of all four human zona proteins may be required to form a structure recognized by human sperm. If successful, such a model would be useful in the clinical study of infertility.

Perinatally, mouse oocytes become surrounded by a single layer of flattened granulose cells to form primordial follicles that represent the full complement of germ cells available to the female. Subsequent, yet to be defined signals, select cohorts to enter into a growth phase after which the eggs undergo meiotic maturation and are ovulated into the oviduct. Exploiting the appearance of the zona pellucida as a biochemical marker of folliculogenesis, a transcription factor, FIGalpha (Factor In the Germline, alpha) required for zona gene expression has been identified. Mice lacking FIGa do not form primordial follicles and are sterile. Additional gene products expressed by the oocytes are required for follicular growth and meiotic progression to metaphase II. After ovulation, eggs are fertilized in the ampulla of the oviduct by capacitated sperm. Although the molecular biology of the zona pellucida is increasingly well understood, the basis of sperm binding to the zona matrix remains enigmatic. Using targeted mutagenesis and transgenesis, mouse lines have been established that lack each of the three mouse zona proteins (Zp1, Zp2, Zp3 null mice) or contain human ZP2, human ZP3 or both in lieu of the endogenous mouse proteins (huZP2, huZP3, huZP2/ZP3 rescue mice). Normally following fertilization, ZP2 is cleaved, sperm no longer bind to embryos, and there is an effective block to sperm penetration of the zona pellucida. Surprisingly, mice with zonae containing human proteins (human ZP2, human ZP3 or both) remain fertile, but do not support human sperm binding. Also unexpected, mouse sperm continue to bind at the two-cell stage to chimeric zonae containing human ZP2, a phenotype that correlates with intact ZP2. These observations support a model in which sperm bind to a three-dimensional zona structure formed ad minimum by ZP2/ZP3, and the cleavage of ZP2 following fertilization alters the matrix so that it is no longer permissive for sperm binding.

After colonization of the gonad, mouse female germ cells enter into the prophase of the first meiotic division as a mid-gestational hallmark of gender. Perinatally, oocytes interact with granulosa cells to form primordial follicles which, with cyclic periodicity, enter into a 3-week growth phase that culminates in meiotic maturation and ovulation. Successful fertilization in the oviduct results in the onset of embryogenesis. Genes expressed in oocytes encode maternal factors that control many of these developmental processes. The establishment of mouse models in which specific genes have been disrupted offers robust insights into molecular mechanisms that control oogenesis, folliculogenesis, fertilization and early embryogenesis. Although relatively few developmental circuits have been characterized in genetic detail, the ongoing revolution in mouse genetics holds great promise. These model systems provide novel information into the molecular basis of the pathways required for oocyte-specific processes as well as for interactions with the temporally changing environment of female germ cells. The similarities between the mouse and human genomes provide assurance that this knowledge will rapidly translate into a better understanding of human reproduction.

Gpbox is a paired-like homeobox gene that colocalizes with two other members of the family, PsxI and Pem, on the proximal portion of the mouse X chromosome. Gpbox is expressed in the extraembryonic placenta and within the germ cells of the embryonic gonad. Beginning with the onset of sexual dimorphism (embryonic day [E]11.5 to 12.5), GPBOX transcripts accumulate faster in female than in male germ cells but disappear later in embryogenesis (E16) and have not been reported in adult tissues. To investigate the function of Gpbox, mouse cell lines lacking GPBOX were established using targeted mutagenesis in embryonic stem cells. Both homozygous Gpbox null female and hemizygous Gpbox null male mice were fertile and reproduced normally. Additionally, the development of male and female gonads in the null background was indistinguishable from that observed in normal littermates. The lack of an obvious phenotype raises the possibility that another member of this homeobox gene family provides the absent Gpbox function.

All vertebrate eggs are surrounded by an extracellular matrix. This matrix is known as the zona pellucida in mammals and is critically important for the survival of growing oocytes, successful fertilization and the passage of early embryos through the oviduct, The mouse zona pellucida is composed of three glycoproteins (ZP1, ZP2 and ZP3), each encoded by a single copy gene, Using targeted mutagenesis in embryonic stem cells, Zp2-null mouse lines have been established, ZP1 and ZP3 proteins continue to be synthesized and form a thin zona matrix in early follicles that is not sustained in pre-ovulatory follicles, The abnormal zona matrix does not affect initial folliculogenesis, but there is a significant decrease in the number of antral stage follicles in ovaries isolated from mice lacking a zona pellucida, Few eggs are detected in the oviduct after stimulation with gonadotropins, and no two-cell embryos are recovered after mating Zp2-null females with normal male mice. The structural defect is more severe than that observed in Zp1-null mice, which have decreased fecundity, but not quite as severe as that observed in Zp3-null mice, which never form a visible zona pellucida and are sterile. Although zona-free oocytes matured and fertilized in vitro can progress to the blastocyst stage, the developmental potential of blastocysts derived from either Zp2- or Zp3-null eggs appears compromised and, after transfer to foster mothers, live births have not been observed. Thus, in addition to its role in fertilization and protection of early embryos, these data are consistent with the zona pellucida maintaining interactions between granulosa cells and oocytes during folliculogenesis that are critical to maximize developmental competence of oocytes.

Primordial follicles are formed perinatally in mammalian ovaries and at birth represent the lifetime complement of germ cells. With cyclic periodicity, cohorts enter into a growth phase that culminates in ovulation of mature eggs, but little is known about the regulatory cascades that govern these events. FIGalpha, a transcription factor implicated in postnatal oocyte-specific gene expression, is detected as early as embryonic day 13. Mouse lines lacking FIGalpha were established by targeted mutagenesis in embryonic stem cells. Although embryonic gonadogenesis appeared normal, primordial follicles were not formed at birth, and massive depletion of oocytes resulted in shrunken ovaries and female sterility. Fig(&agr;) (the gene for FIGalpha null males have normal fertility. The additional observation that null females do not express Zp1, Zp2 or Zp3 indicates that FIGalpha plays a key regulatory role in the expression of multiple oocyte-specific genes, including those that initiate folliculogenesis and those that encode the zona pellucida required for fertilization and early embryonic survival. The persistence of FIGalpha in adult females suggests that it may regulate additional pathways that are essential for normal ovarian development.

XX gonads differentiate into ovaries, a morphologic event evident by embryonic day 13.5 (E13.5) in mice. To identify early markers of oogenesis, sex-specific urogenital ridge cDNA libraries were constructed from E12–13 embryos. After mass excision and isolation of plasmid DNA, approximately 4800 expressed sequence tags were determined and compared to existing databases. Few cDNAs were specifically expressed in the urogenital ridge, but one, designated GPBOX, encodes a 227-amino-acid homeobox protein that is first expressed at E10.5 in the embryo as well as in the extraembryonic tissues. The Gpbox gene is single copy in the mouse genome and is located on the X chromosome in close proximity to two other homeobox genes, Pem and Psx1. Within the embryo, its expression is limited to the gonad, and transcripts are not detected in adult tissues. Although comparable levels are initially present in both sexes, GPBOX transcripts accumulate faster in female germ cells and peak at E12.5 when they are present in fivefold greater abundance than in males. The persistence of GPBOX transcripts in female germ cells until E15.5 and their virtual disappearance in males by E13.5 suggest that Gpbox may play a role in mammalian oogenesis.

All vertebrates have an egg shell that surrounds ovulated eggs and plays critical roles in gamete recognition. This extracellular matrix is known as the zona pellucida in eutherian mammals and consists of three glycoproteins, ZP1, ZP2 and ZP3 in the mouse. To investigate the role of ZP1 in fertilization and early development, we have used targeted mutagenesis in embryonic stem cells to create mouse lines (Zp1(tm/tm)) lacking ZP1. Although a zona pellucida composed of ZP2 and ZP3 was formed around growing Zp1(tm/tm) oocytes, the matrix was more loosely organized than zonae around normal oocytes. In some Zp1 null follicles, this structural abnormality resulted in ectopic clusters of granulosa cells, lodged between the zona matrix and the oolemma, that perturbed normal folliculogenesis. Comparable numbers of eggs were ovulated from Zp1 null females and normal females following hormonal stimulation. However, after mating with males, fewer two-cell embryos were recovered from Zp1 null females, and their litters were significantly smaller than those produced by normal mice. Therefore, although mouse ZP1 is not essential for sperm binding or fertilization, it is required for the structural integrity of the zona pellucida to minimize precocious hatching and reduced fecundity.

Infertility, defined as the inability to conceive after 1 year of unprotected intercourse, affects 1 in 10 couples in the United States (Chandra and Stephen 1998). The etiologies of infertility are diverse. Although many causes, including inflammatory tubal disease, ovulatory dysfunction, azoospermia, and immotile cilia syndrome, are well established, the role of other factors, such as endometriosis, uterine fibroids, and varioceles, is surrounded by intense controversy. During initial clinical appraisals, each partner must be thoroughly evaluated, both because male and female factors contribute roughly equally to the etiology and because more than one cause of infertility may be present.

The mouse zona pellucida is composed of three glycoproteins, ZP1, ZP2 and ZP3, encoded by single-copy genes whose expression is temporally and spatially restricted to oocytes. All three proteins are required for the formation of the extracellular zona matrix and female mice with a single disrupted zona gene lack a zona and are infertile. An E-box (CANNTG), located approximately 200 bp upstream of the transcription start sites of Zp1, Zp2 and Zp3, forms a protein-DNA complex present in oocytes and, to a much lesser extent, in testes. It has been previously shown that the integrity of this E-box in Zp2 and Zp3 promoters is required for expression of luciferase reporter genes microinjected into growing oocytes. The presence of the ubiquitous transcription factor E12 in the complex was used to identify a novel basic helix-loop-helix protein, FIGalpha (Factor In the Germline alpha) whose expression was limited to oocytes within the ovary. The ability of FIGalpha to transactivate reporter genes coupled to each of the three mouse zona promoters in heterologous 10T(1/2) embryonic fibroblasts suggests a role in coordinating the expression of the three zona pellucida genes during oogenesis.

Mammalian oocytes synthesize and secrete a zona pellucida that surrounds the growing oocytes, ovulated eggs and preimplantation embryos. The extracellular zona matrix is composed of three glycoproteins (ZP1, ZP2, ZP3) that are involved in folliculogenesis, species-specific fertilization, and passage of the early embryo down the oviduct. We have established a mouse line in which Zp3 has been inactivated by homologous recombination with an insertional mutation. Neither Zp3 transcripts nor ZP3 protein was detected in female mice homozygous for the mutation (Zp3-/-), whereas both ZP1 and ZP2 were present in mutant oocytes. Homozygous mutant Zp3-/- mice had follicles with germinal-vesicle-intact oocytes but that lacked a zona pellucida matrix and had a disorganized corona radiata. Although mutant oocytes underwent germinal vesicle breakdown (GVBD) prior to ovulation, the cumulus-oocyte complex was markedly disrupted and the oocytes were often separate from the cumulus cells. After hormone-induced ovulation, cumulus masses were present in the oviducts of homozygous mutant mice, but zona-free eggs were observed in only half of the females and, in these, less than 10% of the normal number [correction of mumber] of eggs were detected. No zona-free 2-cell embryos were recovered from homozygous mutant Zp3-/- female mice after mating with males proven to be fertile, and none became visibly pregnant or produced offspring. These results demonstrate that a genetic defect in a zona pellucida gene causes infertility and, given the conserved nature of the zona pellucida, a similar phenotype is expected in other mammals.

The mammalian zona pellucida is an extracellular matrix that surrounds growing oocytes, ovulated eggs and early embryos. The mouse zona is composed of three sulfated glycoproteins: ZP1, ZP2 and ZP3. Each is critically involved in fertilization, the postfertilization block to polyspermy and protection of the preimplantation embryo. We have previously isolated cDNAs encoding mouse ZP2 and ZP3 and now report the isolation of a full-length cDNA encoding ZP1. Mouse ZP1 is composed of a 623 amino acid polypeptide chain with a signal peptide and a carboxyl terminal transmembrane domain, typical of all zona proteins. Sequence comparison demonstrate that mouse ZP1 is an orthologue of a rabbit zona protein, R55. The expression of R55 has been reported previously in both oocytes and granulosa cells. However, by northern analysis and in situ hybridization with 33P-labelled antisense probes to each of the three mouse zona mRNAs, we have determined that the expression of each mouse zona gene is restricted to the oocyte. ZP2 transcripts, but not ZP1 or ZP3, are detected in resting (15 microns diameter) oocytes, and all three zona transcripts coordinately accumulate as oocytes begin to grow. Together they represent approximately 1.5% of the total poly(A)+ RNA in 50-60 microns oocytes. In the latter stages of oogenesis, their abundance declines and each zona transcript is present in ovulated eggs at less than 5% of its maximal level. No zona transcripts were detected above background signal in granulosa cells. We conclude that, in mice, the three zona pellucida genes are expressed in a coordinate, oocyte-specific manner during the growth phase of oogenesis. Our data support the hypothesis that the transcription of the zona genes is controlled, in part, by shared regulatory element(s).