| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
RESEARCH |
1 Laboratory of Developmental Biology and Reproduction, Hyogo College of Medicine, Institute for Advanced Medical Sciences, Nishinomiya, Japan and 2 Department of Obstetrics and Gynecology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya 663-8501, Japan
Correspondence should be addressed to K Koyama; Email: kkoyama{at}hyo-med.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Biochemical analysis has shown that the zona pellucida is composed of three glycoproteins called ZP1 (or ZPB), ZP2 (or ZPA), and ZP3 (or ZPC) in various mammalian species. In mice, it has been reported that sperm initially bind to ZP3, which induces acrosome reaction. Subsequently, the acrosome-reacted sperm penetrates the zona pellucida, maintaining binding to the egg via ZP2. Following the fusion of sperm and egg cell membranes, ZP2 is cleaved proteolytically by enzymes released from cortical granules. Therefore, it is postulated that ZP2 cleavage is involved in blocking polyspermy (Bleil et al. 1981, 1988).
The structural and biological properties of the glycoproteins, however, are different among animal species. Recently, a fourth protein (ZP4) was found in human (Lefievre et al. 2004) and rat zona pellucida (GenBank accession no. MN_172330). The primary structures of pig zona pellucida components are similar to those of mouse and human, but the charge heterogeneity owing to carbohydrate chains is much higher in pigs than in mice and humans (Hedrick & Wardrip 1986, Koyama et al. 1991). In addition, pig zona pellucida glycoprotein 2 (pZP2) is partially cleaved into two components by proteolysis before fertilization (Hatanaka et al. 1992, Hasegawa et al. 1994).
The O-linked carbohydrate in mouse ZP3 is responsible for primary sperm binding to the zona pellucida and induction of acrosome reaction, whereas in pigs, the ortholog of mouse ZP1 has been shown to contribute to sperm binding to the zona pellucida (Yurewicz et al. 1998). Another paper reported that pig ZP2 or the combination of ZP1 and ZP3, induced effective acrosome reaction (Berger et al. 1989). In addition, human ZP4 as well as human ZP3 have been suggested to be involved in sperm–zona interaction in recombinant experiments (Chakravarty et al. 2005). These results indicate that the molecular mechanisms for sperm binding to the zona pellucida are different among mammalian species.
Recently, gene-targeting research has provided informative results. Under an electron microscope, genetically ZP3-knockout mouse lack zona pellucida both in ovarian and ovulated oocytes, completely, while the ZP1-knockout mouse form a zona pellucida, albeit thinner than normal (Liu et al. 1996, Rankin et al. 1996, 1999). The ZP2-knockout mice showed slight zona pellucida formation around the growing oocyte, but the zona pellucida was not detectable in the large antral follicle oocyte (Rankin et al. 2001). Consequently, ZP3 or ZP2-knockout mice are infertile.
Rankin et al. produced transgenic mice expressing human ZP3 in ZP3-knockout mice. Despite the presence of human ZP3 in the mouse zona pellucida, human sperm did not bind to the chimeric zona pellucida (Rankin et al. 1998). Furthermore, they produced a double-knockout mouse of ZP2 and ZP3 reconstituted with human ZP2 and ZP3 (Rankin et al. 2003). The mouse sperm could bind to the zona pellucida of the transgenic mice whose ZP2 and ZP3 were completely replaced with human ZP2 and ZP3, and fertility restored. However, human sperm did not bind to the zona pellucida of these mice. These observations indicated that the primary structures of mouse ZP2 and ZP3 are not responsible for species-specific sperm binding to the zona pellucida.
The ZP domains were conserved among mammalian and non-mammalian egg coat proteins and many other extracellular proteins (Bork & Sander 1992, Jovine et al. 2002). Jovine and his colleagues showed that hydrophobic regions in the domain of ZP2 or ZP3 are associated with intermolecular interaction of each zona protein around the oocyte (Jovine et al. 2004). They also suggested that another hydrophobic region present in the ZP domain in each protein interacts with the former hydrophobic region before their secretion.
Despite intensive research, the molecular details of species-specific egg–sperm binding and mechanisms of zona pellucida assembly are still obscure. Investigations using different animal species are important for proper understanding of the molecular basis of mammalian fertilization and follicular developments. This study is an attempt to examine whether pig ZP2 combines with mouse ZP1 and ZP3 to form a chimeric zona pellucida, and if so, whether pig spermatozoa could bind to the zona pellucida.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
tm1/Dean/J
, ME, USA). The animals were housed in a specific pathogen-free room, and kept in light- and temperature-controlled conditions (12 h light:12 h darkness, 22 ± 2 °C). They were provided with sterile food and water ad libitum. Animal experiments in this study were approved by the Committee on Animal Experimentation of Hyogo College of Medicine. All animals were maintained in accordance with the guidelines of the National Institute of Infectious Diseases.
Transgene construction
The 8.8 kb DNA construct was prepared by first placing a 6.8 kb minigene encoding pig ZP2 downstream of the 1.4 kb mouse promoter sequence. The minigene was prepared as described in our previous paper (Tsubamoto et al. 1999). A 2.1 kb DNA fragment containing 5'-flanking regions and the first two exons of mouse ZP2 cloned into Bluescript II was provided by Dr J Dean (National Institute of Health, Bethesda, ML, USA; Liang et al. 1990). The mouse ZP2 promoter region was obtained as an EcoRI/KpnI fragment of the 2.1 kb DNA and blunt-ligated to the 5'-flanking region of the 6.8 kb minigene. Finally, poly-adenylation and termination signals (0.56 kb) of the rabbit globin gene were placed downstream of the minigene (Fig. 1A
). The construct (~8.8 kb) was then subcloned into Bluescript II. For purification of the transgene construct, the plasmid DNA was digested with XhoI and separated by electrophoresis on 1% agarose gel, purified using QIAEXII Gel Extraction kit (Qiagen), and precipitated with ethanol. The resulting DNA was resuspended at 4 µg/ml in the injection buffer (10 mM Tris–HCl (pH 7.5) and 0.15 mM EDTA) and used for microinjection to fertilized eggs.
|
Production and characterization of transgenic mice
Fertilized eggs were collected from the oviduct of female mice (C57BL/6XDBA/2) mated with males of the same strain after superovulation. The transgene construct was microinjected into the male pronuclei of zygotes under an inverted microscope equipped with a micromanipulator (Narishige, Tokyo). Microinjected eggs were incubated in potassium Simple Optimum Medium (kSOM; Erbach et al. 1994) for 5–8 h until the two nuclei fused and transferred into the oviducts of pseudo-pregnant foster mothers. Detection of transgenic founders was achieved by PCR analysis. A small piece of mouse tail was digested in 50 µl of lysis buffer, incubated at 55 °C overnight, and DNA purified by DNeasy Tissue kit (Qiagen). A pair of primers: 5'-TGGCCATCACCAAAT-GACCA-3' and 5'-ATGAAATCTTTCATGCAG-3' were used for PCR. The reaction was carried out for 45 cycles at 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 90 s with GeneAmp PCR system 9700 (Applied Biosystems, Tokyo, Japan). As a positive control, a 149 bp fragment was amplified from the transgene used for microinjection.
Northern blot analysis
Total RNAs were extracted from 12-day-old mouse ovary, uterus, kidney, liver, spleen, lung, heart and testis using RNeasy (Qiagen). Thirty microgram RNA was electrophoresed on a 1% (w/v) agarose gel containing formaldehyde and transferred onto a nylon membrane (Hybond-N, Amersham). The membrane was washed briefly and baked at 80 °C for 1 h. A pig ZP2 cDNA fragment (307 bp) was excised from the transgene by SmaI/EcoRI and labeled by Random Primer DNA Labeling Kit Ver. 2.0 (TaKaRa, Japan) with [
-32P] dCTP (Amersham) to serve as a probe. The membranes containing total RNA were pre-hybridized at 68 °C for 30 min in ExpressHyb Hybridization Solution (BD Biosciences Clontech). The membranes were then hybridized with 2 x 106 c.p.m. of the radio-labeled probe in the ExpressHyb Hybridization solution at 68 °C for 18 h and washed with 2 x SSC/0.05% SDS and 0.1 x SSC/0.1% SDS solution. The membrane was exposed to an imaging plate for 24 h and analyzed by a BAS 2000 Bio-Image Analyzer (Fujifilm, Tokyo, Japan).
Crossing of the transgenic mice (pZP2 (+/+)) with ZP2-knockout mice (mZP2 (–/–))
The pig ZP2 gene was introduced into a ZP2-knockout mice (mZP2 (–/–)). Genotyping was performed by PCR using two pairs of primers: 5'-ATCTGTAAG-CTCTCCGTGCG-3' and 5'-CGGACTGAGGAAGGCT-TACT-3', 5'-GTGCCCTGAATGAACTGCAG-3' and 5'-CGTCCAGATCATCCTGATCG-3' for amplification of the wild-type mouse ZP2 and targeted (neomycin-resistant) genes respectively. The male transgenic mice of this line were crossed with the female (pZP2 (+/+)) mice to breed mice with the genotype of (pZP2 (+/–), mZP2 (+/–)) in F1 generations. Female mice with the genotype (pZP2 (+/+), mZP2 (–/–)) were obtained in F2 generations in a Mendelian manner.
Ovarian histology and immunocytochemistry
Ovaries were isolated from 4- and 8-week-old normal and transgenic female mice, fixed in 4% formaldehyde in PBS (pH 7.5), for 24 h, incubated in 10% sucrose for 24 h, and transferred into 70% ethanol. Tissues were dehydrated through graded alcohol solutions and embedded in paraffin. Sections (4 µm) were cut and incubated in 3% (v/v) H2O2 in methanol for 30 min, followed by 70% ethanol, distilled water, and PBS. To define the region of the zona pellucida, hematoxylin/eosin and periodic acid Schiff’s reagent stainings were carried out according to standard methods. For immunocytochemical staining, the sections were autoclaved in citrate buffer (pH 5.0) at 120 °C for 5 min and pre-blocked with 3% bovine serum albumen (BSA) in PBS. They were then incubated for 18 h at 4 °C with a MAB (MAb-5H4) specific to pig ZP2 produced using hybridoma techniques (Koyama et al. 1991). The MAB has been revealed to recognize an amino acid sequence 89–96 of pig ZP2 (Shigeta et al. 2000). The primary antibody, hybridoma supernatant was diluted 1:5 in PBS. Subsequent protocols were followed as detailed in Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA) and the color reaction was developed by diaminobenzidine.
Confocal microscopic observation
Pig ZP2 was detected by confocal microscopy using oocytes without fixation (unfixed oocytes). Wild-type or transgenic mice at 6–8 weeks of age were super-ovulated by an injection of 7.5 IU PMS (Teikokuzouki, Tokyo, Japan) followed by 7.5 IU hCG (Mochida, Tokyo, Japan), 48 h later. Mature ovulated eggs were recovered from the oviducts 14–16 h after hCG injection, treated with 1 mg/ml bovine testicular hyaluronidase (Sigma) to remove cumulus cells, and transferred to the primary antibody. Growing germinal vesicle-stage oocytes were isolated from the ovaries by dissecting with needles under an inverted microscope (TS100, Nikon, Tokyo, Japan).
To detect the pig ZP2 protein, the rabbit antiserum to recombinant pig ZP2 peptide was used. An antiserum to a synthetic peptide comprising 121–140 amino acid residues of mouse ZP2, which does not cross-react to pig zona pellucida, was used as a positive control (Sun et al. 1999). Both antisera were diluted at 1:100 (v/v) in PBS containing 0.1% BSA (BSA–PBS).
Live eggs were incubated for 50 min in the diluted antisera followed by washes in PBS containing 0.1% BSA (3 min, four times). The treated eggs were labeled for 40 min with FITC-conjugated anti-rabbit IgG (affinity purified IgG: ICN/Cappel, Aurora, OH, USA) at a 1:200 dilution in PBS. Following washing in BSA–PBS, the eggs were observed under a laser-scanning microscope (Carl Zeiss LSM510, Tokyo, Japan). FITC (fluorescence isothiocyanate) was excited with a 488 nm line from an argon laser, and emissions were imaged through a 505–530 nm filter.
Statistical analysis
Ovulated oocytes from the mice with different genotypes were counted under an inverted light microscope. P values were calculated between the two comparative groups by Student’s t-test and used for the assessment of the significant difference. P values below 0.05 were considered to be statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
). The minigene was comprised of intronless cDNA (exons 1–6), genomic DNA (exons/introns), and cDNA (exon 17–18), followed by polyadenylation and termination signals of rabbit ß-globin and was inserted into pCI-neo Vector to transfect to CHO culture cells to confirm production and secretion of the recombinant pig ZP2. A band of 120–140 kDa was detected with a specific antibody to the pig ZP2 in the supernatant from the transfected cells (Fig. 1B). One hundred and forty-eight fertilized mouse eggs were microinjected with the transgene following which 104 eggs were transplanted into the oviducts of pseudo-pregnant recipients. The pig ZP2 transgene was analyzed by PCR using DNA extracted from the tail of the offspring. Four out of 36 newborn animals were positive in this analysis showing a 149-bp DNA fragment (Fig. 1C). Homozygous transgenic mouse lines were established from three of the positive animals (two males and, one female).
Ovary-specific expression of pig ZP2 transcript
Northern blot analysis was performed to examine the tissue-specific expression of pig ZP2. The totalTotal RNAs were isolated from 12-day-old ovary, uterus, kidney, liver, spleen, lung, heart, and testis. A single -specific transcript was detected in ovarian RNA (Fig. 1D). The probe used in this assay did not hybridize to the wild-type ovary.
Expression of pig ZP2 on the surface of the oolemma
Immunohistochemical studies revealed that pig ZP2 was expressed in the growing oocytes from the transgenic mice at prepuberty and puberty (Fig. 2B and C). pZP2 was detected in the growing oocytes of transgenic mice. In both cases, positive reactions were localized inside the oocytes and at their peripheral regions, suggesting that pZP2 was synthesized in the oocytes and transported towards the cell membrane. At a higher magnification, the reaction was found in a more inward region (Fig. 2F) than that by periodic acid–Schiff’s reagent or the anti-mouse ZP2 antiserum (Fig. 2D and E). No staining area between immuno-positive region and granulosa cells probably corresponds to the zona pellucida. These results, however, do not indicate whether the pig ZP2 protein was secreted from the oocyte or still remains in the peripheral region of the oocyte. To clarify this, live ovarian oocytes were stained by the antibody specific to pig ZP2. A positive reaction was observed in the oocyte cell membrane and the inner portion of the zona pellucida (Fig. 3B), whereas the ovulated eggs did not show the positive reaction (Fig. 3E). The positive control antibody to mouse ZP2 clearly showed the zona pellucida both in growing oocytes and ovulated eggs (Fig. 3C and F). These results indicated that pig ZP2 was synthesized and secreted, but not incorporated into mouse zona pellucida in matured eggs. The protein seems to pass through the zona pellucida during oocyte maturation.
|
|
). The bands of 315, 188, and 149 bp represents were amplified from ZP2-knockout mouse gene, wild-type mouse ZP2, and transgenic pig ZP2 respectively. Ovulated eggs from (pZP2 (+/+), mZP2 (+/+)), (pZP2 (+/+), mZP2 (+/–)), and (pZP2 (+/+), mZP2 (–/–)) mice were observed under an optical microscope (Fig. 4). The zona pellucida was absent around the eggs from the ZP2-knockout mice that expressed the pig ZP2 gene (Fig. 4C). The heterozygous ZP2-targeted mice, on the other hand, formed the zona pellucida (Fig. 4B), although it was thinner than that of the wild-type mice (Fig. 4A).
|
). Ovulation of ZP2-knockout mice was not significantly improved by incorporation of the pig ZP2 gene (a versus b). Heterozygous mice (pZP2 (+/+), mZP2 (+/–)) showed a similar number of ovulated eggs to ZP2 normal mice (pZP2 (+/+), mZP2 (+/+)) with or without pig ZP2 gene. Taken together, these results show that transgenesis of the pig ZP2 gene did not restore zona pellucida formation, granulosa cell proliferation, and oocyte growth.
|
). Not stained area around the oocytes observed in sections A and B but not C probably indicates the zona pellucida. Sections D and E from the (pZP2 (+/+), mZP2 (+/–)) mice were stained with periodic acid–Schiff’s reagent and anti-mouse ZP2 antibody respectively, to show the zona pellucida area. Compared to these clear stainings of zona pellucida, pZP2 protein in the (pZP2 (+/+), mZP2 (–/–)) mouse section was localized in the oocyte cytoplasma and the spaces of the follicular cells (Fig. 5F). These results suggested that the pZP2 protein of the (pZP2 (+/+), mZP2 (–/–)) mouse is diffusely expressed around the oocyte as well as the follicular spaces. This expression pattern of pZP2 resembled that of pig ZP2 transgenic mice (pZP2 (+/+), mZP2 (+/+)) (Fig. 2).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
The present study showed that the transgene, pig ZP2, was expressed in the transgenic mice and ZP2-knockout mice, but could not restore the deficiency of the zona pellucida architecture of the ZP2-knockout mice. The protein was localized around the oocyte cytoplasm, inner part of the zona pellucida, and interspaces of follicular cells. A study reported that mouse ZP2 is present on the plasma membrane of the egg from the ZP3-knockout mice but did not deposit around the oocyte. They argued that the three mouse zona proteins are synthesized and secreted independently and assemble spontaneously to form the zona pellucida outside the oocyte (Qi & Wassarman 1999). This is consistent with the state of pig ZP2 unable to assemble to mouse zona pellucida components in the present study.
Other reports have documented that zona proteins were extensively detected outside the zona pellucida area by immunohistochemical examination (Takagi et al. 1989, Dunbar et al. 1994). In our findings, pZP2 protein was detected in the intercellular spaces of oocyte and granulosa cells, indicating that pZP2 was immersed in the spaces passing through the zona pellucida (Figs 2F and 5F), because ZP2 protein was synthesized by the oocytes but not granulosa cells in mice (Bleil & Wassarman 1980, Shimizu et al. 1983).
Confocal microscopic observations using live oocytes and eggs showed that pig ZP2 was expressed outside of the cell membrane and inner part of the zona pellucida in ovarian oocytes, but was not in ovulated eggs. These results suggest that the pig ZP2 is produced contiguously in the growing oocytes, temporarily stays on the plasma membrane, and diffuses out from oocytes without incorporation into the zona pellucida. Increased fluidity and change in oocyte plasma membrane compositions following oocyte maturation may be due to the complete shed-out of the protein.
Actually, mRNA and protein synthesis of ZP proteins gradually increases during oogenesis and significantly decreases after ovulation (Liang et al. 1990). The foreign pig ZP2 seems to be controlled under this expression regulation. Similar observations have been reported with ZP2-knockout mice (Rankin et al. 2001). They showed that less zona pellucida was formed around mature oocytes in large follicles than in smaller follicles and suggested that the disruption of the zona pellucida compromised the ability of follicles to continue to develop into antral follicles.
The failure of the pig ZP2 assembly to the transgenic mouse zona pellucida was possibly due to the lower affinity to mouse ZP1/ZP3 compared with ZP2. The possibility was tested by introducing the pig ZP2 gene into ZP2-knockout mice, which lack zona pellucida in ovulated eggs. It was found that the pig ZP2 gene did not result in the formation of zona pellucida in ZP2-knockout mouse, indicating that the pig ZP2 did not assemble with mouse ZP1 and ZP3.
In contrast to our result, Rankin et al. have shown that human ZP2 and ZP3 can form a chimeric zona pellucida in mice (Rankin et al. 1998, 2003). Why did pig ZP2 not form a chimeric zona pellucida in polymerization with mouse ZP1 and ZP3? A comparison of the primary structures indicates that 11 cysteine residues are entirely conserved and 6 consensus sequences (Asn–Xaa–Thr) of N-glycosylation sites are also present in ZP2 of mice, humans, and pigs. Comparing each orthologous sequence, the amino acid sequence of pig and human are 54 and 60% identical to that of mouse ZP2 respectively (Harris et al. 1994). The difference (6%) is insufficient as evidence that pig ZP2 did not assemble to mouse zona pellucida. Xenopus laevis zona pellucida could assemble to each mouse zona protein (ZP1, ZP2, and ZP3) with 39–48% amino acid similarity (Doren et al. 1999). The absence or the replacement in critical site(s) probably affected zona assembly of pig ZP2.
Recently, however, hydrophobic segments of the mouse ZP domain in ZP1, ZP2, and ZP3 proteins have been reported to be important for assembly of these zona proteins (Jovine et al. 2004). This domain plays fundamental roles not only in the zona pellucida surrounding the oocyte but also in the various extracellular matrices associated with development, immunity, and cancer (Jovine et al. 2002). They suggested that a hydrophobic amino acid sequence of PGPLVLV (483–489) in mouse ZP2 located in the domain is crucial for protein secretion. The corresponding sequence is replaced by PGPLTLT in pig ZP2. The change in the two sites of hydrophobic amino acid (Valine) to hydroxyl amino acid (Threonine) that is potentially bound to the O-linked carbohydrate may be crucial to form chimeric zona pellucida of mice and pigs.
In conclusion, the present study indicates that pig ZP2 is expressed in transgenic mouse oocytes, but neither participates in mouse zona pellucida architecture nor restores the zona pellucida in ZP2-knockout mice despite the presence of the other two zona proteins. The difference of amino acids in the hydrophobic region of the ZP domain would be crucial for the assembly. Further investigations including site-directed mutagenesis are necessary to find the sequence responsible for incorporation of pig ZP2 into mouse zona pellucida.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Berger T, Davis A, Wardrip NJ & Hedrick JL 1989 Sperm binding to the pig zona pellucida and inhibition of binding by solubilized components of the zona pellucida. Journal of Reproduction and Fertility 86 559–565.
Bleil JD & Wassarman PM 1980 Synthesis of zona pellucida proteins by denuded and follicle-enclosed mouse oocytes during culture in vitro. PNAS 77 1029–1033.
Bleil JD, Beall CF & Wassarman PM 1981 Mammalian sperm–egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2. Developmental Biology 86 189–197.[CrossRef][Web of Science][Medline]
Bleil JD, Greve JM & Wassarman PM 1988 Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs. Developmental Biology 128 376–385.[CrossRef][Web of Science][Medline]
Bork P & Sander C 1992 A large domain common to sperm receptors (Zp2 and Zp3) and TGF-beta type III receptor. FEBS Letters 300 237–240.[CrossRef][Web of Science][Medline]
Chakravarty S, Suraj K & Gupta SK 2005 Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C. Molecular Human Reproduction 11 365–372.
Dean J 2002 Oocyte-specific genes regulate follicle formation, fertility and early mouse development. Journal of Reproductive Immunology 53 171–180.[CrossRef][Web of Science][Medline]
Doren S, Landsberger N, Dwyer N, Gold L, Blanchette-Mackie J & Dean J 1999 Incorporation of mouse zona pellucida proteins into the envelope of Xenopus laevis oocytes. Development Genes and Evolution 209 330–339.[CrossRef][Web of Science][Medline]
Dunbar BS, Avery S, Lee V, Prasad S, Schwahn D, Schwoebel E, Skinner S & Wilkins B 1994 The mammalian zona pellucida: its biochemistry, immunochemistry, molecular biology, and developmental expression. Reproduction, Fertility, and Development 6 331–347.[CrossRef][Medline]
Erbach GT, Lawitts JA, Papaioannou VE & Biggers JD 1994 Differential growth of the mouse preimplantation embryo in chemically defined media. Biology of Reproduction 50 1027–1033.[Abstract]
Harris JD, Hibler DW, Fontenot GK, Hsu KT, Yurewicz EC & Sacco AG 1994 Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC gene families. DNA Sequence 4 361–393.[Web of Science][Medline]
Hasegawa A, Koyama K, Okazaki Y, Sugimoto M & Isojima S 1994 Amino acid sequence of a porcine zona pellucida glycoprotein ZP4 determined by peptide mapping and cDNA cloning. Journal of Reproduction and Fertility 100 245–255.
Hasegawa A, Tsubamoto H, Hamada Y & Koyama K 2000 Blocking effect of antisera to recombinant zona pellucida proteins (r-ZPA) on in vitro fertilization. American Journal of Reproductive Immunology 44 59–64.[CrossRef]
Hatanaka Y, Nagai T, Tobita T & Nakano M 1992 Changes in the properties and composition of zona pellucida of pigs during fertilization in vitro. Journal of Reproduction and Fertility 95 431–440.
Hedrick JL & Wardrip NJ 1986 Isolation of the zona pellucida and purification of its glycoprotein families from pig oocytes. Analytical Biochemistry 157 63–70.[CrossRef][Web of Science][Medline]
Jovine L, Qi H, Williams Z, Litscher E & Wassarman PM 2002 The ZP domain is a conserved module for polymerization of extracellular proteins. Nature Cell Biology 4 457–461.[CrossRef][Web of Science][Medline]
Jovine L, Qi H, Williams Z, Litscher ES & Wassarman PM 2004 A duplicated motif controls assembly of zona pellucida domain proteins. PNAS 101 5922–5927.
Koyama K, Hasegawa A, Inoue M & Isojima S 1991 Blocking of human sperm-zona interaction by monoclonal antibodies to a glycoprotein family (ZP4) of porcine zona pellucida. Biology of Reproduction 45 727–735.[Abstract]
Lefievre L, Conner SJ, Salpekar A, Olufowobi O, Ashton P, Pavlovic B, Lenton W & Afnan M 2004 Four zona pellucida glycoproteins are expressed in the human. Human Reproduction 19 1580–1586.
Liang L & Dean J 1993 Conservation of mammalian secondary sperm receptor gene enables the promoter of the human gene to function in mouse oocyte. Developmental Biology 156 399–408.[CrossRef][Web of Science][Medline]
Liang LF, Chamow SM & Dean J 1990 Oocyte-specific expression of mouse Zp-2: developmental regulation of the zona pellucida genes. Molecular and Cellular Biology 10 1507–1515.
Liu C, Litscher ES, Mortillo S, Sakai Y, Kinloch RA, Stewart CL & Wassarman PM 1996 Targeted disruption of the mZP3 gene results in production of eggs lacking a zona pellucida and infertility in female mice. PNAS 93 5431–5436.
Qi H & Wassarman PM 1999 Secretion of zona pellucida glycoprotein mZP2 by growing oocytes from mZP3(+/+) and mZP3(–/–) mice. Developmental Genetics 25 95–102.[CrossRef][Web of Science][Medline]
Rankin T, Familari M, Lee E, Ginsberg A, Dwyer N, Blanchette-Mackie J, Drago J, Westphal H & Dean J 1996 Mice homozygous for an insertional mutation in the Zp3 gene lack a zona pellucida and are infertile. Development 122 2903–2910.[Abstract]
Rankin TL, Tong ZB, Castle PE, Lee E, Gore-Langton R, Nelson LM & Dean J 1998 Human ZP3 restores fertility in Zp3 null mice without affecting order-specific sperm binding. Development 125 2415–2424.[Abstract]
Rankin T, Talbot P, Lee E & Dean J 1999 Abnormal zonae pellucidae in mice lacking ZP1 result in early embryonic loss. Development 126 3847–3855.[Abstract]
Rankin TL, O’Brien M, Lee E, Wigglesworth K, Eppig J & Dean J 2001 Defective zonae pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development. Development 128 1119–1126.[Abstract]
Rankin TL, Coleman JS, Epifano O, Hoodbhoy T, Turner SG, Castle PE, Lee E, Gore-Langton R & Dean J 2003 Fertility and taxon-specific sperm binding persist after replacement of mouse sperm receptors with human homologs. Developmental Cell 5 33–43.[CrossRef][Web of Science][Medline]
Shigeta M, Hasegawa A, Hamada Y & Koyama K 2000 Analysis of B cell epitopes of a glycoprotein porcine zona pellucida (pZP1). Journal of Reproductive Immunology 47 159–168.[CrossRef][Web of Science][Medline]
Shimizu S, Tsuji M & Dean J 1983 In vitro biosynthesis of three sulfated glycoproteins of murine zonae pellucidae by oocytes grown in follicle culture. Journal of Biological Chemistry 258 5858–5863.
Sun W, Lou YH, Dean J & Tung KS 1999 A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida. Biology of Reproduction 60 900–907.
Takagi J, Araki Y, Dobashi M, Imai Y, Hiroi M, Tonosaki A & Sendo F 1989 The development of porcine zona pellucida using monoclonal antibodies. I. Immunochemistry and light microscopy. Biology of Reproduction 40 1095–1102.[Abstract]
Tsubamoto H, Yamasaki N, Hasegewa A & Koyama K 1999 Expression of a recombinant porcine zona pellucida glycoprotein ZP1 in mammalian cells. Protein Expression and Purification 17 8–15.[CrossRef][Web of Science][Medline]
Wassarman PM 1987 The biology and chemistry of fertilization. Science 235 553–560.
Wassarman PM 1999 Mammalian fertilization: molecular aspects of gamete adhesion, exocytosis, and fusion. Cell 96 175–183.[CrossRef][Web of Science][Medline]
Yanagimachi R 1994 Mammalian fertilization. In The Physiology of Reproduction, 5th edn, pp 189–317. Eds E Knobi & JD Neil. New York: Raven Press.
Yurewicz EC, Sacco AG, Gupta SK, Xu N & Gage DA 1998 Hetero-oligomerization-dependent binding of pig oocyte zona pellucida glycoproteins ZPB and ZPC to boar sperm membrane vesicles. Journal of Biological Chemistry 273 7488–7494.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
and cDNA exons 1–6 and 17–18 (hatched boxes). (B) Western blot analysis of the recombinant protein secreted in the supernatant from transfected CHO cell. The loaded protein was isolated as described in ‘Materials and Methods’. A positive signal of 120–140 kDa was detected by a specific antibody to pig ZP2. (C) PCR analysis of DNA from the transgenic offspring. Genomic DNAwas extractedfromthe tail tips of the tested offspring. A pair of primerswas designed to amplify the cDNA region ofthe transgene. A 149 bp DNA fragment included in the pig ZP2 gene was detected in lanes 6, 10, 11, and 14. Lane 8 is a positive control using the transgene as a template. Lane 7 is a standard size marker. (D) Ovary-specific expression of pig ZP2. Autoradiograph of a northern blot analysis of mouse tissues probed with 32P-labeled cDNA from the transgenic construct. Lane 1, 30 µg total RNAs from wild-type (pZP2 (–/–)) mouse ovary; lanes 2–9, 30 µg total RNAs from ovary, uterus, kidney, liver, spleen, lung, heart, and testis from transgenic (pZP2 (+/+)) mice. Numbers to the left are size markers. (E) Genotyping of F2 generation from transgenic (pZP2 (+/+)) female mice crossed with knockout (mZP2 (–/–)) male mice. Genomic DNA was prepared from tail tips of the tested offspring. Amplified DNA fragments were separated on 1.5% agarose gels to detect bands of 315, 188, and 149 bp corresponding to those from targeted ZP2, wild-type mZP2, and pZP2 respectively; lane M, standard size markers. pZP2 (+/+) shows the pZP2 transgene. mZP2 (+/+), (+/–), and (–/–) indicate the genotypes of wild-type, heterozygous, and homozygous genes from mZP2-targeted mice respectively.
