REPRODUCTIONRESEARCH

Pig 2 (pZP2) does not participate in zona pellucida formation in transgenic mice

Akiko Hasegawa1, Nozomi Kanazawa1, Hideaki Sawai1, Shinji Komori1,2 and Koji Koyama1,2 1Laboratory of Developmental Biology and Reproduction, Hyogo College of Medicine, Institute for Advanced Medical Sciences, Nishinomiya, Japan and 2Department of Obstetrics and Gynecology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya 663-8501, Japan Correspondence should be addressed to K Koyama; Email: [email protected]

Abstract The zona pellucida, an extracellular matrix surrounding mammalian , is composed of three or four . It is well known that the zona pellucida plays several critical roles during fertilization, but there is little knowledge about its formation. The purpose of this study is to examine whether a pig zona pellucida 2 (pZP2) would assemble with mouse zona pellucida. A transgene construct was prepared by placing a minigene encoding pZP2 downstream from the promoter of mouse ZP2. The result showed that the transgenic protein was synthesized in growing oocytes but not incorporated into the zona pellucida. Furthermore, the pZP2 transgene did not rescue the phenotype in ZP2-knockout zona-deficient mice. These results indicate that pZP2 does not participate in mouse zona pellucida formation and the zona pellucida is constituted from its component in a molecular species-specific manner between mice and pigs. Reproduction (2006) 132 455–464

Introduction is postulated that ZP2 cleavage is involved in blocking polyspermy (Bleil et al. 1981, 1988). The mammalian zona pellucida is an extracellular The structural and biological properties of the matrix that surrounds growing oocytes, ovulated eggs glycoproteins, however, are different among animal and pre-implantation embryos, and is known to be species. Recently, a fourth protein (ZP4) was found in involved in several important events during ovarian human (Lefievre et al. 2004) and rat zona pellucida folliculogenesis and fertilization. It has been shown that the zona pellucida mediates relatively species-specific (GenBank accession no. MN_172330). The primary sperm–egg recognition and induction of acrosome- structures of pig zona pellucida components are similar reaction events critical to penetration of the zona. to those of mouse and human, but the charge Following fertilization, the zona pellucida prevents the heterogeneity owing to carbohydrate chains is much penetration of additional sperm and protects the higher in pigs than in mice and humans (Hedrick & pre-implantation embryo passing down the oviduct Wardrip 1986, Koyama et al. 1991). In addition, pig (Wassarman 1987, Yanagimachi 1994). Molecular zona pellucida glycoprotein 2 (pZP2) is partially cleaved details of these events have been investigated over the into two components by proteolysis before fertilization last decade (Wassarman 1999, Dean 2002). (Hatanaka et al. 1992, Hasegawa et al. 1994). Biochemical analysis has shown that the zona The O-linked carbohydrate in mouse ZP3 is respon- pellucida is composed of three glycoproteins called sible for primary sperm binding to the zona pellucida ZP1 (or ZPB), ZP2 (or ZPA), and ZP3 (or ZPC) in various and induction of acrosome reaction, whereas in pigs, the mammalian species. In mice, it has been reported that ortholog of mouse ZP1 has been shown to contribute to sperm initially bind to ZP3, which induces acrosome sperm binding to the zona pellucida (Yurewicz et al. reaction. Subsequently, the acrosome-reacted sperm 1998). Another paper reported that pig ZP2 or the penetrates the zona pellucida, maintaining binding to combination of ZP1 and ZP3, induced effective acro- the egg via ZP2. Following the fusion of sperm and egg some reaction (Berger et al. 1989). In addition, human cell membranes, ZP2 is cleaved proteolytically by ZP4 as well as human ZP3 have been suggested to be enzymes released from cortical granules. Therefore, it involved in sperm–zona interaction in recombinant

q 2006 Society for Reproduction and Fertility DOI: 10.1530/rep.1.01016 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/27/2021 08:03:09PM via free access 456 A Hasegawa and others experiments (Chakravarty et al. 2005). These results ZP2-knockout mouse (mZP2(K/K)) line was provided indicate that the molecular mechanisms for sperm by Jackson Animal Laboratory (JAX Mice & Services, binding to the zona pellucida are different among STOCK Zp2 htm1/Dean/Ji, ME, USA). The animals were mammalian species. housed in a specific pathogen-free room, and kept in Recently, -targeting research has provided infor- light- and temperature-controlled conditions (12 h mative results. Under an electron microscope, geneti- light:12 h darkness, 22G2 8C). They were provided with cally ZP3-knockout mouse lack zona pellucida both in sterile food and water ad libitum. Animal experiments in ovarian and ovulated oocytes, completely, while the this study were approved by the Committee on Animal ZP1-knockout mouse form a zona pellucida, albeit Experimentation of Hyogo College of Medicine. All thinner than normal (Liu et al. 1996, Rankin et al. animals were maintained in accordance with the guide- 1996, 1999). The ZP2-knockout mice showed slight lines of the National Institute of Infectious Diseases. zona pellucida formation around the growing , but the zona pellucida was not detectable in the large antral follicle oocyte (Rankin et al. 2001). Consequently, Transgene construction ZP3 or ZP2-knockout mice are infertile. The 8.8 kb DNA construct was prepared by first placing a Rankin et al. produced transgenic mice expressing 6.8 kb minigene encoding pig ZP2 downstream of the human ZP3 in ZP3-knockout mice. Despite the presence 1.4 kb mouse promoter sequence. The minigene was of human ZP3 in the mouse zona pellucida, human prepared as described in our previous paper sperm did not bind to the chimeric zona pellucida (Tsubamoto et al. 1999). A 2.1 kb DNA fragment (Rankin et al. 1998). Furthermore, they produced a containing 50-flanking regions and the first two double-knockout mouse of ZP2 and ZP3 reconstituted exons of mouse ZP2 cloned into Bluescript II was with human ZP2 and ZP3 (Rankin et al. 2003). The provided by Dr J Dean (National Institute of Health, mouse sperm could bind to the zona pellucida of the Bethesda, ML, USA; Liang et al. 1990). The mouse transgenic mice whose ZP2 and ZP3 were completely ZP2 promoter region was obtained as an EcoRI/KpnI replaced with human ZP2 and ZP3, and fertility restored. fragment of the 2.1 kb DNA and blunt-ligated to the 50- However, human sperm did not bind to the zona flanking region of the 6.8 kb minigene. Finally, poly- pellucida of these mice. These observations indicated adenylation and termination signals (0.56 kb) of the rabbit that the primary structures of mouse ZP2 and ZP3 are not globin gene were placed downstream of the minigene responsible for species-specific sperm binding to the w zona pellucida. (Fig. 1A). The construct ( 8.8 kb) was then subcloned into The ZP domains were conserved among mammalian Bluescript II. For purification of the transgene construct, the and non-mammalian egg coat proteins and many other plasmid DNA was digested with XhoI and separated by extracellular proteins (Bork & Sander 1992, Jovine et al. electrophoresis on 1% agarose gel, purified using QIAEXII 2002). Jovine and his colleagues showed that hydro- Gel Extraction kit (Qiagen), and precipitated with ethanol. phobic regions in the domain of ZP2 or ZP3 are The resulting DNA was resuspended at 4 mg/ml in the associated with intermolecular interaction of each injection buffer (10 mM Tris–HCl (pH 7.5) and 0.15 mM zona protein around the oocyte (Jovine et al. 2004). EDTA) and used for microinjection to fertilized eggs. They also suggested that another hydrophobic region The minigene was confirmed to produce and secrete present in the ZP domain in each protein interacts with pig ZP2 by inserting pCI-neo Mammalian Expression the former hydrophobic region before their secretion. Vector (Promega) and transfecting to a Chinese hamster Despite intensive research, the molecular details of ovarian (CHO) cell line using GeneJammer Transfection species-specific egg–sperm binding and mechanisms of Reagent (Strategene, CA, USA) according to the zona pellucida assembly are still obscure. Investigations manufacturer’s instructions. The culture supernatant 2C using different animal species are important for proper from transfected cells was put through Ni -column understanding of the molecular basis of mammalian chromatography to isolate the histidine-tagged recombi- fertilization and follicular developments. This study is an nant protein, followed by western blot analysis. The pig attempt to examine whether pig ZP2 combines with ZP2 was detected using a rabbit antiserum to recombi- mouse ZP1 and ZP3 to form a chimeric zona pellucida, nant pig ZP2 peptide corresponding to 1–198 amino and if so, whether pig spermatozoa could bind to the acid residues (Hasegawa et al. 2000). zona pellucida. Production and characterization of transgenic mice Materials and Methods Fertilized eggs were collected from the oviduct of female mice (C57BL/6XDBA/2) mated with males of the same Animals strain after superovulation. The transgene construct was C57BL/6XDBA/2 F1 mice were purchased from Japan microinjected into the male pronuclei of zygotes under Shizuoka Laboratory Center (SLC, Japan) and a an inverted microscope equipped with a

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Downloaded from Bioscientifica.com at 09/27/2021 08:03:09PM via free access Transgenic mice incorporated with pig ZP2 gene 457

Figure 1 Transgenic construct, PCR analysis of the transgene, and northern blot analysis. (A) Schematic representation of the pig ZP2 transgene. It consists of a 6.8 kb pig ZP2 minigene flanked by a 1.4 kb region of mouse ZP2 promoter at its 50-end and a 0.56 kb rabbit globin polyadenylation and termination signals at the 30-end. The pig ZP2 minigene contains genomic fragments of exons 7–16 , 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 tipsof the tested offspring.A pair of primerswas designed to amplifythe 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 mg total RNAs from wild-type (pZP2 (K/K)) mouse ovary; lanes 2–9, 30 mg total RNAs from ovary,uterus, kidney, liver,spleen, lung, heart, and testis from transgenic (pZP2 (C/C)) mice. Numbers to the left are size markers. (E) Genotyping of F2 generation from transgenic (pZP2 (C/C)) female mice crossed with knockout (mZP2 (K/K)) 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 (C/C) shows the pZP2 transgene. mZP2 (C/C), (C/K), and (K/K) indicate the genotypes of wild-type, heterozygous, and homozygous from mZP2-targeted mice respectively. micromanipulator (Narishige, Tokyo). Microinjected GeneAmp PCR system 9700 (Applied Biosystems, eggs were incubated in potassium Simple Optimum Tokyo, Japan). As a positive control, a 149 bp fragment Medium (kSOM; Erbach et al. 1994) for 5–8 h until the was amplified from the transgene used for two nuclei fused and transferred into the oviducts of microinjection. pseudo-pregnant foster mothers. Detection of transgenic founders was achieved by PCR analysis. A small piece of Northern blot analysis mouse tail was digested in 50 ml of lysis buffer, incubated at 55 8C overnight, and DNA purified by DNeasy Tissue Total RNAs were extracted from 12-day-old mouse kit (Qiagen). A pair of primers: 50-TGGCCATCACCAAAT- ovary, uterus, kidney, liver, spleen, lung, heart and testis GACCA-30 and 50-ATGAAATCTTTCATGCAG-30 were using RNeasy (Qiagen). Thirty microgram RNA was used for PCR. The reaction was carried out for 45 cycles electrophoresed on a 1% (w/v) agarose gel containing at 95 8C for 30 s, 50 8C for 30 s, and 72 8C for 90 s with formaldehyde and transferred onto a nylon membrane www.reproduction-online.org Reproduction (2006) 132 455–464

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(Hybond-N, Amersham). The membrane was washed USA) and the color reaction was developed by briefly and baked at 80 8C for 1 h. A pig ZP2 cDNA diaminobenzidine. fragment (307 bp) was excised from the transgene by SmaI/EcoRI and labeled by Random Primer DNA 32 Confocal microscopic observation Labeling Kit Ver. 2.0 (TaKaRa, Japan) with [a- P] dCTP (Amersham) to serve as a probe. The membranes Pig ZP2 was detected by confocal microscopy using containing total RNA were pre-hybridized at 68 8C for oocytes without fixation (unfixed oocytes). Wild-type or 30 min in ExpressHyb Hybridization Solution (BD transgenic mice at 6–8 weeks of age were super- Biosciences Clontech). The membranes were then ovulated by an injection of 7.5 IU PMS (Teikokuzouki, hybridized with 2!106 c.p.m. of the radio-labeled Tokyo, Japan) followed by 7.5 IU hCG (Mochida, probe in the ExpressHyb Hybridization solution at Tokyo, Japan), 48 h later. Mature ovulated eggs were 68 8C for 18 h and washed with 2!SSC/0.05% SDS recovered from the oviducts 14–16 h after hCG and 0.1!SSC/0.1% SDS solution. The membrane was injection, treated with 1 mg/ml bovine testicular exposed to an imaging plate for 24 h and analyzed by a hyaluronidase (Sigma) to remove cumulus cells, and BAS 2000 Bio-Image Analyzer (Fujifilm, Tokyo, Japan). transferred to the primary antibody. Growing germinal vesicle-stage oocytes were isolated from the ovaries by dissecting with needles under an inverted microscope Crossing of the transgenic mice (pZP2 (C/C)) with (TS100, Nikon, Tokyo, Japan). K K ZP2-knockout mice (mZP2 ( / )) To detect the pig ZP2 protein, the rabbit antiserum to The pig ZP2 gene was introduced into a ZP2-knockout recombinant pig ZP2 peptide was used. An antiserum to mice (mZP2 (K/K)). Genotyping was performed by PCR a synthetic peptide comprising 121–140 amino acid using two pairs of primers: 50-ATCTGTAAG- residues of mouse ZP2, which does not cross-react to pig CTCTCCGTGCG-30 and 50-CGGACTGAGGAAGGCT- zona pellucida, was used as a positive control (Sun et al. TACT-3 0,50-GTGCCCTGAATGAACTGCAG-30 and 1999). Both antisera were diluted at 1:100 (v/v) in PBS 50-CGTCCAGATCATCCTGATCG-30 for amplification of containing 0.1% BSA (BSA–PBS). the wild-type mouse ZP2 and targeted (neomycin- Live eggs were incubated for 50 min in the diluted resistant) genes respectively. The male transgenic mice antisera followed by washes in PBS containing 0.1% BSA (3 min, four times). The treated eggs were labeled for 40 min of this line were crossed with the female (pZP2 (C/C)) with FITC-conjugated anti-rabbit IgG (affinity purified IgG: mice to breed mice with the genotype of (pZP2 (C/K), ICN/Cappel,Aurora,OH,USA)ata1:200dilutioninPBS. mZP2 (C/K)) in F1 generations. Female mice with the Following washing in BSA–PBS, the eggs were observed genotype (pZP2 (C/C), mZP2 (K/K)) were obtained in under a laser-scanning microscope (Carl Zeiss LSM510, F2 generations in a Mendelian manner. Tokyo, Japan). FITC (fluorescence isothiocyanate) was excited with a 488 nm line from an argon laser, and Ovarian histology and immunocytochemistry emissions were imaged through a 505–530 nm filter. Ovaries were isolated from 4- and 8-week-old normal and transgenic female mice, fixed in 4% formaldehyde in Statistical analysis PBS (pH 7.5), for 24 h, incubated in 10% sucrose for 24 h, Ovulated oocytes from the mice with different genotypes and transferred into 70% ethanol. Tissues were dehy- were counted under an inverted light microscope. P drated through graded alcohol solutions and embedded values were calculated between the two comparative in paraffin. Sections (4 mm) were cut and incubated in 3% groups by Student’s t-test and used for the assessment of (v/v) H2O2 in methanol for 30 min, followed by 70% the significant difference. P values below 0.05 were ethanol, distilled water, and PBS. To define the region of considered to be statistically significant. the zona pellucida, hematoxylin/eosin and periodic acid Schiff’s reagent stainings were carried out according to standard methods. For immunocytochemical staining, Results the sections were autoclaved in citrate buffer (pH 5.0) at 120 8C for 5 min and pre-blocked with 3% bovine serum Production of transgenic animals albumen (BSA) in PBS. They were then incubated for 18 h The w8.8 kb pig ZP2 construct consisted of the 6.8 kb at 4 8C with a MAB (MAb-5H4) specific to pig ZP2 pig ZP2 minigene was ligated to a 1.4 kb mouse ZP2 produced using hybridoma techniques (Koyama et al. promoter (Fig. 1A). The minigene was comprised of 1991). The MAB has been revealed to recognize an amino intronless cDNA (exons 1–6), genomic DNA (exons/ acid sequence 89–96 of pig ZP2 (Shigeta et al. 2000). The introns), and cDNA (exon 17–18), followed by poly- primary antibody, hybridoma supernatant was diluted 1:5 adenylation and termination signals of rabbit b-globin in PBS. Subsequent protocols were followed as detailed in and was inserted into pCI-neo Vector to transfect to CHO Vectastain ABC kit (Vector Laboratories, Burlingame, CA, culture cells to confirm production and secretion of the

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Downloaded from Bioscientifica.com at 09/27/2021 08:03:09PM via free access Transgenic mice incorporated with pig ZP2 gene 459 recombinant pig ZP2. A band of 120–140 kDa was RNAs were isolated from 12-day-old ovary, uterus, detected with a specific antibody to the pig ZP2 in the kidney, liver, spleen, lung, heart, and testis. A single - supernatant from the transfected cells (Fig. 1B). One specific transcript was detected in ovarian RNA hundred and forty-eight fertilized mouse eggs were (Fig. 1D). The probe used in this assay did not hybridize microinjected with the transgene following which 104 to the wild-type ovary. eggs were transplanted into the oviducts of pseudo- pregnant recipients. The pig ZP2 transgene was analyzed Expression of pig ZP2 on the surface of the oolemma by PCR using DNA extracted from the tail of the offspring. Four out of 36 newborn animals were positive Immunohistochemical studies revealed that pig ZP2 was in this analysis showing a 149-bp DNA fragment expressed in the growing oocytes from the transgenic (Fig. 1C). Homozygous transgenic mouse lines were mice at prepuberty and puberty (Fig. 2B and C). pZP2 was established from three of the positive animals (two males detected in the growing oocytes of transgenic mice. In and, one female). both cases, positive reactions were localized inside the oocytes and at their peripheral regions, suggesting that pZP2 was synthesized in the oocytes and transported Ovary-specific expression of pig ZP2 transcript towards the cell membrane. At a higher magnification, Northern blot analysis was performed to examine the the reaction was found in a more inward region (Fig. 2F) tissue-specific expression of pig ZP2. The totalTotal than that by periodic acid–Schiff’s reagent or the

Figure 2 Immunocytochemical localization of pig ZP2 in ovaries from transgenic mice (pZP2 (C/C)). The ovarian sections (A, B and C) were stained by MAb-5H4 that is specifically reactive to pig ZP2. Section A is from a wild-type mouse at 4-weeks old. No positive reaction was observed. B and C are from the transgenic mice at 4- and 8-weeks old respectively. D–F are serial sections from the transgenic mice at 4-weeks old, stained with periodic acid–Schiff’s reagent, anti-mouse ZP2 antibody, and MAb-5H4. D and E show the zona pellucida area. The positive reaction in F was detected inside the oocyte compared with D and E. Bars are 25 mm. www.reproduction-online.org Reproduction (2006) 132 455–464

Downloaded from Bioscientifica.com at 09/27/2021 08:03:09PM via free access 460 A Hasegawa and others anti-mouse ZP2 antiserum (Fig. 2D and E). No staining Introduction of pig ZP2 gene into ZP2-knockout mice area between immuno-positive region and granulosa To produce pig ZP2-expressing and mouse ZP2-deficient cells probably corresponds to the zona pellucida. These mice, ZP2-knockout male mice (mZP2 (K/K)) were results, however, do not indicate whether the pig ZP2 crossed with female mice (pZP2 (C/C)). Progeny having protein was secreted from the oocyte or still remains in the various genotypes were obtained as detected by PCR peripheral region of the oocyte. Toclarify this, live ovarian (Fig. 1D). The bands of 315, 188, and 149 bp represents oocytes were stained by the antibody specific to pig ZP2. were amplified from ZP2-knockout mouse gene, wild- A positive reaction was observed in the oocyte cell type mouse ZP2, and transgenic pig ZP2 respectively. membrane and the inner portion of the zona pellucida Ovulated eggs from (pZP2 (C/C), mZP2 (C/C)), (pZP2 (Fig. 3B), whereas the ovulated eggs did not show the (C/C), mZP2 (C/K)), and (pZP2 (C/C), mZP2 (K/K)) positive reaction (Fig. 3E). The positive control antibody mice were observed under an optical microscope (Fig. 4). to mouse ZP2 clearly showed the zona pellucida both in The zona pellucida was absent around the eggs from the growing oocytes and ovulated eggs (Fig. 3C and F). These ZP2-knockout mice that expressed the pig ZP2 gene results indicated that pig ZP2 was synthesized and (Fig. 4C). The heterozygous ZP2-targeted mice, on secreted, but not incorporated into mouse zona pellucida the other hand, formed the zona pellucida (Fig. 4B), in matured eggs. The protein seems to pass through the although it was thinner than that of the wild-type mice zona pellucida during oocyte maturation. (Fig. 4A).

Figure 3 Confocal microscopic observation of live ovarian oocytes and ovulated eggs from the transgenic mice containing the pig ZP2 gene. Ovarian oocytes (A–C) and ovulated eggs (D–E) were isolated from the transgenic mice with pZP2 (C/C) gene and stained with normal rabbit serum (A and D), MAb-5H4 specific for pig ZP2 (B and E) and the antibody specific for mouse ZP2 (C and F). A and D represent negative controls, and C and F represent positive controls. The positive reactions by MAb-5H4 were detected in the oocyte cell membrane and inside the zona pellucida (B) but not in ovulated eggs (E). Bars are 30 mm.

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Figure 4 Optical microphotographs of ovulated eggs recovered from the ZP2-knockout mice containing pig ZP2 gene. A–C are ovulated eggs collected from mice of three genotypes, wild-type (pZP2 (C/C), mZP2 (C/C)), heterozygous (pZP2 (C/C), mZP2 (C/K)), and homozygous (pZP2 (C/C), mZP2 (K/K)) respectively. Zona pellucida was not observed around the eggs recovered from the (pZP2 (C/C), mZP2 (K/K)) mouse (C). Photographs were taken under an inverted microscope equipped with Hoffman module (original magnification, !100). Bar is 25 mm.

When the numbers of ovulated eggs were examined Discussion after superovulation, those from ZP2-knockout mice This study examined whether a foreign zona protein of pig were significantly fewer than those from wild-type mice, ZP2, an ortholog of mouse ZP2, could be assembled with despite the presence of pig ZP2 gene (a versus c in mouse ZP1 and ZP3 to constitute a chimeric zona Table 1). Ovulation of ZP2-knockout mice was not pellucida. In the four pig ZP2 transgenic mouse lines significantly improved by incorporation of the pig ZP2 C C established, we detected pig ZP2 protein in the growing gene (a versus b). Heterozygous mice (pZP2 ( / ), oocyte and interspaces of granulosa cells in the transgenic mZP2 (C/K)) showed a similar number of ovulated eggs C C C C mouse follicles by immunocytochemical analysis. It was to ZP2 normal mice (pZP2 ( / ), mZP2 ( / )) with or found that the 1.4 kb fragment locating in the 50-flanking without pig ZP2 gene. Taken together, these results show region of mouse ZP2 functioned as a promoter that that transgenesis of the pig ZP2 gene did not restore zona regulated ovary-specific expression of the following pig pellucida formation, granulosa cell proliferation, and ZP2 region as shown by the luciferase reporter assay using oocyte growth. mouse oocytes (Liang & Dean 1993). The present study showed that the transgene, pig ZP2, Immunohistochemical staining of ZP2-knockout and was expressed in the transgenic mice and ZP2-knockout pig ZP2-incorporated mouse ovaries mice, but could not restore the deficiency of the Protein expression of pig ZP2 was detected in the oocyte zona pellucida architecture of the ZP2-knockout mice. peripheral regions from mice of three genotypes: (pZP2 The protein was localized around the oocyte cytoplasm, (C/C), mZP2 (C/C)), (pZP2 (C/C), mZP2 (C/K)), and inner part of the zona pellucida, and interspaces of follicular cells. A study reported that mouse ZP2 is (pZP2 (C/C), mZP2 (K/K)) (Fig. 5A–C). Not stained area present on the plasma membrane of the egg from the around the oocytes observed in sections A and B but not C ZP3-knockout mice but did not deposit around the probably indicates the zona pellucida. Sections D and E from the (pZP2 (C/C), mZP2 (C/K)) mice were stained with periodic acid–Schiff’s reagent and anti-mouse ZP2 Table 1 Number of ovulated oocytes from transgenic mice with different genotypes. antibody respectively, to show the zona pellucida area. Compared to these clear stainings of zona pellucida, Genotype pZP2 protein in the (pZP2 (C/C), mZP2 (K/K)) mouse pZP2 mZP2 ZP formation* MeanGS.D. section was localized in the oocyte cytoplasma and the a spaces of the follicular cells (Fig. 5F). These results C/CK/K No 2.5G4.7 (nZ6) C C C/CC/K Yes 30.3G13.0 (nZ6) suggested that the pZP2 protein of the (pZP2 ( / ), C/CC/C Yes 29.0G17.2 (nZ5) mZP2 (K/K)) mouse is diffusely expressed around the K/KK/K No 4.3G2.7 (nZ6)b K/KC/K Yes 28.0G5.7 (nZ5) oocyte as well as the follicular spaces. This expression K KCC G Z pattern of pZP2 resembled that of pig ZP2 transgenic mice / / Yes 26.8 16.7 (n 4) (pZP2 (C/C), mZP2 (C/C)) (Fig. 2). *Optical microscopic observation. Not significant: t-test (a versus b). www.reproduction-online.org Reproduction (2006) 132 455–464

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Figure 5 Immunocytochemical localization of pig ZP2 protein in ovaries from ZP2-knockout mice containing the pig ZP2 gene. The ovarian sections (A–C) are from the (pZP2 (C/C), mZP2 (C/C)), (pZP2 (C/C), mZP2 (C/K)), and (pZP2 (C/C), mZP2 (K/K)) mice respectively, stained with MAb-5H4 that is specifically reactive to pig ZP2. Positive reactions for pig ZP2 were detected in the oocyte from mice with these three different genotypes. The white area around the oocytes in A and B seem to correspond to zona pellucida. D and E are ovarian sections from the (pZP2 (C/C), mZP2 (C/K)) mice stained with periodic acid–Schiff’s reagent and anti-mouse ZP2 antibody respectively. These two sections show the zona pellucida area in heterologous ZP2-targeted mice. F is an ovarian section from the (pZP2 (C/C), mZP2 (K/K)) mouse stained with MAb-5H4. Positive reactions were observed inside of the oocyte and in the spaces among follicular cells. Bars are 25 mm. oocyte. They argued that the three mouse zona proteins in ovarian oocytes, but was not in ovulated eggs. These are synthesized and secreted independently and results suggest that the pig ZP2 is produced contiguously assemble spontaneously to form the zona pellucida in the growing oocytes, temporarily stays on the plasma outside the oocyte (Qi & Wassarman 1999). This is membrane, and diffuses out from oocytes without consistent with the state of pig ZP2 unable to assemble to incorporation into the zona pellucida. Increased fluidity mouse zona pellucida components in the present study. and change in oocyte plasma membrane compositions Other reports have documented that zona proteins following oocyte maturation may be due to the complete were extensively detected outside the zona pellucida shed-out of the protein. area by immunohistochemical examination (Takagi et al. Actually, mRNA and protein synthesis of ZP proteins 1989, Dunbar et al. 1994). In our findings, pZP2 protein gradually increases during oogenesis and significantly was detected in the intercellular spaces of oocyte and decreases after ovulation (Liang et al. 1990). The foreign granulosa cells, indicating that pZP2 was immersed in pig ZP2 seems to be controlled under this expression the spaces passing through the zona pellucida (Figs 2F regulation. Similar observations have been reported with and 5F), because ZP2 protein was synthesized by the ZP2-knockout mice (Rankin et al. 2001). They showed oocytes but not granulosa cells in mice (Bleil & that less zona pellucida was formed around mature Wassarman 1980, Shimizu et al. 1983). oocytes in large follicles than in smaller follicles and Confocal microscopic observations using live oocytes suggested that the disruption of the zona pellucida and eggs showed that pig ZP2 was expressed outside of compromised the ability of follicles to continue to the cell membrane and inner part of the zona pellucida develop into antral follicles.

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The failure of the pig ZP2 assembly to the transgenic Acknowledgements mouse zona pellucida was possibly due to the lower This work was supported by Grant-in-Aid of Scientific Research affinity to mouse ZP1/ZP3 compared with ZP2. The (No. 14370536) from MEXT (Ministry of Education, Science, possibility was tested by introducing the pig ZP2 gene into Culture, Sports, and Technology), 2002–2004 and by the ‘High- ZP2-knockout mice, which lack zona pellucida in Tech Research Center’ Project for Private Universities, ovulated eggs. It was found that the pig ZP2 gene did matching fund subsidy from MEXT, 2004–2008. We are grateful not result in the formation of zona pellucida in ZP2- to Dr M Okabe (Genome Information Research Center, Osaka knockout mouse, indicating that the pig ZP2 did not University) for advice in producing transgenic mice. The assemble with mouse ZP1 and ZP3. authors declare that there is no conflict of interest that would In contrast to our result, Rankin et al. have shown that prejudice the impartiality of this scientific work. human ZP2 and ZP3 can form a chimeric zona pellucida in mice (Rankin et al. 1998, 2003). Why did References pig ZP2 not form a chimeric zona pellucida in polymerization with mouse ZP1 and ZP3? A compari- Berger T, Davis A, Wardrip NJ & Hedrick JL 1989 Sperm binding to the son of the primary structures indicates that 11 cysteine pig zona pellucida and inhibition of binding by solubilized components of the zona pellucida. Journal of Reproduction and residues are entirely conserved and 6 consensus Fertility 86 559–565. sequences (Asn–Xaa–Thr) of N-glycosylation sites are Bleil JD & Wassarman PM 1980 Synthesis of zona pellucida proteins by also present in ZP2 of mice, humans, and pigs. denuded and follicle-enclosed mouse oocytes during culture in vitro. Comparing each orthologous sequence, the amino PNAS 77 1029–1033. acid sequence of pig and human are 54 and 60% Bleil JD, Beall CF & Wassarman PM 1981 Mammalian sperm–egg interaction: fertilization of mouse eggs triggers modification of the identical to that of mouse ZP2 respectively (Harris et al. major zona pellucida glycoprotein, ZP2. Developmental Biology 86 1994). The difference (6%) is insufficient as evidence 189–197. that pig ZP2 did not assemble to mouse zona pellucida. Bleil JD, Greve JM & Wassarman PM 1988 Identification of a Xenopus laevis zona pellucida could assemble to each secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs. mouse zona protein (ZP1, ZP2, and ZP3) with 39–48% Developmental Biology 128 376–385. amino acid similarity (Doren et al. 1999). The absence Bork P & Sander C 1992 A large domain common to sperm receptors or the replacement in critical site(s) probably affected (Zp2 and Zp3) and TGF-beta type III receptor. FEBS Letters 300 zona assembly of pig ZP2. 237–240. Chakravarty S, Suraj K & Gupta SK 2005 Baculovirus-expressed Recently, however, hydrophobic segments of the recombinant human zona pellucida glycoprotein-B induces acro- mouse ZP domain in ZP1, ZP2, and ZP3 proteins somal exocytosis in capacitated spermatozoa in addition to zona have been reported to be important for assembly of pellucida glycoprotein-C. Molecular Human Reproduction 11 these zona proteins (Jovine et al. 2004). This domain 365–372. Dean J 2002 Oocyte-specific genes regulate follicle formation, fertility plays fundamental roles not only in the zona pellucida and early mouse development. Journal of Reproductive Immunology surrounding the oocyte but also in the various 53 171–180. extracellular matrices associated with development, Doren S, Landsberger N, Dwyer N, Gold L, Blanchette-Mackie J & immunity, and cancer (Jovine et al. 2002). They Dean J 1999 Incorporation of mouse zona pellucida proteins into the suggested that a hydrophobic amino acid sequence of envelope of Xenopus laevis oocytes. Development Genes and Evolution 209 330–339. PGPLVLV (483–489) in mouse ZP2 located in the Dunbar BS, Avery S, Lee V, Prasad S, Schwahn D, Schwoebel E, domain is crucial for protein secretion. The correspond- Skinner S & Wilkins B 1994 The mammalian zona pellucida: its ing sequence is replaced by PGPLTLT in pig ZP2. biochemistry, immunochemistry, molecular biology, and develop- The change in the two sites of hydrophobic amino acid mental expression. Reproduction, Fertility, and Development 6 331–347. (Valine) to hydroxyl amino acid (Threonine) that Erbach GT, Lawitts JA, Papaioannou VE & Biggers JD 1994 Differential is potentially bound to the O-linked carbohydrate growth of the mouse preimplantation embryo in chemically defined may be crucial to form chimeric zona pellucida of media. Biology of Reproduction 50 1027–1033. mice and pigs. Harris JD, Hibler DW, Fontenot GK, Hsu KT, Yurewicz EC & Sacco AG 1994 Cloning and characterization of zona pellucida genes and In conclusion, the present study indicates that pig cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC ZP2 is expressed in transgenic mouse oocytes, but gene families. DNA Sequence 4 361–393. neither participates in mouse zona pellucida architec- Hasegawa A, Koyama K, Okazaki Y, Sugimoto M & Isojima S 1994 ture nor restores the zona pellucida in ZP2-knockout Amino acid sequence of a porcine zona pellucida glycoprotein ZP4 determined by peptide mapping and cDNA cloning. Journal of mice despite the presence of the other two zona Reproduction and Fertility 100 245–255. proteins. The difference of amino acids in the Hasegawa A, Tsubamoto H, Hamada Y & Koyama K 2000 Blocking hydrophobic region of the ZP domain would be crucial effect of antisera to recombinant zona pellucida proteins (r-ZPA) on for the assembly. Further investigations including site- in vitro fertilization. American Journal of Reproductive Immunology directed mutagenesis are necessary to find the sequence 44 59–64. Hatanaka Y, Nagai T, Tobita T & Nakano M 1992 Changes in the responsible for incorporation of pig ZP2 into mouse properties and composition of zona pellucida of pigs during zona pellucida. fertilization in vitro. Journal of Reproduction and Fertility 95 431–440. www.reproduction-online.org Reproduction (2006) 132 455–464

Downloaded from Bioscientifica.com at 09/27/2021 08:03:09PM via free access 464 A Hasegawa and others

Hedrick JL & Wardrip NJ 1986 Isolation of the zona pellucida and Rankin TL, O’Brien M, Lee E, Wigglesworth K, Eppig J & Dean J 2001 purification of its glycoprotein families from pig oocytes. Analytical Defective zonae pellucidae in Zp2-null mice disrupt folliculogen- Biochemistry 157 63–70. esis, fertility and development. Development 128 1119–1126. Jovine L, Qi H, Williams Z, Litscher E & Wassarman PM 2002 The ZP Rankin TL, Coleman JS, Epifano O, Hoodbhoy T, Turner SG, Castle PE, domain is a conserved module for polymerization of extracellular Lee E, Gore-Langton R & Dean J 2003 Fertility and taxon-specific proteins. Nature Cell Biology 4 457–461. sperm binding persist after replacement of mouse sperm receptors Jovine L, Qi H, Williams Z, Litscher ES & Wassarman PM 2004 A with human homologs. Developmental Cell 5 33–43. duplicated motif controls assembly of zona pellucida domain Shigeta M, Hasegawa A, Hamada Y & Koyama K 2000 Analysis of B proteins. PNAS 101 5922–5927. cell epitopes of a glycoprotein porcine zona pellucida (pZP1). Koyama K, Hasegawa A, Inoue M & Isojima S 1991 Blocking of human Journal of Reproductive Immunology 47 159–168. sperm-zona interaction by monoclonal antibodies to a glycoprotein Shimizu S, Tsuji M & Dean J 1983 In vitro biosynthesis of three sulfated family (ZP4) of porcine zona pellucida. Biology of Reproduction 45 glycoproteins of murine zonae pellucidae by oocytes grown in 727–735. follicle culture. Journal of Biological Chemistry 258 5858–5863. Lefievre L, Conner SJ, Salpekar A, Olufowobi O, Ashton P, Pavlovic B, Sun W, Lou YH, Dean J & Tung KS 1999 A contraceptive peptide Lenton W & Afnan M 2004 Four zona pellucida glycoproteins are vaccine targeting sulfated glycoprotein ZP2 of the mouse zona expressed in the human. Human Reproduction 19 1580–1586. pellucida. Biology of Reproduction 60 900–907. Liang L & Dean J 1993 Conservation of mammalian secondary sperm Takagi J, Araki Y, Dobashi M, Imai Y, Hiroi M, Tonosaki A & Sendo F receptor gene enables the promoter of the human gene to function in 1989 The development of porcine zona pellucida using monoclonal mouse oocyte. Developmental Biology 156 399–408. antibodies. I. Immunochemistry and light microscopy. Biology of Liang LF, Chamow SM & Dean J 1990 Oocyte-specific expression of Reproduction 40 1095–1102. Tsubamoto H, Yamasaki N, Hasegewa A & Koyama K 1999 Expression mouse Zp-2: developmental regulation of the zona pellucida genes. of a recombinant porcine zona pellucida glycoprotein ZP1 in Molecular and Cellular Biology 10 1507–1515. mammalian cells. Protein Expression and Purification 17 8–15. Liu C, Litscher ES, Mortillo S, Sakai Y, Kinloch RA, Stewart CL & Wassarman PM 1987 The biology and chemistry of fertilization. Wassarman PM 1996 Targeted disruption of the mZP3 gene results in Science 235 553–560. production of eggs lacking a zona pellucida and infertility in female Wassarman PM 1999 Mammalian fertilization: molecular aspects of mice. PNAS 93 5431–5436. gamete adhesion, exocytosis, and fusion. Cell 96 175–183. Qi H & Wassarman PM 1999 Secretion of zona pellucida glycoprotein Yanagimachi R 1994 Mammalian fertilization. In The Physiology of mZP2 by growing oocytes from mZP3(C/C) and mZP3(K/K) mice. Reproduction, 5th edn, pp 189–317. Eds E Knobi & JD Neil. New Developmental Genetics 25 95–102. York: Raven Press. Rankin T, Familari M, Lee E, Ginsberg A, Dwyer N, Blanchette- Yurewicz EC, Sacco AG, Gupta SK, Xu N & Gage DA 1998 Hetero- Mackie J, Drago J, Westphal H & Dean J 1996 Mice homozygous for oligomerization-dependent binding of pig oocyte zona pellucida an insertional mutation in the Zp3 gene lack a zona pellucida and glycoproteins ZPB and ZPC to boar sperm membrane vesicles. are infertile. Development 122 2903–2910. Journal of Biological Chemistry 273 7488–7494. 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 Received 1 November 2005 affecting order-specific sperm binding. Development 125 2415–2424. Rankin T, Talbot P, Lee E & Dean J 1999 Abnormal zonae pellucidae in First decision 1 December 2006 mice lacking ZP1 result in early embryonic loss. Development 126 Revised manuscript received 12 June 2006 3847–3855. Accepted 22 June 2006

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