Mol. Hum. Reprod. Advance Access originally published online on May 28, 2004
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Molecular Human Reproduction, Vol. 10, No. 7, pp. 481-488, 2004
Molecular Human Reproduction vol. 10 no. 7 © European Society of Human Reproduction and Embryology 2004; all rights reserved
Localization and synthesis of zona pellucida proteins in the marmoset monkey (Callithrix jacchus) ovary
1Center of Dermatology and Andrology, University of Giessen, Gaffkystr. 14, D-35392 Giessen, Germany 2Department of Reproductive Biology, German Primate Center, 37077 Göttingen, Germany 3Center of Urology, University of Marburg, 35043 Marburg, Germany
4 To whom correspondence should be addressed.; Email: klaus-dieter.hinsch{at}derma.med.uni-giessen.de
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Abstract |
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In most species, the zona pellucida (ZP), an extracellular matrix surrounding the mammalian oocyte, is composed of three glycoproteins: ZPA, ZPB and ZPC. Based mainly on results with mice, the site of zona pellucida biosynthesis has been suggested to be exclusively in the oocyte cytoplasm. However, evidence is accumulating that among various species cumulus/granulosa cells may be involved. Because knowledge of ZP biosynthesis in primates is lacking, we used the common marmoset (Callithrix jacchus) to acquire information about the localization and the site of synthesis of ZP proteins in this species. Using antibodies against synthetic ZPA and ZPC peptides, immunoreactivity was found in the marmoset ZP and in surrounding cumulus cells. Interestingly, the amounts of ZPA and ZPC proteins expressed appeared to differ depending on the stage of folliculogenesis. RT–PCR analysis of mRNA from marmoset oocytes and from oocyte-free follicle cells revealed expression of ZPA, ZPB and ZPC in oocytes and in follicle cells of different stages of marmoset monkey folliculogenesis. Our data suggest that the biosynthesis of marmoset ZPA, ZPB and ZPC proteins takes place both in oocytes and in follicle cells of different follicle stages, although the abundance of ZP glycoproteins may differ depending on the individual ZP protein.
Key words: follicle cells/marmoset/zona pellucida/oocyte ZP antisera/ZP mRNA
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Introduction |
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The zona pellucida (ZP) is a translucent glycoprotein matrix enclosing the mammalian oocyte and the early embryo until implantation and plays a critical role in the process of fertilization. For example, the ZP is the site of the sperm acrosome reaction. It provides specific binding sites for sperm and the alteration in structure after sperm–oolemma contact prevents polyspermia (Yanagimachi, 1994
). After fertilization, the ZP is required to maintain the juxtaposition of the precompaction blastomeres as well as to protect the early embryo against physical damage and detrimental external influences (Herrler and Beier, 2000).
Depending on the species, the ZP consists of three to four different glycoproteins encoded by three classes of ZP genes (Hasegawa et al., 1991; Topper et al., 1997; Sinowatz et al., 2001; Spargo and Hope, 2003) that serve different functions. The proteins show considerable micro-heterogeneity due to differences in glycosylation. In mouse and in human, the ZP is mainly composed of three ZP glycoproteins (Bleil and Wassarman, 1980; Bercegeay et al., 1995) whose functions appear to differ, although differences between species may occur. In the mouse, it has been shown that ZPB is a homodimeric filament crosslinker that stabilizes the heterodimeric ZPA/ZPC protein network (Green, 1997). However, binding of recombinant bonnet monkey (Macaca radiata) ZPB (which is different from the mouse ZPB1 gene) to capacitated as well as to acrosome-reacted sperm has been reported and thus may indicate a functional role of ZPB during fertilization (Govind et al., 2001). This is of interest because it has been demonstrated in several species, including mouse and human, that it is ZPC that mediates primary sperm–ZP binding and induction of the acrosome reaction (K.D.Hinsch and E.Hinsch, 1999; Miller et al., 2002; Wassarman, 2002), while ZPA has been shown to serve as a secondary receptor (E.Hinsch et al., 1997; Tsubamoto et al., 1999; Howes et al., 2001; Jansen et al., 2001; Howes and Jones, 2002). Similar to other non-rodent species studied, three ZP proteins have been demonstrated for two Old World non-human primate species [e.g. the bonnet monkey (Macaca radiata) and the cynomolgus monkey (Macaca fascicularis)] (Kolluri et al., 1995; Paterson et al., 1996; Gupta et al., 1997; Jethanandani et al., 1998), as well as for the marmoset (Callithrix jacchus) (EMBL-database, accession no. Y10822, Y10767, S71825). However, several studies in different species suggest a possibility of functional differences of individual glycoproteins during fertilization compared with the mouse (Prasad et al., 2000).
It is not only function that may differ between species groups; although information is still incomplete, the site of de novo synthesis of ZP glycoproteins may also differ. For example, expression of mouse ZP proteins has been shown exclusively for the oocyte (Epifano et al., 1995; El Mestrah et al., 2002), while in other mammals (rabbit, dog, pig and cow), ZP protein expression has also been reported in follicle cells (Sinowatz et al., 2001). For some avian species, in contrast, the synthesis of a mammalian ZPC protein homologue seems to be restricted to granulosa cells (Waclawek et al., 1998; Takeuchi et al., 1999; Sasanami et al., 2002).
For mammals, although information on the sequences of the ZP proteins exists, knowledge about the site and timing of synthesis of the ZP proteins is scarce (Roller et al., 1989; Epifano et al., 1995; Martinez et al., 1996; Jewgenow and Fickel, 1999). In cynomolgus monkeys the contribution of granulosa cells to the expression of ZP genes has been demonstrated by Martinez et al. (1996). ZPA and ZPC mRNA was localized in granulosa cells, whereas in these cells no ZPB mRNA was found at any stage of folliculogenesis (Martinez et al., 1996). To date, in the marmoset monkey only the synthetic site of one single zona pellucida protein, ZPC, has been described. In this study, mRNA expression was shown to be exclusively localized in the oocyte (Thillai-Koothan et al., 1993) in contrast to results reported for the cynomolgus monkey (Martinez et al., 1996).
The aim of the present study was therefore to provide information about ZP gene transcription and ZP protein localization in the marmoset ovary to serve as a basis for studies of factors influencing ZP synthesis throughout folliculogenesis.
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Collection of oocytes and follicle cells
The ovarian cycle of female marmoset monkeys was monitored via cyclic changes in plasma progesterone concentration. Ten to 14 days postovulation, luteolysis was induced by the i.m. application of 0.8 µg of cloprostenol, an analogue of prostaglandin F2
(Estrumate; Pitman–Moore, Germany). Ovary pairs were collected on day 5 or 6 of the follicular phase (day 0 = day of injection of prostaglandin F2) as previously described (Gilchrist et al., 1995) in Leibovitz L-15 medium (GibcoBRL, Germany) with 5% heat-inactivated fetal bovine serum (FBS; Biochrom KG, Germany), supplemented with 0.5 mmol/l sodium pyruvate and 5% gentamycin sulphate (Sigma Aldrich Chemie, Germany). After removal of excess tissue, fat and blood, follicles were dissected in Leibovitz L-15 medium using watchmaker's forceps under a dissecting microscope on a heated stage at 37°C under laminar flow and sorted according to the presence or absence of antrum and size. The diameter of follicles was measured with a calibrated ocular micrometer. Preantral follicles were defined as follicles with a diameter <400 µm (class 1). The periantral and antral follicles were grouped according to size: class 2 (400–599 µm), class 3 (600–999 µm), and class 4 (
1000 µm) as described by Gilchrist et al. (1995). Based on criteria of granulosa cell colour, density, and uniformity of distribution, follicles judged to be atretic were discarded. The selected follicles were then punctured. The oocytes were mechanically stripped of their surrounding cumulus cells and collected and stored separately from cumulus and granulosa cells. Careful microscopical examination of the oocytes ensured that neither cumulus cells nor cumulus cell residues were attached to the cell samples. Both oocytes and follicle cells were stored in RNAlater® (Ambion, UK) at –20°C.
Anti-ZP-peptide antisera
Generation and characterization of the antisera (AS) against synthetic ZPA and ZPC peptides have been described previously (K.D.Hinsch et al., 1994; E.Hinsch et al., 1997, 1998a,b). We also generated antisera against synthetic marmoset ZPB peptides. As a result, we obtained by enzyme-linked immunosorbent assay high antibody titres against the peptide employed, but using immunoblotting and immunohistochemical techniques the antisera did not react with the ZPB protein.
Immunohistochemistry
Intact marmoset ovaries were fixed in Methacarn, cut into sections of 4 µm, and processed as previously described (K.D.Hinsch et al., 1994; E.Hinsch et al., 1999). Specimens were treated with anti-ZPA and -ZPC antibodies (AS ZP2-20 diluted 1:50; AS ZP3-6 diluted 1:100) for 60 min. Antigen–antibody reaction was visualized with anti-rabbit Ig antibodies (dilution 1:100; Dako Diagnostica, Germany) using the peroxidase–antiperoxidase method (Dako) and 3,3'-diaminobenzidine (DAB; Sigma Aldrich Chemie, Germany) as colour substrate. Finally, sections were counterstained with haematoxylin (Sigma Aldrich Chemie). As negative control, sections were incubated with preimmune serum diluted 1:50 and processed as described above.
Immune transmission electron microscopy
Generation of ultrathin sections and immunodetection of ZP proteins has been described previously (E.Hinsch et al., 1998b). Briefly, marmoset ovaries were fixed and subsequently embedded in LR-White (Plano, Germany). Ultrathin sections were cut on an LKB ultramicrotome (Sweden) and collected on nickel grids (Plano). After blocking of non-specific binding sites, sections were exposed to antisera for 75 min (AS ZP2-20 diluted 1:10; AS ZP3-6 diluted 1:5); as negative controls, corresponding preimmune sera in the same dilutions as the antisera were used. Subsequently, sections were incubated with biotinylated anti-rabbit IgG (Sigma Aldrich Chemie; diluted 1:20 for AS ZP2-20 and 1:40 for AS ZP3-6) for 30 min followed by incubation with streptavidin conjugated to colloidal gold (particle size of 20 nm; 1:40 dilution; Bio Cell, UK) for 30 min at room temperature.
Sections were then washed and fixed in 1% (v/v) glutaraldehyde/Tris-buffered saline for 5 min. Finally, the probes were stained for 20 min with 2% (w/v) uranyl acetate and examined and photographed using a Zeiss transmission electron microscope LEO 906.
RT–PCR of ZP mRNA
mRNA from isolated oocytes and oocyte-free follicle cells from marmoset ovaries was prepared using an MPG guanidine direct mRNA purification kit (CPG, USA). The following two types of cell samples were assayed: oocytes derived from follicles with defined diameters (class 1, 2, 3 and 4) and the corresponding oocyte-free follicle cells. For each class, mRNA from seven oocytes and their respective follicle cell masses was used.
An RT reaction was performed with Omniscript RT Kit (Qiagen, Germany) according to the manufacturer's instructions. Subsequently, 2 µl of each RT reaction product (cDNA) was used for PCR in a final volume of 50 µl containing 2.5 IU Taq polymerase (PanScript DNA Polymerase; Pan-Biotech, Germany), NH4-buffer [16 mmol/l (NH4)2SO4, 50 mmol/l Tris–HCl pH 8.8, 0.1% Tween 20], 2 mmol/l MgCl2 (all from Pan-Biotech), 0.25 mmol/l dNTP (Gene-craft, Germany) and 100 pmol of each primer (Table I). After denaturation for 4 min at 94°C, amplification was carried out for 35 cycles at 94°C for 45 s, and primer annealing was performed according to specific annealing temperatures for 45 s, at 72°C for 90 s, and with a final extension at 72°C for 5 min. The concentration of all components as well as the specific annealing temperatures was optimized. As negative control, PCR was performed with distilled water instead of the RT reaction product. Amplification products were sequenced by MWG Biotech (Germany).
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Primer sequences were determined by applying PC/gene software (IntelliGenetics). All three marmoset ZP gene-specific primer pairs were deduced from published marmoset sequences (accession no. Y10822, Y10767, S71824). As positive control, mRNA amplification of the housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase (HGAPDH, accession no. NM_002046) was performed. In addition to the HGAPDH primer pair, a second primer pair (HIGAPDH) based on the human DNA sequence of GAPDH (accession no. J04038) was designed to search for possible genomic DNA (gDNA) contamination within the probes.
Besides these controls, expression of an oocyte-specific protein gene, BMP15 (Table I
), was used in order to determine if contaminating oocyte mRNA was present in the follicle cell preparation (Dube et al., 1998; Braw-Tal, 2002).
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Immunohistochemical localization of ZPA and ZPC proteins
In previous studies, antisera were generated against synthetic peptides corresponding to highly conserved ZPA and ZPC epitopes. Here, the reactivity of these antisera, anti-ZPA antiserum AS ZP2-20 and anti-ZPC antiserum AS ZP3-6, were analysed in sections of marmoset ovaries.
ZPA localized in marmoset ZP and follicle cells varied according to the stage of folliculogenesis (Figure 1A, A1–A3). In the layer of cells that surrounds the primordial follicles, sporadic staining for ZPA was observed (Figure 1, A1, I). Primary follicles displayed immunoreactivity in the early developing ZP as well as in the ooplasm and in the surrounding follicle cell layer (Figure 1, A1, II). For later preantral developmental stages (Figure 1, A2, III and A3, IV) a positive reaction continued to be detected in all three regions. In small preantral follicles (A2, III) as well as in large preantral follicles (A3, IV), staining of the ZP appeared as a homogeneous, brownish ring of substrate deposition surrounding the oocyte. A faint but definitive staining was observed in the ooplasm of the oocytes of these follicles (A1 and A2, asterisk). Additionally, a strong colour development was detected within the marmoset follicle cells of both early and late preantral follicles (A2, A3). An intense color development within the ZP as well as in the surrounding follicle cells was also observed in small antral follicles (not shown).
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= ooplasm; ZP = zona pellucida; FC = follicle cells; I = primordial follicle; II = primary follicle; III = small preantral follicle; IV = large preantral follicle; V = small antral follicle.With the exception of primordial follicles, ZPC protein was mainly found in the ZP of marmoset oocytes independent of the developmental stage (Figure 1, B
), with only faint staining observed in follicle cells of all developmental stages. ZPC was detected in oocytes of primary follicles (B1, II) as well as in small (B2, III) and large preantral follicles (B3, IV) and small antral follicles (B3, V). The ZP was visualized as a distinct brown ring. Compared to ZPA, less immunolabelling was visible in the ooplasm (B2 and B3, asterisks). In the follicle cells of primordial (B1, I), primary (B1, II), and small preantral follicles (B2, III), little colour deposition was observed, which suggested a low level of ZPC protein immunoreactivity. Only faint staining of follicle cells, slightly above background, was visible in large preantral and small antral follicles (B3, IV and V). Sections treated with the preimmune serum did not display any specific colour development (Figure 1, C, C1–C3).
Immune transmission electron microscopy
The ultrastructure of the marmoset zona pellucida and the distribution of ZPA and ZPC were investigated with immunoelectron microscopy using anti-ZP antisera AS ZP2-20 and AS ZP3-6 (Figure 2). In ultrathin sections of preantral follicles from marmoset ovaries, ZPA protein was localized in the zona pellucida (ZP) and in cumulus cells (CC) (Figure 2A) as well as in the ooplasm (not shown). The distribution of gold particles in the ZP matrix (ZP) appeared as a uniform and homogeneous pattern (Figure 2A, arrowheads). Gold particles were also observed in the cytoplasm (CP) of the follicle cells (Figure 2D, arrowheads). Immunoreactive material in the follicle cells was less abundant than in the ZP but clearly above background levels. Only a few gold grains, slightly above background, were observed in the ooplasm (not shown).
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= ZP/cumulus cell area of contact. Arrowheads: (A) note diffuse gold particle distribution throughout the ZP; (B) note concentration of gold particles at the inner part of ZP; (D) note gold particles in the cytoplasm of a cumulus cell.When the anti-ZPC antiserum AS ZP3-6 was applied to sections of preantral marmoset follicles, immunolabelling was also observed in the ZP of the oocyte. However, labelling distribution was much less homogeneous (Figure 2B
). A more intense immunoreaction was apparent in the inner part of the zona pellucida (arrowheads). Only small amounts of immunoreactive material were detected in the outer areas of ZP towards the follicle cells (Figure 2B, asterisk). This faint immunolabelling may be connected with the indentation of ZP and surrounding follicle cells (Figure 2B, rhombus). It is important to note that compared to the distribution of ZPA protein, the surrounding follicle cells (Figure 2E) showed much less immunoreactivity. A little immunoreactive material that was slightly above background was found in the ooplasm (not shown).
In control sections incubated with the preimmune serum, few gold grains could be observed in the ZP (Figure 2C) and in cumulus cells (Figure 2F).
RT–PCR results
RT–PCR revealed that three different ZP mRNA's are expressed in marmoset oocytes (Figure 3). In oocytes from follicles with diameters <400 µm (preantral; Figure 3A), 400–599 µm (periantral; Figure 3B), 600–999 µm (small antral follicles; Figure 3C), and 1000 µm (large antral, Figure 3D), specific intron-spanning primers (Table I) for the marmoset ZPA, ZPB and ZPC genes amplified cDNA fragments of the expected base pair lengths corresponding to ZP mRNA: 465 bp for ZPB (lane 1), 362 bp for ZPA (lane 2) and 388 bp for ZPC (lane 3) were used. Although these were not the results of a quantitative PCR analysis, the intensity of the ZPB and ZPA amplification products appeared to be more pronounced than the bands resulting from ZPC primers. As a positive control, RT–PCR for HGAPDH (Table I) resulted in an amplicon of 300 bp (Figure 3A–D, lane 4).
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Messenger RNA for ZPA, ZPB, and ZPC genes were detected in follicle cells of all follicle groups examined (Figure 4A–D
). In follicle cells from follicles with diameters <400 µm (preantral; Figure 4A), 400–599 µm (periantral; Figure 4B), 600–999 µm (small antral follicles; Figure 4C), and 1000 µm (large antral, Figure 4D), amplified cDNA fragments of the expected base pair lengths corresponding to zona pellucida mRNA were observed: 465 bp for ZPB (lane 1), 362 bp for ZPA (lane 2) and 388 bp for ZPC (lane 3). Interestingly, in follicle cells of classes 2, 3 and 4, a more prominent ZPB band was observed compared to the ZPA and ZPC bands. The positive control experiment, RT–PCR for HGAPDH, resulted in an amplicon of the expected length of 300 bp (Figure 4A–D, lane 4).
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While HIGAPDH was amplified, possible gDNA contamination in the template was excluded by the omission of the reverse trancriptase step yielding no amplification products after PCR (not shown). For the evaluation of possible oocyte mRNA contamination in templates derived from follicle cells, the occurrence of BMP 15 mRNA was examined. The use of BMP 15 primers (Table I
) did not result in amplification of a cDNA fragment (not shown). Control experiments using water instead of the template for RT–PCR revealed no amplification products (not shown). After sequencing the respective ZP amplicons, an identity of 99% for ZPB and ZPA and of 100% for ZPC with the marmoset ZP nucleotide sequences deduced from the NCBI database was observed (not shown).
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Discussion |
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In this study, we have investigated the presence and distribution of ZPA and ZPC proteins in marmoset ovarian tissue using anti-ZP-peptide antisera. At the protein level, ZPB protein could not be localized in marmost ovaries because no appropriate anti-ZPB antisera were available. We generated a number of antisera against marmoset ZPB synthetic peptides; the antibodies, however, reacted with the corresponding synthetic peptides that were used as antigen but did not detect the ZPB polypeptide. We assume that the peptide used for immunization leads to the production of antibodies that react with the linear synthetic peptide. However, the antibodies might not cross-react with the ZPB protein because the epitope is perhaps not available for antibody detection (e.g. through folding of the protein or steric hindrance because of glycosylation). Our efforts to obtain adequate anti-primate ZPB antisera from other groups were, for various reasons, also not successful. However, we succeeded in demonstrating gene expression of all three ZP mRNA (ZPB, ZPA and ZPC) by RT–PCR in the marmoset ovary.
Examination by both immunohistochemical and immune transmission electron microscopy revealed that AS ZP2-20 recognized ZPA protein in the marmoset ZP, both in oocyte cytoplasm as well as in surrounding follicle cells. In spite of apparent structural differences of the inner and outer zona pellucida indicated by the visible bilamellar morphology of marmoset ZP (Gilchrist et al., 1997), distribution of ZPA protein was relatively homogeneous over the entire zona matrix.
The antigenic ZPA detected inside the ooplasm may reflect ZPA protein synthesis and transport of ZPA during oogenesis. These findings are consistent with data from other species generally indicating the presence of immunoreactive material in the ooplasm and in the zona pellucida (Martinez et al., 1996; E.Hinsch et al., 1998b; Eberspaecher et al., 2001).
The localization of ZPA antigenic material in marmoset follicle cells indicates that ZPA protein is synthesized in these cells. This finding contrasts with results from humans (E.Hinsch et al., 1998b) and an Old World non-human primate species (cynomolgus monkey) (Martinez et al., 1996). Our RT–PCR results confirmed that ZP gene transcription appears to take place not only in marmoset oocytes but also in follicle cells, indicating a significant difference from Old World primates in ZPA synthesis. ZPA protein was found in marmoset ZP, in the ooplasm, and in follicle cells, but the abundance of ZPA protein varied depending on the stage of folliculogenesis. With the exception of primordial follicles where the oocyte did not exhibit positive immunostaining, all other stages of oocyte development studied clearly demonstrated the presence of ZPA protein and its de novo synthesis in both oocytes and follicle cells.
Interestingly, the distribution of the ZPC protein differed from ZPA. Anti-ZPC-peptide antiserum AS ZP3-6 produced strong staining in the ZP together with faint staining in the follicle cells. In contrast to Grootenhuis et al. (1996), we have shown in this study that primordial follicles exhibit faint ZPA and ZPC protein immunoreactivity in follicle cells while no circular staining around or within the oocyte could be observed. They reported that by using monoclonal antibodies against recombinant human ZPC in marmosets, small dots of immunoreactive ZP were found in 60% of the primordial follicles but granulosa cells were not stained. In the present study, however, histochemical results that were supported by RT–PCR demonstrate that in the marmoset, the ZPC gene is expressed in the follicle cells as well as in the oocytes, although the follicle cells may be less active in ZPC than in ZPA protein synthesis.
The marmoset ZP was stained in primary follicles for both ZPA and ZPC; furthermore, ZPA- and faint ZPC-stained follicle cells were visible in primary follicles. Immune transmission electron microscopy further revealed a different distribution for ZPC protein within the ZP, with a greater concentration of ZPC molecules in the inner ZP. This contrasted with the uniform distribution of ZPA protein throughout the ZP. E.Hinsch et al. (1999) demonstrated in the murine model bilamellar deposition of ZPC protein using a mouse-specific anti-ZPC antiserum, indicating the possibility of layered arrangements of specific ZP protein, even in species where the intact ZP is not visibly bilaminar and the ZP is entirely synthesized by the oocyte. To various degrees, a bilamellar structure for ZP has been described for several species, including cat (Andrews et al., 1992), mouse (Baranska et al., 1975), hamster (Keefe et al., 1997), pig and rabbit (Dunbar et al., 2001), and human (Nikas et al., 1994). This phenomenon can clearly be observed for the marmoset (Gilchrist et al., 1997). The functional significance of this bilaminar characteristic as well as the differential molecular composition is still unclear. However, in vitro sperm binding studies in the marmoset suggest that firm sperm binding occurs primarily at the interface of the two layers where a higher number of ZPC molecules are located (K.Bogner, unpublished results).
The number of species where ZP gene expression takes place in follicle cells as well as in the oocyte appears to outnumber those where ZP gene expression is found exclusively in oocytes. The only species demonstrating expression of ZPA, ZPB and ZPC mRNA exclusively by the oocyte is the mouse (Epifano et al., 1995; El Mestrah et al., 2002). In other mammalian species studied, although the oocyte is always involved, additional expression of one or more ZP genes in somatic follicular cells was observed (Sinowatz et al., 2001). Lee and Dunbar (1993) have demonstrated that in rabbit, a 55 kDa protein, which is the proposed counterpart of the murine ZPB protein, was expressed in both oocyte and granulosa cells. A similar finding has been reported for bovine ZPC and porcine ZPB (Kolle et al., 1996, 1998). In contrast, for avian species thus far investigated (chicken and the quail), the synthesis of a protein homologous to the ZPC protein can be localized exclusively to the granulosa cells (Waclawek et al., 1998; Takeuchi et al., 1999; Sasanami et al., 2002). With respect to marmoset, the cell types in the ovary that synthesize particular ZP glycoproteins vary between groups, and our knowledge is still incomplete. From Aitkin's laboratory, an oocyte-specific expression of ZPC has been demonstrated for marmosets using in situ hybridization (Thillai-Koothan et al., 1993), a result consistent with their studies of ZPC protein distribution (Grootenhuis et al., 1996). Results of both of these studies contrast with those obtained in the present study. Investigations with other monkey species showed a possible contribution of granulosa cells by the transcription of the ZPA and ZPC genes in the cynomolgus monkey (Macaca fascicularis) while ZPB distribution in this species was restricted to the oocyte (Martinez et al., 1996).
Sequential gene expression during oogenesis and folliculogenesis and communication and cooperation of different follicle cell compartments during ZP formation, as well as allocation of function to the individual ZP protein which serves either as structural and/or as functional protein, are of particular relevance for the understanding of the fertilization process. However, particular information about species-specific differences of ZP protein expression during oogenesis is not only important for the understanding of cellular and molecular events that regulate oocyte development, it also has strong implications for introducing effective, long-term culture systems for oocytes (e.g. for human in vitro fertilization or for retaining genetic diversity in rare species) and for the design of immunocontraceptive vaccines.
In this study, we demonstrated for the marmoset that ZP proteins are transcribed and expressed in almost all stages of folliculogenesis. Although no quantitative estimation of ZP protein expression (e.g. through real time PCR) is provided in this study, our data suggest that the amounts in which ZP proteins are expressed appear to differ depending on the stage of folliculogenesis. Sequential transcription and synthesis of ZP proteins has been demonstrated in several species (Roller et al., 1989; Epifano et al., 1995; Martinez et al., 1996; Jewgenow and Fickel, 1999; Prasad et al., 2000).
In the mouse, ZPB transcripts were detected after growth initiation of the oocytes; ZPA and ZPC proteins are coordinately expressed in mouse oocytes during the growth phase. During ovulation, the level of ZPC mRNA declines and is no longer detectable in fertilized eggs (Roller et al., 1989).
Because of its essential role during fertilization, the ZP is an attractive target for contraceptive vaccination. Active immunization can cause either permanent or reversible infertility. Irreversible infertility might be useful for the control of certain animal populations (e.g. dogs, stray cats, horses, or elephants). Permanent infertility would be induced by the disruption of normal ovarian oogenesis, while interfering at the level of sperm–egg interaction might lead to reversible infertility. Sterilization by vaccination with ZP proteins could be desirable for the development of animal contraceptives, but is detrimental for fertility control in humans or rare animal species. Therefore, it is important to acquire knowledge about the site and time course of expression of individual ZP proteins that are targets for immune contraception during ovarian follicular development.
In the marmoset ovary, we demonstrated that ZPA but not ZPC protein can be detected in the layer of cells that surrounds the primordial follicle, indicating that sequential ZP protein expression occurs at this stage of folliculogenesis. ZPA antigens in marmoset primordial follicles could induce irreversible infertility because the destruction of early follicles would exhaust the resting oocyte pool. If reversible infertility should be desired in this species, ZPC might be a more suitable contraceptive agent.
Sequential expression of zona pellucida protein genes during oogenesis has also been reported for the domestic cat (Jewgenow and Fickel, 1999). In this species, initial expression of ZP proteins was observed in some primary follicles and in secondary follicles. ZPB was expressed in growing primary follicles, while all three ZP mRNA were detectable in secondary follicles. In the cynomolgus monkey, ZPC was expressed as early as the primordial follicle stage, and ZPA was detected in primary follicles; ZPB, however, was detected first in the secondary follicle stage. The results obtained in our study support the hypothesis that species specificity in ZP expression during oogenesis is evident and that the time frames of ZP protein synthesis differ between species.
To the best of our knowledge, this is the first study investigating ZPB, ZPA and ZPC gene expression in the marmoset monkey using RT–PCR. This method has proven to be very effective for determination of qualitative mRNA transcription. Results demonstrated unequivocally that both the oocyte and follicle cells express ZPB, ZPA and ZPC. Because this is not a quantitative method, conclusions about the differential expression of these proteins throughout the different stages of follicular development cannot yet be made. This also holds true for the level of expression of the three ZP proteins in oocytes or follicle cells. In agreement with the immunocytochemical data presented above, however, results appear to suggest a lower level of expression of ZPC compared to the other proteins. We are presently undertaking quantitative PCR analysis to confirm these findings.
In this set of data, intron-spanning ZP primer pairs were used for RT–PCR. Although HIGAPDH primers suggest the presence of gDNA contamination in the RNA preparations, we observed that no ZP gene fragments were amplified using gDNA as a template. These results suggest that the generation of ZP amplicons is specific for ZP mRNA. mRNA expression for all three ZP genes was observed in marmoset oocytes and follicle cells, thus indicating that de novosynthesis of ZP proteins occurs in both cell types. Sequencing of the amplicons obtained by RT–PCR with templates from oocytes and follicle cells confirmed that they definitively represent marmoset ZPA, ZPB and ZPC nucleotide sequences. Purity of follicle cell preparation was proven by negative RT–PCR results with the ‘oocyte-specific’ BMP15 gene (Dube et al., 1998; Braw-Tal, 2002) expression in follicle cells.
In summary, we have presented here the first study to address gene expression in oocytes and follicle cells of all marmoset ZP proteins yet known according to follicular developmental stages. We conclusively demonstrated that ZPB, ZPA and ZPC gene expression was reliably localized in marmoset oocytes as well as in marmoset follicle cells. This is in agreement with our immunohistochemical studies that localized ZPA and ZPC protein in both oocytes and follicle cells. These results indicate that the oocyte is presumably the primary site of production of ZP proteins but that follicle cells are also involved in ZP protein de novosynthesis. Further investigation is warranted to clarify the stage-specific ZP protein synthesis patterns and to investigate whether or not the follicle cell-produced ZP proteins differ structurally from those produced in the oocyte either in the sequence of the protein or in glycosylation pattern, and whether they may have a differential localization and function in the outer layer of the bilamellar marmoset ZP.
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Acknowledgements |
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We thank Nicole Umland for excellent technical assistance and Susanne Rensing for assistance with the surgical procedures. The authors wish to thank Dr E.Martinson and Dr V.A.Aires for the excellent editorial help and valuable discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft GK 533 to K.B.
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Andrews JC, Howard JG, Bavister BD and Wildt DE (1992) Sperm capacitation in the domestic cat (Felis catus) and leopard cat (Felis bengalensis) as studied with a salt-stored zona pellucida penetration assay. Mol Reprod Dev 31, 200–207.[CrossRef][ISI][Medline]
Baranska W, Konwinski M and Kujawa M (1975) Fine structure of the zona pellucida of unfertilized egg cells and embryos. J Exp Zool 192, 193–202.[CrossRef][ISI][Medline]
Bercegeay S, Jean M, Lucas H and Barriere P (1995) Composition of human zona pellucida as revealed by SDS-PAGE after silver staining. Mol Reprod Dev 41, 355–359.[CrossRef][ISI][Medline]
Bleil JD and Wassarman PM (1980) Mammalian sperm-egg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell 20, 873–882.[CrossRef][ISI][Medline]
Braw-Tal R (2002) The initiation of follicle growth: the oocyte or the somatic cells? Mol Cell Endocrinol 187, 11–18.[CrossRef][ISI][Medline]
Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ and Matzuk MM (1998) The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes. Mol Endocrinol 12, 1809–1817.
Dunbar BS, Timmons TM, Skinner SM and Prasad SV (2001) Molecular analysis of a carbohydrate antigen involved in the structure and function of zona pellucida glycoproteins. Biol Reprod 65, 951–960.
Eberspaecher U, Becker A, Bringmann P, van der ML and Donner P (2001) Immunohistochemical localization of zona pellucida proteins ZPA, ZPB and ZPC in human, cynomolgus monkey and mouse ovaries. Cell Tissue Res 303, 277–287.[CrossRef][ISI][Medline]
El Mestrah M, Castle PE, Borossa G and Kan FW (2002) Subcellular distribution of ZP1, ZP2, and ZP3 glycoproteins during folliculogenesis and demonstration of their topographical disposition within the zona matrix of mouse ovarian oocytes. Biol Reprod 66, 866–876.
Epifano O, Liang LF, Familari M, Moos MC and Dean J (1995) Coordinate expression of the three zona pellucida genes during mouse oogenesis. Development 121, 1947–1956.[Abstract]
Gilchrist RB, Nayudu PL, Nowshari MA and Hodges JK (1995) Meiotic competence of marmoset monkey oocytes is related to follicle size and oocyte-somatic cell associations. Biol Reprod 52, 1234–1243.[Abstract]
Gilchrist RB, Nayudu PL and Hodges JK (1997) Maturation, fertilization, and development of marmoset monkey oocytes in vitro. Biol Reprod 56, 238–246.[Abstract]
Govind CK, Gahlay GK, Choudhury S and Gupta SK (2001) Purified and refolded recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B expressed in Escherichia coli binds to spermatozoa. Biol Reprod 64, 1147–1152.
Green DP (1997) Three-dimensional structure of the zona pellucida. Rev Reprod 2, 147–156.[Abstract]
Grootenhuis AJ, Philipsen HL, Breet-Grijsbach JT and van Duin M (1996) Immunocytochemical localization of ZP3 in primordial follicles of rabbit, marmoset, rhesus monkey and human ovaries using antibodies against human ZP3. J Reprod Fertil 50 (Suppl), 43–54.
Gupta SK, Sharma M, Behera AK, Bisht R and Kaul R (1997) Sequence of complementary deoxyribonucleic acid encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP1 and its high-level expression in Escherichia coli. Biol Reprod 57, 532–538.[Abstract]
Hasegawa A, Koyama K and Isojima S (1991) Isolation of four major glycoprotein families (ZP1, ZP2, ZP3, ZP4) of porcine zona pellucida and characterization of antisera raised to each glycoprotein family. Nippon Sanka Fujinka Gakkai Zasshi 43, 221–226.[Medline]
Herrler A and Beier HM (2000) Early embryonic coats: morphology, function, practical applications. An overview. Cells Tissues Organs 166, 233–246.[CrossRef][ISI][Medline]
Hinsch E, Hagele W, Schill WB and Hinsch KD (1997) The zona pellucida "receptors". Adv Exp Med Biol 424, 313–328.[ISI][Medline]
Hinsch E, Hagele W, Bohle RM, Schill WB and Hinsch KD (1998a) Evaluation of ZP2 domains of functional importance with antisera against synthetic ZP2 peptides. J Reprod Fertil 114, 245–251.[Abstract]
Hinsch E, Hagele W, van der Ven H, Oehninger S, Schill WB and Hinsch KD (1998b) Immunological identification of zona pellucida 2 (ZP2) protein in human oocytes. Andrologia 30, 281–287.[ISI][Medline]
Hinsch E, Oehninger S, Schill WB and Hinsch KD (1999) Species specificity of human and murine anti-ZP3 synthetic peptide antisera and use of the antibodies for localization and identification of ZP3 or ZPC domains of functional significance. Hum Reprod 14, 419–428.
Hinsch KD and Hinsch E (1999) The zona pellucida ‘receptors’ ZP1, ZP2 and ZP3. Andrologia 31, 320–322.[ISI][Medline]
Hinsch KD, Hinsch E, Meinecke B, Topfer-Petersen E, Pfisterer S and Schill WB (1994) Identification of mouse ZP3 protein in mammalian oocytes with antisera against synthetic ZP3 peptides. Biol Reprod 51, 193–204.[Abstract]
Howes E, Pascall JC, Engel W and Jones R (2001) Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization. J Cell Sci 114, 4127–4136.
Howes L and Jones R (2002) Interactions between zona pellucida glycoproteins and sperm proacrosin/acrosin during fertilization. J Reprod Immunol 53, 181–192.[CrossRef][ISI][Medline]
Jansen S, Ekhlasi-Hundrieser M and Topfer-Petersen E (2001) Sperm adhesion molecules: structure and function. Cells Tissues Organs 168, 82–92.[CrossRef][ISI][Medline]
Jethanandani P, Santhanam R and Gupta SK (1998) Molecular cloning and expression in Escherichia coli of cDNA encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP2. Mol Reprod Dev 50, 229–239.[CrossRef][ISI][Medline]
Jewgenow K and Fickel J (1999) Sequential expression of zona pellucida protein genes during the oogenesis of domestic cats. Biol Reprod 60, 522–526.
Keefe D, Tran P, Pellegrini C and Oldenbourg R (1997) Polarized light microscopy and digital image processing identify a multilaminar structure of the hamster zona pellucida. Hum Reprod 12, 1250–1252.
Kolle S, Sinowatz F, Boie G, Totzauer I, Amselgruber W and Plendl J (1996) Localization of the mRNA encoding the zona protein ZP3 alpha in the porcine ovary, oocyte and embryo by non-radioactive in situ hybridization. Histochem J 28, 441–447.[CrossRef][ISI][Medline]
Kolle S, Sinowatz F, Boie G and Palma G (1998) Differential expression of ZPC in the bovine ovary, oocyte, and embryo. Mol Reprod Dev 49, 435–443.[CrossRef][ISI][Medline]
Kolluri SK, Kaul R, Banerjee K and Gupta SK (1995) Nucleotide sequence of cDNA encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP3. Reprod Fertil Dev 7, 1209–1212.[CrossRef][Medline]
Lee VH and Dunbar BS (1993) Developmental expression of the rabbit 55-kDa zona pellucida protein and messenger RNA in ovarian follicles. Dev Biol 155, 371–382.[CrossRef][ISI][Medline]
Martinez ML, Fontenot GK and Harris JD (1996) The expression and localization of zona pellucida glycoproteins and mRNA in cynomolgus monkeys (Macaca fascicularis). J Reprod Fertil 50 (Suppl), 35–41.
Miller DJ, Shi X and Burkin H (2002) Molecular basis of mammalian gamete binding. Recent Prog Horm Res 57, 37–73.
Nikas G, Paraschos T, Psychoyos A and Handyside AH (1994) The zona reaction in human oocytes as seen with scanning electron microscopy. Hum Reprod 9, 2135–2138.
Paterson M, Wilson MR, van Duin M and Aitken RJ (1996) Evaluation of zona pellucida antigens as potential candidates for immunocontraception. J Reprod Fertil 50 (Suppl), 175–182.
Prasad SV, Skinner SM, Carino C, Wang N, Cartwright J and Dunbar BS (2000) Structure and function of the proteins of the mammalian zona pellucida. Cells Tissues Organs 166, 148–164.[CrossRef][ISI][Medline]
Roller RJ, Kinloch RA, Hiraoka BY, Li SS and Wassarman PM (1989) Gene expression during mammalian oogenesis and early embryogenesis: quantification of three messenger RNAs abundant in fully grown mouse oocytes. Development 106, 251–261.[Abstract]
Sasanami T, Pan J, Doi Y, Hisada M, Kohsaka T and Toriyama M (2002) Secretion of egg envelope protein ZPC after C-terminal proteolytic processing in quail granulosa cells. Eur J Biochem 269, 2223–2231.[ISI][Medline]
Sinowatz F, Kolle S and Topfer-Petersen E (2001) Biosynthesis and expression of zona pellucida glycoproteins in mammals. Cells Tissues Organs 168, 24–35.[CrossRef][ISI][Medline]
Spargo SC and Hope RM (2003) Evolution and nomenclature of the zona pellucida gene family. Biol Reprod 68, 358–362.
Takeuchi Y, Nishimura K, Aoki N, Adachi T, Sato C, Kitajima K and Matsuda T (1999) A 42-kDa glycoprotein from chicken egg-envelope, an avian homolog of the ZPC family glycoproteins in mammalian Zona pellucida. Its first identification, cDNA cloning and granulosa cell-specific expression. Eur J Biochem 260, 736–742.[ISI][Medline]
Thillai-Koothan P, van Duin M and Aitken RJ (1993) Cloning, sequencing and oocyte-specific expression of the marmoset sperm receptor protein, ZP3. Zygote 1, 93–101.[Medline]
Topper EK, Kruijt L, Calvete J, Mann K, Topfer-Petersen E and Woelders H (1997) Identification of bovine zona pellucida glycoproteins. Mol Reprod Dev 46, 344–350.[CrossRef][ISI][Medline]
Tsubamoto H, Hasegawa A, Nakata Y, Naito S, Yamasaki N and Koyama K (1999) Expression of recombinant human zona pellucida protein 2 and its binding capacity to spermatozoa. Biol Reprod 61, 1649–1654.
Waclawek M, Foisner R, Nimpf J and Schneider WJ (1998) The chicken homologue of zona pellucida protein-3 is synthesized by granulosa cells. Biol Reprod 59, 1230–1239.
Wassarman PM (2002) Sperm receptors and fertilization in mammals. Mt Sinai J Med 69, 148–155.[Medline]
Yanagimachi R (1994) Mammalian Fertilization. In Knobil E and Neill JD (eds), The Physiology of Reproduction. Raven Press, New York, pp. 189–317.
Submitted on April 9, 2004; resubmitted on April 29, 2004; accepted on May 11, 2004.
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