| | Production of monoclonal antibodies against recombinant human zona pellucida glycoproteins: utility in immunolocalization of respective zona proteins in ovarian folliclesReceived 26 April 2007; received in revised form 17 September 2007; accepted 22 October 2007. published online 04 March 2008. Abstract The zona pellucida (ZP) glycoproteins play an important role in oocyte development and gamete biology. To analyze their expression in follicles during various developmental stages, murine monoclonal antibodies (MAbs) were generated against the baculovirus-expressed recombinant human ZP2, ZP3 and ZP4. A panel of MAbs specific for the respective zona protein in ELISA and Western blot, and devoid of cross-reaction with other zona proteins was selected. Immunohistochemistry has shown that ZP2 MAb, MA-1620, did not react with oocytes in resting primordial follicles but showed reactivity with degenerating oocytes in primordial follicles undergoing atresia, and with oocytes in growing and antral follicles. Three MAbs against ZP3 did not react with oocytes in primordial follicles, but reacted only with oocytes in growing and antral follicles. Out of four MAbs against ZP4, three MAbs reacted with oocytes in primordial, growing and antral follicles. No reactivity of these MAbs with other ovarian cell types and other tissues studied (endometrium, uterine cervix, fallopian tubes and kidney) was detected except for a strong reactivity of ZP2 MA-1620 with epithelial cells of the uterine ectocervix or endometrium in some samples investigated. Altogether, these studies document generation of MAbs exhibiting high specificity for human zona proteins, which will be useful reagents to study their immunobiology. 1. Introduction  Zona pellucida (ZP) is an acellular translucent sulfated glycoproteinaceous matrix that surrounds the mammalian oocyte and plays a very important role in mediating critical steps during fertilization. It acts as a species-specific ‘docking site’ for binding of the spermatozoa to the oocyte, induces acrosomal exocytosis in zona-bound spermatozoa, prevents polyspermy and plays an important role in the protection of a pre-implanted blastocyst. The murine ZP is composed of three biochemically and immunologically distinct glycoproteins designated as ZP1, ZP2 and ZP3 based on their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These glycoproteins display extreme heterogeneity in charge and molecular weight due to extensive post-translational modifications involving glycosylation with both N- (asparagine) and O- (serine/threonine) linked oligosaccharides, sulfation and site-specific proteolysis (Wassarman and Litscher, 1995). In the mouse model, ZP3 has been shown to be the primary sperm receptor (Bleil and Wassarman, 1980), while ZP2 binds to acrosome-reacted spermatozoa and thus acts as a secondary sperm receptor (Bleil et al., 1988). ZP1 cross-links the filaments formed by ZP2–ZP3 heterodimers and appears to provide mainly stability and structural integrity to the ZP matrix (Greve and Wassarman, 1985). However, recent studies have shown that human ZP is composed of four ZP glycoproteins designated as ZP1 [amino acid (aa) residues 1–638], ZP2 (aa residues 1–745), ZP3 (aa residues 1–424) and ZP4 (aa residues 1–540) (Conner et al., 2005, Lefievre et al., 2004). In humans, ZP3 is also proposed as the putative primary sperm receptor (Chakravarty et al., 2005, van Duin et al., 1994). In addition, binding of recombinant human ZP4 to capacitated human spermatozoa and its ability to induce acrosomal exocytosis has been documented (Chakravarty et al., 2005, Chakravarty et al., 2008). Studies pertaining to the expression of ZP glycoproteins in the mouse model revealed their presence restricted only to the oocyte (Epifano et al., 1995). The oocyte-specific expression of porcine ZP2 (Taya et al., 1995), rabbit ZP2 (Lee et al., 1993) and marmoset ZP3 (Thillai-Koothan et al., 1993) has also been reported. In the brushtail possum, in situ hybridization studies revealed that expression of ZP4 is restricted to oocytes of the primordial and primary follicles while no expression was detected in the surrounding granulosa cells (GCs) (Haines et al., 1999). On the contrary, in situ hybridization of cynomolgus monkey ovarian sections using digoxigenin-labeled cDNA probes specific for the mRNA encoding ZP2, ZP3 or ZP4 demonstrated the presence of ZP2 in growing follicles at all stages and in the GCs of mature preovulatory follicles (Martinez et al., 1996). ZP3 was detected in oocytes at all stages of folliculogenesis as well as in GCs, while ZP4 was present in secondary follicles and to a lesser extent in tertiary follicles, but was not found in primordial, primary or antral follicles or GCs. In variance with the data from the mouse model, using human ZP3-specific antibodies, ZP3 has been shown to be present in the oocyte as well as the GCs of primordial, primary and secondary follicles of the human ovary (Grootenhuis et al., 1996). Given the above conflicting findings, we sought to investigate the expression of zona proteins in follicles at different stages of development by employing highly specific antibodies as probes, which are devoid of cross-reactivity with antigens present on other tissues. We have reported previously the expression of human ZP2 (aa residues 1–745), ZP3 (aa residues 1–424) and ZP4 (aa residues 1–540) in the baculovirus expression vector system (BEVS: Chakravarty et al., 2005). The availability of recombinant human zona proteins prompted us to develop a panel of murine monoclonal antibodies (MAbs) against the above recombinant proteins so as to generate probes that are specific for individual human ZP glycoproteins. Here, we describe the characteristics of these MAbs with respect to their specificity for respective zona proteins in ELISA and Western blot. Using immunohistochemistry, their specificity in recognizing various human zona proteins in ovarian follicles at various developmental stages and in other ovarian-associated cells has also been investigated. 2. Materials and methods All chemicals, except where specified otherwise, were purchased from Sigma Chemical Co., St. Louis, MO, USA. 2.1. Human tissues In this study, samples from ovaries, oviduct and uterus (endometrium and ectocervix) – hysterectomy specimens from 12 females, ages 26–52, and one kidney (autopsy specimen, age 40) – are included. The study was approved by the Institutional Review Board, and a signed written consent was obtained. 2.2. Baculovirus-expressed recombinant human zona glycoproteins The cloning and expression of recombinant human ZP2 (aa residues 1–745), ZP3 (aa residues 1–424) and ZP4 (aa residues 1–540) as polyhistidine-tagged fusion proteins in the BEVS has recently been reported (Chakravarty et al., 2005). For large scale production of the recombinant proteins, a suspension culture of 50 ×106 Spodoptera frugiperda (Sf21) insect cells growing in a Spinner bottle (Thermolyne, Dubuque, Iowa, USA) was incubated with the respective recombinant virus at a multiplicity of infection (MOI) of 3 at 42 rotations per minute (rpm) on a biological stirrer (Thermolyne) at 27°C. After 96h incubation, cells were pelleted at 1000g for 15min and the respective recombinant protein purified using Ni-NTA resin as described previously (Gahlay and Gupta, 2003). The purified recombinant proteins in 20mM Tris (pH 7.4) were assessed for protein concentration using bicinchoninic acid assay (BCA; Pierce, Rockford, IL, USA) with bovine serum albumin (BSA) as the standard. 2.3. Generation of MAbs against baculovirus-expressed recombinant human ZP glycoproteins The murine MAbs were generated against baculovirus-expressed recombinant human ZP2, ZP3 and ZP4. Male BALB/c mice (8–10 weeks old, Small Experimental Animal Facility, National Institute of Immunology, New Delhi, India) were immunized s.c. with purified recombinant human ZP2, ZP3 and ZP4 (50μg/animal), respectively, emulsified with complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA) after due approval from the Institutional Animal Ethical Committee. Animals were boosted i.p. two times at 4-week intervals by the same amount of respective recombinant proteins emulsified with incomplete Freund's adjuvant (IFA; Difco Laboratories). Immunized animals were used to generate MAbs essentially as described elsewhere (Govind et al., 2000). Hybrid cell clones secreting MAbs were identified by ELISA employing microtitration plates coated with 200ng of the respective recombinant human zona protein per well in 50mM phosphate-buffered saline (PBS), pH 7.4. Plates were washed once with PBS and blocked with 1% BSA in PBS at 37°C for 2h. After blocking, plates were incubated with undiluted culture supernatant obtained by growing hybrid cell clones and processed as described elsewhere (Govind et al., 2000). 2.4. Characterization of MAbs 2.4.1. Enzyme linked immunosorbant assay (ELISA) 2.4.1.1. Reactivity with recombinant human ZP glycoproteins Culture supernatants of the respective hybrid cells secreting MAbs were tested for their reactivity with the respective recombinant proteins used to generate the MAbs and the other zona proteins in an ELISA. Microtitration plates were coated with an optimized concentration of the recombinant zona proteins (200ng/well) for 1h at 37°C and overnight at 4°C. Coated plates were washed once and blocked with 1% BSA for 2h at 37°C. All subsequent incubations were carried out at 37°C and followed by three washings with PBS containing 0.05% Tween-20 (PBST). Post-blocking, the plates were washed and incubated with 100μl of culture supernatant obtained from either the hybrid cells secreting a particular MAb or the SP2/O myeloma cells. In addition, ascites fluid obtained by growing hybrid cell clones in pristane (2,6,10,14-tetramethyl pentadecane, Sigma) primed BALB/c mice was also tested at 1:100 dilution. Bound antibodies were revealed by rabbit anti-mouse immunoglobulins conjugated to HRPO (Pierce, Rockford, IL, USA) at an optimized dilution of 1:2000 for 1h. The enzyme activity was determined by adding 100μl/well of 0.05% orthophenylenediamine (OPD) and 0.06% H2O2 in 50mM citrate phosphate buffer pH 5.0, and the reaction was stopped by adding 50μl/well of 5N H2SO4. The absorbance was read at 490nm with 600nm as the reference filter. Values are presented as the mean of duplicate readings minus the absorbance obtained with uncoated wells. 2.4.2. Western blot The specificity of MAbs for respective zona proteins was analyzed also in a Western blot as described previously (Chakravarty et al., 2005). Baculovirus-expressed recombinant human ZP2, ZP3 and ZP4 (2μg/lane) proteins were boiled for 5min in the sample buffer (62.5mM Tris pH 6.8, 2% SDS, 10% glycerol and 5% β-mercaptoethanol) and resolved on a 0.1% SDS-10% PAGE. The SDS-PAGE resolved proteins were electrophoretically transferred onto a 0.45μm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) at a constant voltage of 15V in Tris glycine buffer with 20% methanol. After transfer, non-specific sites on the membrane were blocked with 3.0% BSA in PBS for 1h at room temperature (RT). All subsequent incubations were carried out for 1h at RT and followed by three washings with PBS containing 0.1% Tween-20. Post-blocking, the membrane was incubated with the culture supernatant (1:5 dilution) of the respective MAb. The bound antibodies were revealed by incubation with goat anti-mouse IgG-HRPO conjugate (Pierce) at an optimized dilution of 1:2000. The blot was developed with 0.6% (w/v) 4-chloro-1-naphthol (Amresco, Solon, Ohio, USA) in PBS containing 25.0% methanol and 0.06% H2O2. The reaction was stopped by extensive washing of the membrane with water. 2.4.3. Immunohistochemistry Human samples were frozen in optimal cutting temperature (O.C.T.) compound (Miles Inc., Elkhart, IN, USA) and stored at −80°C until use. Cryosections (7μm thickness) were fixed in acetone and processed by peroxidase immunohistochemistry as described previously (Bukovsky et al., 1995). Slides were subjected to single color immunohistochemistry for ZP immunoreactivity. All procedures were performed at RT. Briefly, specimens were incubated for 20min with undiluted mouse-anti human ZP MAbs culture supernatant. Alternately, MAbs from ascites were purified by protein-G affinity matrix (Sigma). The pH of the antibody was adjusted by adding 1/10 volume of 1.0M Tris base buffer (pH 8.0; antibody solution). The antibody solution was passed three times through a protein G column. Next, the column was washed with 10 column volumes of 100mM Tris (pH 8.0) and with 10 column volumes of 10mM Tris (pH 8.0). After washing, the column was eluted stepwise with 1/2 column volume per step of 50mM glycine (pH 3.0). The eluate from each step was collected and 1/10 column volume of 1M Tris (pH 8.0) added to bring the pH to neutral. The antibody was dialyzed overnight, lyophilized and resuspended in the original volume. IgG concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA, USA), and the concentration varied from 0.2 to 7.6mg/ml. Affinity-purified MAbs were utilized for immunohistochemistry at the optimized concentration of 10μg/ml in PBS, pH 7.2. Utilization of affinity-purified ascitic ZP MAbs gave results similar to those obtained with culture supernatant of ZP MAbs, where the Ig concentration is known to vary between 5 and 20μg/ml. After washing in PBS, specimens were incubated for 20min with peroxidase conjugated rabbit anti-mouse IgG, Fc fragment specific (Jackson Immunoresearch Laboratories, West Grove, PA, USA) diluted 1:50. After washing, the antigen–antibody complexes were detected by a Vector DAB detection kit according to the supplier's manual (Vector Laboratories, Inc., Burlingame, CA, USA), giving the substrate brown color, followed by hematoxylin counterstain of the nuclei. Dehydrated samples were mounted in Canada balsam. A control procedure consisted of single color immunohistochemistry as above, but the primary antibody was replaced with either PBS or a culture supernatant obtained by growing SP2/O mouse myeloma. For dual color immunohistochemistry, slides were first stained as above, but without the hematoxylin counterstain. After three washes in PBS, slides were incubated for 20min with mouse-anti human CK18, clone CY-90 (Sigma; 5μg/ml in PBS). After washing in PBS, specimens were incubated with secondary antibody as above. After washing, the antigen–antibody complexes were detected by a Vector SG detection kit according to the supplier's manual (Vector Laboratories), giving the substrate blue color, dehydrated and mounted. The diaminobenzidine reaction product masks the antigen and catalytic sites of the first sequence of immunoreagents, preventing interaction with the reagents of the second sequence (Sternberger and Joseph, 1979). Control staining consisted of the same procedure, but primary antibodies were replaced as indicated above. In addition, triple color immunohistochemistry was employed to study simultaneously ZP2 (oocytes), CK18 (granulosa cells) and Thy-1 differentiation protein (fibroblasts/vascular pericytes) expression. Thy-1 expression by stromal fibroblasts is usually low in areas where the cohorts of primordial follicles reside, and its increased local expression by vascular pericytes accompanies initiation of follicular growth (Bukovsky et al., 2004). Dual color immunohistochemistry was performed as above and complemented with the third primary antibody [mouse anti-human Thy-1, clone F15-42-01 (McKenzie and Fabre, 1981), kindly donated by Dr. Rosemarie Dalchau, Institute of Child Health, University of London, England]. Secondary antibodies were utilized as indicated above. Antigen–antibody complexes were detected by a Vector VIP detection kit according to the supplier's manual (Vector Laboratories), giving the substrate a purple color, and slides were dehydrated and mounted. Control staining consisted of the same procedure, but primary antibodies were replaced as indicated above. Differential interference contrast images were captured with a DEI-470 CCD Video Camera System (Optronics Engineering, Goleta, CA, USA) with detail enhancement and CG-7 color frame grabber (Scion Corporation, Frederick, MD, USA) supported by Scion Image public software developed at the National Institutes of Health (Wayne Rasband, NIH, Bethesda, MD, USA). Captured images were compiled using Microsoft® Power-Point® 97 SR-2 (Microsoft Corporation, Redmont, WA, USA) and Microsoft Photo Editor 3.0 (Microsoft Corporation). Immunohistochemical evaluation was performed by two investigators. 3. Results 3.1. MAbs against recombinant human zona proteins Fusion of splenocytes obtained from mice immunized with the baculovirus-expressed recombinant human ZP2 with the SP2/O mouse myeloma cells resulted in 20 hybrid cell clones secreting MAbs reactive with ZP2 in ELISA. Three out of the 20MAbs cross-reacted with baculovirus-expressed recombinant human ZP3 and ZP4 in an ELISA. The remaining 17MAbs were specific for recombinant human ZP2. Out of these 17MAbs, the reactivity profile of two MAbs, namely MA-1615 and MA-1620, both IgG1 isotype, is shown in Table 1. Both MAbs failed to react with human ZP3 and ZP4 in ELISA (Table 1). Fusion of splenocytes from the ZP3 and the ZP4 immunized animals resulted in 11 and 10 hybrid cell clones, respectively, secreting MAbs reactive with baculovirus-expressed recombinant respective human zona proteins. Out of the 11MAbs reactive with ZP3, 2 showed cross-reaction with human ZP2 and ZP4. From the remaining 9MAbs against ZP3, the reactivity of MAbs namely, MA-1552, MA-1556, MA-1558 and MA-1567, are shown in Table 1. MA-1552, MA-1556 and MA-1567 are of the IgG1 isotype, whereas MA-1558 is IgG2a. All the 10 hybrid cell clones secreting antibodies against human ZP4 were specific to ZP4 and were devoid of cross-reactivity with ZP2 and ZP3. The reactivity of the 4MAbs designated as MA-1659, MA-1660, MA-1662 and MA-1671 with baculovirus-expressed recombinant human ZP2, ZP3 and ZP4 is shown in Table 1. Isotype analysis of MAbs revealed that MA-1659 and MA-1671 are IgG1, MA-1660 IgG2b and MA-1662 IgG2a. | | |  | Immunogen | MAb identification number | Isotype | Absorbance at 490nm in ELISA with | Reactivity in Western blot with | |
|---|
| | | | ZP2 | ZP3 | ZP4 | ZP2 | ZP3 | ZP4 | |
|---|
| ZP2 | MA-1615 | IgG1 | 1.47 | 0.02 | 0.02 | + | − | − | | | MA-1620 | IgG1 | 1.20 | 0.01 | 0.02 | + | − | − | | | | | | ZP3 | MA-1552 | IgG1 | 0.02 | 1.38 | 0.02 | − | + | − | | | MA-1556 | IgG1 | 0.01 | 1.31 | 0.01 | − | + | − | | | MA-1558 | IgG2a | 0.01 | 1.38 | 0.01 | − | + | − | | | MA-1567 | IgG1 | 0.03 | 1.46 | 0.01 | − | + | − | | | | | | ZP4 | MA-1659 | IgG1 | 0.05 | 0.01 | 1.56 | − | − | + | | | MA-1660 | IgG2b | 0.06 | 0.02 | 1.51 | − | − | + | | | MA-1662 | IgG2a | 0.10 | 0.02 | 1.48 | − | − | + | | | MA-1671 | IgG1 | 0.08 | 0.03 | 1.48 | − | − | + | | | | |
MA-1615 and MA-1620 against ZP2; MA-1552, MA-1556, MA-1558 and MA-1567 against ZP3; MA-1659, MA-1660, MA-1662 and MA-1671 against ZP4 reacted only with the respective recombinant protein in the Western blot assay. The representative reactivity profiles in Western blot of MAbs against ZP2 (MA-1620), ZP3 (MA-1567) and ZP4 (MA-1660) with all the three recombinant proteins are shown in Fig. 1. 3.2. Reactivity profile of MAbs with human ovaries and other tissues The MA-1620, MAb against human ZP2, did not stain oocytes in resting primordial follicles (arrowhead, Fig. 2A). Weak staining of oocytes appeared in growing primary follicles (arrow). Strong staining of oocytes was observed in primordial follicles undergoing atresia (inset), and in secondary (Fig. 2B) and other growing follicle types (see also beneath). Control procedures gave no non-specific staining (Fig. 2C). The MA-1620 MAb did not react with other tissues investigated, except for epithelium of uterine ectocervix and endometrium during certain menstrual cycle stages (see beneath). The other antibody to ZP2, MA-1615, showed staining of many cell types within the ovary and other tissues (Table 2). MA-1552, MA-1556 and MA-1567 generated against baculovirus-expressed recombinant human ZP3 showed no reactivity with oocytes in primordial follicles (Fig. 2D), but stained ZP of oocytes of growing (Fig. 2E) and antral follicles (Fig. 2F). However, MA-1558 reacted with vascular endothelial cells in ovaries and other tissues studied (Table 2). The MAbs, MA-1660, MA-1662 and MA-1671 generated against baculovirus-expressed recombinant human ZP4 stained oocytes in primordial (Fig. 2G) and other follicle types (Fig. 2H and I). The MA-1659 MAb failed to react with oocytes of follicles of various sizes as well as other tissues (Table 2). Apparent differences in ZP2 expression in resting and degenerating primordial and growing follicles prompted us to investigate further the reactivity of the MA-1620 MAb against human ZP2 with oocytes in distinct ovarian follicles. For that purpose, we employed dual color immunohistochemistry including CK18 (blue) and MA-1620 (brown color), without hematoxylin counterstain (CK/ZP2). Such double color staining allows simultaneous visualization of granulosa cells and ZP2 staining of oocytes. Resting primordial follicles (black asterisk, Fig. 3A) exhibited flat granulosa cells with weak CK18 expression and oocyte containing paranuclear CK18+ Balbiani body (arrowhead, no hematoxylin counterstain). Such follicles showed no MA-1620 reactivity. In early growing primary follicles (red asterisk, panel B), there was an increase in the oocyte and follicle size and enhanced expression of CK18 by cuboidal granulosa cells (solid arrowhead). They still contained the CK18+ Balbiani body (open arrowhead) and showed weak staining of the oocyte ZP with MA-1620 antibody (arrow). Further increase in oocyte and follicle size (red asterisk, panel C) accompanied development of secondary follicles with more layers of granulosa cells (arrowhead). At this stage, staining of the oocyte for ZP2 markedly increased (arrow). Panel D shows ZP2 expression in a medium-sized antral follicle along with hematoxylin counterstain (ZP2/Hx). During the early stage of atresia of primordial follicles (yellow asterisks, Fig. 3E and F), ZP2 staining was not restricted to the oocyte surface, but oocytes were stained throughout the cytoplasm, without a visible unstained nucleus. Advanced atresia of primordial follicles was accompanied by pronounced ZP2 cytoplasmic expression (Fig. 3G). The atresia of secondary and preantral (Fig. 3H) follicles showed a similar pattern of enhanced cytoplasmic ZP2 expression. Observations of the distinct character of ZP2 expression in resting (ZP2−), growing (ZP2+ surface) and degenerating primordial follicles (enhanced cytoplasmic ZP2 staining) led us to investigate the relationship of stromal Thy-1 differentiation protein (Thy-1) expression. Thy-1 expression appears to be associated with initiation of primordial follicle growth, since resting primordial follicles usually lie in Thy-1-depleted areas showing an ‘ovary-in-ovary’ pattern (Bukovsky, 2006, Bukovsky et al., 2004). Fig. 4A shows that resting primary follicles (black asterisks) lie in an area with less pronounced Thy-1 expression (left) compared to the growing (red asterisks) and degenerating follicles (yellow asterisks). This suggests that primordial follicles in the area with more pronounced Thy-1 expression are either stimulated to grow or degenerate. Note that in the given areas about one third of primordial follicles remain in resting stage, one third appears to initiate growth, and one third shows signs of atresia. Once enhanced cytoplasmic expression indicating advanced atresia is attained, the follicles may degenerate despite the low Thy-1 expression (Fig. 4B). In Fig. 4C, it is also apparent in detail that a ZP2-resting primordial follicle (black asterisk) lies in the site with lower Thy-1 expression compared to high expression of Thy-1 in the adjacent stroma containing a primordial follicle undergoing atresia (yellow asterisk). Characteristic localization of resting primordial follicles in Thy-1-depleted areas is shown in Fig. 4D, where the arrowhead indicates the Balbiani body. None of the ZP3 and ZP4 antibodies reacting with oocytes in ovaries reacted with other tissues investigated, but there was a strong reactivity of ZP2 MA-1620 MAb with epithelium in samples of uterine ectocervix or endometrium at certain stages of the menstrual cycle (Table 2). Correlation between endometrium morphology indicated that the staining of ectocervix was associated with the follicular (Fig. 5A and B) and early luteal phase of the menstrual cycle (panels C and D). The ectocervix was, however, unstained during the mid-luteal phase, when the endometrium showed ZP2 expression (panels E and F). No staining for ZP2 was found in the postmenopausal ectocervix and endometrium (panels G and H). 4. Discussion Analyses of the deduced amino acid (aa) sequence of human ZP1, ZP2, ZP3 and ZP4 revealed a considerable degree of sequence homology from different species (for review, see Gupta et al., 2007). Apart from the homology observed within a given ZP protein from different species, a considerable amount of homology is seen also among the aa sequences of different zona proteins from the same species. The mature polypeptide chains of human ZP1 and ZP4 share an identity of 47% at the aa level, which is the highest between any two ZP proteins in humans. A high (40%) sequence identity at the aa level between ZP2 and ZP4 has also been observed. This has often posed a hurdle for generating specific antibodies as probes that recognize a particular zona protein. Hence, the cross-reactivity in ELISA of the three MAbs generated from mice immunized with baculovirus-expressed recombinant human ZP2, and two MAbs generated from mice immunized with ZP3, with the three recombinant human zona proteins may be due to the possibility that these antibodies recognize a conserved epitope present on all three zona proteins. Nonetheless, we succeeded in generating MAbs that showed high specificity both in ELISA as well as Western blots against the three human zona proteins namely ZP2, ZP3 and ZP4. Availability of highly specific MAbs against human ZP2, ZP3 and ZP4 prompted us to examine their reactivity profile with the zona proteins present in oocytes of follicles at various stages of their development and simultaneously evaluate their specificity to recognize the oocyte and not other tissues. The observations presented herein suggest that expression of ZP glycoproteins in human oocytes may be stage-specific. During follicular development, ZP4 appears to be expressed by oocytes earlier (all oocytes in primordial follicles) than ZP2 (oocytes in growing primary follicles and primordial follicles undergoing atresia), and ZP3 emerges as the last, since it is expressed only by oocytes of secondary and other growing follicles. In studies employing both monoclonal and polyclonal antibodies against human ZP3, the expression of ZP3 has been documented in granulosa cells and primordial follicles in the ovaries of rabbit, marmoset, rhesus monkey and human (Grootenhuis et al., 1996). However, the cross-reactivity of antibodies employed in these studies with other human zona proteins was not established. In mice, ZP1 and ZP3 transcripts were detected only in the growing oocytes (Epifano et al., 1995), while the ZP2 transcript could be observed in oocytes before the growth phase of oogenesis and even prior to birth, as early as 16 days of gestation (Millar et al., 1993). As the mouse oocyte grows, all three zona transcripts coordinately accumulate, representing approximately 1.5% of the total mRNA in 50–60μm oocytes. Using immunolabeling technique, association of the mouse ZP proteins was observed with the Golgi apparatus, secretory granules and a complex structure called vesicular aggregate, suggesting the active involvement of these sub-cellular organelles in processing of the three glycoproteins before their secretion to form the ZP matrix (El-Mestrah et al., 2002). An asymmetric spatial distribution of the three ZP glycoproteins in the mouse zona matrix was revealed also at various stages of follicular development. By inhibiting de novo biosynthesis of specific zona proteins with antisense oligonucleotides, it was ascertained that ZP2 and ZP3 are independent of each other in their biosynthesis, but are dependent upon each other for their incorporation into the ZP matrix (Tong et al., 1995). Further, using knockout animals, it was shown that, in ZP1-null mice, the ZP matrix is loosely organized and animals had impaired folliculogenesis and reduced fecundity (Rankin et al., 1999). In ZP2-null mice, ZP1 and ZP3 proteins formed a thin zona matrix in early follicles, which was not sustained in pre-ovulatory follicles (Rankin et al., 2001). This abnormal zona matrix showed no affect on initial folliculogenesis, but a decrease in the number of antral stage follicles was observed. Mice with homologous mutation for ZP3 showed follicles with germinal vesicle-intact oocytes but lacked the zona matrix, had disorganized corona radiata and were sterile (Liu et al., 1996). The implications of these observations in a mouse model for folliculogenesis in humans, however, remains to be elucidated. Various other oocyte-specific proteins have been documented that are expressed at various stages of folliculogenesis. The Factor in the Germline alpha (FIGα), a basic helix–loop–helix transcription factor, is first detected in oocytes, 13.5-day-old embryos and persists in adults. FIGα-null female mice failed to form primordial follicles and also lack expression of zona proteins (Soyal et al., 2000). Expression of growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein 15 (BMP-15), have been documented in the oocytes of primary follicles, which are required for folliculogenesis beyond primary follicle stage (Dong et al., 1996, Galloway et al., 2000). During subsequent stages of folliculogenesis, connexins that are involved in somatic cell interaction [Cnx43; Juneja et al., 1999] or in somatic-germ cell interactions [Cnx37; Simon et al., 1997] play an important role. The reactivity of MA-1620 MAb against human ZP2 is of interest, since it appears not to recognize resting healthy primordial follicles with Balbiani bodies. Studies of Motta et al. (1994) have shown that, in primordial follicles of fetal and adult human ovaries, follicular (granulosa) cell extensions penetrate deep into the ooplasm, much like a sword in its sheath. There may be as many as 3–5 ‘intra-ooplasmic processes’ in one scanning microscopy plane. These intra-oocytic invaginations are closely associated with a variety of organelles. They are in proximity to the nuclear zone, and may help activate growth of the oocyte (Motta et al., 1994). These intra-ooplasmic extensions of granulosa cells constitute a single Balbiani body, which disappear in oocytes of growing follicles in order to release additional organelles, required for oocyte growth (reviewed in Bukovsky et al., 2004). Hence, activation of oocytes by dispersion of organelles during initiation of follicular growth may be associated with ZP2 (and ZP3) expression. MA-1620 MAb also shows enhanced cytoplasmic staining of primordial follicles undergoing atresia, which lack the Balbiani bodies. So far, only morphologic methods have been available to discriminate between healthy and atretic primordial follicles, but characteristic morphological features of primordial follicle atresia are often difficult to identify. With ZP2 expression, the atresia of primordial follicles can be identified from its onset, and follicle/oocyte remnants not detectable morphologically are also apparent (data not shown). This will enable future evaluation of a proportion of healthy resting versus degenerating primordial follicles in human ovaries during distinct periods of life, such as fetal, perinatal, premenarcheal, prime reproductive and premenopausal periods. The relationship of stromal Thy-1 expression to initiation of follicular growth or atresia is also interesting. It remains unclear how it is decided which primordial follicles will resume growth and which will degenerate. Primordial follicles belong to innervated ovarian structures (Peters and McNatty, 1980), and we have suggested that autonomic innervation controls quantitative aspects of tissues and their structures, including ovarian follicles (Bukovsky, 2006). Hence, we speculate that, in the Thy-1+ areas, some follicles are stimulated by autonomic innervation to differentiate and others to degenerate. While the progression of follicular growth is accompanied by increased activity of vascular pericytes [Thy-1 release (Bukovsky et al., 1995, Bukovsky, 2006)], progression of atresia with enhanced cytoplasmic ZP2 expression does not require Thy-1 expression by stromal cells (Fig. 4B). At this stage, the significance of cross-reactivity of the MA-1620 MAb raised against recombinant human ZP2 with differentiating epithelial cells of uterine ectocervix remains unclear. This staining may represent the presence of either ZP2 or immunologically cross-reactive ZP2-like proteins in the ectocervix. To show that human ectocervix indeed synthesize ZP2, it is necessary to characterize the ZP2 transcript in the ectocervix. In fishes, amphibians and chickens, zona proteins are synthesized in liver, secreted and incorporated into the egg envelope (Conner and Hughes, 2003). So far, there is no convincing evidence to suggest that, in humans, zona proteins are secreted from the oocytes/ovaries and incorporated into other reproductive tract organelles. The ectocervix is at the entrance to the uterus, and ZP2 expression there may serve as a sperm signal where to migrate. The expression of ZP2 in ectocervix was apparent during the periovulatory (follicular and early luteal) phases of the menstrual cycle, when sperm have a maximal chance to find and fertilize the oocyte. On the other hand, the mid-luteal phase was associated with ZP2 expression in endometrium and no staining of ectocervix. The endometrial expression of ZP2 may guide the fertilized oocyte where to optimally settle. The epitope(s) recognized by MAbs against ZP3 appear related to oocyte maturation in growing follicles, since they are not apparent in oocytes of primordial follicles, which express ZP4 only. We surmise that premature ovarian failure with primary or secondary hypergonadotropic amenorrhea and a lack of primary follicles could be caused by ZP4 autoantibodies affecting oocytes of the primordial follicular pool. On the other hand, some forms of unexplained human female infertility associated with normo-gonadotropic cycles could be caused by ZP2 or ZP3 IgM autoantibodies not reacting with healthy primordial follicles and unable to pass the blood-follicle barrier of growing follicles for high molecular weight proteins (Shalgi et al., 1973), but preventing sperm from finding or binding to the ovulated eggs. 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a Laboratory of Development, Differentiation and Cancer, The University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, Tennessee 37920, USA b Department of Obstetrics and Gynecology, The University of Tennessee Graduate School of Medicine, Tennessee, USA c Gamete Antigen Laboratory, National Institute of Immunology, New Delhi, India d Department of Pathology, The University of Tennessee Graduate School of Medicine, Knoxville, Tennessee 37920, USA  Corresponding author at: Laboratory of Development, Differentiation and Cancer, The University of Tennessee Graduate School of Medicine, 1924 Alcoa Highway, Knoxville, Tennessee 37920, USA. Tel.: +1 865 544 8969; fax: +1 865 544 6105.
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