Hum. Reprod. Advance Access published online on November 22, 2007
Human Reproduction, doi:10.1093/humrep/dem373
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Detection of zona pellucida proteins during human folliculogenesis
1 Reproductive Services, Royal Women’s Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia 2 Melbourne IVF, 320 Victoria Parade, East Melbourne, Victoria 3002, Australia 3 Department of Obstetrics and Gynaecology, University of Melbourne, Melbourne, Victoria, Australia
4 Correspondence address. Reproductive Services, Royal Women’s Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia. E-mail: debra.gook{at}rwh.org.au
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Abstract |
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BACKGROUND: The stage of folliculogenesis at which the human zona pellucida (ZP) is initiated and the cells responsible for the origin of the ZP continue to be controversial. This study characterizes the development of the ZP during human folliculogenesis using ovarian samples donated from patients requesting ovarian storage.
METHODS: Follicles (from n = 18 patients, 14–40 years old) within fresh tissue and following development in a xenograft system were stained, using immunohistochemical techniques, for the presence of the three human ZP proteins, ZP1, ZP2 and ZP3. Over 500 primordial follicles and >20 follicles at each developmental stage were examined.
RESULTS: All three ZP proteins were detected within the oocyte of the primordial follicle. Presence of ZP1 and ZP3 was observed in the majority of primordial oocytes (93% and 95%, respectively), whereas ZP2 was detected in only 32% of these follicles. The three ZP proteins were detected in the cytoplasm of cuboidal granulosa cells and their distribution correlates with developmental stages throughout folliculogenesis. CONCLUSIONS: ZP proteins were detected in both the oocyte and the granulosa cells as early as the primordial follicle stage in the human. The detection of ZP proteins in the quiescent primordial follicle suggests that these proteins have been present since oogenesis.
Key words: zona pellucida/human follicles/immunohistochemistry/oocyte/granulosa cells
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Introduction |
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The zona pellucida (ZP) is a translucent matrix of glycoproteins that encircles the mammalian oocyte (Wassarman, 1988
). In most species studied, the ZP is a network comprising three cross-linked, heavily glycosylated proteins ZP1, ZP2 and ZP3 (Bleil and Wassarman, 1980a,b). The human ZP is thought to be similar in structure to the murine ZP consisting of long heterodimeric filaments of ZP2 and ZP3 which are cross-linked by homodimers of ZP1 (Greve and Wassarman, 1985; Green, 1997). Recently, however, a fourth protein has been identified in the human ZP (Lefievre et al., 2004).
Although the ZP structure is similar between the species, the diversity is sufficient to prohibit cross-species gamete interaction (Bedford, 1977; Liu De et al., 1991). Functionally, alteration of the ZP structure immediately after fertilization is responsible for preventing further sperm penetration (Wassarman, 1990), and there is general consensus that ZP3 is the receptor and inducer of the acrosome reaction (Moller et al., 1990). However, the origin of the ZP and its development during mammalian folliculogenesis remain controversial.
The appearance of ZP proteins has been reported to be coincidental with initiation of follicular growth in mouse (Epifano et al., 1995; El-Mestrah et al., 2002), cat (Jewgenow and Fickel, 1999) and rabbit (Prasad et al., 1996), but others have detected ZP proteins in primordial follicles of the rabbit (Wolgemuth et al., 1984; Lee and Dunbar, 1993; Grootenhuis et al., 1996), monkey (Grootenhuis et al., 1996; Martinez et al., 1996; Bogner et al., 2004) and human (Grootenhuis et al., 1996). Expression of the gene for at least one of the ZP proteins has been demonstrated in quiescent early porcine (Kolle et al., 1996) and mouse (Epifano et al., 1995) follicles. In the mouse follicle, ZP synthesis is confined solely to the oocyte (Epifano et al., 1995; El-Mestrah et al., 2002). In all other species studied, synthesis is detected in both the oocyte and the granulosa cells.
Knowledge of ZP development during human folliculogenesis is limited to one protein; ZP3 (Grootenhuis et al., 1996; Hinsch et al., 1998), in small numbers of follicles obtained from postmortem material. Comprehensive evaluation of the origin and development of the human ZP requires access to ovarian cortex containing follicles at various developmental stages. In the present study, identification and characterization of ZP proteins in developing human follicles has been achieved predominantly in xenografts of human ovarian cortex which have previously been demonstrated to contain follicles at all developmental stages (Gook et al., 2001, 2005b). In contrast, our evaluation of ZP proteins in primordial follicles has been carried out in diagnostic samples of freshly harvested ovarian tissue which have been shown to contain a very small proportion (
1%) of developing follicles (Gook et al., 1999).
The concurrent availability of follicles at all developmental stages has allowed us to conduct a unique investigation into the development and localization of the human ZP throughout folliculogenesis.
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Approval and consents
The study and associated consent forms were approved by the Human Research and Ethics Committee of The Royal Women’s Hospital. Consent for the use of tissue was obtained from all participants. In the case of minors, consent was obtained from parents.
Controls
Oocytes (n = 8) collected from ovarian stimulation cycles for IVF (n = 3) which, following hyaluronidase (Hyalase; Aventis Pharma, Lane Cove, Australia; 20 IU/ml) treatment, were identified as immature, were fixed and prepared as described below and used as positive controls. These oocytes have a clearly visible ZP. In addition, a portion of cumulus cells, distal to the oocyte, was removed using needles prior to hyaluronidase treatment and fixation (see Fixation and immunohistochemistry section).
Fresh ovarian tissue
Ovarian tissue was donated from patients requesting ovarian tissue harvest and cryopreservation prior to gonadotoxic treatment for malignant disease. Samples were obtained from 18 patients with an age range of 14–40 years. During preparation for ovarian tissue cryopreservation, a small random sample of ovarian cortex was fixed immediately after slicing for histological assessment. These samples were used predominantly to characterize primordial follicles: the proportion of follicles in this category has been reported to range between 89% and 99% (Block, 1952; Gougeon et al., 1994; Gook et al., 1999).
Xenografted ovarian tissue
Identification and characterization of ZP proteins in proliferating human follicles was achieved using xenografts of ovarian cortex which had previously been cryopreserved. The xenografts were from the same samples used for characterization of fresh tissue. The preparation of the ovarian tissue (Gook et al., 2005a), the cryopreservation procedure (Gook et al., 1999) and the xenografting of the cryopreserved tissue into immunodeficient female mice (Gook et al., 2001, 2003) have been previously reported.
Fixation and immunohistochemistry
Fixation for immunohistochemistry was carried out on samples of freshly harvested ovarian cortex (for identification of ZP proteins in primordial and primary follicles) and samples of cryopreserved ovarian cortex which had been xenografted and allowed to develop as previously described (for identification of ZP proteins in proliferating follicles). All samples, including positive control oocytes, were fixed immediately in freshly prepared 4% paraformaldehyde in 0.1 mol/l phosphate buffer for 4 h at room temperature. Fixation was continued overnight at 4°C for tissue samples but not for isolated oocytes. All samples were transferred to phosphate buffer and subsequently embedded in a 2% (w/v) agar block. Blocks were processed and embedded in paraffin wax. Serial sections 4 µm thick were cut and placed on SuperFrost®Plus (Menzel-Glaser, Braunschweig, Germany) slides.
To determine areas of interest in the xenografted tissue, every 10th section was stained with haematoxylin and eosin. Sections were dewaxed and stained using a peroxidase-based EnVision + System followed by serum-free protein block (DakoCytomation, NSW, Australia). At least, two sections were incubated with each of the polyclonal rabbit antibodies (raised against each of the recombinant human ZP proteins), diluted using antibody diluent with background reducing components (DakoCytomation) for 1 h at 37°C. Unbound primary antibody was removed by washing in 0.1 M Tris buffered saline with 0.05% Tween20 (pH 7.6) and sections incubated with peroxidase labelled polymer conjugated to goat antirabbit immunoglobulin (Ig) G (EnVision) for 30 min followed by washing and incubation with the substrate chromogen diaminobenzidine (DAB+, DakoCytomation). Sections were lightly counterstained with haematoxylin. Primary antibody was replaced with rabbit IgG normal fraction (DakoCytomation) in the negative control slides. Consecutive sections were assessed for each antibody followed by the appropriate negative control, i.e. the first section from each block was stained for ZP1, the second for ZP2, the third for ZP3 and the fourth acted as a negative control. This was repeated through each entire block. Therefore, in larger follicles it was common for multiple sections to be stained for each antigen whereas, in smaller follicles (particularly primordial), there was often only a single-stained section for each antigen. The same fixation (but for the shorter 4 h period described above), sectioning and immunohistochemistry were applied to oocytes aspirated in IVF cycles which were used as positive controls. Sections were assessed using a Leica DMB microscope and photographed using a digital camera.
Antibodies
Rabbit polyclonal antibodies to human recombinant ZP1 (ZPB), ZP2 (ZPA) and ZP3 (ZPC) proteins were a gift from Jeffrey Harris (Harris et al., 1994).
Follicle classifications and definitions used in the present study
Follicle classification was in accordance with Gougeon (2003).
Primordial: follicles with a single layer of flattened pre-granulosa cells.
Intermediary primordial: follicles with a single layer of mixed elongated and cuboidal granulosa cells.
Primary: follicles with a single layer of cuboidal granulosa cells.
Proliferating primary: primary follicles in which proliferation of the granulosa cells had been initiated resulting in a partial second layer.
Secondary follicles: follicles with at least two complete layers of granulosa cells but no evidence of a cavity.
Antral: follicles with multiple granulosa cell layers and a distinct cavity.
Theca: the outer cell layers of multilayered follicles immediately below the basement membrane of the follicle.
Corona: cell layer immediately adjacent to the oocyte (Thibault et al., 1975).
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Positive controls
Immature oocytes collected from antral follicles following ovarian stimulation for IVF were stained as positive controls. All three antibodies detected proteins in the ZP surrounding the human oocyte (Fig. 1a, ZP1; b, ZP2 and c, ZP3). Using each of the antibodies, similar variation in thickness and intensity can be identified across the ZP; a thin intense ring surrounding the oocyte followed by a broader less intense region and finally another thin intensely stained ring. The three proteins were also present in the oocyte cytoplasm.
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Negative controls
No positive staining was detected in sections of the oocyte, the ovarian tissue or the xenografted tissue (Fig. 2l and inset) when the primary antibody was omitted. No positive staining was observed, with each of the ZP antibodies, in cumulus cells (Fig. 1a inset ZP1; b inset ZP2; c inset ZP3).
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Follicles
In general, the primordial and primary follicles characterized in this study were from fresh ovarian tissue biopsies and all other stages of follicle development were predominantly observed in xenografted tissue. There was, however, some overlap with a small number of proliferating primary (4), secondary (2) and antral (3) follicles observed in fresh tissue and a small number of primordial (14) and primary (2) follicles observed in xenografted tissue (Table I). The pattern of ZP proteins in follicles from all stages was independent of the tissue source.
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Primordial follicles
Presence of the ZP was confirmed by a faint, incomplete ring around the oocyte surface staining positive for ZP1 (Fig. 1d and g) and ZP3 (Fig. 1f and i) but not ZP2 (Fig. 1e and h). A similar appearance was observed with ZP1 and ZP3 within the oocyte and the pre-granulosa cells when the same follicle was stained with these antibodies (Fig. 1d and f) and this was consistent in tissue from different patients (Fig. 1g and i).
Table II summarizes the detection of ZP proteins in primordial follicles. ZP1 and ZP3 were present within the oocyte cytoplasm in 93% and 95% of the primordial follicles examined, respectively, generally around the germinal vesicle (GV) and at relatively low concentrations. In primordial follicles, the pre-granulosa cells are flattened and elongated (e.g. the two follicles abutting each other in Fig. 1d, e and f). Occasionally, one or two of the pre-granulosa cells appear plump, similar to the cuboidal appearance usually observed in primary follicles. These follicles are in transition from the primordial to the primary follicular stage, referred to as intermediary primordial follicles. ZP1 and ZP3 are present between these cuboidal granulosa cells and the elongated cells in these follicles [arrow Fig. 1d and g (ZP1), and arrow Fig. 1f and i (ZP3)]. In all of the primordial follicles containing at least a single cuboidal granulosa cell (58% of the total primordial follicles), ZP1 was detected in both the oocyte and in some of the cuboidal granulosa cells (Table II). Similarly, in all follicles which had at least a single cuboidal granulosa cell and which were stained for ZP3, positive staining was observed in both the oocyte and the granulosa cells. No follicles were observed with ZP protein in the granulosa cells only and not in the oocyte.
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ZP2 is present within the oocyte cytoplasm in a similar pattern to ZP1 and ZP3, however, at a much lower concentration and in only 32% of primordial follicles examined (Table II, Fig. 1e and h). In contrast to ZP1 and ZP3, the presence of ZP2 in the oocyte cytoplasm was only observed in primordial follicles with some cuboidal granulosa cells (left follicle Fig. 1e and h). Although a similar proportion of primordial follicles with cuboidal granulosa cells was observed (32%) in sections stained for ZP2 as observed with ZP1 and ZP3, ZP2 was rarely observed associated with granulosa cells in this type of follicle (5%; 8/168, Fig. 1h). In the total population of primordial follicles stained with ZP2 antibody, this presence of ZP2 between granulosa cells corresponds to only 1% (8/520) of the total primordial follicles examined.
Although very few primordial follicles were observed in the xenografted tissue (Table I, n = 14, Fig. 1j and k), these showed the same characteristic staining pattern to that of the primordial follicles in fresh tissue. All three ZP proteins were observed within the oocyte cytoplasm in all 14 follicles, predominantly around the GV (Fig. 1j, ZP3; Fig. 1k, ZP2). In primordial follicles, organelles were generally clustered around the GV. Again ZP2 was present in the oocyte cytoplasm but often at a reduced level relative to ZP1 and ZP3. In contrast to fresh tissue, in the xenografted tissue, the majority of the primordial follicles (85%, Fig. 1k) had at least one cuboidal granulosa cell. ZP1 and ZP3 were observed between these cuboidal granulosa cells in all of these follicles and ZP2 in 50% (6/12) of these primordial follicles.
Primary follicles
Very few primary follicles were observed either in the fresh ovarian tissue (n = 27) or after xenografting (n = 2). All three proteins were detected within the oocyte cytoplasm around the GV and as a partial ring around the oocyte surface (Fig. 1l ZP3). Each protein was also present between some of the cuboidal granulosa cells (Fig. 1l arrow). Similar levels of ZP1 and ZP3 were observed within these follicles, although the level of ZP2 appeared to be reduced relative to ZP1 and ZP3.
Proliferating primary follicles
In proliferating primary follicles (Fig. 1j and Fig. 2a), all three proteins were present between some of the granulosa cells (ZP3, Fig. 1j arrow, and ZP1, Fig. 2a arrow) and within the oocyte cytoplasm. More intense staining for all three proteins was observed in the oocyte relative to earlier follicular stages. In contrast to the fine lines of dots which stained in the primordial and primary oocyte cytoplasm, all three ZP proteins were present in large areas of the oocyte cytoplasm and absent in other areas, giving a patchy appearance.
Secondary follicles
This classification spans a large number of stages in follicle development and, therefore, similar stages of development have been grouped together and described according to the number of granulosa cell layers.
Two to three granulosa cell layers
These follicles stained positive with ZP1 antibody (Fig. 2d, top right), ZP2 antibody (Fig. 2e, bottom right) and ZP3 antibody (Fig. 2f, top right and middle right). A slightly thicker band of ZP1 and ZP3 encompasses the oocyte in these follicles. ZP2 is now observed as a complete thin ring around the oocyte. More intense staining of all three of the ZP proteins, with a patchy distribution similar to the proliferating primary stage, is apparent in the oocyte cytoplasm. ZP2 continues to be lower in intensity relative to ZP1 and ZP3 in both the oocyte and the granulosa cells.
In follicles with only two layers of granulosa cells (Fig. 2f, top right), all three proteins were present in the cytoplasm of the granulosa cells and between granulosa cells in the layer abutting the oocyte (first layer). Cytoplasmic localization of protein can be seen in Fig. 2c (see arrows) in the first layer and in other layers as positive staining abutting the granulosa cell nuclei (highlighted in Fig. 2d inset). In the second layer, there are sporadic spots of staining with more intensity at the junction of both layers and this is apparent for all three proteins. The next layer of granulosa cells appears between these two layers initiated at one end of the follicle (Fig. 2b and c). Again, all three proteins are present in the first layer at the granulosa cell cytoplasmic surface, i.e. juxtaposed to the zona and at high intensity in the cytoplasm at the junction of the outer layer of granulosa cells and the new layer (Fig. 2d, top right, and 2f middle right). A sporadic low level of all three proteins is present in this middle (third) layer of granulosa cells, and persists through the completion of this layer and initiation of the next layer. The distribution of positive staining is similar for all three proteins (ZP1, Fig. 2d; ZP2, Fig. 2b; ZP3, Fig. 2c and f).
Four to five layers
Proliferation of the granulosa cell layers continues in a similar fashion between the two static layers; the outer one which is destined to be the theca layer and the inner layer which is to be the corona cell layer. Again at the inner surface of the theca cells (previously the second layer), all three ZP proteins are present (e.g. ZP2, Fig. 2e bottom left; ZP3, Fig. 2f bottom centre). In these follicles, the distribution of ZP positive staining is more predominant in front of the theca layer at the proliferating end of the follicle. Relative to previous stages, the intensity of all three proteins is elevated on the inner side of the theca cells. All three proteins continue to be detected in the corona cells abutting the zona (Fig. 2h) and this persists throughout all developmental stages (Fig. 2g, i, j and k). Only sporadic pale staining of the three ZP proteins was detected in the other granulosa cell layers.
An asymmetry in the follicle is apparent around the stage at which the number of granulosa cell layers exceeds 5; one end of the follicle continues to increase in the number of cell layers whereas, at the other, the formation of further layers has halted. A diminished level of staining for the ZP proteins is observed in the theca cells at the end of the follicle that has halted proliferation at four to five layers relative to the theca cells at the proliferating end. Occasionally, ZP protein is detected in the proliferating layers (Fig. 2h). ZP2 continues to be at a slightly lower intensity in the granulosa cells relative to ZP1 and 3.
A similar pattern of differential staining of the zona is apparent with all three antibodies in the same follicle (Fig. 2h; ZP1= h1, ZP2= h2, ZP3= h3). Abutting the oocyte is a very dark ring followed by a reduced intensity wider band relative to the first, followed by another dark furry intermittent layer abutting the corona cells. This pattern is consistently observed in the zona for all follicles at this and later stages (Fig. 2k). The intensity of all three proteins has increased in the oocyte cytoplasm and ZP2 is now roughly similar in intensity to ZP1 and 3. The oocyte diameter is 90 µm at this stage and the zona is 3 µm thick (Fig. 2 h2).
More than six granulosa cell layers
The intensity of all three proteins in the oocyte cytoplasm appears to reach a maximum at this stage (Fig. 2f, top left). The granulosa cell arrangement has altered from concentric layers to a random distribution interrupted by at least one circular formation of tightly concentrated granulosa cells, generally just below the theca layer (Fig. 2d, centre arrow and Fig. 2e, top arrow). Again, more intense staining is observed at the inner side of the theca layer for all three proteins (ZP1 Fig. 2d arrow, ZP2 Fig. 2e top, ZP3 Fig. 2f arrow). Often, this is concentrated in the region of the follicle with more cell layers, which is also the position of the circular formation of the granulosa cells (Fig. 2 arrow d, e and f). In the top follicle in Fig. 2d, a circular formation was observed in the subsequent section at the position of the arrow and another further through the follicle. It could be postulated that these circular formations of granulosa cells are the initiation of the antrum. Sporadic weak staining is observed throughout the other granulosa cells.
Antral follicles
In follicles with a small cavity (Fig. 2g ZP1), almost no ZP protein is detected in the granulosa cells apart from an occasional small area just below the theca layer (see arrow). In contrast to previous stages where all three ZP proteins were present in only the first layer of corona cells, the staining intensity has now increased for all three proteins and has expanded to the first two to three layers of the corona/cumulus cells. With an increase in the cavity size (Fig. 2i), all previous staining in the mural granulosa cells and below the theca layer has disappeared and only the corona/cumulus pedicles are stained. Slightly lower intensity of all three proteins is now observed in the oocyte cytoplasm and this is maintained through to the preovulatory stage. Similarly, there is no change to the ZP tri-layered appearance. As the follicle increases in size, whether in xenografted (Fig. 2j) or fresh tissue (Fig. 2k), only the first two layers of the corona cells continue to contain all three ZP proteins (see Fig. 2j inset).
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Discussion |
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Although the presence and expression of a number of antigens have been demonstrated in human primordial follicles (Abir et al., 2004a
,b, 2005; Harel et al., 2006), there is currently no information relating to the important oocyte-specific proteins which comprise the ZP. The present study has established that the three proteins required for the formation of the ZP are present within the human primordial oocyte and that the presence of these proteins within the oocyte is the normal manifestation of the initiation of the ZP. The consistent observation of ZP1 and ZP3 proteins in 90% of the primordial oocytes in a random selection of small samples of ovarian cortex from each of 18 patients, together with the knowledge that, in the human ovary, very few follicles enter the growing phase (Gougeon and Chainy, 1987), suggests that this initial synthesis does not occur after recruitment into the growing follicular pool.
This could conflict with the concept that primordial follicles, including the oocyte, are quiescent until selected into the growth pool. Evidence of at least one of the ZP proteins within oocytes of a majority of primordial follicles in other mammalian species (Kang, 1974; Wolgemuth et al., 1984; Epifano et al., 1995; Grootenhuis et al., 1996; Kolle et al., 1996; Martinez et al., 1996) confirms our observation that appearance of these proteins does not signify recruitment.
An alternative hypothesis is that the ZP proteins appeared in the primordial follicle during oogenesis and have been retained throughout reproductive life. There is some evidence of small deposits of ZP matrix shortly after birth in mouse ovaries (Chiquoine, 1960; Odor and Blandau, 1969) and ZP proteins within the oocytes of early rabbit neonates (Lee and Dunbar, 1993). Although protein was not detected until Day 2 in mouse neonates, transcription of one ZP gene was detected just prior to birth (Millar et al., 1993). This stage in the mouse ovary (Chiquoine, 1960), corresponds to the ovary in a 7-month-old human fetus (Ohno et al., 1962).
Although the presence of ZP proteins in human primordial follicles could be demonstrated in fresh biopsies of human ovarian cortex, the detailed study of post-recruitment stages of human follicular development in this study was only possible due to the availability of numerous growing follicles in xenografted tissue. The shift to a higher proportion of co-presence of ZP2 in the oocyte and granulosa cells of primordial follicles in the xenografted tissue, and its association with more morphologically advanced appearance, might suggest that appearance of ZP2 in the granulosa cells is coincidental with recruitment. A similar conclusion was drawn by Martinez et al. (1996) following detection of ZPA (2) expression in the granulosa cells of some primary follicles in cynomologus monkey ovaries.
The present study shows that all three proteins are present within both the granulosa cells and the oocyte, and increase with development. Coexistence in the human oocyte and granulosa cells would concur with observations in a number of mammalian species (Wolgemuth et al., 1984; Lee and Dunbar, 1993; Grootenhuis et al., 1996; Kolle et al., 1996; Martinez et al., 1996; Prasad et al., 2000; Bogner et al., 2004), with the exception of the mouse, where detection is limited to the oocyte (Epifano et al., 1995; El-Mestrah et al., 2002). Although coexistence of ZP protein in both the oocyte and the granulosa cells is associated with developing follicles, the stage at which ZP protein is initially detected varies in the above studies. ZP proteins in granulosa cells are detected consistently at the secondary stage of follicular development (Wolgemuth et al., 1984; Lee and Dunbar, 1993; Grootenhuis et al., 1996; Kolle et al., 1996; Martinez et al., 1996; Prasad et al., 2000; Bogner et al., 2004). When all three proteins were assessed concurrently (Martinez et al., 1996; Bogner et al., 2004), only one protein was detected in granulosa cells in the primary follicle whereas, by the secondary stage, all three proteins were present in granulosa cells. In these studies (Martinez et al., 1996; Bogner et al., 2004), this pattern was mirrored in the oocyte, suggesting that a single protein, at least at the time of recruitment, may not act as an adequate marker of ZP initiation.
During the transition through the secondary follicular stage, the level of all three ZP proteins increases in the oocyte cytoplasm and the granulosa cells abutting the oocyte resulting in an increase in the zona matrix, suggestive of a contribution from both cell types. Two important points, however, should be emphasized. First, the present study does not permit any conclusions relating to site of synthesis of ZP proteins. Second, we cannot exclude the possibility that ZP proteins in granulosa cells have been selectively endocytosed into individual granulosa cells after synthesis elsewhere, although we believe that the localization of positive staining immediately adjacent to granulosa cell nuclei demonstrates cytoplasmic localization. The possibility of some protein also present in interstitial spaces cannot be excluded.
ZP synthesis has been confirmed by the presence of mRNA using in situ hybridization in both the oocyte and the granulosa cells of porcine and Macaca fascicularis secondary follicles (Kolle et al., 1996; Martinez et al., 1996). In the oocyte, the level of ZP mRNA increased throughout the secondary follicular stage peaking in the small antral follicle when the oocyte acquires maximum size (Roller et al., 1989; Epifano et al., 1995; Bogner et al., 2004). A similar correlation was observed with the level of ZP protein in the present study. The diminished ZP protein levels observed in oocytes from large antral follicles in the present study agree with the observations that, although synthesis declines in the antral stage oocyte, it continues up to ovulation (Roller et al., 1989).
The pattern of distribution of granulosa cells positive for ZP proteins associated with developmental changes in the secondary follicles suggests that the granulosa cells are not homogeneous and that some have differentiated during this proliferative stage. The layer of granulosa cells abutting the zona/oocyte (the corona layer), although increasing in cell numbers, maintains high levels of ZP proteins within the cytoplasm through the secondary stage to the large antral stage and has, by this stage, increased to at least two layers of positive cells around the oocyte. In contrast, the other layers of the corona/cumulus complex and the pedicle are negative for the proteins, showing differentiation into at least two populations of cells within the corona/cumulus mass. This distribution, with only the corona cells intimately associated with the oocyte maintaining presence of ZP proteins, has been previously observed in human and monkey antral follicles (Hinsch et al., 1998, 1999; Bogner et al., 2004). The continuity of the proteins between the oocyte surface through the zona and the corona layer throughout development suggest a possible mechanism of communication between the oocyte and corona cells which had previously thought to only involve the processes of the corona cells traversing the ZP. The concomitant morphological changes in the secondary follicles and redistribution of granulosa cells containing ZP proteins into specific regions suggest that there is an organization of mural granulosa cells within these developing follicles which may relate to orientation for antrum formation.
The present study reports the significant finding that ZP proteins are present within the human primordial follicle in both the oocyte and the granulosa cells. The differential patterns of ZP staining in the granulosa cells which correlate with stages of follicular development suggest that these ZP proteins may act as novel markers of human folliculogenesis. The hypothesis that ZP proteins present in the primordial follicles have been conserved from oogenesis through reproductive life in the human is an intriguing concept which requires further investigation.
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References |
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Abir R, Fisch B, Jin S, Barnnet M, Freimann S, Hurk Van den R, Feldberg D, Nitke S, Krissi H, Ao A. Immunocytochemical detection and RT–PCR expression of leukaemia inhibitory factor and its receptor in human fetal and adult ovaries. Mol Hum Reprod (2004) a10:313–319.
Abir R, Fisch B, Jin S, Barnnet M, Kessler-Icekson G, Ao A. Expression of stem cell factor and its receptor in human fetal and adult ovaries. Fertil Steril (2004) b82(Suppl_3):1235–1243.[CrossRef][Web of Science][Medline]
Abir R, Fisch B, Jin S, Barnnet M, Ben-Haroush A, Felz C, Kessler-Icekson G, Feldberg D, Nitke S, Ao A. Presence of NGF and its receptors in ovaries from human fetuses and adults. Mol Hum Reprod (2005) 11:229–236.
Bedford J. Sperm/egg interaction; the specificity of human spermatozoa. Anat Rec (1977) 188:477.[CrossRef][Medline]
Bleil JD, Wassarman PM. Mammalian sperm–egg interaction: identification of a glycoprotein in mouse egg zonae pellucidae possessing receptor activity for sperm. Cell (1980) a20:873–882.[CrossRef][Web of Science][Medline]
Bleil JD, Wassarman PM. Structure and function of the zona pellucida: identification and characterization of the proteins of the mouse oocyte’s zona pellucida. Dev Biol (1980) b76:185–202.[CrossRef][Web of Science][Medline]
Block E. Quantitative morphological investigations of the follicular system in women. Acta Anat (1952) 14:108–123.[Web of Science][Medline]
Bogner K, Hinsch KD, Nayudu P, Konrad L, Cassara C, Hinsch E. Localization and synthesis of zona pellucida proteins in the marmoset monkey (Callithrix jacchus) ovary. Mol Hum Reprod (2004) 10:481–488.
Chiquoine AD. The development of the zona pellucida of the mammalian ovum. Am J Anat (1960) 106:149–169.[CrossRef][Web of Science][Medline]
El-Mestrah M, Castle PE, Borossa G, Kan FW. 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 (2002) 66:866–876.
Epifano O, Liang LF, Familari M, Moos MC Jr, Dean J. Coordinate expression of the three zona pellucida genes during mouse oogenesis. Development (1995) 121:1947–1956.[Abstract]
Gook DA, Edgar DH, Stern C. Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1, 2- propanediol. Hum Reprod (1999) 14:2061–2068.
Gook DA, McCully BA, Edgar DH, McBain JC. Development of antral follicles in human cryopreserved ovarian tissue following xenografting. Hum Reprod (2001) 16:417–422.
Gook DA, Edgar DH, Borg J, Archer J, Lutjen PJ, McBain JC. Oocyte maturation, follicle rupture and luteinization in human cryopreserved ovarian tissue following xenografting. Hum Reprod (2003) 18:1772–1781.
Gook DA, Edgar DH, Borg J, Archer J, McBain JC. Diagnostic assessment of the developmental potential of human cryopreserved ovarian tissue from multiple patients using xenografting. Obstet Gynecol Surv (2005) a60:241–242.[CrossRef]
Gook DA, Edgar DH, Borg J, Archer J, McBain JC. Diagnostic assessment of the developmental potential of human cryopreserved ovarian tissue from multiple patients using xenografting. Hum Reprod (2005) b20:72–78.
Gougeon A. The early stages of follicular growth. In: Biology and Pathology of the Oocyte—Trounson AO, Gosden RG, eds. (2003) Cambridge, UK: Cambridge University Press. 29–43.
Gougeon A, Chainy GB. Morphometric studies of small follicles in ovaries of women at different ages. J Reprod Fertil (1987) 81:433–442.
Gougeon A, Echochard R, Thalabard JC. Age-related changes of the population of human ovarian follicles: increase in the disappearance rate of non-growing and early-growing follicles in aging women. Biol Reprod (1994) 50:653–663.[Abstract]
Green DP. Three-dimensional structure of the zona pellucida. Rev Reprod (1997) 2:147–156.[Abstract]
Greve JM, Wassarman PM. Mouse egg extracellular coat is a matrix of interconnected filaments possessing a structural repeat. J Mol Biol (1985) 181:253–264.[CrossRef][Web of Science][Medline]
Grootenhuis AJ, Philipsen HL, de Breet-Grijsbach JT, van Duin M. Immunocytochemical localization of ZP3 in primordial follicles of rabbit, marmoset, rhesus monkey and human ovaries using antibodies against human ZP3. J Reprod Fertil Suppl (1996) 50:43–54.[Medline]
Harel S, Jin S, Fisch B, Feldberg D, Krissi H, Felz C, Freimann S, Tan SL, Ao A, Abir R. Tyrosine kinase B receptor and its activated neurotrophins in ovaries from human fetuses and adults. Mol Hum Reprod (2006) 12:357–365.
Harris JD, Hibler DW, Fontenot GK, Hsu KT, Yurewicz EC, Sacco AG. Cloning and characterization of zona pellucida genes and cDNAs from a variety of mammalian species: the ZPA, ZPB and ZPC gene families. DNA Seq (1994) 4:361–393.[Web of Science][Medline]
Hinsch E, Hagele W, Ven van der H, Oehninger S, Schill WB, Hinsch KD. Immunological identification of zona pellucida 2 (ZP2) protein in human oocytes. Andrologia (1998) 30:281–287.[Web of Science][Medline]
Hinsch E, Oehninger S, Schill WB, Hinsch KD. 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 (1999) 14:419–428.
Jewgenow K, Fickel J. Sequential expression of zona pellucida protein genes during the oogenesis of domestic cats. Biol Reprod (1999) 60:522–526.
Kang YH. Development of the zona pellucida in the rat oocyte. Am J Anat (1974) 139:535–565.[CrossRef][Web of Science][Medline]
Kolle S, Sinowatz F, Boie G, Totzauer I, Amselgruber W, Plendl J. 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 (1996) 28:441–447.[CrossRef][Web of Science][Medline]
Lee VH, Dunbar BS. Developmental expression of the rabbit 55-kDa zona pellucida protein and messenger RNA in ovarian follicles. Dev Biol (1993) 155:371–382.[CrossRef][Web of Science][Medline]
Lefievre L, Conner SJ, Salpekar A, Olufowobi O, Ashton P, Pavlovic B, Lenton W, Afnan M, Brewis IA, Monk M, et al. Four zona pellucida glycoproteins are expressed in the human. Hum Reprod (2004) 19:1580–1586.
Liu De Y, Lopata A, Pantke P, Baker GHW. Horse and marmoset monkey sperm bind to the zona pellucida of salt-stored human oocytes. Fertil Steril (1991) 56:764–767.[Web of Science][Medline]
Martinez ML, Fontenot GK, Harris JD. The expression and localization of zona pellucida glycoproteins and mRNA in cynomolgus monkeys (Macaca fascicularis). J Reprod Fertil Suppl (1996) 50:35–41.[Medline]
Millar SE, Lader ES, Dean J. ZAP-1 DNA binding activity is first detected at the onset of zona pellucida gene expression in embryonic mouse oocytes. Dev Biol (1993) 158:410–413.[CrossRef][Web of Science][Medline]
Moller CC, Bleil JD, Kinloch RA, Wassarman PM. Structural and functional relationships between mouse and hamster zona pellucida glycoproteins. Dev Biol (1990) 137:276–286.[CrossRef][Web of Science][Medline]
Odor DL, Blandau RJ. Ultrastructural studies on fetal and early postnatal mouse ovaries. II. Cytodifferentiation. Am J Anat (1969) 125:177–215.[CrossRef][Web of Science][Medline]
Ohno S, Klinger HP, Atkin NB. Human oogenesis. Cytogenetics (1962) 1:42–51.[Medline]
Prasad SV, Wilkins B, Skinner SM, Dunbar BS. Evaluating zona pellucida structure and function using antibodies to rabbit 55 kDa ZP protein expressed in baculovirus expression system. Mol Reprod Dev (1996) 43:519–529.[CrossRef][Web of Science][Medline]
Prasad SV, Skinner SM, Carino C, Wang N, Cartwright J, Dunbar BS. Structure and function of the proteins of the mammalian Zona pellucida. Cells Tissues Organs (2000) 166:148–164.[CrossRef][Web of Science][Medline]
Roller RJ, Kinloch RA, Hiraoka BY, Li SS, Wassarman PM. Gene expression during mammalian oogenesis and early embryogenesis: quantification of three messenger RNAs abundant in fully grown mouse oocytes. Development (1989) 106:251–261.[Abstract]
Thibault C, Gerard M, Menezo Y. Preovulatory and ovulatory mechanisms in oocyte maturation. J Reprod Fertil (1975) 45:605–610.
Wassarman PM. Zona pellucida glycoproteins. Annu Rev Biochem (1988) 57:415–442.[CrossRef][Web of Science][Medline]
Wassarman PM. Regulation of mammalian fertilization by zona pellucida glycoproteins. J Reprod Fertil Suppl (1990) 42:79–87.[Medline]
Wolgemuth DJ, Celenza J, Bundman DS, Dunbar BS. Formation of the rabbit zona pellucida and its relationship to ovarian follicular development. Dev Biol (1984) 106:1–14.[CrossRef][Web of Science][Medline]
Submitted on March 2, 2007; resubmitted on October 19, 2007; accepted on October 24, 2007.
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