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Harmful effects of anti-zona pellucida antibodies in folliculogenesis, oogenesis, and fertilization
Journal of Reproductive Immunology 79 (2009) 148–155
Harmful effects of anti-zona pellucida antibodies in folliculogenesis, oogenesis, and fertilization Giannina Calongos a , Akiko Hasegawa b , Shinji Komori a,b , Koji Koyama a,b,∗ a b
Department of Obstetrics and Gynecology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan Laboratory of Developmental Biology and Reproduction, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan Received 27 February 2008; accepted 17 June 2008
Abstract The zona pellucida (ZP) is an extracellular matrix that surrounds the mammalian oocyte and plays an important role in normal folliculogenesis and fertilization. Because of its strong immunogenicity and its possible relation with premature ovarian failure, we conducted the present study to examine whether or not anti-ZP antibodies impaired folliculogenesis. Mouse preantral follicles were cultured with anti-ZP antibodies to evaluate the effects on follicle growth and antral formation. The cultured follicles were also examined by electron microscope and assessed for oocyte maturation, fertilization capacity, and embryo development. The results showed that follicles cultured with anti-ZP antibodies had a smaller diameter than the controls. Also, these antibodies reduced antral formation, mucification, maturation of oocytes (metaphase II), and fertilization rates. Morphologically, ZP thickness was lower in the antiZP antibody groups. The quantity of granulosa cell microvillous processes that transverse the ZP was diminished in follicles cultured with anti-ZP antibodies. In conclusion, anti-ZP antibodies were harmful to the normal development of mouse follicles and oocytes in vitro. These antibodies may be a cause of premature ovarian failure syndrome because they disrupt the gap junctions between the oocyte and granulosa cells and, as a consequence, damage the bidirectional communication necessary for normal folliculogenesis. © 2009 Elsevier Ireland Ltd. All rights reserved. Keywords: Anti-zona pellucida antibodies; Preantral follicles; Folliculogenesis; Oogenesis; Fertilization
1. Introduction The zona pellucida (ZP) is an extracellular coat that surrounds the plasma membrane of mammalian eggs and pre-implantation embryos. It is composed of interconnected filaments of three or four glycoproteins designated ZP1, ZP2, ZP3, and ZP4, which are expressed and assembled by the oocyte (Eberspaecher et al., 2001; Harris et al., 1994; Lefievre et al., 2004). In mice, previous studies suggested that the ZP filaments are ∗
Corresponding author at: Department of Obstetrics and Gynecology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya 663-8501, Japan. Tel.: +81 798 45 6481; fax: +81 798 46 4163. E-mail address: [email protected] (K. Koyama).
polymers of ZP2 and ZP3 cross-linked by ZP1 (Greve and Wassarman, 1985; Wassarman and Mortillo, 1991). The ZP plays an important role during fertilization as it mediates relatively species-specific sperm-egg recognition and induction of acrosome reaction. Also, after that, the ZP protects the early embryo during its passage through the oviduct before implantation (Bronson and MacLaren, 1970; Wassarman, 1987; Yanagimachi, 1994). During ovarian folliculogenesis, an interdependent and close association between the somatic and germ cells is important for the regulation of the preantral growth of the oocyte and the nuclear events before and at the time of ovulation (Anderson and Albertini, 1976; Canipari, 2000). Also, the bi-directional paracrine communication
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at the oocyte–granulosa cell interface is required for the completion of oocyte growth and the acquisition of cytoplasmic meiotic competence necessary for fertilization (Albertini et al., 2001; Carabatsos et al., 2000; Gittens and Kidder, 2005). The ZP physically separates the somatic and the germinal components. However, studies in knockout mice showed that these critical interactions are maintained in the growing follicle via granulosa cell processes that penetrate the ZP and make contact with the oolemma (Koyama and Hasegawa, 2006; Rankin et al., 2001). From a clinical viewpoint, antiovarian autoimmunity can be directed against the somatic (mainly the granulosa and the thecal layer) or the germinal component of the ovarian follicle (oocyte or ZP) and can be associated or not with somatic autoimmune disorders (Forges et al., 2004; Koyama and Hasegawa, 2006). As the ZP has been shown to possess a strong immunogenicity, it may be a target antigen in ovarian autoimmunity (Kelkar et al., 2005; Koyama et al., 2005). Also, previous studies using animal models immunized with ZP proteins demonstrated that among those antiovarian antibodies, anti-ZP antibodies were the most likely to cause premature ovarian failure and infertility (Govind et al., 2002; Skinner et al., 1984). Moreover, anti ZP antibodies are considered to be a cause of infertility because of their blocking effects on sperm–ZP binding (Takamizawa et al., 2007). Despite the many publications that have dealt with the relationship of anti-ZP autoimmunity and infertility, the real significance of these antibodies remains to be established. To evaluate the direct effect of anti-ZP antibodies in ovarian follicles, an in vitro culture system of juvenile mouse follicles would be useful, as their follicle formation occurs after birth and the isolation of a large quantity of homogenous follicles is possible (Demeestere et al., 2005). Culture of individual intact follicles on a collagencoated membrane has been shown to allow an accurate assessment of folliculogenesis by measuring their diameters, as well as evaluating their antral formation (Smitz and Cortvrindt, 2002). Mature oocyte and blastocyst formation after in vitro maturation and fertilization is also obtainable using this culture method (Adam et al., 2004; Calongos et al., 2008). In order to evaluate the effects of anti-ZP antibodies and their relation with ovarian dysfunction, an in vitro culture system of mouse preantral follicles was designed. Folliculogenesis (based on follicle growth, antral formation, and oocyte–granulosa cell connection development), oogenesis (including diameters, maturation, and fertilization capacity), and embryo development were evaluated as parameters.
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2. Materials and methods 2.1. Animals Specific pathogen-free, female (C57BL/6 X DBA/2) F1 mice (Japan SLC Inc., Shizuoka, Japan) were kept in light- and temperature-controlled conditions and provided with sterile food and water ad libitum according to the guidelines set by the Hyogo College of Medicine. All animal experiments were approved by the Committee on Animal Experimentation of the Hyogo College of Medicine. 2.2. Isolation of preantral follicles Sixteen-day-old mice were anesthetized with diethylether and killed by cervical dislocation. Preantral follicles were isolated and selected as described in a previous paper (Calongos et al., 2008). Ovaries were removed and transferred to a 200-L drop of Leibovitz’s 15 with L-glutamine (Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS; Gibco, NY, USA), 50 IU/mL of penicillin and 50 g/mL of streptomycin (Sigma–Aldrich) in a 60-mm Falcon dish (Becton Dickinson, NJ, USA). Preantral follicles were obtained by mechanical isolation with 30G needles and transferred to a new drop for evaluation. Selected preantral follicles were 110–130 m in diameter with a centrally located oocyte and no damage to the basal membrane. Ten to fifteen follicles with these characteristics were obtained from each ovary. 2.3. In vitro growth of preantral follicles with anti-ZP antibodies Antiserum was prepared by immunization of rabbits with synthetic peptides of mouse ZP2 and ZP3 conjugated with diphtheria toxoid, as described in previous papers (Hasegawa et al., 2002; Koyama et al., 2005; Millar et al., 1989). The growth medium was prepared as described in previous studies carried out in our laboratory with some modifications (Calongos et al., 2008; Hasegawa et al., 2004, 2006). The growth medium was composed of ␣-minimum essential medium with 5% FCS, ITS (insulin 1 mg/mL, transferrin 0.55 mg/mL, selenium 0.5 g/mL), penicillin–streptomycin solution, L-glutamine 2 mM (all these products were obtained from Sigma–Aldrich), and 100 mIU/mL of FSH (Fertinorm, Serono, Tokyo, Japan). Antisera were treated by heating at 56 ◦ C for 30 min to inactivate the complement and added to the growth medium at a 10% concentration. Pre-immune serum was used as a control.
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Selected preantral follicles were randomly distributed in the control, anti-ZP2, and anti ZP3 groups, and cultured in 24-well cell clusters (Iwaki, Chiba, Japan) on membrane inserts (Transwell-COL 3.0-m pore size, Corning, NY, USA) in 700 L of the respective growth medium at 37 ◦ C in a humidified atmosphere of 5% CO2 in air. Three hundred microliters of culture medium per well was replaced with fresh medium every other day. For a measurement of follicle diameters without impairment of the culture conditions, pictures of the preantral follicles were taken every day using an inverted microscope (Eclipse TE300 Nikon, Japan) with a digital camera (Hamamatsu C4742-95, Japan) at 40× magnification. Two diameters (width and length) were measured and the average of these two values was documented as the diameter of each follicle. 2.4. In vitro maturation of antral follicles cultured with anti-ZP antibodies and oocyte diameter measurement Following in vitro growth, the follicles were transferred to a maturation medium, as described in a previous paper (Calongos et al., 2008). The maturation medium was composed of growth medium without antibodies supplemented with 2.5 IU/mL of human chorionic gonadotropin (HCG, Mochida, Tokyo, Japan) and 10 ng/mL of mouse epithelial growth factor (EGF, Upstate Biotechnology, Inc, NY, USA). Eighteen hours later, follicle mucification was evaluated under an inverted microscope. Then, the oocytes were mechanically denuded from the mucified granulosa cells for assessment of the maturation stage. Germinal vesicles (GV) containing oocytes and first polar body extruded oocytes were assessed as the GV stage and the MII (metaphase II) stage respectively. The intermediate oocytes between GV and MII stages were considered to be MI stage oocytes. For the measurements of oocyte, ooplasm, and zona pellucida diameters, pictures of the denuded oocytes were taken using an inverted microscope (Eclipse TE300 Nikon, Japan) with a digital camera (Hamamatsu C474295, Japan) at 80× magnification. Two diameters were measured and the average of these two values was documented as the respective oocyte diameters. 2.5. In vitro fertilization and further embryo development Sperm were collected from the cauda epididymis of 12-week-old F1 male mice and suspended in a drop
(200 L) of TYH medium (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan) under mineral oil. The composition of TYH medium is 119.37 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl2 , 1.19 mM KH2 PO4 , 1.19 mM MgSO4 , 25.07 mM NaHCO3 , 1.0 mM sodium pyruvate, 5.56 mM glucose, 4 mg/mL of bovine serum albumin, 0.075 mg/mL of penicillin, and 0.005 mg/mL of streptomycin. Sperm capacitation was made by incubation at 37 ◦ C in a humidified atmosphere of 5% CO2 in air for 1 h. Mature oocytes were obtained from preantral follicles that were grown and matured in vitro. Then, these MI and MII stage oocytes were transferred to a drop of TYH medium. Capacitated sperm were mixed with mature oocytes at a concentration of 0.3 × 106 sperm/mL and incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 in air for 6 h. Fertilized oocytes were assessed for the presence of two pronuclei under a phase-contrast inverted microscope and transferred to a drop of fresh, potassium simplex-optimized medium (KSOM; Erbach et al., 1994). Embryo stages were assessed at 6, 24, 48, 72, and 96 h after insemination. 2.6. Electron microscopy At the end of in vitro growth, follicles from each group were transferred to different centrifuge tubes and centrifuged for 3 min at 200 × g. Follicles were then fixed with 1.25% glutaraldehyde in 0.1 M phosphate buffer for 1 h at 4 ◦ C, followed by buffer rinses. After that, the samples were treated for 1 h at 4 ◦ C with 1% osmium tetroxide in 0.1 M phosphate buffer and rinsed with buffer. Then, they were dehydrated in a graded series of ethanol (50%, 70%, 80%, 90%, 95%, and 100%) and embedded in Epon 812. Between all the steps, samples were centrifuged for 5 min at 200 × g. Sections of 80 nm were cut with a diamond knife on an ultramicrotome (Leica Ultracut UCT, Leica, Austria) and mounted on copper grids. The sections were stained with uranyl acetate and Reynold’s lead before examination in an electron microscope (JEM-1220 JEOL, Japan). 2.7. Statistical analysis Antral formation, mucification, oocyte maturation, fertilization, and embryo development results were expressed in percentages and statistically analyzed based on the χ2 test. Follicle diameter and oocyte diameter were expressed in mean ± S.D. and analyzed based on Student’s t test. P values below 0.05 were considered to be statistically significant.
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3. Results 3.1. Effects of anti-ZP antibodies on follicle growth and antral formation Twenty preantral follicles per group were used for the study. During in vitro growth, granulosa cells differentiated and an antrum was noticeable from day 4 in the control group (Fig. 1). At this time, in both anti-ZP2 and anti-ZP3 groups, antral formation was not evident. At day 7, preantral follicles in the control group were clearly differentiated from antral ones, with granulosa cells tightly joined around the oocyte as a corona and the presence of the antrum (Fig. 1d). However, antral follicles in anti-ZP2 and anti-ZP3 groups were smaller and not as well developed as those in the control group. This type of follicle was more evident in the anti-ZP3 group with oocytes loosely surrounded by granulosa
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cells, resulting in the extrusion of the oocyte and degeneration of the remaining granulosa cells (Fig. 1l). Preantral follicle diameters in the control group increased continuously and uniformly during the seven days of in vitro growth (Fig. 2). On the contrary, growth of follicles cultured with anti-ZP antibodies was retarded from day 2. 3.2. Effects of anti-ZP antibodies on antral formation, mucification, and oocyte maturation rates and diameters For this part of the study, 60 preantral follicles per group and their corresponding oocytes obtained after in vitro maturation were used. After seven days of invitro growth, the antral formation rate was lower in the anti-ZP2 (68.3%) and anti-ZP3 groups (63.3%) than in the control group (93.3%; Fig. 3). Then, all follicles were transferred to the maturation medium to deter-
Fig. 1. Effects of anti-ZP2 and anti-ZP3 antibodies on the growth and development of preantral follicles during in vitro growth. Cultured follicles in the control group (a–d), the anti-ZP2 group (e–h), and the anti-ZP3 group (i–l) at day 0, day 3, day 5, and day 7 are shown. The progressive increase in size of the follicles in the control is noticeable, while follicles cultured with anti-ZP antibodies stop growing normally. Antral formation (arrow) can be seen in the control group with the increase in the size of follicles and the proliferation of granulosa cells. In the anti-ZP3 group, the extrusion of the oocyte with the degeneration of the remaining granulosa cells is shown. Scale bars represent 150 m.
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G. Calongos et al. / Journal of Reproductive Immunology 79 (2009) 148–155 Table 1 Effects of anti-ZP2 and anti-ZP3 antibodies on oocyte diameters after in vitro growth and maturation.
Oocyte Ooplasm Zona pellucida
Control (m)
Anti-ZP2 (m)
Anti-ZP3 (m)
83.38 ± 1.49 69.03 ± 0.66 5.95 ± 0.37
82.62 ± 2.29 68.49 ± 1.71 5.54 ± 0.53a
78.97 ± 2.12b 67.73 ± 2.82c 4.17 ± 0.61d
Data were expressed as mean ± S.D. Superscripts a, b, c, and d denote a significant difference between the study and control groups. P < 0.05.
the control group (Table 1). Only the ZP diameter was reduced in the anti-ZP2 group. Fig. 2. Effects of anti-ZP2 and anti-ZP3 antibodies on follicle diameter and growth profile during in vitro growth. Data represent diameters (mean ± S.D.). Asterisks (*) indicate a significant difference among anti-ZP2 or anti-ZP3 and the control on each day of culture (P < 0.05 Student’s t-test).
mine the mucification and oocyte maturation rates. The mucification rate was reduced in the anti-ZP2 (51.7%) and anti-ZP3 groups (43.3%) compared with the control group (90%; Fig. 3). MII oocyte percentages were also lower in the anti-ZP2 (33.3%) and anti-ZP3 groups (3.3%) than in the control group (51.7%). MI oocyte rates were 41.4%, 59.6%, and 58.8% for control, anti-ZP2, and anti-ZP3 groups respectively. The incidence of GV stage oocytes was 3.4%, 3.5%, and 3.3% in the control, anti-ZP2 and anti-ZP3 groups respectively. Degenerated oocyte values were higher in the anti-ZP3 group (35%) compared with the other two groups (3.4%; Fig. 3). Oocyte and ooplasm diameters as well as ZP thickness were smaller in the anti-ZP3 group compared with
3.3. Effects of anti-ZP antibodies on fertilization and embryo development rates Nineteen mature oocytes (MI and MII stages) per group were used for the study. The fertilization rate was 94.7% in the control group and 0% in both the anti-ZP2 and anti-ZP3 groups (Fig. 3). After 24 and 96 h of culture, the two-cell stage embryo rate and the blastocyst formation rate were 89.5% and 57.9% respectively for the control group. Both anti-ZP2 and anti-ZP3 groups failed to develop embryos during the culture. 3.4. Electron microscopic observation After 7 days of in vitro growth, follicles from each group were fixed and treated for electron microscopic observation. The ZP was thinner in the anti-ZP antibody groups than in the control group, similar to the optical
Fig. 3. Effects of anti-ZP2 and anti-ZP3 antibodies on antral formation, mucification, oocyte maturation, and fertilization rates. Significant values are written above the columns based on the χ2 test.
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microscopic observation shown in Table 1. At higher magnification, the granulosa cell processes that transverse the ZP are present in great quantities and make contact with the oolemma in the control group. In contrast, in follicles cultured with anti-ZP antibodies the quantity is dramatically decreased (Fig. 4b and c). 4. Discussion
Fig. 4. Electron microscopic observation of follicles cultured with pre-immune rabbit serum (control), and with anti-ZP2 and anti-ZP3 antibodies. Cultured follicles after 7 days of in vitro growth in the control (a), the anti-ZP2 (b), and the anti-ZP3 groups (c) are shown at ×5000 magnification. The presence of the ZP between the oocyte (O) and the granulosa cells (GC) is noticeable. Also, the granulosa cell projections that traverse the ZP can be identified (arrows). There were fewer of these projections in the anti-ZP antibody groups compared with the control group.
The relationship between anti-ZP antibodies and infertility is suggested in serum, peritoneal fluid, and follicular fluid (Ivanova et al., 1999; Szczepanska et al., 2001). Also, previous studies showed that anti-ZP antibodies are an important etiology in autoimmune premature ovarian failure (POF; Kelkar et al., 2005; Koyama et al., 2005; Takamizawa et al., 2007). POF is a disorder that affects 1% of women and is characterized by amenorrhea with elevated FSH levels because of the cessation of ovarian function. It has a multicausal etiology, such as genetic, autoimmune, iatrogenic, environmental, and idiopathic (Goswani and Conway, 2005). Autoimmune causes account for up to 30% of cases of POF (Conway et al., 1996). The cellular targets of the immune reaction can be the somatic or the germinal component of the follicle, mainly the oocyte itself or the ZP (Forges et al., 2004). Because of the importance of autoimmunity in POF and infertility etiology, a culture system of mouse follicles was designed to evaluate the effects of ZP antibodies in vitro. Preantral follicles cultured with both anti-ZP2 and anti-ZP3 antibodies showed smaller diameters and antral formation compared with the control group. This observation correlates with in vivo experiments in which immunization of different animals with natural source or recombinant ZP proteins caused ovarian dysfunction (Govind et al., 2002; Koyama et al., 2005; Skinner et al., 1984). During in vitro growth, follicles cultured with anti-ZP3 had granulosa cells that were not as tightly attached to the oocyte as those in the control group, which resulted in the extrusion of the oocyte. As a result, the degenerated follicle rate was higher in the anti-ZP3 group (Fig. 3). This growth pattern was also observed in ZP3 knockout mice follicles that developed a corona radiata that was less uniformly arrayed than in normal animals and as a result the degree of contact between the oocyte and granulosa cells was diminished (Liu et al., 1996; Rankin et al., 1996). Moreover, the ovarian histology of ZP2 and ZP3 knockout mice showed that in preovulatory follicles the cumulus cells were poorly organized and in some cases were completely separated from the oocyte (Rankin and Dean, 1996; Rankin et al., 1996, 2001).
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We observed that the mucification and MII oocyte rates were significantly lower in the anti-ZP antibody groups than in the control group as a result of the abnormal folliculogenesis. These results correlate with a previous study that reported impairment of folliculogenesis as well as inhibition of the growth and maturation of oocytes after in vitro culture of oocyte–granulosa cell complexes and intact follicles with anti-ZP antibodies (Koyama et al., 2005). In the present study, anti-ZP3 had a more deleterious effect in folliculogenesis than anti-ZP2, with the highest rate of degenerated follicles and oocytes. As ZP2 knockout mice form a thin ZP in early follicles while ZP3 knockout mice completely lack ZP (Rankin et al., 1996, 2001), it is possible that the function of ZP3 in ZP formation and structure is essential. ZP2 and ZP3 are synthesized and traffic independently through the oocyte; however, ZP3 presence is necessary for incorporation of ZP2 into the ZP during oogenesis (Hoodbhoy et al., 2006; Wassarman et al., 1997). The direct effect of antibody binding to ZP3 during in vitro growth may not only damage this glycoprotein, but also avoid the assembly of normally synthesized ZP2, resulting in a more harmful effect. Oocyte diameters were also reduced after in vitro growth and maturation in the anti-ZP3 group and ZP diameter was reduced in both anti-ZP groups. Nascent ZP2 and ZP3 are incorporated into only the innermost layer of the ZP as the assembly of ZP glycoproteins may be concentration-dependent and the concentration is highest near the site of secretion (Qi et al., 2002). The continuous effect of anti-ZP antibodies during in vitro growth may hinder the formation of the ZP, making it thinner. Also, the damage to the ZP, and as a result to the bidirectional communication between the oocyte and granulosa cells that is necessary for oogenesis, may produce oocytes with a smaller diameter than normal. In the present study, after in vitro fertilization, the oocytes from both the anti-ZP2 and the anti-ZP3 groups failed to fertilize. Bidirectional communication between oocytes and granulosa cells has been shown to be essential for fertilization and embryo development because the growth and meiotic regulation of oocytes are dependent on granulosa cells (Liu et al., 2007). As this communication is made through the granulosa cell processes present in the ZP, the anti-ZP antibodies may interfere with the normal folliculogenesis and oocyte development resulting in oocytes with impaired fertilization capacity. Also, these antibodies may avoid sperm binding and penetration. The electron microscopic observation revealed a lower quantity of granulosa cell projections in follicles
cultured with anti-ZP antibodies compared with the control group. Previous studies using electron microscopy showed that in a normal follicle the granulosa cell projections are observed in the ZP, and the number and complexity of the granulosa cell projections increase during folliculogenesis, only to disappear shortly before either ovulation or atresia (Gilula et al., 1978; Zamboni, 1974). During follicle development, the plasticity and remodeling of the ZP may allow the establishment and maintenance of functional granulosa cell projections for the regulation of paracrine signaling and information exchange in the follicle (Albertini et al., 2001). The direct binding of the anti-ZP antibodies to the ZP in follicle culture seems to be harmful enough to stop and involute the development of the granulosa cell projections. In conclusion, anti-ZP antibodies had a harmful effect on folliculogenesis and oogenesis of mouse preantral follicles. Anti-ZP antibodies may have a direct action on the ZP structure and thereby impair the normal development of the oocyte–granulosa cell gap junctions. As bidirectional communication between the oocyte and somatic cells in the ovarian follicle is necessary for folliculogenesis and oocyte growth, its damage results in follicle atresia and impairment of oocyte maturation and fertilization. Acknowledgments This work was supported by Grant-in-Aid of Scientific Research (No. 18390453) from the Ministry of Education, Science, Culture, Sports and Technology, Japan, 2006–2008 and by MEXT.HAITEKU, Japan, 2004–2008, and supported in part by a Grant-in-aid for Graduate Students from Hyogo College of Medicine, 2007–2008 (for Dr. Calongos). The authors want to thank Ms. Fujimoto for her technical assistance with the electron microscopic studies. References Adam, A., Takahashi, Y., Katagiri, S., Nagano, M., 2004. In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes. J. Reprod. Dev. 50, 579–586. Albertini, D., Combelles, C., Benecchi, E., Carabatsos, M., 2001. Cellular basis for paracrine regulation of ovarian follicle development. Reproduction 121, 647–653. Anderson, E., Albertini, D., 1976. Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J. Cell. Biol. 71, 680–686. Bronson, R., MacLaren, B., 1970. Transfer to the mouse oviduct of mouse eggs with and without the zona pellucida. J. Reprod. Fertil. 22, 129–137. Calongos, G., Hasegawa, A., Komori, S., Koyama, K., 2008. Comparison of urinary and recombinant follicle-stimulating hormone
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microsomal antigen in women with premature ovarian failure. J. Reprod. Immunol. 11, 53–67. Koyama, K., Hasegawa, A., 2006. Premature ovarian failure syndrome may be induced by autoimmune reactions to zona pellucida proteins. J. Reproduktionsmed. Endokrinol. 3, 94–97. Koyama, K., Hasegawa, A., Mochida, N., Calongos, G., 2005. Follicular dysfunction induced by autoimmunity to zona pellucida. Reprod. Biol. 5, 269–278. Lefievre, L., Conner, S., Salpekar, A., Olufowobi, O., Ashton, P., Pavlovic, B., Lenton, W., Afnan, M., Brewis, I., Monk, M., et al., 2004. Four zona pellucida glycoproteins are expressed in the human. Hum. Reprod. 19, 1580–1586. Liu, C., Litscher, E., Mortillo, S., Sakai, Y., Kinloch, R., Stewart, C., Wassarman, P., 1996. Targeted disruption of the mZP3 gene results in production of eggs lacking a zona pellucida and infertility in female mice. Dev. Biol. 93, 5431–5436. Liu, L., Rajareddy, S., Reddy, P., Du, C., Jagarlamudi, K., Shen, Y., Gunnarsson, D., Selstam, G., Goman, K., Liu, K., 2007. Infertility caused by retardation of follicular development in mice with oocyte-specific expression of Foxo3a. Development 134, 199–209. Millar, S., Chamow, S., Baur, A., Oliver, C., Robey, F., Dear, J., 1989. Vaccination with a synthetic zona pellucida peptide produces longterm contraception in female mice. Science 246, 935–938. Qi, H., Williams, Z., Wassarman, P., 2002. Secretion and assembly of zona pellucida glycoproteins by growing mouse oocytes microinjected with epitope-tagged cDNAs for mZP2 and mZP3. Mol. Biol. Cell. 13, 530–541. Rankin, T., Dean, J., 1996. The molecular genetics of the zona pellucida: mouse mutations and infertility. Mol. Hum. Reprod. 2, 889–894. Rankin, T., Familari, M., Lee, E., Ginsberg, A., Dwyer, N., BlanchetteMackie, J., Drago, J., Westphal, H., Dean, J., 1996. Mice homozygous for an insertional mutation in the Zp3 gene lack a zona pellucida and are infertile. Development 122, 2903–2910. Rankin, T., O’Brien, M., Lee, E., Wigglesworth, K., Eppig, J., Dean, J., 2001. Defective zona pellucidae in Zp2-null mice disrupt folliculogenesis, fertility and development. Development 128, 1119–1126. Skinner, S., Mills, T., Kirchick, H., Dunbar, B., 1984. Immunization with zona pellucida proteins results in abnormal ovarian follicular differentiation and inhibition of gonadotropin-induced steroid secretion. Endocrinology 115, 2418–2432. Smitz, J., Cortvrindt, R., 2002. The earliest stages of folliculogenesis in vitro. Reproduction 123, 185–202. Szczepanska, M., Skrzypczak, J., Kamieniczna, M., Kurpisz, M., 2001. Antizona and antisperm antibodies in women with endometriosis and/or infertility. Fertil. Steril. 75, 97–105. Takamizawa, S., Shibahara, H., Shibayama, T., Suzuki, M., 2007. Detection of antizona pellucida antibodies in the sera from premature ovarian failure patients by a highly specific test. Fertil. Steril. 88, 925–932. Wassarman, P., 1987. The biology and chemistry of fertilization. Science 235, 553–560. Wassarman, P., Mortillo, S., 1991. Structure of the mouse egg extracellular coat, the zona pellucida. Int. Rev. Cytol. 130, 85–110. Wassarman, P., Qi, H., Litscher, E., 1997. Mutant female mice carrying a single mZP3 allele produce eggs with a thin zona pellucida, but reproduce normally. Proc. Biol. Sci. 264, 323–328. Yanagimachi, R., 1994. Mammalian fertilization. In: Knobi, E., Neil, J.D. (Eds.), The Physiology of Reproduction, 5th edn. Raven Press, New York, pp. 189–317. Zamboni, L., 1974. Fine morphology of the follicle wall and follicle cell-oocyte association. Biol. Reprod. 10, 125–149.
Harmful effects of anti-zona pellucida antibodies in folliculogenesis, oogenesis, and fertilization Giannina Calongos a , Akiko Hasegawa b , Shinji Komori a,b , Koji Koyama a,b,∗ a b
Department of Obstetrics and Gynecology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan Laboratory of Developmental Biology and Reproduction, Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan Received 27 February 2008; accepted 17 June 2008
Abstract The zona pellucida (ZP) is an extracellular matrix that surrounds the mammalian oocyte and plays an important role in normal folliculogenesis and fertilization. Because of its strong immunogenicity and its possible relation with premature ovarian failure, we conducted the present study to examine whether or not anti-ZP antibodies impaired folliculogenesis. Mouse preantral follicles were cultured with anti-ZP antibodies to evaluate the effects on follicle growth and antral formation. The cultured follicles were also examined by electron microscope and assessed for oocyte maturation, fertilization capacity, and embryo development. The results showed that follicles cultured with anti-ZP antibodies had a smaller diameter than the controls. Also, these antibodies reduced antral formation, mucification, maturation of oocytes (metaphase II), and fertilization rates. Morphologically, ZP thickness was lower in the antiZP antibody groups. The quantity of granulosa cell microvillous processes that transverse the ZP was diminished in follicles cultured with anti-ZP antibodies. In conclusion, anti-ZP antibodies were harmful to the normal development of mouse follicles and oocytes in vitro. These antibodies may be a cause of premature ovarian failure syndrome because they disrupt the gap junctions between the oocyte and granulosa cells and, as a consequence, damage the bidirectional communication necessary for normal folliculogenesis. © 2009 Elsevier Ireland Ltd. All rights reserved. Keywords: Anti-zona pellucida antibodies; Preantral follicles; Folliculogenesis; Oogenesis; Fertilization
1. Introduction The zona pellucida (ZP) is an extracellular coat that surrounds the plasma membrane of mammalian eggs and pre-implantation embryos. It is composed of interconnected filaments of three or four glycoproteins designated ZP1, ZP2, ZP3, and ZP4, which are expressed and assembled by the oocyte (Eberspaecher et al., 2001; Harris et al., 1994; Lefievre et al., 2004). In mice, previous studies suggested that the ZP filaments are ∗
Corresponding author at: Department of Obstetrics and Gynecology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya 663-8501, Japan. Tel.: +81 798 45 6481; fax: +81 798 46 4163. E-mail address: [email protected] (K. Koyama).
polymers of ZP2 and ZP3 cross-linked by ZP1 (Greve and Wassarman, 1985; Wassarman and Mortillo, 1991). The ZP plays an important role during fertilization as it mediates relatively species-specific sperm-egg recognition and induction of acrosome reaction. Also, after that, the ZP protects the early embryo during its passage through the oviduct before implantation (Bronson and MacLaren, 1970; Wassarman, 1987; Yanagimachi, 1994). During ovarian folliculogenesis, an interdependent and close association between the somatic and germ cells is important for the regulation of the preantral growth of the oocyte and the nuclear events before and at the time of ovulation (Anderson and Albertini, 1976; Canipari, 2000). Also, the bi-directional paracrine communication
0165-0378/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2008.06.003
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at the oocyte–granulosa cell interface is required for the completion of oocyte growth and the acquisition of cytoplasmic meiotic competence necessary for fertilization (Albertini et al., 2001; Carabatsos et al., 2000; Gittens and Kidder, 2005). The ZP physically separates the somatic and the germinal components. However, studies in knockout mice showed that these critical interactions are maintained in the growing follicle via granulosa cell processes that penetrate the ZP and make contact with the oolemma (Koyama and Hasegawa, 2006; Rankin et al., 2001). From a clinical viewpoint, antiovarian autoimmunity can be directed against the somatic (mainly the granulosa and the thecal layer) or the germinal component of the ovarian follicle (oocyte or ZP) and can be associated or not with somatic autoimmune disorders (Forges et al., 2004; Koyama and Hasegawa, 2006). As the ZP has been shown to possess a strong immunogenicity, it may be a target antigen in ovarian autoimmunity (Kelkar et al., 2005; Koyama et al., 2005). Also, previous studies using animal models immunized with ZP proteins demonstrated that among those antiovarian antibodies, anti-ZP antibodies were the most likely to cause premature ovarian failure and infertility (Govind et al., 2002; Skinner et al., 1984). Moreover, anti ZP antibodies are considered to be a cause of infertility because of their blocking effects on sperm–ZP binding (Takamizawa et al., 2007). Despite the many publications that have dealt with the relationship of anti-ZP autoimmunity and infertility, the real significance of these antibodies remains to be established. To evaluate the direct effect of anti-ZP antibodies in ovarian follicles, an in vitro culture system of juvenile mouse follicles would be useful, as their follicle formation occurs after birth and the isolation of a large quantity of homogenous follicles is possible (Demeestere et al., 2005). Culture of individual intact follicles on a collagencoated membrane has been shown to allow an accurate assessment of folliculogenesis by measuring their diameters, as well as evaluating their antral formation (Smitz and Cortvrindt, 2002). Mature oocyte and blastocyst formation after in vitro maturation and fertilization is also obtainable using this culture method (Adam et al., 2004; Calongos et al., 2008). In order to evaluate the effects of anti-ZP antibodies and their relation with ovarian dysfunction, an in vitro culture system of mouse preantral follicles was designed. Folliculogenesis (based on follicle growth, antral formation, and oocyte–granulosa cell connection development), oogenesis (including diameters, maturation, and fertilization capacity), and embryo development were evaluated as parameters.
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2. Materials and methods 2.1. Animals Specific pathogen-free, female (C57BL/6 X DBA/2) F1 mice (Japan SLC Inc., Shizuoka, Japan) were kept in light- and temperature-controlled conditions and provided with sterile food and water ad libitum according to the guidelines set by the Hyogo College of Medicine. All animal experiments were approved by the Committee on Animal Experimentation of the Hyogo College of Medicine. 2.2. Isolation of preantral follicles Sixteen-day-old mice were anesthetized with diethylether and killed by cervical dislocation. Preantral follicles were isolated and selected as described in a previous paper (Calongos et al., 2008). Ovaries were removed and transferred to a 200-L drop of Leibovitz’s 15 with L-glutamine (Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum (FCS; Gibco, NY, USA), 50 IU/mL of penicillin and 50 g/mL of streptomycin (Sigma–Aldrich) in a 60-mm Falcon dish (Becton Dickinson, NJ, USA). Preantral follicles were obtained by mechanical isolation with 30G needles and transferred to a new drop for evaluation. Selected preantral follicles were 110–130 m in diameter with a centrally located oocyte and no damage to the basal membrane. Ten to fifteen follicles with these characteristics were obtained from each ovary. 2.3. In vitro growth of preantral follicles with anti-ZP antibodies Antiserum was prepared by immunization of rabbits with synthetic peptides of mouse ZP2 and ZP3 conjugated with diphtheria toxoid, as described in previous papers (Hasegawa et al., 2002; Koyama et al., 2005; Millar et al., 1989). The growth medium was prepared as described in previous studies carried out in our laboratory with some modifications (Calongos et al., 2008; Hasegawa et al., 2004, 2006). The growth medium was composed of ␣-minimum essential medium with 5% FCS, ITS (insulin 1 mg/mL, transferrin 0.55 mg/mL, selenium 0.5 g/mL), penicillin–streptomycin solution, L-glutamine 2 mM (all these products were obtained from Sigma–Aldrich), and 100 mIU/mL of FSH (Fertinorm, Serono, Tokyo, Japan). Antisera were treated by heating at 56 ◦ C for 30 min to inactivate the complement and added to the growth medium at a 10% concentration. Pre-immune serum was used as a control.
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Selected preantral follicles were randomly distributed in the control, anti-ZP2, and anti ZP3 groups, and cultured in 24-well cell clusters (Iwaki, Chiba, Japan) on membrane inserts (Transwell-COL 3.0-m pore size, Corning, NY, USA) in 700 L of the respective growth medium at 37 ◦ C in a humidified atmosphere of 5% CO2 in air. Three hundred microliters of culture medium per well was replaced with fresh medium every other day. For a measurement of follicle diameters without impairment of the culture conditions, pictures of the preantral follicles were taken every day using an inverted microscope (Eclipse TE300 Nikon, Japan) with a digital camera (Hamamatsu C4742-95, Japan) at 40× magnification. Two diameters (width and length) were measured and the average of these two values was documented as the diameter of each follicle. 2.4. In vitro maturation of antral follicles cultured with anti-ZP antibodies and oocyte diameter measurement Following in vitro growth, the follicles were transferred to a maturation medium, as described in a previous paper (Calongos et al., 2008). The maturation medium was composed of growth medium without antibodies supplemented with 2.5 IU/mL of human chorionic gonadotropin (HCG, Mochida, Tokyo, Japan) and 10 ng/mL of mouse epithelial growth factor (EGF, Upstate Biotechnology, Inc, NY, USA). Eighteen hours later, follicle mucification was evaluated under an inverted microscope. Then, the oocytes were mechanically denuded from the mucified granulosa cells for assessment of the maturation stage. Germinal vesicles (GV) containing oocytes and first polar body extruded oocytes were assessed as the GV stage and the MII (metaphase II) stage respectively. The intermediate oocytes between GV and MII stages were considered to be MI stage oocytes. For the measurements of oocyte, ooplasm, and zona pellucida diameters, pictures of the denuded oocytes were taken using an inverted microscope (Eclipse TE300 Nikon, Japan) with a digital camera (Hamamatsu C474295, Japan) at 80× magnification. Two diameters were measured and the average of these two values was documented as the respective oocyte diameters. 2.5. In vitro fertilization and further embryo development Sperm were collected from the cauda epididymis of 12-week-old F1 male mice and suspended in a drop
(200 L) of TYH medium (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan) under mineral oil. The composition of TYH medium is 119.37 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl2 , 1.19 mM KH2 PO4 , 1.19 mM MgSO4 , 25.07 mM NaHCO3 , 1.0 mM sodium pyruvate, 5.56 mM glucose, 4 mg/mL of bovine serum albumin, 0.075 mg/mL of penicillin, and 0.005 mg/mL of streptomycin. Sperm capacitation was made by incubation at 37 ◦ C in a humidified atmosphere of 5% CO2 in air for 1 h. Mature oocytes were obtained from preantral follicles that were grown and matured in vitro. Then, these MI and MII stage oocytes were transferred to a drop of TYH medium. Capacitated sperm were mixed with mature oocytes at a concentration of 0.3 × 106 sperm/mL and incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 in air for 6 h. Fertilized oocytes were assessed for the presence of two pronuclei under a phase-contrast inverted microscope and transferred to a drop of fresh, potassium simplex-optimized medium (KSOM; Erbach et al., 1994). Embryo stages were assessed at 6, 24, 48, 72, and 96 h after insemination. 2.6. Electron microscopy At the end of in vitro growth, follicles from each group were transferred to different centrifuge tubes and centrifuged for 3 min at 200 × g. Follicles were then fixed with 1.25% glutaraldehyde in 0.1 M phosphate buffer for 1 h at 4 ◦ C, followed by buffer rinses. After that, the samples were treated for 1 h at 4 ◦ C with 1% osmium tetroxide in 0.1 M phosphate buffer and rinsed with buffer. Then, they were dehydrated in a graded series of ethanol (50%, 70%, 80%, 90%, 95%, and 100%) and embedded in Epon 812. Between all the steps, samples were centrifuged for 5 min at 200 × g. Sections of 80 nm were cut with a diamond knife on an ultramicrotome (Leica Ultracut UCT, Leica, Austria) and mounted on copper grids. The sections were stained with uranyl acetate and Reynold’s lead before examination in an electron microscope (JEM-1220 JEOL, Japan). 2.7. Statistical analysis Antral formation, mucification, oocyte maturation, fertilization, and embryo development results were expressed in percentages and statistically analyzed based on the χ2 test. Follicle diameter and oocyte diameter were expressed in mean ± S.D. and analyzed based on Student’s t test. P values below 0.05 were considered to be statistically significant.
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3. Results 3.1. Effects of anti-ZP antibodies on follicle growth and antral formation Twenty preantral follicles per group were used for the study. During in vitro growth, granulosa cells differentiated and an antrum was noticeable from day 4 in the control group (Fig. 1). At this time, in both anti-ZP2 and anti-ZP3 groups, antral formation was not evident. At day 7, preantral follicles in the control group were clearly differentiated from antral ones, with granulosa cells tightly joined around the oocyte as a corona and the presence of the antrum (Fig. 1d). However, antral follicles in anti-ZP2 and anti-ZP3 groups were smaller and not as well developed as those in the control group. This type of follicle was more evident in the anti-ZP3 group with oocytes loosely surrounded by granulosa
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cells, resulting in the extrusion of the oocyte and degeneration of the remaining granulosa cells (Fig. 1l). Preantral follicle diameters in the control group increased continuously and uniformly during the seven days of in vitro growth (Fig. 2). On the contrary, growth of follicles cultured with anti-ZP antibodies was retarded from day 2. 3.2. Effects of anti-ZP antibodies on antral formation, mucification, and oocyte maturation rates and diameters For this part of the study, 60 preantral follicles per group and their corresponding oocytes obtained after in vitro maturation were used. After seven days of invitro growth, the antral formation rate was lower in the anti-ZP2 (68.3%) and anti-ZP3 groups (63.3%) than in the control group (93.3%; Fig. 3). Then, all follicles were transferred to the maturation medium to deter-
Fig. 1. Effects of anti-ZP2 and anti-ZP3 antibodies on the growth and development of preantral follicles during in vitro growth. Cultured follicles in the control group (a–d), the anti-ZP2 group (e–h), and the anti-ZP3 group (i–l) at day 0, day 3, day 5, and day 7 are shown. The progressive increase in size of the follicles in the control is noticeable, while follicles cultured with anti-ZP antibodies stop growing normally. Antral formation (arrow) can be seen in the control group with the increase in the size of follicles and the proliferation of granulosa cells. In the anti-ZP3 group, the extrusion of the oocyte with the degeneration of the remaining granulosa cells is shown. Scale bars represent 150 m.
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G. Calongos et al. / Journal of Reproductive Immunology 79 (2009) 148–155 Table 1 Effects of anti-ZP2 and anti-ZP3 antibodies on oocyte diameters after in vitro growth and maturation.
Oocyte Ooplasm Zona pellucida
Control (m)
Anti-ZP2 (m)
Anti-ZP3 (m)
83.38 ± 1.49 69.03 ± 0.66 5.95 ± 0.37
82.62 ± 2.29 68.49 ± 1.71 5.54 ± 0.53a
78.97 ± 2.12b 67.73 ± 2.82c 4.17 ± 0.61d
Data were expressed as mean ± S.D. Superscripts a, b, c, and d denote a significant difference between the study and control groups. P < 0.05.
the control group (Table 1). Only the ZP diameter was reduced in the anti-ZP2 group. Fig. 2. Effects of anti-ZP2 and anti-ZP3 antibodies on follicle diameter and growth profile during in vitro growth. Data represent diameters (mean ± S.D.). Asterisks (*) indicate a significant difference among anti-ZP2 or anti-ZP3 and the control on each day of culture (P < 0.05 Student’s t-test).
mine the mucification and oocyte maturation rates. The mucification rate was reduced in the anti-ZP2 (51.7%) and anti-ZP3 groups (43.3%) compared with the control group (90%; Fig. 3). MII oocyte percentages were also lower in the anti-ZP2 (33.3%) and anti-ZP3 groups (3.3%) than in the control group (51.7%). MI oocyte rates were 41.4%, 59.6%, and 58.8% for control, anti-ZP2, and anti-ZP3 groups respectively. The incidence of GV stage oocytes was 3.4%, 3.5%, and 3.3% in the control, anti-ZP2 and anti-ZP3 groups respectively. Degenerated oocyte values were higher in the anti-ZP3 group (35%) compared with the other two groups (3.4%; Fig. 3). Oocyte and ooplasm diameters as well as ZP thickness were smaller in the anti-ZP3 group compared with
3.3. Effects of anti-ZP antibodies on fertilization and embryo development rates Nineteen mature oocytes (MI and MII stages) per group were used for the study. The fertilization rate was 94.7% in the control group and 0% in both the anti-ZP2 and anti-ZP3 groups (Fig. 3). After 24 and 96 h of culture, the two-cell stage embryo rate and the blastocyst formation rate were 89.5% and 57.9% respectively for the control group. Both anti-ZP2 and anti-ZP3 groups failed to develop embryos during the culture. 3.4. Electron microscopic observation After 7 days of in vitro growth, follicles from each group were fixed and treated for electron microscopic observation. The ZP was thinner in the anti-ZP antibody groups than in the control group, similar to the optical
Fig. 3. Effects of anti-ZP2 and anti-ZP3 antibodies on antral formation, mucification, oocyte maturation, and fertilization rates. Significant values are written above the columns based on the χ2 test.
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microscopic observation shown in Table 1. At higher magnification, the granulosa cell processes that transverse the ZP are present in great quantities and make contact with the oolemma in the control group. In contrast, in follicles cultured with anti-ZP antibodies the quantity is dramatically decreased (Fig. 4b and c). 4. Discussion
Fig. 4. Electron microscopic observation of follicles cultured with pre-immune rabbit serum (control), and with anti-ZP2 and anti-ZP3 antibodies. Cultured follicles after 7 days of in vitro growth in the control (a), the anti-ZP2 (b), and the anti-ZP3 groups (c) are shown at ×5000 magnification. The presence of the ZP between the oocyte (O) and the granulosa cells (GC) is noticeable. Also, the granulosa cell projections that traverse the ZP can be identified (arrows). There were fewer of these projections in the anti-ZP antibody groups compared with the control group.
The relationship between anti-ZP antibodies and infertility is suggested in serum, peritoneal fluid, and follicular fluid (Ivanova et al., 1999; Szczepanska et al., 2001). Also, previous studies showed that anti-ZP antibodies are an important etiology in autoimmune premature ovarian failure (POF; Kelkar et al., 2005; Koyama et al., 2005; Takamizawa et al., 2007). POF is a disorder that affects 1% of women and is characterized by amenorrhea with elevated FSH levels because of the cessation of ovarian function. It has a multicausal etiology, such as genetic, autoimmune, iatrogenic, environmental, and idiopathic (Goswani and Conway, 2005). Autoimmune causes account for up to 30% of cases of POF (Conway et al., 1996). The cellular targets of the immune reaction can be the somatic or the germinal component of the follicle, mainly the oocyte itself or the ZP (Forges et al., 2004). Because of the importance of autoimmunity in POF and infertility etiology, a culture system of mouse follicles was designed to evaluate the effects of ZP antibodies in vitro. Preantral follicles cultured with both anti-ZP2 and anti-ZP3 antibodies showed smaller diameters and antral formation compared with the control group. This observation correlates with in vivo experiments in which immunization of different animals with natural source or recombinant ZP proteins caused ovarian dysfunction (Govind et al., 2002; Koyama et al., 2005; Skinner et al., 1984). During in vitro growth, follicles cultured with anti-ZP3 had granulosa cells that were not as tightly attached to the oocyte as those in the control group, which resulted in the extrusion of the oocyte. As a result, the degenerated follicle rate was higher in the anti-ZP3 group (Fig. 3). This growth pattern was also observed in ZP3 knockout mice follicles that developed a corona radiata that was less uniformly arrayed than in normal animals and as a result the degree of contact between the oocyte and granulosa cells was diminished (Liu et al., 1996; Rankin et al., 1996). Moreover, the ovarian histology of ZP2 and ZP3 knockout mice showed that in preovulatory follicles the cumulus cells were poorly organized and in some cases were completely separated from the oocyte (Rankin and Dean, 1996; Rankin et al., 1996, 2001).
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We observed that the mucification and MII oocyte rates were significantly lower in the anti-ZP antibody groups than in the control group as a result of the abnormal folliculogenesis. These results correlate with a previous study that reported impairment of folliculogenesis as well as inhibition of the growth and maturation of oocytes after in vitro culture of oocyte–granulosa cell complexes and intact follicles with anti-ZP antibodies (Koyama et al., 2005). In the present study, anti-ZP3 had a more deleterious effect in folliculogenesis than anti-ZP2, with the highest rate of degenerated follicles and oocytes. As ZP2 knockout mice form a thin ZP in early follicles while ZP3 knockout mice completely lack ZP (Rankin et al., 1996, 2001), it is possible that the function of ZP3 in ZP formation and structure is essential. ZP2 and ZP3 are synthesized and traffic independently through the oocyte; however, ZP3 presence is necessary for incorporation of ZP2 into the ZP during oogenesis (Hoodbhoy et al., 2006; Wassarman et al., 1997). The direct effect of antibody binding to ZP3 during in vitro growth may not only damage this glycoprotein, but also avoid the assembly of normally synthesized ZP2, resulting in a more harmful effect. Oocyte diameters were also reduced after in vitro growth and maturation in the anti-ZP3 group and ZP diameter was reduced in both anti-ZP groups. Nascent ZP2 and ZP3 are incorporated into only the innermost layer of the ZP as the assembly of ZP glycoproteins may be concentration-dependent and the concentration is highest near the site of secretion (Qi et al., 2002). The continuous effect of anti-ZP antibodies during in vitro growth may hinder the formation of the ZP, making it thinner. Also, the damage to the ZP, and as a result to the bidirectional communication between the oocyte and granulosa cells that is necessary for oogenesis, may produce oocytes with a smaller diameter than normal. In the present study, after in vitro fertilization, the oocytes from both the anti-ZP2 and the anti-ZP3 groups failed to fertilize. Bidirectional communication between oocytes and granulosa cells has been shown to be essential for fertilization and embryo development because the growth and meiotic regulation of oocytes are dependent on granulosa cells (Liu et al., 2007). As this communication is made through the granulosa cell processes present in the ZP, the anti-ZP antibodies may interfere with the normal folliculogenesis and oocyte development resulting in oocytes with impaired fertilization capacity. Also, these antibodies may avoid sperm binding and penetration. The electron microscopic observation revealed a lower quantity of granulosa cell projections in follicles
cultured with anti-ZP antibodies compared with the control group. Previous studies using electron microscopy showed that in a normal follicle the granulosa cell projections are observed in the ZP, and the number and complexity of the granulosa cell projections increase during folliculogenesis, only to disappear shortly before either ovulation or atresia (Gilula et al., 1978; Zamboni, 1974). During follicle development, the plasticity and remodeling of the ZP may allow the establishment and maintenance of functional granulosa cell projections for the regulation of paracrine signaling and information exchange in the follicle (Albertini et al., 2001). The direct binding of the anti-ZP antibodies to the ZP in follicle culture seems to be harmful enough to stop and involute the development of the granulosa cell projections. In conclusion, anti-ZP antibodies had a harmful effect on folliculogenesis and oogenesis of mouse preantral follicles. Anti-ZP antibodies may have a direct action on the ZP structure and thereby impair the normal development of the oocyte–granulosa cell gap junctions. As bidirectional communication between the oocyte and somatic cells in the ovarian follicle is necessary for folliculogenesis and oocyte growth, its damage results in follicle atresia and impairment of oocyte maturation and fertilization. Acknowledgments This work was supported by Grant-in-Aid of Scientific Research (No. 18390453) from the Ministry of Education, Science, Culture, Sports and Technology, Japan, 2006–2008 and by MEXT.HAITEKU, Japan, 2004–2008, and supported in part by a Grant-in-aid for Graduate Students from Hyogo College of Medicine, 2007–2008 (for Dr. Calongos). The authors want to thank Ms. Fujimoto for her technical assistance with the electron microscopic studies. References Adam, A., Takahashi, Y., Katagiri, S., Nagano, M., 2004. In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes. J. Reprod. Dev. 50, 579–586. Albertini, D., Combelles, C., Benecchi, E., Carabatsos, M., 2001. Cellular basis for paracrine regulation of ovarian follicle development. Reproduction 121, 647–653. Anderson, E., Albertini, D., 1976. Gap junctions between the oocyte and companion follicle cells in the mammalian ovary. J. Cell. Biol. 71, 680–686. Bronson, R., MacLaren, B., 1970. Transfer to the mouse oviduct of mouse eggs with and without the zona pellucida. J. Reprod. Fertil. 22, 129–137. Calongos, G., Hasegawa, A., Komori, S., Koyama, K., 2008. Comparison of urinary and recombinant follicle-stimulating hormone
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