Effect of human ZP3 monoclonal antibody on expression of GDF-9 and number of theca cells in ovary of mice (Mus musculus)

Objectives This study investigated the effects of a human ZP3 monoclonal antibody (mAb hZP3) on the expression of growth differentiation factor-9 (GDF-9) and number of theca cells in the ovaries of mice (Mus musculus). Methods Our study employed a true experiment posttest-only control group design of 48 mice that were divided into the control and three mAb hZP3-treatment groups (20, 40, and 60 μg). Mice in each group were terminated on days 10, 15, and 20. GDF-9 expression was measured by immunohistochemistry and the number of theca cells was counted. Results Analysis of the effects of mAb hZP3 (at 20–60 μg) on the expression of GDF-9 and amount of theca cells did not show significant differences. Similar findings were observed throughout the study period (at 10–20 days). Therefore, mAb hZP3 had no effect on GDF-9 expression and theca cells. Conclusion This study showed that mAb hZP3 can be considered to be an effective and safe immunocontraception.


Abstract
Objectives: This study investigated the effects of a human ZP3 monoclonal antibody (mAb hZP3) on the expression of growth differentiation factor-9 (GDF-9) and number of theca cells in the ovaries of mice (Mus musculus).
Methods: Our study employed a true experiment posttest-only control group design of 48 mice that were divided into the control and three mAb hZP3-treatment groups (20, 40, and 60 mg). Mice in each group were terminated on days 10, 15, and 20. GDF-9 expression was measured by immunohistochemistry and the number of theca cells was counted.
Results: Analysis of the effects of mAb hZP3 (at 20e60 mg) on the expression of GDF-9 and amount of theca cells did not show significant differences. Similar findings were observed throughout the study period (at 10e20 days). Therefore, mAb hZP3 had no effect on GDF-9 expression and theca cells.
Conclusion: This study showed that mAb hZP3 can be considered to be an effective and safe immunocontraception.

Introduction
The population of Indonesia was estimated to be a quarter billion in 2015. The government has suppressed population growth by establishing Keluarga Berencana (KB) throughout Indonesia. Although many kinds of contraception have been used, hormonal contraception is the most common. However, hormonal contraception can have serious side effects, including an increased risk of cardiovascular diseases, such as hypertension, myocardial infarction, and stroke, as well as reproductive cancer. 1 As an alternative to hormonal contraception, immunocontraception methods are currently being developed, which are considered to be safe, effective, and reversible. The zona pellucida (ZP) is a strong candidate target for immunocontraception due to its glycoprotein membrane, which is required for fertilization. Most species contain three ZP proteins (ZP1, ZP2, and ZP3); however, in humans, there is the additional ZP protein, ZP4. 2e4 Anti-ZP3-antibodies have been developed in several species, including rabbit and pig, 5 mouse, 6 cow, 7 and human. 8 In a study by Naz, a human monoclonal ZP3 antibody (mAb hZP3) was produced, that has 73% amino acid similarity to a mAb produced in mice. 9 This ZP3 antibody binds to glucose on the surface of the ZP oocyte, which is close to the sperm receptor, inducing an early cortical reaction, toughening the ZP, and inhibiting sperm penetration. 10 ZP3 antibody has been confirmed to effectively suppress fertilization in several animals, such as mice, rats, cats, and rabbits. 11e13 The immunocontraceptive effect of ZP3 has been well studied. In addition, Araujo et al. reported that treatment with a murine ZP antibody had no effect on embryo development and follicle quantity. 6 In contrast, Borillo et al. reported that a treated ZP seemed to be more transparent, swollen, and looser by immunohistochemical assessment, and that the ZP antibody inhibited the synthesis of gap junctions. 11 It has been shown that depletion of the ZP leads to incomplete ZP synthesis, disturbing the establishment of gap junctions and decreasing the number of gap junctions. Altered gap junctions leads to decreased intracellular communication during folliculogenesis. 14,15 Growth differentiation factor-9 (GDF-9) is a growth factor involved in follicle development. GDF-9-BMPRII binding activates AKT and suppresses pro-apoptotic factors, leading to activation of the follicle and oocyte. 16 Previous studies showed that a deficiency in GDF-9 inhibits granulosa cell proliferation and theca cell recruitment. 17 Theca cells are derived from stromal cells and express LH receptor in the pre-antral stage. In response to LH, theca cells secrete androstenedione, an androgen, which is transferred to the granulosa through the basal lamina, leading to the production of testosterone. Along with aromatase enzyme and FSH, androstenedione is converted to estradiol, further promoting ovulation. Therefore, inhibition of theca cell proliferation decreases androstenedione production. 18e20 A mAb against the ZP was developed about 30 years ago. However, as studies of hZP3 are limited, additional studies of its effect on folliculogenesis are needed. Thus, we aimed to determine the effect of mAb hZP3 on the expression of GDF-9 and the amount of theca cells in the ovary of mice (M. musculus).

Mice
Mice weighing 20e25 g that had given birth were acclimated and synchronized for 3 weeks. Mice were randomly divided into the control group (injected with 50 mL of adjuvant Al(OH) 3 þ 50 mL of TriseHCl) and three treatment groups (injected with mAb hZP3 at 20 mg [P1], 40 mg [P2], or 60 mg [P3]). Each group was sampled based on proestrus cycle 2, 3, and 4.

Measurement of GDF-9 expression
The ovary was removed, and GDF-9 expression was measured by immunohistochemical staining with a primary antibody against GDF-9 (catalog no. bs-4720R) Bioss Inc., USA. GDF-9 expression was measured via semi-quantitative assessment based on Remmele score.

Measurement of theca cells
The ovary was removed, and the number of theca cells was determined by histopathology assessment of hematoxylin and eosin (HE) staining. The numbers of cells in each layer were counted manually. 21

Results
Saphiro-Wilk and Levene analyses of the data yielded p values > 0.05, indicating that the data were normally distributed and homogeneous ( Table 1). Figure 1 shows the expression of GDF-9 as a brown chromogen, which is indicated with a black arrow in panel a.

Effect of mAb hZP3 at various doses on the expression of GDF-9
ANOVA showed that there was a significant interaction effect between mAb hZP3 and time on the expression of GDF-9 (p ¼ 0.017). However, based on a histogram of the effect of mAb hZP3 at various doses and times on the expression of GDF-9, the average GDF-9 expression in the control group was similar to that in all mAb hZP3 treatment groups. The only significant difference was between the control group on day 20 and the 20 mg mAb hZP3 treatment  Figure 2).

Characteristics of the theca cells in secondary follicles
The characteristics of the theca cells and granulosa proliferation cells is shown in Figure 1b.

Effect of mAb hZP3 on the number of theca cells
ANOVA showed there was no interaction effect between the various doses of mAb hZP3 and observation times on the number of theca cells on days 10, 15, and 20 (p ¼ 0.713). As shown in Figure 2, the average number of theca cells in the control group was similar to that in all mAb hZP3 treatment groups. Average number of theca cells was lowest in the control on day 10. However, although there was an increase in the number of theca cells following exposure to mAb hZP3 at various doses, the difference was not significant (Figure 2).

Correlation between the number of theca cells and GDF-9 expression
Pearson correlation analysis showed that there was no significant correlation between the number of theca cells and GDF-9 expression in oocytes treated with various doses of mAb hZP3 (correlation coefficient ¼ À0.024; p ¼ 0.869; Table 2).

Effect of mAb hZP3 at various doses on the expression of GDF-9
Growth Differentiation Factor-9 (GDF-9), a member of the transforming growth factor b (TGF-b) family, is an oocyte-secreted factor. 22,23 GDF-9 is found in most species, exclusively in the oocyte at nearly every stage of follicle development, except in the primordial oocyte, and at ovulation. After fertilization, GDF-9 mRNA levels decrease, and at 1.5 days postfertilization, it is undetected. 24 The role of GDF-9 was demonstrated in previous studies; it is a strong mitogen that stimulates the proliferation of granulosa and theca cells, the oocyte, and embryo development.
In this study, we showed that treatment with mAb hZP3 did not affect the expression of GDF-9. According to Anifandis et al., the variable domain of ZP3 IgG binds to the surface of the ZP epitope and promotes the early cortical reaction, causing ZP hardening, which leads to inhibition of sperm penetration. 25 Such an alteration might be caused by the monospecificity of mAb hZP3. A variety of amino acids in the variable domain of the ZP3 antibody, which is produced by a single specific B-cell clone, only recognizes a single ZP3 epitope. Abbas et al. reported that an antibody with high specificity can recognize different antigens, even in the presence of a different amino acid. 26 Simply put, antibody specificity is determined by the amount of binding epitope that least recognizable epitope is associated with higher specificity. This indicates that ZP3 antibody ability in mice did not disrupt the ZP synthesis. Stabilization of the ZP is associated with gap junctions, and an intact ZP is associated with properly functioning gap junctions. Therefore, there is no disturbance of gap junctions, as indicated by proper transport of GDF-9 from the oocyte to granulosa cells, leading to proper folliculogenesis. Borillo et al. reported that the alteration of the conformation of the ZP depends on antibody binding during follicle development. 11 Antibody binding to the oocyte in the antral phase and ovulation maintain the structure of the ZP and disrupt the synthesis and secretion of ZP protein when it binds in the preantral phase.

Effect of mAb hZP3 at various doses on the number of theca cells
Theca cells surround the granulosa cells. At the early stage of oocyte differentiation, theca cells are present in a thin layer of polyhedral, lengthwise cells with large nuclei and cytoplasm vacuolization; additional lengthwise cells are then formed, in about 3e5 layers. 27 Data show that mAb hZP3 had no effect on the number of theca cells, as the average numbers of theca cells in the control and treatment groups were not significantly different. As described above, the antibody did not affect follicle development. The high specificity of this antibody means that there is likely no cross reactivity, thus inflammation is avoided. 26 Previous studies also showed that a mZP3 mAb does not disrupt follicle or embryo  development, as the average numbers of small, medium, and large follicles were similar. 28 The primary follicle developed into the secondary follicle, demonstrating proper recruitment and proliferation of theca cells. A similar finding was reported in pZP3-injected monkeys, in which there was no indication of lymphocyte inflammation and infiltration in the ovary, and no increase in follicular atresia. 29 A peroxidaselabeled secondary antibody was used by Greenhouse et al. to characterize the effects of mAb mZP3 and mAb hZP3 30 ; the antibody was detected in the cells surrounding the oocyte ZP and the follicles appeared normal, displaying all stages of follicle development, including secondary follicles, tertiary follicles, and the corpus luteum. 11,31 Antibody-ZP binding of a specific, mAb during pre-implantation does not negatively affect embryo development. Thus, the embryo can undergo normal development. 30 Correlation between the number of theca cells and GDF-9 expression The GDF-9 signal relies on type 1 and 2 receptors. BMP receptor type II (BMPRII) is the main receptor that is expressed in the oocyte, granulosa cells, and theca cells. 24,32 GDF-9-BMPRII binding mediates activation of the type 1 receptor, ALK 5, followed by phosphorylation of intracellular transcription factors, Smad2, and Smad3. 21,33 This mechanism activates the follicle, causing increased cell proliferation and survival.
Dong et al. analyzed the ovaries of mice with a GDF-9 deficiency, which blocked development at the primary follicle stage. Although oocyte growth and the ZP were normal, the ultrastructure between the oocyte and granulosa was abnormal. 34 In a similar study, it was reported and oocyte growth was faster than normal, while the proliferation and differentiation of theca and granulosa cells was inhibited. 35 In an in vitro study, treatment with GDF-9 increased the amount of secondary follicles and prevented atresia. 37 This is in line with Alves studies, which showed an increase in the diameters of oocytes and follicles in culture medium containing GDF-9. 38 It also describes the role of GDF-9 in triggering the development and survival of the follicle.
This study showed the different results than expected, as the statistical analysis showed there was no significant correlation between GDF-9 and theca cells. Nilsson and Skiner treated mice with GDF-9, and observed no effect on follicle quantity, but only detected in its composition. Besides, GDF-9 also had no effecton the transition from primordial to primary follicle but indirectly stimulated the proliferation of granulosa and theca cells. 36 The GDF-9 signal likely affects growth factor production by increasing expression of KL mRNA. KL promotes the recruitment of theca cells from the stroma and increases their proliferation. In addition to the Kit ligand, IGF, activin, and BMP-15 are also involved in regulating the recruitment and proliferation of theca and granulosa cells and oocyte growth. 16

Conclusion
In this study, we conclude that treatment of mice with a mAb hZP3 antibody does not affect the expression of GDF-9 or the number of theca cells.