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Synergistic Regulatory Effect of Inhibin and Anti-Müllerian Hormone on Fertility of Mice

Inhibin (INH) and anti-Müllerian hormone (AMH) are essential in ovarian folliculogenesis and play an inhibitory role in mammalian fertility. However, the interactive effect of INH and AMH on the animal reproduction remains unknown. This study aimed to determine the possible interaction and synergy between INH and AMH in steroidogenesis by primary granulosa cells, and investigate their synergistic effect on fertility in mice. In in vitro granulosa cell culture system, we found that the treatment of either INHA or AMH had no significant effect on basal estradiol and progesterone production, whereas both significantly attenuated FSH-induced steroid hormone secretion. Importantly, combined treatment with INHA and AMH showed additive inhibitory effect on FSH-induced estradiol and progesterone production, accompanying a significant downregulation in the expression of FSH-stimulated CYP19A1, HSD3B, CYP11A1, StAR transcripts. The interrelationship of INH and AMH combinations was further investigated through active immune neutralization strategy. Female mice were immunized against INH and AMH eukaryotic expression plasmids, and the litter size was recorded after successfully mating. We observed that both INH and AMH plasmids were able to induce either anti-AMH or anti-INH antibodies in the immunized mice. In comparison with the control group, co-immunization with INH and AMH plasmids induced higher levels of estradiol, resulting in more litter size. Moreover, there was no significant difference on the offspring's weight between each group. Collectively, the results of the present study suggest that INH and AMH have synergistic effect in regulating steroidogenesis and the litter size in mice.


INTRODUCTION
The fundamental functions of the ovary are to produce oocytes and steroid hormones, which are mainly under the control of two glycoprotein hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Within the microenvironment of the ovary, local growth factors seem to play a crucial role in regulation of granulosa cell proliferation and differentiation, oocyte maturation, as well as steroidogenic activity. Among these factors, inhibin (INH) and anti-Müllerian hormone (AMH) are the members of the transforming growth factor β (TGF-β) superfamily, which is known to be the prime regulators of ovarian function in both autocrine or paracrine manner.
INH is a heterodimer glycoprotein consisting of two subunits, an α-subunit and either of two related β-subunits (βA and βB), thus, forming INHA (α-βA) and INHB (α-βB) (1)(2)(3). INHA expresses mainly in pre-ovulatory follicles, while INHB exists in the intermediately antral follicles (4). INH has long been regarded as a suppressor of FSH secretions through pituitarygonadal negative feedback to regulate ovarian functions (5,6). In transgenic mice model, it has been demonstrated that INH α-subunit leads to the disruption of the normal INH-to-activin ratio and reproductive deficiencies (7). In theca cells, INH has been reported to decrease the inhibitory effects of activin on the expression of StAR and HSD3B and androgen production (8). However, its role in steroidogenesis of granulosa cells remains equivocal. INH has been revealed to decrease FSH-induced estradiol production and aromatase activity by granulosa cells in rat (6,9) and cattle (10). In contrast, INH A promotes the estradiol production in sheep granulosa cells, and the antiserum of INH can attenuate FSH-stimulated estradiol production (11).
On the other hand, anti-Müllerian hormone (AMH), another member of the TGF-β superfamily, is mainly expressed in the gonads and plays an important role in the sexual differentiation and gonadal function. In the ovary, the expression of AMH begins in the primary follicles, which is the highest in the preantral follicles and small follicles, and decreases with the increase in antral follicle diameter in mice (12,13), rats (14), humans (15,16), cattle (17), and buffaloes (18). AMH has been clearly demonstrated to be involved in inhibiting the primordial follicle recruitment (19) and the growth of preantral and antral follicles through regulating the sensitivity of FSH (20). Furthermore, AMH abolishes FSH-dependent aromatase and estradiol production through its specific type II receptor (AMHRII) by sharing BMP signaling pathway in granulosa cells (21)(22)(23). In addition, AMH has been recognized as a reliable marker for ovarian reserve (24,25), superovulation response (17,26), and outcome of in vitro embryos (27)(28)(29).
In the clinics, both AMH and INHA are used as markers to diagnose polycystic ovary syndrome (PCOS), and the sensitivity reached 96.2% when both AMH and INH are detected in combination (30), indicating that AMH and INHA may have complementary roles in abnormal ovarian function. However, the possible interactive action of AMH and INH in ovarian function has not yet been determined. In the current study, we aimed to explore the synergistic regulatory effect of AMH and INH on steroidogenesis in the presence/absence of FSH by primary granulosa cells and the fertility including litter size and steroid hormones in female mice.

Cell Culture
Three-week-old female mice were intraperitoneally injected with 10 IU of pregnant mare serum gonadotropin (PMSG, Ningbo Sansheng Pharmaceutical Corporation, China) to stimulate granulosa cell proliferation. The mice were then sacrificed 48 h later by cervical dislocation. Ovaries were carefully removed and placed in a sterile 35-mm cell culture dish (five to six ovaries per dish) containing 2 ml of DMEM/F12 medium (HyClone, USA). The granulosa cells (GCs) were harvested by repeatedly puncturing the antral follicles with a sterile 25-gauge needle. Follicular fluid was filtered by a 40-µm nylon cell strainer, and then centrifuged at 700 × g for 5 min to obtain the GCs in the form of pellets. GC pellets were then washed twice in 6-ml DMEM/F12 followed by centrifugation at 700 × g for 5 min. After a final wash, the cells were seeded into 6-well plates at a density of 1 × 10 6 cells/ml in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 IU/ml of penicillin (Gibco, USA), and 100 µg/ml of streptomycin (Gibco, USA), and incubated in a humidified 5% CO 2 at 37 • C.

Detection of Estradiol and Progesterone in the Supernatant
For the detection of estradiol production, 100 nM androstenedione (Sigma-Aldrich, USA), a substrate of CYP19, was supplemented to each GC culture medium. Granulosa cells were cultured with INHA or AMH alone or in combination with INHA and AMH in the presence or absence of FSH for 48 h. The levels of steroid hormones in the supernatant were measured according to estradiol (Cusabio, CSB-E05109m, China) and progesterone (Cusabio, CSB-E05104m, China) ELISA kit instructions. The assay sensitivity was 40 pg/ml for estradiol and 0.2 ng/ml for the progesterone.

Reverse Transcription and Quantitative PCR
After treatment with INHA and AMH alone or in combination for 48 h, total RNA was extracted with E.Z.N.A Total RNA Kit I (Omega, R6834-02, USA). cDNA was synthesized from 500 ng RNA of each samples by Quanti TectReverse Transcription Kit (QIAGEN, Germany). The quantitative PCR was performed using Quantinova SYBR Green PCR Kit (QIAGEN, Germany) on CFX384 real-time PCR detection system (Bio-Rad, USA). Specific primers were designed by Primer Premier 5.0 and shown in Table 1

Plasmid Preparation and Immunization
The recombinant plasmids, pVAX-tPA-SINH-asd and pVAX-tPA-SAMH-asd were constructed and preserved by our laboratory. Briefly, pVAX-tPA-SINH-asd encodes SINH gene fusing with INH subunit α (1-32) and hepatitis B surface antigen S gene, while pVAX-tPA-SAMH-asd expresses SAMH gene fusing with AMH and hepatitis B surface antigen S gene. A large number of plasmids were extracted by the large-scale endotoxin removal kit (Tiangen Biotech, China) under the guide of instruction. A total of 92 5-week-old female Kunming mice were purchased from the laboratory animal center of Huazhong Agricultural University, and raised in air-conditioned room at a constant temperature of 25 • C with 12-h light/dark cycles, provided ad libitum with water and food. After 1 week of prefeeding period, mice were randomly divided into four groups with 23 mice in each group. As shown in Table 2, mice from all the four groups were intramuscularly injected with 50 µg of pVAX-tPA-SAMH-asd (T1), 50 µg of pVAX-tPA-SINH-asd (T2), 25 µg of pVAX-tPA-SAMH-asd + 25 µg of pVAX-tPA-SINH-asd (T3), and 100 µl of physiological saline (C), respectively. A booster immunization was performed after 2 weeks of primary immunization. All mice were weighed and recorded before immunization, and 2 and 4 weeks following primary immunization. Blood was subsequently collected from the tail vein of each mouse and then centrifuged at 3,000 rpm for 10 min. The upper plasma was carefully separated and stored at −20 • C for further use.

Determination of Estrogen and Progesterone in the Plasma
Vaginal smears of mice were employed to determine estrous cycle at 2 weeks after booster immunization. Three mice in estrus period were selected from each treatment group to perform blood collection and ovary removal. The levels of steroid hormones in the plasma were detected by using estradiol (Cusabio, CSB-E05109m, China) and progesterone (Cusabio, CSB-E05104m, China) ELISA kits. The assay sensitivity was 40 pg/ml for estradiol and 0.2 ng/ml for progesterone.

Evaluation of Litter Size and Offspring Birth Weight
After 4 weeks of primary immunization, all female mice were mated with healthy male mice. Mice were checked for the presence of a vaginal plug in the next morning. The male mice were picked out until the female mice were all pregnant. The litter size and offspring birth weight were carefully recorded.

Calculation of Organ Index
At the end of the experiment, the mice were immediately sacrificed by cervical dislocation, and the major organs including heart, liver, spleen, lung, and kidney were collected and weighed. The organ indexes (organ weight/body weight) of these organs were calculated.

Statistical Analysis
Each experiment was conducted at least in triplicate. Data analysis was performed using GraphPad Prism version 8.0 software and included one-way ANOVA followed by either Tukey or Bonferroni post hoc test.

Effect of Inhibin A and Anti-Müllerian Hormone Alone on Estradiol and Progesterone Production in Granulosa Cells
The results showed that INHA had no influence on the basal estradiol and progesterone production, whereas INHA in all concentrations significantly attenuated FSH-induced estradiol and progesterone production (p < 0.01; Figures 1A,B).
Accordingly, INHA significantly decreased the mRNA expression of FSH-induced steroidogenic-related genes of CYP19A1, HSD3B, CYP11A1, and StAR (p < 0.01), but there was no significant effect of INHA on basal mRNA expression of CYP19A1, HSD3B, CYP11A1, and StAR transcripts (Figures 1C-F). Similarly, AMH significantly decreased the FSH-stimulated estradiol and progesterone production in a concentration-dependent manner (p < 0.01) but had no effect on basal estradiol and progesterone release (Figures 2A,B). In addition, AMH significantly reduced FSH-induced CYP19A1, HSD3B, and StAR mRNA expression, but not CYP11A1 (Figures 2C-F).

The Synergistic Effect of Inhibin A and Anti-Müllerian Hormone on Estradiol and Progesterone Production in Granulosa Cells
The results revealed that the treatment of AMH and INHA in combination or alone significantly inhibited FSH-stimulated estradiol and progesterone production (p < 0.01), but unaffected estradiol and progesterone production in the absence of FSH.
Importantly, we found that AMH and INHA have a significant additive action in the inhibitory effect of FSH-induced estradiol and progesterone levels (Figures 3A,B). Furthermore, we also investigated the interaction between the expression of INHA and AMH showing that there was no significant difference on AMH levels in the supernatant after treatment with INHA, and vice versa (data not shown). Accordingly, we observed that the treatment of AMH and INHA in combination or alone significantly downregulated FSHstimulated mRNA expression of CYP19A1, HSD3B, CYP11A1, and StAR transcripts (p < 0.01; Figures 3C-F), but had no effect on basal expressions. In particular, real-time PCR results showed that the combined treatment of AMH and INHA significantly diminished FSH-induced mRNA expression of CYP19A1 gene in granulosa cells than AMH or INHA alone (p < 0.01). Moreover, the FSH-induced mRNA expressions of HSD3B, CYP11A1 genes in AMH and INHA combination group were significantly downregulated when compared with AMH alone group.

The Synergistic Effect of Inhibin A and Anti-Müllerian Hormone on cAMP Levels in Granulosa Cells
The results of cAMP synthesis showed that there was no significant difference in each group at the basal level. However, FSH treatment significantly increased the intracellular cAMP level, while AMH and INHA combined treatment significantly  All data are represented in mean ± SD.

Evaluation of Antibodies After Co-immunization of Inhibin and Anti-Müllerian Hormone Plasmids
After immunization with plasmids of pVAX-tPA-SINH-asd and pVAX-tPA-SAMH-asd, the levels of anti-AMH or anti-INH antibodies in plasma were detected at 4 weeks after the first immunization. As shown in Table 3, we found that all plasmids induced, respectively, specific antibodies. In particular, both anti-AMH and anti-INH antibodies were observed in T3 group, suggesting immunization program is successful in this experiment.

Effect of Co-immunization of Inhibin and Anti-Müllerian Hormone Plasmids on Estradiol and Progesterone Secretion
We confirmed that the immunized mice indeed exhibited estrous as detected by vaginal smears (Figures 5A-D), and estradiol contents in all immunized group were significantly higher than that in the control group ( Figure 5E). As for progesterone level (Figure 5F), there was no significant difference among all experimental groups, even though the decreasing trend was observed in the AMH plus INH immunized group.

Effect of Co-immunization of Inhibin and Anti-Müllerian Hormone Plasmids on Litter Size in Mice
As shown in Table 4, the mean litter size in pVAX-tPA-SINHasd and pVAX-tPA-SAMH-asd co-immunization group was significantly higher than that in control group. More specifically, the combined group obtained the highest litter size of 15.45, which increased by 1.6 compared with the control group. In addition, there was no significant difference in the offspring's birth weight.

Effect of Co-immunization of Inhibin and Anti-Müllerian Hormone Plasmids on Organ Index
At the end of the experiment, the organ indexes (organ weight/body weight) of the heart, liver, spleen, lungs, and kidneys were calculated and compared. As shown in Table 5, there was no significant difference in the organ indexes between immunized group and control group, suggesting that the vaccines did not cause any adverse effect to mice.

DISCUSSION
The growth and development of ovarian follicles are wellknown to be modulated by gonadotropins, steroids, and local intraovarian signaling system. In response to FSH, granulosa cells secrete serial growth factors as coregulators of follicular development. Among them, both AMH and INH are members of the TGF-β superfamily and play crucial roles in folliculogenesis, acting in an autocrine or paracrine manner. It has been reported that AMH or INHA alone are able to inhibit the biosynthesis and secretion of estradiol induced by FSH in granulosa cells (6,(21)(22)(23), but the results are inconsistent (11), which are likely due to differences in species, culture medium, and hormonal concentrations. Moreover, there is compelling evidence of negative correlation between AMH and INH in human follicular fluid (31). Based on the above observations for similarity and specificity of AMH and INHA, we assume that INH may interact with AMH in regulating ovarian function. By combining both in vitro and in vivo studies, the present study reveals the synergistic effect of INH and AMH on follicular FSH responsiveness and fertility in mice.
In primary granulosa cell culture system, we found that both AMH and INHA alone significantly inhibited the secretion of FSH-induced estradiol and progesterone, and such inhibitory effect was exerted by inhibiting the mRNA expression of FSHinduced CYP19A1, CYP11A1, HSD3B, and StAR, which are important enzyme genes involved in steroid biosynthesis. These results are consistent with the previous studies from human granulosa cells (32,33), which showed that AMH decreases sensitivity to FSH through reduction of the P450 aromatase activity, as well as, studies on cultured rat granulosa cells showing that INHA inhibits FSH-induced progesterone and estradiol production by decreasing P450 aromatase and P450 scc levels (6). Interestingly, it was found for the first time that INHA and  AMH in combination had an additive inhibitory effect on FSHinduced estradiol and progesterone release, indicating that AMH and INHA play synergistic roles on steroidogenesis at the cellular level. We further demonstrated that the synergistic effect might be due to the downregulation of intracellular cAMP levels, which attenuates FSH-dependent cAMP/PKA steroidogenic pathway (34,35). Within TGF-β superfamily, it has been demonstrated that AMH and BMP15 have the synergistic action on FSHinduced steroidogensis in human luteinizing granulosa cells possibly via smad5 protein phosphorylation (22). Apart from targeting gonads, AMH has been reported to synergistically cooperate with activin but not with BMP2 increasing FSHb expression in LβT2 cells (36,37). In this respect, it would be interesting in the near future to analyze interaction of AMH and INHA in gonadotropin cells. Immunological neutralization of reproductive hormone has been considered as a promising approach to regulate fertility. Our previous work has shown that INH DNA vaccine effectively reduced the biological activity of INH, thereby increasing the litter size (38,39). Likewise, the synthesized AMH peptide can also activate the immune system of sheep, resulting in a significant decrease in the number of gonadotropin responsive preantral and small antral follicles, while significantly increasing the number and ovulation rate of gonadotropin dependent antral follicles (40). To our knowledge, this is the first report on coimmunization of AMH and INH DNA vaccine for improving the fertility in mice, even though there has been published a novel DNA vaccine harboring INH and the RFRP genes (39). We found that co-immunization with AMH and INH plasmids were able to induce both anti-AMH and anti-INH antibodies, which were proposed to neutralize endogenous AMH and INH, resulting in higher litter size. In Dan's study (39), the litter size of the first generation in mice increased by 14.71 after immunization with INH and RFRP dual expression plasmids p-TPA-SINH/TPA-SRFRP, which is less than the litter size in the current study. This can be explained by the fact that the mice used in our study have higher basal fertility rate, which could also be the possible reason for no significant change in fertility after INH immunization. In contrast to an earlier study on ewe (41), where significantly a higher number of lambs was born with low birth weight after INH immunization, we presented the increased litter size in the co-immunization group with no significant difference in offspring birth weights.
The plasma estradiol concentration in co-immunization of AMH and INHA group was significantly higher than that of control group, which is in agreement with a previous study in the rats indicating that INH DNA immunization induced higher estradiol concentrations than the control group (39). On the other hand, the changes in estradiol concentration in vivo are basically consistent with the synergistic regulation of AMH and INHA on steroid production at the cellular level. However, this finding does not apply to progesterone hormone since these mice were in estrus phase, that is to say, mature follicles did not ovulate, and CL formation did not occur (39). It is worth noting that AMH has been reported to induce FSHb expression and FSH secretion only in immature females, without any significant change in estradiol (37). It seems that AMH acts directly on FSH secretion specifically in females before puberty. Further studies in this direction are necessary in order to determine the effect of AMH on FSH expression and secretion in mature mice.

CONCLUSION
We uncover that INHA and AMH synergistically inhibit FSH-induced estradiol and progesterone production by decreasing intracellular cAMP levels in primary granulosa cells. Furthermore, such synergistic action has been further confirmed through co-immunization with AMH and INH plasmids, showing higher estradiol levels and litter size in mice. Further studies on the underlying mechanism between AMH and INH signaling are essential for the elucidation of synergistic action in fertility of mice.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by Scientific Ethics Committee of Huazhong Agricultural University (HZAUMO-2018-021).