Decreased growth differentiation factor 9, bone morphogenetic protein 15, and forkhead box O3a expressions in the ovary via ulipristal acetate

Rats were euthanized by intracardiac blood collection after experiments. Serum AMH levels were measured by ELISA in these blood samples. The blood samples were centrifuged at 4,500 rpm for 15 min. The obtained serum samples were stored at −20°C before analysis. Serum AMH levels were determined using the Rat AMH ELISA kit (E0456Ra, BTLAB, China) following the manufacturer's kit procedures. Statistical analysis was performed using an SPSS program (IBM SPSS Statistics 20). The Kruskal-Wallis method with the Dunn-Bonferroni post-hoc test was used for statistical analysis of immunoreactivity density among nonparametric groups. ANOVA test was used for statistical analysis of serum AMH levels. A p-value less than 0.05 was considered statistically significant.

Folliculogenesis occurs in the ovaries which is a crucial organ of the female reproductive system. This mechanism is a complex process involving various growth factors and signaling molecules. Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are the factors involved in the development of the preantral follicle from the primary follicle 1 . These two factors not only improve the developmental competence of the oocyte but also act directly on the granulosa cells. GDF9 and BMP15 have critical effects on granulosa cell proliferation, differentiation, apoptosis, and cumulus expansion 2 . Forkhead box O3a (FOXO3a) is involved in various processes such as cell proliferation, apoptosis, differentiation, and metabolism. Activation of the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signaling pathway inhibits these transcription factors. Akt hyperphosphorylation results in the nuclear export of FOXO3a and primordial follicle activation 3 . Active FOXO3a causes infertility with insufficient oocyte and follicular development leading to anovulation. It has also been shown that inhibition of Akt leads to FOXO3a-dependent apoptosis 4 . Another factor, anti-Müllerian hormone (AMH), is an inhibitory regulator of primary and preantral follicle development 1 . Progesterone is one of the hormones involved in the control of ovulation. It is synthesized from the corpus luteum and follicle. The progesterone receptor (PR) is a nuclear receptor and is expressed in the granulosa cells of the Graffian follicle. Another progesterone-binding protein, PGR membrane component 1 (PGRMC1), is synthesized in the granulosa cells of developing follicles. PGRMC1 controls the antiapoptotic and antimitotic effects of progesterone on granulosa cells 5 . Progesterone regulates follicle growth through PI3K/Akt and mitogen-activated protein kinase (MAPK) signaling pathways 6 . According to previous research, progesterone at periovulation concentration was shown to stimulate primary follicle development 5 . UPA is a 19-norprogesterone derivative and a selective PR modulator. Selective PR modulators act as antagonists when the progesterone level increases, preventing the LH peak and ovulation. UPA inhibits ovulation possibly by suppressing the expression of PR-dependent genes critical for the process. Antiproliferative and apoptotic effects are the researched effects of UPA 7 . However, there are limited studies on the effect of folliculogenesis. According to a study conducted in 2000, the single dose administration of UPA in the mid-follicular phase suppressed lead follicle growth. They presented that high dose causes luteinized unruptured follicles 8 . In the literature, UPA effects on folliculogenesis markers are not elucidated. Effects via these factors are significant to understand the underlying mechanism. Vitamin D3 (VitD3) is converted from 7-dehydrocholesterol in the skin under UV light. VitD3 regulates cellular functions via the VitD3 receptor (VitD3R). VitD3R binds to the vitamin response element region in DNA. These binding sites are involved in the regulation of many genes 9 . VitD3R is expressed in many organs, including the ovary. Previous studies have reported that VitD3 plays a significant role in the functions of the ovaries, including follicular development. VitD3 supplementation increases preantral follicle survival, antral follicle growth, and survival 10 . In VitD3-deficient rats, follicular development is stalled 11 . From the analyzed data, it is determined that VitD3 has a fundamental role in ovarian functions. However, VitD3 and folliculogenesis-associated mechanism is still not elucidated. It requires comprehensive research regarding the mechanism of action on ovarian paracrine signals 12 . In the literature, it was reported that 600,000 IU VitD3 single-dose administration led to an increment of BMP15 and GDF9 levels in follicular fluid 13 . The relationship between VitD3 and AMH according to a recent meta-analysis is controversial 14 . Ovarian paracrine factors originating from different compartments in the ovary play roles in follicular developmental stages. Considering progesterone and VitD3 significance for follicular development, we investigated the effects of recommended dietary VitD3 supplementation and emergency contraceptive UPA on ovarian factors. To contribute to the literature, the variations of GDF9, BMP15, FOXO3a, and AMH by UPA and VitD3 are analyzed. The observed changes and inferences of each group are given from a statistical perspective.

We used 18 adult female Wistar albino rats (180–240 g, 6–8 weeks). The rats were housed in cages under a 12 h light/12 h dark cycle at a temperature of 22 ± 2°C with free access to food and water. The experimental procedures were approved by the Ethics Committee of Gazi University (G.Ü.ET-20.055), and the study was conducted at Gazi University Experimental Research Center. The rats were randomly divided into three groups: Control group (n = 6): 0.05 mL/rat/day saline (peroral) per day, VitD3 group (n = 6): 3 weeks oral VitD3 (300 ng/day) administration 15 , UPA group (n = 6): 5 weeks oral UPA (3 mg/kg/day) administration 16 . Saline, VitD3, and UPA gavage administrations for each group continued once a day and at a certain time (10.00–11.00 a.m.) throughout the week continuously. UPA and VitD3 doses were prepared to be nontoxic in accordance with the literature. As VitD3 is given to healthy rats, the application time is shorter to avoid toxic effects in rats 15 , 16 . VitD3 300 ng/day for a 200 g body-weighted rat is equivalent to 15.81 μg/day for a 60 kg human 17 . The recommended dietary allowance of VitD3 for adults through to 70 years is 600 IU daily (15 μg) 18 . UPA 3 mg/kg/day for a 200 g body-weighted rat is equivalent to 31.62 mg/day UPA for a 60 kg human 17 . UPA's daily dose in adults as an emergency contraceptive is 30 mg 19 . After the experiments, rats were anesthetized. Rats were euthanized by intracardiac blood collection. Ovarian tissue samples were fixed in neutral buffered 10% formalin for 48 h. Samples were dehydrated through an ascending alcohol series, cleared with xylene, and embedded in paraffin. Paraffin-embedded ovarian samples were then sliced into 5 μm sections, rehydrated, and stained with hematoxylin and eosin. Ovarian sections were examined under a bright-field microscope. Images were acquired using Leica DCM 4000 (Germany). For immunohistochemical analyses, the sections were deparaffinized, rehydrated, and incubated with pH 6.0 citrate buffer. Endogenous peroxidase activity was denatured with hydrogen peroxide. After Ultra V block (Thermo Scientific), the sections were incubated with primary antibody at a dilution of 1:200 in BMP15 (E-AB-62302, Elab, USA), 1:100 in GDF9 (bs4720R, Bioss, USA), and 1:100 in FOXO3A (bs-1548R, Bioss, USA) for overnight. The sections were then washed and incubated with the biotinylated secondary antibodies (Thermo Scientific). Immunoreactive signals were detected using streptavidin-HRP (Thermo Scientific) and diaminobenzidine (Thermo Scientific). Sections were also counterstained with hematoxylin. Images were acquired using Leica DCM 4000 (Germany). For quantification of IHC staining, the mean immunoreactivity intensity for an ovarian section was measured. After color deconvolution via ImageJ software (ImageJ), images are inverted. The mean density was measured with the MATLAB software (MATLAB). Six ovaries from each group and five areas from each ovary were evaluated 20 .

In the histological examination of the control group, cortex and medulla structures were observed. Normal follicles were detected through all developmental stages in the cortex ( Figures 1A , B ). Follicles at various developmental stages in the VitD3 group were normal in the cortex ( Figures 1C , D ). In the UPA group, hemorrhagic cyst structures were observed ( Figure 1E ). Follicles in their normal developmental stage have become atretic ( Figure 1F ). The number of these atretic follicles relatively increased in the UPA group compared with the other groups. Immunohistochemical studies were performed to evaluate the expression of BMP15, GDF9, and FOXO3a in the ovary. BMP15 expression was predominantly localized in the oocyte cytoplasm and granulosa cells of follicles. BMP15 expression was also detected in the corpus luteum. It was noticed that BMP15 expression decreased in the corpus luteums of the UPA group ( Figure 2A ). The mean pixel density of BMP15 throughout the ovary decreased in the UPA group compared with the other groups (p<0.05) ( Figure 2D ). The GDF9 expression was mainly detected in the oocyte cytoplasm. The GDF9 immunoreactivity was also detected in the corpus luteum structures. The GDF9 immunoreactivity decreased in the corpus luteums and preantral and antral follicles of the UPA group ( Figure 2B ). The mean pixel density of GDF9 decreased throughout the ovary in the UPA group compared with the other groups (p<0.05) ( Figure 2D ). FOXO3a expression was predominantly detected in the oocyte cytoplasm of the preantral follicles. It was observed that its expression decreased in antral follicles. UPA group shows weak FOXO3a immunoreaction in the antral and preantral follicles ( Figure 2C ). The mean pixel density of FOXO3a decreased throughout the ovary in the UPA group compared with the VitD3 group (p0.05) ( Table 1 ). One-way ANOVA test. Statistical significance: p<0.05.

Studies on follicular development will provide comprehensive data on female reproductive life and facilitate the development of new therapeutic approaches against reproductive aging 1 . In the scope of this study, the effects of UPA and VitD3 on ovarian paracrine factors were examined immunohistochemically and biochemically. Progesterone affects granulosa cells via the PGRMC1 receptor, and its concentration correlates with follicle development. Komatsu et al. showed that when the variation of the progesterone concentration level was imitated, periovulation concentration stimulated primary follicle development 5 . According to a study conducted in 2000, Stratton et al. found that the single dose administration of antiprogestin UPA in the mid-follicular phase suppressed follicular growth 8 . In our study, we observed atretic follicles in the UPA group. By considering related studies, it was deduced that the specified situation presumably is related to the inhibition of required progesterone concentration by UPA for proper follicle development. In 2009, Tamura et al. demonstrated that mifepristone, a PR antagonist, caused luteal cysts and large corpus luteum structures at doses of 20 mg/kg or more in rats 21 . Large hemorrhagic cyst structures were also detected in our UPA group. BMP15 is involved in normal follicular development as well as being mitogenic for somatic cells. Another crucial factor for preantral follicle development is GDF9 22 . In 2018, Xu et al. showed that VitD3 causes increased expression of BMP and GDF9 in preantral follicles 10 . There is no information in the literature about UPA effects on folliculogenesis markers. In our study, GDF9 and BMP15 expressions did not show a significant difference with recommended dietary VitD3 dose (approximately 15 μg/day for an adult human) in healthy cases 18 . UPA administration with an emergency contraceptive dose (approximately 30 mg) suppressed these expressions 19 . The phosphorylated FOXO3a by PI3K-Akt is an inactive form and localized in the cytoplasm. Its active nonphosphorylated form is localized in the nucleus and induces apoptosis 23 . In this study, decreased cytoplasmic FOXO3a expression in the UPA group represents inactive form and it is parallel with atretic follicle findings. We conclude that UPA causes atresia, possibly by inducing FOXO3a-dependent apoptosis. In the literature, it was concluded that activated FOXO3a reduces the expression of BMP15 24 . In our study, decreased expressions of BMP15 and inactive FOXO3a in the UPA group were consistent with the association of these factors. The relationship between vitamin D and AMH in the literature is controversial 14 . In our study, recommended dietary VitD3 supplementation did not change serum AMH levels. In 2017, Dennis et al. indicated that the amount of AMH produced per follicle is only a minor determinant of circulating AMH level 25 . In this study, the absence of a significant change in serum AMH values between the groups was attributed to this deduction.

The effects of UPA and VitD3 on ovarian paracrine factors were studied in this work by considering studies addressing the corresponding topic. During histological analyses, UPA decreased inactive form of FOXO3a expression and caused follicular atresia. The recommended dietary VitD3 supplementation in healthy cases did not cause significant changes in GDF9 and BMP15 expressions. However, UPA suppressed these expressions. It was concluded that UPA negatively affects folliculogenesis via ovarian paracrine factors.