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

SUMMARY

OBJECTIVE:

Folliculogenesis is a complex process involving various ovarian paracrine factors. During folliculogenesis, vitamin D3 and progesterone are significant for the proper development of follicles. This study aimed to investigate the effects of vitamin D3 and selective progesterone receptor modulator ulipristal acetate on ovarian paracrine factors.

METHODS:

In the study, 18 female Wistar-albino rats were randomly divided into three groups: control group (saline administration, n=6), vitamin D3 group (300 ng/day vitamin D3 oral administration, n=6), and UPA group (3 mg/kg/day ulipristal acetate oral administration, n=6). Ovarian tissue was analyzed by histochemistry and immunohistochemistry. For quantification of immunohistochemistry, the mean intensities of growth differentiation factor 9, bone morphogenetic protein 15, and forkhead box O3a expressions were measured by Image J and MATLAB. Blood samples were collected for the analysis of serum anti-Müllerian hormone levels by ELISA.

RESULTS:

Atretic follicles and hemorrhagic cystic structures were observed in the UPA group. After immunohistochemistry via folliculogenesis assessment markers, growth differentiation factor 9, bone morphogenetic protein 15, and cytoplasmic forkhead box O3a expressions decreased in the UPA group (p<0.05). Anti-Müllerian hormone level did not differ significantly between the experimental groups (p>0.05).

CONCLUSION:

Ulipristal acetate negatively affects folliculogenesis via ovarian paracrine factors. The recommended dietary vitamin D3 supplementation in healthy cases did not cause a significant change.

KEYWORDS:
Ovarian follicles; Immunohistochemistry; Progesterone; Vitamin D

INTRODUCTION

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 follicle¹. 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 expansion². 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 activation³. 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 apoptosis⁴. Another factor, anti-Müllerian hormone (AMH), is an inhibitory regulator of primary and preantral follicle development¹.

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 cells⁵. Progesterone regulates follicle growth through PI3K/Akt and mitogen-activated protein kinase (MAPK) signaling pathways⁶. According to previous research, progesterone at periovulation concentration was shown to stimulate primary follicle development⁵. 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 UPA⁷. 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 follicles⁸. 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 genes⁹. 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 survival¹⁰. In VitD3-deficient rats, follicular development is stalled¹¹. 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 signals¹². 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 fluid¹³. The relationship between VitD3 and AMH according to a recent meta-analysis is controversial¹⁴.

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.

METHODS

Animals

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.

Experimental design

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) administration¹⁵,

UPA group (n = 6): 5 weeks oral UPA (3 mg/kg/day) administration¹⁶.

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 rats¹⁵,¹⁶. VitD3 300 ng/day for a 200 g body-weighted rat is equivalent to 15.81 μg/day for a 60 kg human¹⁷. 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 human¹⁷. UPA's daily dose in adults as an emergency contraceptive is 30 mg¹⁹. After the experiments, rats were anesthetized. Rats were euthanized by intracardiac blood collection.

Histological examination

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).

Immunohistochemistry

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 evaluated²⁰.

ELISA

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

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.

RESULTS

Histomorphological findings

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.

Figure 1
Hematoxylin & eosin stained sections belonging to the groups. Control group: (A) Primordial follicle (black arrowhead) and preantral follicles (black thin arrow). Scale bar: 100 μm. (B) Antral follicle (*). Scale bar: 100 μm. Vitamin D3 group: (C) Normal follicles at various developmental stages in the cortex. Scale bar: 200 μm. (D) Primordial follicle (black arrowhead) and preantral follicle (black thin arrow). Scale bar: 100 μm. UPA group: (E) Hemorrhagic cyst (Ct) structures. Scale bar: 500 μm. (F) Atretic follicles (black thick arrow). Scale bar: 100 μm.

Immunohistochemical results

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 (p<0.05) (Figure 2D).

Figure 2
Bone morphogenetic protein 15, growth differentiation factor 9, and forkhead box O3a immunohistochemical staining of the groups and mean pixel density assessment. The first panels of immunohistochemical stains present negative controls (a,d,g). Immunoreactivity is represented in the ooplasm of preantral follicle with a black arrow and antral follicle with a black arrowhead. (A) Bone morphogenetic protein 15 immunohistochemical staining; (b) bone morphogenetic protein 15 expression in the oocyte cytoplasm (black arrowhead). (c) Predominant bone morphogenetic protein 15 expression is localized in the oocyte cytoplasm (black arrow) and granulosa cells. (e) Vitamin D3 group shows strong immunoreaction in the corpus luteum. (f) Bone morphogenetic protein 15 expression in the oocyte cytoplasm (black arrowhead, black arrow). (h) UPA group shows weak immunoreaction in the corpus luteum. (j) Bone morphogenetic protein 15 expression in the oocyte cytoplasm (black arrowhead). Scale bars: Panel (c)—50 μm, panels (d,e)—200 μm, and others—100 μm. (B) Growth differentiation factor 9 immunohistochemical stain. (b) Growth differentiation factor 9 expression in the oocyte cytoplasm (black arrowhead). (c) Predominant growth differentiation factor 9 expression is localized in the oocyte cytoplasm (black arrow). (e) Growth differentiation factor 9 immunoreaction in the corpus luteum of the vitamin D3 group. (f) Growth differentiation factor 9 expression in the oocyte cytoplasm (black arrow). (h) UPA group shows weak immunoreaction in the antral follicle (black arrowhead). (j) UPA group shows weak immunoreaction in the preantral follicle (black arrow). Expression of growth differentiation factor 9 in corpus luteums decreased in this group. Scale bars: Control (c,g,j)—50 μm, (e)—200 μm, and others—100 μm. (C) Forkhead box O3a immunohistochemical stain. (b) Control group shows strong immunoreaction in the preantral follicle (black arrow). (c) Control group shows weaker immunoreaction in the antral follicle (black arrowhead) compared with preantral follicles. (e,f) Forkhead box O3a expression in the oocyte cytoplasm (black arrowhead, black arrow). (h) UPA group shows weak immunoreaction in the antral follicle (black arrowhead). (j) UPA group shows weak immunoreaction in the preantral follicles (black arrow). Scale bars: Panels (c,f,g)—50 μm and others—100 μm. (D) Immunoreactivity assessment via mean pixel density of the groups throughout the ovary; Kruskal-Wallis test and post-hoc Dunn-Bonferroni. Bone morphogenetic protein 15, growth differentiation factor 9, and forkhead box O3a mean pixel density decreased in the UPA group. (a) significant difference compared with the control and vitamin D3 groups. (b) Significant difference compared with the vitamin D3 group. Statistical significance: p<0.05.

Serum anti-Müllerian hormone levels

After the evaluation of serum AMH levels with ELISA, there was no statistically significant difference between the groups (p>0.05) (Table 1).

Table 1
Comparison of serum anti-Müllerian hormone levels (ng/mL).

AMH (ng/mL)
Groups	Mean	Std. deviation	Std. error
Control	6.245	4.14	1.85129
VitD3	4.963	2.072	0.92645
UPA	5.574	2.762	1.23535

Source of variation	Sum of squares	Degree of freedom	Mean square	F	p-value
Between groups	4.111	2	2.055	0.212	0.812
Within groups	116.233	12	9.686
Total	120.344	14

• One-way ANOVA test. Statistical significance: p<0.05.

DISCUSSION

Studies on follicular development will provide comprehensive data on female reproductive life and facilitate the development of new therapeutic approaches against reproductive aging¹. 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 development⁵. 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 growth⁸. 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 rats²¹. 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 GDF9²². In 2018, Xu et al. showed that VitD3 causes increased expression of BMP and GDF9 in preantral follicles¹⁰. 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 cases¹⁸. UPA administration with an emergency contraceptive dose (approximately 30 mg) suppressed these expressions¹⁹.

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 apoptosis²³. 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 BMP15²⁴. 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 controversial¹⁴. 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 level²⁵. In this study, the absence of a significant change in serum AMH values between the groups was attributed to this deduction.

CONCLUSION

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.

• Funding: Scientific Research Project of Gazi University (TGA-2021-6954).

ACKNOWLEDGMENTS

This study was supported by the Scientific Research Project of Gazi University (TGA-2021-6954).

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Publication Dates

• Publication in this collection
  14 Aug 2023
• Date of issue
  2023

History

• Received
  05 May 2023
• Accepted
  20 May 2023