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

Abstract

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.

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.

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.