Increased Formation of Follicular Antrum in Aquaporin-8-Deficient Mice Is Due to Defective Proliferation and Migration, and Not Steroidogenesis of Granulosa Cells.

Dejiang Wang,Meiyan Sun,Yaxin Hou,Miao Li,Tonghui Ma,Weiheng Su,He Zhang,Xiangjun Di,Xiangyu Meng,Di Zhang,Jie Wang,Ge Zhang,Chunlai Jiang,Bo Sun,Jiao Hu

doi:10.3389/fphys.2018.01193

Abstract

Aquaporin-8 (AQP8) is a water channel protein expressed exclusively in granulosa cells (GCs) in mouse ovary. Our previous studies of AQP8-deficient (AQP8-/-) mice demonstrated that AQP8 participates in folliculogenesis, including in the formation of follicles, ovulation, and atresia. However, its physiological function in formation of the antral follicle is still largely unknown. In the present study, we observed significantly increased numbers of antral follicles in AQP8-/- ovaries as well as significantly increased follicular antrum formation in in vitro 3D culture of AQP8-/- follicles. Functional detection of AQP8-/- GCs indicated that cell proliferation is impaired with FSH treatment, and wound healing and Transwell migration are also impaired with or without FSH treatment, compared with that in WT. However, the biosynthesis of estradiol and progesterone as well as the mRNA levels of key steroidogenic enzyme genes (CYP19A1 and StAR) in AQP8-/- GCs did not change, even with addition of FSH and/or testosterone. In order to estimate the influence of the impaired proliferation and migration on the density of GC mass, preantral follicles were injected with FITC-dextran, which distributes only in the intercellular space, and analyzed by confocal microscopy. The micrographs showed significantly higher transmission of fluorescence in AQP8-/- follicles, suggesting increased intercellular space of GCs. Based on this evidence, we concluded that AQP8 deficiency leads to increased formation of follicular antra in vivo and in vitro, and the mechanism may be associated with increased intercellular space of GCs, which may be caused by defective proliferation and migration of GCs. This study may offer new insight into the molecular mechanisms of the formation of follicular antrum.

Highlights

The formation and expansion of the fluid-filled antrum, as well as the development of the structural foundation of follicular differentiation and ovulation, are important processes during ovarian folliculogenesis
More antra had formed in AQP8−/− follicles compared with that in WT follicles on day 8, which lasted until the end of culture (Figure 2C)
In our 3D culture of preantral follicles, AQP8−/− follicles showed a significantly increased survival rate compared with that of WT (Figure 2B). This result is consistent with our previous study, in which the apoptosis rate of granulosa cells (GCs) was decreased by AQP8 deficiency (Su et al, 2010), because the atresia or death of a follicle is primarily dependent on the apoptosis of GCs (Hughes and Gorospe, 1991; Jiang et al, 2003)

Summary

Introduction

The formation and expansion of the fluid-filled antrum, as well as the development of the structural foundation of follicular differentiation and ovulation, are important processes during ovarian folliculogenesis. As the antrum begins to appear, specific growth-supporting factors begin to express, including IGF-1, ERa/b subtypes, FSH receptor, and cyclin D2 (Richards, 2001). These expressed proteins lead to expansion of the antrum. Follicular fluid consists of water, low-molecular-weight components, proteoglycan, and enzymes and contains a variety of soluble regulatory factors, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, insulin-like growth factor-1, tumor necrosis factor α, interleukin-6 (IL-6), and stem cell factor, which are essential for oocyte maturation and fertilization, GC proliferation, differentiation, luteinization, and follicle ovulation (Zachut et al, 2016)

Methods

Results

Discussion

Conclusion