Effects of Estradiol on the Paracrine Regulator Expression of In Vitro Maturated Murine Ovarian Follicles

Abstract

The preservation of female germ cells is important in the individuals with ovarian dysfunction and failure. For this purpose, ovarian follicle in vitro maturation (OFIVM) is an important technology for the retrieval of mature oocytes. In the in vivo follicular development, paracrine factors such as angiotensin (AT) and anti-Müllerian hormone (AMH) play important roles. We attempted to add estrogen during the OFIVM and to assess their expression on the follicular cells. The ovaries and pre-antral follicles were collected from 13-day C57BL/6 mice and cultured in vitro with estradiol (E₂) treatment for up to two weeks. In the whole ovaries, the expression of AT II was decreased and the expression of AMH was similar between control and E₂-treated ovaries after in vitro culture. Although there was no difference in the survival, ovulation, maturation and fertilization rates between control and E₂-treated groups, the expression of AT II in the follicular cells was down-regulated after E₂ treatment at mRNA level, and AMH showed similar expression. In conclusion, adding E₂ in OFIVM may regulate paracrine factors and their receptors that are related to follicular development. Further investigations are necessary to elucidate the roles of various sex hormones in the regulation of AT and AMH expression during the OFIVM.

Keywords: Angiotensin; Anti-Müllerian hormone; Estrogen; Ovary.

Fig. 1

The expression of paracrine factors in E₂-treated mouse ovary (Western blot). CTL: control, E2: estradiol, AT II: angiotensin II, AMH: anti-Müllerian hormone

Fig. 2

Murine follicular morphology after E₂ treatment. A control group, B E₂-treated group. a Preantral follicle, b culture day 5, c culture day 12 (hCG treatment), d ovulated follicle. Typical development, antral formation and ovulation of follicle were shown in both groups during OFIVM. E2: estradiol, hCG: recombinant human chorionic gonadotropin, OFIVM: ovarian follicle in vitro maturation

Fig. 3

The expression of AT II and FSH-R on the follicular cells after OFIVM (immunocytochemical staining). AT II expression on granulosa cells of in vitro matured follicle was down-regulated in the E₂-treated group compared to control group. FSH-R expression was similar between the two groups. E2: estradiol, AT II: angiotensin II, DAPI: 4′,6-diamidino-2-phenylindole, dihydrochloride, FSH-R: follicle stimulating hormone receptor, OFIVM: ovarian follicle in vitro maturation

Fig. 4

Relative expression of receptor and paracrine factor genes on dominant follicle of E₂-treated group compared to control group (Quantitative RT-PCR). A The expression of receptor genes on dominant follicle of E₂-treated group. B The expression of paracrine factor genes on dominant follicle of E₂-treated group (*P < 0.05). AT II: angiotensin II, AMH: anti-Müllerian hormone, LH: luteinizing hormone, FSH: follicle-stimulating hormone, IGF-R: insulin-like growth factor receptor, VEGF: vascular endothelial growth factor, RT-PCR: reverse transcription polymerase chain reaction

Fig. 5

Relative expression of receptor genes and paracrine factor genes on atretic follicles of E₂-treated group compared to control group (Quantitative RT-PCR). A The expression of receptor genes on the atretic follicles of E₂-treated group. B The expression of paracrine factor genes on the atretic follicles of E₂-treated group (*P < 0.05)

Fig. 6

Relative expression of receptor genes on dominant follicle compared to atretic follicle in E₂-treated group (Quantitative RT-PCR). A Relative expressions of receptor genes on dominant follicle compared to atretic follicle in E₂-treated group. B Relative expression of paracrine factors on dominant follicle compared to atretic follicle in E₂-treated group (*P < 0.05). AT II: angiotensin II, AMH: anti-Müllerian hormone, LH: luteinizing hormone, FSH: follicle-stimulating hormone, IGF-R: insulin-like growth factor receptor, VEGF: vascular endothelial growth factor, RT-PCR: reverse transcription polymerase chain reaction