Steroid Production and Follicular Development of Neonatal Mouse Ovary during in vitro Culture

Abstract

Background: The aim of this study was to investigate follicular growth and steroid production in neonatal mouse ovary during in vitro culture.

Materials and methods: In this experimental study, 7-day-old mouse whole ovaries were cultured in α-MEM (medium supplemented with 100 mIU/ml recombinant follicle stimulating hormone, 1% insulin, transferrin and selenium (ITS), 5% fetal bovine serum (FBS), 100 IU/ml penicillin and 50 μg/ml streptomycin for 7 days. The size of whole ovary was determined as mean area during culture. The survival rates of isolated preantral follicles after culture were assessed using trypan blue staining after being mechanically isolated. Histological evaluation of whole ovary was done by hematoxylin and eosin staining. 17-β estradiol, progesterone and dehydroepiandrosterone concentrations in the medium were measured during culture.

Results: :The mean area of ovary increased after culture (1.47 vs. 0.21 mm(2)). The survival rate of isolated follicles in ovary after culture was 99.2%. There was a significant decline in the percentage of primordial follicles after seven days of culture (91.8 ± 0.2% vs. 65.1 ± 1.1%), whereas the rate of preantral follicles increased significantly (4.6 ± 0.4% vs. 29.2 ± 0.5%). The levels of estradiol, progesterone and dehydroepiandrosterone also increased significantly after culture (p<0.001).

Conclusion: These results show that the growth and development of primordial follicles in contrast with hormonal production decreased during in vitro culture of neonatal mouse ovaries.

Keywords: Organ Culture; Ovary; Primordial Follicles; in vitro.

Fig 1

Photomicrographs of 7-day-old mouse ovary viewed under the invert microscope in non-cultured fresh samples (A) and during one week of culture (B-D).

Fig 2

Hematoxylin and eosin staining of fresh and cultured mouse ovarian organ. Non-cultured fresh one week old mouse ovary which contains mainly primordial follicles with a few primary and secondary follicles (A), one week old mouse ovary after 7 days of culture (B). Higher magnification of one week old mouse ovary after 7 days, more secondary follicles were observed (C). Non-cultured fresh 14-day-old mouse ovary (D).

Fig 3

Normality rates of follicles after 7 days of in vitro ovarian culture. *; significantly different with one week non-cultured ovary.

Fig 4

The trypan blue staining of preantral follicles. A. The survived follicle was not stained and B. degenerated follicle was stained intensively.

Fig 5

Area of mouse ovary during 7 days of culture. *; There were significant differences between lengths of culture

Fig 6

The level of estradiol (A), progesterone (B) and dehydroepiandrosterone (C) in collected culture media of mouse ovary. *; There were significant differences between lengths of culture.