Current Issue

Volume 19, Number 3, Oct-Dec (Autumn) 2017, Serial Number: 75 Pages: 332-342

Morphological and Molecular Aspects of In Vitro Culture of Preantral Follicles Derived from Vitrified Ovarian Tissues Using A Two-Step Culture


Mahboobeh Amoushahi, Ph.D, 1, Mojdeh Salehnia, Ph.D, 1, *, Seyed Javad Mowla, Ph.D, 2, Nassim Ghorbanmehr, Ph.D, 3,
Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Department of Biotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran
*Corresponding Address: P.O. BOX: 14115-111 Department of Anatomy Faculty of Medical Sciences Tarbiat Modares University Tehran Iran Email:salehnim@modares.ac.ir

Abstract

Objective

This study aimed to evaluate the expression of the genes related to folliculo-genesis after vitrification of mouse ovarian tissues using a two-step in vitro culture.

Materials and Methods

In this experimental study, vitrified and non-vitrified ovaries from 7- day old (neonate) female mice were cultured using alpha-Minimum Essential Medium (α-MEM) supplemented with 5% fetal bovine serum (FBS) for 7 days. Morphology, surface area of ovaries and percentage of normal follicles were evaluated and compared in both groups. After one-week culture, in non-vitrified group, preantral follicles of cultured ovaries were isolated and cultured in a three-dimensional alginate culture system for 12 days. Then, the collected metaphase (M) II oocytes were inseminated with capacitated spermatozoa derived from 7-8-week old (adult) male NMRI mice. Follicular diameter, oocyte maturation, fertilization, embryo development and the expression of genes related to follicular development (Pcna, Fshr and Cyp17a1,) using real time reverse transcription-polymerase chain reaction (RT-PCR) were assessed at the end of last culture period in both groups.

Results

The ovarian area in vitrified group (162468.20 703.78) was less than non-vitrified group (297211.40 6671.71), while the percentage of preantral follicles in vitrified group (18.40%) was significantly lower than those of non-vitrified group (24.50%) on day 7 of culture (P<0.05). There were no significant differences between the two groups in terms of follicular diameter, expression of genes related to development of follicles, oocyte maturation, fertilization, as well as embryo development (P>0.05).

Conclusion

The results of this study showed that vitrification of ovarian tissue following in vitro culture had negative impact on the survival and development of follicles within the tissue. However, no significant alterations were observed in development, gene expression and hormonal production of in vitro culture of isolated follicles derived from vitrified ovarian tissues as compared to the non-vitrified samples.