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Morphological and Molecular Aspects of In Vitro Culture of Preantral Follicles Derived from Vitrified Ovarian.

ABSTRACT: OBJECTIVE:This study aimed to evaluate the expression of the genes related to folliculogenesis 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 (?-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.

SUBMITTER: Amoushahi M

PROVIDER: S-EPMC5570399 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Morphological and Molecular Aspects of In Vitro Culture of Preantral Follicles Derived from Vitrified Ovarian.

Amoushahi Mahboobeh M Salehnia Mojdeh M Mowla Seyed Javad SJ Ghorbanmehr Nassim N

Cell journal 20170819 3

<h4>Objective</h4>This study aimed to evaluate the expression of the genes related to folliculogenesis after vitrification of mouse ovarian tissues using a two-step in vitro culture.<h4>Materials and methods</h4>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 follicle ...[more]

PMID: 28836396

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Project description:ObjectiveVitrification of the ovarian tissue is one of the techniques recommended for preserving the fertility of women who are dealing with infertility. Despite its benefits, our information about the molecular aspects of ovarian follicles vitrification is somehow ambiguous. Therefore, the aim of this study was to evaluate the expression pattern of DNA repair genes in vitrified preantral follicles.Materials and methodsIn this experimental study, the isolated preantral follicles (n=906) from 14-16 days old mice (n=12) were divided into three groups: fresh, toxic and vitrified which were cultured in vitro for 12 days. Preantral follicles were vitrified using cryotop followed by exposure to equilibration solution for five minutes and vitrification solution (VS) for 30 seconds. In the toxic group, preantral follicles were only placed in equilibration and vitrification media and they were then placed in the warming solutions without exposure to liquid nitrogen. On the second and sixth days of the culture period, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out to evaluate expression of the selected genes involved in DNA repair, including Msh6 (MutS homolog 6), Mre11 (Meiotic recombination 11), Brca1 (Breast cancer type 1), Rad51 (RAD51 recombinase), Pcna (Proliferating cell nuclear antigen) and Atm (ATM serine/threonine kinase). In addition, developmental parameters including growth, survival rate, antrum cavity formation and ovulation were analyzed.ResultsThe relative mRNA expression of Msh6, Mre11, Brca1, Rad51, Pcna and Atm on the second and sixth days of the culture period in vitrified group was significantly higher than those of the control and toxic groups, but there was no significant difference between the toxic and control groups. In addition, developmental parameters of follicles were similar in both toxic and control groups, while both were significantly higher than that of vitrified group.ConclusionVitrification changes the expression pattern of DNA repair genes of the mouse preantral follicles.

Project description:The ovarian follicle (each contains a single oocyte) is the fundamental functional tissue unit of mammalian ovaries. In humans, it has been long held true that females are born with a maximum number of follicles (or oocytes) that are not only nonrenewable, but also undergoing degeneration with time with a sharply decreased oocyte quality after the age of ?35. Therefore, it is of importance to isolate and bank ovarian follicles for in vitro culture to obtain fertilizable oocytes later, to preserve the fertility of professional women who may want to delay childbearing, young and unmarried women who may lose gonadal function because of exposure to environmental/occupational hazards or aggressive medical treatments, such as radiation and chemotherapy, and even endangered species and breeds. Although they contributed significantly to the understanding of follicle science and biology, most studies reported to date on this topic were done using the man-made, unnatural inbred animal species. It was found in this study that the conventional two-dimensional microliter drop and three-dimensional hanging drop (HD) methods, reported to be effective for in vitro culture of preantral follicles from inbred mice, are not directly transferrable to outbred deer mice. Therefore, a modified HD method was developed in this study to achieve a much higher (>5 times compared to the best conventional methods) percentage of developing early secondary preantral follicles from the outbred mice to the antral stage, for which, the use of an ovarian cell-conditioned medium and multiple follicles per HD were identified to be crucial. It was further found that the method for in vitro maturation of oocytes in antral follicles obtained by in vitro culture of preantral follicles could be very different from that for oocytes in antral follicles obtained by hormone stimulation in vivo. Therefore, this study should provide important guidance for establishing effective protocols of in vitro follicle culture to preserve the fertility of wildlife and humans outbred by nature.

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Morphological and Molecular Aspects of In Vitro Culture of Preantral Follicles Derived from Vitrified Ovarian.

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Morphological and Molecular Aspects of In Vitro Culture of Preantral Follicles Derived from Vitrified Ovarian.

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