Amoushahi Mahboobeh, Salehnia Mojdeh, Mowla Seyed Javad, Ghorbanmehr Nassim
Department of Anatomy, Tarbiat Modares University, Tehran, Iran.
Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Cell J. 2017 Oct;19(3):332-342. doi: 10.22074/cellj.2017.4264. Epub 2017 Aug 19.
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.
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.
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).
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.
本研究旨在通过两步体外培养评估小鼠卵巢组织玻璃化冷冻后卵泡发生相关基因的表达。
在本实验研究中，使用添加5%胎牛血清（FBS）的α-最低必需培养基（α-MEM）对7日龄（新生）雌性小鼠的玻璃化冷冻和未玻璃化冷冻的卵巢进行7天培养。评估并比较两组卵巢的形态、表面积和正常卵泡百分比。培养一周后，在未玻璃化冷冻组中，分离培养卵巢的窦前卵泡并在三维海藻酸盐培养系统中培养12天。然后，将收集的中期（M）II期卵母细胞与来自7 - 8周龄（成年）雄性NMRI小鼠的获能精子进行受精。在最后一个培养期结束时，评估两组卵泡直径、卵母细胞成熟、受精、胚胎发育以及卵泡发育相关基因（Pcna、Fshr和Cyp17a1）的表达，采用实时逆转录-聚合酶链反应（RT-PCR）进行检测。
玻璃化冷冻组卵巢面积（162468.20±703.78）小于未玻璃化冷冻组（297211.40±6671.71），而在培养第7天，玻璃化冷冻组窦前卵泡百分比（18.40%）显著低于未玻璃化冷冻组（24.50%）（P>0.05）。两组在卵泡直径、卵泡发育相关基因表达、卵母细胞成熟、受精以及胚胎发育方面无显著差异（P>0.05）。
本研究结果表明，体外培养后卵巢组织的玻璃化冷冻对组织内卵泡的存活和发育有负面影响。然而，与未玻璃化冷冻样本相比，源自玻璃化冷冻卵巢组织的分离卵泡在体外培养的发育、基因表达和激素产生方面未观察到显著变化。
