The E-modulus of the oocyte is a non-destructive measure of zona pellucida hardening.

After fertilization, the oocyte-specific metalloproteinase ovastacin is released and cleaves the zona pellucida protein 2 (ZP2), making the zona pellucida impermeable to sperm. Before fertilization, the zona remains permeable because previously released ovastacin is inhibited by fetuin-B. Consequently, in the absence of fetuin-B, ZP2 cleavage occurs prematurely and leads to infertility of female fetuin-B deficient mice. In contrast, fetuin-B/ovastacin double-deficient oocytes show a permanently permeable zona with intact ZP2. In this study, we asked if the elastic modulus of the zona pellucida informs about ZP2 cleavage and thus could serve as a new reference of oocyte fertility. Therefore, we determined the elastic modulus of mouse oocytes by nanoindentation as a direct measure of mechanical zona hardening. The elastic modulus reflects ZP2 cleavage, but with more than double sensitivity compared to immunoblot analysis. The elastic modulus measurement allowed to define the range of zona hardening, confined by the extreme states of the zona pellucida in fetuin-B and ovastacin-deficient oocytes with cleaved and uncleaved ZP2, respectively. We present here nanoindentation as a method to quantify the effect of potential contributing factors on the zona hardening of individual oocytes. To demonstrate this, we showed that mechanical hardening of the zona pellucida is forced by recombinant ovastacin, inhibited by additional administration of fetuin-B, and unaffected by zinc. Since the change in elastic modulus is induced by ZP2 cleavage, an automated elastic modulus measurement of oocytes may serve as a novel sensitive, non-destructive, marker-free, and observer-unbiased method for assessing individual oocyte quality.

Utilizing Fibrin-Alginate and Matrigel-Alginate for Mouse Follicle Development in Three-Dimensional Culture Systems.

In vitro culture of ovarian follicles is a new technique in reproductive technology, which helps in understanding the process of folliculogenesis. The in vitro culture of follicles could be carried out using three-dimensional (3D) natural scaffolds that mimic the ovarian tissue stroma. Selection of the right matrix and culture media in these scaffolds could increase the survival and maturation of the follicles. In this work, the applicability of matrigel-alginate (MA) and fibrin-alginate (FA) 3D scaffolds for folliculogenesis was assessed. The ovaries of 13-day-old Naval Medical Research Institute (NMRI) mice were isolated and distributed into control and vitrification groups. Preantral follicles (mean diameter: 120-140 µm) were mechanically isolated from control and vitrified-warmed ovaries, encapsulated in MA or FA scaffold and cultured for 12 days. Follicle survival, growth, maturation, and quantitative expression of oocyte maturation genes (Gdf9, Bmp15, Fgf8, KitL, Kit, and Amh) and proteins (GDF9 and BMP15) were assessed. Survival rate of culture preantral follicles in control groups was found to be significantly higher than vitrified follicles. Antrum formation was similar in all groups. Follicle diameters were significantly increased in all groups during culture period. A decreasing pattern of gene expression was seen for all genes in all groups. This trend was verified through evaluation of protein expression, during which there was strong staining in antral follicles from all groups in the last day of in vitro culture. The better survival and maturation rate of follicles in the MA compared to FA scaffold indicates that the MA matrix, being rich in extracellular matrix components, could mimic the ovarian condition better and presents a good environment for follicle development.

Mouse preantral follicle development in two-dimensional and three-dimensional culture systems after ovarian tissue vitrification.

OBJECTIVES: This work on follicle culture and evaluation of expression of oocyte maturation genes helps to better understand the complicated processes of folliculogenesis and to develop new approaches for infertility treatment. STUDY DESIGN: Ovaries of 12-day-old female NMRI mice were divided into control and vitrification groups. After vitrification and warming procedures, ovarian tissue morphologies were histologically evaluated and compared to those of the control group. In the second stage, preantral follicles were mechanically isolated from non-vitrified and vitrified ovaries and cultured for 12 days in two-dimensional (2D) and three-dimensional (3D) systems. Finally, the survival and growth rate of follicles and quantitative expression of oocyte maturation genes (Gdf9, Bmp15 and Bmp6) were studied. RESULT: Morphological integrity of ovarian tissue in vitrification group was well preserved. Survival rates of cultured preantral follicles in control group during 2D and 3D systems were somewhat similar, but were significantly different between 2D and 3D systems in vitrification group. Although the growth rate of follicles was similar in the 3D system in both groups, substantially higher growth rate was observed for the control group in the 2D system. Expressions of oocyte maturation genes were, to some extent, similar between control and vitrification groups. There was a remarkable reduction in expression pattern of genes in 3D compared to 2D system in both experimental groups, during the 12th day of culture period. CONCLUSIONS: 3D in-vitro culture system could be appeared more appropriate than 2D culture system for preservation of follicles in terms of spatial morphology, growth rate and expression reduction of maturation genes.