A way for in vitro/ex vivo egg production in mammals

Abstract

Eggs are female germ cells that are required for producing offspring through sexual reproduction. In mammals, eggs are produced in the ovary and ovulated into the oviduct. It is well known that over 99% of eggs are degenerated without ovulation, so that many studies have attempted in vitro folliculogenesis to produce many eggs in different species for a few decades. Although many methods have been developed, a success of in vitro egg production with the resultant live birth of offspring has been limited, especially in livestock animals. More recently, we have succeeded in producing live pups derived from in vitro/ex vivo egg production in mice. This review aims to introduce our recent findings with a brief history of in vitro/ex vivo culture systems for follicles and ovaries.

Keywords: Folliculogenesis; In vitro egg production; Oogenesis.

Fig. 1.

A schematic overview of in vitro/ex vivo egg production in the mouse and livestock animals [5, 13,14,15,16,17, 26, 56,57,58,59,60,61,62,63]. In the mouse, the culture methods have been developed in all follicular stages as well as in primordial germ cells with successful live birth of offspring, whereas in livestock animals, the methods have been developed only in the late secondary and antral follicles with a low yield of embryo production. The 2-step protocol consists of ex vivo organ culture and in vitro follicle culture. PGCs; primordial germ cells, OGCs; oocyte-granulosa cell complexes.

Fig. 2.

An overview of the 2-step culture of mouse PGCs ovaries collected at 12.5 embryonic day (E12.5). In step 1, the ovaries are subjected to ex vivo organ culture on a Transwell-COL membrane for 17 days in α-MEM medium supplemented with 10% FBS. Follicle formation is assisted by the addition of an inhibitor for estrogen receptors using ICI182,780 at day 5 to 11 after culture to facilitate oocyte cyst breakdown. In step 2, secondary follicles isolated after 17 days of culture are cultured on the membrane with 5% FBS, 0.1 IU/ml follicle stimulating hormone (FSH), and 2% PVP supplemented α-MEM. In the course of culture, the follicles are treated with collagenase on day 20 after culture to remove the theca cell layers and basement membrane, both of which suppress in vitro follicle growth, and further cultured to day 31, after culture followed by IVMFC and embryo transfer to produce offspring. (A) Illustration of the 2-step culture. (B)–(H) Micro photos of cultures; PGCs ovary before culture (B), PGCs ovary after 17 days of culture (C), secondary follicles after 20 days of culture (D), in vitro follicle growth at day 26 (E) and day 30 (F) after culture, respectively, cumulus cell-oocyte complexes (G) collected from in vitro grown follicles and matured oocytes (H) produced after IVM. Each bar represents 100 µm.