Reconstitution in vitro of the Entire Cycle of the Mouse Female Germ Line

Hikabe et al., 2016. Hayashi Lab.

To reconstitute the oogenesis process, they divided the cultures into three groups
(1) in vitro differentiation (IVDi)
(2) in vitro growth (IVG)
(3) un vitro maturation (IVM)

Female ESC line, BVSCH18 (129+Ter/svj(agouti)xC57BL/6) harboring Blimp1-mVenus (BV) and Stella-ECFP (SC) constructs differentiated to PGCLCs. In IVDi medium, these PGCLCs aggregated with E12.5 female gonadal somatic cells of albino ICR strain to form reconstitute ovaries (rOvaries). The oOvaries was changed to Transwell-COL. And at d4, the medium was replaced with StemPro34-based medium. To prevent multiple oocytes follicle formation , oestrogen inhibitor ICI182780 was added to the culture, so each follicle only possessed one oocyte.  During the IVDi culture, BV (marker for early PGCs) was detected at d3 and appeared to be weaker after 1 week. At 2 weeks, BV disappeared and SC (marker for oocytes and PGCs) were prominent in rOvaries. At 3 weeks, SC-positive primary oocytes were abundant in rOvaries. Cluster of PGCLCs were formed by d5 and follicles were formed around d11 of culture. Foxl2 (marker for grandulosa cells) was present in cells surrounding the oocytes at d21. The IVDi culture yielded a large number of secondary follicle-like structure (2FLs) at 3 weeks. These results demonstrated that a number of primary oocytes were induced from ESCs under these culture conditions.

Oocyte growth accompanied by follicular growth is regulated by gonadotropins. FSH plays a central role in proliferation and maturation of granuolsa cells. IVG medium containing FSH proliferation of granulosa cells layer was limited in 2FLs located at the edge of the rOvaries, indicating that 2Fls in the center portion lack signaling space for cell growth. So individual 2FLs were separated from rOvaries. At 11d, primary oocytes in 2FLs grew to germinal vesicle oocytes. On average, 55 fully grown oocytes were collected from 1 rOvary. When transferring them under the IVM culture, 29% of the germinal vesicle oocytes extruded a 1st polar body. The contamination rate of endogenous oocytes was low. The diameters of SC-positive MII oocytes generated in vitro was comparable to those from in vivo. 78% of the MII oocytes generated in vitro has correct number of chromosomes. These results demonstrated that the MII oocytes were successfully produced from pluripotent stem cells in vitro.

To evaluate oogenesis in vitro, the global transcriptome was analyzed. The data showed that oogenesis in vitro recapitulated the differentiation process in vivo with similar dynamics gene expressions. Repetitive elements were repressed in oocytes and the long terminal repeat (LTR) transposons were upregulated both in vitro and in vivo, demonstrating that the trascripts of transposons enriched in growing oocytes are tightly related to oocyte-specific transcriptional regulation. However, there were some differential gene expression between oogenesis in vitro and in vivo. The DEGs upregulated in MII oocytes in vitro were downregulated from primary oocytes to MII oocytes in vivo. In the other hand, DEGs downregulated in MII oocytes in vitro were upregulated from primary oocytes to MII oocytes in vivo. These results suggested that oocyte growth during IVG and IVM culture was compromised in a subset, or perhaps all, of the oocytes.

To test if the in vitro generated MII oocytes could be used for offspring production, they did in vitro fertilization (IVF) with wild-type sperm of albino ICR strain. They collected viable and colored-eye pups. These pups grew normally without any developmental complications and were fertile in both females and males. Their epigenetics of the imprinted genes were comparable to those from in vivo. These results demonstrated that BVSC18 ESC-derived oocytes were capable of producing viable and fertile offspring. The only down point was that the full-term development of  embryos from in vitro generated oocytes was 3.5% while it was 61.7% using in vivo generated oocytes.

To  verify the success of in vitro oogenesis using this system, they tested 5 other cell lines using the same protocol. The 5 lines included BVSCH14 (another ESC from the same mouse strain BVSCH), 2 iPSC lines from 10-week old tail tip fibroblasts (TTFs), 2 iPSC lines from MEFs.  All of these lines gave rise to fully potent oocytes. These oocytes produced colored-eye pups. These pups grew normally, expressed retroviral genes as expected, and were fertiles in both males and females.

Finally, to demonstrate that these in vitro derived oogenesis could complete the entire cycle of female germ line. They nourished the blastocysts formed by BVSCH18-derived oocytes on embryonic fibroblasts in medium containing CHI99021, PD0325901, and LIF. 72.5% of these blastocysts gave rise to ESCs, called regenerated ESCs (rESCs) to distinguish them from in vivo ESCs. The BCSV reporter genes and  sex chromosomes were segregated to each ESC in the Medelian manner. Then, they extracted these ESCs to while-type blastocysts, they observed that these ESCs contributed to multiple tissues including PGCs of the chimera embryos. The rESC-derived MII oocytes underwent second round of meiosis from the initial ESCs. In conclusion, they successfully found a method to recapulate the entire female germline development in vitro using this system.

[Since they had to use embryonic gonadal somatic cells to form aggregates with the derived in vitro derived PGCs, they can one day substitute these somatic cells with gonadal cell-like generated from pluripotent stem cells.]


[Combined Bisulfite Restriction Analysis (or COBRA) is a molecular biology technique that allows for the sensitive quantification of DNA methylation levels at a specific genomic locus on a DNA sequence in a small sample of genomic DNA.]