In vitro gametogenesis

Question 1

What is IVG?

Answer 1

In vitro gametogenesis

Question 2

How does IVG work? (2)

Answer 2

• Recreates germ cell development in vitro using induced pluripotent stem cells (iPSCs) from somatic cells
• Would theoretically allow people to reproduce who are currently unable to make eggs/sperm

Question 3

What are the potential benefits of IVG? (3)

Answer 3

• Understanding germ cell development
• Preserving endangered species
• New possibilities in medicine for fertility treatments and new assisted reproductive technologies (ART)

Question 4

How did Hikabe et al 2016 make mature mouse oocytes from skin cells? (4)

Answer 4

• Made induced stem cells from mouse tail fibroblasts and cultured them to become primordial germ cell-like cells
• Cultured PGC-like cells with ovarian cells from a different mouse foetus to make a reconstituted ovary in vitro
• Developed into a secondary then tertiary follicle and eventually the PGC-like cells matured into oocytes
• The mature oocytes were fertilised and implanted into a surrogate mother and resulted in healthy offspring

Question 5

What was the drawback of the Hikabe et al 2016 technique? (2)

Answer 5

• Making the reconstituted ovary required cells from another mouse foetus (essentially using a foetus to make a foetus)
• Next aim is to make these cells synthetically too

Question 6

What were the main outcomes of Hikabe et al 2016? (3)

Answer 6

• A culture system for generating fertilisation-competent mouse oocytes from fibroblast-derived pluripotent stem cells in about 30 days
• Fully in vitro method where PGC-like cells are co-cultured with female gonadal somatic cells, hormones, inhibitors administered at key stages to simulate follicular development
• Only 3.5% oocytes went on to make pups (compared to 60% when cells were implanted into surrogate mice to develop mature oocytes) so not ready for human application

Question 7

What happened to imprinted loci in Hikabe et al 2016? (3)

Answer 7

• Maternal pattern of DNA methylation in the differentially methylated region (DMR) of H19 and Igf2r was almost complete
• Imprints were sufficiently maintained to generate fertile mice (both male and female)
• No evidence of premature death

Question 8

How far has artificial gamete research come in humans? (3)

Answer 8

• 2014: immature human eggs grown from stem cells in the lab
• 2018: complete in vitro development of human oocytes from primordial follicles taken from adult women
• To date no human live birth has resulted from artificial gametes

Question 9

What was achieved by Yamashiro et al 2018? (4)

Answer 9

• Human PGC-like cells differentiated into oogonia-like cells during a long-term in vitro culture (4 months) in xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells
• The hPGCLC-derived oogonia displayed hallmarks of epigenetic reprogramming such as genome-wide DNA demethylation, imprint erasure and partial demethylation, REactivation of the inactive X chromosome
• Concluded that they established the germline competence of human pluripotent stem cells and provided a critical step towards human IVG
• Didn’t prove that the gametes would work because didn’t attempt to fertilise them (ethical constraints in humans)

Question 10

What was achieved by Gyobu-Motani 2023?

Answer 10

Used mouse embryos for the embryonic cells to make a reconstituted ovary to support development of monkey induced PGC-like cells

Question 11

Why is there less focus on spermatogonial stem cells? (3)

Answer 11

• Maybe because there isn’t a shortage of sperm
• There has been a study which shows in vitro derivation of mouse germline stem cell-like cells (GSCLCs) from embryonic stem cells
• GSCLCs colonised adult testes (need an animal for this still) and contribute to spermatogenesis and fertile offspring but show aberrant DNA methylation

Question 12

What are the ethical considerations associated with human IVG? (3)

Answer 12

• Need to asses the safety and ‘normality’ of offspring born using IVG gametes ideally using primate models before human applications
• Evaluation of the genetic and epigenetic makeup of stem cells used and the resulting gametes
• Societal discussions once the technology is established to be safe to address the ethical implications considering the potential impact on our understanding of human origins and the continuation of life