In vitro Production of Functional Sperm in Cultured Neonatal Mouse Testes

Takya Sato et al., 2011. Takehiko Ogawa Lab.

"Neonatal mouse testes [containing] gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media."  Why serum free? Spermatogenesis can be maintained in the gas-liquid interphase for up to 2 months. Healthy and reproductively competent offspring are born from microinsemination ICSI using these in-vitro-grown spermatids and sperm even when using using thawed cryopreserved neonatal mouse testis.

Sertoli cells are like feeder cells for spermatogonial stem cells.

In the gas-liquid interphase organ culture method, 1-3 mm testis tissue fragment is placed on a piece of agar half-soaked in medium. Gsg2-GFP and Acr-GFP transgenic mouse lines were used to monitor the expression of GFP specifically only in meiosis and  haploid cells via Gsg2 and Acr promoters. In vitro spermatogenesis was proceeded at 34C in aMEM + 10% FBS. FBS is neccessary/indispensible for spermatogenesis in organ culture, specifically round spermatids and haploid cells were observed. However, FBS might contains repressing factors for Gsg2-GFP and Acr-GFP expression. Therefore, they replaced FBS with KSR, which allowed expression of Gsg2-GFP and Acr-GFP. KRS induced stronger and more prolong GFP signal than did FBS. To ensure GFP signal reflects meiosis, meitotic factors SYCP1 and SYCP3 were tested. SYCP1 is colocalized with Acr-GFP during pachytene stage of spermatocytes, but SYCP1 is only colocalized with outer side of Gsg2-GFP positive cells which are finishing meiosis. In somatic cells in this 2.5 dpp mouse testis tissue, Sertoli cells and myoid cells also expressed androgen receptors (AR), a mediator of testosterone effects and essential for spermatogenesi. This result demonstrate that spermatogenesis proceeded in this organ culture condition.

They found spermatids in 6 out of 7 samples (23-50 days) and sperm in 5 out of 11 samples (27-45 days. The sperm formation (1N) was also tested by flow cytometry gating 1N, 2N and 4N. These sperm  were next tested for fertilization via microinsemination. Round spermatids from 3.5 dpp testis cultured for 23 days was used for round spermatid injection technique. Sperm from 2.5 dpp testis cultured for 42 days were used for intracytoplasmic sperm injection (ICSI). Using 23 oocytes for ROSI, 7 live offspring was produced; using 35 oocytes for ICSI, 5 live offspring were produced. 4 out of 12 carried GFP gene because of heterozygous Gsg2-GFP cultured testis. All of these progeny mice were fertile. Cryoreserved testis tissue (4-25 days) grown in the same in-vitro gas-liquid interphase condition as above, 1 out of 5 samples produced results the same as fresh testis tissue cultured in this condition.

It was demonstrated that FBS does not contain factors that inhibit spermatogenesis. They found that lipid-rich bovine serum albumin AlbuMAX was critical in KRS to allow normal spermatogenesis. Addition of only AlbuMAX instead of KRS yielded the same results as observed when using KRS, and using FBS with AlbuMAX also yielded normal spermatogenesis.