Assesment of spermatogenesis on differently designed three dimentional mouse epididymal white adipose tissue - testis co-culture

Gonadotoxic treatments in childhood cancer patients damage the testicular germ cells and microenvironment, causing 46% of the patients to become infertile. Air-liquid interphase (ALI) and U-plate (UP) techniques are three-dimensional culture platforms and can support in vitro spermatogenesis and production functional sperm. The epididymal white adipose tissue (EWAT) produces adipokines required for spermatogenesis and spermatogenesis is inhibited when it is removed. Epididymal white adipose tissue can preserve the spermatogonial stem cell (SSC) pool in number and function, and support spermatogenesis in testicular coculture with three-dimensional UP and ALI techniques in vitro. This thesis evaluated the in vitro spermatogenesis histomorphometrically and immunohistochemically from week 1 to 6, in neonatal testicular cocultures treated with syngenic C57BL/6 mouse adult EWAT by using ALI and UP techniques. The ALI platform with and without EWAT increased the seminiferous tubular epithelial area up to week 3 (p=0,0001), while no change was observed with UP culture and coculture platforms from week 1 to 6. Tubular lumen area was higher in EWAT applied ALI coculture platform on weeks 1 (p<0,0001), 3 (p=0,0009), 4 (p=0,0001) and 6 (p<0,0001) compared to UP with EWAT. The seminiferous tubule luminal area was higher with the ALI technique on weeks 3 and 4 compared to UP (p<0,0001). The ALI technique increased the number of ID4(+) SSCs on weeks 3 and 6 (p<0,0001) when compared to UP. The protective effect of the EWAT on the ID4(+) SSC pool was observed from week 3 in the ALI coculture platform. The number of c-Kit(+) differentiating spermatogonia increased from weeks 1 to 6 with the ALI technique when compared to UP, and the positive effect of EWAT was observed in ALI coculture platform on week 6 and the UP platform on week 4. While EWAT did not change the number of SCP3(+) spermatocytes in ALI coculture platforms from week 1 to 6 when compared control, EWAT decreased the number of SCP3(+) spermatocytes on week 3 in UP coculture platform compared to control (p= 0,0348). EWAT increased the number of ACR(+) spermatids in ALI coculture platform on weeks 3 (p=0,0006) and 4 (p=0,0203); and increased the number of ACR(+) spermatids in UP coculture platforms compared to control. UP technique decreased the number of ACR(+) spermatids on weeks 3, 4 and 6 compared to the ALI (p<0,0001). The overall data revealed the positive effect of EWAT on the SSC pool and in vitro spermatogenesis by ALI technique, which could be a good model for three-dimensional testicular organ cultures in terms of providing sperm production for childhood cancer patients.