Honaramooz, Ali2023-08-1620232023-082023-08-16August 202https://hdl.handle.net/10388/14885The studies presented in this thesis were designed to find optimal conditions to produce fertile haploid male germ cells using the neonatal piglet model. The first study aimed to optimize the physical attributes of testicular tissue fragments for long-term in vitro maintenance. Our finding showed that the integrity of small (~2 mg) intact testicular tissue fragments cultured in KSR could be maintained for up to four weeks. However, the number of germ cells in the culture decreased with every passing week. Therefore, we designed the second study to check the effects of different growth factors (GDNF, bFGF, SCF, and EGF; alone or in combination) on in vitro maintenance of tissue integrity and germ cell number over the period of culture and potential induction of in vitro spermatogenesis (IVS). We found that GDNF, bFGF, and SCF were superior at maintaining the number of gonocytes over the culture period. Furthermore, we observed that the Sertoli cells matured over the culture period in the latter-mentioned groups. Interestingly, we observed differentiating germ cells (differentiating spermatogonia and spermatocytes) in all groups. Overall, GDNF, bFGF, and SCF had a higher percentage of seminiferous with spermatocytes. However, we did not observe any haploid male germ cells. Therefore, we designed our third study to optimize the culture conditions further to induce complete IVS. Our third study consisted of two experiments. In the first experiment, we checked the effects of retinoic acid (RA). In the second experiment, we checked the effects of antioxidants (DL-ATA and L-GSH) and testosterone on complete induction of IVS. We observed that adding RA to the culture media increased the number of early germ cells (gonocytes/spermatogonial stem cells) over the culture period. Furthermore, Sertoli and germ cells' maturation was observed in all RA supplemented (50 μM, 100 μM, or 200 μM). Overall, the group supplemented with 100 μM had the highest percentage of seminiferous tubules with spermatocytes. Moreover, haploid male germ cells (confirmed with H&E and ACR staining) were observed in a few seminiferous tubules. The aim of the second experiment of the third study was to increase the number of haploid male germ cells. We observed that with RA, adding antioxidants and testosterone dramatically increased the number of haploid male germ cells. The group supplemented with antioxidants and testosterone in combination had ~8% of fertile (confirmed with ROSI) haploid male germ cells. At week 22 of culture, we also observed the presence of sperm. Equally important, our developed IVS also supported the maturation of cryopreserved testicular tissues. Thus, these results have important implications for overcoming the infertility issues related to childhood cancer therapy in the future.application/pdfenSpermatogonial stem cellsIn vitro spermatogenesismale infertilitytesticular tissue culturegrowth factorsporcine testicular tissuesThesis2023-08-16