Epigenetic status and expression of SALL4 in normal and impaired testicular development

Abstract

Uloga gena SALL4, koji kontrolira razvoj spermatogonija, nije dovoljno istražena u razvoju sjemenika sisavaca, a u neplodnosti čovjeka je potpuno neistražena. Cilj disertacije bio je analizirati izražaj Sall4/SALL4 u razvoju sjemenika štakora in vivo i in vitro te u ljudskim sjemenicima s različitim dijagnozama neopstruktivne azoospermije (NOA) in vivo, kao i njegov metilacijski status u in vivo uzorcima. Pirosekvenciranjem je pokazano da je SALL4/Sall4 uvijek hipometiliran u sjemeniku. qRT-PCR-om je pokazan značajno veći izražaj Sall4 mRNA u fetalnom štakorskom sjemeniku nego u postnatalnom; a u ljudskim sjemenicima sa sindromom samo Sertolijevih stanica (SCOS) SALL4 mRNA je snižena u odnosu na hipospermatogenezu (HS) i zastoj u sazrijevanju (MA). Osim u spermatogonijama, poboljšanim IHC i IF protokolima je po prvi put pokazan SALL4 u XY tjelešcu primarnih spermatocita. Također, pokazan je pojačan izražaj SALL4 u pre-pahitenskim spermatocitama i citoplazmi intersticijskih stanica u neplodnom sjemeniku u odnosu na zdravi i HS sjemenik čovjeka. Nadalje, uspostavljen je novi 3D in vitro sustav za uzgoj tkiva sjemenika štakora, a primjena siRNA snizila je izražaj Sall4 na mRNA (za 42 %) i proteinskoj razini uzrokujući smanjeni rast i narušenu histologiju. Ovi rezultati ukazuju na SALL4 kao novi potencijalni biljeg u dijagnostici NOA te podrobnije rasvjetljavaju njegovu ulogu u procesu spermatogeneze.The role of the SALL4 gene which controls the development of spermatogonia, has been insufficiently investigated in the development of the mammalian testis, and is completely unexplored in human infertility. The doctoral thesis aimed to analyze the expression of Sall4/SALL4 in the rat testis development in vivo and in vitro as well as in the human testis with a different diagnosis of nonobstructive azoospermia (NOA) and its DNA methylation status. The pyrosequencing showed that SALL4/Sall4 is always hypomethylated in the testis. The qRT-PCR has shown significantly higher expression of the Sall4 mRNA in fetal rat testis than in postnatal; while SALL4 mRNA was lower in human testis with Sertoli cell only syndrome (SCOS) than in hypospermatogenesis (HS) and maturation arrest (MA). Except in spermatogonia, improved IHC and IF protocols showed the expression of the SALL4 protein in the XY body of primary spermatocytes of human testes. Also, increased expression of SALL4 in pre-pachytene spermatocytes and cytoplasm of interstitial cells in infertile compared to healthy human testis was demonstrated. Furthermore, an original 3D in vitro system for the cultivation of rat testis tissue was established, and the application of siRNA decreased Sall4 at the mRNA (by 42 %) and protein levels, causing reduced growth and impaired histology. These results indicate SALL4 as a new potential marker in the diagnosis of NOA and shed more light on its role in the process of spermatogenesis