RT reactions were performed using the High Capacity RNA to cDNA kit. Gene specific primers were designed using Primer Express software. ABI Prism 7900HT Sequence Detec tion System was used for qRT PCR analysis. Power SYBR Green PCR Master Mix was used for PCR quantification. Actin from C. sativus was used as an endogenous control and for normalization. Each qRT experiment was repeated three times. PCR products from each gene were quantified with reference to corre sponding standard curves. Background Spermatogenesis, a complex yet highly regulated develop mental process of male germ cells, consists of three stages the proliferation of spermatogonia, the meiosis of spermatocytes and the morphogenesis of spermatids.
The formation of the terminally differentiated germ cells, the spermatozoa, requires a series of important processes that are unique to spermatogenic cells, including nuclear condensation, mitochondrial rearrangement, histone selleckchem ON-01910 re placement by transition proteins and protamin, the shed ding of residual bodies, and the formation of acrosomes. It is not surprising that a large number of testis specific genes are required for these events. In mice and rats, several high throughput gene expression datasets have revealed that a large number of genes are expressed to fuel spermatogenesis. It is also estimated that about 4% of protein coding genes in the mouse genome are specifically expressed in the mouse testis. Ubiquitination of protein is an indispensable post translational modification that serves as a component of the protein quality control system.
It is also involved in diverse biological processes such as signal transduction, DNA repair, transcriptional regulation in a protein degradation independent way. In principle, ubiquiti nation selleck p53 inhibitors is a process containing three ubiquitin transfer ring reactions catalyzed by three corresponding enzymes. At first, the 8. 5 kDa ubiquitin polypeptide is activated by the ubiquitin activating enzyme in an ATP dependent way. Subsequently, this activated ubiqui tin is transferred to the ubiquitin conjugating enzyme via a thioester bond. In the end, the ubiquitin is transferred to a lysine residue of the substrate catalyzed by the ubiquitin protein ligase. Substrate specifi city is conferred by E3s, implying a much larger number of E3s in the genome than E1s and E2s. The mammalian genome encodes 1 2 E1s, 10 20 E2s and several hun dred E3s.
There are three typical E3 families includ ing the RING finger family, HECT domain family and the U box family. To our knowledge, approximate 29 E3s have been reported to be expressed in mammalian testes and most of them execute diverse functions at different stages of spermatogenesis. A dozen of E3s as well as associated complex play import ant roles in DNA double strands break and his tone modifications during early meiosis.