Study Of Spermatogonia Differentiation Mechanism And C-kit

IntroductionSpermatogenesis in mammalian is a complicated and a precise process. Many regulators participate in generation, proliferation and differentiation of germ cells. Spermatogonial stem cells are germ cells with stem cell function, and it is source head of spermatogenesis. Understanding the differentiation mechanism in spermatogonia will be helpful in revealing spermatogenesis origination, utilization and reforming of SSCs. It will promote the development in medical science, biology and animal husbandryAlready confirmed:Germ cells experience a series differentiation in spermatogenesis, including A0 spermatogonia, A1-A4 spermatogonia, Intermediate (In), B spermatogonia, primary spermatocytes, secondary spermatocyte, spermatid and spermatozoa. In all kinds of subtype of cells only A0 have the stem cell function, and it are spermatogonial stem cells. These cells are source head of spermatogenesis. The cell is kind of undifferentiated spermatogonia, and it can differentiate type A1-A4 spermatogonia and other germ cells subsequently. Obviously, it is a very important period when germ cells differentiate from A0 to A1. Cryptorchidism, vitamin A deficiency, Sertoli cell intoxication and radiation damage can cause a blockage of this differentiation step. As yet there is no clue how the differentiation of undifferentiated spermatogonia is regulated and how this process becomes arrested in these situations.C-kit is the transmembrane tyrosine kinase receptor for stem cell factor (SCF, product of steel locus), encoded by proto-oncogene White spotting locus. C-kit and its ligand are required in proliferation and survival of germ cells. Spermatogonia can not differentiate without c-kit presence. Some study show, undifferentiated spermatogonia doesn't express c-kit, and differentiating spermatogonia has the expression of c-kit. The association of differentiation process in spermat ogonia and c-kit become hot in research field. Investigation and utilization of the c-kit expression feature in development of spermatogonia will be very important to illumination the differentiation of spermatogonia.Spermatogonia stem cells have the property of dissymmetry division. It means the cells can differentiate downward and self-renew. Plzf is noticeable in the studies of maintain spermatogonia stem cells self-renew. It was confirmed that male plzf deficient mouse appear gradualness infertility after birth. It shows spermatogenic cells exhaustion. The capability of self-renew in spermatogonia stem cells was absent. As a inhibit transcription factor, plzf will provide us a new angle to research spermatogonia stem cells development process by investigating its expression feature in develope testes and contrasting them with c-kit which represent cell differentiation.In this study, c-kit expression level in nucleic acid and protein were investigated in different developmental stage testes of rat, and approaching the associativity between c-kit and spermatogonia. Based on this, we sorted A1-A4 subtype spermatogonia by utilization of c-kit, and detected the proportion of spermatogonia in differentiating. Then, the expression of plzf in these testes was detected, which has important relationship with spermatogonia renew, and contrasted with c-kit expression feature, in order to reveal the destiny choosing characteristic in undifferentiated spermatogonia go to differentiation or renew, and investigated its mechanism.I. Expression of c-kit in developmental testesSD rats of 1 day after birth, 9 days after birth and 30 days after birth were chosen for these experiments. Immunohistochemistry and western-blot were performed to detect c-kit protein expression, semiquantitative RT-PCR to detect c-kit mRNA expression, and flow cytometry was carried out in 9 days testes to investigate c-kit positive cells proportion. Here are the results:1. c-kit protein and mRNA were expression persistently in rat testes, but there are some variance in expression location and level. There is a higher expression level in 9 days testes, and mRNA together protein changing conformity.2. Together with the phase feature reported in reference about testes growth, this detection result suggests that c-kit express higher in 9 days testes have correlation with much differentiation of spermatogonia in this stage.3. Spermatogonia and Sertoli cells are the only cell types in 9 days testes in light microscope. Detected these cells by flow cytometry, together with cell type in this stage, we can think: some spermatogonia in this stage occurring differentiation, and there are (3.16±0.84)% cells in testes express c-kit, and it is differentiating A1-A4 spermatogonia.This result provides evidence for isolation these cells further. Above results suggest:Expression of c-kit has closely relationship with differentiation of spermatogonia. When the differentiation process increase, expression of c-kit become higher. C-kit is a marker for differentiation of spermatogonia, and its expression level can represent the variance of differentiation in spermatogonia.II. Isolation of A1-A4 subtype spermatogoniaFive SD rats 9 days after birth were killed and their testes were removed. After decapsulation, the testes were dispersed into cell suspension with a two-step enzymatic digestion. Spermatogonia were separated the with percoll discontinuous density gradient centrifugation. Type A1-A4 spermatogonia were isolated from the purified spermatogonia using c-kit as the marker with fluorescence-activated cell sorting (FACS). Here are results:1. Differentiating spermatogonia appears in rat testes of 9 days after birth. And they are A1-A4 subtype spermatogonia, and the percentage of these cells in test is about (3.16±0.84) %.2. Cells separated after percoll discontinuous density gradient centrifugation, proportion of A1-A4 subtype cells increased to (18.65±1.69) %. It shows a conspicuous purification effect.3. A1-A4 subtype cells were isolated successfully by FACS with c-kit as the marker, and these isolated cells have good cytoactive and eumorphism.Above results suggest:Using c-kit as marker, FACS combining discontinuous density gradient centrifugation can enrich type A1-A4 spermatogonia from 9 days old rats'testes sufficiently. They provide good experience for other spermatogonia subtype cells isolation strategy in the future. III. Regulators in proliferation and differentiation of spermatogonia SD rats of 1 day after birth, 9 days after birth and 30 days after birth were chosen for these experiments. Western-blot and semiquantitative RT-PCR were performed to detect plzf expression, and the results were contrast to the c-kits'. Here are the results:1. Plzf was expressed in developmental rat testes. There is a higher expression level in 1 day testes, and the level decreases accompany the age raise.2. Contrasted with the expression of c-kit, plzf is decrease and c-kit is increase accompany the age raise before 9 days old. And both decrease after this age.3. Expression level of plzf decrease apparently from 1 day old to 9 day old and c-kit expression increase simultaneously. In this period, the development process in spermatogonia is the proliferation decrease but differentiation increase. That indicates plzf play an important role in maintaining SSCs self-renew and inhibiting differentiation.Above results suggest:Plzf correlate with maintaining SSCs self-renew closely, and that is proliferation of SSCs. C-kit correlate with differentiation of SSCs closely. Both of them are important for spermatogonia development mode chosen. Expression level of c-kit and plzf represent the directions of spermatogonia development, and it provides suitable targets and experience for regulation of spermatogonia development in vivo and intervention of spermatogenesis by man-made.Conclusion1. C-kit is important for differentiation of spermatogonia, and it is the marker of spermatogonia differentiation. Its expression level can reflect the level of spermatogonia differentiation. A1-A4 subtype cells can be separated with c-kit as the marker.2. Plzf is important for spermatogonia self-renew, and its expression level is opposite toward c-kit in some period. Plzf can reflect the level of spermatogonia self-renew.3. This study provides experimental evidence for regulation of spermatogonia development in future.