The Study Of SSCs Regulation-associated Cytokines Gene And Marker Antigen Expression In Immature Porcine Testis

Spermatogonial stem cells, as progenitor cells in mammalian male spermatogenesis system, are of utmost importance in study on male reproductive development, inducing pluripotent stem cell and transgenic animal production.In rodents and primates, owing to the uncover of antigens and intensive investigation of relevant cytokine, knowledge about identifying and in-vitro culturing spermatogonial stem cells is increased greatly, but associated study of livestock SSCs is unsatisfactory.In this experiment, porcine testis from different age were analyzed for CSF1r and CSF1expression by immunofluorescence.Next GDNF, EGF, bFGF, CSF1, LIF mRNA derived from porcine tistis tissue before sexual maturation was examined by semi-quantitative RT-PCR.Finally, in order to explore the morphological development dynamic of porcine testis, we employed H&E staining and PGP9.5immunofluorescence. Results are as follows:1Localization of CSF1and CSF1r in porcine testisTissues collected from4-day-old and3-month-old porcine testis were paraffin-sectioned and stained by immunofluorescence, the results showed that CSF1r antigen was expressed in leydig cells but not in peritubular myoid cells, and the antigen was also fund expressing in gonocytes and spermatogonia within the seminiferous tubes.At day4postnatal, CSF1r positive gonocytes was distributed in the centre of seminiferous tubes, single-cell, big cell body, high nuclear-cytoplasmic ratio’while3month after birth, a large number of positive spermatogonias attached to basement membrane of seminiferous tubes, showing a single-cell or short-chain distribution.Using double-immunofluorescence between PLZF and CSFlr, the results indicated that PLZF was expressed in part of CSF1r positive cells,and co-expression cell was observed single-cell distribution.As described above, CSF1r expressed in gonocytes and leydig cells, simultaneously a part of positive cells co-expressed PLZF.Tissues obtained from14-day-old,3-month-old,7-month-old porcine testis were stained by immunofluorescence.The results denoted:CSF1was expressed in all samples we collected, CSF1positive cells were testis leydig cells and peritubular myoid cells.2Expression analysis of SSCs regulation-associated cytokines encoding gene in porcine testis before sexual maturationExpression of GDNF, EGF, bFGF,CSF1and LIF was analyzed in4-day-old,14-day-old,1-month-old,2-month-old porcine testis tissue by RT-PCR.The results suggested that the genes we detected existed in porcine testis from different age.The comparison analysis denoted that the mRNA level of GDNF and bFGF took a declined tendency, while the level of LIF took a increased tendency and the level of EGF as well as CSF1had no significant difference.The above results suggested that the SSCs regulation-associated cytokines encoding genes existed in testis from different age, but the difference of expression tendency was found among genes.3Dynamic change of porcine testis morphological development.The tissues derived from4-day-old,7-day-old,14-day-old,1-month-old,2-month-old,3-month-old,7-month-old and old-sterile porcine testis were paraffin-sectioned, next, stained by H&E and PGP9.5immunofluorescence, the results were as follows:(1) Various cell morphology was found in interstitum region at4-day-old testis and a quite number of mesenchymal cells(precursor cells of leydig) were distributed around the seminiferous tubes.From day4to1month postnatal, the tubes were mainly composed of gonocytes or spermatogonia, while the primary spermatocytes was rare.From2month to3month postnatal, the number of primary spermatocytes was significantly increased and the secondry spermatocytes had always been found.When7month postnatal, a large number of mature spermatids appeared in tubes.The number of primary spermatocytes in some tubes was extremely little.(2) The relative area (parenchma area/figure area) calculation of each parenchyma type and the diameter measurement of seminiferous tube was carried by ImageJ software.The results show that before3month postnatal, both the relative area of seminiferous tube and blood vessel took on a declined tendency, although the interstitum relative area still occupied dominance and the diameter of the seminiferous tubes did not increase significantly.After2month postnatal, interstitum area decreased significantly and the diameter of tubes took on a rapid increase tendency.At7month postnatal, Compared with3month postnatal, both the relative area of seminiferous tubes and interstitum had not significant difference, but the relative area of blood vessel and diameter of tube took on a increase tendency.(3) The results of PGP9.5immunofluorescence show that before2month old, the PGP9.5positive cells, single-cell or paired, consistent and strong fluorescence intensity, were distributed close to the basement membrane.The number of PGP9.5positive cell had no significant change before1month old.Compared with1月龄nth old, the number of positive cell increased significantly in2month old, meanwhile, strong positive colony-like cells could be found at this age.At3month old, the PGP9.5positive colony-like cells not only distributed along the membrane basement but also in the centre of tube, and the fluorescence intensity gradually fainted from basement membrane to centre of tube.Compared with2month old, the number of positive cells in3month old increased significantly.The number and distribution of PGP9.5positive cells at7month old didn’t change significantly compared with3month old, reversely, the number of positive at old-sterlie reduced significantly compared with7month old.Above results suggested that the number of PGP9.5positive cells growed sustainedly from newborn to3month old, and the fluorescence intensity as well as location of positive cells were different at distinct age.In conclusion, the development of various testicular cell was dyssynchrony from newborn to sexual maturation.Before1month old, the proliferation of germ cell was slow so the type of the germ cell was monadical, but the somatic cells occurred proliferation as well as maturation in tubes.After1month old, the diameter of tube gradually bigger, simultaneously, a rapid increasement in number of germ cells and their layers were found in tubes.PGP9.5was expressed in gonocytes and subsequent spermatogonia, and the expression intensity showed heterogeneity with the proliferation of spermatogonia, meanwhile, the fluorescence intensity gradually fainted from basement membrane to the centre of tubes.