Enrichment And In Vitro Culture Of The Ovine Spermatogonial Stem Cell

Spermatogonial stem cells (SSCs) can self-renew and produce large numbers of differentiating germ cells that become spermatozoa throughout postnatal life and transmit genetic information to the next generation. Testis germ cell transplantation is emerging as a novel reproductive technology with application in animal breeding systems, including the potential for use as1) an alternative to artificial insemination (AI),2) an alternative to cloning,3) as part of an approach to reducing generation intervals, or4) as an approach towards development of interspecies hybrids. The present study was designed to investigate the isolation, purification and long term culture conditions of sheep spermatogonial stem cells.In the first set of experiments, partial cDNA fragment of the ovine SSCs self-renewal, proliferation and differentiation related genes including PLZF, SCF, c-Kit, PGP9.5, GFRα1, VASA, GATA4were successfully sequenced. Result shown that SCF, c-Kit sequence perfectly match published ovine SCF and c-Kit sequence and other five ovine genes sequence didn't published in Gene Bank yet, but these genes had high homology to bovine related genes that published in pubmed.In the second set of experiments, testes from four distinct developmental stages were analysed. Scrotal circumference, testis weight and tubule diameter all increased with age. PLZF staining was confined to the nuclei of gonocytes in Stage1testis, whereas immunoreactivity was detected in the nuclei of spermatogonia, located adjacent to the basement membrane, in testes at Stage2or onwards (Stages3-4). At Stage1, PGP9.5-positive cells were randomly distributed in the central area of the tubule, as testis development progressed from gonocytes to spermatogonia. PGP9.5-positive cells migrated from a predominantly central to a basal position. At Stage1, PGP9.5-positive cells were randomly distributed in the central area of the tubule. But this stage, more then three aligned cells were observed from one tubule cross section. At stage2, about80%of the cross section at least had four PGP9.5-positive cells. All four stages usually two or three aligned PGP9.5-positive cells were observed. VASA-positive cells were distributed in the cytoplasm of the gonocyte at stage1and then were observed from spermatogonia adjacent to the basement membrane and spermatocytes located to the centre areas of the tubule. After that VASA-positive cells number increase rapidly at Stage3-4and immunoreactivity was detected from all spermatogonia, spermatocyte, elongated spermatids and spermatozoa. Vimentin staining in Sertoli cells was predominantly perinuclear. Germ cells were negative. Weak immunoreactivity was observed in a few interstitial cells and blood vessel walls in all samples tested. The intensity of the Sertoli cell staining appeared to become stronger as the age of testis increased.The coincidences seen in merged images from double staining of testis sections with PLZF and PGP9.5demonstrated that PLZF immunoreactivity was localised in the nuclei of gonocytes or spermatogonia, whereas immunostaining for PGP9.5was seen in the cytoplasm of these cells. At Stage1, most of the PGP9.5-positive cells also stained positive to PLZF antibody.However, at Stage2, some of the PGP9.5-positive cells were negative for PLZF expression. The number of cells that were double positive for PGP9.5and PLZF decreased progressively with age. It is known that VASA and vimentin antibodies will stain Sertoli cells and germ cells, respectively. In the present study, VASA staining was cytoplasmic, whereas vimentin staining was predominantly perinuclear. Merged images from double staining demonstrated that VASA-positive cell were negative for vimentin. Absolute number of VASA-positive cells in tubule increased significantly with testis maturity, but absolute number of vimentin-positive cells in tubule was relatively stable. Double staining of testis sections with PGP9.5and vimentin demonstrated that PGP9.5-povitive cell is not vimentin-positive. Vimentin-positive cell had irregular cell type and PGP9.5-positive cell was round or oval shape. Gonocytes were double positive for PLZF and VASA at stage1and some VASA-positive cells became PLZF-negative at stage2, after that VASA-positive cell increased dramatically at stage3-4, whereas PLZF and VASA double positive cell become very rare.In the Stage1testis, the tubules were composed mainly of Vimentin-positive Sertoli cells (93%), whereas gonocytes comprised6.4%of tubular cells.The number of PLZF-and PGP9.5-positive cells increased significantly from6.4%to12.2%and from8.3%to19.8%, respectively, in Stage2testis, whereas the percentage of Sertoli cells dropped from93%to64%. Because of the significant increase in the number of VASA-positive cells at Stages3and4(P<0.05), the proportion of PLZF-positives cells decreased froml2.2%to4.6%for Stage3and from12.2%to3.1%in Stage4. The percentage of Sertoli cells also decreased significantly with testis maturity, with37.3%and17.9%vimentin-positive cells in Stage3and Stage4testis, respectively (P <0.05).In the third set of experiments, on average,19.2±4.3×107cells were isolated from1g tissue from Stage1testis, whereas only8.0±0.6×107cells were recovered from Stage2testis. The percentages of different cell types in the initial isolation samples and supernatants collected from differential plating were analysed by immunostaining with PLZF, VASA and vimentin. In Stage1testis the percentages of PLZF-positive cells in2h differential plating (42.9±6.2%) and2+16h enrichment (75.1±4.0%) were significantly higher than those in the initial isolation (7.8±1.3%; P <0.05). Similar enrichment efficiency was obtained for Stage2testis. In smear staining, dark-and light-staining populations existed among the PLZF-positive cells and their numbers were recorded. The dark-stained cells were smaller in size compared with the light-stained cells. The dark stained cells were significantly enriched after2h differential plating in Stage1and Stage2testes, but the numbers did not differ significantly between2h and2+16h differential plating. During the process of enrichment for both stages, the proportion of VASA-positive cells increased significantly, whereas the number of vimentin-positive cells decreased significantly (P<0.05).In the fourth set of experiments, ovine SSCs were cultured on type Ⅰand type II feeder with DMEM/F12+10%FBS media. After1-2d of culture, SSCs started attaching to the flask and began to proliferate during next3-4d. SSCs colonies become visible after7days culture. Spermatogonia were present in the form of small clusters and, later on, colonies. Small clones comprised of pairs (Apr) or chains (Aal) of A spermatogonia (3-64cells), spermatogonial clones aggregated into clumps of spermatogonia in which no cytoplasmatic bridges could be discerned and cell proliferation appeared plocoid. The colony numbers from the type I feeder group were significantly higher than of the type II feeder group. At day10of culture, the size of spermatogonial colonies increased and became round bird nest-like shape, with tight compaction in the central area.After14d cultures, the formed colonies showed strong PLZF-and VASA-positive, but feeder cells were negative to PLZF and VASA staining. In the colonies with larger size, more VASA-positive cells were observed than PLZF-positive cells. The individual cells were prepared from mechanically isolated colonies and dissociated with trypsin. The smears prepared with these cells were stained with different antibodies to determine the cell types. In the type I feeder group, there was about30%cells stained positive for PLZF, but the cells from the type II feeder group only had10%PLZF-positive cells. Whereas the majority of the cells were VASA-positive, indicating that some SSCs have started differentiating in the current culture condition.In order to fabricate a long-term culture system, we cultured the isolated cells with DMEM/F12+B27+GDNF+LIF+β FGF+1%FBS on the0.2%gelatin coated flask. Chains of spermatogonia (4-8cells) were observed after3days culture and the numbers of the spermatogonia chains increase dramatically after5days, and appeared32-cells chains, but most of cells did not attach the flask. After passaged SSC colonies, most of the cells began to attach and number of the SSCs colonies became less and less duting the passages. Results from RT-PCR showed that the expression of PLZF, GFRal and PGP9.5were detected in the samples collected at two and four weeks. The expression level of c-Kit (a marker for the differentiated spermatogonia) and VASA (a marker for germ cells) were lower in the samples collected at two weeks and not detected in the samples collected at4weeks.