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Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats.

ABSTRACT: The development of germ cells has not been entirely documented in the cat especially the transition phase of the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes were divided into 3 groups according to donor age (I, < 4 months; II, 4-6 months; and III, > 6 months). In Exp. 1, we studied testicular development by histology, transmission electron microscopy and immunohistochemistry. In Exp. 2, we determined the expression of GFR?-1, DDX-4 and c-kit and performed flow cytometry. The SSCs isolated from groups II and III were characterized by RT-PCR and TEM (Exp. 3). Chronological changes in the G/SSC transition were demonstrated. The size, morphology and ultrastructure of SSCs were distinguishable from those of gonocytes. The results demonstrated that group II contained the highest numbers of SSCs per seminiferous cord/tubule (17.66 ± 2.20%) and GFR?-1(+) cells (14.89 ± 5.66%) compared with the other groups. The findings coincided with an increased efficiency of SSC derivation in group II compared with group III (74.33 ± 2.64% vs. 23.33 ± 2.23%). The colonies expressed mRNA for GFRA1, ZBTB16, RET and POU5F1. Our study found that the G/SSC transition occurs at 4-6 months of age. This period is useful for isolation and improves the establishment efficiency of cat SSCs in vitro.

SUBMITTER: Tiptanavattana N

PROVIDER: S-EPMC4685225 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats.

Tiptanavattana Narong N Radtanakatikanon Araya A Hyttel Poul P Holm Hanne H Buranapraditkun Supranee S Setthawong Piyathip P Techakumphu Mongkol M Tharasanit Theerawat T

The Journal of reproduction and development 20150927 6

The development of germ cells has not been entirely documented in the cat especially the transition phase of the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes were divided into 3 groups according to donor age (I, < 4 months; II, 4-6 months; and III, > 6 months). In Exp. 1, we studied testicular development by histology, transmission electron micr ...[more]

PMID: 26411537

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Project description:BACKGROUND:Spermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment. METHODS:A mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n?=?10), MSCs (MSCT, n =?10), a combination of SSCs and MSCs (MS-SSCT, n?=?10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n?=?10). RESULTS:The best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P?<?0.001). TFI after MSi-SSCT (83.3?±?19.5%) was significantly higher compared to MS-SSCT (71.5?±?21.7%, P?=?0.036) but did not differ statistically compared to SSCT (78.2?±?12.5%). In contrast, TFI after MSCT (50.2?±?22.5%) was significantly lower compared to SSCT (P?<?0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8?±?8.0%) compared to SSCT (1.9?±?1.1%; P?<?0.001), MSCT (0.0?±?0.0%; P?<?0.001), and MS-SSCT (3.4?±?1.9%; P?<?0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs. CONCLUSION:Co-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven.

Project description:The spermatogenic lineage is established after birth when gonocytes migrate to the basement membrane of seminiferous tubules and give rise to spermatogonial stem cells (SSC). In adults, SSCs reside within the population of undifferentiated spermatogonia (A(undiff)) that expands clonally from single cells (A(single)) to form pairs (A(paired)) and chains of 4, 8 and 16 A(aligned) spermatogonia. Although stem cell activity is thought to reside in the population of A(single) spermatogonia, new research suggests that clone size alone does not define the stem cell pool. The mechanisms that regulate self-renewal and differentiation fate decisions are poorly understood due to limited availability of experimental tools that distinguish the products of those fate decisions. The pluripotency factor SALL4 (sal-like protein 4) is implicated in stem cell maintenance and patterning in many organs during embryonic development, but expression becomes restricted to the gonads after birth. We analyzed the expression of SALL4 in the mouse testis during the first weeks after birth and in adult seminiferous tubules. In newborn mice, the isoform SALL4B is expressed in quiescent gonocytes at postnatal day 0 (PND0) and SALL4A is upregulated at PND7 when gonocytes have colonized the basement membrane and given rise to spermatogonia. During steady-state spermatogenesis in adult testes, SALL4 expression overlapped substantially with PLZF and LIN28 in A(single), A(paired) and A(aligned) spermatogonia and therefore appears to be a marker of undifferentiated spermatogonia in mice. In contrast, co-expression of SALL4 with GFRα1 and cKIT identified distinct subpopulations of A(undiff) in all clone sizes that might provide clues about SSC regulation. Collectively, these results indicate that 1) SALL4 isoforms are differentially expressed at the initiation of spermatogenesis, 2) SALL4 is expressed in undifferentiated spermatogonia in adult testes and 3) SALL4 co-staining with GFRα1 and cKIT reveals distinct subpopulations of A(undiff) spermatogonia that merit further investigation.

Project description:BACKGROUND:Spermatogonial stem cell transplantation (SSCT) is a promising therapy in restoring the fertility of childhood cancer survivors. However, the low efficiency of SSCT is a significant concern. SSCT could be improved by co-transplanting transforming growth factor beta 1 (TGF?1)-induced mesenchymal stem cells (MSCs). In this study, we investigated the reproductive efficiency and safety of co-transplanting spermatogonial stem cells (SSCs) and TGF?1-induced MSCs. METHODS:A mouse model for long-term infertility was used to transplant SSCs (SSCT, n?=?10) and a combination of SSCs and TGF?1-treated MSCs (MSi-SSCT, n?=?10). Both transplanted groups and a fertile control group (n?=?7) were allowed to mate naturally to check the reproductive efficiency after transplantation. Furthermore, the testes from transplanted males and donor-derived male offspring were analyzed for the epigenetic markers DNA methyltransferase 3A (DNMT3A) and histone 4 lysine 5 acetylation (H4K5ac). RESULTS:The overall tubular fertility index (TFI) after SSCT (76?±?12) was similar to that after MSi-SSCT (73?±?14). However, the donor-derived TFI after MSi-SSCT (26?±?14) was higher compared to the one after SSCT (9?±?5; P?=?0.002), even after injecting half of the number of SSCs in MSi-SSCT. The litter sizes after SSCT (3.7?±?3.7) and MSi-SSCT (3.7?±?3.6) were similar but differed significantly with the control group (7.6?±?1.0; P?<?0.001). The number of GFP+ offspring per litter obtained after SSCT (1.6?±?0.5) and MSi-SSCT (2.0?±?1.0) was also similar. The expression of DNMT3A and H4K5ac in germ cells of transplanted males was found to be significantly reduced compared to the control group. However, in donor-derived offspring, DNMT3A and H4K5ac followed the normal pattern. CONCLUSION:Co-transplanting SSCs and TGF?1-treated MSCs results in reproductive efficiency as good as SSCT, even after transplanting half the number of SSCs. Although transplanted males showed lower expression of DNMT3A and H4K5ac in donor-derived germ cells, the expression was restored to normal levels in germ cells of donor-derived offspring. This procedure could become an efficient method to restore fertility in a clinical setup, but more studies are needed to ensure safety in the long term.

Dataset Information

Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats.

Publications

Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats.

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