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Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat.

ABSTRACT: The tumor suppressor protein p53 (TP53) has many functions in cell cycle regulation, apoptosis, and DNA damage repair and is also involved in spermatogenesis in the mouse. To evaluate the role of p53 in spermatogenesis in the rat, we characterized testis biology in adult males of a novel p53 knockout rat (SD-Tp53tm1sage ). p53 knockout rats exhibited variable levels of testicular atrophy, including significantly decreased testis weights, atrophic seminiferous tubules, decreased seminiferous tubule diameter, and elevated spermatocyte TUNEL labeling rates, indicating a dysfunction in spermatogenesis. Phosphorylated histone H2AX protein levels and distribution were similar in the non-atrophic seminiferous tubules of both genotypes, showing evidence of pre-synaptic DNA double-strand breaks in leptotene and zygotene spermatocytes, preceding cell death in p53 knockout rat testes. Quantification of the spermatogonial stem cell (SSC) proliferation rate with bromodeoxyuridine (BrdU) labeling, in addition to staining with the undifferentiated type A spermatogonial marker GDNF family receptor alpha-1 (GFRA1), indicated that the undifferentiated spermatogonial population was normal in p53 knockout rats. Following exposure to 0.5 or 5 Gy X-ray, p53 knockout rats exhibited no germ cell apoptotic response beyond their unirradiated phenotype, while germ cell death in wild-type rat testes was elevated to a level similar to the unexposed p53 knockout rats. This study indicates that seminiferous tubule atrophy occurs following spontaneous, elevated levels of spermatocyte death in the p53 knockout rat. This phenomenon is variable across individual rats. These results indicate a critical role for p53 in rat germ cell survival and spermatogenesis.

SUBMITTER: Dai MS

PROVIDER: S-EPMC5673550 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat.

Dai Matthew S MS Hall Susan J SJ Vantangoli Policelli Marguerite M MM Boekelheide Kim K Spade Daniel J DJ

Andrology 20170822 6

The tumor suppressor protein p53 (TP53) has many functions in cell cycle regulation, apoptosis, and DNA damage repair and is also involved in spermatogenesis in the mouse. To evaluate the role of p53 in spermatogenesis in the rat, we characterized testis biology in adult males of a novel p53 knockout rat (SD-Tp53<sup>tm1sage</sup> ). p53 knockout rats exhibited variable levels of testicular atrophy, including significantly decreased testis weights, atrophic seminiferous tubules, decreased semini ...[more]

PMID: 28834365

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Project description:ObjectiveApplications of biological scaffolds for regenerative medicine are increasing. Such scaffolds improve cell attachment, migration, proliferation and differentiation. In the current study decellularised mouse whole testis was used as a natural 3 dimensional (3D) scaffold for culturing spermatogonial stem cells.Materials and methodsIn this experimental study, adult mouse whole testes were decellularised using sodium dodecyl sulfate (SDS) and Triton X-100. The efficiency of decellularisation was determined by histology and DNA quantification. Masson's trichrome staining, alcian blue staining, and immunohistochemistry (IHC) were done for validation of extracellular matrix (ECM) proteins. These scaffolds were recellularised through injection of mouse spermatogonial stem cells in to rete testis. Then, they were cultured for eight weeks. Recellularised scaffolds were assessed by histology, real-time polymerase chain reaction (PCR) and IHC.ResultsHaematoxylin-eosin (H and E) staining showed that the cells were successfully removed by SDS and Triton X-100. DNA content analysis indicated that 98% of the DNA was removed from the testis. This conﬁrmed that our decellularisation protocol was efﬁcient. Masson's trichrome and alcian blue staining respectively showed that glycosaminoglycans (GAGs) and collagen are preserved in the scaffolds. IHC analysis confirmed the preservation of fibronectin, collagen IV, and laminin. MTT assay indicated that the scaffolds were cell-compatible. Histological evaluation of recellularised scaffolds showed that injected cells were settled on the basement membrane of the seminiferous tubule. Analyses of gene expression using real-time PCR indicated that expression of the Plzf gene was unchanged over the time while expression of Sycp3 gene was increased significantly (P=0.003) after eight weeks in culture, suggesting that the spermatogonial stem cells started meiosis. IHC confirmed that PLZF-positive cells (spermatogonial stem cells) and SYCP3-positive cells (spermatocytes) were present in seminiferous tubules.ConclusionSpermatogonial stem cells could proliferate and differentiated in to spermatocytes after being injected in the decellularised testicular scaffolds.

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Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat.

Publications

Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat.

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