Radiation effects on gene expression in rat testes (Rattus norvegicus)
ABSTRACT: Analysis of LBNF1 rat testes from controls, containing both somatic and all germ cell types and from irradiated rats in which all cells germ cells except type A spermatgogonia are eliminated. Results provide insight into distinguishing germ and somatic cell genes and identification of somatic cell genes that are upregulated after irradiation. Overall design: We studied 10 Sample replicates of control rat testes and 5 Sample replicates of irradiated rat testes.
INSTRUMENT(S): [Rat230_2] Affymetrix Rat Genome 230 2.0 Array
Project description:Analysis of LBNF1 rat testes from controls, containing both somatic and all germ cell types and from irradiated rats in which all cells germ cells except type A spermatgogonia are eliminated. Results provide insight into distinguishing germ and somatic cell genes and identification of somatic cell genes that are upregulated after irradiation. Experiment Overall Design: We studied 10 Sample replicates of control rat testes and 5 Sample replicates of irradiated rat testes.
Project description:Analysis of hormone effects on irradiated LBNF1 rat testes, which contain only somatic cells except for a few type A spermatgogonia. Rats were treated for 2 weeks with either sham treatment (group X), hormonal ablation (GnRH antagonist and the androgen receptor antagonist flutamide, group XAF), testosterone supplementation (GnRH antagonist and testosterone, group XAT), and FSH supplementation ((GnRH antagonist, androgen receptor antagonist, and FSH, group XAFF). Results provide insight into identifying genes in the somatic testis cells regulated by testosterone, LH, or FSH. Overall design: 14 Sample replicates of sham treatment, 11 Sample replicates of hormonal ablation, 5 Sample replicates of testosterone supplementation, and 5 Sample replicates of FSH supplementation were studied.
Project description:The pim-1 proto-oncogene encodes a serine/threonine protein kinase and is expressed in cells of hematolymphoid origin and in the germ cell lineages. In somatic cells, the pim-1 gene is expressed as a 2.8 kb transcript while a shorter sized transcript (2.3 kb) is expressed in rat testes. We have determined that the shorter testes-specific pim-1 transcript arises through the use of an alternate polyadenylation signal present in the 3' untranslated region of the gene. This alternate polyadenylation event results in the removal of an A/U-rich regulatory element located in the 3' untranslated region of the pim-1 gene. This A/U-rich motif has been shown by a number of laboratories to destabilize the transcripts of genes that contain this sequence. Consistent with these findings, we have demonstrated that the shortened testes-specific pim-1 transcript is more stable than the longer A/U-rich containing somatic transcript. We suggest that the functional significance of different sized pim-1 transcripts may be directly related to their different stabilities and that the greater stability of the testes-specific transcript may be essential for the translational delay observed in post-meiotic male germ cells.
Project description:Exposure to excess retinoic acid (RA) disrupts the development of the mammalian testicular seminiferous cord. However, the molecular events surrounding RA-driven loss of cord structure have not previously been examined. To investigate the mechanisms associated with this adverse developmental effect, fetal rat testes were isolated on gestational day 15, after testis determination and the initiation of cord development, and cultured in media containing all-trans RA (ATRA; 10-8 to 10-6 M) or vehicle for 3?days. ATRA exposure resulted in a concentration-dependent decrease in the number of seminiferous cords per testis section and number of germ cells, assessed by histopathology and immunohistochemistry. Following 1?day of culture, genome-wide expression profiling by microarray demonstrated that ATRA exposure altered biological processes related to retinoid metabolism and gonadal sex determination. Real-time RT-PCR analysis confirmed that ATRA enhanced the expression of the key ovarian development gene Wnt4 and the antitestis gene Nr0b1 in a concentration-dependent manner. After 3?days of culture, ATRA-treated testes contained both immunohistochemically DMRT1-positive and FOXL2-positive somatic cells, providing evidence of disrupted testicular cell fate maintenance following ATRA exposure. We conclude that exogenous RA disrupts seminiferous cord development in ex vivo cultured fetal rat testes, resulting in a reduction in seminiferous cord number, and interferes with maintenance of somatic cell fate by enhancing expression of factors that promote ovarian development.
Project description:Spermatogonial stem cells (SSC) are essential for spermatogenesis and male fertility. In addition, these adult tissue stem cells can be used as vehicles for germline modification in animal models and may have application for treating male infertility. To facilitate the investigation of SSCs and germ lineage development in rats, we generated a DEAD-box helicase 4 (DDX4) (VASA) promoter-enhanced green fluorescent protein (EGFP) reporter transgenic rat. Quantitative real-time polymerase chain reaction and immunofluorescence confirmed that EGFP was expressed in the germ cells of the ovaries and testes and was absent in somatic cells and tissues. Germ cell transplantation demonstrated that the EGFP-positive germ cell population from DDX4-EGFP rat testes contained SSCs capable of establishing spermatogenesis in experimentally infertile mouse recipient testes. EGFP-positive germ cells could be easily isolated by fluorescence-activated cells sorting, while simultaneously removing testicular somatic cells from DDX4-EGFP rat pup testes. The EGFP-positive fraction provided an optimal cell suspension to establish rat SSC cultures that maintained long-term expression of zinc finger and BTB domain containing 16 (ZBTB16) and spalt-like transcription factor 4 (SALL4), two markers of mouse SSCs that are conserved in rats. The novel DDX4-EGFP germ cell reporter rat described here combined with previously described GCS-EGFP rats, rat SSC culture and gene editing tools will improve the utility of the rat model for studying stem cells and germ lineage development.
Project description:Sertoli cells (Sc) are unique somatic cells of testis that are the target of both FSH and testosterone (T) and regulate spermatogenesis. Although Sc of neonatal rat testes are exposed to high levels of FSH and T, robust differentiation of spermatogonial cells becomes conspicuous only after 11-days of postnatal age. We have demonstrated earlier that a developmental switch in terms of hormonal responsiveness occurs in rat Sc at around 12 days of postnatal age during the rapid transition of spermatogonia A to B. Therefore, such "functional maturation" of Sc, during pubertal development becomes prerequisite for the onset of spermatogenesis. However, a conspicuous difference in robust hormone (both T and FSH) induced gene expression during the different phases of Sc maturation restricts our understanding about molecular events necessary for the spermatogenic onset and maintenance. Here, using microarray technology, we for the first time have compared the differential transcriptional profile of Sc isolated and cultured from immature (5 days old), maturing (12 days old) and mature (60 days old) rat testes. Our data revealed that immature Sc express genes involved in cellular growth, metabolism, chemokines, cell division, MAPK and Wnt pathways, while mature Sc are more specialized expressing genes involved in glucose metabolism, phagocytosis, insulin signaling and cytoskeleton structuring. Taken together, this differential transcriptome data provide an important resource to reveal the molecular network of Sc maturation which is necessary to govern male germ cell differentiation, hence, will improve our current understanding of the etiology of some forms of idiopathic male infertility.
Project description:Many studies on various organs have concluded that venous congestion (VC) causes severe organ dysfunction with elevation of oxidative stress relative to that of arterial ischaemia (AI). However, a comparison of the pathological effects of AI and VC on the testes has not been conducted. In this study, models of AI and VC and their reperfusion in rat testes, respectively, were developed and analysed. Testicular arteries or veins were interrupted for 6?h, re-perfused and kept for 4 weeks; the effects on the testes were then evaluated. Severe spermatogenic disturbances were observed at 4 weeks after reperfusion in AI but not in VC. At 6?h after blood flow interruption, oxidative stress was significantly increased and germ cells were severely damaged in AI compared with those in VC. RT-PCR analyses revealed that haem oxygenase-1, which exhibits anti-oxidative effects, and vascular endothelial growth factor-A, which exhibits vasculogenic effects, were significantly increased in VC but not in AI. Surprisingly, the results of our experiment in rat testes differed from those of experiments in previous studies performed in other organs. Oxidative stress in testes was more easily elevated by AI than it was by VC, explainable by the different experimental conditions.
Project description:Spermatogenesis is a process by which haploid cells differentiate from germ cells in the seminiferous tubules of the testes. TLE3, a transcriptional co-regulator that interacts with DNA-binding factors, plays a role in the development of somatic cells. However, no studies have shown its role during germ cell development in the testes. Here, we examined TLE3 expression in the testes during spermatogenesis. TLE3 was highly expressed in mouse testes and was dynamically regulated in different cell types of the seminiferous tubules, spermatogonia, spermatids, and Sertoli cells, but not in the spermatocytes. Interestingly, TLE3 was not detected in Sertoli cells on postnatal day 7 (P7) but was expressed from P10 onward. The microarray analysis showed that the expression of numerous genes changed upon TLE3 knockdown in a Sertoli cell line TM4. These include 1597 up-regulated genes and 1452 down-regulated genes in TLE3-knockdown TM4 cells. Ingenuity Pathway Analysis (IPA) showed that three factors were up-regulated and two genes were down-regulated upon TLE3 knockdown in TM4 cells. The abnormal expression of the three factors is associated with cellular malfunctions such as abnormal differentiation and Sertoli cell formation. Thus, TLE3 is differentially expressed in Sertoli cells and plays a crucial role in regulating cell-specific genes involved in the differentiation and formation of Sertoli cells during testicular development.
Project description:BACKGROUND:Murine kobuviruses (MuKV) are newly recognized picornaviruses first detected in murine rodents in the USA in 2011. Little information on MuKV epidemiology in murine rodents is available. Therefore, we conducted a survey of the prevalence and genomic characteristics of rat kobuvirus in Guangdong, China. RESULTS:Fecal samples from 223 rats (Rattus norvegicus) were collected from Guangdong and kobuviruses were detected in 12.6% (28) of samples. Phylogenetic analysis based on partial 3D and complete VP1 sequence regions showed that rat kobuvirus obtained in this study were genetically closely related to those of rat/mouse kobuvirus reported in other geographical areas. Two near full-length rat kobuvirus genomes (MM33, GZ85) were acquired and phylogenetic analysis of these revealed that they shared very high nucleotide/amino acids identity with one another (95.4%/99.4%) and a sewage-derived sequence (86.9%/93.5% and 87.5%/93.7%, respectively). Comparison with original Aichivirus A strains, such human kobuvirus, revealed amino acid identity values of approximately 80%. CONCLUSION:Our findings indicate that rat kobuvirus have distinctive genetic characteristics from other Aichivirus A viruses. Additionally, rat kobuvirus may spread via sewage.