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Long-term vitamin A deficiency induces alteration of adult mouse spermatogenesis and spermatogonial differentiation: direct effect on spermatogonial gene expression and indirect effects via somatic cells.

ABSTRACT: The objective of this study was to further understand the genetic mechanisms of vitamin A deficiency (VAD) induced arrest of spermatogonial stem-cell differentiation. Vitamin A and its derivatives (the retinoids) participate in many physiological processes including vision, cellular differentiation and reproduction. VAD affects spermatogenesis, the subject of our present study. Spermatogenesis is a highly regulated process of differentiation and complex morphologic alterations that leads to the formation of sperm in the seminiferous epithelium. VAD causes early cessation of spermatogenesis, characterized by degeneration of meiotic germ cells, leading to seminiferous tubules containing mostly type A spermatogonia and Sertoli cells. These observations led us to the hypothesis that VAD affects not only germ cells but also somatic cells. To investigate the effects of VAD on spermatogenesis in mice we used adult Balb/C mice fed with Control or VAD diet for an extended period of time (6-28 weeks). We first observed the chronology, then the extent of the effects of VAD on the testes. Using microarray analysis of isolated pure populations of spermatogonia, Leydig and Sertoli cells from control and VAD 18- and 25-week mice, we examined the effects of VAD on gene expression and identified target genes involved in the arrest of spermatogonial differentiation and spermatogenesis. Our results provide a more precise definition of the chronology and magnitude of the consequences of VAD on mouse testes than the previously available literature and highlight direct and indirect (via somatic cells) effects of VAD on germ cell differentiation.

SUBMITTER: Boucheron-Houston C

PROVIDER: S-EPMC3610847 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Long-term vitamin A deficiency induces alteration of adult mouse spermatogenesis and spermatogonial differentiation: direct effect on spermatogonial gene expression and indirect effects via somatic cells.

Boucheron-Houston Catherine C Canterel-Thouennon Lucile L Lee Tin-Lap TL Baxendale Vanessa V Nagrani Sohan S Chan Wai-Yee WY Rennert Owen M OM

The Journal of nutritional biochemistry 20121217 6

The objective of this study was to further understand the genetic mechanisms of vitamin A deficiency (VAD) induced arrest of spermatogonial stem-cell differentiation. Vitamin A and its derivatives (the retinoids) participate in many physiological processes including vision, cellular differentiation and reproduction. VAD affects spermatogenesis, the subject of our present study. Spermatogenesis is a highly regulated process of differentiation and complex morphologic alterations that leads to the ...[more]

PMID: 23253600

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Project description:The objective of this study was to understand the genetic mechanisms of Vitamin-A-Deficiency (VAD)-induced arrest of spermatogonial stem-cell differentiation. Vitamin A and its derivatives (the retinoids) participate in many physiological processes including vision, cellular differentiation and reproduction. VAD affects spermatogenesis, the subject of our present study. Spermatogenesis is a highly regulated process of differentiation and complex morphologic alterations that, in the postnatal testis, leads to the formation of sperm in the seminiferous epithelium. VAD causes early cessation of spermatogenesis, characterized by degeneration of meiotic germ cells, leading to seminiferous tubules containing mostly type A spermatogonia and Sertoli cells. In this study, we investigated the molecular basis of VAD on spermatogenesis in mice. We used adult Balb/C mice fed with a Control or VAD diet for an extended period of time (8-28 weeks) and selected two time points (18 and 25 weeks) for microarray analysis. To understand the effect of VAD on the spermatogonial stem cell transcriptome, we studied isolated pure populations of spermatogonia from control and vitamin-A-deficient mice from two representative time points (18 and 25 weeks) using Affymetrix GeneChip microarrays. We identified target genes involved in the arrest of spermatogonial differentiation and spermatogenesis. Our results establish a better understanding of the chronology and magnitude of the consequences of VAD on mouse testes and add to the current knowledge of the molecular regulatory mechanisms of germ cell development. Spermatogonia were isolated by the STATPUT procedure with minor modifications. VAD mice were sacrificed at 18 and 25 weeks of VAD treatment for spermatogonia isolation. Decapsulated testes from 2 mice were suspended in RPMI medium containing collagenase (0.6 mg/ml), Hyaluronidase (0.12 mg/ml) and DNAse I (1.25 mg/ml) and incubated at 37ºC for 30 minutes in a shaking water bath. The tissues were then allowed to come down the tube and the supernatant (containing interstitial cells) was removed. The pellet was incubated with 3 ml of 0.25% Trypsin /EDTA (GIBCO, Invitrogen, USA), in the presence of DNAse I (0.2 mg/ml) at 37ºC for 15 minutes in a shaking water bath. The dispersed cells were washed twice with RPMI medium containing 10% heat inactivated FBS to neutralize the protease activity, and filtered through a sterile 0.22 nylon to remove any undigested fragments. Cells of the dissociated seminiferous epithelium were then plated overnight in a 34ºC 3% CO2 incubator. The following day, germ cells, in suspension, were separated by sedimentation with use of a 2-4% BSA gradient. The cells were allowed to sediment for a standard period of 2.5 h, and fractions of 2-ml volume were collected. The cells of each fraction were examined under a phase contrast microscope, and fractions containing cells of similar size and morphology were pooled and spun down by low-speed centrifugation. Purity of spermatogonia was estimated and was routinely higher than 90%. Total RNA was extracted from the isolated germ cells using TRIzol ® Reagent, and cleaned with RNeasy minicolumns. RNA content was determined by measurement of optical density at 260 nm. Only the RNA samples showing an OD 260/280 ratio higher than 1.8 were used for microarray hybridization. Raw expression values in Affymetrix CEL file format were generated by GeneChip Operating Software.

Project description:Camellia oleifera Abel. is an important woody oil species; however, the shortage of rapid and industrialized seedling culture is a large constraint on the development of the tea oil industry. Somatic embryogenesis (SE) is one of the main powerful biotechnological tools for plant mass regeneration, but the largely unknown SE in C. oleifera limits the scale production of clonal plants. In this study, we described a high-efficiency SE system via direct and indirect pathways in C. oleifera and investigated the effect of genotype, explant age and phytohormones on SE. In the direct pathway, somatic embryos were highly induced from immature seeds 220 days after full blossom, and the development of embryoids was achieved with a combination of 0.19 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/L thidiazuron (TDZ). In the indirect pathway, embryogenic calli were induced from the same explants in medium containing 1.5 mg/L 2,4-D, while 0.75 mg/L 2,4-D treatment led to high proliferation rates for embryogenic calli. The addition of 0.19 mg/L 2,4-D alone stimulated the production of globular embryos while causing a 75% loss of the induction rate in the heart embryo stage. Upon transfer of the globular embryos to phytohormone-free medium, an optimal induction rate of 62.37% from globular embryos to cotyledonary embryos was obtained. These data suggest that the subsequent differentiation process after the globular embryo stage in ISE is more similar to an endogenous phytohormones-driven process. Mature embryos germinated to produce intact plantlets on half-strength MS basal medium with a regeneration rate of 63.67%. Histological analysis confirmed the vascular bundle isolation of embryoids from the mother tissue. We further studied the different varieties and found that there were no significant genotype differences for SE induction efficiency in C. oleifera. Thus, we established a high-efficiency induction system for direct and indirect somatic embryogenesis (ISE) in C. oleifera and regenerated intact plantlets via SE, not organogenesis. ISE has a more complicated induction and regulatory mechanism than direct somatic embryogenesis. The improved protocol of SE would benefit mass propagation and genetic manipulation in C. oleifera.

Dataset Information

Long-term vitamin A deficiency induces alteration of adult mouse spermatogenesis and spermatogonial differentiation: direct effect on spermatogonial gene expression and indirect effects via somatic cells.

Publications

Long-term vitamin A deficiency induces alteration of adult mouse spermatogenesis and spermatogonial differentiation: direct effect on spermatogonial gene expression and indirect effects via somatic cells.

Similar Datasets

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