Systemic L-Kynurenine sulfate administration disrupts object recognition memory, alters open field behavior and decreases c-Fos immunopositivity in C57Bl/6 mice

L-Kynurenine (L-KYN) is a central metabolite of tryptophan degradation through the kynurenine pathway (KP). The systemic administration of L-KYN sulfate (L-KYNs) leads to a rapid elevation of the neuroactive KP metabolite kynurenic acid (KYNA). An elevated level of KYNA may have multiple effects on the synaptic transmission, resulting in complex behavioral changes, such as hypoactivity or spatial working memory deficits. These results emerged from studies that focused on rats, after low-dose L-KYNs treatment. However, in several studies neuroprotection was achieved through the administration of high-dose L-KYNs. In the present study, our aim was to investigate whether the systemic administration of a high dose of L-KYNs (300 mg/bwkg; i.p.) would produce alterations in behavioral tasks (open field or object recognition) in C57BI/6j mice. To evaluate the changes in neuronal activity after L-KYNs treatment, in a separate group of animals we estimated c-Fos expression levels in the corresponding subcortical brain areas. The L-KYNs treatment did not affect the general ambulatory activity of C57BI/6j mice, whereas it altered their moving patterns, elevating the movement velocity and resting time. Additionally, it seemed to increase anxiety-like behavior, as peripheral zone preference of the open field arena emerged and the rearing activity was attenuated. The treatment also completely abolished the formation of object recognition memory and resulted in decreases in the number of c-Fos-immunopositive-cells in the dorsal part of the striatum and in the CA1 pyramidal cell layer of the hippocampus. We conclude that a single exposure to L-KYNs leads to behavioral disturbances, which might be related to the altered basal c-Fos protein expression in C57BI/6j mice

Identification and Characterization of RBM44 as a Novel Intercellular Bridge Protein

Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44) as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 null male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14

Characterization of Spermatogonial Stem Cells Lacking Intercellular Bridges and Genetic Replacement of a Mutation in Spermatogonial Stem Cells

Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14) null spermatogonial stem cells lacking intercellular bridges but also a trial application of genetic correction of a mutation in spermatogonial stem cells as a model for future gene therapy. In TEX14 null testes, some genes important for undifferentiated spermatogonia as well as some differentiation-related genes were activated. TEX14 null spermatogonial stem cells, surprisingly, could form chain-like structures even though they do not form stable intercellular bridges. TEX14 null spermatogonial stem cells in culture possessed both characteristics of undifferentiated and differentiated spermatogonia. Long-term culture of TEX14 null spermatogonial stem cells could not be established likely secondary to up-regulation of CDK4 inhibitors and down-regulation of cyclin E. These results suggest that intercellular bridges are essential for both maintenance of spermatogonial stem cells and their proliferation. Lastly, a mutation in Tex14+/− spermatogonial stem cells was successfully replaced by homologous recombination in vitro. Our study provides a therapeutic potential of spermatogonial stem cells for reproductive medicine if they can be cultured long-term

H3K27 Demethylase, JMJD3, Regulates Fragmentation of Spermatogonial Cysts

<div><p>The spermatogonial stem cell (SSC) compartment is maintained by self-renewal of stem cells as well as fragmentation of differentiating spermatogonia through abscission of intercellular bridges in a random and stochastic manner. The molecular mechanisms that regulate this reversible developmental lineage remain to be elucidated. Here, we show that histone H3K27 demethylase, JMJD3 (KDM6B), regulates the fragmentation of spermatogonial cysts. Down-regulation of <i>Jmjd3</i> in SSCs promotes an increase in undifferentiated spermatogonia but does not affect their differentiation. Germ cell-specific <i>Jmjd3</i> null male mice have larger testes and sire offspring for a longer period compared to controls, likely secondary to increased and prolonged maintenance of the spermatogonial compartment. Moreover, JMJD3 deficiency induces frequent fragmentation of spermatogonial cysts by abscission of intercellular bridges. These results suggest that JMJD3 controls the spermatogonial compartment through the regulation of fragmentation of spermatogonial cysts and this mechanism may be involved in maintenance of diverse stem cell niches.</p> </div

Expression profiles of SSC markers and cell cycle regulators in <i>Tex14^+/−</i> and <i>Tex14^−/−</i> SSCs.

<p>Relative expressions of SSC markers (A) and cell cycle regulators (B) in <i>Tex14^+/−</i> (blue) and <i>Tex14^−/−</i> (red) SSCs were quantitatively analyzed. All of the expression levels were normalized to <i>Gapdh</i> expression. * indicates that there is significant difference (P<0.05).</p

Detection of intercellular bridge formation and establishment of spermatogonial stem cells in culture.

<p>A. Detection of intercellular bridge proteins in <i>Tex14^+/−</i> and <i>Tex14^−/−</i> testis. Immunofluorescence images of CEP55 (red) and TEX14 (green) and nucleus (DAPI) in 8-week old <i>Tex14^+/−</i> (+/−) and <i>Tex14^−/−</i> (KO) testis are shown. Insets indicate higher magnification images. B. Detection of intercellular bridges in short term culture of <i>Tex14^+/−</i> and <i>Tex14^−/−</i> spermatogonia. GFP (green) positive spermatognia were cultured and stained with TEX14 antibody (red) at second passage. Arrows indicate TEX14 positive intercellular bridges. C. Cytoskelton of short-term culture of <i>Tex14^+/−</i> and <i>Tex14^−/−</i> spermatogonia. Immunofluorescence images of TUBA (alpha-tubulin: green) and ZBTB16 (red) in <i>Tex14^+/−</i> (+/−) and <i>Tex14^−/−</i> (KO) spermatogonia are shown. D. Morphology of established spermatogonial stem cells in culture. Typical morphologies of cultured wildtype and <i>Tex14^+/−</i> SSCs at 3 months of culture are shown.</p

Characteristics of JMJD3 null undifferentiated spermatogonia.

<p>A. Relative expressions of SSC markers in control (F/-) and JMJD3 cKO (cKO) EPCAM-positive testicular cells were quantitatively analyzed. All of the expression levels were normalized to <i>Gapdh</i> expression. * indicates that there is significant difference (P<0.05). B. Flow cytometric analysis of EPCAM and intracellular staining of PLZF in the EPCAM-positive JMJD3 F/- (upper) and JMJD3 cKO (lower) spermatogonia. Ratio of PLZF positive cells is significantly different (P<0.01). C. Flow cytometric analysis of side scatter and CD9 in the JMJD3 F/- and the JMJD3 cKO testicular cell (Left panels). Ratio of side scatter-low and CD9+ cells is statistically different (P<0.001). Right panels: KIT expression in the side scatter-low and CD9+ JMJD3 F/- and JMJD3 cKO cells. Ratio of KIT-negative cells is significantly different (P<0.001).</p

Effect of <i>Jmjd3</i> knockdown in germline stem cells.

<p>A. Immuno-localization of H3K27me3 and H3K27 modifiers in germline stem (GS) cells. Colocalization of H3K27me3, EZH2, and JMJD3 with PLZF in GS cell are shown. Notably, PLZF strongly localizes in clusters and weakly localized in chains of GS colony. Arrow and solid line indicate chains of GS cells. B. The number of colonies formed by <i>Jmjd3</i> and mock knockdown. The number of colonies at 1 week after replating of mock (green) and <i>Jmjd3</i> (red) knockdown GS cells were counted. Bars indicate mean ± se. * indicates that there is significant difference (P<0.05). C. Representative morphology of mock and JMJD3 KD GS cell colony. D. Relative expression of spermatogonial stem cell markers and cell cycle regulators in mock and JMJD3 KD GS cells. Relative expression of indicated genes are quantitatively analyzed after normalization with <i>Gapdh</i>. * indicates that there is significant difference (P<0.05).</p

Alterations of cell cycle regulators in spermatogonial stem cells by lack of intercellular bridges.

<p>Protein expression of cell cycle regulators in <i>Tex14^+/−</i> and <i>Tex14^−/−</i> SSCs were analyzed by immunoblot (A) and immunostaining (B). Immunofluorescence images (B) of indicated cell cycle regulators (green) and ZBTB16 (red) in <i>Tex14^+/−</i> (+/−) and <i>Tex14^−/−</i> (KO) SSCs are shown.</p