Abnormal chromatin remodeling caused by ARID1A deletion leads to malformation of the dentate gyrus.

ARID1A, an SWI/SNF chromatin-remodeling gene, is commonly mutated in cancer and hypothesized to be a tumor suppressor. Recently, loss-of-function of ARID1A gene has been shown to cause intellectual disability. Here we generate Arid1a conditional knockout mice and investigate Arid1a function in the hippocampus. Disruption of Arid1a in mouse forebrain significantly decreases neural stem/progenitor cells (NSPCs) proliferation and differentiation to neurons within the dentate gyrus (DG), increasing perinatal and postnatal apoptosis, leading to reduced hippocampus size. Moreover, we perform single-cell RNA sequencing (scRNA-seq) to investigate cellular heterogeneity and reveal that Arid1a is necessary for the maintenance of the DG progenitor pool and survival of post-mitotic neurons. Transcriptome and ChIP-seq analysis data demonstrate that ARID1A specifically regulates Prox1 by altering the levels of histone modifications. Overexpression of downstream target Prox1 can rescue proliferation and differentiation defects of NSPCs caused by Arid1a deletion. Overall, our results demonstrate a critical role for Arid1a in the development of the hippocampus and may also provide insight into the genetic basis of intellectual disabilities such as Coffin-Siris syndrome, which is caused by germ-line mutations or microduplication of Arid1a.

The histone modification reader ZCWPW1 is required for meiosis prophase I in male but not in female mice.

Meiosis is a specialized type of cell division that creates haploid germ cells and ensures their genetic diversity through homologous recombination. We show that the H3K4me3 reader ZCWPW1 is specifically required for meiosis prophase I progression in male but not in female germ cells in mice. Loss of Zcwpw1 in male mice caused a complete failure of synapsis, resulting in meiotic arrest at the zygotene to pachytene stage, accompanied by incomplete DNA double-strand break repair and lack of crossover formation, leading to male infertility. In oocytes, deletion of Zcwpw1 only somewhat slowed down meiosis prophase I progression; Zcwpw1-/- oocytes were able to complete meiosis, and Zcwpw1-/- female mice had normal fertility until mid-adulthood. We conclude that the H3K4me3 reader ZCWPW1 is indispensable for meiosis synapsis in males but is dispensable for females. Our results suggest that ZCWPW1 may represent a previously unknown, sex-dependent epigenetic regulator of germ cell meiosis in mammals.

[LNK Gene Single Nucleotide Polymorphisms and Acute Leukemia Susceptibility].

To investigate the relationship between the LNK(SH2B3) gene single nucleotide polymorphism and risk of acute leukemia (AL) in Chinese population. METHODS: The bone marrow and peripheral blood samples from 31 cases of acute lymphoblastic leukemia, 70 cases of acute myeloid leukemia and 130 healthy persons as the controls were collected. Genotype of LNK SNP Rs3184504(c.784T>C) and Rs78894077(c.724C>T) were determined by PCR-RFLP, and were confirmed by gel electrophoresis and sequencing. The NB4, THP-1 and Raji leukemia cell line models were cultured, the leukemia cell line LNK Rs3184504 and Rs78894077 polymorphism were detected by using direct sequencing. RESULTS: The CC genotype frequencies of Rs3184504 SNP were higher in ALL and AML patients than those in control (P<0.01), but there was no different between the groups in AML and ALL. The frequency of LNK gene Rs3184504 C allele was higher in AL as compared with control (P<0.01). The LNK gene Rs78894077 locus genotype distribution was not significantly different between the AL and the normal control group (P>0.05). Both Rs3184504 and Rs78894077 sites were detected as CC genotype in NB4, THP-1 and Raji cells. CONCLUSION: The persons carrying C allele of LNK gene Rs3184504 are more prone to develop acute leukemia.

Confocal Microscopic Analysis of the Spindle and Chromosome Configurations of in vitro-Matured Oocytes from Different Types of Polycystic Ovary Syndrome Patients.

OBJECTIVE: To evaluate the meiotic spindle and chromosome distribution of in vitro-matured oocytes from infertile nonobese or obese women with polycystic ovary syndrome (PCOS) and to compare in vitro maturation (IVM) rates between groups. DESIGN: Prospective study. SETTING: Hospital-based IVF center. PATIENTS: A total of 99 patients (26 obese women with PCOS, 25 nonobese women with PCOS, and 48 controls) undergoing stimulated cycles for intracytoplasmic sperm injection had immature oocytes for IVM. INTERVENTIONS: Immature oocytes (germinal vesicle and metaphase I stages) were collected from obese and nonobese PCOS patients and controlled infertile patients. The meiotic spindle and chromosome configurations in oocytes matured in vitro were studied by confocal microscopy, with fluorescent labeling techniques for visualization of both microtubules and chromosomes. MAIN OUTCOME MEASURES: Meiotic spindle and associated chromosome configurations. RESULTS: There were no significant differences between the different types of PCOS and the control group with respect to IVM rates (61.8, 63.8, and 63.2%, respectively), the percentage of spindle abnormalities in metaphase II oocytes (40.6, 42.9, and 37.5%, respectively) or chromosome abnormalities in metaphase II oocytes (31.2, 34.3, and 33.3%, respectively). CONCLUSIONS: In vitro-matured oocytes obtained from stimulated cycles had a high ratio of meiotic abnormalities. The different types of PCOS had the same ratio of meiotic abnormalities.

[Role of LNK gene mutation in pathogenesis of myeloproliferative neoplasms-review].

Myeloproliferative neoplasms ( MPN ) is a class of clonal hematopoietic stem cell disease. Studies found that the JAK-STAT signaling pathway is closely related to the pathogenesis of MPN. The lymphocyte-specific adaptor protein (LNK) gene negatively regulates Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling and may play an important role in the pathogenesis of MPN. Especially in JAK2 mutation-negative MPN, LNK gene specific mutations may be the key to cause MPN subtypes. Certain single nucleotide polymorphism of LNK gene regulation of hematopoietic cells in different directions may also be important influence factors of MPN performance for different subtypes. LNK gene functional changes lead to abnormal activation of the JAK-STAT signaling pathway, and may be a new mechanism of MPN. In this review, the role of LNK gene in MPN pathogenesis is briefly summarized.

Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors.

Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem or progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1ß and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bidirectional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage conversion into bipotential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering.

[Role of signal transducer and activator of transcription 3 in the development of tumors].

Signal transducer and activator of transcription 3 (STAT3) take part in cell proliferation, differentiation, survival, apoptosis, transformation, cellular immunity and some other important physiological and pathological processes. Among STAT3 signaling pathways, the JAK-STAT signaling pathway has been comprehensively studied. Abnormal activation of STAT3 is frequently detected in various tumors, and the abnormal activation is closely related with the tumorigenesis. Recent studies have found that mutations and several specific genotypes of single nucleotide polymorphisms in STAT3 gene may be involved in tumor formation, also suggesting the important role of STAT3 in tumor biology. In this review, the role of STAT3 in the development of tumors is briefly summarized.

Expression of inhibin-alpha is regulated synergistically by Wilms' tumor gene 1 (Wt1) and steroidogenic factor-1 (Sf1) in sertoli cells.

Wt1 encodes a zinc finger nuclear transcriptional factor, which is specifically expressed in testicular Sertoli cells and knockdown of Wt1 in Sertoli cells causes male mice subfertility. However, the underlying mechanism is still unclear. In this study, we found that expression of inhibin-α is significantly reduced in Wt1-deficient Sertoli cells. Luciferase assays using the inhibin-α promoter indicated that the inhibin-α promoter is transactivated by the Wt1 A, and B isoforms (-KTS), but not the C, and D isoforms (+KTS). Analysis of the Wt1 responsive element of the inhibin-α promoter region using site-directed mutagenesis showed that the nucleotides between -58 and -49 are essential for Wt1-dependent transactivation of the inhibin-α promoter. ChIP assays indicated that Wt1 directly interacts with the inhibin-α promoter. In addition, the inhibin-α promoter is activated synergistically by Wt1 and Sf1. Mutation of the ligand binding domain (LBD) of Sf1 (residues 235-238) completely abolished the synergistic action between Wt1 and Sf1, but did not affect the physical interaction between these two proteins, suggesting that other factor(s) may also be involved in the regulation of inhibin-α in Sertoli cells. Further studies demonstrated that ß-catenin enhances the synergistic activation of Wt1 and Sf1 on the inhibin-α promoter. Given the fact that inhibin-α, a subunit of inhibin, is known to be involved in the regulation of spermatogenesis and testicular steroidogenesis, this study reveals a new regulatory mechanism of inhibin-α in Sertoli cells and also sheds light on the physiological functions of Wt1 in gonad development and spermatogenesis.

Mouse Fem1b interacts with and induces ubiquitin-mediated degradation of Ankrd37.

Ankyrin repeat domain 37 (Ankrd37), a protein containing ankyrin repeats (ARs) and a putative nuclear localization signal (NLS), is highly conserved from zebrafish to humans. In mouse testes, Ankrd37 protein was initially present in the cytoplasm of elongating spermatids, and finally restricted to the nuclei of spermatozoa during spermatogenesis. Ankrd37 bound to feminization 1 homolog b (Fem1b) as indicated by yeast two-hybrid screening and co-immunoprecipitation assays. Ankrd37 facilitated the transport of Fem1b protein from cytoplasm to nuclei in co-transfected CHO cells. In addition, the protein level of Ankrd37 was decreased in a Fem1b dose-dependent manner as shown by the transfection experiments, and Ankrd37 was ubiquitinated in the presence of Fem1b. As the nematode Fem-1 has been shown to target its downstream effector TRA-1 for ubiquitin-mediated degradation, we report in the present study that mouse Fem1b targets Ankrd37 for degradation in the same manner.

Male germ cell-specific protein Trs4 binds to multiple proteins.

Temperature-related sequence 4 (Trs4) has been identified as a testis-specific gene with expression sensitive to the abdominal temperature changes induced by artificial cryptorchidism. In murine testes, Trs4 mRNA was detected in round spermatids and its protein was localized mainly in the elongating spermatids as well as in the acrosomes and tails of mature spermatozoa. Using a yeast two-hybrid screening system, we identified Rshl-2, Gstmu1, and Ddc8 as putative binding partners of the Trs4 protein in mouse testes. Their interactions were confirmed by in vivo and in vitro binding assays. Further studies demonstrated that Ddc8, a newly identified gene with unknown functions, displayed a similar expression pattern with Trs4 in mouse testes. In particular, Trs4, Ddc8, and Rshl-2 proteins were co-localized to the tails of mature spermatozoa. These results suggested that Trs4 might be involved in diverse processes of spermiogenesis and/or fertilization through interactions with its multiple binding partners.

[Neuromodulation for treatment for neurogenic bowel dysfunction].

OBJECTIVE: To explore the efficacy of neuromodulation (including sacral neuromodulation and dorsal penile/clitoral nerve neuromodulation) for the treatment to neurogenic bowel dysfunction due to spinal cord injury. METHODS: From January 2006 to April 2008, 9 patients with neurogenic constipation after spinal cord injury underwent the therapy of neuromodulation, 1 patient underwent the therapy of sacral neuromodulation, 8 patients underwent the therapy of dorsal penile/clitoral nerve neuromodulation. The therapeutic efficacy was evaluated and followed up by means of Wexner constipation score. RESULTS: One patient received permanent electrode and neurostimulator implantation and constipation were improved continuously. A significant improvement in the Wexner constipation score was observed compared with the preoperative baseline level (preoperative baseline: median 22; after implantation: median 9). Four patients were effective after the therapy of dorsal penile/clitoral nerve neuromodulation. Wexner constipation score decrease from 19 to 11 after 12 weeks dorsal penile/clitoral nerve neuromodulation. Patients also showed a significant improvement in their symptoms and quality of life during follow up. CONCLUSIONS: Sacral neuromodulation and dorsal penile/clitoral nerve neuromodulation may be effective for some neurogenic constipation. However there are no methods successfully identify the candidate who will be beneficial before the procedure. Good quality research data are needed to evaluate the effects of sacral neuromodulation and dorsal penile/clitoral nerve neuromodulation for these conditions.

[Video-urodynamic studies on 1800 patients with neurogenic bladder].

OBJECTIVE: To explore the Video-urodynamic characteristics of various neurogenic bladder. METHODS: A total of 1800 patients with neurogenic bladder were included in our study from December 2002 to June 2008. All patients underwent Video-urodynamic studies. Urodynamic data was collected and analyzed. RESULTS: Urodynamic study showed detrusor overactivity in 71%, of which 60% with uninhibited sphincter relaxation, and acontractile detrusor in 29% stroke patients. No upper urinary tract deterioration was found in all 42 stroke patients. Detrusor overactivity without sphincter dyssynergia was found in 70% patients with head trauma. Seven patients with Parkinson disease showed detrusor overactivity, of which 3 with delayed sphincter relaxation. Detrusor overactivity was found in 91% and detrusor sphincter dyssynergia in 83% supra-sacral spinal cord injured patients. Acontractile detrusor was found in 73% patients with conus medullaris and cauda equina injury. Overall, upper urinary tract changes were found in 12% and vesicoureteral reflux in 4% spinal cord injured patients. Urodynamic study showed acontractile detrusor in 81%, reduced compliance in 86%, upper urinary tract changes in 55% and vesicoureteral reflux in 33% patients with myelodysplasia. Most patients (92%) with protruded lumbar disc showed detrusor areflexia. Normal bladder compliance was found in 88% patients with protruded lumbar disc. Urodynamic study showed reduced bladder sensation in 81% and detrusor under-activity in 76% patients with diabetic urinary bladder disease. CONCLUSIONS: Video-urodynamic study can provide the most detailed information about the bladder dysfunction. It is the most valuable examination before treatment of patients with neurogenic bladder.

Afaf, a novel vesicle membrane protein, is related to acrosome formation in murine testis.

As a cell-specific organelle, acrosome (Acr) and its formation are an important event for spermiogenesis. However, the Acr formation is far more complicated than has been proposed. In this study, we have cloned a novel membrane protein Afaf (Acr formation associated factor) that was expressed abundantly in the round spermatids, localized in the inner and outer membrane of forming Acrs, and declined in the maturing Acrs. In the transfected Hela cells, Afaf protein was localized in the plasma membrane, EEA1-positive early endosomes (EEs) and occasionally in the nuclei. Therefore, we propose that EEs and plasma membrane may be also directly involved in the Acr biogenesis.

In vitro propagation of spermatogonial stem cells from KM mice.

Spermatogonial stem cells (SSCs) are a unique type of stem cells in that they transmit genetic information to the next generation by producing sperms. Studies of SSC proliferation and differentiation have been hampered by the inability of reconstructing these processes in vitro, particularly in a serum-free culture system. Several groups have reported the long term culture of SSCs during which SSCs self-renew and restore spermatogenesis when transplanted back to recipient testes. However, different protocols and mice with particular genetic background have been used by different laboratories, and the techniques have not been adopted widely. In the present study, we first established a SSC isolation and culture system composed of differential adherence selection of SSCs, serum-free medium and mouse embryonic fibroblast (MEF) feeder cells. SSCs from KM pups could be cultured on MEF feeders in StemPro-34 SFM Medium supplemented with glial cell line-derived neurotrophic factor (GDNF), soluble GDNF family receptor alpha-1 (GFRa1) and basic fibroblast growth factor (bFGF) for 1 month. These SSCs were characterized morphologically and by examining the expression of marker genes. Expression of Oct4 and Sox2, which are crucial factors in embryonic stem cell (ESC) self-renewal, were detected in our cultured SSCs, suggesting that SSCs may share with ESCs some common mechanisms in self-renewal regulation. We also found that LIF had no effect on the proliferation of cultured SSCs derived from KM mice.

Cloning and characterization of a novel spermiogenesis-related gene, T6441, in rat testis.

We report in the present study the cloning and characterization of a novel gene, named T6441, initially derived by the suppressive subtracted hybridization (SSH) cDNA library. The full-length T6441cDNA was 664 bp long, containing a complete open-reading frame for a protein of 149 amino acids (aa). The protein bears no homology to any reported genes. It is predicted that the molecular mass was about 16.7 kDa. Northern blot analysis showed that the T6441 gene had about 4 transcripts in adult rat testis and was temporally regulated in a stage-dependent manner in the testis. In situ hybridization showed that T6441 mRNA was specifically localized in spermatids, and its expression level varied in the cells at different stages of the testicular development, with the highest level at steps 7-14. RT-PCR results showed that the T6441 mRNA was transcribed in most of the tested tissues with its strongest signal in the testis. Recombinant T6441 protein was prepared, purified, and was used to raise rabbit. Western blot analysis using the antiserum revealed four possible testicular specific proteins with their molecular weights being about 22, 25, 50 and 55 kDa respectively. The T6441 protein was expressed mainly in the cytoplasm of spermatids with the maximal levels at steps 12-19. At step 19 spermatid, the T6441 was mainly localized in the residual bodies. The cytoplasm localization of T6441 protein was supported by transient over expression of GFP-fusion protein in Hela cells. Interestingly, the expression of T6441 caused death of transfected cells within 48 h. Our preliminary experimental results suggest that the T6441 gene may play a role in cytoplasm movement and removal during spermiogenesis.

Activation of the p38 MAPK pathway by follicle-stimulating hormone regulates steroidogenesis in granulosa cells differentially.

In the present study, we started out to test whether the follicle-stimulating hormone (FSH)-activated p38 MAPK signaling cascade was involved in the regulation of steroidogenesis in granulosa cells (GCs). GCs were prepared from the ovaries of DES-treated immature rats and cultured in serum-free medium. Treatment of GCs with FSH (50 ng/ml) induced the phosphorylation of p38 MAPK rapidly with the phosphorylation being observed within 5 min and reaching the highest level at 30 min. Such activation was protein kinase A-dependent as indicated by the results using specific inhibitors. FSH stimulated the production of progesterone and estradiol as well as the expression of the steroidogenic acute regulatory protein (StAR) in a time-dependent manner, with a maximum level being observed in the production of progesterone and StAR at 48 h. Moreover, the potent p38 MAPK inhibitor SB203580 (20 microM) augmented FSH-induced progesterone and StAR production, while reduced FSH-induced estradiol production at the same time (P<0.01). RT-PCR data showed that inclusion of SB203580 in the media enhanced FSH-stimulated StAR mRNA production, while decreased the FSH-stimulated P450arom mRNA expression (P<0.05). Immunocytochemical studies showed that FSH treatment together with the inhibition of p38 MAPK activity resulted in a higher expression of StAR in mitochondria than FSH treatment alone. FSH also significantly up-regulated the protein level of LRH-1, a member of the orphan receptor family that activates the expression of P450arom in ovaries and testes. p38 MAPK inactivation down-regulated the basal and FSH-induced LRH-1 expression significantly. The intra-cellular level of DAX-1, another orphan receptor that inhibits StAR expression, also decreased upon p38 MAPK being inactivated. For the first time, the present study suggests that FSH-activated p38 MAPK signal pathway regulates progesterone and estrogen production in GCs differentially, and that the transcription factors LRH-1 and DAX-1 might play important roles in the process.

Expression of HSP105 and HSP60 during germ cell apoptosis in the heat-treated testes of adult cynomolgus monkeys (macaca fascicularis).

To confirm that transient increase in temperature of the testis (43C for 30 minutes once daily for 2 consecutive days) could induce apoptosis of germ cells in non-human primates and to investigate the possible roles of Hsp105 and Hsp60 in regulation of germ cell loss, we conducted the study on eight cynomolgus monkeys. The sperm concentration on day 28 after heat shock decreased to 8.4% of pretreatment levels and recovered to baseline on day 144. Using the TUNEL assay, increased numbers of apoptotic spermatocytes and round spermatids were detected on days 3, 8, and 30 post heat treatment. Hsp105 and Hsp60 mRNA and protein levels were analyzed using in situ hybridization, RT-PCR, immunohistochemical and Western blot methods. Hsp105 was confined to nuclei of spermatids before treatment, decreased dramatically with the loss of spermatids on days 3, 8, and 30, before returning to baseline levels on days 84 and 144. The expression of Hsp60 was high on days 3, 8, 30 and was only detected in Sertoli cells and spermatogonia. These results suggested that exposure of the testis to heat resulted in selective, but reversible damage to the seminiferous epithelium via increased germ cell apoptosis. Temporal changes in the expression pattern of Hsp105 and Hsp60 in relation to germ cell death suggests they may be involved in key processes in regulation of germ cell apoptosis.

Stem cell factor modulates the expression of steroidogenesis related proteins and FSHR during ovarian follicular development.

Stem cell factor (SCF) is essential for the development of primordial follicles. By using cultured ovaries from neonatal rats, the effect of SCF on early follicular development was investigated. Steroidogenesis is a hallmark of follicular development. Expressions of three key protein factors in steroidogenesis, SF-1, StAR, and P450arom, were investigated using immunohistochemistry and in situ hybridization. SF-1 and StAR proteins were expressed in all ovarian cells. P450arom mRNA was localized exclusively in oocytes implying that estrogen might be synthesized by oocytes at this stage. SCF up-regulated the mRNA and protein expression of these proteins, suggesting SCF might promote the production of estrogen during this period of time. To study the differentiation status of follicular cells, the expression of FSHR and its response to SCF treatment was examined by using semi-quantitative RT-PCR. The results showed that SCF inhibited the expression of FSHR mRNA. It was also observed that SCF stimulated the expression of basic fibroblast growth factor (bFGF) in oocytes. Inactivation of bFGF by its neutralizing antibody resulted in a reversal of the inhibitory effect of SCF on the expression of FSHR. Therefore, the FSHR inhibitory effect of SCF could be mediated by bFGF. In summary, it seems that, at the early stages of follicular development, SCF might stimulate oocytes to produce estrogen while it inhibits the differentiation of granulosa cells that are the major sources of estrogen at the late stages of follicular development.