Seungil Ro

Tissue-dependent paired expression of miRNAs

It is believed that depending on the thermodynamic stability of the 5′-strand and the 3′-strand in the stem-loop structure of a precursor microRNA (pre-miRNA), cells preferentially select the less stable one (called the miRNA or guide strand) and destroy the other one (called the miRNA* or passenger strand). However, our expression profiling analyses revealed that both strands could be co-accumulated as miRNA pairs in some tissues while being subjected to strand selection in other tissues. Our target prediction and validation assays demonstrated that both strands of a miRNA pair could target equal numbers of genes and that both were able to suppress the expression of their target genes. Our finding not only suggests that the numbers of miRNAs and their targets are much greater than what we previously thought, but also implies that novel mechanisms are involved in the tissue-dependent miRNA biogenesis and target selection process.

A Ca2+-activated Cl― conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity

Interstitial cells of Cajal (ICC) are unique cells that generate electrical pacemaker activity in gastrointestinal (GI) muscles. Many previous studies have attempted to characterize the conductances responsible for pacemaker current and slow waves in the GI tract, but the precise mechanism of electrical rhythmicity is still debated. We used a new transgenic mouse with a bright green fluorescent protein (copGFP) constitutively expressed in ICC to facilitate study of these cells in mixed cell dispersions. We found that ICC express a specialized ‘slow wave’ current. Reversal of tail current analysis showed this current was due to a Cl− selective conductance. ICC express ANO1, a Ca2+‐activated Cl− channel. Slow wave currents are not voltage dependent, but a secondary voltage‐dependent process underlies activation of these currents. Removal of extracellular Ca2+, replacement of Ca2+ with Ba2+, or extracellular Ni2+ (30 μm) blocked the slow wave current. Single Ca2+‐activated Cl− channels with a unitary conductance of 7.8 pS were resolved in excised patches of ICC. These are similar in conductance to ANO1 channels (8 pS) expressed in HEK293 cells. Slow wave current was blocked in a concentration‐dependent manner by niflumic acid (IC50= 4.8 μm). Slow wave currents are associated with transient depolarizations of ICC in current clamp, and these events were blocked by niflumic acid. These findings demonstrate a role for a Ca2+‐activated Cl− conductance in slow wave current in ICC and are consistent with the idea that ANO1 participates in pacemaker activity.

Many X-linked microRNAs escape meiotic sex chromosome inactivation

Meiotic sex chromosome inactivation (MSCI) during spermatogenesis is characterized by transcriptional silencing of genes on both the X and Y chromosomes in mid-to-late pachytene spermatocytes. MSCI is believed to result from meiotic silencing of unpaired DNA because the X and Y chromosomes remain largely unpaired throughout first meiotic prophase. However, unlike X-chromosome inactivation in female embryonic cells, where 25–30% of X-linked structural genes have been reported to escape inactivation, previous microarray- and RT-PCR–based studies of expression of >364 X-linked mRNA-encoding genes during spermatogenesis have failed to reveal any X-linked gene that escapes the silencing effects of MSCI in primary spermatocytes. Here we show that many X-linked miRNAs are transcribed and processed in pachytene spermatocytes. This unprecedented escape from MSCI by these X-linked miRNAs suggests that they may participate in a critical function at this stage of spermatogenesis, including the possibility that they contribute to the process of MSCI itself, or that they may be essential for post-transcriptional regulation of autosomal mRNAs during the late meiotic and early postmeiotic stages of spermatogenesis.

Sertoli cell Dicer is essential for spermatogenesis in mice

Spermatogenesis requires intact, fully competent Sertoli cells. Here, we investigate the functions of Dicer, an RNaseIII endonuclease required for microRNA and small interfering RNA biogenesis, in mouse Sertoli cell function. We show that selective ablation of Dicer in Sertoli cells leads to infertility due to complete absence of spermatozoa and progressive testicular degeneration. The first morphological alterations appear already at postnatal day 5 and correlate with a severe impairment of the prepubertal spermatogenic wave, due to defective Sertoli cell maturation and incapacity to properly support meiosis and spermiogenesis. Importantly, we find several key genes known to be essential for Sertoli cell function to be significantly down-regulated in neonatal testes lacking Dicer in Sertoli cells. Overall, our results reveal novel essential roles played by the Dicer-dependent pathway in mammalian reproductive function, and thus pave the way for new insights into human infertility.

Catsper3 and Catsper4 Are Essential for Sperm Hyperactivated Motility and Male Fertility in the Mouse

Abstract Catsper3 and Catsper4 are two recently identified testis-specific genes homologous to Catsper1 and Catsper2 that have been shown to play an essential role in sperm hyperactivated motility and male fertility in mice. Here we report that Catsper3 and Catsper4 knockout male mice are completely infertile due to a quick loss of motility and a lack of hyperactivated motility under capacitating conditions. Our data demonstrate that both CATSPER3 and CATSPER4 are required for hyperactivated sperm motility during capacitation and for male fertility. The present study also demands a revisit to the idiopathic male infertility patients who show normal sperm counts and normal initial motility for defects in sperm hyperactivated motility and for potential CATSPER gene mutations. The CATSPER channel also may be an excellent drug target for male contraceptives.

Kit signaling is essential for development and maintenance of interstitial cells of Cajal and electrical rhythmicity in the embryonic gastrointestinal tract

Interstitial cells of Cajal (ICC) are specialized cells in smooth muscle organs that generate and propagate pacemaker activity, receive inputs from motor neurons, and serve as mechanosensors. In the gastrointestinal tract, development and maintenance of the ICC phenotype have been linked to intracellular signaling via Kit, but its role in development of ICC during embryogenesis is controversial. Here we have studied the development of functional ICC‐MY during the late gestational period in mice. Blocking Kit with a neutralizing antibody before and after development of spontaneous electrical activity (E17 to P0) caused loss of ICC‐MY networks and pacemaker activity. ICC‐MY and pacemaker activity developed normally in W/+ and WV/+ heterozygotes, but failed to develop between E17 to P0 in W/WV embryos with compromised Kit function. Muscles treated with Kit neutralizing antibody or the tyrosine kinase inhibitor, imatinib mesylate (STI571), from E17‐P0 for 3 days caused loss of functionally developed ICC‐MY networks, but ICC‐MY and pacemaker activity recovered within 9 days after discontinuing treatment with neutralizing antibody or imatinib mesylate. These data suggest that Kit signaling is an important factor in lineage decision and in the development of functional ICC in late gestation. ICC‐MY demonstrate significant plasticity in gastrointestinal tissues. Manipulation of the ICC phenotype might provide useful therapies in gastrointestinal disease where the Kit‐positive cell population is either lost or amplified. Developmental Dynamics, 2006.

The mitochondrial genome encodes abundant small noncoding RNAs

Small noncoding RNAs identified thus far are all encoded by the nuclear genome. Here, we report that the murine and human mitochondrial genomes encode thousands of small noncoding RNAs, which are predominantly derived from the sense transcripts of the mitochondrial genes (host genes), and we termed these small RNAs mitochondrial genome-encoded small RNAs (mitosRNAs). DICER inactivation affected, but did not completely abolish mitosRNA production. MitosRNAs appear to be products of currently unidentified mitochondrial ribonucleases. Overexpression of mitosRNAs enhanced expression levels of their host genes in vitro, and dysregulated mitosRNA expression was generally associated with aberrant mitochondrial gene expression in vivo. Our data demonstrate that in addition to 37 known mitochondrial genes, the mammalian mitochondrial genome also encodes abundant mitosRNAs, which may play an important regulatory role in the control of mitochondrial gene expression in the cell.

Birth of Mice after Intracytoplasmic Injection of Single Purified Sperm Nuclei and Detection of Messenger RNAs and MicroRNAs in the Sperm Nuclei

We have developed a method that effectively removes all of the perinuclear materials of a mouse sperm head, including the acrosome, plasma membrane, perinuclear theca, and nuclear envelope. By injection of a single purified sperm head into a metaphase II mouse oocyte followed by activation with strontium chloride, 93% of the zygotes developed into two-cell embryos. Although only approximately 17% of the transferred two-cell embryos were born alive, all live pups developed into adults, and they appeared to be normal in reproduction and behavior. We detected RNA species, including mRNAs and miRNAs from the purified sperm heads. Our data demonstrate that pure membrane-free sperm heads are sufficient to produce normal offspring through intracytoplasmic sperm injection and that at least part of the RNA molecules are deeply embedded in the sperm nucleus.

A Model to Study the Phenotypic Changes of Interstitial Cells of Cajal in Gastrointestinal Diseases

BACKGROUND & AIMS Interstitial cells of Cajal (ICC) express the receptor tyrosine kinase, KIT, the receptor for stem cell factor. In the gastrointestinal (GI) tract, ICC are pacemaker cells that generate spontaneous electrical slow waves, and mediate inputs from motor neurons. Absence or loss of ICC are associated with GI motility disorders, including those consequent of diabetes. Studies of ICC have been hampered by the low density of these cells and difficulties in recognizing these cells in cell dispersions. METHODS Kit(+/copGFP) mice harboring a copepod super green fluorescent protein (copGFP) complementary DNA, inserted at the Kit locus, were generated. copGFP(+) ICC from GI muscles were analyzed using confocal microscopy and flow cytometry. copGFP(+) ICC from the jejunum were purified by a fluorescence-activated cell sorter and validated by cell-specific markers. Kit(+/copGFP) mice were crossbred with diabetic Lep(+/ob) mice to generate compound Kit(+/copGFP);Lep(ob/ob) mutant mice. copGFP(+) ICC from compound transgenic mice were analyzed by confocal microscopy. RESULTS copGFP in Kit(+/copGFP) mice colocalized with KIT immunofluorescence and thus was predominantly found in ICC. In other smooth muscles, mast cells were also labeled, but these cells were relatively rare in the murine GI tract. copGFP(+) cells from jejunal muscles were Kit(+) and free of contaminating cell-specific markers. Kit(+/copGFP);Lep(ob/ob) mice displayed ICC networks that were dramatically disrupted during the development of diabetes. CONCLUSIONS Kit(+/copGFP) mice offer a powerful new model to study the function and genetic regulation of ICC phenotypes. Isolation of ICC from animal models will help determine the causes and responses of ICC to therapeutic agents.

Novel voltage-dependent non-selective cation conductance in murine colonic myocytes

Two components of voltage‐gated, inward currents were observed from murine colonic myocytes. One component had properties of L‐type Ca2+ currents and was inhibited by nicardipine (5 × 10−7m). A second component did not ‘run down’ during dialysis and was resistant to nicardipine (up to 10−6m). The nicardipine‐insensitive current was activated by small depolarizations above the holding potential and reversed near 0 mV. This low‐voltage‐activated current (ILVA) was resolved with step depolarizations positive to ‐60 mV, and the current rapidly inactivated upon sustained depolarization. The voltage of half‐inactivation was ‐65 mV. Inactivation and activation time constants at ‐45 mV were 86 and 15 ms, respectively. The half‐recovery time from inactivation was 98 ms at ‐45 mV. ILVA peaked at ‐40 mV and the current reversed at 0 mV. I lva was inhibited by Ni2+ (IC50= 1.4 × 10−5m), mibefradil (10−6 to 10−5m), and extracellular Ba2+. Replacement of extracellular Na+ with N‐methyl‐d‐glucamine inhibited ILVA and shifted the reversal potential to ‐7 mV. Increasing extracellular Ca2+ (5 × 10−3m) increased the amplitude of ILVA and shifted the reversal potential to +22 mV. ILVA was also blocked by extracellular Cs+ (10−4m) and Gd3+ (10−6m). Warming increased the rates of activation and deactivation without affecting the amplitude of the peak current. We conclude that the second component of voltage‐dependent inward current in murine colonic myocytes is not a ‘T‐type’ Ca2+ current but rather a novel, voltage‐gated non‐selective cation current. Activation of this current could be important in the recovery of membrane potential following inhibitory junction potentials in gastrointestinal smooth muscle or in mediating responses to agonists.