Yung Ming Lin

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.

Transitional Cell Carcinoma in Dialysis Patients

Objective: The aim of our study was to determine whether there is an increased incidence of urothelial cancer, especially transitional cell carcinoma (TCC), in uremic patients on dialysis.Methods: Retrospective chart analyses were completed for 1,910 uremic patients undergoing maintenance dialysis between January 1987 and December 1997. The incidence of urinary tract cancer was assessed. Only the patients with cancers diagnosed after start of dialysis were enrolled in the study.Results: Of the 1,910 patients, 70 had concomitant urinary tract cancers. Nineteen patients (0.99%), including 17 patients with TCC and 2 patients with renal cell carcinoma, were diagnosed after the initiation of dialysis. The average duration from dialysis to TCC diagnosis was 38.3 (range 2–144) months. Painless gross hematuria was the cardinal symptom in 16 of the 17 patients with TCC. In the 17 patients with TCC, no distant metastases were found at the time of diagnosis. Fourteen patients (82.3%) were stage 0 or A, and 1 patient was stage B1.Conclusions: The 0.89% incidence of TCC in our dialysis patients was high as compared with that of the general population. The risks of developing urinary TCC in dialysis patients were examined, and we suggest that immunosuppressive stage, dialysis procedure, and chronic bladder irritation (decreased urinary wash effect) may play a part in the development of urinary TCC in dialysis patients. Early detection of hematuria due to regular visits and decreased exposure of urinary tract epithelium to carcinogens from urine may explain why early–stage TCC was seen in most of our patients.

The Expression Level of Septin12 Is Critical for Spermiogenesis

Septins belong to a family of polymerizing GTP-binding proteins that are required for many cellular functions, such as membrane compartmentalization, vesicular trafficking, mitosis, and cytoskeletal remodeling. One family member, septin12, is expressed specifically in the testis. In this study, we found septin12 expressed in multiple subcellular compartments during terminal differentiation of mouse germ cells. In humans, the testicular tissues of men with either hypospermatogenesis or maturation arrest had lower levels of SEPTIN12 transcripts than normal men. In addition, increased numbers of spermatozoa with abnormal head, neck, and tail morphologies lacked SEPT12 immunostaining signals, as compared with normal spermatozoa. To elucidate the role of septin12, we generated 129 embryonic stem cells containing a septin12 mutant allele with a deletion in the exons that encode the N-terminal GTP-binding domain. Most chimeras derived from the targeted embryonic stem cells were infertile, and the few fertile chimeras only produced offspring with a C57BL/6 background. Semen analysis of the infertile chimeras showed a decreased sperm count, decreased sperm motility, and spermatozoa with defects involving all subcellular compartments. The testicular phenotypes included maturation arrest of germ cells at the spermatid stage, sloughing of round spermatids, and increased apoptosis of germ cells. Electron microscopic examination of spermatozoa showed misshapen nuclei, disorganized mitochondria, and broken acrosomes. Our data indicate that Septin12 expression levels are critical for mammalian spermiogenesis.

Adrenal myelolipoma with translocation (3;21)(q25;p11)

Adrenal myelolipoma (ML) is a rare, benign, nonfunctioning tumor-like lesion composed of mature adipose tissue interspersed with bone marrow-like hematopoietic elements in various proportions. It occurs usually in adults and is frequently asymptomatic in about half of cases. The histogenesis of adrenal ML is not clear and this lesion has been found to be associated with endocrine disorders, other adrenal dysfunction and tumors, and hyperstimulation with adrenocorticotropic hormone. Specific chromosomal abnormalities, however, have not been observed in such cases. Herein, we report a typical case of adrenal ML found incidentally in a 26-year-old man. Conventional cytogenetic techniques demonstrated balanced translocation between bands 3q25 and 21p11 in 9 of 20 metaphases analyzed in cultured tumor cells. To the best of our knowledge, this is the first reported case of adrenal ML showing chromosomal abnormality. This finding would indicate that adrenal ML is a bona fide neoplasm and the possibility of derivation from misplaced hematopoietic cells may be alternatively taken into consideration in view of the similar genetic changes in hematopolietic neoplasms.

Gene-based screening for Y chromosome deletions in Taiwanese men presenting with spermatogenic failure

OBJECTIVE To develop a simple and rapid protocol for detecting deletions of the Y chromosome and to evaluate the feasibility of gene-based screening in men with spermatogenic failure. DESIGN Prospective case study. SETTING University-based reproductive clinics and genetics laboratory. PATIENT(S) Two hundred two infertile men presenting with severe oligozoospermia and nonobstructive azoospermia. INTERVENTION(S) Fifteen gene-specific primers were used to detect deletions of Y chromosome genes in men with spermatogenic failure. A multiplex polymerase chain reaction amplification system was developed to facilitate rapid screening. Another 24 markers for sequence-tagged sites (STS) were used to ensure the adequacy of gene-based screening. MAIN OUTCOME MEASURE(S) Detection of deletions of Y chromosome genes. RESULT(S) Of 180 patients evaluated, 19 (10.6%) had deletions of one or more genes, including DFFRY, DBY, RBM1, DAZ, CDY1, and BPY2. A second round of STS-based screenings did not show an increase in the deletion rate but more clearly defined the extent of deletion in 14 of the 19 patients. In most patients, deletions detected by gene-based screening were similar to those detected by STS markers. CONCLUSION(S) Gene-based screening with multiplex polymerase chain reaction is a rational alternative for detecting deletions of Y chromosome genes in infertile men.

Presence of DAZL transcript and protein in mature human spermatozoa

OBJECTIVE To identify the DAZL transcript and protein location in human spermatozoa. DESIGN In vitro experiment. SETTING University-based reproductive genetics laboratory. PATIENT(S) A fertile volunteer. INTERVENTION(S) Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunostaining for DAZL. MAIN OUTCOME MEASURE(S) Expression of DAZL in human spermatozoa. RESULT(S) The DAZL-specific primers yield a 128 bp product in ejaculate. A protein of approximately 33.5 kDa was detected by Western blot analysis. Immunofluorescence staining showed strong homogeneous staining in the midpiece of spermatozoa and weak staining in the principal piece. A speckled-type distribution was found in the head region. CONCLUSION(S) The DAZL transcript and protein are present in human spermatozoa. The roles of DAZL protein in sperm motility and in the sperm-oocyte interaction await further investigation.

Incomplete cre-mediated excision leads to phenotypic differences between Stra8-iCre; Mov10l1lox/lox and Stra8-iCre; Mov10l1lox/Δ mice

In the Cre–loxp system, expression level and activity of Cre recombinase in a Cre deleter line are critical because these determine not only the cell specificity of gene knockout (KO), but also the efficiency of Cre‐mediated excision in a specific cell lineage. Although the spatiotemporal expression pattern of a Cre transgene is usually defined upon the generation of the mouse line, the Cre excision efficiency in a specific targeted cell lineage is rarely evaluated and often assumed to be 100%. Incomplete excision can lead to highly variable phenotypes owing to mosaicism (i.e., coexistence of cells with the flox or the recombined flox allele) and this problem has long been overlooked. Here, we report that Stra8‐codon‐improved Cre recombinase (iCre), a transgenic allele expressing iCre under the control of the male germ cell‐specific Stra8 promoter, could efficiently delete one Mov10l1 flox allele in spermatogenic cells, whereas the excision was incomplete when two Mov10l1 flox alleles were present. The incomplete Cre‐mediated excision led to a testicular phenotype that was much less severe than that in the true conditional KO (inactivation, 100%) mice. Our findings suggest that it is essential to determine the efficiency of Cre excision when Cre–loxp system is used for deleting genes in a specific cell lineage and the Cre; genelox/Δ genotype should be used to evaluate phenotypes instead of Cre; genelox/lox owing to the fact that the latter usually bears incomplete deletion of the flox allele(s). genesis 51:481–490.

Fibroblast growth factor 9 stimulates steroidogenesis in postnatal Leydig cells.

Fibroblast growth factor 9 (FGF9) is a potent mitogen and survival factor required for morphogenesis during embryonic development and numerous biological functions at adulthood. The reproductive phenotype of mice lacking Fgf9 gene exhibits male to female sexual reversal, suggesting a crucial role of Fgf9 in male sex determination. Our previous study showed that polymorphic microsatellite of FGF9 genes is associated with 46XY female with ambiguous genitalia, implying that the aberrant expression of FGF9 might affect androgen secretion. In this study, we aimed to investigate the effect of FGF9 on testosterone production in mouse Leydig cell and to study the signalling pathways by which FGF9 modulate steroidogenesis. Our results show that mRNAs of Fgf9 and Fgfr isoforms (Fgfr2IIIc, Fgfr3 and Fgfr4) were all expressed in mouse Leydig cells. FGF9 significantly stimulates mouse Leydig cell testosterone production in a dose- and time-dependent manner. Ras-MAPK, PI3K and PKA signalling pathways are involved in the FGF9-induced steroidogenesis. These results provide supportive evidence linking the aberrant expression of FGF9 to human gonadal dysgenesis and suggest a role of FGF9 in postnatal testicular development.

Y-chromosome microdeletion and its effect on reproductive decisions in Taiwanese patients presenting with nonobstructive azoospermia

OBJECTIVES To investigate the position, extent, and frequency of Y chromosome microdeletions in Taiwanese patients presenting with nonobstructive azoospermia, and to investigate the effect of microdeletions on reproductive decisions. METHODS We studied 176 consecutive men with azoospermia in our urology clinic. Polymerase chain reaction tests were performed in 94 patients with nonobstructive azoospermia, and a series of 27 sequence-tagged sites (STSs) mapped within intervals 5 and 6 of Yq11 was selected for analysis. Clinical genetics counseling was provided to couples with microdeletions, and these couples made their own choices about further treatment modalities. RESULTS Among 94 patients screened for microdeletion, 11 (11.7%) showed microdeletions of one or more STSs. One had a deletion confined to the azoospermia factor b (AZFb) region (encompassing the RBM gene). Two were found to have deletions of both the AZFb and AZFc regions. Eight patients had deletions in the AZFc region (encompassing the DAZ gene). Five had deletions distal to the DAZ gene family. One had multiple, noncontiguous deletions. In 8 patients with testicular histology available, a lack of genotype/phenotype correlation was noted. Of the 11 couples with deletions, 3 thought microdeletion was a serious defect and opted for an artificial insemination of donor or adoption, 5 chose intracytoplasmic sperm injection, and the other 3 decided to undergo treatment with Chinese medicinal herbs. CONCLUSIONS The most commonly deleted region in the Taiwanese population is AZFc. The genes implicated in Taiwanese spermatogenesis defects are the DAZ and RBM gene families. Twenty-seven percent of couples with microdeletions deferred assisted reproductive technologies because of concern about their underlying genetic defects.

The prophylactic protective effect of sesamol against ferric–nitrilotriacetate-induced acute renal injury in mice

The aim of this study was to examine the prophylactic protective effects of 3,4-methylenedioxyphenol (sesamol) on ferric-nitrilotriacetate (Fe-NTA)-induced acute renal damage in mice. We induced acute renal injury in mice by treating them with 4 mg/kg of Fe-NTA for 3h. We used blood biochemistry, creatinine clearance, and histological examinations to assess renal function. With a high-performance chemiluminescence analyzer, we also determined the hydroxyl radical and superoxide anion levels (free radicals) generated. Renal xanthine oxidase activities were also assessed. Sesamol inhibited Fe-NTA-induced acute renal injury, renal lipid peroxidation, the levels of renal hydroxyl radical and superoxide anion generated, and the activity of xanthine oxidase in mice. Therefore, we concluded that sesamol protected mice against Fe-NTA-induced oxidative-stress-associated acute renal injury by at least partially inhibiting the production of reactive oxygen species.