Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility.

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

A de novo paradigm for male infertility.

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10-5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10-4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Prostate involvement during sexually transmitted infections as measured by prostate-specific antigen concentration.

BACKGROUND: We investigated prostate involvement during sexually transmitted infections by measuring serum prostate-specific antigen (PSA) as a marker of prostate infection, inflammation, and/or cell damage in young, male US military members. METHODS: We measured PSA before and during infection for 299 chlamydia, 112 gonorrhoea, and 59 non-chlamydial, non-gonococcal urethritis (NCNGU) cases, and 256 controls. RESULTS: Chlamydia and gonorrhoea, but not NCNGU, cases were more likely to have a large rise (40%) in PSA than controls (33.6%, 19.1%, and 8.2% vs 8.8%, P<0.0001, 0.021, and 0.92, respectively). CONCLUSION: Chlamydia and gonorrhoea may infect the prostate of some infected men.

mRNA nuclear export.

The export of mRNA from the nucleus to the cytoplasm is an essential step in the expression of genetic information in eukaryotes. It is an energy-dependent process and involves transport across the nuclear pores. It requires both cis-acting ribonucleoprotein particle signals and specific trans-acting factors. Although much remains to be learned, recent information has begun to define this pathway at both the cellular and biochemical levels and indicates that it is used as a key regulatory step by several viruses.

The tumour-suppressor protein ASPP1 is nuclear in human germ cells and can modulate ratios of CD44 exon V5 spliced isoforms in vivo.

The ASPP1 (Apoptosis Stimulating Protein of p53) protein is an important tumour-suppressor. We have detected a novel protein interaction between the human ASPP1 (hASPP1) protein and the predominantly nuclear adaptor protein SAM68. In the human testis, full-length endogenous hASPP1 protein is located in the nucleus like SAM68, predominantly within meiotic and postmeiotic cells. Mouse ASPP1 (mASPP1) protein is mainly expressed in the brain and testis. The interaction with nuclear SAM68 is likely to be restricted to human germ cells, since endogenous mASPP1 protein is exclusively cytoplasmic. The C-terminal region of hASPP1 efficiently targeted a fused GFP molecule to the nucleus, whereas the N-terminus of hASPP1 targeted GFP to the cytoplasm. In the context of the full-length molecule this cytoplasmic targeting sequence is dominant in HEK293 and Saos-2 cells, since full-length hASPP1-GFP is almost exclusively cytoplasmic. Despite its predominantly cytoplasmic location, we show that ASPP1-GFP expression in HEK293 cells can regulate the ratio of alternative spliced isoforms derived from a pre-mRNA regulated downstream of cytoplasmic signalling pathways, and our data suggest that ASPP1 may operate in this case downstream or parallel to RAS signalling pathways.

Mutually exclusive synthetic pathways for sea urchin mitochondrial rRNA and mRNA.

The structure and abundance of mitochondrial transcripts in sea urchin embryos were investigated by a combination of RNA blot-hybridization, S1 mapping, and primer extension assays. Between the egg and blastula stages, the relative abundance of mitochondrial rRNAs declined slightly, while that of mitochondrial mRNAs increased up to 10-fold. Fine mapping of the termini of the rRNAs and of the adjacent transcripts indicated that, although they appeared to be butt-joined at their 5' ends to the upstream transcripts, tRNA-Phe 5' to the small subunit (12S) rRNA and NADH dehydrogenase subunit 2 mRNA 5' to the large subunit (16S) rRNA, respectively, their 3' ends were found to overlap the 5' ends of the downstream transcripts. 12S rRNA was found to extend 7 to 13 nucleotides into the sequence of tRNA-Glu; 16S rRNA was shown to terminate 3 to 5 nucleotides inside the coding region of cytochrome oxidase subunit 1 (COI) and 8 to 10 nucleotides from the mapped 5' end of COI mRNA. The rRNAs and the downstream transcripts must therefore be synthesized by distinct pathways, either by alternative processing of the same primary transcript(s) or by processing of different precursors. In either case, the events which select the ribosomal 3' ends preclude the production of functional transcripts of the downstream genes from the same precursor molecule. No developmental alterations in transcript structure were detected. We propose that mitochondrial RNA levels are regulated in early development by the selection of alternate and mutually exclusive RNA-processing pathways.

Longitudinal assessment of urinary PCA3 for predicting prostate cancer grade reclassification in favorable-risk men during active surveillance.

BACKGROUND: To assess the utility of urinary prostate cancer antigen 3 (PCA3) as both a one-time and longitudinal measure in men on active surveillance (AS). METHODS: The Johns Hopkins AS program monitors men with favorable-risk prostate cancer with serial PSA, digital rectal examination (DRE), prostate magnetic resonance imaging and prostate biopsy. Since 2007, post-DRE urinary specimens have also been routinely obtained. Men with multiple PCA3 measures obtained over ⩾3 years of monitoring were included. Utility of first PCA3 score (fPCA3), subsequent PCA3 (sPCA3) and change in PCA3 were assessed for prediction of Gleason grade reclassification (GR, Gleason score >6) during follow-up. RESULTS: In total, 260 men met study criteria. Median time from enrollment to fPCA3 was 2 years (interquartile range (IQR) 1-3) and from fPCA3 to sPCA3 was 5 years (IQR 4-6). During median follow-up of 6 years (IQR 5-8), 28 men (11%) underwent GR. Men with GR had higher median fPCA3 (48.0 vs 24.5, P=0.007) and sPCA3 (63.5 vs 36.0, P=0.002) than those without GR, while longitudinal change in PCA3 did not differ by GR status (log-normalized rate 0.07 vs 0.06, P=0.53). In a multivariable model including age, risk classification and PSA density, fPCA3 remained significantly associated with GR (log(fPCA3) odds ratio=1.77, P=0.04). CONCLUSIONS: PCA3 scores obtained during AS were higher in men who underwent GR, but the rate of change in PCA3 over time did not differ by GR status. PCA3 was a significant predictor of GR in a multivariable model including conventional risk factors, suggesting that PCA3 provides incremental prognostic information in the AS setting.

Nucleotide sequence and gene organization of sea urchin mitochondrial DNA.

The 15,650 base-pair mitochondrial genome of the sea urchin Strongylocentrotus purpuratus has been cloned and sequenced. It exhibits a novel organization that suggests the primacy of post-transcriptional gene regulation. The same 13 polypeptides, two rRNAs and 22 tRNAs are encoded as in other animal mitochondrial DNAs, but are organized with extreme economy; non-coding information between genes is almost completely absent, some stop codons are generated post-transcriptionally and tRNA sequences are interspersed between only a minority of other structural genes. The genome uses a variant genetic code, in which AAA specifies asparagine, ATA isoleucine, TGA tryptophan and AGN serine, and has an unusual pattern of codon bias. The order of genes shows several differences from that of vertebrates. The genes for the large (16 S) ribosomal RNA and for NADH dehydrogenase subunit 4L (ND4L) are in different positions, located respectively between those encoding ND2 and cytochrome oxidase subunit I (COI) and between COI and COII. This organization is conserved amongst at least four regular echinoids diverging by some 225 million years. Most tRNA genes are also in different positions. The only long unassigned sequence in the genome (121 base-pairs) is located within a cluster of 15 tRNA genes. It contains elements resembling some of those found in the displacement (D) loop of vertebrate mtDNAs, notably polypurine/polypyrimidine tracts that may play a role in regulating transcription and the initiation of replication. The separation of the ribosomal RNA genes from each other and from the putative control region imposes special demands on the transcription of the genome.

Does Rbmy have a role in sperm development in mice?

The Y(d1) deletion in mice removes most of the multi-copy Rbmy gene cluster that is located adjacent to the centromere on the Y short arm (Yp). XY(d1) mice develop as females because Sry is inactivated, probably because it is now juxtaposed to centromeric heterochromatin. We have previously produced XY(d1)Sry transgenic males and found that they have a substantially increased frequency of abnormal sperm. Staining of testis sections with a polyclonal anti-RBMY antibody appeared to show a marked decrease of RBMY protein in the spermatids of XY(d1)Sry males compared to control males, which led us to suggest that this may be responsible for the increase in sperm anomalies. In the current study we sought to determine whether augmenting Rbmy expression specifically in the spermatids of XY(d1)Sry males would ameliorate the sperm defects. An expressing Rbmy transgene driven by the spermatid-specific mouse protamine 1 promotor (mP1Rbmy) was therefore introduced into XY(d1)Sry males. This failed to reduce the frequency of abnormal sperm. In the course of this study, a new RBMY antibody was generated that, in contrast to the original antibody, failed to detect RBMY in spermatid stages by immunostaining. The lack of RBMY was confirmed by western blotting of lysates from purified round spermatids and elongating spermatids. The implications of these results for the proposed role for RBMY in sperm development are discussed.

The contribution of RNA-binding motif (RBM) antibody to the histopathologic evaluation of testicular biopsies from infertile men.

Testicular biopsies of infertile men are often characterized by mixed histologic patterns, with different types of spermatogenic impairments being found in adjacent seminiferous tubules. RNA-binding motif (RBM) is a nuclear protein expressed exclusively in the male germ cell line. We reasoned that RBM might be a useful marker to identify germ cells in testicular sections, particularly in biopsies with mixed histologic phenotype and small focal concentrations of spermatogenesis. Testicular biopsies from azoospermic men were immunohistochemically evaluated for RBM expression. RBM expression was detectable in spermatogonia, spermatocytes, and round spermatids in biopsies of men with obstructive azoospermia and normal spermatogenesis. No specific cell staining was shown in cases of Sertoli-cell-only (SCO) syndrome. In biopsies of patients with spermatogenic disorders, all the germ cells were stained up to and including the stage level of the arrest in spermatogenesis. This approach enabled identification of small focal concentrations of spermatogenesis in a biopsy previously classified as being SCO by hematoxylin and eosin staining. Thus, RBM can be a useful immunohistochemical marker for the specific identification of germ cells and provide greater accuracy in the histopathologic evaluation of testicular biopsies.

Absence of RBM expression as a marker of intratubular (in situ) germ cell neoplasia of the testis.

RBM (RNA-binding motif) protein is a marker of male germ cells. This protein is encoded by the Azoospermia factor region-b (AZF-b) of the human Y chromosome and is expressed exclusively in the male germ cell line, that is, spermatogonia, spermatocytes, and round spermatids. The authors analyzed the expression of the RBM gene in germ cell tumors and in the seminiferous tubules in the vicinity of these tumors to identify the presence of IGCN. Sections from testicular germ cell tumors of 21 patients were stained with anti-RBM antibody by using an immunohistochemical method. Distal tubules showing spermatogenesis were immunopositive for RBM protein. All of the germ cell tumors studied were completely immunonegative for RBM. Defined areas of IGCN also showed an absence of RBM expression. Tubules with spermatocyte-like cells, which were expected to express RBM, did not express this protein. This result enabled the identification of tubules as being IGCN. RBM is a novel marker consistently expressed in normal male germ cells but not in malignant germ cell tumors or IGCN. Thus, the absence of RBM expression in germ cells provides a new diagnostic tool of preinvasive malignancy of the testis.

Splicing and the single cell.

The selection of alternative splice sites is an important component of cell-specific gene regulation in eukaryotic cells. Use of splice sites can be positively and negatively regulated, and often physiologically appropriate splice site choice is achieved by a balance of the two. RNA elements controlling splice site choice are found in both exons and introns, and these determine management by the cellular splicing machinery. However, the molecular basis of how the splicing machinery responds to these signals in different cells is somewhat of a paradox. Thus far the identified proteins which bind to tissue/cell-specific regulatory elements in mammals are expressed in many different tissues, and not just in the regulating tissue. Potential tissue-specific splicing regulators have been identified by non-biochemical means. However, alternative splicing choices are likely to be affected by subtle differences in the splicing machinery in different cells. In this review I suggest that one important factor is the ratio of proteins in different nuclear compartments, which might be established in a cell type specific fashion.

Understanding the genes involved in spermatogenesis: a progress report.

OBJECTIVE: To review the current literature on genes known to affect fertility in the human and mouse. DESIGN: A literature review was performed and key articles were chosen for focus in the areas of genes with effects only on spermatogenesis and oogenesis, with an emphasis on Y-chromosome-encoded gene families and spermatogenesis. In addition, studies describing genes deleted in transgenic mice were incorporated. RESULT(S): Several gene families on the Y chromosome are implicated in spermatogenic failure, but the link between the genetic lesion and the resulting defect is unclear. Many mouse genes involved in repair and DNA damage monitoring have specific effects on gametogenesis in and around meiosis. CONCLUSION(S): Many genes are involved only in gametogenesis, and some of these are beginning to be understood in terms of their functions. An even larger number of genes is required for gametogenesis, and other functions and mouse models give insights important for human disease.

Intratubular germ cell neoplasia: associated infertility and review of the diagnostic modalities.

The incidence of testicular neoplasia has increased, and its early detection has become a pressing clinical issue. The strong association between male subfertility and risk of testicular neoplasia is consistent with the existence of common pathogenetic factors. Most forms of testicular germ tumors are believed to stem from a common precursor, intratubular germ cell neoplasia (ITGCN), also known as testicular carcinoma in situ. Identification of ITGCN cells in testicular biopsies, however, is a diagnostic challenge and markers are sorely needed to assist in the accurate identification of the lesion.

Variable photosynthetic characteristics in waste stabilisation ponds.

Algae play several key roles in waste stabilisation ponds. A model has been developed to predict algal concentration in waste stabilisation ponds, in which the relationship between photosynthesis and underwater light availability is central. One equation was selected from several alternative expressions that describe this relationship. The selected equation consisted of four photosynthetic parameters. A field sampling programme was designed to investigate the relationships between the photosynthetic parameters and the pond environment. Although initial regression analyses were unsuccessful, distinct diurnal variations were revealed in two key photosynthetic parameters, related to an inverse variation in chlorophyll a concentration. This led to the derivation of a dynamic feedback hypothesis which challenges the classic assumption in algal modelling of constant photosynthetic parameters.

The RNA-binding and adaptor protein Sam68 modulates signal-dependent splicing and transcriptional activity of the androgen receptor.

The RNA-binding protein Sam68 has been reported to be up-regulated in clinical cases of prostate cancer (PCa), where it is thought to contribute to cell proliferation and survival. Consistent with this, we observed over-expression of Sam68 in a panel of clinical prostate tumours as compared with benign controls. Since Sam68 is implicated in a number of signalling pathways, we reasoned that its role in PCa may involve modulation of the androgen receptor (AR) signalling cascade, which drives the onset and progression of PCa. We found that Sam68 interacts with the AR in vivo in LNCaP cells, and is dynamically recruited to androgen response elements within the promoter region of the prostate-specific antigen (PSA) gene. Based on its known functions and nuclear location, Sam68 might either: (a) co-regulate AR-dependent transcription positively or negatively; or (b) modulate AR-dependent alternative splicing by enhancing incorporation of a Sam68-responsive exon transcribed under the control of an androgen-responsive promoter. We tested these possibilities using functional assays. Both wild-type Sam68 protein and the Sam68(V229F) mutant, which is impaired in RNA binding, functioned as a ligand-dependent AR co-activator on an androgen-regulated reporter gene. In contrast, splicing of a Sam68-responsive variable exon, transcribed under control of an androgen-responsive promoter, was strongly repressed in the presence of AR and androgens. This splicing inhibition was reversed by ectopic expression of Sam68 but enhanced by Sam68(V229F). These results demonstrate that Sam68 has separable effects on AR-regulated transcriptional activity and alternative splicing, both of which may affect PCa phenotypes.

RBMY genes and AZFb deletions.

Microdeletions of the AZFb region of the human Y chromosome usually result in severe consequences for spermatogenesis. AZFb contains at least four kinds of genes/gene families. These include a number of RBMY genes, which are clustered in the AZFb deletion interval. They are amongst the oldest genes on the mammalian Y chromosome, and are related to the gene encoding hnRNPG (RBMX) on the X chromosome. A retroposon-derived version of these genes is found on chromosome 11 that might replace the function of these genes during meiosis, during which time the X and Y chromosomes are transcriptionally inactivated. Each of these genes encodes proteins with an RNA binding motif, and interacts with more ubiquitously expressed proteins involved in pre-mRNA splice site selection. These findings imply that important pre-mRNA processing pathways might be disrupted in the germ cells of AZFb men.

Control of energy absorption rate in transmission line radiofrequency exposure systems.

A frequent problem in the radiofrequency (RF) irradiation of experimental animals in health effects studies is the temporal variation of the specific absorption rate (SAR) with animal movement. An RF power controller that regulates the energy absorption rate has been designed for use with transmission line exposure systems that utilize the power difference method to monitor the SAR. The controller operates by altering the incident power to the exposure cell in order to compensate for the change in RF energy absorption rate that is due to animal motion. A circuit diagram is presented as well as experimental data under three conditions of exposure. The controller is effective in maintaining the mean value of energy absorption rate at the setpoint value even for the case of a highly active animal.

Depressive characteristics of sexually abused children.

Allen and Tarnowski (1988) identified a pattern of depressive characteristics that distinguished physically abused from nonabused children. The present study attempted to extend the generality of these findings to sexually abused children. Sexually abused and nonabused children were compared on measures of depression, hopelessness, and self-esteem. Results indicated that while sexually abused children self-reported more depressive symptoms in comparison to nonabused controls, that these differences were not statistically significant and failed to replicate the findings noted for physically abused children.