LRGUK-1 Is Required for Basal Body and Manchette Function during Spermatogenesis and Male Fertility

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.     

A novel protein,sperm head and tail associated protein (SHTAP), interacts with cysteine‐rich secretory protein 2 (CRISP2) during spermatogenesis in the mouse

Background information. CRISP2 (cysteine‐rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca²⁺ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2‐binding partner, which we have designated SHTAP (sperm head and tail associated protein). Results. Using yeast two‐hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2‐binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (～20–87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the ～26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two‐hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related‐1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri‐acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co‐localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP—CRISP2 complex appeared to be redistributed within the head. Conclusions. The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP—CRISP2 complex play a role in the attainment of sperm functional competence.     

An essential role for katanin p80 and microtubule severing in male gamete production

Katanin is an evolutionarily conserved microtubule-severing complex implicated in multiple aspects of microtubule dynamics. Katanin consists of a p60 severing enzyme and a p80 regulatory subunit. The p80 subunit is thought to regulate complex targeting and severing activity, but its precise role remains elusive. In lower-order species, the katanin complex has been shown to modulate mitotic and female meiotic spindle dynamics and flagella development. The in vivo function of katanin p80 in mammals is unknown. Here we show that katanin p80 is essential for male fertility. Specifically, through an analysis of a mouse loss-of-function allele (the Taily line), we demonstrate that katanin p80, most likely in association with p60, has an essential role in male meiotic spindle assembly and dissolution and the removal of midbody microtubules and, thus, cytokinesis. Katanin p80 also controls the formation, function, and dissolution of a microtubule structure intimately involved in defining sperm head shaping and sperm tail formation, the manchette, and plays a role in the formation of axoneme microtubules. Perturbed katanin p80 function, as evidenced in the Taily mouse, results in male sterility characterized by decreased sperm production, sperm with abnormal head shape, and a virtual absence of progressive motility. Collectively these data demonstrate that katanin p80 serves an essential and evolutionarily conserved role in several aspects of male germ cell development.     

Declining Trend of Hepatitis A Seroepidemiology in Association with Improved Public Health and Economic Status of Thailand

Hepatitis A virus (HAV) is transmitted via the fecal-oral route from contaminated food or water. As part of the most recent survey of viral hepatitis burden in Thailand, we analyzed the current seroprevalence of HAV in the country and compared with data dating back to 1971. From March to October, 2014, a total of 4,260 individuals between one month and 71 years of age from different geographical regions (North = 961; Central = 1,125; Northeast = 1,109; South = 1,065) were screened for anti-HAV IgG antibody using an automated chemiluminescent microparticle immunoassay. Overall, 34.53% (1,471/4,260) possessed anti-HAV IgG antibody, and the age-standardized seroprevalence was 48.6%. Seroprevalence rates were 27.3% (North), 30.8% (Central), 33.8% (Northeast) and 45.8% (South) and were markedly lower than in the past studies especially among younger age groups. The overall trend showed an increase in the age by which 50% of the population were anti-HAV IgG antibody: 4.48 years (1971–1972), 6 (1976), 12.49 (1990), 36.02 (2004) and 42.03 (2014).This suggests that Thailand is transitioning from low to very low HAV endemicity. Lower prevalence of HAV correlated with improved healthcare system as measured by decreased infant mortality rate and improved national economy based on increased GDP per capita. The aging HAV immuno-naïve population may be rendered susceptible to potential HAV outbreaks similar to those in industrialized countries and may benefit from targeted vaccination of high-risk groups.     

Development of a Rapid,Simple Method for Detecting Naegleria fowleri Visually in Water Samples by Loop-Mediated Isothermal Amplification (LAMP)

Naegleria fowleri is the causative agent of the fatal disease primary amebic meningoencephalitis. Detection of N. fowleri using conventional culture and biochemical-based assays is time-consuming and laborious, while molecular techniques, such as PCR, require laboratory skills and expensive equipment. We developed and evaluated a novel loop-mediated isothermal amplification (LAMP) assay targeting the virulence-related gene for N. fowleri. Time to results is about 90 min and amplification products were easily detected visually using hydroxy naphthol blue. The LAMP was highly specific after testing against related microorganisms and able to detect one trophozoite, as determined with spiked water and cerebrospinal fluid samples. The assay was then evaluated with a set of 80 water samples collected during the flooding crisis in Thailand in 2011, and 30 natural water samples from border areas of northern, eastern, western, and southern Thailand. N. fowleri was detected in 13 and 10 samples using LAMP and PCR, respectively, with a Kappa coefficient of 0.855. To the best of our knowledge, this is the first report of a LAMP assay for N. fowleri. Due to its simplicity, speed, and high sensitivity, the LAMP method described here might be useful for quickly detecting and diagnosing N. fowleri in water and clinical samples, particularly in resource-poor settings.     

Al<Superscript>3+</Superscript> and Fe<Superscript>2+</Superscript> toxicity reduction potential by acid-resistant strains of <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis> isolated from acid sulfate soils under acidic conditions

This research aimed to evaluate the capacity of acid-resistant purple nonsulfur bacteria, Rhodopseudomonas palustris strains VNW02, TLS06, VNW64, and VNS89, to resist Al³⁺ and Fe²⁺ and to investigate their potential to remove both metals from aqueous solutions using exopolymeric substances (EPS) and biomasses. Based on median inhibition concentration (IC₅₀), strain VNW64 was the most resistant to both metals under conditions of aerobic dark and microaerobic light; however, strain TLS06 was more resistant to Al³⁺ under aerobic dark conditions. High metal concentrations resulted in an altered cellular morphology, particularly for strain TLS06. Metal accumulation in all tested PNSB under both incubating conditions as individual Al³⁺ or Fe²⁺ was in the order of cell wall?>?cytoplasm?>?cell membrane. This was also found in a mixed metal set only under conditions of aerobic dark as microaerobic light was in the degree of cytoplasm?>?cell wall?>?cell membrane. Of all strains tested, EPS from strain VNW64 had the lowest carbohydrate and the highest protein contents. Metal biosorption under both incubating conditions, EPS produced by strains VNW64 and TLS06, achieved greater removal (80 mg Al³⁺ L^?1 and/or 300 mg Fe²⁺ L^?1) than their biomasses. Additionally, strain VNW64 had a higher removal efficiency compared to strain TLS06. Based on the alteration in cellular morphology, including biosorption and bioaccumulation mechanisms, R. palustris strains VNW64 and TLS06 demonstrated their resistance to metal toxicity. Hence, they may have great potential for ameliorating the toxicity of Al³⁺ and Fe²⁺ in acid sulfate soils for rice cultivation.     

Humanized beta-thalassemia mouse model containing the common IVSI-110 splicing mutation

Splicing mutations are common causes of beta-thalassemia. Some splicing mutations permit normal splicing as well as aberrant splicing, which can give a reduced level of normal beta-globin synthesis causing mild disease (thalassemia intermedia). For other mutations, normal splicing is reduced to low levels, and patients are transfusion-dependent when homozygous for the disease. The development of therapies for beta-thalassemia will require suitable mouse models for preclinical studies. In this study, we report the generation of a humanized mouse model carrying the common IVSI-110 splicing mutation on a BAC including the human beta-globin ((hu)beta-globin) locus. We examined heterozygous murine beta-globin knock-out mice ((mu)beta(th-3/+)) carrying either the IVSI-110 or the normal (hu)beta-globin locus. Our results show a 90% decrease in (hu)beta-globin chain synthesis in the IVSI-110 mouse model compared with the mouse model carrying the normal (hu)beta-globin locus. This notable difference is attributed to aberrant splicing. The humanized IVSI-110 mouse model accurately recapitulates the splicing defect found in comparable beta-thalassemia patients. This mouse model is available as a platform for testing strategies for the restoration of normal splicing.