The dynamics of gene expression during and post meiosis sets the sperm agenda.

Meiosis is the defining event of spermatogenesis. Spermatocytes undergo meiosis to give rise to round spermatids, which in turn metamorphose to flagellated spermatozoa that mature in the epididymis. To characterize the dynamics of gene expression during these important stages of spermatogenesis, we undertook transcriptome analysis in >90% pure pachytene spermatocytes and round spermatids, and pure mature sperm of rat by massive parallel deep sequencing. The study has identified 10,719 total transcripts expressed in meiotic and postmeiotic cells, out of which 7,641 were present in all the three cell types. Most abundant transcripts were related to gametogenesis in spermatocytes and spermatids, and mitochondrial energy metabolism in sperm. Importantly, 108 transcripts were specific to spermatocytes, including Cpeb2, Dpf3, H2afy, Haus7, Plcb1, Taf9, and Tdrd7 strongly linked with meiosis. Similarly, 323 transcripts unique to round spermatids included Arpc5, Apoa1, Cntrob, Dcaf17, Ift88, and Ly6k that play essential roles in spermiogenesis. Likewise, 178 transcripts unique to sperm included Camta1, Hoxb1, and Prdx6 having assigned roles in fertility and/or embryonic development. Levels of ~16% transcripts declined from spermatocytes to sperm while two (Cd300e and Ddx17) increased. New candidate genes with possible roles in meiosis (91), spermiogenesis (298), and sperm function (171), have been identified. This study has provided new potential targets for contraception and/or treatment of male infertility. (CDRI communication number 9889).

The thermo-sensitive gene expression signatures of spermatogenesis.

BACKGROUND: Spermatogenesis in most mammals (including human and rat) occurs at ~ 3 °C lower than body temperature in a scrotum and fails rapidly at 37 °C inside the abdomen. The present study investigates the heat-sensitive transcriptome and miRNAs in the most vulnerable germ cells (spermatocytes and round spermatids) that are primarily targeted at elevated temperature in a bid to identify novel targets for contraception and/or infertility treatment. METHODS: Testes of adult male rats subjected to surgical cryptorchidism were obtained at 0, 24, 72 and 120 h post-surgery, followed by isolation of primary spermatocytes and round spermatids and purification to > 90% purity using a combination of trypsin digestion, centrifugal elutriation and density gradient centrifugation techniques. RNA isolated from these cells was sequenced by massive parallel sequencing technique to identify the most-heat sensitive mRNAs and miRNAs. RESULTS: Heat stress altered the expression of a large number of genes by ≥2.0 fold, out of which 594 genes (286↑; 308↓) showed alterations in spermatocytes and 154 genes (105↑; 49↓) showed alterations in spermatids throughout the duration of experiment. 62 heat-sensitive genes were common to both cell types. Similarly, 66 and 60 heat-sensitive miRNAs in spermatocytes and spermatids, respectively, were affected by ≥1.5 fold, out of which 6 were common to both the cell types. CONCLUSION: The study has identified Acly, selV, SLC16A7(MCT-2), Txnrd1 and Prkar2B as potential heat sensitive targets in germ cells, which may be tightly regulated by heat sensitive miRNAs rno-miR-22-3P, rno-miR-22-5P, rno-miR-129-5P, rno-miR-3560, rno-miR-3560 and rno-miR-466c-5P.

Mercury Concentrations in the Fish Community from Indrawati River, Nepal.

This study quantified concentrations of mercury (Hg) and its trophic transfer along the fish community in the Indrawati River, Nepal. Stable isotope ratios of nitrogen (δ15N) and carbon (δ13C), complemented by stomach contents data were used to assess the food web structure and trophic transfer of Hg in 54 fishes; 43 Shizothorax richardsonii and 11 Barilius spp. [B. bendelisis (1), B. vagra (3) and B. barila (7)]. Sixty-one muscle samples (including six replicates) were used for the analysis of total mercury (THg) and stable isotopes. Mean THg concentrations in B. spp. and the more common species S. richardsonii was observed to be 218.23 (ng/g, ww) and 90.82 (ng/g, ww), respectively. THg versus total length in both S. richardsonii and B. spp. showed a decreasing tendency with an increase in age. Regression of logTHg versus δ15N among the fish species showed a significant positive correlation only in S. richardsonii indicating biomagnification along the trophic level in this species.

Energy Utilization for Survival and Fertilization-Parsimonious Quiescent Sperm Turn Extravagant on Motility Activation in Rat.

Quiescent sperm survive in cauda epididymis for long periods of time under extreme crowding conditions and with a very limited energy substrate, while after ejaculation, motile sperm live for a much shorter period with an unlimited energy resource and without crowding. Thus, the energy metabolism in relation to the energy requirement of the two may be quite different. A simple physiological technique was evolved to collect viable quiescent sperm from rat cauda epididymis to compare its energy metabolism with motile sperm. Quiescent sperm exhibited 40%-60% higher activities of mitochondrial electron transport chain complexes I-IV and ATP synthase in comparison to motile sperm and accumulated Ca(2+) in the midpiece mitochondria to enhance oxidative phosphorylation (OxPhos). In contrast, motile sperm displayed up to 75% higher activities of key glycolytic enzymes and secreted more than two times the lactate than quiescent sperm. Quiescent sperm phosphorylated AMPK and MAPK-p38, while motile sperm phosphorylated AKT and MAPK/ERK. Glycolytic inhibitor iodoacetamide prevented motility activation of quiescent rat sperm and inhibited conception in rabbits more effectively than OxPhos uncoupler 2,4-dinitrophenol. Apparently, quiescent sperm employ the most energy efficient OxPhos to survive for extended periods of time under extreme conditions of nutrition and crowding. However, on motility initiation, sperm switch predominantly to glycolysis to cater to their high- and quick-energy requirement of much shorter periods. This study also presents a proof of concept for targeting sperm energy metabolism for contraception.

Combining a synthetic spermicide with a natural trichomonacide for safe, prophylactic contraception.

STUDY QUESTION: Can a specifically acting synthetic spermicide (DSE-37) be combined with a natural microbicide (saponins) for safe, prophylactic contraception? SUMMARY ANSWER: A 1:1 (w/w) combination of DSE-37 and Sapindus saponins can target sperm and Trichomonas vaginalis precisely without any noticeable off-target effects on somatic cells at effective concentrations. WHAT IS KNOWN ALREADY: Broad-spectrum vaginal agents like nonoxynol-9 (N-9) and cellulose sulfate have failed clinically as microbicides due to non-specific off-target effects, whereas agents that specifically target retroviruses have shown promise in clinical trials. DSE-37 and Sapindus saponins, respectively, specifically target human sperm and T. vaginalis in vitro. STUDY DESIGN, SIZE, DURATION: A comprehensive study of efficacy and safety was undertaken using in vitro (human cells) and in vivo (rabbit) models. The 1:1 combination of DSE-37 and Sapindus saponins was based on the in vitro spermicidal and anti-Trichomonal activities of the two components. N-9, the spermicide in clinical use, served as reference control. Free sperm thiols were fluorescently glinted to reveal differences in the targets of the test agents. PARTICIPANTS/MATERIALS, SETTING, METHODS: On/off-target effects were evaluated in vitro against human sperm, T. vaginalis, HeLa, Vk2/E6E7, End1/E6E7 and Lactobacillus jensenii, using standard assays of drug susceptibility, cell viability, flow cytometric assessment of cell apoptosis and qPCR for expression of pro-inflammatory cytokine mRNAs. The spermicidal effect was also recorded live and free thiols on sperm were fluorescently visualized using a commercial kit. In vivo contraceptive efficacy (pregnancy/fertility rates) and safety (vaginal histopathology and in situ immune-labeling of inflammation markers VCAM-1, E-selectin and NFkB) were evaluated in rabbits. MAIN RESULTS AND THE ROLE OF CHANCE: A 0.003% drug 'combination' containing 0.0015% each of DSE-37 and Sapindus saponins in physiological saline irreversibly immobilized 100% human sperm in ∼30 s and eliminated 100% T. vaginalis in 24 h, without causing any detectable toxicity to human cervical (HeLa) cells and Lactobacilli in 24-48 h, in vitro. N-9 at 0.003% exhibited lower microbicidal activity against Trichomonas but failed in spermicidal assays while causing severe toxicity to HeLa cells and Lactobacilli in 12-24 h. The 'combination' of DSE-37 and Sapindus saponins completely prevented pregnancy in rabbits at a vaginal dose of 20 mg (1% in K-Y Jelly), while application of 5% 'combination' in K-Y Jelly for 4 consecutive days caused negligible alterations in epithelial lining of rabbit vagina with only minor changes in levels of inflammation markers. N-9 at a 20 mg vaginal dose prevented pregnancy in 33% animals and a 4-day repeat application of 2% N-9 gel caused severe local toxicity to vaginal epithelium with molecular expression of acute inflammation markers. LIMITATIONS, REASONS FOR CAUTION: The number of animals used for the in vivo efficacy study was limited by the approval of the animal ethics committee. WIDER IMPLICATIONS OF THE FINDINGS: Anti-Trichomonal contraceptives with specifically acting synthetic component and clinically-proven safe natural component may define a new concept in empowering women to control their fertility and reproductive health. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by CSIR-Network Project 'PROGRAM' (BSC0101) and partly by the Ministry of Health and Family Welfare, Government of India (GAP0001). The funding agencies did not play any role in this study and none of the authors had any competing interest(s).

Mupirocin resistance in nasal carriage of Staphylococcus aureus among healthcare workers of a tertiary care rural hospital.

INTRODUCTION: Mupirocin (pseudomonic acid A) is a topical antimicrobial agent with excellent antistaphylococcal and antistreptococcal activity. A nasal formulation is approved by the United States Food and Drug Administration for eradicating nasal carriage in adult patients as well as in health care personnel. Resistance to mupirocin has already been reported worldwide. The increasing prevalence of mupirocin resistance among Staphylococcus aureus and coagulase-negative Staphylococcus (CoNS) species could be an important threat to the future use of mupirocin against methicillin-resistant S. aureus (MRSA). Thus, this study was carried out to find the prevalence of mupirocin resistance in S. aureus and CoNS by disc diffusion and to determine the rates of high-level and low-level mupirocin resistance in S. aureus and CoNS by disc diffusion. MATERIALS AND METHODS: A total of 140 healthcare workers (HCWs) (doctor, nursing staff, housekeeping staff) were randomly selected. S. aureus and CoNS isolates were tested for mupirocin resistance by the disk diffusion method using 5 µg and 200 µg mupirocin discs. MRSA isolates were tested for antibiotics by Kirby-Bauer disc-diffusion method as per Clinical and Laboratory Standards Institute guidelines. RESULTS: Out of 140 nasal swabs collected from HCWs, S. aureus was isolated in 38 (27.14%), and CoNS was isolated in 73 (52.14%). MRSA was isolated in 20 (14.28%) and methicillin-resistant coagulase-negative Staphylococci (MRCoNS) in 34 (24.29%. Methicillin-sensitive S. aureus (MSSA) and MSCoNS isolates were 100% sensitive to mupirocin, but two isolates from MRSA (1.43%) and five from MRCoNS (3.57%) were mupirocin resistant. CONCLUSION: The presence of mupirocin resistance in MRSA and MRCoNS is a cause for concern. It could be limited by regular surveillance and effective infection control initiatives so to inform health care facilities to guide therapeutic and prophylactic use of mupirocin.

Concentration, seasonality, and sources of trace elements in atmospheric aerosols from Godavari in the southern Himalayas.

Atmospheric pollution has detrimental effects on human health and ecosystems. The southern region of the Himalayas, undergoing rapid urbanization and intense human activities, faces poor air quality marked by high aerosol loadings. In this study, we conducted a two-year PM10 sampling in the suburban area (Godavari) of Kathmandu, a representative metropolis situated in the southern part of the central Himalayas. The trace elements were measured to depict aerosol-bound element loadings, seasonality, and potential sources. The mean concentrations of trace elements varied considerably, ranging from 0.27 ± 0.19 ng m-3 for Tl to 1252.78 ng m-3 for Zn. The average concentration of Co and Ni was 1.2 and 22.4 times higher, respectively, than those in Lhasa city in Tibet in the northern Himalayas. The concentration of Pb was 38 times lower than that in Lahore, Pakistan, and 9 times lower than urban sites in India. For the seasonality, the trace element concentrations displayed remarkable variation, with higher concentrations during the non-monsoon seasons and lower concentrations during the monsoon season. This trend was primarily influenced by anthropogenic activities such as low-grade fuel combustion in vehicles, coal combustion in brick kilns, and biomass burning, along with seasonal rainfall that induced aerosol washout. The enrichment factors (EFs) analysis revealed that Cd, Zn, Sb, Ni, Cu, Cr, and Pb had higher EFs, indicating their significant contributions from anthropogenic sources. In contrast, elements like Tl, Co, V, Cs, U, Ba, Th, and Sr, characterized by lower EFs, were mainly associated with natural sources. The Pb isotopic ratio profiles exhibited the Pb in PM10 are derived major contribution from legacy lead. Biomass burning contributed to the Pb source in winter. These findings provide policymakers with valuable insights to develop guidelines and strategies aimed at improving air quality and mitigating the impact of aerosol pollution on human health in the Himalayan region.

miRNA Profiling of Major Testicular Germ Cells Identifies Stage-Specific Regulators of Spermatogenesis.

Spermatogenesis is tightly controlled at transcriptional, post-transcriptional, and epigenetic levels by various regulators, including miRNAs. This study deals with the identification of miRNAs critical to the three important stages of germ cell development (spermatocytes, round spermatids, and mature sperm) during spermatogenesis. We used high-throughput transcriptome sequencing to identify the differentially expressed miRNAs in the pachytene spermatocytes, round spermatids, and mature sperm of rat. We identified 1843 miRNAs that were differentially expressed across the three stages of germ cell development. These miRNAs were further categorized into three classes according to their pattern of expression during spermatogenesis: class 1 - miRNAs found exclusively in one stage and absent in the other two stages; class 2 - miRNAs found in any two stages but absent in the third stage; class 3 - miRNAs expressed in all the three stages. Six hundred forty-six miRNAs were found to be specific to one developmental stage, 443 miRNAs were found to be common across any two stages, and 754 miRNAs were common to all the three stages. Target prediction for ten most abundant miRNAs specific to each category identified miRNA regulators of mitosis, meiosis, and cell differentiation. The expression of each miRNA is specific to a particular developmental stage, which is required to maintain a significant repertoire of target mRNAs in the respective stage. Thus, this study provided valuable data that can be used in the future to identify the miRNAs involved in spermatogenic arrest at a particular stage of the germ cell development.

Heavy metals in surface sediments in the trans-Himalayan Koshi River catchment: Distribution, source identification and pollution assessment.

Rivers flowing across the Himalayas are important water resources and deliver large amounts of sediment to regional and downstream ecosystems. However, the geochemistry of Himalayan river sediments has been less studied. Surface sediment samples collected from a typical trans-Himalayan river, the Koshi River (KR), were used to investigate the distribution, pollution status and potential sources of heavy metals. Heavy metals did not show significant spatial differences between the upstream and downstream areas of the river, but Cd and Pb displayed higher values in the upstream area. The average heavy metal concentrations in the KR sediments are comparable to the natural background values and are lower than the sediment guidelines. Pollution assessment using the geo-accumulation index (Igeo), enrichment factor (EF) and pollution load index (PLI) suggested negligible anthropogenic disturbances except for slight contamination by Cd, Pb and Cu at a few sites. Principal component analysis revealed that Cr, Co, Ni and Zn were primarily from the parent rock and that Cu, Cd and Pb were derived from both natural and anthropogenic sources. Despite contrasting environmental settings and human activities in the upper and lower reaches of the river, the heavy metals concentrations in the KR sediments showed consistency with natural backgrounds and negligible contamination. The geochemistry of river sediments is a useful indicator of environmental changes, and long-term observations of the geochemistry of trans-Himalayan river sediments are needed to understand the impacts of intensified climate change and human activities on the Himalayan environment.

Mercury variation and export in trans-Himalayan rivers: Insights from field observations in the Koshi River.

Strengthening the research of riverine mercury (Hg) export is of great significance for understanding the regional and global Hg cycle, especially for the data lacking trans-Himalayan rivers. In this study, three systematic sampling campaigns were conducted in the Koshi River Basin (KRB) during the post-monsoon, pre-monsoon and monsoon seasons. Hg speciation and distribution of river water were analyzed among the different seasons for a total of 88 water samples. The total Hg (THg) concentration of surface water in the KRB ranged from 0.64 to 32.96 ng·L-1 with an average of 5.83 ± 6.19 ng·L-1 and decreased in the order of post-monsoon (8.79 ± 7.32 ng·L-1) > monsoon (6.68 ± 6.12 ng·L-1) > pre-monsoon (2.18 ± 1.29 ng·L-1). Particulate Hg (PHg) accounted for 63% of THg on average and had a positive correlation with THg among all the three sampling seasons, indicating that the differences in PHg concentration were likely one of the main factors leading to the seasonal and spatial variations in THg in the KRB surface water. The annual Hg exports and fluxes were estimated to be 339.04 kg and 3.88 µg·m-2·yr-1, respectively. Furthermore, Hg export from the KRB had significant seasonal variation and decreased in the order of monsoon (259.47 kg) > post-monsoon (61.18 kg) > winter (9.31 kg) > pre-monsoon (9.08 kg), and this pattern was mainly related to seasonal changes in river runoff. The annual Hg export is projected to increase in the future, especially in the post-monsoon season. Therefore, more attention should be paid to river runoff observations and riverine Hg research for water resources management in the Himalaya.

Ammonium salts of carbamodithioic acid as potent vaginal trichomonacides and fungicides.

Chemical attenuation of the reactive oxygen species (ROS)-sensitive anaerobes Trichomonas vaginalis, which is the most prevalent non-viral sexually transmitted infection, and two often coexisting vaginal infections, namely Candida albicans and Staphylococcus aureus, which are opportunistic reproductive tract infections, was attempted with novel ammonium salts of carbamodithioic acid through inhibition of free thiols. In vitro and in vivo efficacies of the designed compounds were evaluated as topical vaginal microbicides. Five compounds showed exceptional activity against drug-resistant and -susceptible strains with negligible toxicity to host (HeLa) cells in vitro in comparison with the standard vaginal microbicide nonoxynol-9 (N-9), without disturbing the normal vaginal flora (i.e. Lactobacillus). The compounds significantly inhibited the cytopathic effects of Trichomonas on HeLa cells in vitro with efficacies comparable with metronidazole (MTZ); however, their efficacy to rescue host cells from co-infection (protozoal and fungal) was greater than that of MTZ. The compounds inhibited ß-haemolysis of red blood cells caused by Trichomonas and were found to be active in vivo in the mouse subcutaneous abscess assay. Some compounds rapidly immobilized human sperm. A mechanism involving inhibition of free thiols and consequently the cysteine proteases of T. vaginalis by the new compounds has been proposed. Thus, a unique scaffold of antimicrobial agents has been discovered that warrants further investigation for development as contraceptive vaginal microbicides.