Late Spermatogenesis Research Articles

Beneficial effect of residential greenness on sperm quality and the role of air pollution: A multicenter population-based study

BackgroundPoor sperm quality is a major cause of male infertility. However, evidence remains scarce on how greenness affects male sperm quality. ObjectivesTo assess the associations of residential greenness with male sperm quality and the modification effect of air pollution exposure on the relationship. MethodsA total of 78,742 samples from 33,184 sperm donors from 6 regions across China during 2014–2020 were included and analyzed. Individual residential greenness exposures of study subjects were estimated using the Normalized Difference Vegetation Index (NDVI) during the entire (0–90 lag days) and two key stages (0–37, and 34–77 lag days) of sperm development. Contemporaneous personal exposure levels to air pollutants were estimated using a spatio-temporal deep learning method. Linear mixed models were employed to assess the impact of greenspace in relation to sperm quality. The modification effect of air pollution on the greenspace-sperm quality relationship was also estimated. ResultsPer IQR increment in NDVI exposure throughout spermatogenesis were statistically associated with increasing sperm count by 0.0122 (95 % CI: 0.0007, 0.0237), progressive motility by 0.0162 (95 % CI: 0.0045, 0.0280), and total motility by 0.0147 (95 % CI: 0.0014, 0.0281), respectively. Similar results were observed when the model added air pollutants (PM1, PM2.5 or O3) for adjustment. Additionally, specific air pollutants, including PM1, PM2.5, and O3, were found to modify this association. Notably, the protective effects of greenness exposure were more pronounced at higher concentrations of PM1 and PM2.5 and lower concentrations of O3 (all Pinteraction < 0.05). Statistically significant positive effects of NDVI were observed on sperm motility in early spermatogenesis and sperm count in late spermatogenesis. ConclusionsExposure to residential greenness may have beneficial effects on sperm quality and air pollution modifies their relationship. These findings highlight the importance of adopting adaptable urban greenspace planning and policies to safeguard male fertility against environmental factors.

Alterations of the hormones follitropin and lutropin in the blood of young mice dosed with lindane

BackgroundThe gonadotropins lutropin and follitropin stimulate steroid synthesis in the gonads during gonadal maturation by promoting oogenesis and spermatogenesis. Endocrine disruptors such as lindane (γHCH) can alter the reproductive stage so and could alter puberty by interfering with lutropin and follitropin signaling. γHCH was classified by World Health Organization as “moderately restricted”, and in 2009 it is production and agricultural use was prohibited, under the Stockholm Convention, however, its use as a pharmaceutical treatment against lice and scabies is still allowed (and mainly, for use by the health sector). This study aimed to examine the effects of single-dose exposure to γHCH in young mice (Mus musculus) on lutropin and follitropin concentration, and to correlate gonadal maturity status and phenotypic characteristics.MethodsYoung γHCH-treated mice (16 females and 16 males) received a single dose of 25, 10, 5, or 0 µg/ml γHCH per gram of body weight, ocular route. Once secondary sexual characteristics were observed, the gonads were dissected and examined using histological techniques and the ovarian follicles were classified as dependent and independent of gonadotropins; the testicles were classified as inactive, early and late spermatogenesis; and the blood was processed with the lutropin and follitropin ELISA kit.ResultsThe results indicate significant differences in the concentration of lutropin and follitropin between males and females (Fisher p < 0.05). The lutropin and follitropin levels showed a tendency to decrease in females, whereas, in males they tended to increase as the dose of γHCH increased. In females, it was observed that at the follitropin and lutropin decreased 42.3% and 83.7%, respectively at dose of 25 µg/ml; while in males, follitropin increased 51.9% at the dose of 25 µg/ml, and at the dose of 10 µg/ml lutropin increased by 242.5%. Contrary to that, gonadal maturity increased in females and decreased in males with increasing γHCH dose, disagreeing with the idea that gonadotropins coincide with the onset of puberty and gonadal maturity.ConclusionsThe results allow us to infer that exposure to γHCH could promote non-monotonic responses; however, this does not seem to alter puberty in the doses and conditions of the present experiment.

The Role of Chorein Deficiency in Late Spermatogenesis.

VPS13A, also known as chorein, whose loss of function causes chorea-acanthocytosis (ChAc), is characterized by Huntington's-disease-like neurodegeneration and neuropsychiatric symptoms in addition to acanthocytosis in red blood cells. We previously reported that ChAc-model mice with a loss of chorein function exhibited male infertility, with asthenozoospermia and mitochondrial dysmorphology in the spermatozoa. Here, we report a novel aspect of chorein dysfunction in male fertility, particularly its role in spermatogenesis and mitochondrial integrity. An increase in anti-malondialdehyde antibody immunoreaction within the testes, predominantly observed at the advanced stages of sperm formation in chorein-deficient mice, suggests oxidative stress as a contributing factor to mitochondrial dysfunction and impaired sperm maturation. The chorein immunoreactivity in spermatids of wild-type mice accentuates its significance in sperm development. ChAc-model mice exhibit mitochondrial ultrastructural abnormalities, specifically during the late stages of sperm maturation, suggesting a critical timeframe for chorein's action in spermiogenesis. We observed an increase in TOM20 protein levels, indicative of disrupted mitochondrial import mechanisms. The concurrent decrease in metabolic enzymes such as IDH3A, LDHC, PGK2, and ACAT1 suggests a complex chorein-mediated metabolic network that is essential for sperm vitality. Additionally, heightened separation of cytoplasmic droplets from sperm highlights the potential membrane instability in chorein-deficient spermatozoa. Metabolomic profiling further suggests a compensatory metabolic shift, with elevated glycolytic and TCA-cycle substrates. Our findings suggest that chorein is involved in anti-ferroptosis and the maturation of mitochondrial morphology in the late stages of spermatogenesis, and its deficiency leads to asthenozoospermia characterized by membrane instability, abnormal cytosolic glycolysis, abnormal mitochondrial function, and a disrupted TCA cycle. Further analyses are required to unravel the molecular mechanisms that directly link these findings and to elucidate the role of chorein in spermatogenesis as well as its broader implications.

Derepression of Y-linked multicopy protamine-like genes interferes with sperm nuclear compaction in D. melanogaster

Across species, sperm maturation involves the dramatic reconfiguration of chromatin into highly compact nuclei that enhance hydrodynamic ability and ensure paternal genomic integrity. This process is mediated by the replacement of histones by sperm nuclear basic proteins, also referred to as protamines. In humans, a carefully balanced dosage between two known protamine genes is required for optimal fertility. However, it remains unknown how their proper balance is regulated and how defects in balance may lead to compromised fertility. Here, we show that a nucleolar protein, modulo, a homolog of nucleolin, mediates the histone-to-protamine transition during Drosophila spermatogenesis. We find that modulo mutants display nuclear compaction defects during late spermatogenesis due to decreased expression of autosomal protamine genes (including Mst77F) and derepression of Y-linked multicopy Mst77F homologs (Mst77Y), leading to the mutant's known sterility. Overexpression of Mst77Y in a wild-type background is sufficient to cause nuclear compaction defects, similar to modulo mutant, indicating that Mst77Y is a dominant-negative variant interfering with the process of histone-to-protamine transition. Interestingly, ectopic overexpression of Mst77Y caused decompaction of X-bearing spermatids nuclei more frequently than Y-bearing spermatid nuclei, although this did not greatly affect the sex ratio of offspring. We further show that modulo regulates these protamine genes at the step of transcript polyadenylation. We conclude that the regulation of protamines mediated by modulo, ensuring the expression of functional ones while repressing dominant-negative ones, is critical for male fertility.

Potential role for the germ cell-specific Rad21 in early meiosis of oocyte and spermatocyte in the Chinese mitten crab Eriocheir sinensis

Rad21/Rec8 family proteins are vital for sister chromatid segregation in mitosis and homologous recombination in meiosis, but no molecular data are available in crustacean species. In this study, a germ cell-specific Rad21 named EsRad21 was identified in the crab Eriocheir sinensis. EsRad21 mRNA has an open reading frame of 2310 base pairs (bp) encoding a 769 amino acids (aa) protein. RT-PCR showed that EsRad21 mRNA was particularly expressed in testis and ovary. The RT-qPCR results further revealed that the EsRad21 mRNA exhibited similar expression pattern in gonads at various developmental stages. EsRad21 mRNA expression level was the highest in testis at early spermatogenesis stage and ovaries at previtellogenesis stage, thereafter decreased significantly at middle spermatogenesis and vitellogenesis, and finally reach the lowest level at late spermatogenesis and vitellogenesis. In situ hybridization (ISH) analysis showed that EsRad21 mRNA was exclusively expressed in germline cells, but not in gonadal somatic cells. Notably, hybridized signal was detected on chromosomes of metaphase spermatocytes. EsRad21 is thus an underlying helpful indicator of the early phases of germ cell development. RNAi knockdown of EsRad21 downregulated the expression of other meiosis-related genes like Smc5-Smc6 and SPO11 and resulted in high mortality of individuals after 24 h post injection of EsRad21 dsRNA. Taken together, our results showed a potential role for EsRad21 in early meiosis of oocytes and spermatocytes in E. sinensis. This is the first report on the molecular characterization of the Rad21 transcript in a crustacean species.

Transcriptional and mutational signatures of the Drosophila ageing germline.

Ageing is a complex biological process that is accompanied by changes in gene expression and mutational load. In many species, including humans, older fathers pass on more paternally derived de novo mutations; however, the cellular basis and cell types driving this pattern are still unclear. To explore the root causes of this phenomenon, we performed single-cell RNA sequencing on testes from young and old male Drosophila and genomic sequencing (DNA sequencing) on somatic tissues from the same flies. We found that early germ cells from old and young flies enter spermatogenesis with similar mutational loads but older flies are less able to remove mutations during spermatogenesis. Mutations in old cells may also increase during spermatogenesis. Our data reveal that old and young flies have distinct mutational biases. Many classes of genes show increased postmeiotic expression in the germlines of older flies. Late spermatogenesis-biased genes have higher dN/dS (ratio of non-synonymous to synonymous substitutions) than early spermatogenesis-biased genes, supporting the hypothesis that late spermatogenesis is a source of evolutionary innovation. Surprisingly, genes biased in young germ cells show higher dN/dS than genes biased in old germ cells. Our results provide new insights into the role of the germline in de novo mutation.

LncRNA 1700101O22Rik and NONMMUG030480.1 Are Not Essential for Spermatogenesis in Mice.

Many testis-specific lncRNAs are highly expressed in late spermatogenesis, especially in spermiogenesis. However, their functions and the underlying mechanisms in male fertility are largely unknown. Here, we screened two highly expressed lncRNAs, 1700101O22Rik (O22Rik) and NONMMUG030480.1 (NM480) in testes, to investigate the roles in spermatogenesis using lncRNA knockout (KO) mouse generated by CRISPER/Cas9 technology. Both testis-specific lncRNAs were mainly expressed from secondary spermatocytes to round spermatids, suggesting that they might be involved in spermiogenesis. Phenotypic analysis showed that the deletion of O22Rik or NM480 did not affect the development of testis and epididymis or spermatogenesis. These results were confirmed in both young and middle-aged male mice. In addition, there was no significant difference in sperm morphology and other parameters including concentration and motility between wild type (WT) and KO mice. Fertility tests showed that litter size was significantly lower in O22Rik KO mice compared with WT controls. Although O22Rik did not exert dramatic roles in spermatogenesis, on molecular levels, its surrounding gene expression was disturbed significantly. Gm32773 was decreased; however, Gm32828 was increased in KO mice. In conclusion, lncRNA O22Rik and NM480 are not individually essential for spermatogenesis in mice.

PRSS37 deficiency leads to impaired energy metabolism in testis and sperm revealed by DIA-based quantitative proteomic analysis.

Our previous studies have reported that a putative trypsin-like serine protease, PRSS37, is exclusively expressed in testicular germ cells during late spermatogenesis and essential for sperm migration from the uterus into the oviduct and sperm-egg recognition via mediating the interaction between PDILT and ADAM3. In the present study, the global proteome profiles of wild-type (wt) and Prss37-/- mice in testis and sperm were compared employing data independent acquisition (DIA) technology. Overall, 2506 and 459 differentially expressed proteins (DEPs) were identified in Prss37-null testis and sperm, respectively, when compared to control groups. Bioinformatic analyses revealed that most of DEPs were related to energy metabolism. Of note, the DEPs associated with pathways for the catabolism such as glucose via glycolysis, fatty acids via β-oxidation, and amino acids via oxidative deamination were significantly down-regulated. Meanwhile, the DEPs involved in the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation (OXPHOS) were remarkably decreased. The DIA data were further confirmed by a markedly reduction of intermediate metabolites (citrate and fumarate) in TCA cycle and terminal metabolite (ATP) in OXPHOS system after disruption of PRSS37. These outcomes not only provide a more comprehensive understanding of the male fertility of energy metabolism modulated by PRSS37 but also furnish a dynamic proteomic resource for further reproductive biology studies.

Chronological characterization of sperm morpho-functional damage and recovery after testicular heat stress in Nellore bulls

Heat stress (HS) affects spermatogenesis and sperm maturation, decreasing sperm quality. Yet sperm morpho-functional changes caused by HS in Nellore bulls are not fully elucidated. This study aimed to show the chronological effects on sperm quality of HS during spermatogenesis and sperm maturation until recovery of the seminiferous epithelium in Nellore bulls. Nine Nellore bulls were distributed into control and heat stress (HS-scrotal bags/96 h) groups. The study was divided into five Periods: 1. Control (14-7 days before HS); 2. Stored sperm (0–7 days after HS); 3. Sperm maturation and late spermatogenesis (14–42 days after HS); 4. Early spermatogenesis (49–63 days after HS), and 5. Recovery (70–77 days after HS). Semen was collected once a week and evaluated for sperm motility, morphology, plasma, acrosome, and mitochondrial membranes, lipid peroxidation, and DNA fragmentation. Sperm characteristics were similar between groups in Periods 1 (control). During Period 2, HS increased detached normal head defect and decreased mitochondrial membrane potential, denoting effects on the sperm stored at the epididymis cauda. In Period 3, HS decreased sperm motility, plasma membrane integrity, and mitochondrial membrane potential and increased abnormal sperm, lipid peroxidation, and DNA fragmentation; reflecting the effects on sperm that were in the epididymis body and head and late spermatogenesis (spermiogenesis and meiosis). In Period 4, HS maintained a reduction in the mitochondrial membrane potential and an increase in abnormal sperm; injuries that could occur during early spermatogenesis (mitosis). Finally, in Period 5, the groups were similar, confirming the recovery of the seminiferous epithelium after HS. This study provides insights on the effects of HS on the complete process of sperm maturation and spermatogenesis, until recovery in sperm from Nellore bulls.

Molecular Evolution across Mouse Spermatogenesis.

Genes involved in spermatogenesis tend to evolve rapidly, but we lack a clear understanding of how protein sequences and patterns of gene expression evolve across this complex developmental process. We used fluorescence-activated cell sorting (FACS) to generate expression data for early (meiotic) and late (postmeiotic) cell types across 13 inbred strains of mice (Mus) spanning ∼7 My of evolution. We used these comparative developmental data to investigate the evolution of lineage-specific expression, protein-coding sequences, and expression levels. We found increased lineage specificity and more rapid protein-coding and expression divergence during late spermatogenesis, suggesting that signatures of rapid testis molecular evolution are punctuated across sperm development. Despite strong overall developmental parallels in these components of molecular evolution, protein and expression divergences were only weakly correlated across genes. We detected more rapid protein evolution on the X chromosome relative to the autosomes, whereas X-linked gene expression tended to be relatively more conserved likely reflecting chromosome-specific regulatory constraints. Using allele-specific FACS expression data from crosses between four strains, we found that the relative contributions of different regulatory mechanisms also differed between cell types. Genes showing cis-regulatory changes were more common late in spermatogenesis, and tended to be associated with larger differences in expression levels and greater expression divergence between species. In contrast, genes with trans-acting changes were more common early and tended to be more conserved across species. Our findings advance understanding of gene evolution across spermatogenesis and underscore the fundamental importance of developmental context in molecular evolutionary studies.

On the Origin and Evolution of Drosophila New Genes during Spermatogenesis.

The origin of functional new genes is a basic biological process that has significant contribution to organismal diversity. Previous studies in both Drosophila and mammals showed that new genes tend to be expressed in testes and avoid the X chromosome, presumably because of meiotic sex chromosome inactivation (MSCI). Here, we analyze the published single-cell transcriptome data of Drosophila adult testis and find an enrichment of male germline mitotic genes, but an underrepresentation of meiotic genes on the X chromosome. This can be attributed to an excess of autosomal meiotic genes that were derived from their X-linked mitotic progenitors, which provides direct cell-level evidence for MSCI in Drosophila. We reveal that new genes, particularly those produced by retrotransposition, tend to exhibit an expression shift toward late spermatogenesis compared with their parental copies, probably due to the more intensive sperm competition or sexual conflict. Our results dissect the complex factors including age, the origination mechanisms and the chromosomal locations that influence the new gene origination and evolution in testes, and identify new gene cases that show divergent cell-level expression patterns from their progenitors for future functional studies.

The \u03b22Tubulin, Rad50-ATPase and enolase cis-regulatory regions mediate male germline expression in Tribolium castaneum

Genetics-based pest management processes, including the sterile insect technique, are an effective method for the control of some pest insects. However, current SIT methods are not directly transferable to many important pest insect species due to the lack of genetic sexing strains. Genome editing is revolutionizing the way we conduct genetics in insects, including in Tribolium castaneum, an important genetic model and agricultural pest. We identified orthologues of β2Tubulin, Rad50-ATPase and enolase in T. castaneum. Using RT-PCR, we confirmed that these genes are predominantly expressed in the testis. PiggyBac-based transformation of T. castaneum cis-regulatory regions derived from Tc-β2t, Tc-rad50 or Tc-eno resulted in EGFP expression specifically in the T. castaneum testis. Additionally, we determined that each of these regulatory regions regulates EGFP expression in different cell types of the male gonad. Cis-regulatory regions from Tc-β2t produced EGFP expression throughout spermatogenesis and also in mature sperms; Tc-rad50 resulted in expression only in the haploid spermatid, while Tc-eno expressed EGFP in late spermatogenesis. In summary, the regulatory cis-regions characterized in this study are not only suited to study male gonadal function but could be used for development of transgenic sexing strains that produce one sex in pest control strategies.

PD62-10 THE ROLE OF MTOR IN DIFFERENTIATION OF SPERMATOZOA

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology (PD62)1 Sep 2021PD62-10 THE ROLE OF MTOR IN DIFFERENTIATION OF SPERMATOZOA Karen Doersch, Kadijah Abston, Hannah Kim, Amanda Pereira, Xiaoyang Su, and Xin Zhiguo Li Karen DoerschKaren Doersch More articles by this author , Kadijah AbstonKadijah Abston More articles by this author , Hannah KimHannah Kim More articles by this author , Amanda PereiraAmanda Pereira More articles by this author , Xiaoyang SuXiaoyang Su More articles by this author , and Xin Zhiguo LiXin Zhiguo Li More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002099.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The mammalian target of rapamycin (mTOR) is an evolutionarily preserved protein that regulates homeostatic processes, including growth-factor response, autophagy, cytoskeletal organization, and metabolism. Rapamycin, an inhibitor of mTOR, is well-established to extend organisms’ lifespan from yeast to mice; however, its application in humans causes male infertility. Because mTOR pathway function in germ cells is not well-understood, the objective of this study was to examine the role of mTOR in germ cell development. METHODS: A testis-specific mTor-conditional knockout (CKO) mouse was created using a lox-cre system driven by Neurog3-cre, which is expressed in late spermatogenesis. Testis were examined by H&E, electron microscopy, and immunofluorescence microscopy. RNA-seq and small RNA-seq were also used. Metabolomic analysis of testis tissue with mass spectroscopy was performed. RESULTS: The CKO mutants are completely infertile with arrest of spermiogenesis at the round spermatid stage on H&E and electron microscopy. RNA-seq demonstrated decreases in markers of mature spermatozoa CKO compared to controls, corroborating the finding of arrest at the round spermatid. Furthermore, analysis of micro-RNAs (miRNAs) demonstrated aberrant miRNA expression, which may play a role in halting spermatic development. Metabolomic analysis demonstrated increased amino acids, such as threonine, glutamine, and isoleucine, in the CKO mouse compared with controls. CKO testis had altered nucleic acid levels, with elevated guanosine but decreased nucleic acid-monophosphate species. Aberrant guanosine likely represents alterations in the guanosine-triphosphate-dependent signaling via the mTORC1 pathway while decreased nucleotide monophosphates likely represent decreased energy production and translation. CONCLUSIONS: This study demonstrates a germ cell specific CKO of mTOR signaling leads to arrest of spermiogenesis at the round spermatid phase. Metabolomic analysis demonstrated decreased mRNA transcription and protein translation in the CKO. Our results demonstrate a direct role for mTOR-mediated mRNA and protein regulation in spermatozoa differentiation and metabolism. Source of Funding: None © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1076-e1076 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Karen Doersch More articles by this author Kadijah Abston More articles by this author Hannah Kim More articles by this author Amanda Pereira More articles by this author Xiaoyang Su More articles by this author Xin Zhiguo Li More articles by this author Expand All Advertisement Loading ...

Environmental temperature and relative humidity differently affect the sperm characteristics in Brown Swiss and Belgian Blue bulls.

In a temperature-increasing scenario, due to global warming, the individual thermic resilience of the male assumes a crucial role in the reproductive efficiency of a male since the thermic stress, such as the inability of the male to reduce body or regional temperature on a physiological level, impairs testicular function. In this study, the effect of the environmental conditions on the fresh semen quality, in terms of volume, concentration, total sperm in the ejaculate, total motility, normal morphology, membrane integrity, and discarding rate, were compared longitudinally in Belgian Blue (BB) and Brown Swiss (BS) bulls. The environmental conditions, summarized in the mean temperature-humidity index (THI), were calculated on the day of collection, as well as 7days (epididymal maturation), 35days (late spermatogenesis), and 70days (early spermatogenesis) before the collection, to reflect spermatogenesis time. Our findings showed that limited seasonal effects were present in the semen quality of BS bulls. On the other hand, in BB bulls lower semen quality was found between July and November, with a different timing depending on the seminal parameter. This effect of the season on BB semen parameters appears to be related to the THI. The data presented in this study shows that the temperature and humidity, summarized in THI, could affect the semen quality of the bull on breed basis,given that volume, concentration, total sperm in the ejaculate, total motility, membrane integrity, and sperm normal morphology were significantly reduced by an increasing THI in the Belgian Blue bulls, but not in Brown Swiss bulls.

Polyubiquitin gene Ubb is required for upregulation of Piwi protein level during mouse testis development

Testis development, including early embryonic gonad formation and late postnatal spermatogenesis, is essential for the reproduction of higher metazoans to generate fertile gametes, called sperm. We have previously reported that the polyubiquitin gene Ubb is required for fertility in both male and female mice. In particular, the Ubb-null male mice showed an azoospermia phenotype due to arrest of spermatogenesis at the pachytene stage. Here, we analyzed the whole testis proteome at postnatal day 20 to define the molecular mediators of the male-infertility phenotype caused by Ubb knockout. From the identified proteome, 564 proteins were significantly and differentially expressed in Ubb-knockout testes and, among these, 36 downregulated proteins were involved at different stages of spermatogenesis. We also found that levels of piRNA metabolic process-related proteins, including Piwil2 and Tdrd1, were downregulated in Ubb-null testes through functional gene ontology analysis. Further, protein–protein interaction mapping revealed that 24 testis development-related proteins, including Hsp90aa1, Eef1a1, and Pabpc1, were directly influenced by the depletion of ubiquitin. In addition, the reduced mRNA levels of these proteins were observed in Ubb-knockout testes, which closely resembled the global downregulation of piRNA-metabolic gene expression at the transcriptional and post-transcriptional levels. Together with proteomic and transcriptional analyses, our data suggest that Ubb expression is essential for the maintenance of testicular RNA-binding regulators and piRNA-metabolic proteins to complete spermatogenesis in mice.

Modulation of Ago2 Loading by Cyclophilin 40 Endows a Unique Repertoire of Functional miRNAs during Sperm Maturation in Drosophila

In gene silencing, Hsp90 chaperone machinery assists Argonaute (Ago) binding and unwinding of silencing small RNA (sRNA) duplexes. This enables the formation of effector RNA-induced silencing complex (RISC) that often displays cargo preferences. Hence, in Drosophila, microRNAs (miRNAs) and small-interfering RNAs (siRNAs) are differentially sorted into Ago1-RISC and Ago2-RISC, respectively. Here, we identify fly Cyclophilin 40 (Cyp40) as a testis-specialized Hsp90 co-chaperone essential for spermatogenesis and for modulating Ago2-RISC formation. We show that testis-distinctive Ago-sorting and strand-selection mechanisms accumulate a unique set of miRNAs on Ago2. Cyp40 interacts with duplex-incorporating Ago2 through Hsp90 invitro and selectively promotes the build-up of Ago2-bound miRNAs, but not endogenous siRNAs, invivo. Moreover, one of Cyp40-dependent Ago2-sorted miRNAs is required for late spermatogenesis, unraveling the physiological relevance of the unconventional yet conserved Drosophila miRNA-Ago2 sorting pathway. Collectively, these results identify RISC-regulatory roles for Hsp90 machinery and, more generally, highlight the tissue-specific adaptation of sRNA pathways through chaperone diversification.

Particulate Pollution and Male Reproductive Health: Differential Impacts in Early and Late Spermatogenesis

Particulate Pollution and Male Reproductive Health: Differential Impacts in Early and Late SpermatogenesisBackground/Aim: Despite increasing evidence that particulate air pollution has adverse effects on human semen quality, few studies examine clinically relevant thresholds typically employed by clinicians to diagnose male fertility problems. Furthermore, exposure is often assessed using average air pollution levels in a geographic area rather than individualized measures. Finally, physiology-driven selection exposure windows are inconsistent.Methods: We examined a previous cohort of 132 healthy males seeking to become parents, using spermatogenesis-relevant exposure windows of 77-34 days and 37-0 days prior to semen collection. Exposure to PM2.5 and PM10 was individualized by selecting multiple air pollution sensors within participants’ geographic air basins and employing inverse distance weighting to calculate mean daily exposure levels. We used multiple logistic regression to assess the association between pollution, temperature, and dichotomized World Health Organization semen parameters.Results: During the early phase of spermatogenesis, air pollution exposure is associated with 1.563 (95% CI, 1.049 to 2.452) times greater odds of &lt;30% normal heads per 1-unit increase in IQR for PM2.5. In the late phase of spermatogenesis, air pollution exposure is associated with 0.368 (95% CI, 0.114 to 0.767) times greater odds of semen concentration &lt;15 million/mL per 1-unit increase in IQR for PM2.5, and 0.208 (95% CI, 0.042 to 0.653) for PM10.Conclusions: Particulate exposure has a differential and more deleterious impact on early-phase spermatogenesis than late-phase, especially for PM2.5.This suggests that smaller particles breathed earlier in the spermatogenic cycle are likelier to affect sperm head formation.

Modulation of Ago2 Loading by Cyclophilin 40 Endows a Unique Repertoire of Functional miRNAs During Sperm Maturation in Drosophila

In gene silencing, Hsp90 chaperone machinery assists Argonaute(Ago)-binding and unwinding of silencing small (s)RNA duplexes. This enables formation of effector RNA-induced silencing complex (RISC) often displaying cargo preferences. Hence, in Drosophila, micro(mi)RNAs and small-interfering (si)RNAs are differentially sorted into Ago1-RISC and Ago2-RISC, respectively. Here, we identify fly Cyclophilin 40 (Cyp40) as a testis-specialized Hsp90 co-chaperone essential for spermatogenesis and for modulating Ago2-RISC formation. We show that testis-distinctive Ago-sorting and strand-selection mechanisms accumulate a unique set of miRNAs on Ago2. Cyp40 interacts with duplex-incorporating Ago2 through Hsp90 in vitro, and selectively promotes the build-up of Ago2-bound miRNAs, but not endogenous siRNAs, in vivo. Moreover, one of Cyp40-dependent Ago2-sorted miRNAs is required for late spermatogenesis, unraveling the physiological relevance of the unconventional yet conserved Drosophila miRNA-Ago2 sorting pathway. Collectively, these results identify hitherto unacknowledged RISC-regulatory roles for Hsp90 machinery and, more generally, highlight the tissue-specific adaptation of sRNA pathways through chaperone diversification.

Distinct roles of two eIF4E isoforms in the germline of Caenorhabditis elegans.

Germ cells use both positive and negative mRNA translational control to regulate gene expression that drives their differentiation into gametes. mRNA translational control is mediated by RNA-binding proteins, miRNAs and translation initiation factors. We have uncovered the discrete roles of two translation initiation factor eIF4E isoforms (IFE-1, IFE-3) that bind 7-methylguanosine (m7G) mRNA caps during Caenorhabditiselegans germline development. IFE-3 plays important roles in germline sex determination (GSD), where it promotes oocyte cell fate and is dispensable for spermatogenesis. IFE-3 is expressed throughout the germline and localizes to germ granules, but is distinct from IFE-1 and PGL-1, and facilitates oocyte growth and viability. This contrasts with the robust expression in spermatocytes of IFE-1, the isoform that resides within P granules in spermatocytes and oocytes, and promotes late spermatogenesis. Each eIF4E is localized by its cognate eIF4E-binding protein (IFE-1:PGL-1 and IFE-3:IFET-1). IFE-3 and IFET-1 regulate translation of several GSD mRNAs, but not those under control of IFE-1. Distinct mutant phenotypes, in vivo localization and differential mRNA translation suggest independent dormant and active periods for each eIF4E isoform in the germline.

RNA-Guided Genomic Localization of H2A.L.2 Histone Variant.

The molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open question. Our previous investigations showed that in postmeiotic male germ cells, the genome-scale incorporation of histone variants TH2B-H2A.L.2 allows a controlled replacement of histones by protamines to occur. Here, we highlight the intrinsic ability of H2A.L.2 to specifically target the pericentric regions of the genome and discuss why pericentric heterochromatin is a privileged site of histone retention in mature spermatozoa. We observed that the intranuclear localization of H2A.L.2 is controlled by its ability to bind RNA, as well as by an interplay between its RNA-binding activity and its tropism for pericentric heterochromatin. We identify the H2A.L.2 RNA-binding domain and demonstrate that in somatic cells, the replacement of H2A.L.2 RNA-binding motif enhances and stabilizes its pericentric localization, while the forced expression of RNA increases its homogenous nuclear distribution. Based on these data, we propose that the specific accumulation of RNA on pericentric regions combined with H2A.L.2 tropism for these regions are responsible for stabilizing H2A.L.2 on these regions in mature spermatozoa. This situation would favor histone retention on pericentric heterochromatin.