Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.Miesten sukusolujen kehittyminen, spermatogeneesi, on tärkeä ja monella tavoin poikkeuksellinen kehitysprosessi, joka tuottaa suunnattomia määriä geneettisesti yksilöllisiä siittiöitä. Spermatogeneesi on monimutkainen, tarkasti säädelty tapahtumasarja, mikä myös aiheuttaa teknisiä haasteita spermatogeneesin molekyylimekanismien tutkimisessa. Näin ollen prosessin yksityiskohdat ovat vielä pitkälti tuntemattomia. Eräs siittiön haploidien esiasteiden (pyöreät spermatidit) erityisominaisuus on niiden ainutlaatuisen runsas transkriptionaalinen aktiivisuus. Genomin aktiivinen ilmentyminen puolestaan edellyttää tehokkaita ja täsmällisiä RNA:n säätelymekanismeja. Pyöreiden spermatidien solulimassa sijaitsee epätavallisen suuri RNA:ta ja proteiineja sisältävä rakenne, kromatoidikappale (chromatoid body, CB), joka ilmaantuu juuri voimakkaimman transkriptioaallon aikana ja osallistuu RNA-säätelyyn. Tutkimuksen tavoitteena oli selvittää CB:n toimintaa siittiönkehityksen aikana. Tärkeänä osana tutkimusta kehitimme menetelmän, jonka avulla CB:t voidaan eristää hiiren kiveksestä. Menetelmä on yksinkertainen, nopea ja tehokas, ja sen avulla saadaan eristettyä toistettavasti rakenteeltaan ehjiä CB:ta, joiden puhtaus on hyvä ja määrä riittävä molekyylitason analyysiin. Jatkotutkimukset paljastivat, että suurin osa CB:n sisältämistä proteiineista on erilaisilla RNA-säätelyreiteillä toimivia RNA:ta sitovia proteiineja. Useat CB:n proteiineista pysyvät rakenteessa stabiilisti, mutta näytimme myös, että RNA:ta sitova proteiini SAM68 vierailee CB:ssa vain hyvin hetkellisesti tarkasti määritellyssä kehitysvaiheessa. Poistogeenisen hiirimallin avulla saimme selville, ettei SAM68 proteiinia kuitenkaan tarvita CB:n muodostumiseen. Tulostemme mukaan CB:een kulkeutuu suuri määrä RNA:ta kaikissa pyöreiden spermatidien kehitysvaiheissa. RNA-sekvenointi osoitti, että pienet piRNA (PIWI-interacting RNA) molekyylit ovat rikastuneet CB:ssa. Lisäksi CB sisältää suuren joukon erilaisia lähetti-RNA:ita ja aivan tuntemattomia intergeenisiä eikoodaavia RNA:ita. Analyysimme mukaan hallitsevin CB:n molekyylireiteistä on piRNAvälitteinen RNA-säätelyreitti, mutta myös lähetti-RNA:n prosessointiin liittyvät tekijät ovat vahvasti edustettuina. CB ei selvästikään ole vain passiivinen RNA:n varastointipaikka, vaan näytimme sen kykenevän ATP:stä riippuvaiseen RNA prosessointiin in vitro Kaikki tutkimuksen tulokset osoittavat, että CB on keskeinen RNA:n säätelykeskus, joka koordinoi sukusolujen erittäin monimuotoista transkriptomia. Tällä toiminnallaan CB osallistuu tärkeänä tekijänä miesten hedelmällisyyden ja sukusolujen geneettisen ja epigeneettisen informaation säätelyyn.Siirretty Doriast

piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis.

MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.This work was supported by the National Institutes of Health R37 grant GM062534-14 to G.J.H. iTRAQ was performed with assistance from the Cold Spring Harbor Laboratory Proteomics Shared Resource, which is supported by Cancer Center support grant 5P30CA045508. W.S.S.G. is a McClintock Fellow of the Watson School of Biological Sciences and is supported by the NSS Scholarship from the Agency for Science, Technology and Research, Singapore. O.H.T. is supported by a fellowship of the Human Frontier Science Program. R.B. is supported by the Starr Centennial Scholarship from the Watson School of Biological Sciences. G.J.H. is a Howard Hughes Medical Institute Investigator.This is the final version of the article. It first appeared from Cold Spring Harbor Laboratory Press via http://dx.doi.org/10.1101/gad.260455.11

Enrichment of Pachytene Spermatocytes and Spermatids from Mouse Testes Using Standard Laboratory Equipment

To characterize each step of spermatogenesis, researchers must separate different subpopulations of germ cells from testes. However, isolating discrete populations is challenging, because the adult testis contains a complex mix of germ cells from all steps of spermatogenesis along with certain populations of somatic cells. Over the past few decades, different techniques such as centrifugal elutriation, fluorescence-activated cell sorting (FACS), and STA-PUT have been successfully applied to the isolation of germ cells. A drawback is that they all require dedicated devices and specialized training. Following principles underlying the STA-PUT method, a simple protocol has been developed for the isolation of pachytene spermatocytes, round spermatids, and elongating spermatids from mouse testes. After preparing a single cell suspension of testicular cells, specific cell populations are enriched by gravity sedimentation through a discontinuous bovine serum albumin (BSA) density gradient. The cell fractions are then manually collected and microscopically analysed. This modified density gradient for round spermatids (MDR) sedimentation protocol can be widely applied, because it requires only standard laboratory equipment. Furthermore, the protocol requires minimal starting materials, reducing its cost and use of laboratory animals

An atlas of chromatoid body components

The genome of male germ cells is actively transcribed during spermatogenesis to produce phase-specific protein-coding mRNAs and a considerable amount of different noncoding RNAs. Ribonucleoprotein (RNP) granule-mediated RNA regulation provides a powerful means to secure the quality and correct expression of the requisite transcripts. Haploid spermatids are characterized by a unique, unusually large cytoplasmic granule, the chromatoid body (CB), which emerges during the switch between the meiotic and post-meiotic phases of spermatogenesis. To better understand the role of the CB in male germ cell differentiation, we isolated CBs from mouse testes and revealed its full RNA and protein composition. We showed that the CB is mainly composed of RNA-binding proteins and other proteins involved RNA regulation. The CB was loaded with RNA, including pachytene piRNAs, a diverse set of mRNAs, and a number of uncharacterized long noncoding transcripts. The CB was demonstrated to accumulate nascent RNA during all the steps of round spermatid differentiation. Our results revealed the CB as a large germ cell-specific RNP platform that is involved in the control of the highly complex transcriptome of haploid male germ cells

The RNA Binding Protein SAM68 Transiently Localizes in the Chromatoid Body of Male Germ Cells and Influences Expression of Select MicroRNAs

The chromatoid body (CB) is a unique structure of male germ cells composed of thin filaments that condense into a perinuclear organelle after meiosis. Due to the presence of proteins involved in different steps of RNA metabolism and of different classes of RNAs, including microRNAs (miRNAs), the CB has been recently suggested to function as an RNA processing centre. Herein, we show that the RNA binding protein SAM68 transiently localizes in the CB, in concomitance with the meiotic divisions of mouse spermatocytes. Precise staging of the seminiferous tubules and co-localization studies with MVH and MILI, two well recognized CB markers, documented that SAM68 transiently associates with the CB in secondary spermatocytes and early round spermatids. Furthermore, although SAM68 co-immunoprecipitated with MVH in secondary spermatocytes, its ablation did not affect the proper localization of MVH in the CB. On the other hand, ablation of the CB constitutive component MIWI did not impair association of SAM68 with the CB. Isolation of CBs from Sam68 wild type and knockout mouse testes and comparison of their protein content by mass spectrometry indicated that Sam68 ablation did not cause overall alterations in the CB proteome. Lastly, we found that SAM68 interacts with DROSHA and DICER in secondary spermatocytes and early round spermatids and that a subset of miRNAs were altered in Sam68−/−germ cells. These results suggest a novel role for SAM68 in the miRNA pathway during spermatogenesis

Dicer1 Depletion in Male Germ Cells Leads to Infertility Due to Cumulative Meiotic and Spermiogenic Defects

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.Principal Findings: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.Conclusions/Significance: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis

Poly(A)-binding proteins are required for diverse biological processes in metazoans

PABPs [poly(A)-binding proteins] bind to the poly(A) tail of eukaryotic mRNAs and are conserved in species ranging from yeast to human. The prototypical cytoplasmic member, PABP1, is a multifunctional RNA-binding protein with roles in global and mRNA-specific translation and stability, consistent with a function as a central regulator of mRNA fate in the cytoplasm. More limited insight into the molecular functions of other family members is available. However, the consequences of disrupting PABP function in whole organisms is less clear, particularly in vertebrates, and even more so in mammals. In the present review, we discuss current and emerging knowledge with respect to the functions of PABP family members in whole animal studies which, although incomplete, already underlines their biological importance and highlights the need for further intensive research in this area

The Learned Gardeners of the Botanical Gardens of the University of Tartu and Their Activities (1803–1918)

The paper discusses the individual learned gardeners and assistant learned gardeners of the Botanical Garden of the University of Tartu against the backdrop of the development of botanical research areas at the university and species diversity in the botanical garden in 1803–1918. It also addresses the university’s botany professors / garden directors and assistant directors, focusing in more detail on the learned gardeners, who were more notable for their activities prior to commencing work in Tartu or during or after their Tartu period. A total of 22 learned gardeners and 14 assistant learned gardeners have been identified. Among them were persons from Germany , as well as representatives of other nationalities, including Estonians, Poles, Russians and local Germans. The employment duration of the learned gardeners at the botanical garden lasted from a few months to 42 years. The longest serving learned gardener was Wilhelm Eduard Stelling, a local German. Among the learned gardeners, the career and activities of Johann Anton Weinmann, Ludwig Riedel and Jan Muszynski stood out. After leaving Tartu, Weinmann and Muszynski became outstanding researchersin St. Petersburg and Poland, respectively, and Riedel emerged as a sucessful plant collector, scientific organiser and developer of park culture in Brazil

Sada aastat emakeelset akadeemilist metsandusharidust Eestis

SaateksTänavu möödub sada aastat päevast, kui Eesti Vabariigi Tartu Ülikoolis alustas tegevust metsaosakond, mida peetakse emakeelse metsandushariduse ja metsateaduse hälliks. Selle saja aasta sisse mahub kiiret arengut, spurte ja hoovõtte, aga ka seisakuid ja suunamuutusi. Kuigi metsaosakonna sünni tingis noore vabariigi terav ja kiireloomuline vajadus haritud metsandusspetsialistide järele, sai see siiski teoks Eesti Vabariigi alguspäevil loodud emakeelse ülikooli avamisega Tartus 1. detsembril 1919. Ehkki vastloodud rahvusülikoolis ei kujunenud metsaosakonna asutamine 1920. aastal kergeks ettevõtmiseks, rajas see tugeva baasi süsteemsele metsanduslikule uurimis- ja katsetegevusele ning metsanduslikule kõrgharidusele Eestis. Tänu metsaosakonna loomisele suurenes kiiresti kõrgharidusega metsaametnike hulk, kes rakendasid oma värskelt omandatud teadmised ja energia kodumaa metsamajanduse teenistusse. Lisaks metsandusspetsialistide kasvatamisele alustati metsaosakonnas rahvusliku metsandusliku teadusloomega, mis jõudis kiiresti ka rahvusvahelisele areenile. Metsandushariduse viljakatele algusaastatele järgnesid keerulised sõjaaastad. Paljud metsandusõppejõud ja –teadlased emigreerusid kodumaalt, arreteeriti või hukkusid. Järgnenud riigikorra muutus jättis paratamatult korvamatu jälje ka metsandusõppele ja -teadusele. Sellest hoolimata taasalustati uute lootustega vastses ülikoolis 1951. aastal, mil loodi Eesti Põllumajanduse Akadeemia. Ka taasiseseisvumise pöördelised aastad tõid akadeemilisse metsandusharidusse suunamuutusi. Viimastel aastakümnetel on akadeemilist metsandusõpet ja -teadust Eesti Põllumajandusülikoolis ja Eesti Maaülikoolis mõjutanud nii muutused ühiskonnas kui ka üldine haridus- ja keskkonnapoliitika. Tänase emakeelse metsandushariduse ja metsateaduse kestvat head tervist näitab see, et saja aasta jooksul on Eestis metsandusspetsialisti diplomini ja bakalaureuse- või rakendusmagistrikraadini jõudnud 4048 eestimaalast. Metsanduslike teadusmagistritööde kaitsjaid on sel perioodil olnud 83, kandidaadi- ja doktoritöid on kaitstud vastavalt 67 ja 56. Eestis tehtud töö on pälvinud ka kõrge tunnustuse – rahvusvahelises ülikoolide pingereas kuulub maaülikool täna maailma viiekümne parima ülikooli hulka just metsateaduse ja metsandusõppe alal. Noorte õppejõudude juurdekasv ning Eesti metsateaduse ja metsandushariduse jätkuvalt hea tase ja rahvusvaheline haare annavad kindlust, et Eesti metsanduslik kõrgharidus elab, kasvab ja õitseb ka järgmised sada aastat.Vivat, crescat, floreat scientia academica rerum forestalium in aeternum!Ivar SibulRaamatu väljaandmist on toetanud SA Keskkonnainvesteeringute Kesku