sperm chromatin

IN THE NAME OF ALLAH

Mohammad Salehi Ph.D Embryologist

Chromatin structure in somatic cell DNAProteinsHistones: rich of lys & arg H1, H2A, H2B, H3, H4Non HistonesAcidic proteinsHertons Enzymes Topoisomerase ,PolymerasesHelicase Neutral proteinsScarcely RNA

DNA structureNucleotideBase (A,T,C,G) Pentose sugar (deoxy ribose)Phosphoric acid

B DNA

Organization of chromatin structureFirst levelNucleosome: cylindrical shape ( diameter: 10 nm , height: 6nm) Core particle contains an octamer of 2 each of the core histones (H2A, H2B, H3 and H4) 146 bp of DNA wrapped 1.75 turns 10 nm fiber: Nucleosome + linker DNA (0-50bp)

Nucleosome

nucleosome- building blocks of chromosomesH2A: redH2B: yellowH3: purpleH4: greenHistones are small, positively-charged proteins

The nucleosome has an approximate twofold axis of symmetry

Higher-order chromatin structureH binds to linker DNA at one end ofThe nucleosome and the central DNA helix

The core Histone N-terminal tails are required for the formation of the 30-nm fiber

The tail of H2A, H3 and H4 interact with adjacent nucleosome

Organization of chromatin structureSecond level30 nm fiberSolenoid modelZigzag (crossed linker) modelNucleomer (super bead) modelThird levelSuper coil

The addition of H1 leads to more compact nucleosomal DNAWithout H1

30-nm fiberSuperhelix, 6 nucleosome per turn, supported by EM and X-ray studiesBased on zigzag pattern upon H1 addition, requires linker DNA to pass through central axis,

Higher compaction of DNA involves large loops ofnucleosomal DNA Nuclear scaffold (Topo II, SMC)

How histones chaperones facilitate the assembly of nucleosomeduring DNA replication

(sliding clamp)

Chromatin organization in spermDuring spermatogenesis spermatogonia undergo modification to produce spermatozoa that include:Mitosis (spermatocytogenesis)MeiosisSpermiogenesis Golgi phaseCap phaseAcrosomal phase (chromatin condensation)Maturation phase

Causes of condensation in sperm chromatinReduction of size Facilitate of sperm penetration into oocyteProtection of genome from physical, chemical and biological damagesReprogram of paternal genomeCoordination of cell cycle between sperm and oocyte

Protein replacement in spermiogenesisTransitional ProteinsSomatic HistonesTesticular HistonesProtamines

Increase basic property

Testicular HistonesAbsence of H1H2AH2AZ H2AXH3 and H4 (hyperacetylation)15-20% remains in mature sperm

Mechanism of replacementHistones acetylation(H4)

H2AX Phosphorylation

Ubiquitination

Transitional proteinsType of proteinMW (KDa)Amino acid lengthProperties HPI1(TP1)654Rich of arg, lys, hisDNA destabilization and promote of repairHPI2(TP2)13137Rich of cys, arg, lysBond to CG and cessation transcriptional activity HPS1N/A69P2 precursor HPS2N/A66P2 precursor

ProtaminesType of ProtamineMW (Da)Amino acid length Properties HP16692.250Rich of arg, cysHP27652.457Rich of his

HP3N/A54Rich of arg, cys, hisHP4N/A58Rich of arg, cys, his

Amino acid sequence of P1&P2

Human P2 and P1Bovine P1Anchoring domains

His, Arg, Arg sequences

P2 family: zinc finger proteinsContain cys2/his2 motifInhibition of transcription at the end of spermiogenesisStabilization of sperm chromatin by zinc P2 family possibly attached to major groove of DNA

Comparison P1 and P2 P1/P2 ratio= 0.98 0.12P1 rich of cysP2 rich of his and few cys compare to P1P2 is more basic P2 has more affinity to DNA

Mechanism of condensationIn bull and fish P1 attached to major groove of DNAAnchoring domains attached to major grooveAfter the synthesis of protamine, and before its deposition on DNA, the serine and threonine residues in protamine are phosphorylatedAfter attachment, this residues dephosphorylatedFormation of intra and inter protamine disulfide bond

Mechanism of condensationIn humans and other mammalian which have two or more protamine there are contradictory evidence:P1 & P2 lie down in major grooveP1 & P2 attached to both minor and major grooveP1 & P2 attached to external surface of the DNA chain

Evaluation of nuclear maturity Chromatin decondensation induced in vitro DNA staining after chromatin denaturationAniline blue stainingAssessment of thiol- disulfide status of nucleiUltra structural examinationBiochemical analysisComet assayTUNEL assaySperm chromatin dispersion test (SCD test)