sperm chromatin

Mohammad Salehi Ph.D Embryologist

Chromatin structure in somatic cell • DNA• Proteins

• Histones: rich of lys & arg • H1, H2A, H2B, H3, H4

• Non Histones• Acidic proteins

• Hertons • Enzymes

• Topoisomerase ।,॥• Polymerases• Helicase

• Neutral proteins• Scarcely RNA

DNA structure Nucleotide

Base (A,T,C,G) Pentose sugar (deoxy

ribose) Phosphoric acid

B DNA

Organization of chromatin structure

• First level• Nucleosome: 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 chromosomes

H2A: redH2B: yellowH3: purpleH4: green

Histones are small, positively-charged proteins

The nucleosome has an approximate twofold axis of symmetry

Higher-order chromatin structure

H 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 structure

• Second level• 30 nm fiber

• Solenoid model• Zigzag (crossed linker)

model• Nucleomer (super bead)

model

• Third level• Super coil

The addition of H1 leads to more compact nucleosomal DNA

Without H1

30-nm fiber

Superhelix, 6 nucleosome per turn, supported by EM and X-ray studies

Based 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 sperm

During spermatogenesis spermatogonia undergo modification to produce spermatozoa that include:

• Mitosis (spermatocytogenesis)• Meiosis• Spermiogenesis

• Golgi phase• Cap phase• Acrosomal phase (chromatin condensation)• Maturation phase

Causes of condensation in sperm chromatin

• Reduction of size • Facilitate of sperm penetration into oocyte• Protection of genome from physical,

chemical and biological damages• Reprogram of paternal genome• Coordination of cell cycle between sperm

and oocyte

Protein replacement in spermiogenesis

Transitional Proteins

Somatic H

istones

Testicular Histones

Protamines

Increase basic property

Testicular Histones• Absence of H1• H2A

• H2AZ • H2AX

• H3 and H4 (hyperacetylation)• 15-20% remains in mature sperm

Mechanism of replacement

• Histones acetylation(H4)

• H2AX Phosphorylation

• Ubiquitination

Transitional proteinsType of protein MW (KDa) Amino acid

lengthProperties

HPI1(TP1) 6 54 Rich of arg, lys, hisDNA destabilization and promote of repair

HPI2(TP2) 13 137 Rich of cys, arg, lysBond to CG and cessation transcriptional activity

HPS1 N/A 69 P2 precursor

HPS2 N/A 66 P2 precursor

ProtaminesType of Protamine

MW (Da) Amino acid length

Properties

HP1 6692.2 50 Rich of arg, cys

HP2 7652.4 57 Rich of his

HP3 N/A 54 Rich of arg, cys, his

HP4 N/A 58 Rich of arg, cys, his

Amino acid sequence of P1&P2

Human P2 and P1

Bovine P1Anchoring domains

His, Arg, Arg sequences

P2 family: zinc finger proteins• Contain cys2/his2 motif• Inhibition of

transcription at the end of spermiogenesis

• Stabilization of sperm chromatin by zinc

• P2 family possibly attached to major groove of DNA

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

Mechanism of condensation• In bull and fish P1 attached to major groove

of DNA• Anchoring domains attached to major groove• After the synthesis of protamine, and before its

deposition on DNA, the serine and threonine residues in protamine are phosphorylated

• After attachment, this residues dephosphorylated

• Formation of intra and inter protamine disulfide bond

Mechanism of condensation• In humans and other mammalian which

have two or more protamine there are contradictory evidence:

• P1 & P2 lie down in major groove• P1 & P2 attached to both minor and major

groove• P1 & P2 attached to external surface of the

DNA chain

Evaluation of nuclear maturity • Chromatin decondensation induced in vitro • DNA staining after chromatin denaturation• Aniline blue staining• Assessment of thiol- disulfide status of nuclei• Ultra structural examination• Biochemical analysis• Comet assay• TUNEL assay• Sperm chromatin dispersion test (SCD test)