This document discusses the structure and organization of chromatin in somatic cells and sperm cells. In somatic cells, DNA is wrapped around histone proteins to form nucleosomes, which further condense to form the 30nm fiber. In sperm cells, histones are replaced by transitional proteins and protamines to heavily compact the DNA for protection and transport. This compaction involves acetylation, phosphorylation and ubiquitination of histones, followed by replacement with basic transitional proteins and protamines containing cysteine residues that form disulfide bonds, further condensing the DNA for sperm function. Various assays are used to evaluate the maturity and integrity of sperm chromatin structure.

1. Mohammad Salehi Ph.D
Embryologist

2. 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

3. DNA structure
 Nucleotide
 Base (A,T,C,G)
 Pentose sugar (deoxy
ribose)
 Phosphoric acid
B DNA

4. 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)

5. Nucleosome

6. nucleosome- building blocks of chromosomes
H2A: red
H2B: yellow
H3: purple
H4: green
Histones are small, positively-charged proteins

8. The nucleosome has an approximate twofold axis of symmetry

9. Higher-order chromatin structure
H binds to linker DNA at one end of
The nucleosome and the central DNA helix

10. 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

11. Organization of chromatin
structure
• Second level
• 30 nm fiber
• Solenoid model
• Zigzag (crossed linker)
model
• Nucleomer (super bead)
model
• Third level
• Super coil

12. The addition of H1 leads to more compact nucleosomal DNA
Without H1

14. 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,

16. Higher compaction of DNA involves large loops of
nucleosomal DNA
Nuclear scaffold (Topo II, SMC)

17. How histones chaperones facilitate the assembly of nucleosome
during DNA replication
(sliding clamp)

18. 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

19. 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

20. Protein replacement in
spermiogenesis
TransitionalProteins
SomaticHistones
TesticularHistones
Protamines
Increase basic property

21. Testicular Histones
• Absence of H1
• H2A
• H2AZ
• H2AX
• H3 and H4 (hyperacetylation)
• 15-20% remains in mature sperm

22. Mechanism of replacement
• Histones acetylation(H4)
• H2AX Phosphorylation
• Ubiquitination

23. Transitional proteins
Type of protein MW (KDa) Amino acid
length
Properties
HPI1(TP1) 6 54 Rich of arg, lys, his
DNA destabilization
and promote of repair
HPI2(TP2) 13 137 Rich of cys, arg, lys
Bond to CG and
cessation transcriptional
activity
HPS1 N/A 69 P2 precursor
HPS2 N/A 66 P2 precursor

24. Protamines
Type 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

26. Amino acid sequence of
P1&P2
Human P2 and P1
Bovine P1
Anchoring domains
His, Arg, Arg sequences

27. 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

28. 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

30. 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

31. 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

33. 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)