**5. Nuclear basic proteins**

Transition proteins TNP1 and TNP2 are involved in transition of histones to protamines. TNP2 was responsible for the condensation of DNA through interacting with PRM2 by methylating events. TNP1 and 2 are the basic proteins rich in lysine and arginine residues where as protamines consists of lysine and cysteines residues and loss of protein interactions between TNP2 and PRM2 results in prevention of histone replacement with protamines proved by spatiotemporal orientation of these genes on chromosome [16]. TNP1 is involved in decondensation of chromatin [17] at nucleosome core and relaxing DNA with the help of topoisomerase which removes positive supercoiling. TNP1 is also involved in repair of ss breaks of DNA generated during the chromatin remodelling. Histones are present as nucleosome core up to 11–12 step and replaced by TPs at 12–13 steps and by protamines in 13–15 steps. TNP1 and TNP2 are loaded on to DNA at apposition ends [18, 19] and the mutants of TNPs does not alter the expression of protamines PRM1 [20] and 2 (**Figure 2**).

**49**

**Figure 2.**

*Understanding the Epigenetic Modifications in Sperm Genome*

Histone to protamine transition involves replacement of histones by protamines. From the previous reports, transition proteins play an important role in guiding this process. But recent report on male infertility has proved that ubiquitination of histones by RNF8 a ring domain containing proteins in the nucleus [21]. This protein mainly interacts with MIWI a PIWI protein and sequesters the protein RNF 8 in the cytoplasm. This interaction mainly prevents histone replacement by protamines even though the expression of protamines and transition proteins are normal. In this case not only the replacement of histones, but also the posttranslational modifications in protamines also play a major role in male fertility [22]. For example incorporation of Prm2 on chromatin requires phosphorylation of Prm2 to attain maturity of the protein so that it can interact with DNA. Phosphorylation of

From the recent reports RNF8 is required for ubiquitination of H2A and H2B followed by H4 acetylation. Ubiquitinated H2 and H2B acts as platform for binding of MOF (Males absent of first) necessary for dosage compensation in drosophila fly and adds acetyl groups to H4 leading to free chromatin. The role of RNF8 is limited to spermatid stage and post meiotic, and plays an important role in male fertility. But how the ubiquitinated histones are protected from degradation by proteasome is a major question. But it is solved by spatial and temporal expression of protea-

Males defective in genes Tnp1 and Tnp2 does not effect the expression of proteins either protamines or Tnps. But abnormal retention of Tnp1 and 2 is the main reason for elevated levels in both Tnp−/− and as well as Tnp+/− mutants. The mutations would not affect the transition from histones to protamines. Histone to Protamine transition follows inter and intra disulfide bond formation in between

Prm2 is carried out by CAMK, loss of which leads to male infertility.

*Factors responsible for maintenance of genome integrity from parent to offspring.*

somal proteins especially in the late spermatid state.

*DOI: http://dx.doi.org/10.5772/intechopen.88506*

*Understanding the Epigenetic Modifications in Sperm Genome DOI: http://dx.doi.org/10.5772/intechopen.88506*

**Figure 2.**

*Innovations in Assisted Reproduction Technology*

required for replacement of histones with protamines [12].

Methylation is one of the main PTM to be considered during spermiogenesis. Lysine methylation is the most common methylation in histones during spermiogenesis. To some extent arginine methylation by type iii arginine methylases was also one of the common methylation that occurs in protamines like PRM2. Methylations like H2BK117 and H2BK121 is required for TH2B replacement in germ cells. Methylation in H3K9 was required for maintaining chromatin in repressed state [9], and it helps in maintaining some residual nucleosome core histones in paternal genome seen in sperm. Phosphorylation of H2A byTSSK6 is required for histone acetylation followed by condensation of chromatin [10, 11]. May be DNA double strand breaks activate TSSK6 in late spermatids forming ɤH2AX foci

Poly ADP-ribosylation is one of the events involved in gene regulation and Cell proliferation. ADP-ribosylation was another important modification to be considered during meiotic programme and also during DNA condensation [13]. Both the steps requires ds break formation and as per recent reports poly ADP ribosylation was seen during stages of spermatocytes and spermatids but not in case of mature spermatids. Ds breaks activates enzyme Poly ADP ribosyl polymerase which was taken as a score of DNA damage that proceeds with ds break formation [14, 15]. Methylation, Ubiquitination, acetylation were some of the common modifications seen in sperm chromatin during late spermatid stages. However sumoylation is attributed to centromere and telomeric heterochromatin regions. Presence of canonical histones was necessary for the identification of the paternal genome by embryo. In late spermatid stage most of the genome mRNA is kept in repressed state where as Protamine and transition proteins synthesis occurs in early spermatocytes. Phosphorylation is one of the PTM that protamine undergoes before its exchange with transition proteins. After loading on DNA dephosphorylation and disulfide bond formation of protamine occurs. Protamine P2 is synthesised as precursor and processed later on in order to get a functional protein. Disulfide bond formation mainly occurs during the transit of spermatozoa from caput to cauda. Thiol content has positive relation with tyrosine phosphorylation and infertility. Some of the residual Histones must be required in the sperm cells as it is necessary for gene expression and genetic imprinting of certain genes in paternal genome. Phosphorylation of protamines was effectively seen at some places and necessary for effective binding with DNA. Zinc stabilises the sperm chromatin mainly due to binding to protamines. Protamines interacts with toxic metals so pesticides used, damages DNA integrity by binding to protamines and some of them may also cause alkylation of protamines.

Transition proteins TNP1 and TNP2 are involved in transition of histones to protamines. TNP2 was responsible for the condensation of DNA through interacting with PRM2 by methylating events. TNP1 and 2 are the basic proteins rich in lysine and arginine residues where as protamines consists of lysine and cysteines residues and loss of protein interactions between TNP2 and PRM2 results in prevention of histone replacement with protamines proved by spatiotemporal orientation of these genes on chromosome [16]. TNP1 is involved in decondensation of chromatin [17] at nucleosome core and relaxing DNA with the help of topoisomerase which removes positive supercoiling. TNP1 is also involved in repair of ss breaks of DNA generated during the chromatin remodelling. Histones are present as nucleosome core up to 11–12 step and replaced by TPs at 12–13 steps and by protamines in 13–15 steps. TNP1 and TNP2 are loaded on to DNA at apposition ends [18, 19] and the mutants of TNPs

does not alter the expression of protamines PRM1 [20] and 2 (**Figure 2**).

**48**

**5. Nuclear basic proteins**

*Factors responsible for maintenance of genome integrity from parent to offspring.*

Histone to protamine transition involves replacement of histones by protamines. From the previous reports, transition proteins play an important role in guiding this process. But recent report on male infertility has proved that ubiquitination of histones by RNF8 a ring domain containing proteins in the nucleus [21]. This protein mainly interacts with MIWI a PIWI protein and sequesters the protein RNF 8 in the cytoplasm. This interaction mainly prevents histone replacement by protamines even though the expression of protamines and transition proteins are normal. In this case not only the replacement of histones, but also the posttranslational modifications in protamines also play a major role in male fertility [22]. For example incorporation of Prm2 on chromatin requires phosphorylation of Prm2 to attain maturity of the protein so that it can interact with DNA. Phosphorylation of Prm2 is carried out by CAMK, loss of which leads to male infertility.

From the recent reports RNF8 is required for ubiquitination of H2A and H2B followed by H4 acetylation. Ubiquitinated H2 and H2B acts as platform for binding of MOF (Males absent of first) necessary for dosage compensation in drosophila fly and adds acetyl groups to H4 leading to free chromatin. The role of RNF8 is limited to spermatid stage and post meiotic, and plays an important role in male fertility. But how the ubiquitinated histones are protected from degradation by proteasome is a major question. But it is solved by spatial and temporal expression of proteasomal proteins especially in the late spermatid state.

Males defective in genes Tnp1 and Tnp2 does not effect the expression of proteins either protamines or Tnps. But abnormal retention of Tnp1 and 2 is the main reason for elevated levels in both Tnp−/− and as well as Tnp+/− mutants. The mutations would not affect the transition from histones to protamines. Histone to Protamine transition follows inter and intra disulfide bond formation in between

cysteines of protamines there by centering the amino and carboxy terminal ends with DNA binding domain. Disulfide bonds are necessary for further compaction of DNA and four of seven cysteines in protamines were involved in disulfide bond formation. Reduction of disulfide bonds leads to decondensation of chromatin proved by X-ray crystallography. Intra and inter linking of protamines through disulfide bond formation occurs at spermatid stage especially during maturation in epididymis [23], before to 14 days of their release.

Inhibin B activates mainly five pathways namely GPCR signalling, calcium pathway [24], MAP kinase pathway, PI3 pathway, Phospholipase A2 pathway. In which GPCR signalling, calcium Pathway and MAP kinase pathway causes phosphorylation of CAMP Responsive Element Modulator (CREM). This will leads to decreased expression of Prm2 causing male infertility. As earlier discussed histone— Protamine replacement requires certain modifications in histones like acetylation. In mouse CHD5 promotes H4 acetylation and ablation of this gene leads to retention of H3 and decreased condensation of nuclear DNA in head. This gene is expressed in both brain and testes but with higher in testes. Haploid insufficiency of gene does not have any effect on male fertility but the DNA condensation is affected further from step 9 of spermiogenesis [25] with out affecting brain tissue. Abnormality of sperm was mainly due to defects in condensation of chromatin [26] but not due to other defects like cytoplasmic retention, apical epistatic specialisation, acrosomes biogenesis, flagella movement and mitochondrial sheath formation.
