**3. Testes-specific histones**

*Innovations in Assisted Reproduction Technology*

compared with histones.

**2. Protamines are originated from histone H1**

In humans and mammals histones was replaced by either protamines or histone variants in sperm cell. But recent discovery of protamine P1 show both N- terminal and C- terminal sequence similarity between each other, and where as histone variant precursor resembled to that of protamine P2. Histones are basic proteins rich in Arg and Lys residues where as protamines in Styela contain polyarginine tracts than

Sperm condensation is one of the important criteria to be considered during assisted reproductive technologies in order to prevent genetic defects in upcoming generation. Spermatid formation involves 12–14 steps, and finally elongation of nuclear material leads to elongated sperms with acrosome. In sperm, histones are replaced by protamines and in humans histone variants are present leading to sperm condensation. Disulfide bonds contribute additional stability to the condensation as it results in interlinking of protamines [2, 3]. Certain PTMs of histones was also one of the epigenetic modifications required for condensation. Decondensation of Sperm chromatin takes place after sperm penetration in to oocyte which leads to exposure of sperm DNA [4] to oocyte reducing agents like glutathione. As known, glutathione reduces disulfide bonds between the protamines and relaxing the sperm DNA. From the previous reports, agents like SDS, EDTA reduces the disulfide bonds in combined state. Glycosaminoglycans like heparin sulphate binds to receptors on sperm membrane and

promotes decondensation instead with nonsulfated form heparin (**Figure 1**).

*Flow cytometry of spermatozoa showing DNA fragmentation. Where (a) is control, (b) is testes treated with* 

*Metosartan (c) with RNaseA +aspirin and (d) with RNaseA+metosartan.*

**46**

**Figure 1.**

H1T1 was one of the variant of linker histone found during pachytene of the cell division. H1T2 was the other linker variant found in apical pole of spermatids and responsible for male fertility. In case of humans HilS1 is necessary for condensation in elongated spermatids. Certain Core histone variants like TH2A and TH2B are found to be elevated during elongation of spermatids and whereas the normal core histones show low amounts of expression. Gene knock outs of TH2A and TH2B induced male infertility but PTMs on core histones compensate the loss of these variants in the spermatids [5]. TH2A was found to be present at transcriptional start site [6] and where as TH2B is required during leptotene of the cell division.

Other histone variant is with core histone H3 which include H3.3, H3T and CenP. H3.3 is present at the stage of spermatids [7] and where as H3T was specifically expressed in testes and both differ by 5 amino acid residues from core histone H3.1. H3.3 represents actively transcripted regions where as H3.1 represents transcriptionally repressive regions and may be involved in replacement of Histones by protamines. H3.1 may be required for maintaining the repression of X and Y chromosomes where as H3.1 is involved in activating the genes required for transition protein and protamine synthesis.

Recently, humans contain histone variants instead of canonical histones which are subjected to acetylation at H4K5, H4K8, H4K12 and H4K16. In Drosophila H3 is also acetylated especially at H3 K9 by Plant domain containing protein PYGO2 and responsible for open confirmation of chromatin. Presence of histone chaperones as identified recently in Drosophila named CAF1 (Chromatin Assembly Factor) and in humans was normal Heat shock protein variant namely HSPA2 (HSP 70.2). These chaperones acts as chromatin remodellers and responsible for exchange of transition proteins with protamines. ATP dependent remodeller proteins that act as histone chaperones was not known up to now, but above mentioned are some of the remodellers that help in chromatin remodelling in testes.

## **4. Various histone modifications during spermiogenesis**

Histone modifications like phosphorylation, acetylation and ubiquitination play an important role in spermiogenesis [8]. Phosphorylation is found to be common on the four core histone proteins, and acetylation was mostly seen in H4, whereas ubiquitination was with respect to H2A and B. Phosphorylation and acetylation leads to neutralisation of charges on histones whereas ubiquitylation causes Rnf 8 mediated recruitment of transition proteins and found to be normal in gene knock out animals with respect to meiosis and repression of expression in germ cells.

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 required for replacement of histones with protamines [12].

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.
