**6. ABP as the main molecule for chromatin condensation and decondensation**

Androgen Binding Protein (ABP) acts as carrier for steroid hormone [27] Dihydrotestosterone the potent form of testosterone which acts as acceptor of hydrogen atoms. It oxidises disulfide bonds in the nucleus of sperm at the time of

**51**

up to shorter time periods.

*Understanding the Epigenetic Modifications in Sperm Genome*

maturation in caput epididymis. 3- Hydroxy steroid dehydrogenase is the enzyme that has both oxidase and reductase activities but oxidase activity requires activation by female sex hormones like oestrogens or Progesterone. The enzyme requires NADPH as reducing equivalent and later glutathione after oocyte penetration to induce decondensation [2]. The process requires 3- diol instead of dihydrotestosterone. The steps of condensation are ABP > ABP—Dihydrotestosterone > Nuclear Localisation > Release of Dihydrotestosterone > Condensation > Penetration in to oocyte by sperm > Decondensation > Decondensation further By Glutathione >

In S phase the dye uptake is more compared to G1 phase, where as similar in M phase as that of the S phase because immature sperms are stained by aniline blue and in case of aneuploid and apoptotic cells the dye uptake is low. By aniline blue we cannot distinguish the dividing cells from the cells undergoing segregation of the nuclear content followed by cytokinesis. The maximum absorption spectrum of aniline blue is 234.5 nm which can be used to detect the dye by flow cytometry using detector at this wavelength. It is low cost effective compared to propidium iodide but it has yet to be used and by this, we can study the aneuploidy, sperm maturity and apoptosis of sperm cells by using ejaculated semen sample. Flow cytometry analysis of sperm suspension collected from epididymis can be performed after staining with aniline blue as protamines exclude aniline blue, only immature sperms takes aniline blue so, this property of dye can be used to study sperm maturity by flow cytometry.

Spermatocyte recombination mainly occurs by double strand break formation and involves interactions between chromosome domains by forming nuclear territories. The telocentric chromosomes along with NORs form bouquet like structure at the periphery of the nucleus where as metacentric chromosomes at centre of the nucleus. If 2n = 24, 8 bivalents are formed and attached to form bouquet structure. There was little exchange seen between the nonhomologous chromosomes in Mus species so evolutionary importance is less in these species. Heterochromatin surrounding the centromere form the basis for absence of recombination as it encodes

Constitutive heterochromatin in nucleolus makes the rDNA arrangement with difference in different species. Together with proximal arrangement with respect to telomere and metacentric chromosomes with NORs in long arms serve as excellent models for study of both NORs and bouquet formation, seen in maintenance of topology. Bouquet formation is necessary for interaction of chromosomal domains in nuclear territories. Two telocentric chromosome forms a single metacentric

EtBr is normally used as intercalating dye to study DNA and also to stain DNA. Increased concentrations of EtBr causes decondensation of DNA as the bonds between the intercalated bases rearrange and results in loop formation. After certain concentration of dye, the decondensed DNA will be condensed again, i.e., biphasic kinetics was seen with the epididymal sperms. In late elongated spermatids histones are already replaced by protamines as cysteines present in protamines provide extra stability and these sperms are proven to resistant to DNase I digestion,

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

Reducing disulfide bonds in DNA (**Figure 3**).

**7. Cell cycle analysis in spermatozoa using aniline blue**

**8. Understanding the topology of spermatocytes**

proteins that keep the centromere integrity.

chromosome which forms the basis of recombination.

**Figure 3.** *Overall schematic representation of condensation and decondensation of sperm DNA.*

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

*Innovations in Assisted Reproduction Technology*

epididymis [23], before to 14 days of their release.

**decondensation**

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

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—

biogenesis, flagella movement and mitochondrial sheath formation.

**6. ABP as the main molecule for chromatin condensation and** 

Androgen Binding Protein (ABP) acts as carrier for steroid hormone [27] Dihydrotestosterone the potent form of testosterone which acts as acceptor of hydrogen atoms. It oxidises disulfide bonds in the nucleus of sperm at the time of

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

**50**

**Figure 3.**

*Overall schematic representation of condensation and decondensation of sperm DNA.*

maturation in caput epididymis. 3- Hydroxy steroid dehydrogenase is the enzyme that has both oxidase and reductase activities but oxidase activity requires activation by female sex hormones like oestrogens or Progesterone. The enzyme requires NADPH as reducing equivalent and later glutathione after oocyte penetration to induce decondensation [2]. The process requires 3- diol instead of dihydrotestosterone. The steps of condensation are ABP > ABP—Dihydrotestosterone > Nuclear Localisation > Release of Dihydrotestosterone > Condensation > Penetration in to oocyte by sperm > Decondensation > Decondensation further By Glutathione > Reducing disulfide bonds in DNA (**Figure 3**).

### **7. Cell cycle analysis in spermatozoa using aniline blue**

In S phase the dye uptake is more compared to G1 phase, where as similar in M phase as that of the S phase because immature sperms are stained by aniline blue and in case of aneuploid and apoptotic cells the dye uptake is low. By aniline blue we cannot distinguish the dividing cells from the cells undergoing segregation of the nuclear content followed by cytokinesis. The maximum absorption spectrum of aniline blue is 234.5 nm which can be used to detect the dye by flow cytometry using detector at this wavelength. It is low cost effective compared to propidium iodide but it has yet to be used and by this, we can study the aneuploidy, sperm maturity and apoptosis of sperm cells by using ejaculated semen sample. Flow cytometry analysis of sperm suspension collected from epididymis can be performed after staining with aniline blue as protamines exclude aniline blue, only immature sperms takes aniline blue so, this property of dye can be used to study sperm maturity by flow cytometry.

## **8. Understanding the topology of spermatocytes**

Spermatocyte recombination mainly occurs by double strand break formation and involves interactions between chromosome domains by forming nuclear territories. The telocentric chromosomes along with NORs form bouquet like structure at the periphery of the nucleus where as metacentric chromosomes at centre of the nucleus. If 2n = 24, 8 bivalents are formed and attached to form bouquet structure. There was little exchange seen between the nonhomologous chromosomes in Mus species so evolutionary importance is less in these species. Heterochromatin surrounding the centromere form the basis for absence of recombination as it encodes proteins that keep the centromere integrity.

Constitutive heterochromatin in nucleolus makes the rDNA arrangement with difference in different species. Together with proximal arrangement with respect to telomere and metacentric chromosomes with NORs in long arms serve as excellent models for study of both NORs and bouquet formation, seen in maintenance of topology. Bouquet formation is necessary for interaction of chromosomal domains in nuclear territories. Two telocentric chromosome forms a single metacentric chromosome which forms the basis of recombination.

EtBr is normally used as intercalating dye to study DNA and also to stain DNA. Increased concentrations of EtBr causes decondensation of DNA as the bonds between the intercalated bases rearrange and results in loop formation. After certain concentration of dye, the decondensed DNA will be condensed again, i.e., biphasic kinetics was seen with the epididymal sperms. In late elongated spermatids histones are already replaced by protamines as cysteines present in protamines provide extra stability and these sperms are proven to resistant to DNase I digestion, up to shorter time periods.

### **9. Molecular chaperones are necessary for male fertility**

Molecular chaperones are the proteins, which help in folding of proteins and prevent their aggregation. So after spermiogenesis, post testicular maturation in requires proteins like chaperones in addition to several other proteins in epididymis. Those are the proteins like HSP60, HSP70 family including HSPA2, HSPA5, and other than HSP 70 family include chaperonin containing t- complex protein and HSP90. HSP 70 plays an important role in maintaining proteins in partially folded state and facilitates in transport of those proteins in the membranes. HSPa2 is responsible for spermiogenesis and also aids in maturation of sperms. It binds to the plasma membrane of sperm along with its co chaperones and forms heterocomplex with CDC2 and cyclin B during G1- S phase and G2- M phase transitions. This protein is also present in ovial secretions along with HSPA5 and is necessary for capacitation of sperms.

As discussed previous the thiol content reflects the tyrosine phosphorylation. HSPa2 promotes condensation analogous to decondensation factor and terminates signalling by tyrosine phosphorylation. Chaperonin containing t- complex protein (CCT) binds to sperm plasma membrane and promotes ZP binding as it promotes binding to ZP receptors. CCT protein along with its co chaperones enters the sperm through endocytosis of receptor and ligand coated with caveolin protein in membrane rafts. This protein is mainly present in t-complex of sperm and prevents protein aggregation or misfolding. CCT is mainly composed of 8 subunits in which 3 subunits form the domains and substrate recognition was through electrostatic and hydrophobic interactions, and then transported to the central cavity of the protein.

HSP60 was the other molecular chaperone found in mitochondria and helps in transport of proteins in to mitochondria. HSPA5 also known as Bip aids in folding of proteins in endoplasmic reticulum and participates in ER stress signalling pathway. Like HSPA5 calcium binding protein Calmigen also express in ER and become arrested after late spermatid stage. HSPA8 the one of the member of HSP70 and was expressed ubiquitously and facilitates binding of sperm to ovum. So, injection of this protein with IVF may be useful as it is important for enhanced survival of sperm in vivo.

Calmigen and Calsporin were the variants of Calnexin and Calreticulin seen in testes and along with ADAM1A, ADAM2 and ADAM 3 were required for sperm capacitation by interacting with zona of oocyte and helps in sperm—oocyte interaction. Calreticulin is mainly necessary for calcium oscillations inside the sperm cells for hyper activation and capacitation. PDI (Protein Disulfide Isomerase) along with Calmigen is necessary for disulfide bond formation and remodelling in sperm.

### **10. Epigenetic modulations in sperm genome**

Histone H3 undergoes demethylation at K9 due to loss of lysine demethylase in primordial germ cells after E7.5 days. In meiotic recombination histone mono, di tri methylations are responsible for DSBs and recombination. Methylation at pericentric regions [28] is responsible for preventing homologous recombination. Retrotransposons restrict methylating events and responsible for maintenance of genome imprinting. Reduced DNA methylation in retrotransposons leads to gene expression in LINE 1 transposons and proteins like PIWI and AUBERGINE undergoes methylation in repetitive elements of PGC and responsible for gene silencing mechanisms [29]. This pattern was found to be conserved in mice and in animals and mammals, in addition to mutational hotspots there are some recombination hotspots characterised by H3K4me3 and H3K9 acetylation.

**53**

*Understanding the Epigenetic Modifications in Sperm Genome*

Paternal genomes are hypomethylated compared to maternal genome and in Drosophila the protamine eviction was done by DHD (Dead Head) by reducing the disulfide bonds with the help of thioredoxin and NADPH. Protamines form oligomers by intrachain disulfide bonds. Molecular chaperone TAP/p32 acts on monomeric protamines and causes their eviction and found to be inactive on oligomers. DHD interacts physically with TAP/P32 to cause the eviction of protamines from DNA and TAP/P32 binds poorly to DNA. DHD normally found in sperm nucleus and was degraded after fertilisation. DHD domain was necessary for the reduction of disulfide bonds, DNA decondensation, and protamine eviction and also functions as chaperone. It shares some common properties with thioredoxins and also has unique properties necessary for the embryo formation. Mutation in DHD domain is non tolerant and results in haploid embryo formation with the absence of

Proteomic profile of testes sperm was one of the important researches in progress nowadays. Normally in DNA condensing proteins the disulfide bonds are oxidised spontaneously and does not require any enzymatic source. There are certain proteins in sperm which require enzymes like thioredoxin/Glutathione reductase which acts on glutathione peroxidise and induces disulfide bond formation. Thioredoxin glutathione reductase catalyses isomerisation of disulfide bonds in these proteins and the protein was mainly localised at mid piece of mitochondrial sheath and was required for structural maintenance of mid piece and tail proteins. Those include proteins like mitochondrial capsule selenoprotein, two outer dense fibre (ODF) and glutathione-s-transferase M5. Differences in maternal and paternal chromosomes mainly occur at pericentric chromosomes, through which epigenetic

**11. Differentiation in meiotic events of sperm and oocyte in** *C. elegans*

In oocyte, synaptonemal complex (SC) is formed along the lateral and central axis. In sperms condensation plate is formed after diplotene. Formation of karyosomes, [the aggregation of chromosomes] is characterised in both oocytes and sperms of *C. elegans* and other mammals and referred as karyosome stage in *C. elegans*. The meiotic process mainly differs in microtubule dynamics. In oocytes chromosome dependent spindle formation is necessary for chromosome segregation, where as in case of sperms centrioles are present. So, the spindle formation is

There are some of the kinetochore differences observed in oocytes and sperms. In sperms, outer kinetochore proteins attract the inner proteins. CENP –c is present in spermatocytes and CENP- A is present in oocytes. Some of the kinases like AIR-2 is present in spermatocytes and PLK-1 is found in oocytes. SYP-1 is necessary protein of central element in SC and recruits AIR-2. Mutation in SYP-1 protein leads to random distribution of metaphase bivalents. Some of the kinases like AIR-2 phosphorylates Rec-8 protein which is required for cohesion of sister chromatids. Cytokinesis after meiosis −1 is incomplete in both sperms and oocyte without any

necessity for re condensation of chromatin after diplotene.

**12. Oxidative potential in testes and epididymis is required for** 

GPRX 4 and PXRX4 are the two enzymes which are mainly required for compaction of chromatin in sperm mainly by sulfoxidation of protamines attached to

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

male pronucleus [30].

message is conveyed to embryo.

mediated by centrioles.

**spermiogenesis**

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

*Innovations in Assisted Reproduction Technology*

capacitation of sperms.

sperm in vivo.

**9. Molecular chaperones are necessary for male fertility**

Molecular chaperones are the proteins, which help in folding of proteins and prevent their aggregation. So after spermiogenesis, post testicular maturation in requires proteins like chaperones in addition to several other proteins in epididymis. Those are the proteins like HSP60, HSP70 family including HSPA2, HSPA5, and other than HSP 70 family include chaperonin containing t- complex protein and HSP90. HSP 70 plays an important role in maintaining proteins in partially folded state and facilitates in transport of those proteins in the membranes. HSPa2 is responsible for spermiogenesis and also aids in maturation of sperms. It binds to the plasma membrane of sperm along with its co chaperones and forms heterocomplex with CDC2 and cyclin B during G1- S phase and G2- M phase transitions. This protein is also present in ovial secretions along with HSPA5 and is necessary for

As discussed previous the thiol content reflects the tyrosine phosphorylation. HSPa2 promotes condensation analogous to decondensation factor and terminates signalling by tyrosine phosphorylation. Chaperonin containing t- complex protein (CCT) binds to sperm plasma membrane and promotes ZP binding as it promotes binding to ZP receptors. CCT protein along with its co chaperones enters the sperm through endocytosis of receptor and ligand coated with caveolin protein in membrane rafts. This protein is mainly present in t-complex of sperm and prevents protein aggregation or misfolding. CCT is mainly composed of 8 subunits in which 3 subunits form the domains and substrate recognition was through electrostatic and hydrophobic interactions, and then transported to the central cavity of the protein. HSP60 was the other molecular chaperone found in mitochondria and helps in transport of proteins in to mitochondria. HSPA5 also known as Bip aids in folding of proteins in endoplasmic reticulum and participates in ER stress signalling pathway. Like HSPA5 calcium binding protein Calmigen also express in ER and become arrested after late spermatid stage. HSPA8 the one of the member of HSP70 and was expressed ubiquitously and facilitates binding of sperm to ovum. So, injection of this protein with IVF may be useful as it is important for enhanced survival of

Calmigen and Calsporin were the variants of Calnexin and Calreticulin seen in testes and along with ADAM1A, ADAM2 and ADAM 3 were required for sperm capacitation by interacting with zona of oocyte and helps in sperm—oocyte interaction. Calreticulin is mainly necessary for calcium oscillations inside the sperm cells for hyper activation and capacitation. PDI (Protein Disulfide Isomerase) along with Calmigen is necessary for disulfide bond formation and remodelling in sperm.

Histone H3 undergoes demethylation at K9 due to loss of lysine demethylase in primordial germ cells after E7.5 days. In meiotic recombination histone mono, di tri methylations are responsible for DSBs and recombination. Methylation at pericentric regions [28] is responsible for preventing homologous recombination. Retrotransposons restrict methylating events and responsible for maintenance of genome imprinting. Reduced DNA methylation in retrotransposons leads to gene expression in LINE 1 transposons and proteins like PIWI and AUBERGINE undergoes methylation in repetitive elements of PGC and responsible for gene silencing mechanisms [29]. This pattern was found to be conserved in mice and in animals and mammals, in addition to mutational hotspots there are some recombination

**10. Epigenetic modulations in sperm genome**

hotspots characterised by H3K4me3 and H3K9 acetylation.

**52**

Paternal genomes are hypomethylated compared to maternal genome and in Drosophila the protamine eviction was done by DHD (Dead Head) by reducing the disulfide bonds with the help of thioredoxin and NADPH. Protamines form oligomers by intrachain disulfide bonds. Molecular chaperone TAP/p32 acts on monomeric protamines and causes their eviction and found to be inactive on oligomers. DHD interacts physically with TAP/P32 to cause the eviction of protamines from DNA and TAP/P32 binds poorly to DNA. DHD normally found in sperm nucleus and was degraded after fertilisation. DHD domain was necessary for the reduction of disulfide bonds, DNA decondensation, and protamine eviction and also functions as chaperone. It shares some common properties with thioredoxins and also has unique properties necessary for the embryo formation. Mutation in DHD domain is non tolerant and results in haploid embryo formation with the absence of male pronucleus [30].

Proteomic profile of testes sperm was one of the important researches in progress nowadays. Normally in DNA condensing proteins the disulfide bonds are oxidised spontaneously and does not require any enzymatic source. There are certain proteins in sperm which require enzymes like thioredoxin/Glutathione reductase which acts on glutathione peroxidise and induces disulfide bond formation. Thioredoxin glutathione reductase catalyses isomerisation of disulfide bonds in these proteins and the protein was mainly localised at mid piece of mitochondrial sheath and was required for structural maintenance of mid piece and tail proteins. Those include proteins like mitochondrial capsule selenoprotein, two outer dense fibre (ODF) and glutathione-s-transferase M5. Differences in maternal and paternal chromosomes mainly occur at pericentric chromosomes, through which epigenetic message is conveyed to embryo.
