Introduction

Sperm genome contributes to the one-half of the whole genome to the developing embryo The development of sperm involves stages of controlled meiotic division throughout maintaining its genomic integrity and appropriate epigenetic modifications (Allegrucci et al. 2005, Kimmins & Sassone-Corsi 2005, Seki et al. 2005, Seki et al. 2007). Sperm chromatin modification involves the replacement of histones with protamine to form a highly organized compact structure (Ward & Coffey 1991, Balhorn 2007). Sperm morphological and nuclear alteration including chromatin aneuploidy and DNA strand break results in pregnancy loss (Evenson et al. 1999, Rubio et al. 1999, Gopalkkrishnan et al. 2000, Larson et al. 2000, Spano et al. 2000, Carrell et al. 2003, Bernardini et al. 2004).

Association studies in humans and studies in mouse models have helped us to better understand the role of paternal genetic and epigenetic factors in embryonic development and in the outcome of pregnancy. Understanding genetic and epigenetic reprogramming of sperm is also important to improve the outcome of Assisted reproductive technology (ART). Here we aim to discuss in detailed the process of genetic and epigenetic modifications that occur in sperm.

Male Gametogenesis

The process of differentiation of a spermatogonium into a sperm is known as spermatogenesis (Roosen-Runge & Holstein 1978) which starts at puberty and continues throughout the entire lifespan of the individual. It is a complex process that involves differentiation of diploid spermatogonial cells to primary and secondary spermatocytes and then to spermatids which culminates in the production of mature spermatozoa in approximately 75 days (Clermont 1963). Spermatogenesis can be divided into two major phases: (1) proliferation and differentiation of spermatogonial and meiosis which is combined known as spermatocytogenesis and (2) spermiogenesis, is a process of differentiation of spermatids into sperm cells called the spermatozoon which undergoes cell remodeling and DNA compaction to form motile sperm. Structural and nutritional support to the developing germ cells is provided by Sertoli cells. Cellular interactions between the germ line and sertoli cells of the testis are very important for normal spermatogenesis. Aberrant spermatogenesis and mutation of the genes involved in the process affects specific testicular cell types and reproductive function, resulting in reduced sperm count and its quality. 

Sperm Chromatin Organization

Spermatozoa maturation involves a series of mitotic and meiotic changes with the replacement of histone to protamines P1 and P2 at approximately a 1:1 ratio leading to highly packaged DNA which enhances sperm motility and protect DNA damage (Balhorn et al. 1988, Hecht 1990, Oliva & Dixon 1990, Dadoune 1995). Majority of the DNA in human sperm chromatin is bound by protamines for e.g. post-natal expressed B-globin gene is protamine enriched (Gardiner-Garden et al. 1998). However, a small percentage retains a histone component like genes involved in early embryogenesis for e.g. Embryonic-specific E- and G-globin genes are histone enriched (Gardiner-Garden et al. 1998). Hence the distribution of histone and protamine may also play role in early development of embryo and pregnancy outcome.  Remodeling of gametic chromatin after fertilization is followed by the decondensation and epigenetic modifications. (Mayer et al. 2000, Dean et al. 2003). Alteration in the process of decondensation of sperm chromatin has been associated with the failure of fertilization (Kren et al. 2003, Lee et al. 2003).  DNA in sperm chromatin is organized into looped domains attached at their bases to the nuclear matrix (Ward et al. 1989). Spermatozoa with a disrupted nuclear matrix were found to produce non- viable offspring which suggest that the spatial organization of the sperm genome provides important epigenetic information critical for both sperm function and early embryonic development [Ward et al. 1999, Sotolongo Ward 2000).

Sperm Epigenome

Epigenetic landscape in the sperm plays a very important role in the development of the embryo. Epigenetic state of mature sperm is determined by histone retention and modification, protamine incorporation into the chromatin, DNA methylation, and spermatozoa RNA transcripts [Tanphaichitr et al. 1978, Gatewood et al. 1987, Wykes & Krawetz 2003, Oakes et al. 2007).  Proper establishment and maintenance of the paternal epigenetic program is associated with gamete and embryonic development (Figure 2) (Jaenisch & Jahner 1984, Surani 1998, Ng & Bird 1999). Epigenetic modifications of sperm involve chromatin remodeling, DNA methylation and imprinting.

 a) Sperm chromatin remodeling: Chromatin remodeling in human spermatogenesis takes place by the replacement of histones with transition proteins followed by protamines in spermatids (Yu et al. 2000, Cho et al. 2001, Zhao et al. 2001). Upon fertilization, these protamines in sperm chromatin are rapidly replaced with histones. Chromatin remodeling factors play an important role in the process of sperm chromatin remodeling (Rousseaux et al. 2008) which include SWI/SNF complex components, polycomb-group genes (PcGs), bromodomain proteins, chromodomain/helicase/DNA-binding domain (CHD) proteins, plant homeodomain (PHD) proteins, chromobox/heterochromatin protein 1 (HP1) homologues, nucleosome remodeling and histone deacetylase (NuRD) complex components, inhibitor of growth (ING) family members, methyl-CpG DNA-binding domain (MBD) proteins and the CCCTC-binding factor (zinc finger protein). Differential expression of these proteins has been found to alter sperm formation (Steilmann et al. 2010).

b) DNA methylation and histone modification: On fertilization, the paternal genome undergoes rapid changes with the replacement of protamines with histones, DNA demethylation, and various histone modifications (Li 2002, Morgan et al. 2005). The maternal genome during these events is protected epigenetically (Li 2002, Morgan et al. 2005). The histones H3.3 preferentially associates with the paternal chromatin facilitates progressive demethylation (Bramlage et al. 1997, McKittrick et al. 2004) whereas the maternal pronucleus is associated with repressive histone modification marks. This demethylation is completed before DNA replication begins in the paternal pronucleus (Mayer et al. 2000, Oswald et al. 2000). Paternal demethylation is needed to regain the totipotency property of the embryo and is needed for early transcriptional activity of genes for the development of embryo (Reik et al. 2001). During preimplantation development, the difference in DNA methylation between the paternal and maternal genome seems to disappear (Monk et al. 1987, Howlett & Reik 1991).

c) Imprinting: Epigenetic information is passed on to a child through the sperm or the egg. This epigenetic phenomenon of being ‘imprinted’ according to the paternal or maternal origin of a gene copy is called ‘genomic imprinting’. Genomic imprinting as a result of differential methylation of cytosine in CpG islands is a mechanism of gene regulation, by which only one of two parental alleles is expressed (Neumann et al. 1995). Inherited imprints are erased in embryonic germ cells, and reset later according to the sex of the embryo during gametogenesis or after fertilization (Sanford et al. 1987, Kafri et al. 1992). Many imprinted genes are involved in embryonic or placental growth by regulating the cell cycle (Labosky et al. 1994, Tada et al. 1998).

Conclusion

In summary, the paternal genetic and epigenetic factor plays an important role in pregnancy loss. Pregnancy loss like RPL is considered a multifactorial disease in which females are primarily evaluated. Male factors are however poorly defined and hardly evaluated in RPL patients. Paternal genetic and epigenetic alteration may affect fertilization, early embryo development, genomic activation and ultimately pregnancy outcome. Recent studies’ associating the role of paternal factors suggests and both male and female partners must undergo clinical and genetic evaluation to determine the cause of pregnancy loss. Moreover, before performing IUI (Intrauterine insemination) and IVF sperm genetic and epigenetic status should be determined as it has been found to be one of the causes of pregnancy loss.

Sperm chromatin is a very complex structure. DNA integrity in sperm is essential for the accurate transmission of genetic information. Besides routine semen analysis, sperm function tests such as Hypo-osmotic swelling test, Acrosome reaction test, Nuclear condensation and decondensation test, Sperm chromatin structure assay (SCSA), Leukocytospermia may be an informative tool in cases of idiopathic RPL. Culture and sensitivity study of semen should be done and any infection should be treated with appropriate antibiotics. Hence appropriate treatment of both male and female partner may overcome RPL.

This review shed light on the role of genetic and epigenetic effect of paternal factors in pregnancy loss. From various IVF studies sperm is found to be a potential vehicle for transmitting paternal methylation abnormalities to the embryo. Detailed study of male sperm factors other than DNA damage such as epigenetic basis which could be responsible for RPL should be studied.

Future studies could address the fundamental molecular basis of DNA damage and involved genes so that insights might be gained into the mechanisms responsible for the aberrant spermiogenesis seen in male partners of RPL patients. Sperm DNA damage or defect in sperm genetic and epigenetic functions may lead to impaired spermatogenesis may not only have the consequences like male infertility but also pregnancy loss. Thus it is imperative to analyze the differential expression of the genes in case of RPL and in male infertility patients to delineate sets of genes in sperm which are responsible for embryo development. Comparative microarray analysis of the sperm transcriptome of both male infertile patients and of male partners of recurrent pregnancy loss patients will provide information about the set of genes which are differently expressed in both diseases.  This review provides a better understanding of genetic and epigenetic alterations that could hinder embryo development and have consequences like pregnancy loss.

References

Allegrucci C, Thurston A, Lucas E & Young L (2005) Epigenetics and the germline. Reproduction, 129:137–149.

Balhorn R (2007) The protamine family of sperm nuclear proteins. Genome Biol, 8:227.

Balhorn R, Reed S & Tanphaichitr N (1988) Aberrant protamine 1/protamine 2 ratios in sperm of infertile human males. Experientia, 44:52-55.

Bernardini LM, Costa M, Bottazzi C, Gianaroli L, Magli MC, Venturini PL, Francioso R, Conte N & Ragni N (2004) Sperm aneuploidy and recurrent pregnancy loss. Reprod Biomed Online, 9:312–320.

Bramlage B, Kosciessa U & Doenecke D (1997) Differential expression of the murine histone genes H3.3A and H3.3B. Differentiation, 62:13–20.

Carrell DT, Liu L, Peterson CM, Jones KP, Hatasaka HH & Erickson L (2003) Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl, 49:49 –55.

Cho C, Willis A, Goulding E, Jung-Ha H, Choi Y, Hecht N & Eddy E (2001) Haploinsufficiency of protamine-1 or -2 causes infertility in mice. Nat Genet, 1:82–86.

Clermont Y (1963) The cycle of the seminiferous epithelium in man. Am J Anat, 112: 35–51.

Dadoune JP (1995) The nuclear status of human sperm cells. Micron, 26: 323-345.

Dean W, Santos F & Reik W (2003) Epigenetic reprogramming in early mammalian development and following somatic nuclear transfer. Semin Cell Dev Biol, 14: 93–100.

DeBaun MR, Niemitz EL & Feinberg AP (2003) Association of in vitro fertilization with Beckwith–Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet, 72: 156–160.

Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, de Angelis P & Claussen OP (1999) Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod, 14: 1039–1049.

Gardiner-Garden M, Ballesteros M, Gordon M & Tam PP (1998) Histone and protamine-DNA association: conservation of different patterns within the beta-globin domain in human sperm. Mol Cell Biol, 18:3350-3356.

Gatewood J, Cook G,  Balhorn R, Bradbury E & Schmid C (1987) Sequence-specific packaging of DNA in human sperm chromatin. Science, 236: 962–964.

Gopalkkrishnan K, Padwal V & Meherji PK (2000) Poor quality of sperm as it affects repeated early pregnancy loss. Arch Androl, 45: 111–117.

Hecht NB (1990) Regulation of 'haploid expressed genes' in male germ cells. J Reprod Fertil, 88: 679-693.

Holliday R (1989) DNA methylation and epigenetic mechanisms. Cell Biophys,  15: 15–20.

Howlett SK & Reik W (1991) Methylation levels of maternal and paternal genomes during preimplantation development. Development, 113:119–127.

Jaenisch R & Jahner D (1984) Methylation, expression and chromosomal position of genes in mammals. Biochim Biophys Acta, 782:1-9.

Kafri T, Ariel M, Brandeis M, Shemer R, Urven L, McCarrey J, Cedar H & Razin A (1992) Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev, 6: 705–714.

Kimmins S & Sassone-Corsi P (2005) Chromatin remodeling and epigenetic features of germ cells. Nature, 434: 583-589.

Kren R, Kikuchi K, Nakai M, Miyano T, Ogushi S, Nagai T, Suzuki S, Fulka J & Fulka J Jr (2003) Intracytoplasmic sperm injection the pig: where is the problem? J Reprod Develop, 49: 271–273.

Labosky PA, Barlow DP & Hogan BLM (1994) Mouse embryonic germ (EG) cell-lines—transmission through the germline and differences in the methylation imprint of insulin-like growth-factor 2 receptor (IGF2R) gene compared with embryonic stem (ES) cell lines. Development, 120: 3197–3204.

Larson KL, DeJonge CJ, Barnes AM, Jost LK & Evenson DP( 2000) Sperm chromatin  structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques. Hum Reprod, 15: 1717–1722.

Lee JW, T XC & Yang XW (2003) Failure of male pronucleus formation is the major cause of lack of fertilization and embryo development in pig oocytes subjected to intracytoplasmic sperm injection. Biol Reprod, 68(4): 1341-1347.

Li E (2002) Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet, 3: 662-673.

Mayer W, Niveleau A, Walter J, Fundele R & Haaf T (2000)  Demethylation of the zygotic paternal genome. Nature, 403: 501–502.

McKittrick E, Gafken PR, Ahmad K & Henikoff S (2004) Histone H3.3 is enriched in covalent modifications associated with active chromatin. Proc Natl Acad Sci USA, 101: 1525–1530.

Monk M, Boubelik M & Lehnert S (1987) Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development, 99: 371-382.

Morgan HD, Santos F, Green K, Dean W & Reik W (2005) Epigenetic reprogramming in mammals. Hum Mol Genet, 14(1): 47–58.

Neumann B, Kubicka P & Barlow DP (1995) Characteristics of imprinted genes Nature Genet, 9: 12-13.

Ng HH & Bird A (1999) DNA methylation and chromatin modification. Curr Opin Genet Dev, 9: 158-163.

Oakes CC, Kelly TL, Robaire B & Trasler JM (2007) Adverse effects of 5-aza-2'- deoxycytidine on spermatogenesis include reduced sperm function and selective inhibition of de novo DNA methylation. J Pharmacol Exp Ther, 322:1171-1180.

Oliva R & Dixon GH (1990) Vertebrate protamine gene evolution I. Sequence alignments and gene structure. J Mol Evol, 30:333-346.

Oswald J, Engemann S, Lane N, Mayer W, Olek A, Fundele R, Dean W, Reik W & Walter J (2000) Active demethylation of the paternal genome in the mouse zygote. Curr Biol, 10(8): 75-78

Reik W, Dean W & Walter J (2001) Epigenetic reprogramming in mammalian development. Science, 293: 1089- 1092.

Rhind SM, Taylor JE, De Sousa PA, King TJ, McGarry M & Wilmut I (2003) Human cloning: can it be made safe? Nature Rev Genet, 4:855–864.

Roosen-Runge EC & Holstein A (1978) The human rete testis. Cell Tissue Res, 189:409–433.

Rubio C, Simon C, Blanco J, Vidal F, Minguez Y & Egozcue J (1999) Implications of sperm chromosome abnormalities in recurrent miscarriage.  J Assist Reprod Genet, 16: 253–258.

Sanford JP, Clark HJ, Chapman VM & Rossant J (1987)  Differences in DNA methylation during oogenesis and spermatogenesis and their persistence during early embryogenesis in the mouse. Genes Dev, 1: 1039–1046.

Sassone-Corsi P (2002) Unique chromatin remodeling and transcriptional regulation in spermatogenesis. Science, 296:2176–2178.

Seki Y, Hayashi K, Itoh K, Mizugaki M, Saitou M & Matsui Y (2005) Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice. Dev Biol,  278: 440-458

Seki Y, Yamaji M, Yabuta Y, Sano M, Shigeta M, Matsui Y, Saga Y, Tachibana M, Shinkai Y & Saitou M (2007) Cellulary dynamics associate with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice. Development, 134: 2627–2638

Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E &  Leter G (2000) Sperm chromatin damage impairs human fertility, The Danish First Pregnancy Planner Study Team.  Fertil Steril, 73:43–50.

Steilmann C, Cavalcanti MC, Bergmann M, Kliesch S, Weidner W & Steger K (2010) Aberrant mRNA expression of chromatin remodeling factors in round spermatid maturation arrest compared with normal human spermatogenesis. Mol Hum Reprod, 16: 726-733.

Surani MA (1998) Imprinting and the initiation of gene silencing in the germ line. Cell, 93:309-312.

Tada T, Tada M, Hilton K,  Barton SC, Sado T, Takagi N &  Surani MA (1998) Epigenotype switching of imprintable loci in embryonic germ cells. Dev Genes Evol,  207:551–56.

Tanphaichitr N, Sobhon P, Taluppeth N & Chalermisarachai P (1978) Basic nuclear proteins in testicular cells and ejaculated spermatozoa in man. Exp Cell Res, 479:347-356.

Ward WS & Coffey DS (1991) DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells. Biol Reprod, 44:569–574.

Ward WS, Kimura Y & Yanagimachi R (1999) An intact sperm nuclear matrix may be necessary for the mouse paternal genome to participate in embryonic development.  Biol Reprod, 60:702–706.

Ward WS, Partin A & Coffey D (1989) DNA loop domains in mammalian spermatozoa. Chromosoma, 98:153–159.

Wykes SM & Krawetz SA (2003) The structural organization of sperm chromatin. J Biol Chem, 278: 29471-29477.

Yu YE, Zhang Y, Unni E, Shirley CR, Deng JM, Russell LD, Weil MM, Behringer RR & Meistrich ML (2000) Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice.  Proc Natl Acad Sci USA,  97:4683– 4688.

Zhao M, Shirley CR, Yu YE, Mohapatra B, Zhang Y, Unni E, Deng JM, Arango NA, Terry NH, Weil MM, Russell LD, Behringer RR,  Meistrich ML (2001) Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice. Mol Cell Biol, 21:7243– 7255.