EPIGENETICS: the key to healthy female sperm


Epigenetics. A key biological processes that needs to be fully controlled in order to make healthy female sperm is a phenomenon known as epigenetics, which refers to genetic information that exists in your chromosomes in addition to the DNA sequences that make up your genes. The phenomenon affects about one percent of your genes, with the main form of epigenetics, imprinting, leading to some of your genes being turned off (and in woman, an entire chromosome, one of their X chromosomes being turned off, an epigenetic/imprinting process referred to as X-inactivation). Prof C. David Allis of Rockefeller University has an excellent animation on gene silencing and epigenetics.

Before you delve deeply into the biology of epigenetics, let's first consider a common occurrence of this phenomenon in cats, in particular, Calico cats, that is, female cats of many species that have patches of different colors in their hair. The cat pictured below is a classic example of a Calico cat, having multiple patches of black and orange hair mixed in with her generally white fur. How does this happen?

The genes for black and orange colored hair are located on the cat's Y chromosome. Let's suppose a female cat has one X chromosome with the black color gene (say, from her mother), and one X chromosome with the orange color gene (from her father). What happens in all females, including humans, is that one X chromosome is randomly inactivated in all of their cells (they are referred to as being mosaic). If the same X chromosome (say the one from her mother) is inactivated, then all of her patches would be orange. Or, if all of the X chromosomes from her father are inactivated, then all of her patches would be black. But X chromosomes are randomly inactivated in each cell, so some groups of cells inactivate the mother's X chromosome (creating orange patches) while others inactivate the father's X chromosome (creating black patches). So when you see a Calico cat, you are seeing a very subtle biological phenomenon occuring, as well as a cute cat.

Image:Katze in Tunesien.jpg

Epigenetics in Making Babies - When a sperm and egg meet during fertilization to form a zygote and then embryo, epigenetic mechanisms play a crucial role. It is very necessary that some of the genes from the sperm's DNA have the epigenetic marks of males, while some of the genes from the egg's DNA have the epigenetic marks of females. Fuse two eggs together, and the fertilization eventually fails, because the male markings are absent. Combine two sperm (in an empty egg), and the fertilization eventually fails, because the female markings are absent.


It is very important to remember that epigenetic phenomenon such as imprinting and X-inactivation, when faulty, can lead to serious diseases. Consider, for example, Turner syndrome, a phenomenon in which a women is born with just one X chomosome instead of the usual two. In a sense, having Turner syndrome is equivalent to having the same (either mother's or father's) X-chromosome inactivated in every cell. Women with Turner syndrome have a variety of diseases (especially being infertile), even though the X chromosome they have inherited is perfectly fine. Scientists have evidence that the reason that Turner syndrome woman are short is that the have only one copy of the SHOX gene which is important for bone development.


What follows is a list of fulltext articles on epigenetics, imprinting and X-inactivation available on the Internet, followed by a discussion of epigenetics as it affects female sperm, and ending with a list of links to abstracts of medical journal articles dealing with this phenomenon. Keep in mind that adequately controlling epigenetics is crucial to making healthy female sperm. Fortunately, there is much research into the reproductive aspects of epigenetics. Much of this research is tracked at the Genomic Imprinting website. The European Union has a large-scale joint research center effort focusing on Epigenetics.

General Introductory Articles to Epigenetics and Imprinting

What is Epigenetics - an excellent and often humorous 56 page guide prepared by the European Union Epigenome project in 2006. (full text)

Silence of the Genes - Prof C. David Allis of Rockefeller University has an excellent animation on gene silencing and epigenetics.

Pulling Genes' Strings: genes would be hardly more than molecular dead weight if not for the 'epigenetic' context that turns them on and off by Ivan Amato, Chemical and Engineering News, July 2006. (full text)

Epigenetics: the science of change by Bob Weinhold, Environmental Health Perspectives (NIH), March 2006. (full text)

Imprinted and More Equal: why silence perfectly good copies of inherited diseases? by Randy Jirtle and Jennifer Weidman, American Scientist, March-April 2007. (full text)

The silence of the genes: is genomic imprinting the software of evolution or just a battleground for gender conflict? by Philip Hunter, EMBO Reports, May 2007. (full text)

Epigenetics in Reproductive Medicine by A. Giacobino, Pediatric Research, 2007. (full text)

Genomic imprinting and assisted reproduction by A. Giacobino and J. Richard Chaillet, Reproductive Health Journal, October 2004. (full text)

What good is genomic imprinting: the function of parent-specific gene expression, Nature Reviews: Genetics, May 2003. (full text)

Medical Research Articles on Epigenetics and Imprinting

Metastable Epialleles, Imprinting and the Fetal Origins of Adult Diseases by Randy Jirtle et al., Pediatric Research, 2007. (full text)

The emerging science of epigenomics by Pauline Callinan and Andrew Feinberg, Human Molecular Genetics, 2006. (full text)

Genomic imprinting and methylation: epigenetic canalization and conflict by Jon Wilkins, Trends in Genetics, June 2005. (full text)

Epigenetics - 10 articles in special issue of Human Molecular Genetics, April 2005. (table of contents), including the article Epigenetic reprogramming in mammals by Reik et al. (full text)

Epigentic regulation of mammalian genome imprinting by Katia Delaval and Robert Feil, Current Opinion in Genetics & Development, 2004. (full text)

[Green] Tea Polyphenol(-)-Epigallocatechin-3-Gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines by Fang et al., Cancer Research, November 2003 - some research results suggesting a chemical in green tea reactivate good genes that are turned off in cancer cells. (full text)

Basics of Genomic Imprinting by A. Ruvinsky, Journal of Animal Science, 1999. (full text)

Epigenetics: regulation through repression by Alan Wolffe and Marjori Matzke, Science, October 1999. (full text)

Medical Research Articles on Epigenetics and Sperm

Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm by Victoria Cortessis et al., PLoS ONE, December 2007. (full text)

Genomic imprinting and reproduction by Swales and Spears, Reproduction, October 2005. (full text)

Epigenetics and the germ line by Allegrucci et al., Reproduction, 2005. (full text)

Genetic and epigenetic properties of mouse male germline stem cells during long-term culture by Shinohara et al., Development, 2005. (full text)

Establishment of the paternal methylation imprint of the human H19 and MEST/PEG1 genes during spermatogenesis by Antoine Kerjean et al., Human Molecular Genetics, 2000. (full text)


(From U.S. Patent Application)


[0012] One major difference between male and female gametogenesis is imprinting. Imprinting is an epigenetic, gamete-of-origin-, and therefore parent-of-origin-,dependent modification of the genome, i.e., changes in DNA function without changes in DNA sequences. Typically, all humans have pairs of genes, one on each chromosome inherited from each parent, that are both expressed in cells. One class of imprinted genes are those that are parentally imprinted. When an autosomal gene at an imprintable location on a chromosome passes through gametogenesis of one sex, its ability to be expressed is unaffected. However, when this gene passes through gametogenesis of the opposite sex, it becomes inactivated - it cannot be expressed. Such an inactivated gene is termed "imprinted". Approximately one percent of human genes are imprinted. Some imprinted genes control fetal growth (such as the IGF2/H19 pair), as evidenced by gynogenetic embryos (no male imprints) having poor placental development but normal embryonic development, while androgenetic embryos (no female imprints) having the reverse. The results of parental imprinting have been long recognized by phenotypical effects, as far back as 3000 years by mule breeders in Asia Minor. Other epigenetic modifications include histone modifications and use of non-coding RNAs, but imprinting dominates. Epigenetics can be viewed as an evolutionary gift from our sexually liberal female placental ancestors, who survived better by always having generally healthy babies (averaged sized embryos) from multiple fathers (who survived better by having their children start out as larger embryos at the expense of imperiling future offspring from other fathers).

[0013] Only in recent years, however, have the genetic processes underlying imprinting been gradually brought to light, in particular, medical problems caused by faulty imprinting. For example, one of the major problems with nuclear-transfer cloning is that the cloned DNA is not properly imprinted, leading to cloned animals with abnormal phenotypes, assuming the cloned embryos survive at all to birth (only a low percentage so survive). In recent years, biologists have similarly noted that the imprinting problems that arise with cloning also arise in the creation of artificial sperm and eggs. In general, mastering epigenetic reengineering is the key to clinical acceptance of therapeutic uses of regenerated adult stem cells. Methods to compensate for transsexual gametogenesis problems due to differences in male and female imprinting and other epigenetic phenomenon are disclosed herein. A second class of imprinted genes involves the entire X chromosome in women, where early in development, one X chromosome in each cell of a woman is randomly imprinted ("X-inactivation").


[0076] Imprinting is one type of process involving the epigenome, i.e., modifications to genes that change the functioning of the gene without changing the DNA sequences that comprises the gene. At least three types of epigenetic modifications have been observed: imprinting via DNA methylation of cytosine residues in CpG dinucleotides (forming differentially methylated regions - DMRs), modification of core histones (a "histone code"), and gene silencing due to non-coding RNAs. There are three types of imprinting modifications: 1) in women, the random total inactivation of one X chromosome in each cell ("X-inactivation"); 2) in men and women, random inactivation of some autosomal genes; and 3) in men and women, modification of a small percentage (100 to 200) of one of each of a pair of autosomal genes depending on the person's sex. Sex-specific imprinting is imparted in the germ line, where inherited maternal and paternal imprinting is erased and new imprinting established according to the individual's sex.

[0077] Imprinting partly is a process of functional inactivation, with inactive regions of chromatin having altered methylation patterns at the 5-position of cytosine. DNA methyltransferases (such as the DNMT family) are the only known mammalian enzymes that catalyze the formation of 5-methyl cytosine (i.e., adding a methyl group CH3- to a cytosine); and thus play a key role in establishing and maintaining methylation patterns during male spermatogenesis, as seen in measurements of substantial levels of DNA methyltransferase mRNA and protein expression in mitotic types A and B spermatogonia, spermatocytes and round spermatids [JUE94]. This is consistent with the presence of DNA methyltransferase during the frequent cell divisions of spermatogonia, where methylation patterns are maintained on each daughter strand of DNA following semiconservative DNA replication. Such DNA methyltransferases also help maintain methylation patterns during DNA replication for somatic cell divisions.

[0078] In some embodiments of the methods disclosed herein, spermatogenic female germ cells formed as described above (e.g., by cloning) have their imprints erased, and are then imprinted as male germ cells. Many imprints are the result of gene site-specific DNA methylation, so that before male imprints can be made for female cells (or female imprints for male cells), the old imprints have to be erased, e.g., actively using DNA demethylases or passively during replication if methylation is interfered with (e.g., to remove any DNMT1 that typically maintains methylation patterns during replication), and/or by using histone-modifying enzymes. Experiments show imprinting to be cell-autonomous, with cells having at least one Y chromosome (for example, from XY or XXY males or sex-reversed XY females) able to acquire male imprints.

[0079] Much erasure of the previous gametic imprint occurs by the time of genital ridge colonization, and with a relatively late re-establishment in the gametes [SZA95], [BAR97]. After fertilization, much of the paternally inherited DNA is erased much more quickly then the maternally inherited DNA, and remains erased as the germ cells migrate from the yolk sac to the genital ridge, at which point complete erasure starts, since Onyango et al. report that human embryonic germ cells from the gonadal ridge still have imprints [II.4]. Thus germ cells extracted from a cloned female embryo at the time of genital ridge colonization can be made ready for a male imprinting pattern (and other epigenetic modifications) when transplanted to a male testis to experience spermatogenesis.

[0080] The results of Kerjean [II.3] suggest that some human male germ cell imprints remain erased until imprinting occurs in adult spermatogenesis. Male germ cells undergo mitosis starting from the fetal stage until death. Male germ cells undergo meiosis primarily post-puberty, with about 80% of the germ cells in the adult testis being in the (post) meiotic stages. Imprinting of the male genome starts during fetal stages of mitosis, with the bulk of germ-cell specific methylation patterns being acquired prior to the type A spermatogonia stage. Any epigenetic mitotic/meiotic modifications to male germ cells in the testes that occur post-puberty (e.g., histone-to-protamine exchange, methylation of the maternally-expressed non-coding RNA gene H19 [SOL98], etc.) will also happen to altered female germ cells when transplanted into a post-puberty testicular environment (or in vitro equivalents). To some extent, male germ line imprinting may be a waste of energy (e.g., for any imprinted genes only active in somatic cells), and may not need to be fully replicated, since paternal imprints in the paternal pronucleus are quickly erased shortly after fertilization at the zygote stage, and experience additional erasure as the embryo undergoes two waves of demethylation. Similarly, Kerjean reports that the imprinted gene MEST/PEG1 remains unimprinted throughout spermatogenesis even until the mature spermatozoa stage.

[0081] For male germ cell epigenetic modifications that are fully erased in the embryo by the fetal stage, or by the time of adult spermatogenesis post-puberty, one can erase, in vitro, the imprints of a prepared female diploid germ cell (preparation including the addition of an artificial Y chromosome), so that when the prepared germ cells are transplanted into the testicular environment, the female's chromosomes acquire the necessary male epigenetic modifications naturally. Any imprinting establishment that occurs from the fetal period to pre-puberty can be achieved, for example, using the mitotic steps of procedures for in vitro conversion of male embryonic stem cells into embryonic germ cells to similarly cultivate, in vitro, female diploid germ cells that have been reset epigenetically, before the female germ cells undergo testicular transplantation Chuma and colleagues [CHU05] report on extracting mouse primordial germ cells and transplanting them into a natural testicular environment to produce sperm resulting in normal fertile offspring, supporting the idea that epigenetic modifications in the male germ line during mitotic stages of spermatogenesis pre-puberty do not to be exactly established, if at all, but rather are amenable to in vitro engineering. Kono and colleagues [KON07] report on the successful combination of two mouse eggs to produce healthy offspring by deleting part of the H19 gene in one egg to make it more masculine. While the procedure has a low frequency of success, it suggests much of male epigenetics post-paternal-pronucleus erasure is optional, including epigenetic modifications during the pre-puberty mitotic stages of spermatogenesis, despite the large number of fetal mitotic divisions during which methylation patterns are maintained. Some fetal imprinting, such as for the Oct4 and Nanog genes which promote pluripotency, can be blocked from any expression in altered female germ cells. The work of Chuma et al. and Kono et al. lend support to the preference of testicular injection of (mostly) epigenetically-reset Y-chromosome-compensated female diploid germ cells to achieve sufficient epigenetic modifications for normal fertilization.

Abstracts to Medical Research Articles on Epigenetics and Imprinting