Human Reproduction Update, Vol.7, No.2 pp. 211±216, 2001
Epigenetic and experimental modi®cations in early mammalian development: Part I Preface Susan Benoff1,2,3,4 and Ian R.Hurley1 1
Center for Molecular and Cell Biology, North Shore-Long Island Jewish Research Institute, Manhasset, New York, 2Department of Obstetrics and Gynecology, North Shore University Hospital, Manhasset, New York, and 3Departments of Obstetrics and Gynecology and Cell Biology, New York University School of Medicine, New York, USA 4
To whom correspondence should be addressed at: NS-LIJ Research Institute, 350 Community Drive, Room 125, Manhasset, New York 11030, USA. E-mail:
[email protected]
Preface IVF was ®rst introduced for the treatment of severe female tubal infertility but was rapidly utilized for treatment of cervical factor infertility, polycystic ovarian disease, endometriosis, immunological infertility, unexplained infertility and male factor infertility. Programmes for sperm donation and/or donation of oocytes were established allowing couples unable (e.g. azoospermic males; females without endogenous ovarian function) or unwilling (e.g. due to hereditary disease carrier status) to use their gametes to become biological parents. Cryopreservation of gametes and embryos has been shown to increase the chances of pregnancy in IVF patients and to reduce their costs. Micromanipulation techniques, e.g. intracytoplasmic sperm injection (ICSI), were subsequently developed which now allow treatment of severe male factor cases not amenable to conventional IVF insemination by recovery of mature spermatozoa from the epididymis or testis. Micromanipulation (to remove polar bodies or cells from the developing embryo) when combined with molecular biological protocols, e.g. reverse transcription±polymerase chain reaction (RT±PCR) and ¯uorescence in-situ hybridization (FISH), also aids in preimplantation genetic diagnosis, thereby preventing transfer of embryos at risk of inheritable genetic disease (e.g. sex-linked or autosomal recessive) or chromosomal anomalies. The increase in recent years in older female partners (>40 years old) and in severe male factor cases seeking infertility treatment has led to the use of immature gametes in assisted reproductive technologies (ART) procedures. Such patients often reject the use of donor gametes both because they desire their own (`genetic') child (Tsai et al., 2000) and because of social issues about whether or not to inform the child about his/her genetic background (e.g. van Berkel et al., 1999). The use of immature gametes has also been spurred by shortage of oocyte, but not semen, donors (Van den Hurk et al., 2000). Genetic history or the results of genetic testing preclude acceptance into an oocyte donor programme of a large fraction of candidate women (Wallerstein et al., 1998). Ó European Society of Human Reproduction and Embryology
Animal studies ®rst demonstrated that immature haploid male germ cells, such as round and elongating spermatids, could produce normal offspring and therefore could be used as gametes (Ogura and Yanagimachi, 1995). Rodent primary and secondary spermatocyte nuclei are able to complete meiosis after oocyte injection and give rise to live offspring, albeit at lower frequencies than with haploid germ cells (Kimura and Yanagimachi, 1995; Kimura et al., 1998; Sasagawa et al., 1998). Human studies have primarily focused on the use of haploid germ cell precursors. Human pregnancies and live births were produced following ICSI with round or elongating spermatids retrieved from testicular tissue (Tesarik et al., 1995; Tessarik, 1996; Araki et al., 1997; Kahraman et al., 1998; AlHasani et al., 1999; Choavaratana et al., 1999; Schoysman et al., 1999). However, fertilization rates, embryo morphology, implantation and pregnancy rates are lower, blastocyst development is delayed, and spontaneous abortion rates are higher with round spermatids as the male gamete, compared with elongated spermatids or mature spermatozoa (Fishel et al., 1997; Vanderzwalmen et al., 1997, 1998; Kahraman et al., 1998; AlHasani et al., 1999; Ghazzawi et al., 1999; Schoysman et al., 1999; Balaban et al., 2000; Levran et al., 2000). This may be due, at least in part, to dif®culties in accurate identi®cation of round spermatids (Vanderzwalmen et al., 1998; Vereyen et al., 1998; Schoysman et al., 1999). In addition, the reduced fertility potential of round spermatids may be related to the timing of oocyte activating ability (Tesarik et al., 1998), which, though the study of homologous and heterologous spermatid injection into mouse oocytes, is thought to occur between the round and elongating stages of sperm development (Yanagida et al., 2000). Other factors may also come into play. These include nuclear maturity (Antinori et al., 1997), whether the sperm calcium oscillation-promoting activity of round spermatid nuclei (Tesarik et al., 2000), which develops between the secondary spermatocyte and round spermatid stage (Sousa et al., 1996), is suf®cient to induce a rapid inactivation of the oocyte's metaphase-promoting factor (Tesarik, 1998) and whether or not the round spermatids are
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S.Benoff and I.R.Hurley affected by the genetic defects producing the azoospermic state (Vanderzwalmen et al., 1998). For example, spermatids from men with non-obstructive azoospermia present with high rates of DNA fragmentation (Tesarik et al., 1998; Jurisicova et al., 1999). As a way of an aside, genetic background also impacts on ICSI fertilization rates of mature testicular or epididymal spermatozoa (Palermo et al., 1999). Animal studies also provided the necessary background information for in-vitro maturation of oocyte-cumulus complexes from antral follicles in the clinical setting. In-vitro maturation was ®rst implemented in 1965 (Edwards, 1965), with the ®rst live birth in 1991 (Cha et al., 1991). Antral follicles contain germinal vesicle (GV) stage oocytes. A recent review summarizes success rates as well as the problems associated with the de®nition of optimal in-vitro culture conditions with this technique (Van den Hurk et al., 2000). Cryopreservation of GV stage oocytes is generally more successful than that of mature oocytes because of the absence of the spindle structure (Paynter, 2000) and when combined with in-vitro maturation could serve as source of oocytes for low responders to controlled ovarian hyperstimulation, for women with damaged ovaries, and women undergoing premature menopause. However, it is important to recognize that the developmental capacity of immature oocytes is linked to the ovulatory status (regular cycling versus irregular cycling or anovulatory) of the oocyte donor (Cha and Chian, 1998), that many of the in-vitro matured oocytes fail to develop normal calcium signalling mechanisms (Herbert et al., 1997), and that embryos produced from in-vitro matured GV stage oocytes have a high incidence of multinuclear blastomeres and aneuploidy (Nogueira et al., 2000). The overall poor outcome when immature gametes are employed in ART procedures raises its own set of concerns. As gametes undergo epigenetic modi®cations (which orchestrate reversible changes in gene expression in the absence of mutation; Lewin, 1998) as they mature (Tycko et al., 1997; Surani, 1998), the question arises as to whether the reduced pregnancy rates with immature gametes could be attributed to perturbations in epigenetic processes. The current mini-symposium seeks to explore this issue.
Imprinting Imprinting is the major form of epigenetic modi®cation of the mammalian genome. Imprinting, which renders a gene transcriptionally silent, results in uniparental expression of a set of alleles at a given locus. Imprinting is generally established during gametogenesis, is maintained through embryogenesis, and is erased and re-established in the germline based on the sex of the embryo (Latham, 1999). Imprinted genes often occur in clusters, demonstrate sex-speci®c recombination rates, and are primarily involved in growth control (Tycko et al., 1997). The most extensively studied clusters are located on human chromosomes 11 (region 11p15.3±15.5, which encodes the oppositely imprinted genes Igf2 and H19) and 15 (region 15q11-13, which encodes at least six imprinted genes including a putative brain mRNA splicing factor) (Tycko et al., 1997). Imprinting is required for normal development (Tycko et al., 1997; Villar and Pedersen, 1997). The consequences of the functional nonequivalence of parental chromosomes may be
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observed in parthenogenetic and androgenetic embryos. The former give rise to relatively normal embryos that fail to implant while the later are able to implant but are primarily composed of extra-embryonic tissues. These ®ndings are supported by the examination of dosage effects in hydatiform moles (diploid premalignant placental tumours lacking maternal chromosomes) and in benign diploid ovarian teratomas (containing exclusively maternal chromosomes and a variety of somatic cell types in the absence of extra-embryonic elements) (Tycko et al., 1997). Dosage effects may also be observed in cases of chromosomal duplications wherein duplications of maternal chromosomes result in retarded embryonic growth while duplications of paternal chromosomes have the opposite effect (Ferguson-Smith et al., 1990). Imprinting aberrations underlie a variety of disease states, including those resulting in mental retardation. For example, deletion of 15q11-q13 from the paternal chromosome 15 produces Prader±Willi syndrome (PWS) while deletion of the same region from the maternal chromosome results in Angelman syndrome (AS) (Donlon, 1997; Lalande, 1997). Beckwith±Wiedermann syndrome (BWS) is linked to chromosome 11p15.5. BWS is observed after passage of an abnormal chromosome or disease gene through the maternal germline and in cases of paternal disomy (loss of heterozygosity [LOH]) or in cases of biallelic IGF2 expression in the absence of paternal disomies (Tycko et al., 1997; Smilinich et al., 1999). A two-fold increase in IGF2 protein is observed in BWS and is associated with organomegaly. It is thought that disruption of a tumour suppressor gene underlies BWS. Consistent with this, a signi®cant fraction of BWS subjects display a predisposition to Wilms or other tumours, and Wilms tumours often exhibit LOH (Okamato et al., 1997). LOH may also contribute to the progression of some adult onset cancers (Rennie and Nelson, 1999). Imprinted genes differ from their non-imprinted genes on the basis of methylation status of CpG repeats (high) (Turker and Bestor, 1997), on histone acetylation status (low) (Cheung et al., 2000), on chromatin structure (condensed) (Banerjee and Smallwood, 1998), on timing of replication (late) (Kitsberg et al., 1993), and on proximity of homologues during the S-phase (LaSalle and Lalande, 1996). Methylation control has been the subject of the majority of studies into the mechanism underlying imprinting. In somatic cells, DNA methylation and replication are coupled. In contrast, methylation is a post-replicative event in germ cells. A wave of demethylation and remethylation also occurs after fertilization (Constancia et al., 1998; Oswald et al., 2000). In all cases, methylation is implemented through the action of a single enzyme, cytosine DNA methyltransferase. Alternate splicing of sex-speci®c 5¢ exons regulates production and localization of DNA methyltransferase during speci®c stages of gametogenesis (Mertineit et al., 1998). Recent studies suggest that maternally and paternally derived alleles can be distinguished in the mouse embryo male germline in the absence of methylation (Davis et al., 2000). In addition, deletions of upstream DNA sequences can disrupt imprinting in the absence of changes in methylation states (Drewell et al., 2000). Thus, methylation appears to be a secondary event, that is responsible for maintenance of the imprint (e.g., Sado et al., 2000). Rather, imprinting is now known to be controlled by imprinting control centres, which may be activated by binding of
Preface proteins or RNAs (Constancia et al., 1998; Goto and Monk, 1998; Reik and Murrell, 2000). Imprinting is unaffected in conventional IVF (Tycko et al., 1997) or after ICSI with mature spermatozoa (Manning et al., 2000). A pilot study in rodents suggests that imprinting of the male genome has been completed by the round spermatid stage (Shamanski et al., 1999). These results are supported by a study demonstrating that post-meiotic male germ cells are resistant to the in-vivo effects of a DNA methylation inhibitor (Doerksen and Trasler, 1996). However, round spermatids do contain residual DNA methytransferase activity and the methylation status of some genes is apparently modi®ed in the epididymis (Ariel et al., 1994). This means that further studies are required and that monitoring the outcome of ART procedures employing immature gametes is warranted. Environmental toxicants can also have epigenetic effects (Jirtle et al., 2000). Nickel exposure of CHO cells containing a reporter gene affected gene function differentially, depending upon whether the site of reporter gene integration responded to nickel exposure by changes in global methylation and heterochromatin levels (Lee et al., 1998). Diethylstilboestrol, a stand-in for the much less toxic phyto-oestrogens, changes levels of CpG methylation in a lactoferrin promoter, and can, in combination with oestrogen, can produce uterine tumours in mice (Li et al., 1997). Mechanisms leading to tumour formation downstream of global methylation changes have been explored in tumours induced by a variety of toxicants, including a nitrosamine, a metal (beryllium) radiation (X-rays and plutonium) and diesel exhaust components (presumably polyaromatic hydrocarbons). This has implicated the promoter region of the p16 cell cycle regulator, whose methylation levels are correlated with cancer stage (Swafford et al., 1997). Conjectural mechanisms connecting toxicant exposures to methylation changes might, in the case of metal ions, involve DNA binding proteins. It has long been recognized that replacing magnesium (Mg2+) by other divalent ions such as cobalt (Co2+) and manganese (Mn2+) alters the binding speci®city and products of terminal transferase and deoxyribonuclease I action (Sambrook et al., 1989). Zinc (Zn2+) ®nger DNA binding proteins offer potential targets for replacement by divalent transition metal and heavy metal ions. Alternatively, secondary signalling pathways involving calcium (Ca2+) ions might be disrupted by divalent metal toxicants. Other groups of toxicants may target oestrogen or progesterone regulation pathways or aromatic hydrocarbon response elements.
Nuclear transfer The technique of nuclear transfer has been used to demonstrate the existence of imprinting (Surani and Barton, 1983; Barton et al., 1984; Surani, 1999). More recently, nuclear transfer has been employed to determine the timing of primary maternal epigenetic modi®cation, which is now believed to be completed by the time of the latter half of oocyte growth before cytoplasmic maturation is completed (Bao et al., 2000). Nuclear transfer at the GV stage has been offered as an alternative to oocyte donation for the treatment of women aged >40 years (Zhang et al., 1998,1999). These women display a
decline in implantation and clinical pregnancy rates, which as been attribute to chromosomal aneuploidies presumably arising at metaphase II (Moore and Orr-Weaver, 1998; Zhang et al., 1998). That GV transfer from oocytes from older women to enucleated oocytes of younger women restores normal meiotic competence (Zhang et al., 1998) implies that epignenetic modi®cations can be directed by trans-acting factors present in the ooplasm. This hypothesis is supported by ®ndings that somatic nuclei can be haploidized by transfer to enucleated GV stage oocytes (Kubelka and Moore, 1997; Tsai et al., 2000). It is also supported by the facts that the maturity of the host oocyte cytoplasm determines meiosis competence (Liu et al., 1999) and that better results are obtained if the donor and recipient are at the same stage of the cell cycle (Kono, 1997). The cytoplasmic factors of interest potentially include the components of the meiotic spindle. Structural abnormalities in the meiotic spindle can produce aneuploidy (Plachot and Crozet, 1992; Battaglia et al., 1996). The sperm deposition site relative to the meiotic spindle during ICSI clearly affects fertilization rates and embryo quality (Blake et al., 2000). It is easy to envisage how environmental toxicants, such as metal ion could also have epigenetic effects on chromosome segregation. Metal toxicants will alter the level of microtubule assembly both in cell culture and in vitro. Zn2+ (Hesketh, 1982), Mn2+ and Co2+ (Buttlaire et al., 1980) favour reassembly, while cadmium (Cd2+) (Perrino et al., 1986) and lead (Pb2+) (Faulstich et al., 1984) favour disassembly. The physiological metal ions involved are Mg2+ in assembly, and Ca2+ in disassembly. There is evidence that some toxicants may act by direct replacement of the physiological ions. The thermodynamics of manganese binding to tubulin is consistent with this hypothesis. There appears to be one high af®nity Mn2+ binding site per tubulin molecule. The physiological ion Mg2+ can displace Mn2+ from this site, as can the other toxicants that can promote microtubule assembly, Co2+ and Zn2+. However, the physiological ion promoting disassembly, Ca2+, cannot (Buttlaire et al., 1980). Electron paramagnetic resonance spectra of bound Mn2+ are consistent with the binding site lying on the surface of the assembled microtubule. Microtubule disassembly by toxicants appears to require intervention of a Ca2+ binding protein, calmodulin (Perrino et al., 1986), since calmodulin inhibitors can block disassembly by both Ca2+ and Cd2+. The closeness of the match of ionic radii of these two ions has been suggested to be responsible for the action of Cd2+. Since calmodulin binding Cd2+ is effective in activating an enzyme involved in signal transduction, calmodulin-dependent cAMP phosphodiesterase (Nimura et al., 1987) a route is open through which Cd2+ may affect a variety of other cellular processes. However, as Cd2+ may also directly activate at least one other Ca2+-requiring enzyme, Ca2+-sensitive myosin ATPase, it is premature to conclude that toxic effects of Cd2+ are transmitted principally through calmodulin. Pb2+, another metal ion capable of disassembling microtubules appears to directly bind to a fraction of the available sulphydral groups of tubulin, since adding sulphydrals to the medium can reverse disassembly. This raises the question as to what extent sulphydral binding by other metal ions capable of forming somewhat weaker bonds to sulphydrals, such as Cd2+ and Zn2+, may be a factor in their interactions with tubulin.
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S.Benoff and I.R.Hurley Telomerase may be considered as another trans-acting factor with potential epigenetic effects. Telomere shortening has now been associated with premature ageing (Lange, 1998). The mRNA encoding the catalytic subunit of telomerase undergoes alternate splicing in both testes (Killian et al., 1997) and oocytes (Brenner et al., 1999). Many of the splicing variants in the germline and in somatic tissues direct the synthesis of truncated proteins (Killian et al., 1997; Meyerson et al., 1997). It has been suggested that telomerase mRNA splicing variants may contribute to oocyte senescence and/or chromosome abnormalities (Brenner et al., 1999). The same may be true in the male germline as some mouse offspring produced with primary spermatocytes have been reported to have an unexpectedly shortened life span (Tsai et al., 2000).
Ooplasmic transplantation The above discussion of potential trans-acting factors is an obvious lead in to the topic of ooplasmic transfer. The major issue in ooplasmic transfer involves the potential inheritance of `foreign' (donor) mitochondrial DNA. Mitochondrial DNA is maternally inherited (Zeviani and Antozzi, 1997). The mitochrondrial DNA of the embryo is derived by clonal expansion of a limited number of randomly selected mitochondria (`bottleneck' theory; Jenuth et al., 1996; Zeviani and Antozzi, 1997). Mitochondrial DNA rearrangements accumulate more rapidly in non-dividing than dividing somatic tissues (Barritt et al., 1999). An increase in oocyte mitochondrial DNA deletions has been reported to occur with increasing age of the female (Keefe et al., 1995). It has been proposed that aberrations in mitochondrial metabolism result in the production of aneuploid oocytes (Zenses et al., 1992). The effect of transmission of the mitochondrial DNA rearrangements depends on dosage. Small amounts of mutated mitochondrial DNA appear to be without clinical impact (Kagawa and Hayashi, 1997; Rinaudo et al., 1999). As for epigenetic effects, it appears that mitochondria may be unequally distributed in the blastomeres of developing embryos, resulting in reduced developmental competence (Van Blerkom et al., 2000). Preliminary ®ndings in a small population indicate that mitochondrial heteroplasmy after human ooplasmic transplantation occurs >50% of the time in embryos and in in newborns (Brenner et al., 2000). This means that oocyte donors will have to be screened for carrier status of both nuclear and mitochondrial diseases. However, a recent report suggests that the majority of oocytes carrying mitochondrial DNA rearrangements are eliminated prior to the metaphase II stage of oocyte development (Barritt et al., 1999). In spite of this, the activity of the respiratory chain enzymes may decline with age (Muller-Hocker et al., 1996). ATP levels are thought to regulate the development competence of oocytes (Van Blerkom et al., 1995, 2000). Animal studies suggest that mitochondrial transfer results in a net increase in recipient ATP production (Van Blerkom et al., 1998). We propose that occupational and/or environmental insults may contribute to epigenetic changes in mitochondrial function. Reactive oxygen species (ROS) are created by redox-cycling metal ions, e.g. chromium (Cr2+ ± Cr3+) (Stohs et al., 2000) and iron (Fe2+ ± Fe3+) in vitro and in isolated brain mitochondria complexes (Bautista et al., 2000), so a direct role of toxicants
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creators of ROS can not be ruled out. However, as Ozawa (1995) pointed out, mitochondria contain in cytochrome C a superior catalyst for formation of hydroxyl radicals that they cope with very well. It seems likely that metal toxicant concentrations high enough to increase intrinsic mitochondrial ROS levels to the point of producing mitochondrial DNA deletions would have more serious consequences elsewhere in a cell. As mechanisms of ROS formation are elucidated, various potential targets for metal toxicants emerge. The involvement of secondary messenger systems that increase ROS by triggering release of Ca2+ from intracellular stores adjacent to mitochondria (Nassar and Simpson, 2000) and by triggering Zn2+ entry through cell membrane ion channels (Sensi et al., 1999) suggest that toxicants may act by modulating ¯uxes of physiological ions present in relative abundance. Poisoning of critical enzyme systems, e.g. the calcium-sensitive dehydrogenases (Territo et al., 2000) or the ROS scavenging system (Stohs et al., 2000) is another possibility.
Future considerations The ethics of using immature gametes and/or reconstituting oocytes is matter of continuing debate (e.g. Robertson, 1998). The major issues involve: (i) possible ill effects on offspring; (ii) symbolic altering of the germline through mitochondrial DNA transfer; and (iii) fear of exploitation for ®nancial gain. While we chose not to address the latter here, assuming that the safety of these procedures is validated, the use of immature male germ cells and of donor oocyte cytoplasm will allow biological parents to also be genetic parents and will be a great stride forward in the treatment of infertility.
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