Hypothesis: sex determination and germline formation are committed at the pronucleate stage in mammalian embryos

Sex determination in mammals has been defined as those events that fix the nature of the germ cell and the gonad, while other processes influencing sexual form and function such as hormone action constitute sex differentiation (Austin et al., 1981). Such determination involves the separation of germline and soma, controlled initially by maternal genes and then by the zygotic genome after fertilization and early cleavage (Braude et al., 1988). A cascade of events in the genital ridge initiates the differentiation of the gonads and secondary sex characteristics. In this article, we correlate embryological and genetic evidence and propose that germline could be allocated by the 4-cell stage and regulated by oocyte polarity, controlled cleavage planes and early transcription of sex-determining genes from pronuclear stages. Subsequent steps including germline segregation in early embryos and migration of primordial germ cells, and their possible role in the initiation of gonadal sex differentiation in the genital ridge are discussed. Maternal germplasm, an ooplasmic region containing polarized determinants for germline, is fundamental to germ cell determination in many animals. Its maternal regulation is achieved via the translation of vegetally-localized mRNAs (Micklem, 1995; Grunert and St Johnston, 1996; Macdonald and Smibert, 1996). Genes associated with vegetal germplasm in oocytes of three different animals and their known homologies are shown in Table I. A continuous sequence of development from germplasm to germcell can be traced from the unfertilized egg to the genital ridge of the differentiating embryo in Zebrafish, Drosophila and other species using markers such as vasa protein in oocytes and migratory primordial germ cells (Figure 1) (Komiya et al., 1994; Warrior, 1994; Yoon et al., 1997). This clearcut sequence is not apparent in mammals, where germ cells positive for alkaline phosphatase emerge in epiblast (Pedersen, 1986; Ginsburg et al., 1990; Lawson and Hage, 1994), leaving a lineage gap in the cleavage stages when the absence of recognizable germ cell markers leaves the early origins of germline an open question. Genes regulating germplasm and germline in Drosophila and amphibians have been classified into class I (specifying germplasm formation and spatial distribution), class II (assembling germplasm) and class III (specifying germ cell segregation) (Wakahara, 1996). Since there is no clear evidence for germplasm in mammalian oocytes (Gardner 1996; Edwards and Beard 1997), class II genes may be inactive in mammals, a concept urgently in need of verification. Recently it has been proposed that vasa proteins may be involved throughout the life cycle in a common mechanism of germ cell segregation in organisms with or without germplasm (Ikenishi, 1998). A clear marker for germline in Caenorhabditis elegans, Xenopus, Drosophilia and Zebrafish (Komiya et al., 1994; Warrior, 1994; Ikenishi and Tanaka, 1997; Yoon et al., 1997), homologues of vasa protein have now been identified in germline of mice (Fujiwara et al., 1994) and rats (Komiya and Tanigawa, 1995). Further work is obviously needed in these species to trace protein and mRNA expression in developing embryos. It is not clear how germplasm controls germline development, whether through germline `determinants' (Davidson, 1986) or by protecting it from somatic differentiation (Smith, 1966; Dixon, 1981). Germplasm is damaged and sexual determination and differentiation are impaired in Xenopus by exposing embryos to UV irradiation (Dixon, 1981), fertility being restored by transferring putative germplasm (Wakahara, 1978). Irradiating Drosophila pole plasm also invokes sterility unless donor pole plasm is given (Okada et al., 1974), and when oskar mRNA is injected at the opposite pole in the presence of vasa and tudor proteins germplasm forms ectopically (Ephrussi and Lehmann, 1992). Oocyte cortex is essential for the polarization of regulatory maternal transcripts (Kloc and Etkin, 1995). X-cat2 is one clear germline marker in Xenopus, homologous to the Drosophila gene nanos, and it encodes an RNA-binding protein which associates with the mitochondrial cloud, migrates to vegetal cortex, then forms islands of germplasm. Its movements are probably regulated by microfilaments and microtubules (Forristall et al., 1995; Micklem, 1995) and the kinesin superfamily of motor proteins (Mowry, 1996; Robb et al., 1996). Oocyte cortex may be the site of post-translational modifications, as in the activation of Vg1 protein by bone matrix protein (BMP) (Vize and Thomsen, 1994). Many aspects of early development in mammals resemble that of lower animal forms. In mammals an anterior/posterior axis extends meridionally from the polar body (Gardner, 1996, 1997), and an anterior/posterior polarity was identified in the distribution of the leptin (cytokine), and STAT3 (signal transducer and activator of transcription), in a thin cortical rim on either side of the meiotic spindle at the animal pole of unfertilized and newly fertilized mouse and human oocytes (Figure 2) (Antczak and Van Blerkom, 1997). Products of several other genes with widely differing functions, including Bax and Bclx (apoptosis proteins), TGF-α2 (transforming growth factor), VEGF (vascular endothelial growth factor), c-kit (stem cell factor receptor) and c-erB (epidermal growth factor receptor), each had similar distributions to those of leptin and STAT3 (Antczak and Van Blerkom, 1999), an interesting finding as yet unexplained. Other maternal proteins are radially distributed in cortex of mouse oocytes, including α1 and α3 subunits of Na/K–ATPase (Betts et al., 1998), and Dazla protein (Ruggiu et al., 1997). The symmetry of Dazla protein, an autosomal gene product essential for germ cell differentiation, must be confirmed since oocyte sections did not pass through germinal vesicle (Ruggiu et al., 1997). This gene is a homologue of the Drosophila gene boule although it has a slightly different action since the fruit fly gene is essential for spermatogenesis but not oogenesis (Eberhardt et al., 1996). Recent studies identified a cortical equatorial polarization of calcium-channel regulating receptors for type I inositol 1, 4, 5-triphosphate in human oocytes (Goud et al., 1999), and the astonishing tight distribution of myosin IIA to the cortex overlying the meiotic spindle in human and rhesus oocytes which may even mark the exact location of the animal pole, (Hewitson et al., 1999). Transcripts of other regulatory factors, such as the nuclear multipotency factor oct-4, are not polarized (Palmieri et al., 1994). Cytoplasmic movements imposed by microtubules from the sperm centriole occur soon after fertilization in human eggs, just as in C.elegans (Van Blerkom et al., 1995; Goldstein and Hird, 1996). Two membrane-bound pronuclei form some hours later. A larger paternal pronucleus forms initially followed by a smaller maternal pronucleus located near the polar body and marking the animal segment (Dieguez et al., 1995). In video sequences of human eggs after intracytoplasmic sperm injection (ICSI), rotation placed paternal pronuclei adjacent to vegetal cortex in some cases (Edwards and Beard, 1997; Payne et al., 1997). Further video sequences showed how rotation to the polar axis occurred in other eggs as the first cleavage spindle was being formed (Payne, 1998). Rotation could have important consequences in realigning the polar axis preparatory to germline formation. We postulate that, following fertilization (Figure 3a–c), rotation positions the paternal pronuclei adjacent to cortically localized proteins (Figure 3d,e). At this time, chromatin packaging is reorganized and protamines are replaced by histones in the paternal pronuclei, which outcompete maternal pronuclei for the hyperacetylated histone H4 pool until close to syngamy (Thompson et al., 1998). Paternal pronuclei are accordingly 5-fold more active transcriptionally throughout virtually all the 1-cell in mice (Figure and et al., 1997; et al., 1994). syngamy (Figure all then be distributed to by and on a C.elegans and equatorial in a germline at the 4-cell stage (Edwards and Beard, maternal are replaced by zygotic transcripts zygotic gene expression et al., 1995; and 1997). Genes which active in the first of zygotic transcription in the egg class I and et al., et al., et al., 1994; et al., 1995; et al., et al., 1996). is to be a sex located on of a into pronuclei development in to with no of et al., Its transcription in genital ridge as a of a is regulated by a different to that regulating transcripts in The transcripts not function in genital ridge et al., et al., 1995; et al., 1995). may have an role in sex and for role in development 1998). Its of a function may be by role as a transcription absence in some and the that in in or into can germ and with differing to the as and a homologous sequence of in a known as the et al., 1996). is with in mouse embryos et al., but role in sex determination at these early stages is the and is located on and encodes a protein with with at the region et al., Its homologue has similar and while protein of similar with at the and may the The role of sex-determining genes early in development is In and transcripts identified by in mRNA from of mouse embryos the and stage et al., Transcripts of sex-determining gene and autosomal gene not identified in by as by the stages not The gene or for was not identified et al., postulate that sexual in mammals gonadal differentiation as in et al., and transcripts identified the and stage in mouse embryos and 1994), with the product and being in also on an early regulatory role for in sex et al., did not transcripts in unfertilized eggs or mouse but using with an identified some in early and cell in a distribution and cleavage stage embryos not and of the was to and transcripts in human embryos and in et al., 1994). Maternal transcripts identified in oocytes, but and transcripts not and zygotic transcripts from the 1-cell to the and those of from the to the It was not possible to the of transcripts in their embryos. on the possible expression and the growth of embryos. using on human embryos was to or human transcripts not in unfertilized oocytes, or but identified in of eggs at for transcription in et al., 1995). transcripts in of three and 4-cell and in of embryos the and stages et al., 1995). of embryos the and stages from eggs a to be transcripts in oocytes and and transcripts in embryos identified at et al., 1996). transcripts at all stages even when in with et al., et al., 1995) to in mouse of these in Table could from in transcripts in or species gene expression may be by different initiation in et al., or by the of and forms and 1994). studies are in the that mRNA expression and not protein such positive of and in embryos by The of sex-determining genes in embryos may be from their in genital although it has been 1997), and that their expression in embryos is of sex differentiation, mammals to embryos of several species are known to et al., et al., 1994) and the that and other genes may as growth has been et al., 1994; et al., 1995). In expression of the which controls growth in mice and is to the region of the is not by the sex of the embryo et al., 1998). possible function of these early sex-determining genes be a role in development et al., this showed development in mouse embryos by pronuclear We that these sex-determining genes are more to be with events which the germline, and the in this is whether their early expression may be with a function in development, germ cell in stages. The segregation of germline from embryo development is fundamental to the of the species and be to be an early The concept of in early cleavage stages as no since many early that as as cells formed germline in mice 1974), in genetic mice with marker in mice et al., and in mice and In animals where expression of germline genes can be traced from germplasm in the oocyte through early cleavage and development to an early segregation of germline is In germplasm is to a germline cell at the first regulated by microfilaments and the while germline segregation with et al., 1996). In Xenopus, segregation of germplasm at the stage can and and a similar was for the segregation of the germline gene X-cat2 et al., 1995). A type of segregation in Zebrafish where vasa mRNA to the cleavage planes the first in in the first where it in development, to germline cells while other cells to et al., 1997). Germplasm or early germline markers in mammals, although segregation at the stage has been proposed in a of cell on the of the of oocyte polarization and maternal of early development in mammals (Edwards and Beard, 1997). of polarization in mammalian oocytes and embryos (Gardner, 1996; Edwards and Beard, has been by the of proteins in and 4-cell mouse and human embryos (Figure and Blerkom, 1997). these proteins are in the developing and are but slightly polarized in the The first cleavage slightly from the polarity of the that leptin and STAT3 are the first (Figure and cleavage (Figure Further evidence for polarization in the cortex of the mammalian egg from studies on in human embryos as similar to those for leptin and STAT3 identified for several other regulatory genes including c-kit and (Antczak and Blerkom, 1999). The similar for all these proteins may a common cortical receptors since many of these from the Such a is by a (Goud et al., which the polarization of the cortical regulating type I inositol receptors in and human Recently to role of polarity in the regulation of early mouse vegetal or of from pronuclear eggs to with cell without their development to at embryo This to It that polarized are located on the polar of being and from one pole to the other as for leptin (Figure Such markers not to the exact in these mouse was on the and of pronuclear stages. The of did not the of early development but was not to The all embryos was to cells in and could be restored by embryos. The of a of polar determinants to their was not A form of to be the for this work with a of following there to be evidence to of early events in mammalian (Edwards and Beard, with some in germline to one at the first cleavage and to one at the 4-cell stage following spindle rotation in the cell at the We this the since it is with the of distribution in mouse and human where one equatorial and one cleavage at the stage in a cell of in 4-cell (Figure This cell is a possible germline since cell these markers (Antczak and Van Blerkom, 1997). different could be there is evidence for controlled cleavage planes in the germline of C.elegans the in mammalian cells is not The in 4-cell embryos of many species including and and Edwards and Beard, involves rotation the polar axis but it is not whether this is spindle rotation or rotation as cleavage and 1978). is it known whether segregation of maternal determinants is regulated by controlled cleavage planes as it is in other animals. The distribution of germline markers such as X-cat2 mRNA in Xenopus is an active of the (Forristall et al., 1995) and similar processes could be active in mammalian It is possible to several in which mammalian germline in cells or even a in this cleavage in common with other animals from C.elegans to Xenopus and germline are by the distribution of maternal determinants when to A of mice by with epiblast cells that some cells from mouse of and germline (Gardner et al., is and in differentiation in embryos. as we the 4-cell stage is the stage for to in mammals et al., 1995). is more after this some somatic cells can and in is the of cells in when placed on containing and some genes and as into cells et al., et al., 1996). The of and other cell on may some and as form such as and for human et al., 1997). events could nuclear steps which could to as occurred the of et al., 1997). In of this allocated but not in of differentiation may the to in to as are for early germline segregation in different of Xenopus, as by the segregation of et al., 1995) and by the of germ cell formation with germplasm with of germplasm in and the multipotency gene allocated germ cell embryos until are in as primordial germ cells in epiblast close to in and stages (Pedersen, 1986; Lawson and Hage, 1994). of embryological events to germline formation is the of germ cell from the 4-cell stage be by the and the of it to one Drosophila germline more et al., 1998). germline cells in mouse embryos as by cells in (Palmieri et al., 1994), not a at mouse germline has to alkaline cells and Hage, 1994). of germline that all of the germline form germ cells, and that cleavage is or after the 4-cell germ cells to genital ridge their regulated by and and by of and on early germline formation must be to the of in in mouse expression is until the when it regulated by a It then for as it the maternal and in mice et al., 1997; and 1998). many genes on the are including and which are homologous with the genes and et al., et al., from may be to associated with in function of of islands was to the of and several of and species and 1998). the where was This also the homologous sequences in other and it has been proposed that this finding an these and genes and 1998). Such species in may in expression of sex-determining genes in embryos in and human embryos. is controlled in germinal and somatic cells at different of development and 1998), and work may to into for early germline formation. is a of the of The protein is and of of which are located in which to the in unfertilized eggs, protein is in at one pronucleus and distributed in from embryos to as zygotic transcripts maternal transcripts et al., et al., 1994; et al., 1995; and 1998). It is not to cells in embryos as (Edwards and Beard, 1997). is in and in a cells of the cell et al., et al., 1996). It is then to to of and to germline and primordial germ cells after (Palmieri et al., 1994; and 1998). It is regulated by a in germline and a in epiblast et al., 1996). A location in germline that is a germline expression and are more of a multipotency It a has transcription and close to the mouse and the human et al., and 1998). The protein is a activator of the growth factor gene and a of the human gene in It transcription of the with the regulatory genes and is with in mouse embryos proteins to in the gene to the cell et al., 1995; et al., 1995). In by has with and an which in by to et al., 1998). via several could cells to from somatic This role was for germline genes more three (Smith, 1966; Dixon, 1981). and germ cell differentiation and et al., 1990; et al., 1990; et al., 1997), as by the that transcripts are in mouse genital ridge until migratory germ cells et al., The expression of in mouse genital ridge at cell differentiation et al., and expression of factor et al., 1995; et al., 1995; et al., 1998). not in primordial germ cells in the genital expression in human but not et al., 1995; et al., 1995). and genes are proposed to sex determination 1998). and as sex-determining genes in somatic cells of the genital ridge sex differentiation, and may be there with et al., 1996). a transcription may of in cells et al., and may expression in the genital is then in and and in after is et al., 1996). genes are also active in somatic has many of a Its in genital ridge that of This gene of and to et al., 1994; et al., 1998), while the protein has no or other yet to and the transcription of the regulatory protein gene et al., 1997). It has not been in embryos. In to it is in the differentiating but in the and as these genes in the genital and from and other of this have been or by their of et al., sex differentiation and a expression of is associated with in some et al., 1998). is known genetic sexual differentiation and gonadal formation. is placed on sex-determining of genes as in This concept be since it that primordial germ cells are in the genital at these stages may have been their migration from the to genital ridge some of their via a close being et al., 1994). as cells then in as the cells into the genital by the separation of from and the absence of a and cells with the of germ cells into the developing as some are cell processes as at while and oogenesis and 1988). for primordial germ cells in sex differentiation have been on their sex can be as in mice where germ cells may form oocytes in the and germ cells form in the et al., et al., the of germ cells on their 1995) and after cells in the must to et al., from such evidence that germ cells are for sex and other proposed that germ cells to the genital ridge 1995; et al., 1998). The role of cells and was also by their apparent formation in genital ridge even germ cells on the of sexual differentiation was accordingly to or somatic cells, and primordial germ cells to be as to these The sex-determining must be as germ cells of opposite from the stages. from germ cells from cells could migration or a clear can be for a role of germ cells in gonadal evidence of an formation of without need for germ cells, on the formation of in and mice and in mouse embryos to early stages that primordial germ cells in the early genital ridge in in mice et al., 1981), in mouse embryos and also in those et al., 1997). The germinal cells then to The is for mouse for the since some primordial germ cells in the genital ridge by their of c-kit cells could the all and et al., evidence also a regulatory role for germ cells, and oocytes as of the from formation to and et al., 1997; et al., The associated with primordial germ cell migration in the of the and in including human which adjacent et al., 1998). these oocytes could in a clear sequence and are at This the of distribution in et al., 1997), and similar to those in This mechanism may sequences in formation and migration of from the several and It also the of in human et and oocytes not cell in et al., et al., 1998). can be from the In a germ cells genital ridge and the developing the of and in have been but a early form of This could also occur after and is known to in some at and their of germ cells Such forms could from in of migratory germ cells, to germplasm or germ cell formation In in genes such as or in eggs or early cleavage could how sexual differentiation and be impaired (Edwards and 1997). in early germline cells could also how a a forms of et al., evidence that mammalian germline is pronuclear and cleavage stages in mammalian embryos as genes involved in sex determination and differentiation, including and and and are aspects of sex determination could germline formation but species and other in expression of these genes for The early expression of these genes from pronuclear stages be with cleavage in or the of although with either of these processes have not been is needed mammalian germplasm and germline, and primordial germ cells and gonadal such as sex-determining genes on the are in paternal pronuclei, and whether the paternal is active in paternal pronuclei of must be The of the of genes in embryos and of in germline germ cells not to be migration and may have an essential role in gonadal Since germ cells are the of the genetic for the and in the first cleavage in many lower we whether mammals can to until the cell or epiblast germline, and for a close of the regulation of early mammalian Genes to the and function of in Caenorhabditis elegans, pole plasm in Drosophila and germplasm in Xenopus Genes to the and function of in Caenorhabditis elegans, pole plasm in Drosophila and germplasm in Xenopus from of and expression in embryos from of and expression in embryos cells in Drosophila can be traced from their polar location in the fertilized egg to the genital ridge in the developing using to vasa are from vasa in A embryo germ cells at the At after germ cells at the of the At stage germ cells are in a three on either side of the At stage the germ cells have into the A newly fertilized mouse egg with an to leptin and using a with the of Van Blerkom and (Antczak and Van Blerkom, 1997). polar leptin of leptin of polarization on germline determination in the 4-cell mammalian This the stages fertilization and early segregation of germline in the 4-cell sperm at of II body shown at and formation of polar the pronucleus is slightly of transcription of the paternal shown and spindle formation. that the axis of the cleavage is Products of the first cleavage and axis for of the first of the cleavage Products of the and axis for of the of the cleavage cleavage to distribution of germline that the axis of cleavage is slightly to the polar in the location of germline of leptin in a mouse embryo distribution of leptin to an region of cortical 4-cell embryo in which one is are and one is leptin with the of Van Blerkom and and Van Blerkom, 1997). polar