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1 Departments of Pathology, 2 Molecular and Human Genetics and 3 Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
Correspondence should be addressed to Martin M Matzuk at the Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; Email: mmatzuk{at}bcm.tmc.edu
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Abstract |
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 Top Abstract IntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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Historically, two major obstacles have impeded the study of reproductive biology in mammals (reviewed by Staub 2001). First, germ cells in mammals develop in a microenvironment of supporting stromal cells of somatic origin that interacts with the former through autocrine/paracrine mechanisms as well as direct cell-to-cell interactions (Matzuk et al. 2002). Devoid of this support, isolated germ cells in culture fail to survive and maintain their characteristics, making it highly difficult to study molecular interactions and pathways that are unique to these cells. The second, but related, hindrance has been the inability to develop a proper cellular context in vitro that would allow undifferentiated germ cells to differentiate into specialized gametes that retain their ability to fertilize and produce healthy offspring. Recently, interest has exploded in the field of in vitro germ cell differentiation (Lacham-Kaplan 2004, West & Daley 2004) due to exciting findings by several investigators who have been able to generate meiosis-competent germ cells from undifferentiated embryonic stem cells (Hubner et al. 2003, Toyooka et al. 2003, Geijsen et al. 2004). Although these gametes have been shown to fertilize oocytes and produce blastocysts, no data are available yet to suggest that these can progress to produce live offspring. In addition, Feng and coworkers (2002) have generated an undifferentiated spermatogonial cell line immortalized by the ectopic expression of the catalytic component of telomerase. The cell line was also shown to differentiate in vitro to morphologically similar haploid round spermatids but the authors did not investigate whether these haploid germ cells were competent to initiate fertilization and zygotic development when injected into oocytes. So, although the future looks promising, more careful analysis of the physiological state of these in vitro-formed gametes, including the identification of parent-of-origin-specific imprints, would be critical for enhancing the utility of these techniques.
Our understanding of the molecular mechanisms of mammalian reproduction therefore has been largely dependent on loss-of-function mutagenesis in mice. Ever since researchers developed techniques to culture mouse embryonic cells in vitro (Evans & Kaufman 1981) and methods were developed to inject cultured embryonic pluripotential cells into blastocysts to produce germ-line chimeras (Bradley et al. 1984), the field of mouse transgenic technology has undergone a veritable explosion. Around the same time, researchers were successfully targeting exogenous DNA into endogenous loci based on limited amounts of homology between the two DNA sequences in cultured mammalian cells (Smithies et al. 1985). Simultaneously, Thomas and colleagues (Thomas et al. 1986, Thomas & Capecchi 1987) had developed a highly efficient method of targeted mutagenesis of cultured embryonic stem cells and other mammalian cells by using homologous recombination between endogenous genes and exogenously introduced DNA (targeting vector), a technique that was shown to be highly specific by Doetschman and coworkers (Doetschman et al. 1987, 1988). Our present-day knockout technology, a cornerstone of functional genomic studies, got its final form when these two technologies were harnessed together to generate the first targeted knockout mouse model through homologous recombination in embryonic stem cells (Koller et al. 1989).
A knockout mouse model is typically designed to produce a null mutation in the target gene and thus uncover an essential function. In dissecting gene functions, a hypomorphic mutation or the generation of an allelic series can at times be beneficial to researchers testing for genetic interactions between candidate genes. However, a null mutation is preferred in identifying non-redundant and essential functions of genes, as exemplified by the generation of approximately 300 mouse knockouts that manifest defects in reproduction (see below and online table that accompanies Matzuk & Lamb 2002).
Among the earliest mouse knockouts generated that were found to affect different aspects of reproduction were Inha (inhibin 
), Lif (leukemia inhibitory factor), and estrogen receptor (Matzuk et al. 1992, Stewart et al. 1992, Lubahn et al. 1993). While LIF-deficient females had defects in implantation, knockout males and females of inhibin were infertile and had gonadal tumors. Female mice null for estrogen receptor were infertile, and males had reduced fertility. Over the last decade, several hundred mouse knockouts have been created that affect different stages of reproduction (reviewed in Matzuk & Lamb 2002), including primordial germ cell (PGC) formation and migration into the gonads, gonad development and sex determination, postnatal gametogenesis, sperm–egg binding and fertilization, and early embryonic development. For more comprehensive background on these topics and the mouse models that have shed light on these stages of reproductive development, readers are referred to these excellent reviews: Bendel-Stenzel et al.(1998), Matzuk & Lamb (2002), Matzuk et al.(2002), Primakoff & Myles (2002), Zhao & Garbers (2002), de Rooij & de Boer (2003), Brennan & Capel (2004) and Park & Jameson (2005).
In this review, we focus, in a stage-specific manner, on specific knockouts that have proved pivotal in our understanding of the biology of reproduction, with special emphasis on a few in vivo models that have clarified or refuted conflicting findings from in vitro experiments. We then briefly describe the principle of conditional mutagenesis and highlight its use in reproductive studies. Finally we discuss some of the current deficits of these technologies that have tempered the enthusiasm of researchers embracing them as their primary tools in functional genomics.
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Germ-line specification: what seals the fate? |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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In mice, PGCs are the founder cells of the germ line that are allocated from a pool of proximal epiblast cells at around embryonic day 7.2 (E 7.2; reviewed in Zhao & Garbers 2002). By using mice that were deficient for bone morphogenetic protein (BMP), member 4 (Bmp4 –/– mice), Lawson and colleagues (1999) provided clear evidence that germ-cell competence is induced in a subset of proximal epiblast cells in response to BMP4 secreted from the extra-embryonic ectoderm. In Bmp4 –/– mice PGCs were completely absent, and in Bmp4 heterozygotes the PGC number was either greatly reduced or completely absent, suggesting that the effect of BMP4 is dose-dependent (Lawson et al. 1999). Deficiency of the related transforming growth factor ß (TGFß ) superfamily members, BMP8b and BMP2, led to a very similar phenotype of reduced or absent PGCs on a mixed genetic background (Ying et al. 2000; Ying & Zhao 2001). Interestingly, mice null for SMAD5 or SMAD1 (Chang & Matzuk 2001, Tremblay et al. 2001), the intracellular effectors of BMP signaling, have pronounced defects in PGC formation as well. More recently, knockout mice carrying a targeted mutation in the type I BMP receptor, activin receptor-like kinase 2 (ALK2), were found to have a complete absence of PGCs, further highlighting the importance of this signaling pathway in germ-cell specification (de Sousa Lopes et al. 2004).
While these studies suggest that an intact BMP signaling system in the embryonic stages is necessary for PGC fate specification, what is not clear is whether such a signaling mechanism is actually sufficient to induce PGC fate. What are the primary targets of BMP4 and BMP8b signaling in the epiblast? Do they signal directly to the PGC precursors? Is there a master regulator of PGC fate? These are some of the questions that remain unsolved. However, a recent study, utilizing a knockout in the mouse zinc-finger-containing transcriptional repressor PRDM1 (PR domain containing 1, with zinc-finger domain), has uncovered an essential role for this protein in PGC specification. Embryos lacking the PRDM1 protein have a complete absence of PGCs while heterozygotes have a drastic reduction in PGC number (Vincent et al. 2005). Since Prdm1 transcripts are expressed in PGC precursors as early as E 7.0, this is the first transcriptional factor to be identified that plays an essential role in PGC specification. So, is PRDM1 the master regulator that seals the fate of PGC precursors into entering a germ-cell lineage? The answer to that question awaits further studies, which would address key questions regarding the sufficiency of PRDM1 in specifying a germ-cell fate upon non-committed epiblast cells and whether its action is cell-autonomous in these precursors.
Studying gene function through knockouts has proved highly significant in the field of reproduction not only because of the uncovering of new genes and pathways but also because these in vivo studies on many occasions served to prove or refute several models that were based on either cell-culture-based assays or on evidence from forward-genetics approaches. One instance is noteworthy: by analyzing the expression pattern of genes from solitary nascent PGCs, Saitou and colleagues (2002) proposed a mechanism for acquisition of germ-cell competence and specification in PGC precursors in the epiblast which was based on essential roles for two highly expressed and unique cDNAs (named fragilis and stella) in PGC fate determination. However, when the stella gene was knocked out in mice, there appeared to be no defects in the specification, maintenance or proliferation of PGCs (Payer et al. 2003, Bortvin et al. 2004); instead, knockout males were fertile although null females failed to sire normal numbers of offspring as the gene fulfilled an essential role in the pre-implantation development of embryos as a maternally derived factor. In this context it would be interesting to know the phenotype of mice with a null mutation in fragilis.
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PGC proliferation, migration and survival |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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Through knockout approaches, researchers have identified several new genes that play essential roles in some or all of these processes. Targeted disruption of a peptidylprolyl isomerase-encoding gene, Pin1, uncovered an unexpected role for this gene in the regulation of the cell cycle and PGC proliferation. On an isogenic C57BL/6J background, mice null for Pin1 were found to have reduced PGC numbers during the mid-to-late migratory phases (E 9.5–12.5) compared with wild-type mice (Atchison et al. 2003); by analyzing BrdU labeling, this reduction seemed to be due to a delay in progression through the cell cycle rather than due to a cell-cycle arrest or enhanced apoptosis in these cells.
Specific genes regulate the survival of the migratory germ cells: the POU domain transcription factor, Oct4 (Pou5f1; Mouse Genome Informatics), is a critical determinant of pluripotency in the inner cell mass that causes embryonic death when mutated (Nichols et al. 1998, Niwa et al. 2000). To circumvent the early embryonic death and to examine the effect of deletion of Oct4 from PGCs, investigators have used TNAP Cre (tissue non-specific alkaline phosphatase – Cre), which encodes the Cre recombinase only in the PGC population prior to E 10.5 (Kehler et al. 2004; Table 1
). Surprisingly, loss of OCT4 protein from PGCs leads not to differentiation but to enhanced apoptosis in these cells prior to their colonization of the gonads.
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PGCs that successfully navigate to the genital ridges enter cell-cycle arrest, although at different stages in the two sexes, as mentioned above. Heterozygosity for the transcription factor zinc-finger protein 148 (Zfp148) has been shown to increase apoptosis and cause complete loss of germ cells from the male gonad at the E 13.5 stage (Takeuchi et al. 2003), an effect that is probably due to improper regulation of the p53 protein in these cells. Thus Zfp148 is one more addition to the growing list of genes that seem to affect PGC specification and maintenance in a dosage-dependent manner in mice.
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Sex determination |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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It is critical to note, however, that the role of DAX1 is highly dependent on the genetic background of the mice under investigation. A recent study by Eicher and colleagues demonstrated that although DAX1 function is essential on the C57BL/6 (B6) background for testis development in XY mice, it is completely dispensable on the DBA/2J background (Bouma et al. 2005). In B6 Dax1 –/Y mice, there was a failure of up-regulation of Sox9, which is another important gene in the sex-determination pathway (see below), even though Sry expression initiated normally.
The homeobox gene Sox9 is the earliest marker of pre-Sertoli cells following Sry expression. Through the use of a conditional knockout model SOX9 has been shown to be essential for normal testis differentiation and sex-cord formation in the testis (Chaboissier et al. 2004). SOX9 regulates the differentiation of Sertoli cells and the production of male-specific markers Amh (anti-Müllerian hormone) and P450scc (cytochrome P450 side-chain cleavage), both of which are decreased in homozygous knockout mice. Indeed, this finding is a corroboration of earlier studies in which targeted disruption of the SOX9-binding site in the mouse Amh promoter caused a failure of transcription of Amh (see Table 2) that resulted in non-regression of the Müllerian duct structures in XY mice and the formation of pseudo-hermaphrodites, confirming that SOX9-mediated up-regulation of AMH is a critical and specific step that is essential in the male sex-determination pathway (Arango et al. 1999). Precisely how AMH mediates regression of Müllerian duct structures in vivo has been characterized by the generation of a conditional knockout for one of the type I BMP receptors, Bmpr1a (Jamin et al. 2002); male mice lacking these receptors in mesenchymal cells of the Müllerian duct fail to respond to AMH and have persistent uteri and oviducts.
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). Since Sry expression was reduced in the KTS-ablated WT1 homozygotes, it can be inferred that WT1 + KTS is important for Sry-mediated XY gene program establishment in the bipotential gonad; whether this down-regulation is a direct result of WT1 + KTS loss or as a result of decreased numbers of Sry-expressing pre-Sertoli cells awaits further experiments. Another gene involved in ovary development is Wnt4, which is required for Müllerian duct formation in both sexes prior to sex determination (Vainio et al. 1999); female mice lacking Wnt4 exhibit partial female–male sex reversal. Since Fst-null ovaries have a similar defect and since Fst expression is absent in Wnt4-null XX gonads, Fst, operating downstream of Wnt4, has been implicated in the suppression of BMP and/or activin signaling to suppress the male-specific coelomic vessel (Yao et al. 2004).
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Molecular control of pre-antral folliculogenesis |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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The formation of primordial follicles in the newborn mouse ovary is preceded by the development of germ-cell clusters in the embryonic ovary in which the dividing germ cells undergo incomplete cytokinesis to remain connected to each other through intercellular bridges and their subsequent breakdown perinatally (Pepling & Spradling 2001). The search for active players that regulate the formation of primordial follicles in mammals has resulted in the discovery of a bHLH (basic helix-loop-helix) transcription factor called FIG (factor in the germ line ; Figla) present specifically in the female germ cells. Mice carrying a targeted disruption of the Figla gene (Figla –/–) had comparable number of germ cells in their ovaries with respect to wild-type ovaries at the E 18–19 stage (Soyal et al. 2000). However, by postnatal day 1 (PD 1) the oocytes in the Figla –/– ovaries failed to grow in size and began to disappear; by PD 2 the null ovaries were completely devoid of primordial follicles. The Figla –/– oocytes do progress through meiosis I, but fewer cells survive beyond the diplotene stage, suggesting that FIG regulates the expression of survival factors/growth factors at this stage.
While Figla represents an oocyte-derived factor critical for the formation of primordial follicles, data from another mutant provide evidence that primordial follicle formation is regulated by the somatic components as well. Mutants lacking the Wnt4 gene product die shortly after birth due to defects in kidney development; however, at day 1 the mutant ovaries have a dramatic reduction in the number of meiotic-stage oocytes and most oocytes degenerate shortly thereafter (Vainio et al. 1999). A similar finding has emerged from mice with follistatin-deficient ovaries (Yao et al. 2004), highlighting the functional interactions between these two proteins (see above).
Shortly after birth a subset of primordial follicles is selected from a resting pool of quiescent follicles to undergo the follicular activation and growth. Follicular activation is characterized by oocyte growth and the transition of squamous to cuboidal granulosa cells, which signals the formation of a primary follicle followed by granulosa cell proliferation. Work from our group and others has recently identified several proteins believed to be critical at this step. A homeobox transcription factor, NOBOX, which is specific to the oocyte in the newborn ovary (Rajkovic et al. 2004), is essential in this process. In Nobox –/– ovaries, primordial follicles do not develop into primary follicles and beyond; instead, by PD 14, the majority of the primordial follicles are lost leading to ovarian failure and infertility. Future studies will illustrate the target genes for this transcription factor and uncover other oocyte-specific genes that play critical roles in this process.
Primordial follicles are recruited in cohorts for growth, differentiation, and maturation through a series of highly regulated processes. However, primordial follicles in Foxo3a –/– ovaries undergo global activation so that by PD 14 the null ovaries do not have any quiescent primordial follicles but have an almost 2-fold increase in the number of primary and secondary follicles as compared with wild-type ovaries. Significantly, the null ovaries also have an almost 4-fold increase in atretic follicles and an approx. 14-fold increase in the amount of zona pellucida remnants, suggesting that the majority of the growing follicles undergo atresia following the formation of the zona pellucida by the oocyte. Not surprisingly, therefore, this sequence of global activation and atresia leads to a premature depletion of the pool of primordial follicles and secondary infertility by 15 weeks of age (Castrillon et al. 2003, Hosaka et al. 2004).
Mutations in the human forkhead transcription factor FOXL2 cause an autosomal dominant condition referred to as Blepharophimosis/ptosis/epicanthus inversus syndrome (BPES), associated with premature ovarian failure (Crisponi et al. 2001, De Baere et al. 2001). Mice with a targeted disruption in the murine ortholog (Foxl2 –/– mice) of the human gene are infertile and provide a mechanism to explain the etiology of BPES. In Foxl2 –/– mice granulosa cells in the mutant ovaries do not make the squamouscuboidal transition and consequently the mutants lack any secondary follicles and are infertile (Schmidt et al. 2004). Another granulosacell derived factor that regulates the rate of follicle recruitment, in a subtler manner, is the anti-Müllerian hormone (AMH), which, when secreted from the granulosa cells of secondary follicles, acts in a paracrine fashion within the follicle to inhibit the recruitment of primordial follicles. In Amh –/– females there is a derepression of the follicular activation process causing mutant ovaries to be depleted of primordial follicles earlier than in wild-type ovaries (Durlinger et al. 1999).
Growth differentiation factor 9 (Gdf9) is an oocyte-derived mitogenic paracrine factor that plays a critical role in folliculogenesis by regulating the somatic components of the follicle. Gdf9 encodes for a member of the TGFß superfamily of secreted proteins that play important roles in cell proliferation and differentiation in mammals (Chang et al. 2002). Members of this family, such as GDF9, BMP15, AMH, and activins, exquisitely regulate early stages of folliculogenesis. In mice, a null mutation in Gdf9 causes complete infertility in female mice (Dong et al. 1996). Histopathological examination of null ovaries demonstrate a total lack of normal appearing secondary follicles indicating that GDF9 secreted from oocytes acts upon the granulosa cell population to augment the proliferation/differentiation of these follicular supporting cells. Thus, GDF9 is essential for the primary-to-secondary follicle transition in mice. The oocyte that has already entered the growth phase following activation continues to grow in these follicles (Carabatsos et al. 1998); however, devoid of the close support from the granulosa cell mass there is atresia of both the oocyte and eventually the somatic cells.
Another member of the TGFß superfamily of proteins that appears to play an important role in pre-antral stages of folliculogenesis is BMP15. BMP15 is secreted from oocytes and regulates the proliferation and differentiation of the granulosa cell population. Analysis of a null mutation in Bmp15 generated in our laboratory revealed that Bmp15 –/– mice are subfertile and display defects in ovulation and fertilization (Yan et al. 2001). However, studies in sheep have shown that BMP15 may also play important roles at the primary to secondary follicle transition (Galloway et al. 2000) or, alternatively, the mutant BMP15 may antagonize the functions of GDF9 (Liao et al. 2004).
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Spermatogenesis |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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Among the first mutations that were identified to have a defect in spermatogonia were the W and Sl mutations (Russell et al. 1979) that affect the Kit receptor, which is present on differentiating spermatogonia and its ligand, the Stem cell factor, which is produced in the Sertoli cells. However, mutations in the Kit signaling system affect only the differentiated spermatogonia as the receptor Kit is not expressed on earlier spermatogonial populations (Donovan & de Miguel 2001). Recently, glial cell-derived neurotrophic factor (GDNF), another Sertoli cell-derived factor distantly related to the TGFß family of growth factors, was shown, in a dose-dependent manner, to control the cell-fate decision of undifferentiated spermatogonia; reduced levels of GDNF caused spermatogonial differentiation and depletion of the stem-cell pool while high levels in vivo caused a differentiation block and increase in the numbers of spermatogonia (Meng et al. 2000). Germ-cell factors that play equally important roles in the proliferation of undifferentiated spermatogonia have also been identified through mouse knockouts; prominent has been the identification of a zinc-finger-containing transcriptional repressor, Plzf, that is essential for stem-cell self-renewal in the adult testis (Buaas et al. 2004, Costoya et al. 2004). Although the precise genes that are regulated by this transcription factor are not yet known, future studies utilizing transcriptome analysis of spermatogonia similar to the approach used by Costoya and colleagues (2004) should lead to further insights into the molecular mechanisms of germ-line stem-cell biology.
Knockouts have been created that identify essential genes at almost all steps of spermatogenesis (reviewed in Matzuk & Lamb 2002, de Rooij & de Boer 2003); literally hundreds of mutant mice have been characterized with arrest at various stages such as a spermatogonial differentiation block, pachytene stage meiotic arrest, and defects in spermiogenesis and sperm function.
Several genes that are expressed in post-meiotic cells have been found to be essential for spermiogenesis in a dose-sensitive manner. Notable among these are mutations in the genes encoding protamines 1 and 2 (Prm1 and Prm2), Kelch-like 10 (Klhl10) and sperm antigen 16 (Spag16), haploinsufficiency of which are incompatible with male fertility (Cho et al. 2001, Yan et al. 2004, Zhang et al. 2004). Male mice that are high-percentage chimeras of these genes are unable to transmit the mutant alleles through the germ line due to either severe morphological defects (as in the Prm1 or Prm2 mice) or severe reduction of sperm count and defects in sperm morphology and function (Klhl10 or Spag16). During spermiogenesis, chromatin packaging in the sperm head requires the replacement of histones by the transition proteins and protamines. Similar to protamines, deficiency of both transition proteins 1 and 2 also leads to severe defects in sperm morphology and infertility (Zhao et al. 2004), thereby highlighting the importance of proper chromatin remodeling during spermatogenesis.
Defects in sperm motility also lead to infertility; analysis of knockouts of CatSper1 (Ren et al. 2001) and CatSper2 (Quill et al. 2003), which are cationic channels present on the sperm membrane, a sperm-specific isoform of a glycolytic enzyme, Gapdhs (Miki et al. 2004), and of structural components of the axoneme (Spag6, Tekt2) have revealed interesting models of asthenozoospermia (Sapiro et al. 2002, Tanaka et al. 2004) that lead to infertility in male mice. Finally, knockouts have uncovered genes that play essential functions in the final step of sperm function, sperm–egg binding (reviewed in Evans 2001, Primakoff & Myles 2002). The plethora of single-knockout models that impair spermatogenesis at so many discrete steps is in addition to numerous models that cause a hitherto undefined or non-specific block in spermatogenesis and as a whole serve to illustrate both the complexity of this process and the huge number of potential targets for development of contraceptives in humans.
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Endocrine, paracrine and autocrine regulation of gametogenesis |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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Sex hormones (estrogen and progesterone in females, testosterone in males) have been long believed to play essential roles in the development, differentiation and proper function of female and male reproductive systems. Targeted knockouts of their respective receptors have revealed precisely how these steroid hormones function and have uncovered hitherto unsuspected roles for estrogen signaling in males and testosterone in females. Estrogen acts through two nuclear hormone receptors, ER and ERß ; whereas deletion of ERß leads to subfertility (Krege et al. 1998) due to defects in granulosa cell development (Dupont et al. 2000), disruption of ER results in female infertility due to a complete lack of ovulation with the null ovaries displaying hemorrhagic cysts (Lubahn et al. 1993, Dupont et al. 2000). Interestingly, ER-knockout males are infertile as well due to a defect in fluid reabsorption in the epididymis that leads to degeneration of seminiferous tubules and disruption of spermatogenesis (Eddy et al. 1996, Hess et al. 1997), thereby identifying a male-specific role for estrogen. Progesterone, which also acts through two receptor isoforms (PR-A and PR-B), was believed to be important for the establishment and maintenance of the pregnant state; however, deletion of the progesterone receptor in females led to infertility due to defects in ovulation with associated uterine inflammation and disruption of mammary gland morphogenesis (Lydon et al. 1995). Identifying which of these effects were mediated by the two receptor isoforms required some subtle manipulations because both isoforms were generated from the same gene from alternative promoters and translation initiation sites. In elegant experiments, Mulac-Jericevic and colleagues (Mulac-Jericevic et al. 2000, 2003) mutated the two AUG initiation codons in embryonic stem cells and generated PR-A and PR-B knockouts (Table 2). Analysis of these mutants revealed essential roles for PR-A in ovulation and uterine physiology while PR-B was important for mammary gland development.
Studies from the spontaneous tfm (testicular feminized) mutant and from androgen receptor-knockout mice (Yeh et al. 2002) have shown androgen signaling to be essential for the descent of testes, development of male accessory sex organs, and spermatogenesis beyond the pachytene spermatocyte stage. Recently, generation of Sertoli cell-specific knockout of the androgen receptor have clarified the roles of testosterone signaling by demarcating androgen signaling in these cells as essential for spermatogenesis but not for non-gonadal functions of testosterone (Chang et al. 2004, De Gendt et al. 2004). Interestingly, signaling through the androgen receptor has also been found to have important roles in female fertility as null females show longer estrous cycles and reduced fertility (Hu et al. 2004).
Studies from our group and several others have also discovered the essential roles played by multiple TGFß family members in reproduction (reviewed by Pangas & Matzuk 2004, Jorgez et al. 2005; see Fig. 1). Among these, the Inha mouse model revealed unexpectedly important roles for the protein in gonadal cell-cycle regulation as null animals developed gonadal tumors of granulosa cell/Sertoli cell origin (Matzuk et al. 1992). As identified by our group and others, also important in reproduction are the activin signaling components; activin receptor type II mutants develop testicular hypoplasia and activin ß B subunit mutants have defective parturition (Vassalli et al. 1994, Matzuk et al. 1995). In order to analyze the functional interaction between activin ß A and activin ß B, our group also created a knockin allele of ß B into the ß A locus (Table 2). The knockin rescued the neonatal lethality of the ß A-knockout mice, thus allowing dissection of the reproductive phenotype (Brown et al. 2000). While male knockin mice had delayed fertility, female mice had severely reduced fertility, thus highlighting the important roles played by activin ß A in mammalian reproduction. Also significant is the follistatin conditional knockout model in which the activin/BMP/GDF antagonist follistatin is deleted only in the granulosa cells of the ovary. Female mice with a granulosa cell-specific deletion of follistatin have severely reduced fertility due to reduced numbers of ovarian follicles and defects in ovulation and fertilization (Jorgez et al. 2004). These and other models (reviewed by Chang et al. 2002) have identified an enormous number of proteins belonging to the TGFß superfamily with essential roles in reproduction.
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Early embryo: the maternal effect |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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). Methyltransferases that are implicated in initiation of methylation at imprinted loci (Dnmt3 l and Dnmt3a) have also been demonstrated to be essential maternally supplied proteins for embryonic viability (Bourc’his et al. 2001, Kaneda et al. 2004). Recently our group and others have added to the list of maternal effect genes in mice. First, female mice null for an oocyte-specific gene Zar1 (zygote arrest 1) were found to be infertile due to a failure of progression of fertilized embryos from the one- to two-cell stage (Wu et al. 2003); in the arrested embryos the parental genomes remain separated and there is a marked reduction in the synthesis of the transcription-requiring complex. Thus Zar1 seems to act at an earlier time-point than Mater in embryonic development. Next, a mouse homolog of Xenopus nucleoplasmin (nucleoplasmin 2, Npm2) was identified to be critical in the process as well. Embryos from Npm2-null females had defects in nuclei and nucleolar organization specifically evident in the loss of heterochromatin from these organelles (Burns et al. 2003). Although, some embryos are able to proceed through the two-cell stage and diminished numbers of offspring are born from null females, most embryos show reduced cleavage to the two-cell stage. Another recent maternal-effect factor to be identified is Stella or Dppa3 (developmental pluripotency-associated 3). Embryos from Dppa3 –/– females progress to the four-cell stage but are defective in further cleavage and in pre-implantation development (Payer et al. 2003, Bortvin et al. 2004). Future experiments utilizing in silico approaches to identifying early-embryo-enriched genes would provide more candidate genes that might play important roles during this phase of embryonic development. |
Conditional mutagenesis |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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; for a list of available floxed alleles, go to www.mshri.on.ca/nagy/floxed.html.
The binary Cre-loxP method is so designed that the bacteriophage recombinase Cre is driven by a tissue- or stage-specific promoter in a restricted spatio-temporal manner; the deletion of gene function in that tissue is then dependent on the Cre recombinase to bind to its cognate recognition sequences (loxP) sites and effect a DNA recombination event between any two of such loxP sites, which can lead to deletion, inversion and other genomic rearrangements based on the position and orientation of such sites. The gene (or more commonly, the gene region) to be deleted is flanked by loxP (‘floxed’) sites in a targeting event similar to a conventional knockout; floxed mice are then bred with mice expressing Cre under the regulation of a promoter of choice to obtain tissue-specific knockouts in the offspring. Using this method several conditional knockouts have been generated that display reproductive defects (see Table 1); indeed, some of them were created after conventional knockouts resulted in embryonic or post-natal lethality (Oct4 and Fst).
One apparent deficit of this technology in reproduction is the paucity of available Cre- driver lines that are specifically expressed in reproductive tissues of interest. Another problem relates to the fact that our restricted knowledge of gene-regulatory elements results in use of promoter regions that prove to be ‘leaky’, meaning that they drive Cre expression and consequently gene deletion outside of their specific spatio-temporal zone (for example the Sf1-Cre line), thus defeating the very purpose of creating a conditional knockout. The last problem, and not a trivial one, is the issue of the efficiency of Cre-mediated recombination. Cre lines often express the recombinase at levels that are not sufficient to achieve complete recombination between loxP sites, thus resulting in the generation of a hypomorph. Attention has been focused on producing more efficient deleter Cre lines (Shimshek et al. 2002) but we are still some distance from achieving mastery over this elegant but at times difficult technology. Nevertheless, with the intensive efforts of various groups and the advent of newer technologies, for example inducible Cre lines, the Flp-Frt system, it is conceivable that in the future, instead of one cure for all ills, the field will witness the use of a multitude of technologies each best tailored in a particular situation to deliver the proper result: that of generation of a null mutation in the tissue of interest.
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In the future |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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In this review we have illustrated some examples of how knockouts in mice have greatly enhanced our understanding of this complex biological process. This is not meant to be a comprehensive survey of all knockouts with reproductive defects but rather an illustrative essay of the utility of the knockout approach towards understanding a process. Challenges remain plenty in both our understanding of the process and in the use of this technology itself, but the emergence of exciting technologies and projects such as RNA interference in mammals (reviewed in Mittal 2004), the MICER project for genome-wide large-scale insertional mutagenesis from indexed targeting vectors (Adams et al. 2004), and the recent conceptualization of a whole-genome knockout mouse project, or KOMP, by an international collaboration (Austin et al. 2004) will broaden the scope and increase the enthusiasm of researchers in the power of knockouts and knockdowns in evaluating the roles of additional proteins in the reproductive axis in vivo.
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Acknowledgements |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionGerm-line specification: what...PGC proliferation, migration and...Sex determinationMolecular control of pre-antral...SpermatogenesisEndocrine, paracrine and...Early embryo: the maternal...Conditional mutagenesisIn the futureAcknowledgementsReferences |
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 S. M. Kehoe, M. Oka, K. E. Hankowski, N. Reichert, S. Garcia, J. R. McCarrey, S. Gaubatz, and N. Terada A Conserved E2F6-Binding Element in Murine Meiosis-Specific Gene Promoters Biol Reprod, November 1, 2008; 79(5): 921 - 930. [Abstract] [Full Text] [PDF]
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 S. Natesampillai, J. Kerkvliet, P. C. K. Leung, and J. D. Veldhuis Regulation of Kruppel-like factor 4, 9, and 13 genes and the steroidogenic genes LDLR, StAR, and CYP11A in ovarian granulosa cells Am J Physiol Endocrinol Metab, February 1, 2008; 294(2): E385 - E391. [Abstract] [Full Text] [PDF]
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 C. Hoekstra, Z. Z. Zhao, C. B. Lambalk, G. Willemsen, N. G. Martin, D. I. Boomsma, and G. W. Montgomery Dizygotic twinning Hum. Reprod. Update, January 1, 2008; 14(1): 37 - 47. [Abstract] [Full Text] [PDF]
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 R. Zachow and M. Uzumcu The hepatocyte growth factor system as a regulator of female and male gonadal function J. Endocrinol., December 1, 2007; 195(3): 359 - 371. [Abstract] [Full Text] [PDF]
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 S. Ro, R. Song, C. Park, H. Zheng, K. M. Sanders, and W. Yan Cloning and expression profiling of small RNAs expressed in the mouse ovary RNA, December 1, 2007; 13(12): 2366 - 2380. [Abstract] [Full Text] [PDF]
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 A. Bettegowda, J. Yao, A. Sen, Q. Li, K.-B. Lee, Y. Kobayashi, O. V. Patel, P. M. Coussens, J. J. Ireland, and G. W. Smith JY-1, an oocyte-specific gene, regulates granulosa cell function and early embryonic development in cattle PNAS, November 6, 2007; 104(45): 17602 - 17607. [Abstract] [Full Text] [PDF]
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 T. Hassold, H. Hall, and P. Hunt The origin of human aneuploidy: where we have been, where we are going Hum. Mol. Genet., October 15, 2007; 16(R2): R203 - R208. [Abstract] [Full Text] [PDF]
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F. Chalmel, A. D. Rolland, C. Niederhauser-Wiederkehr, S. S. W. Chung, P. Demougin, A. Gattiker, J. Moore, J.-J. Patard, D. J. Wolgemuth, B. Jegou, et al. The conserved transcriptome in human and rodent male gametogenesis PNAS, May 15, 2007; 104(20): 8346 - 8351. [Abstract] [Full Text] [PDF]
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 F. K. Hamra, K. M. Chapman, D. Nguyen, and D. L. Garbers Identification of Neuregulin as a Factor Required for Formation of Aligned Spermatogonia J. Biol. Chem., January 5, 2007; 282(1): 721 - 730. [Abstract] [Full Text] [PDF]
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 W. J. Racki and J. D. Richter CPEB controls oocyte growth and follicle development in the mouse Development, November 15, 2006; 133(22): 4527 - 4537. [Abstract] [Full Text] [PDF]
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