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A profound collaboration between the germline and somatic cells of an organism is the creation of a functional gonad. Here we establish a foundation for studying molecular gonadogenesis in the sea urchin by use of RNA-seq, quantitative mRNA measurements, and in-situ hybridizations throughout the life cycle of the variegated sea urchin, Lytechinus variegatus (Lv). We found through three distinct analyses that the ovary and testis of this echinoderm expresses unique transcripts involved in gametogenesis, and also discovered uncharacterized gene products unique to each gonad. We further developed a pipeline integrating timepoint RNA-seq data throughout development to identify hallmark gene expression in gonads. We found that meiotic and candidate genes involved in sex determination are first expressed surprisingly early during larval growth, and well before metamorphosis. We further discovered that individual larvae express varying amounts of male- or female-hallmarks before metamorphosis, including germline, oocyte, sperm, and meiotic related genes. These distinct male- or female-gonad gene profiles may indicate the onset of, and commitment to, development of a bipotential gonad primordium, and may include metabolic differences, supported by the observation that transcripts involved in glycolysis are highly enriched in the ovary compared to the testis. Together these data support a hypothesis that sex determination is initiated prior to metamorphosis in the sea urchin and that the many uncharacterized genes unique to each gonad type characterized herein may reveal unique pathways and mechanisms in echinoderm reproduction., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Gametogenesis, the process of forming mature germ cells, is an integral part of both an individual's and a species' health and well-being. This chapter focuses on critical male and female genetic and epigenetic processes underlying normal gamete formation through their differentiation to fertilization. Finally, we explore how knowledge gained from this field has contributed to progress in areas with great clinical promise, such as in vitro gametogenesis., (© 2019 Elsevier Inc. All rights reserved.)

Piwi proteins play an important regulatory role in germ cell division during gametogenesis and gonad development. In order to understand the function of Piwi genes in the reproductive process of the dark sleeper, we identified and characterized Piwil1 and Piwil2 from gonad tissue. The tissue distribution demonstrated that Piwils were highly expressed in the gonad of the dark sleeper. During gonad development, higher expression was observed in stage I of both the testes and ovaries than in subsequent stages at mRNA and protein levels. The results of immunohistochemistry demonstrated that Piwils were predominantly distributed in the spermatogonia, spermatocytes, and early oocytes. When treated with the HPG axis hormone (HCG and LHRH-A2), the expression of Piwils was significantly decreased in the testes and ovaries at mRNA and protein levels. All of these results indicated that Piwils play a vital role in gonad development and gametogenesis. Our findings provide valuable evidence to further clarify the underlying modulation mechanism of Piwils in teleosts., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Polycomb group proteins and the related histone modification H3K27me3 can maintain the silencing of key developmental regulators and provide cellular memory. However, how such an epigenetic state is reprogrammed and inherited between generations is poorly understood. Using an ultra-sensitive approach, STAR ChIP-seq, we investigated H3K27me3 across 14 developmental stages along mouse gametogenesis and early development. Interestingly, highly pervasive H3K27me3 is found in regions depleted of transcription and DNA methylation in oocytes. Unexpectedly, we observed extensive loss of promoter H3K27me3 at Hox and other developmental genes upon fertilization. This is accompanied by global erasure of sperm H3K27me3 but inheritance of distal H3K27me3 from oocytes. The resulting allele-specific H3K27me3 patterns persist to blastocysts before being converted to canonical forms in postimplantation embryos, where both H3K4me3/H3K27me3 bivalent promoter marks are restored at developmental genes. Together, these data revealed widespread resetting of epigenetic memory and striking plasticity of epigenome during gametogenesis and early development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Epigenetics is an important mechanism of gene regulation that is dependent on the chromatin structure, which is determined by the epigenetic chemical modification of DNA and histone proteins. It is known that the failure of epigenetic mechanisms causes congenital neurodevelopmental disorders (NDs), and that early life exposure to mental stress and endocrine disrupting chemicals, such as phthalates, bisphenol A, and tobacco, can change epigenetic mechanism and gene expression in the brain and cause NDs. Moreover, environmentally induced epigenetic changes are not erased during gametogenesis and are transmitted to subsequent generations, leading to changes in behavior phenotypes. However, epigenetics has a reversible nature because it is based on the addition or removal of chemical residues, and thus the original epigenetic status may be restored. Indeed, several drugs used for mental disorders and NDs restore the epigenetic state and gene expression. Improved epigenetic understanding of NDs will provide important clues for the development of new drugs that take advantage of epigenetic reversibility.

Postnatal spermatogonial stem cells (SSCs) progress through proliferative and developmental stages to populate the testicular niche prior to productive spermatogenesis. To better understand, we conducted extensive genomic profiling at multiple postnatal stages on subpopulations enriched for particular markers (THY1, KIT, OCT4, ID4, or GFRa1). Overall, our profiles suggest three broad populations of spermatogonia in juveniles: (1) epithelial-like spermatogonia (THY1(+); high OCT4, ID4, and GFRa1), (2) more abundant mesenchymal-like spermatogonia (THY1(+); moderate OCT4 and ID4; high mesenchymal markers), and (3) (in older juveniles) abundant spermatogonia committing to gametogenesis (high KIT(+)). Epithelial-like spermatogonia displayed the expected imprinting patterns, but, surprisingly, mesenchymal-like spermatogonia lacked imprinting specifically at paternally imprinted loci but fully restored imprinting prior to puberty. Furthermore, mesenchymal-like spermatogonia also displayed developmentally linked DNA demethylation at meiotic genes and also at certain monoallelic neural genes (e.g., protocadherins and olfactory receptors). We also reveal novel candidate receptor-ligand networks involving SSCs and the developing niche. Taken together, neonates/juveniles contain heterogeneous epithelial-like or mesenchymal-like spermatogonial populations, with the latter displaying extensive DNA methylation/chromatin dynamics. We speculate that this plasticity helps SSCs proliferate and migrate within the developing seminiferous tubule, with proper niche interaction and membrane attachment reverting mesenchymal-like spermatogonial subtype cells back to an epithelial-like state with normal imprinting profiles., (© 2015 Hammoud et al.; Published by Cold Spring Harbor Laboratory Press.)

Telomeres are ribonucleoprotein structures at the end of chromosomes composed of telomeric DNA, specific-binding proteins, and noncoding RNA (TERRA). Despite their importance in preventing chromosome instability, little is known about the cross talk between these three elements during the formation of the germ line. Here, we provide evidence that both TERRA and the telomerase enzymatic subunit (TERT) are components of telomeres in mammalian germ cells. We found that TERRA colocalizes with telomeres during mammalian meiosis and that its expression progressively increases during spermatogenesis until the beginning of spermiogenesis. While both TERRA levels and distribution would be regulated in a gender-specific manner, telomere-TERT colocalization appears to be regulated based on species-specific characteristics of the telomeric structure. Moreover, we found that TERT localization at telomeres is maintained throughout spermatogenesis as a structural component without affecting telomere elongation. Our results represent the first evidence of colocalization between telomerase and telomeres during mammalian gametogenesis., (© 2014 by the Society for the Study of Reproduction, Inc.)

The European clam, Ruditapes decussatus is a species with a high commercial importance in Portugal and other Southern European countries. Its production is almost exclusively based on natural recruitment, which is subject to high annual fluctuations. Increased knowledge of the natural reproductive cycle of R. decussatus and its molecular mechanisms would be particularly important in providing new highly valuable genomic information for better understanding the regulation of reproduction in this economically important aquaculture species. In this study, the transcriptomic bases of R. decussatus reproduction have been analysed using a custom oligonucleotide microarray representing 51,678 assembled contigs. Microarray analyses were performed in four gonadal maturation stages from two different Portuguese wild populations, characterized by different responses to spawning induction when used as progenitors in hatchery. A comparison between the two populations elucidated a specific pathway involved in the recognition signals and binding between the oocyte and components of the sperm plasma membrane. We suggest that this pathway can explain part of the differences in terms of spawning induction success between the two populations. In addition, sexes and reproductive stages were compared and a correlation between mRNA levels and gonadal area was investigated. The lists of differentially expressed genes revealed that sex explains most of the variance in gonadal gene expression. Additionally, genes like Foxl2, vitellogenin, condensing 2, mitotic apparatus protein p62, Cep57, sperm associated antigens 6, 16 and 17, motile sperm domain containing protein 2, sperm surface protein Sp17, sperm flagellar proteins 1 and 2 and dpy-30, were identified as being correlated with the gonad area and therefore supposedly with the number and/or the size of the gametes produced.

Cyclin-dependent kinases (cdks) are key regulators of the cell cycle. In mammals, cdk2 plays an essential role in the meiosis of spermatocytes and oocytes. To investigate the role of cdk2 kinase during gametogenesis in crustaceans, we cloned a complete cDNA sequence of cdk2 from the freshwater giant prawn, Macrobrachium rosenbergii, and examined its localization and expression in the developing gonads. The prawn cdk2 cDNA is 1,745 bp in length and encodes a putative protein of 305 amino acids. The deduced protein contains a conserved cyclin binding motif PSTAIRE and shares high homology with reported cdk2 kinases of other species. RT-PCR analysis showed a wide distribution of the cdk2 mRNA in all tested organs including the testis, ovary, heart, muscles, hepatopancreas and gills, and the highest level of expression in the ovary and testis. Localization by in situ hybridization of cdk2 mRNA in the ovary showed high expression in the ooplasm of previtellogenic and the nuclei of late vitellogenic oocytes. In testicular sections, cdk2 transcript is low in spermatogonia, high in spermatocytes, but reduced in spermatids and sperm. The high expression of the cdk2 transcripts in meiotic spermatocytes and oocytes indicated that the cdk2 gene has the conservative function in the germ cells meiosis during gametogenesis.

The small ubiquitin-like modifier (SUMO) pathway in eukaryotes is an essential post-translational modification required for a variety of cellular processes, development and organelle biogenesis. SUMO-conjugating enzyme (Ubc9) is an important conjunction enzyme in the SUMO pathway. SUMO-1 and Ubc9 have been found in vertebrates; however, their expression in crustaceans was poorly characterized. In this study, the SUMO-1 and Ubc9 genes were identified in the developing testis and ovary of Chinese mitten crab, Eriocheir sinensis, and designated EsSUMO-1 and EsUbc9, respectively. Quantitative real-time PCR demonstrated the expression level of both mRNAs varied significantly during testis and ovary development. In the testis, EsSUMO-1 and EsUbc9 were expressed at moderate levels in stage II-1, increased at stage II-2, and then gradually declined in stage IV. In the ovary, EsSUMO-1 and EsUbc9 expression were low in the early stage, reach the highest level at stage III-2, and then gradually decreased in stage IV. Transcripts from both genes were detected using in situ hybridization throughout the testis and ovary, in the spermatids and oocytes. The pattern of EsSUMO-1 and EsUbc9 expression in the testis and ovary suggests that SUMOylation may play an important role in spermatogenesis and oogenesis in E. sinensis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

VASA is one of the important regulatory factors that determine the development of the reproductive system. However, no information on vasa gene from Pleocyemata Brachyura is available. By using Race, we obtained a full-length cDNA of Sp-vasa of the green mud crab Scylla paramamosain. The full-length (2,851 bp) cDNA of vasa encodes a peptide of 631 amino acids. Real-time PCR results indicated that the expression level of Sp-vasa in the growth stage of ovary was higher than in the maturation stage, and in stage I and II of testis, the expression level of Sp-vasa were higher than in stage III. By using in situ hybridization, Sp-vasa RNAs were detected in the large part of oocyte plasm in stage I, nucleus zone in stage III and perinuclear zone in stage V. As the size of oocytes increases during oogenesis, the signals change from strong to weak. In addition, in stage I and II of testis, the expression levels of Sp-vasa were higher than in stage III, and the hybridization intensity of Sp-vasa gene gradually increased during spermatogenesis from spermatogonia to spermatids. However, no hybridization signal was detected in spermatozoon. Real-time PCR and in situ hybridization were consistent. These findings suggest that Sp-vasa is likely to serve as a useful and specific marker for germ cell development of S. paramamosain.

Microgametogenesis is a complex process that involves numerous well-coordinated cell activities, ending with the production of pollen grains. Pollen development has been studied at the cytological level in Arabidopsis and other plant species, where its temporal time course has been defined. However, the molecular mechanism underlying this process is still unclear, since a relative small number of genes and/or processes have been identified as essential for pollen development. We have designed a methodology to select candidate genes for functional analysis, based on transcriptomic data obtained from different stages of pollen development. From our analyses, we selected At2g22950 as a candidate gene; this gene encodes a protein belonging to the auto-regulated Ca(2+)-ATPase family, ACA7. Microarray data indicate that ACA7 is expressed exclusively in developing pollen grains, with the highest level of mRNA at the time of the second pollen mitosis. Our RT-PCR experiments showed that ACA7 mRNA is detected exclusively in developing flowers. Confocal microscopy experiments showed a plasma membrane localization for the recombinant GFP:ACA7 protein. We identified two different insertional mutant lines, aca7-1 and aca7-2; plants from both mutant lines displayed a normal vegetative development but showed large amounts of dead pollen grains in mature flowers assayed by Alexander's staining. Histological analysis indicated that abnormalities are detected after the first pollen mitosis and we found a strong correlation between ACA7 mRNA accumulation and the severity of the phenotype. Our results indicate that ACA7 is a plasma membrane protein that has an important role during pollen development, possibly through regulation of Ca(2+) homeostasis., (© Springer-Verlag 2011)

Small RNAs derived from repetitive sequences appear to play essential roles in mammalian gametogenesis and early development. In this study we focused on the short interspersed nucleotide element B1 (SINE/B1) small RNAs, which were zygotically expressed in pre-implantation mouse embryos; and we investigated whether the SINE/B1 small RNAs played an active role in gene silencing during early mouse development. The results indicated that the level of silencing activity involving the SINE/B1 small RNAs as mediators was significantly reduced in Dicer-knockdown mouse embryos. In addition, when the SINE/B1 small RNAs were mapped to a full-length SINE/B1 sequence, phase-distribution of the small RNAs appeared, suggesting possible enzymatic involvement. Therefore, our present study suggested that the zygotically expressed SINE/B1 small RNAs in pre-implantation mouse embryos contain active small RNAs, which were presumably processed by Dicer and involved in gene silencing.

Embryo development requires a series of cell fate decisions; cell lineages are established early during development and must be "remembered" through multiple cell divisions. It is increasingly evident that epigenetic marks, DNA methylation, histone modifications, and noncoding RNAs, have a critical role in this cell memory during development. During gametogenesis, epigenetic programming results in the production of spermatozoa and oocytes with distinctive chromatin. The goal of this article is to review what is known about the epigenetic marks in mature gametes and how these marks change during early embryo development. An understanding of the role of epigenetic programming during normal development will lay the basis for the elucidation of its role when development goes awry and the consequence is a birth defect., (Copyright © 2011 Wiley-Liss, Inc.)

A Foxl2 ortholog has been identified in a lophotrochozoa, the pacific oyster, which is a successive irregular hermaphrodite mollusc. Its cDNA has been called Cg-Foxl2 (Crassostrea gigas Foxl2) and the deduced protein sequence is 367aa long. This sequence contains the conserved domain Forkhead box and its gene is devoid of intron at least in the first 926 bp of the cDNA, as found for Foxl2 factors. Real time PCR and in situ hybridization have shown a gonadic male and female Cg-Foxl2 expression which increases during the adult gametogenetic cycle for both sexes, but with a significant increase occurring earlier in females than in males. In females this increase corresponds to the vitellogenetic stage. During development, a peak of Cg-DMl (a potential factor of the male gonadic differentiation) and Oyvlg (a germ cell marker) expression and a significant decrease of Cg-Foxl2 expression were observed after metamorphosis in 1-1.5-month-old spats, a period of development when primordial germ cells may differentiate into germinal stem cells during the first gonadic establishment.

Early seed development of sexually reproducing plants requires both maternal and paternal genomes but is prominently maternally influenced. A novel gametophytic maternal-effect mutant defective in early embryo and endosperm development, glauce (glc), has been isolated from a population of Arabidopsis Ds transposon insertion lines. The glc mutation results from a deletion at the Ds insertion site, and the molecular identity of GLC is not known. glc embryos can develop up to the globular stage in the absence of endosperm and glc central cells appear to be unfertilized. glc suppresses autonomous endosperm development observed in the fertilization-independent seed (fis) class mutants. glc is also epistatic to mea, one of the fis class mutants, in fertilized seeds, and is essential for the biparental embryonic expression of PHE1, a repressed downstream target of MEA. In addition, maternal GLC function is required for the paternal embryonic expression of the ribosome protein gene RPS5a and the AMP deaminase gene FAC1, both of which are essential for early embryo and endosperm development. These results indicate that factors derived from the female gametophyte activate a subset of the paternal genome of fertilized seeds.

The Deleted in azoospermia family consists of RNA-binding proteins Boule, Daz, and Daz-like (Dazl) that are expressed in the germline. Here, we report the cloning and expression of the medakafish (Oryzias latipes) dazl gene (odazl). Interestingly, although the predicted medaka Dazl protein (oDazl) contains a RRM motif and a DAZ repeat characteristic of its mammalian homologs, it lacks 80 aa at the C-terminus. By RT-PCR, RNA in situ hybridization, Western blotting and fluorescent immunohistochemistry using a rabbit anti-Dazl antibody (alphaDazl), we analyzed the expression patterns of odazl and its protein. The odazl transcript persists throughout embryogenesis and delineates with primordial germ cells. In adults, the expression of odazl RNA and its protein is restricted to germ cells of both the testis and ovary. We observed differential expression of RNA and protein at critical stages of gametogenesis. In the testis, the odazl RNA is low at premeiotic stages, abundant at meiotic stages, but absent in postmeiotic stages; whereas the oDazl protein is rich in premeiotic stages, reduced at meiotic stages, becomes barely detectable or absent in postmeiotic round spermatids or sperm, respectively. This is in sharp contrast to the human situation where the Dazl transcript and protein are present in mature spermatozoa. In the ovary, the odazl RNA and protein persist throughout oogenesis and also show differential expression at premeiotic, meiotic and postmeiotic stages. Thus, the odazl or its protein is a marker for germ cells during embryogenesis and at critical stages of gametogenesis in both sexes of medaka.

Using in situ hybridization on tissue sections with DIG antisense RNA probe, the distribution of zebrafish (Danio rerio) nanos1 mRNA during oogenesis and spermatogenesis was detected, and the expression and function of nanos 1 during zebrafish gametogenesis was preliminarily studied. The results were as follows: nanos 1 mRNA uniformly dispersed throughout the cytoplasm of oocytes at all stages. Strong expression of nanos1 mRNA was observed in oogonia and stage I, II oocytes, but the signal became weaker in later stage oocytes. Strong expression of nanos 1 mRNA was detected in spermatogonia and relatively weaker signal was found in primary spermatocytes but no nanos 1 mRNA expression was observed in spermatids. The results suggested that nanos 1 may play an important role in maintenance and functioning of the germline stem cell-oogonia and spermatogonia.

rck/p54 is a DEAD-box RNA helicase protein with ATP-dependent RNA-unwinding activity. Its ortholog is required for sexual reproduction in yeast and for oocyte survival and sperm fertility in Caenorhabditis elegans. In the current study, we investigated the expression of rck/p54 in mouse gametogenesis and early embryogenesis. Western blot analysis revealed that rck/p54 was highly expressed in both the ovary and testis. In the ovary, maturing oocytes strongly expressed rck/p54 in their cytoplasm. In contrast, in the testis, spermatogonia and primary spermatocytes highly expressed rck/p54 in their cytoplasm, but its expression decreased in the spermatids. Interestingly, rck/p54 was concentrated in the heads of spermatozoa; and then its expression gradually decreased as these cells matured along the epididymal duct. After fertilization, rck/p54 protein and its mRNA remained present in the pronucleus phase; and then their expression levels slightly but definitely decreased in morulae and blastocytes. The injection of a CMV-based rck/p54 expression vector into the pronuclei of fertilized eggs caused a delay in early embryogenesis. In generating RCK transgenic mice, the birth rate of the mice was significantly lower than those of other gene transgenic mice. These findings indicate that rck/p54 may play an important role in gametogenesis and early embryogenesis in mammals.

Imprinted genes are characterized by predominant expression from one parental allele and differential DNA methylation. Few imprinted genes have been found to acquire a methylation mark in the male germ line, however, and only one of these, H19, has been studied in detail. We examined methylation of the Rasgrf1 and Gtl2 differentially methylated regions (DMR) to determine whether methylation is erased in male germ cells at e12.5 and when the paternal allele acquires methylation. We also compared their methylation dynamics with those of H19 and the maternally methylated gene Snrpn. Our results show that methylation is erased on Rasgrf1, H19, and Snrpn at e12.5, but that Gtl2 retains substantial methylation at this stage. Erasure of methylation marks on Gtl2 appears to occur later in female germ cells to give the unmethylated profile seen in mature MII oocytes. In the male germ line, de novo methylation of Rasgrf1, Gtl2, and H19 occurs in parallel between e12.5 and e17.5, but the DMR are not completely methylated until the mature sperm stage, suggesting a methylation dynamic different from that of IAP, L1, and minor satellite sequences, which have been shown to become fully methylated by e17.5 in male germ cells. This study also indicates important differences between different imprinted DMR in timing and extent of methylation in the germ cells.

With the Plasmodium falciparum genome sequencing near completion, functional analysis of individual parasite genes has become the major task of the postgenomic era. Understanding the expression patterns of individual genes is the initial step toward this goal. In this report, we have examined gene expression during gametocytogenesis of the malaria parasite, P. falciparum, using a modified differential display (DD) method. The modifications of this method include adjusting the dNTP mix, using upstream primers with higher AT contents, and reducing the extension temperature of the polymerase chain reaction (PCR). With a combination of 16 arbitrary upstream primers and 3 one-base-anchored oligo(dT) primers, we have successfully cloned 80 unique cDNA tags from stage IV-V gametocytes. Further analysis by dot blots and semiquantitative reverse transcriptase-PCR showed that at least 49 cDNAs had induced or elevated levels of expression in gametocytes. These results indicate that this modified DD procedure is suitable for large-scale identification of developmentally regulated genes in the AT-rich Plasmodium genome., (Copyright 2001 Elsevier Science (USA).)

The salmon gonadotropin-releasing hormone (sGnRH) is the major form of GnRH decapeptide expressed in the salmonid brain and it acts as a gonadotropin releaser. In rainbow trout, sGnRH-1 and sGnRH-2 mRNA forms were found in brain and gonads. We analyzed the expression of both forms in trout gonads at different stages of gametogenesis. Northern blot demonstrated that sGnRH-2 mRNA was the major sGnRH form in testis and ovary. In testis but not in ovary, brain or pituitary, alternatively spliced sGnRH-2 transcripts which coded for prepro-sGnRH with a truncated GnRH-associated peptide due to a premature stop codon in retained intron 2 were detected. In testis, sGnRH mRNA was highly expressed before the onset of spermatogenesis, it disappeared at stage II and then increased progressively up to stage VI. In ovary, the expression of sGnRH was high in immature pre-vitellogenic fish and progressively decreased throughout vitellogenesis. At ovulation it reached its maximum and came down again after stripping. The decrease of sGnRH mRNA expression during the period of active spermatogonial proliferation in testis and increase during meiosis occurrence in testis and ovary suggest an anti-proliferative and meiosis-stimulating effect of sGnRH during rainbow trout gametogenesis.

Differences in DNA methylation distinguish the maternal and paternal alleles of many imprinted genes. Allele-specific methylation that is inherited from the gametes and maintained throughout development has been proposed as a candidate imprinting mark. To determine how methylation is established in the germline, we have analyzed the allelic methylation patterns of the maternally expressed, paternally methylated H19 gene during gametogenesis in the mouse embryo. We show here that both parental alleles are devoid of methylation in male and female mid-gestation embryonic germ cells, suggesting that methylation imprints are erased in the germ cells prior to this time. In addition, we demonstrate that the subsequent hypermethylation of the paternal and maternal alleles in the male germline occurs at different times. Although the paternal allele becomes hypermethylated during fetal stages, methylation of the maternal allele begins during perinatal stages and continues postnatally through the onset of meiosis. The differential acquisition of methylation on the parental H19 alleles during gametogenesis implies that the two unmethylated alleles can still be distinguished from each other. Thus, in the absence of DNA methylation, other epigenetic mechanism(s) appear to maintain parental identity at the H19 locus during male germ cell development.

H19 and Igf2 are expressed in a monoallelic fashion from the maternal and paternal chromosomes, respectively. A region upstream of H19 has been shown to regulate such imprinted expression of both genes in cis. We have taken advantage of a loxP/cre recombinase-based strategy to delete this region in mice in a conditional manner to determine the temporal requirement of the upstream region in initiating and maintaining the imprinted expression of H19 and Igf2. Analysis of allele-specific expression of H19 and Igf2 and DNA methylation at the H19 promoter demonstrates that this region controls the monoallelic expression of the two genes in different ways, suggesting that it harbors two functionally distinct regulatory elements. Continued presence of the region is required to silence maternal Igf2 in accordance with its proposed role as an insulator. However, it does not have a direct role in keeping the paternal H19 promoter silenced. Instead, on the paternal chromosome, the upstream element mediates epigenetic modifications of the H19 promoter region during development, leading to transcriptional silencing of H19. Thereafter, its presence is redundant for preventing transcription. Presently, this temporal requirement of the silencing element appears to be a unique cis activity in the mammalian system. However, it is likely that other cis-acting elements, positive and negative, have the ability to effect stable changes in the chromatin structure and are not constantly required to give signals to the transcriptional machinery.

The Caenorhabditis elegans germline is composed of mitotically dividing cells at the distal end that give rise to meiotic cells more proximally. Specification of the distal region as mitotic relies on induction by the somatic distal tip cell and the glp-1 signal transduction pathway. However, the genetic control over the transition from mitosis to meiosis is not understood. In this paper, we report the identification of a gene, gld-2, that has at least two functions in germline development. First, gld-2 is required for normal progression through meiotic prophase. Second, gld-2 promotes entry into meiosis from the mitotic cell cycle. With respect to this second function, gld-2 appears to be functionally redundant with a previously described gene, gld-1 (Francis, R., Barton, M. K., Kimble, J. and Schedl, T. (1995) Genetics 139, 579-606). Germ cells in gld-1(o) and gld-2 single mutants enter meiosis at the normal time, but germ cells in gld-2 gld-1(o) double mutants do not enter meiosis. Instead, the double mutant germline is mitotic throughout and forms a large tumor. We suggest that gld-1 and gld-2 define two independent regulatory pathways, each of which can be sufficient for entry into meiosis. Epistasis analyses show that gld-1 and gld-2 work downstream of the glp-1 signal transduction pathway. Therefore, we hypothesize that glp-1 promotes proliferation by inhibiting the meiosis-promoting functions of gld-1 and gld-2.

The autosomal homologs of the human Y-chromosomal DAZ gene (DAZH and Dazh in human and mouse, respectively) are strong candidate for Azoospermia factor and encode a testis specific RNA-binding proteins. We studied the expression pattern of the mouse Dazh during embryonic development by using Northern-blotting of developing gonads. In the mouse, we have detected 3.5 kb and 4.5 kb transcripts in male and female embryonic gonads at 12.5 dpc (days post coitum). During this period, the only germ cells present in the gonad are primordial germ cells. Dazh transcripts were not detected in embryonic gonads of mice that lack germ cells because of mutation in W gene, suggesting that expression is limited to germ-cells. In females, oogonia enter meiosis at 13.5-14.5 dpc: at this time Dazh transcription levels are similar to those of the male (when prospermatogonia are in the male gonad). Transcription levels decrease steadily after birth as the number of oocytes is depleted and is hardly detectable by puberty. A human DAZH transcript was also detected by Northern-blotting in the human ovary in levels which are of about 100 fold lower than those observed in the human testis. The expression of the Dazh in male and female gonad before germ cell sex differentiation suggests that these genes may act at the first phase of male and female gametogenesis.

Replacement-variant H3.3 histones have been isolated and sequenced in different eukaryotes, but no functional H3.3A gene has been characterized in the mouse so far. We have cloned an H3.3A cDNA from a mouse fetal ovary library, differentially screened with testis versus somatic cDNA probes. We showed this gene contains a region homologous to the reverse and complementary alpha-globin gene. We believe such a structure could have been generated by retroposition during the evolution of both globin and histone gene families. The sequence coding for H3.3A is 76.6% homologous to the mouse H3.3B gene at the nucleotide level and differs in only one amino acid at the protein level. The high degree of homology between these genes and the H3.3 variant histones from other eukaryotes reveals the conservation of these replication-independent class of histones throughout evolution. Analysis of gene expression reveals a developmental regulation concurrent with meiotic progression, with the highest level of transcript detection coincident with meiotic onset during both oogenesis and spermatogenesis.