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Crosses between the two North American rodent species Peromyscus polionotus (PO) and Peromyscus maniculatus (BW) yield parent-of-origin effects on both embryonic and placental growth. The two species are approximately the same size, but a female BW crossed with a male PO produces offspring that are smaller than either parent. In the reciprocal cross, the offspring are oversized and typically die before birth. Rare survivors are exclusively female, consistent with Haldane's rule, which states that in instances of hybrid sterility or inviability, the heterogametic sex tends to be more severely affected. To understand these sex- and parent-of-origin-specific patterns of overgrowth, we analysed reciprocal backcrosses. Our studies reveal that hybrid inviability is partially due to a maternally expressed X-linked PO locus and an imprinted paternally expressed autosomal BW locus. In addition, the hybrids display skewing of X-chromosome inactivation in favour of the expression of the BW X chromosome. The most severe overgrowth is accompanied by widespread relaxation of imprinting of mostly paternally expressed genes. Both genetic and epigenetic mechanisms underlie hybrid inviability in Peromyscus and hence have a role in the establishment and maintenance of reproductive isolation barriers in mammals.

Expansion of trinucleotide repeat tracts has been shown to be associated with numerous human diseases. The mechanism and timing of the expansion events are poorly understood, however. We show that CTG repeats, associated with the human DMPK gene and implanted in two homologous yeast artificial chromosomes (YACs), are very unstable. The instability is 6 to 10 times more pronounced in meiosis than during mitotic division. The influence of meiosis on instability is 4.4 times greater when the second YAC with a repeat tract is not present. Most of the changes we observed in trinucleotide repeat tracts are large contractions of 21 to 50 repeats. The orientation of the insert with the repeats has no effect on the frequency and distribution of the contractions. In our experiments, expansions were found almost exclusively during gametogenesis. Genetic analysis of segregating markers among meiotic progeny excluded unequal crossover as the mechanism for instability. These unique patterns have novel implications for possible mechanisms of repeat instability.

Genetic causes of infertility are probably not rare. Today only a fraction of genes directly or indirectly involved in reproduction including sex determination and differentiation are known. Nevertheless, the list of well-defined genetic disorders impairing fertility is impressing already today and growing rapidly. Gonosomal aneuploidy and structural rearrangements represent a significant portion of the genetic causes of infertility in both sexes. Other chromosomal conditions include autosomal balanced structural changes (e.g. translocations), probably due to pairing disturbances of the affected chromosomes during meiosis. Some fundamental mechanisms in sex determination and differentiation have been characterized in recent years. Mutations in some of the genes involved in this process may lead to familial infertility. Genetic defects in gametogenesis of both sexes are currently being investigated using mouse models. Male specific causes of infertility include microdeletion within the AZF region of the euchromatic part of the long arm of the Y chromosome and obstructive azoospermia due congenital aplasia of the vas deferens in the presence of mutations in the CFTR gene.

Interaction between germ cells and the supporting somatic cells guides many of the differentiative processes of gametogenesis. The expression pattern of the Pem homeobox gene suggests that it may mediate specific inductive events in murine reproductive tissues. During gestation, Pem is expressed in migrating and early postmigratory primordial germ cells, as well as in all embryo-derived extraembryonic membranes. Pem expression ceases in the germline after Embryonic Day 14 in both sexes and then reappears postnatally in the supporting cells of the gonad. In mature mice, Pem is produced by testicular Sertoli cells during stages VI-VIII of spermatogenesis and transiently by ovarian granulosa cells lining periovulatory follicles. Despite this tightly regulated reproductive expression pattern, mice with a targeted mutation in Pem have normal fecundity, with no detectable alteration in extraembryonic testicular or ovarian development or function. We also show that Pem is expressed throughout embryonic and adult development in a subset of a tissue-specific class of macrophages, Kupffer cells, as well as in a localized fraction of cells in macrophage cell lines. Although the number of Pem-positive Kupffer cells increases in mice treated with lipopolysaccharide, loss of Pem does not detectably interfere with the cells' ability to induce iNOS expression, demonstrating this Kupffer cell function does not require Pem. No differences were observed between Pem-knockout mice in 129, C57BL6/J, or mixed genetic backgrounds. Together, these data show that Pem is dispensable for embryonic and postnatal development, gonadal function, and Kupffer cell activation, perhaps due to compensatory expression of a similar homeobox gene., (Copyright 1998 Academic Press.)

Infertility is a common feature of the human disorder ataxia-telangiectasia and Atm-deficient mice are completely infertile. To gain further insight into the role of ATM in meiosis, we examined meiotic cells in Atm-deficient mice during development. Spermatocyte degeneration begins between postnatal days 8 and 16.5, soon after entry into prophase I of meiosis, while oocytes degenerate late in embryogenesis prior to dictyate arrest. Using electron microscopy and immunolocalization of meiotic proteins in mutant adult spermatocytes, we found that male and female gametogenesis is severely disrupted in Atm-deficient mice as early as leptonema of prophase I, resulting in apoptotic degeneration. A small number of mutant cells progress into later stages of meiosis, but no cells proceed beyond prophase I. ATR, a protein related to ATM, DMC1, a RAD51 family member, and RAD51 are mislocalized to chromatin and have reduced localization to developing synaptonemal complexes in spermatocytes from Atm-deficient mice, suggesting dysregulation of the orderly progression of meiotic events. ATM protein is normally present at high levels primarily in ova cytoplasm of developing ovarian follicles, and in the nucleus of spermatogonia and to a lesser extent in spermatoctyes, but without localization to the synaptonemal complex. We propose a model in which ATM acts to monitor meiosis by participation in the regulation or surveillance of meiotic progression, similar to its role as a monitor of mitotic cell cycle progression.

A long-term, quantitative analysis was conducted on the gametogenesis of autotriploid rainbow trout (Oncorhynchus mykiss) to quantify their degree of germline development and reproductive potential. Triploid and diploid (control) trout siblings were raised separately under identical conditions and sampled randomly for histological analysis. Triploid males underwent testicular development and proliferation of germ cells by mitosis and meiosis, progressing through initial phases of spermatogenesis at a similar pace to diploid controls. The effects of triploidy on males were most evident during the final stages of spermatogenesis, when all diploid males contained free spermatozoa in the lumen of most tubules (average relative frequency, ARF = 68.5%), whereas triploid males contained predominantly spermatocytes (ARF = 36.3%) and morphologically abnormal spermatozoa (ARF = 31.8%). In contrast, the gonadal development of triploid females was affected during its early stages; the major patterns observed were the arrest of the oogonia within oogonial clusters (ARF = 30.4-71.1%), the appearance of small numbers (ARF = 1.5-6.0%) of previtellogenic and early vitellogenic follicles, and the proliferation of non-follicular elements (vascular lacunae, fibrosis and tubular adenomas). In agreement with previous reports on the ovarian development of chromosomally female (3A:ZZW) triploid chickens, male-differentiating areas (ARF = 0.2-12.2%) were observed in most triploid females examined, which by the end of the sampling period appeared as gonadal hermaphrodites. It is hypothesized that the lack of proper somatic-to-germ cell interactions prevents the segregation of the oocytes from the gonial clusters and may explain the early blockage observed during the gonadal morphogenesis of autotriploid female rainbow trout.

We previously showed that a 383 bp (-274 to approximately +109) promoter of a pollen-specific gene, GBAN215-6, had a property of a late gene in pollen development in transgenic tobacco plants. It drove GUS gene expression from uninucleate microspores to pollen tube growth of trinucleated cells. To more precisely characterize the specificity of the promoter, we placed the diphtheria toxin A-chain (DTx-A) coding region under the control of the GBAN215-6 promoter. Transgenic tobacco plants containing the GBAN215-6/DTx-1 were phenotypically normal until an early stage of flowering. The dehisced anthers do not contain pollen grains and the filament length of stamen was shorter than that of normal plants. Microscopic examination showed that ablation of pollen by the expression of DTx-A was variable. The transgenic tobacco plants containing one copy of the DTx-A gene show 50% aborted and 50% normal pollen, which suggests that this gene acts gametophytically. However, most of the transgenic plants with high copy number were male-sterile. When these male-sterile tobacco plants were backcrossed as female with pollen from wild-type tobacco plants, the fruit capsule sizes and seed yields of the next generation (BC1 lines) were severely reduced and the segregation of male-sterile to fertile plants in BC1 seeds was not Mendelian.

As a strategy for the identification of T-DNA-tagged gametophytic mutants, we have used T-DNA insertional mutagenesis based on screening for distorted segregation ratios by antibiotic selection. Screening of approximately 1000 transgenic Arabidopsis families led to the isolation of eight lines showing reproducible segregation ratios of approximately 1:1, suggesting that these lines are putative gametophytic mutants caused by T-DNA insertion at a single locus. Genetic analysis of T-DNA transmission through reciprocal backcrosses with wild type showed severe reductions in genetic transmission of the T-DNA through the male and/or female gametes. Direct evidence for mutant phenotypes in these lines was investigated by DAPI staining of mature pollen grains and by the analysis of seed set and embryo sac morphology in cleared ovules. One line, termed limpet pollen, showed a novel pollen phenotype in that the generative cell failed to migrate inward after pollen mitosis I, such that the generative or sperm cells remained against the pollen wall. Two other lines, andarta and tistrya, were defective in female transmission and showed an early arrest of embryo sac development with the viable megaspore not initiating the nuclear division cycles. These data demonstrate the efficacy of a segregation ratio distortion strategy for the identification of T-DNA-tagged gametophytic mutants in Arabidopsis.

The actual and effective number of gametophytic self-incompatibility alleles maintained at mutation-drift-selection equilibrium in a finite population subdivided as in the island model is investigated by stochastic simulations. The existing theory founded by Wright predicts that for a given population size the number of alleles maintained increases monotonically with decreasing migration as is the case for neutral alleles. The simulation results here show that this is not true. At migration rates above Nm = 0.01-0.1, the actual and effective number of alleles is lower than for an undivided population with the same number of individuals, and, contrary to Wright's theoretical expectation, the number of alleles is not much higher than for an undivided population unless Nm < 0.001. The same pattern is observed in a model where the alleles display symmetrical overdominant selection. This broadens the applicability of the results to include proposed models for the major histocompatibility (MHC) loci. For a subdivided population over a large range of migration rates, it appears that the number of self-incompatibility alleles (or MHC-alleles) observed can provide a rough estimate of the total number of individuals in the population but it underestimates the neutral effective size of the subdivided population.

Male gametogenesis was cytologically analyzed in wheat lines homozygous or hemizygous for gametocidal (Gc) factors with different modes of action. The first and second meiotic divisions in all lines were cytologically normal. The postmeiotic mitoses were normal in the homozygous lines; however, chromosome fragments and bridges were observed in the mitoses of the hemizygous lines. The morphology of the chromosome fragments suggests that the Gc genes induce chromosome breaks in the G1 phase prior to DNA synthesis of the first postmeiotic mitosis. The age of an anther was correlated with the frequency of aberrant second mitosis. Younger anthers contained a higher number of pollen undergoing normal second mitosis. This observation suggests that the arresting of the cell cycle occurs as the result of chromosome breaks during the first mitosis. Because chromosome bridges were more frequent than fragments in the second mitosis, breakage-fusion-bridge cycles possibly occurred during gametogenesis, which led to further chromosomal rearrangements. The Gc factors located on chromosomes 2S of Aegilops speltoides and 4Ssh of Ae. sharonensis induce severe chromosome breakage in pollen lacking them. However, the Gc factor on telosome 2CcL of Ae. cylindrica only induced chromosome breaks at a low frequency. The observed partial fertility of Gc lines is presumably due to cell cycle arrest and the competition among gametes with and without chromosome breakage.

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.

NIMA protein kinase is a major regulator of progression into mitosis in Aspergillus nidulans. Dominant negative forms of NIMA protein prevent entrance into mitosis in HeLa cells, suggesting that mammals have a similar pathway. We have reported previously the isolation of a murine NIMA-related kinase, designated Nek1, and more recently several additional NIMA-related human kinases have been cloned. The existence of several mammalian NIMA-related genes raises the questions of whether the different mammalian members have redundant, overlapping or distinct functions, and whether these functions are related to the role of NIMA in controlling mitosis. To address these questions we have studied the expression patterns of the different murine nek genes. To this end, we isolated a murine nek2 cDNA and compared its patterns of expression, during both gametogenesis and embryogenesis, to those of nek1. Both genes were highly expressed in developing germ cells, albeit in distinct patterns. In both females and males, nek1 is expressed much earlier than nek2, suggesting only limited ability for functional redundancy. Surprisingly, a striking specificity of nek1 expression was found: high levels of nek1 RNA were observed in distinct regions of the nervous system, most notably in neurons of the peripheral ganglia. These patterns suggest that the different mammalian NIMA-related kinases participate in different phases of the meiotic process and may also have functions other than cell cycle control.

Caenorhabditis elegans hermaphrodites switch from making sperm to oocytes. This switch involves repression of fem-3 mRNA, mediated by a protein that binds RNA through a conserved motif; a similar motif mediates RNA binding by the Drosophila pattern-regulatory protein Pumilio.

The female gametophyte is an absolutely essential structure for angiosperm reproduction. It produces the egg cell and central cell (which give rise to the embryo and endosperm, respectively) and mediates several reproductive processes including pollen tube guidance, fertilization, the induction of seed development, and perhaps also maternal control of embryo development. Although much has been learned about these processes at the cytological level, specific molecules mediating and controlling megagametogenesis and female gametophyte function have not been identified. A genetic approach to the identification of such molecules has been initiated in Arabidopsis and maize. Although genetic analyses are still in their infancy, mutations affecting female gametophyte function and specific steps of megagametogenesis have already been identified. Large-scale genetic screens aimed at identifying mutants affecting every step of megagametogenesis and female gametophyte function are in progress; the characterization of genes identified in these screens should go a long way toward defining the molecules that are required for female gametophyte development and function.

In order to modify the early stages of pollen development in a transgenic context microspore-specific promoters are required. We tested two putatively microspore-specific promoters, the Bp4 promoter from rapeseed and the NTM19 promoter from tobacco. Expression of the gus and barnase reporter genes under the control of these two promoters was studied in transgenic tobacco. Contrary to expectations, the Bp4 promoter became active only after the first pollen mitosis, and not in the microspores. The NTM19 promoter turned out to be highly microspore-specific and directed very high levels of gus expression to the unicellular microspores. The NTM19-barnase transgene caused cell-autonomous death at the mid-unicellular microspore stage, whereas Bp4-barnase induced cell ablation of early to mid-bicellular pollen. Both promoter-barnase transgenes did not affect the sporophyte and were inherited through the female germline. These results show that both the NTM19 and Bp4 promoters are expressed only in the male germline, and that the NTM19 promoter is an excellent tool to direct high levels of transgene expression exclusively to the microspores. This may have important biotechnological applications.

Plants, in general, have a high proportion of their CpG and CpNpG nucleotide motifs modified with 5-methylcytosine (5mC). Developmental changes in the proportion of 5mC are evident in mammals, particularly during gametogenesis and embryogenesis, but little information is available from flowering plants due to the intimate association of gametes with sporophytic tissues. In ferns, sperm are uninucleate and free-swimming and thus are easily isolated. We have examined 5mC in DNA isolated from fern sperm and other tissues with methylation-sensitive and -insensitive restriction enzyme isoschizomers, Southern blots probed with chloroplast and nuclear ribosomal RNA genes and end-labeled restriction fragments. We conclude that fern sperm DNA is methylated to a similar or greater degree than DNA isolated from either sporophytes or gametophytes.

Ribosomal proteins contribute to the regulation and activity of ribosomes, and hence, the translational activity of the cell. Aberrant expression of ribosomal proteins has been linked to certain pathological conditions such as neoplasms. We have isolated and characterized a cDNA for the ribosomal protein (rp) S19 from a marine bivalve, the soft-shell clam (Mya arenaria), and we have examined its pattern of mRNA expression in the ovary and testis. The S19 cDNA contains a 450 nucleotide (nt) open reading frame (ORF), flanked by 89 nt and 26 nt of 5' and 3' untranslated regions, respectively. Probes synthesized from the S19 cDNA recognize a single transcript of approximately 550 nt in four different tissues. The predicted amino acid sequence from the ORF exhibits 58% identity with human and rat S19. Southern analysis of genomic DNA suggests that M. arenaria may have multiple copies of S19, a feature that is more similar to vertebrate than invertebrate rp genes. Expression of S19 mRNA in both ovary and testis was elevated throughout gametogenesis until after spawning, when a decrease in S19 message was observed. A comparison of S19 mRNA levels in post-spawn animals revealed a trend of elevated expression in ovaries and testes affected by a gonadal neoplasm, indicating that S19 may be a useful molecular marker for the pathological condition.

RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins containing RNA-binding motifs, and both have been described as candidate human spermatogenesis genes. Transmission of deletions from father to son has been observed in the case of DAZ, but neither gene family has been shown to be essential for spermatogenesis in human males. The DAZ/SPGY genes are particularly amenable to a knockout approach, as they are found on the Y chromosome in Old World primates and apes, but in other mammals, they are represented only by an autosomal gene, DAZLA, which is also present in Old World primates and apes. It has also been shown that a Dazla homologue is essential for spermatogenesis in Drosophila. Here we show that Dazla protein is cytoplasmic in male and female germ cells, unlike the nuclear RBM protein. Disruption of the Dazla gene leads to loss of germ cells and complete absence of gamete production, demonstrating that Dazla is essential for the differentiation of germ cells.

The Chlamydomonas reinhardtii mutant lrg2 exhibits a partial defect in the blue-light activated signal transduction chain that controls gametogenesis. By genomic complementation, a cosmid clone that corrects the lrg2 phenotype was isolated. The smallest fragment of this cosmid that gave complementation was used in RFLP analysis, which revealed that the cloned gene was not linked to the LRG2 locus. The sequence of the genomic clone and a cDNA of this gene, which we named LRG5, was determined. Although no significant homology with sequences in the databases was found, the nuclear localization signal and the presence of negatively and positively charged domains suggests a function for the LRG5 protein in the regulation of transcription. The correction of the lrg2 phenotype is probably caused by overexpression of the LRG5 gene in the transformants, as these expressed a level of LRG5 approximately twofold higher than wild-type or mutant strains. Southern blot analysis provided evidence for the existence of homologous sequences in other green algae as well as in higher plants, suggesting a conserved function for the LRG5 protein.

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.

Homozygosity mapping is a very powerful method for mapping rare recessive diseases in humans. In many applications, it is often desirable to compute prior (or unconditional) multilocus probability of autozygosity for inbred pedigrees. This paper proposes a simple yet powerful method for computing the prior multilocus autozygosity probability for complex inbred pedigrees. The method has an added feature of providing explicit multilocus autozygosity probability in terms of recombination fractions, if desired. An example is presented to illustrate the method.

Transcription of the early gametocyte-specific gene pfg27/25, and genomic structure and polymorphism of its upstream region were studied in the human malaria parasite Plasmodium falciparum. The upstream genomic sequence of the pfg27/25 gene is characterised by a repetitive region that contains five direct and one inverted repeats of a unit constituted by a perfectly conserved sequence flanked by a poly-dT and a poly-d(AT) tract. Sequences further upstream from the repetitive region are polymorphic in distantly related parasite lines. Nuclear 'run off' experiments indicated that transcription of pfg27/25 is developmentally regulated. Transcription of the gene, undetectable in asexual parasites, is activated at the onset of gametocytogenesis. Te gene is transcribed in a 2.5 kb mature mRNA for the first 2-3 days of sexual differentiation, while transcription is down-regulated in more mature gametocytes.

With few exceptions, the determination of unconditional probability of genes shared identical by descent (IBD) by relatives can be very difficult, especially if the relationship is complex or if multiple loci are involved. It is particularly difficult if one needs the IBD probability in a explicit form, expressed in terms of interlocus recombination fractions. In this paper, I will further extend the concept of gametogenesis process introduced elsewhere and indicate that it completely determines the gene IBD events of interest in pedigrees. I will demonstrate that the gametogenesis process not only serves as a convenient conceptual framework in considering IBD events in pedigrees but also provides a simple yet powerful tool to solve a wide range of seemingly difficult problems. In particular, I consider the problem of multilocus IBD probability for relative pairs, k siblings, and a group of pedigree members. In addition, I consider the problem of multilocus autozygosity probability and the problem of gene preservation in close relatives.

Isolation and initial characterization of clones from a mouse fetal ovary cDNA library are reported. In mammals, the development of female gametogenesis, including the prophase of the first meiotic division, begins in the fetus. A parallel conserved process occurs in males, from puberty onwards. We cloned cDNAs preferentially expressed in both sexes during gametogenesis considering both the meiotic programme and the non-germ cells participating in the developmental regulation of gametogenesis. From 183 clones that showed positive hybridization with adult testis and negligible hybridization with somatic probes, 32 clones were selected. After partial sequencing, 16 were found to correspond to known genes that are expressed during other programmes of differentiation and development. Sixteen clones represent novel sequences. The accumulation of corresponding transcripts was analysed by developmental northern blots during spermatogenesis, and genes expressed during gametogenesis were characterized together with others showing pleiotropic expression in different tissues. Additional specific, gametic transcripts were also detected.

Understanding the genetic program that controls the regulation of cells progressing through the mitotic and meiotic cell cycles, whether in response to growth factors, other extracellular signals, or intrinsic programs, is critical to problems in infertility and in gonadal neoplasias. In this study, we present a consideration of important stages of cell cycle control in mammalian germ cells and data illustrating the experimental approaches that can be used to determine the pattern of such genes which may be involved in these events.

In Arabidopsis thaliana, a mutation in the SIN 1 gene causes aberrant ovule development and female-specific sterility. The effect of the sin 1 mutation is polymorphic and pleiotropic in different genetic backgrounds. The polymorphism concerns morphology of the mutant ovules. The pleiotropism involves internodal distance and inflorescence initiation time. The particular ovule phenotype and the length of internodes are dependent on an interaction of sin 1 with a second recessive gene, which we term mod 1. The recessive mod 1 allele in a homozygous sin 1 mutant plant reduces internode length and ovule integument size. The mutation sin 1, but not mod 1, has a demonstrable effect on ovule morphology when acting independently. In our crosses mod 1 was inseparably linked to the well known mutation erecta that is known to cause a reduction in internode and pedicle lengths.

The c-mos proto-oncogene encodes a 37-39K cytoplasmic serine/threonine kinase implicated in the meiotic maturation events during murine spermatogenesis and oogenesis. In Xenopus, ectopic expression of pp39mos can promote both the meiotic maturation of oocytes and also arrest the cleavage of blastomeres. To elucidate the role of pp39mos we have generated homozygous mutant mice by gene targeting in embryonic stem cells. These mice are viable and mutant males are fertile, demonstrating that pp39mos is not essential for spermatogenesis. In contrast, mutant females, have a reduced fertility because of the failure of mature eggs to arrest during meiosis. c-mos-/- oocytes undergo germinal vesicle breakdown and extrusion of both polar bodies followed in some cases by progression into cleavage. Mutant females also develop ovarian cysts. These results demonstrate that a major role for pp39mos is to prevent the spontaneous parthenogenetic activation of unfertilized eggs.

Previous studies were focussed on the attempt to correlate observable variations in the size of Plasmodium berghei chromosomes with the loss of ability to produce viable gametocytes. A temporal coincidence between the appearance of a subtelomeric deletion on P. berghei chromosome 5 and the loss of the ability to produce viable gametocytes was observed in a clone (HPE) directly derived from the high gametocyte-producer clone 8417 during mechanical passages. Interestingly enough, three P. berghei sexual-specific genes have already been mapped on internal fragments of this chromosome. A novel gene, clone 150, isolated from a genomic library of clone 8417 using a probe enriched for sexual-specific transcripts, maps on chromosome 5 within 100kb from the telomere. Subtelomeric deletions of chromosome 5 affecting two non-producer clones involve part of the transcribed region of this gene.

The maize cell cycle regulation mutant polymitotic (po) progresses through abnormal cell cycles, characterized by premature cell divisions without chromosome duplication of the daughter cells produced by meiosis during microsporogenesis and macrosporogenesis. There are three recessive alleles of the Po gene; po, ms4, and ms6. A new method of permeabilizing cells based on freeze-fracture technology was used to study the distribution of microtubules in wild-type and mutant microspores. Here we show that an abnormal distribution of microtubules is correlated with changes in chromosome morphology in a cell cycle-dependent manner in po, ms4 and ms6 mutant alleles. After meiosis II, the cell cycle is complete and becomes progressively less synchronous in po homozygotes compared with wild-type cells. During microsporogenesis, the distribution of microtubules is abnormal, and chromosome morphology is altered in both po, ms4 and ms6 mutants. However, more chromosome fragments or micronuclei associated with minispindles are present in ms6 than po and ms4. After microspores are released from the tetrads, disruptions in structure and organization of chromosomes and microtubules continues in subsequent abnormal cell cycles. However, these cell cycles are incomplete since phragmoplasts are not formed. During these incomplete cell cycles, abnormal spindles and microtubule arrays are induced and extra microtubule arrays are associated with irregularly distributed chromosome fragments. States corresponding to interphase, prophase, metaphase and anaphase can be recognized in the mutant microspores. Abnormal cell cycles also occur after female meiosis during ms4 macrospore development. Since only the cell that normally undergoes embryo sac development (the chazal-most cell) undergoes supernumerary divisions this suggests that the po phenotype can be characterized as premature haploid divisions rather than repetition of meiosis II.

Four genes specifically expressed during gametogenesis of Chlamydomonas reinhardtii have been cloned and their expression patterns analyzed. mRNAs encoded by these gamete-specific genes (gas) were absent or present only at very low levels in vegetative cells and mature zygotes. In young zygotes 2 h after gamete fusion, the mRNAs of three gas genes still persisted. The gas mRNAs accumulated during gametic differentiation. The temporal patterns of accumulation of individual mRNAs differed; some started to increase early during gametogenesis, others accumulated in the late phase. The accumulation of one of the late mRNAs (gas28) was strictly light-dependent. To illustrate the utility of the genes cloned in the analysis of sexual differentiation in Chlamydomonas reinhardtii we show that in a gametogenesis-defective mutant, the expression of late genes is prevented while that of early genes is normal.

The c-kit receptor tyrosine kinase belongs to the PDGF/CSF-1/c-kit receptor subfamily. The kit-ligand, KL, also called steel factor, is synthesized from two alternatively spliced mRNAs as transmembrane proteins that can either be proteolytically cleaved to produce soluble forms of KL or can function as cell-associated molecules. The c-kit receptor kinase and KL are encoded at the white spotting (W) and steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl locus cause deficiencies in gametogenesis, melanogenesis and hematopoiesis. The c-kit receptor is expressed in the cellular targets of W and Sl mutations, while KL is expressed in their microenvironment. In melanogenesis, c-kit is expressed in melanoblasts from the time they leave the neural crest and expression continues during embryonic development and in the melanocytes of postnatal animals. In gametogenesis c-kit is expressed in primordial germ cells, in spermatogonia, and in primordial and growing oocytes, implying a role at three distinct stages of gametogenesis. Many mutant alleles are known at W and Sl loci and their phenotypes vary in the degree of severity in the different cellular targets of the mutations. While many W and Sl alleles severely affect primordial germ cells (PGC), several mild Sl alleles have weak effects on PGCs and exhibit differential male or female sterility. Steel Panda (Sl(pan)) is a KL expression mutation in which KL RNA transcript levels are reduced in most tissues analyzed. In female Sl(pan)/Sl(pan) mice, ovarian follicle development is arrested at the one layered cuboidal stage as a result of reduced KL expression in follicle cells, indicating a role for c-kit in oocyte growth. Wsh is a c-kit expression mutation, which affects mast cells and melanogenesis. While the mast cell defect results from lack of c-kit expression, the pigmentation deficiency appears to stem from ectopic c-kit receptor expression in the somitic dermatome at the time of migration of melanoblasts from the neural crest to the periphery. It is proposed that the ectopic c-kit expression in Wsh mice affects early melanogenesis in a dominant fashion. The "sash" or white belt of Wsh/+ animals and some other mutant mice is explained by the varying density of melanoblasts along the body axis of wild-type embryos.

The small (18-kDa) heat shock proteins (hsps) of maize are encoded by a complex multigene family. In a previous report, we described the genetic information from cDNAs encoding two different members of the family. In this communication, we report the isolation and characterization of cDNA and genomic clones encoding information for a third member of this hsp family (c/gMHSP18-1). DNA fragments containing nucleotide sequences common to, or specific for, each of these characterized 18-kDa genes were prepared and used as probes to assess the expression of these genes during microsporogenesis and development of the gametophyte in an inbred line of maize (Oh43). Our results demonstrate (1) that mRNA transcripts encoding the 18-kDa hsps are expressed and/or accumulate during microsporogenesis, and (2) that genes encoding two of the characterized 18-kDa hsps are expressed and/or accumulate independently, in a stage-specific manner during microsporogenesis. These observations imply that the stage-specific expression of particular 18-kDa hsp genes results from gene-specific regulation during microsporogenesis and gametophyte development rather than from an overall activation of the heat shock or stress response.

Genomic and cDNA clones of the anther-specific APG gene from Arabidopsis thaliana and Brassica napus, which encodes a novel proline-rich protein, were isolated and characterized. Southern blotting and Northern analysis of male fertile and cytoplasmic male sterile varieties of B. napus showed that the APG gene is present as a single copy in the Arabidopsis genome, and that the B. napus APG gene is a member of a small anther-specific gene family. Analysis of developmentally staged B. napus flower buds indicated that APG transcript is confined to the anther during the period of microspore development. Reporter gene fusions established that the APG promoter directs expression in a number of cell types in anthers of transformed plants. This expression is consistent with the temporal pattern of mRNA accumulation in B. napus buds and follows a complex developmental pattern. Most significantly, the promoter is active in both sporophytic and gametophytic cell types, with activity of the transgene in each cell type being delineated by various cytological markers.

The methylation status of a mouse metallothionein-I/human transthyretin fusion gene was studied during gametogenesis in transgenic mice. In the adult tissues of this mouse line, the promoter region of the transgene on chromosome 11 is methylated when it is maternally inherited and undermethylated when it is paternally inherited. Germ cells from various developmental stages of gametogenesis were isolated, and their DNAs were assayed using methylation-sensitive restriction endonucleases and the polymerase chain reaction. Only low to nonexistent levels of transgene methylation were detected in germ cells from 14.5-day-old male and female fetuses irrespective of the parental origin of the transgene. This undermethylated state persisted in oocytes from newborn females as well as in testicular spermatogenic cells and sperm. By contrast, the transgene promoter was completely methylated in fully grown oocytes arrested at the first meiotic prophase. The endogenous metallothionein-I gene promoter, located on a different chromosome, remained undermethylated at all stages examined, consistent with previous findings reported for a typical CpG island. Taken together, the results suggest that parental-specific adult patterns of transgene methylation are established during gametogenesis.

The c-fos proto-oncogene has been implicated as a central regulatory component of the nuclear response to mitogens and other extracellular stimuli. Embryonic stem cells targeted at the c-fos locus have been used to generate chimeric mice that have transmitted the mutated allele through the germline. Homozygous mutants show reduced placental and fetal weights and significant loss of viability at birth. Approximately 40% of the homozygous mutants survive and grow at normal rates until severe osteopetrosis, characterized by foreshortening of the long bones, ossification of the marrow space, and absence of tooth eruption, begins to develop at approximately 11 days. Among other abnormalities, these mice show delayed or absent gametogenesis, lymphopenia, and altered behavior. Despite these defects, many live as long as their wild-type or heterozygous littermates (currently 7 months). These data indicate that c-fos is not required for the growth of most cell types but is involved in the development and function of several distinct tissues.

Fertility of interspecific hybrids between Oryzias latipes and O. curvinotus was examined. F1 females were able to lay eggs but males were sterile. Histological examination of the ovaries of hybrids revealed that oogenesis does not proceed normally in spite of the apparent fertility. Most oocytes degenerated at the pachytene stage of the meiotic prophase, and only a few entered the diplotene stage to develop into ova. Hybrid males could induce females to spawn eggs, an indication that they had differentiated completely into true males. However, they did not produce fertile sperm. Most germ cells in testes of hybrids passes through almost the entire process of spermatogenesis, but deviations from the normal course of events were observed during spermiogenesis. The condensation of chromatin in spermatids occurred, but the diameters of sperm heads were about 1.5-fold larger than those of normal ones. Prominent abnormalities were apparent in the quantity and arrangement of microtubules in the cytoplasm. Abnormal spermatozoa were phagocytized by Sertoli cells. These observations indicate that the mechanisms of impaired gametogenesis in these interspecific hybrids are sexually differentiated.

Germinal mosaicism is a major problem in risk estimation for an X-linked disease. A mutation can happen anytime in germ cell development, and the proportion of germ cells bearing the mutated gene is twice the probability of recurrence of the mutation. This proportion could be either very low in late mutations or very high in germinal and somatic mosaicism. When this heterogeneity is taken into consideration, the distribution of the recurrence risk is conveniently represented as a set of discrete classes that may be derived either from models of gametogenesis or from empirical data. A computer program taking into account germinal mosaicism has been devised to calculate the probability of a possible carrier belonging to any of these classes, in order to settle the origin of the mutation of a given family. Germinal mosaicism increases the probability of inheriting the mutation, but this effect is always lowered by the possibility of heterogeneity. When the mother of a possible carrier is not herself a carrier, the risk of her daughter being a carrier is approximately halved, even under the assumption of a high recurrence risk from mosaicism.

Man appears to be unique among mammals with respect to very high level of reproductive wastage. It seems reasonable to suppose that many early losses attribute to cytogenetical abnormalities. However, this stage of pregnancy is difficult to be studied. The investigation of gametes is one of the most reliable ways to estimate the cytogenetical disorders in early life. The results from cytogenetical analysis of human male gametes are summarized.

Oncogenes are identified functionally by their ability to induce neoplastic transformation of susceptible cells. The first oncogenes to be characterized were isolated from acutely transforming retroviruses. Subsequently, it was determined that the retroviral oncogenes were formed from normal, progenitor genes. These cellular homologs of the viral oncogenes are termed proto-oncogenes. The derivation of oncogenes from proto-oncogenes is the consequence of mutations that remove regulatory constraints from the proto-oncogene. The ability of oncogenes to induce transformation implies that proto-oncogenes may function in growth and differentiation pathways in normal cells. Although many proto-oncogenes have been defined, the normal physiological function of most is not known. Studies of proto-oncogene expression during normal gametogenesis have determined that some genes are expressed in a stage-specific manner. The use of germ cells to provide homogeneous and defined normal cell populations facilitates identifying the roles proto-oncogenes have in regulating cell growth and differentiation.

We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in vivo asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in vivo. Gametocyte production and karyotype pattern were established at regular intervals during prolonged periods of asexual multiplication of clone 8417 of P. berghei. We found that karyotype mutants and mutants which do not produce gametocytes can replace the original high-producer parasites of clone 8417 within several weeks. The time at which mutants became predominant in the population in different experiments, however, differed greatly. Mutants with intermediate or low gametocyte production were not found. In experimentally mixed infections, containing parasites from two clones from different strains (clone 8417 of the ANKA strain; clone 1 of the K173 strain), high-producer parasites of clone 8417 were overgrown by parasites of the nonproducer clone. Nonproducer mutants from the originally high-producer clone 8417, however, were able to coexist with parasites of the nonproducer clone. These results demonstrate that in our experiments nonproducer parasites had a strong selective advantage during asexual multiplication compared to high producers. All karyotype mutants which became predominant in our experiments were nonproducers. In two experiments a change in karyotype coincided with the loss of gametocyte production which may suggest a causal relationship between these events.

Pairing failure at meiosis has been postulated as a cause of gametogenic arrest in both heterozygous translocation carriers and males whose spermatocytes exhibit univalent X and Y chromosomes. The present investigation is a survey of pachytene translocation configurations, at the electron microscopic level, in six stocks of mice, comprising a total of 464 spermatocytes and 343 oocytes. Univalence of the X and Y chromosomes was studied in the same stocks, as well as in three additional homozygous translocation stocks. Fully paired as well as asynaptic configurations were found in all translocation stocks, and the proportions of each configuration differed considerably between spermatocytes and oocytes of mice carrying the same translocation. In both spermatocytes and oocytes, other pairing anomalies were more frequent in cells with asynapsed than with fully synapsed configurations, and spermatocytes with univalent sex chromosomes had a higher proportion of autosomal anomalies than did spermatocytes with XY bivalents. It is concluded that pairing failure at meiosis is primarily a symptom, rather than a cause, of gametogenic arrest, and that chromosome rearrangements, even if they appear to be balanced, may affect the rate of atresia by interfering with the normal rate of meiotic progression. Once pairing failure is established, it could secondarily increase the probability of gametogenic failure.

The female germ line (germ cell lineage, Keimbahn) is provided with only one proliferation wave, the oogenic, whereas male gametogenesis involves two successive waves: prespermatogenic, which corresponds to the female proliferation wave, and spermatogenesis, which is responsible for the immense number of male gametes produced in mature testes. Both male proliferation systems are linked by the transitional or T prospermatogonia. Using the reverse percentage of labelled metaphases method, it has been shown that the first differences between female and male germ cells can be identified by the end of the first wave, when oogonia and multiplying or M prospermatogonia are proliferating. This prenatal first wave of proliferation of male germ cells was also demonstrated in man and ceases around the 22nd week of pregnancy. Spermatogenesis involves a stock of stem cells (stem spermatogonia), a flexibly reacting pool of undifferentiated spermatogonia and several generations of differentiating spermatogonia, which proliferate almost exponentially. Furthermore, it consists of spermatocytes and haploid spermatids transforming into spermatozoa. The oocytes pass through the preleptotene stage, synthesizing DNA, and thereafter traverse the meiotic prophase up to the diplotene stage. In mammals they act as 'pre-embryos' in a similar but to a lesser degree than oocytes of amphibia and insects. The maternal chromosomes are largely responsible for the development of the embryo, the paternal genome for the development of the extra-embryonic tissue. The synthesis of transgenic animals is a powerful weapon in the armoury of geneticists, as has recently been demonstrated: a 14 kb genomic DNA fragment (Sry) is sufficient to induce testis differentiation and subsequent male development when introduced into chromosomally female mouse embryos.

Reproduction in plants rivals the complexity of the process in animals. Recently, several genes that are expressed at specific stages during male gametogenesis have been cloned. These anther-specific genes are providing tools with which to study the cis- and trans-acting factors that regulate gene expression during pollen formation. Sequence analysis of the coding regions of some of these abundantly expressed anther genes is providing unexpected insights into the cell-cell interactions occurring during gamete formation and fertilization.

Genomic imprinting is a regulatory process that requires a cell to recognize the parental origin of alleles. To understand how these alleles are distinguished, we have assessed changes in the DNA methylation of an imprinted transgene as it switches from one inheritance pattern to another while moving through gametogenesis and embryogenesis. We find that both maternally and paternally inherited methylation patterns are erased in primordial germ cells and that distinctive patterns emerge during germ cell maturation. In the case of the maternal allele, the methylation pattern is fully acquired during oogenesis. In the case of the paternal allele, the methylation pattern found in sperm undergoes further modification during embryogenesis. Thus, the distinction between "erased" maternal and paternal alleles is first established during their residence in different germ cells and then may be maintained by the recognition of the distinctive patterns that each allele displays in the zygote.

The gene encoding the gametocyte specific cytoplasmic protein Pfg27/25 of the human malaria parasite Plasmodium falciparum has been cloned. The gene encodes a highly hydrophilic non-repetitive protein which does not share obvious homologies with other polypeptides. The stage specificity of Pfg27/25 is controlled at the stage of the production of stable mRNA, which is detectable only in the sexual stages of the parasite, and contains long additional sequences outside the Pfg27/25 coding region. As the activation of Pfg27/25 gene expression occurs at an early stage of gametocytogenesis, the study of its regulation might provide information on the molecular events occurring after the parasite commitment to sexual differentiation and at the beginning of gametocyte formation.