Zygote Maturation Research Articles

Rapid Selective Proliferation of Mitochondria during Zygote Maturation in the Uniparental Inheritance of <i>Physarum polycephalum</i>

Mitochondrial DNA (mtDNA) is uniparentally inherited in most sexual eukaryotes called maternal/uniparental inheritance. It is well known that selective elimination of paternal mitochondria and digestion of paternal mtDNA are important to this process. However, little is known about the control of maternal mitochondrial proliferation. In the isogamous Physarum polycephalum, haploid myxamoebae act as gametes. When two myxamoebal strains with different mating types are mated, mtDNA is inherited from only one strain, defined as the maternal strain. Here, we developed a method to distinguish uniparental mitochondria in zygotes of P. polycephalum by chemical staining and investigated the proliferation of maternal and paternal mitochondria during the selective digestion of mitochondrial nucleoids (mt-nucleoids) in paternal mitochondria. We stained the myxamoebae with MitoTracker Red CMXRos (MTR) or MitoTracker Green FM (MTG). MTG-stained mitochondria more specifically than MTR. When the AI35 strain was crossed with the MTG-stained DP246 strain and mtDNA was stained with N-aryl pyrido cyanine 3, mt-nucleoids in mitochondria derived from DP246 became shorter in the zygotes 3 h after crossing. There was no significant difference in the number of mitochondria derived from both AI35 and DP246 in early zygotes, but the number of mitochondria derived from AI35 proliferated 1.7-fold from 3 to 5 h after crossing. Similar results were obtained when MTG-stained AI35 crossed with DP246. These results indicate that the proliferation of mitochondria derived from AI35 rapidly occurred after the initiation of digestion of mt-nucleoids derived from DP246. Our results suggest the possibility of controlling the mechanism of the selective proliferation of maternal mitochondria during zygote formation for uniparental inheritance.

Produção in vitro de embriões ovinos

In vitro embryo production (IVP) comprises oocyte maturation, sperm capacitation, oocyte fertilization and culture of zygotes up to morula or blastocyst stag...

수염녹두말속(Chlamydomonas) 단세포 녹조의 유성생식

이 논문은 다세포 녹색식물의 조상으로 생각되는 단세포인 수염녹두말속(Chlamydomonas) 녹조의 복잡한 유성생식 과정을 종합적으로 이해하기 위해서 기술되었다. 모델생물로 알려진 클래미도모나스 레인하르티(Chlamydomonas reinhardtii)의 유성생식 생활사는 배우자발생, 배우자 활성화, 세포융합, 접합자 성숙, 감수분열과 발아 등의 다섯 시기로 구별된다. 배우자발생은 서식지에서 질소가 결핍되어 시작된다. 그 결과, 교배형 유전자자리(<TEX>$MT^+$</TEX> 또는 <TEX>$MT^-$</TEX>)에 의해 조절된 두 가지 교배형(<TEX>$mt^+$</TEX> 및 <TEX>$mt^-$</TEX>)의 배우자들이 발생된다. 유성생식을 위한 두 번째 자극원인 청색광이 교배능력을 갖고 있지 않는 예비배우자 세포에 조사되면 교배능력을 가진 배우자로 분화된다. 배우자발생의 초기에, 두 배우자 세포들은 응집소(agglutin) 분자들을 합성한다. 즉, <TEX>$mt^+$</TEX> 응집소 및 <TEX>$mt^-$</TEX> 응집소는 상염색체에 있는 성응집(SAG1) 및 성접착(SAD1) 유전자들이 발현되어 각각 합성된다. 응집소들에 의해서 두 배우자 세포들의 편모가 접착되면 cAMP가 신호전달 경로를 작동시켜서 편모의 끝 부부을 활성화시킨다. cAMP의 신호에 반응하여 두 배우자들의 세포벽이 벗겨지고 2개의 편모 사이에서 각각의 교배구조(mating structure)가 발달한다. 교배구조의 원형질막에는 <TEX>$mt^+$</TEX> 및 <TEX>$mt^-$</TEX> 배우자-특이 융합 단백질인 Fus1 및 Hap2/Gcs1이 있다. 이 단백질들의 상호작용으로 두 교배구조가 접촉되면, 두 배우자 사이에 세포질이 연결되어 수정세관(fertilization tubule)이 발달한다. <TEX>$mt^+$</TEX> 및 <TEX>$mt^-$</TEX> 배우자들의 핵과 엽록체가 융합되면 2배체의 접합자가 형성된다. 그 후 배우자들의 특성은 사라지며 융합단백질들(Fus1 및 Hap2)이 분해되고 접합자-특이(zygote-specific) 유전자들이 활성화 된다. 접합자는 24시간에 걸쳐 두꺼운 세포벽을 가진 접합포자(zygospore)로 발달한다. 어린 접합자에서 교배 후 60분 이내에 <TEX>$mt^-$</TEX> 엽록체 DNA와 <TEX>$mt^+$</TEX> 미토콘드리아 DNA가 선택적으로 제거된다. 따라서 이 두 소기관들은 각각 <TEX>$mt^-$</TEX> 미토콘드리아 DNA와 <TEX>$mt^+$</TEX> 엽록체 DNA만 남아서 단친유전(uniparental inheritance)이 이루어진다. 적당한 조건이 되면 접합포자가 감수분열과 발아 과정을 거쳐 반수체 영양세포가 방출되어 새로운 세대가 다시 시작된다. The sexual reproduction of the unicellular green alga Chlamydomonas is reviewed for a comprehensive understanding of the complex processes. The sexual life cycle of C. reinhardtii is distinguished into five main stages: gametogenesis, gamete activation, cell fusion, zygote maturation, and meiosis and germination. Gametogenesis is induced by nitrogen starvation in the environment. C. reinhardtii has two mating types: mating type plus (<TEX>$mt^+$</TEX>) and mating type minus (<TEX>$mt^-$</TEX>), controlled by a single complex mating type locus (<TEX>$MT^+$</TEX> or <TEX>$MT^-$</TEX>) on linkage group VI. In the early gametogenesis agglutinins are synthesized. The <TEX>$mt^+$</TEX> and <TEX>$mt^-$</TEX> agglutinins are encoded by the autosomal genes SAG1 (Sexual AGglutination1) and SAD1 (Sexual ADhesion1), respectively. The agglutinins are responsible for the flagellar adhesion of the two mating type of gametes. The flagellar adhesion initiates a cAMP mediated signal transduction pathways and activates the flagellar tips. In response to the cAMP signal, mating structures between two flagella are activated. The <TEX>$mt^+$</TEX> and <TEX>$mt^-$</TEX> gamete-specific fusion proteins, Fus1 and Hap2/Gcs1, are present on the plasma membrane of the two mating structures. Contact of the two mating structures leads to develop a fertilization tubule forming a cytoplasmic bridge between the two gametes. Upon fusion of nuclei and chloroplasts of <TEX>$mt^+$</TEX> and <TEX>$mt^-$</TEX> cells, the zygotes become zygospores. It is notable that the young zygote shows uniparental inheritance of chloroplast DNA from the <TEX>$mt^+$</TEX> parent and mitochondrial DNA from the <TEX>$mt^-$</TEX> parent. Under the favorable conditions, the zygospores divide meiotically and germinate and then new haploid progenies, vegetative cells, are released.

5′-methylcytosine and 5′-hydroxymethylcytosine Each Provide Epigenetic Information to the Mouse Zygote

Covalent modification of cytosine nucleotides within the genome encode essential epigenetic information, with methylation (5meC) and hydroxymethylation (5hmC) having received most attention. It has been proposed that the formation of 5hmC is an intermediate in the active demethylation of 5meC. Some reports show that global loss of 5meC in the newly fertilised embryo is accompanied by increased 5hmC, but others have failed to confirm this finding. These analyses have relied on immuno-localization of these modifications. In this study we have established the conditions required for equilibrium binding of antibodies to 5meC and 5hmC in zygotes. Simultaneous detection of these antigens required denaturation of chromatin by acid treatment followed by antigen retrieval by tryptic digestion. Equilibrium binding then required incubation at 4°C for greater than 6 h. These are more demanding conditions than generally reported and resulted in the consistent detection of 5meC and 5hmC in both male and female pronuclei throughout zygotic maturation. No dynamic reciprocal change in the level of 5meC relative to 5hmC was observed. Both 5meC and 5hmC accumulated within the peri-nucleolar regions and this was more pronounced in the male pronucleus. Staining of 5meC was relatively more intense within the cortical and 5hmC in the central regions of pronuclei. The results are not consistent with a role for 5hmC in global demethylation in the zygote. The persistence of both modifications throughout zygotic maturation, and their differing patterns of localization and solvent exposure infer each modification provides its own epigenetic information to the early embryo.

Methylation and hydroxymethylation of CpG display dynamic landscapes in early embryo development and define differentiation into embryonic and placental lineages

Covalent modifications to cytosine provide important epigenetic information required for normal embryo development. 5’methylcytosine (5mC) has received most experimental attention while 5’hydroxymethyl cytosine (5hmC) has assumed recent prominence. Extant gold-standard methods of analysis of do not discriminate between 5mC and 5hmC hence most published methylomes have limits to their interpretation. Immunolocalization allows discrimination between the range of modifications, provides a genome-wide level of analysis, allows differential assessment of nuclear localisation, and is compatible with the limited DNA available within the early embryo. Using newly established methodology for full retrieval of these two antigens [1] we have reassessed the patterns of expression of 5mC and 5hmC across preimplantation development. Both the male and the female pronuclei show extensive and relatively stable staining of both 5mC and 5hmC across all stages of zygotic maturation. The analysis does not provide support for the oft claimed active global demethylation of the paternally-derived genome relative to that of the maternal-derived genome, and also provide no support for a role of 5hmC as an intermediate in such an active demethylation step. During the cleavage stage of development (2-cell to 8-cell) mC became progressively more associated with heterochromatic regions of the nucleus while 5hmC staining was the dominant modification in euchromatin. By the morulae stage several cells with inner positions in the embryo showed some reduced euchromatic staining of 5hmC and 5mC. Relative to trophectodermal cells, the pluripotent inner cell mass of blastocysts show a marked overall reduction in euchromatic of both 5hmC and 5mC staining, and a progressive reduction of mC staining from heterochromatic foci. The distinctively different landscapes of 5mC and 5hmC on the embryonic genome indicate that these modifications may provide different epigenetic information to the early embryo, and shows that differential changes in this landscape define the first differentiation events in the early embryo. Current claims of extensive remodelling of CpG in the zygote seem to be largely caused by dynamic changes in the conformation of chromatin in early development leading to extensive antigenic masking of these modifications. When this masking is removed a much greater level of stability of these modifications at a global level becomes evident.

Chronological transition of mitochondrial morphology in Chlamydomonas reinhardtii (Chlorophyceae) poststationary phase growth(1).

In Chlamydomonas reinhardtii P. A. Dangeard, mitochondrial morphology has been observed during asexual cell division cycle, gamete and zygote formation, zygote maturation, and meiotic stages. However, the chronological transition of mitochondrial morphology after the stationary phase of vegetative growth, defined as the poststationary phase, remains unknown. Here, we examined the mitochondrial morphology in cells cultured for 4months on agar plates to study mitochondrial dynamics in the poststationary phase. Fluorescence microscopy showed that the intricate thread-like structure of mitochondria gradually changed into a granular structure via fragmentation after the stationary phase in cultures of about 1week of age. The number of mitochondrial nucleoids decreased from about 30 per cell at 1week to about five per cell after 4months of culture. The mitochondrial oxygen consumption decreased exponentially, but the mitochondria retained their membrane potential. The total quantity of mitochondrial DNA (mtDNA) of cells at 4months decreased to 20% of that at 1week. However, the mitochondrial genomic DNA length was unchanged, as intermediate lengths were not detected. In cells in which the total mtDNA amount was reduced artificially to 16% after treatment with 5-fluoro-2-deoxyuridine (FdUrd) for 1week, the mitochondria remained as thread-like structures. The oxygen consumption rate of these cells corresponded to that of untreated cells at 1week of culture. This suggests that a decrease in mtDNA does not directly induce the fragmentation of mitochondria. The results suggest that during the late poststationary phase, mitochondria converge to a minimum unit of a granular structure with a mitochondrial nucleoid.

Persistence of Cytosine Methylation of DNA following Fertilisation in the Mouse

Normal development of the mammalian embryo requires epigenetic reprogramming of the genome. The level of cytosine methylation of CpG-rich (5meC) regions of the genome is a major epigenetic regulator and active global demethylation of 5meC throughout the genome is reported to occur within the first cell-cycle following fertilization. An enzyme or mechanism capable of catalysing such rapid global demethylation has not been identified. The mouse is a widely used model for studying developmental epigenetics. We have reassessed the evidence for this phenomenon of genome-wide demethylation following fertilisation in the mouse. We found when using conventional methods of immunolocalization that 5meC showed a progressive acid-resistant antigenic masking during zygotic maturation which gave the appearance of demethylation. Changing the unmasking strategy by also performing tryptic digestion revealed a persistence of a methylated state. Analysis of methyl binding domain 1 protein (MBD1) binding confirmed that the genome remained methylated following fertilisation. The maintenance of this methylated state over the first several cell-cycles required the actions of DNA methyltransferase activity. The study shows that any 5meC remodelling that occurs during early development is not explained by a global active loss of 5meC staining during the cleavage stage of development and global loss of methylation following fertilization is not a major component of epigenetic reprogramming in the mouse zygote.

Splice Variants of the Dual Specificity Tyrosine Phosphorylation-regulated Kinase 4 (DYRK4) Differ in Their Subcellular Localization and Catalytic Activity

Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P -3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs.

Effect of oxidative stress during repeated ovulation on the structure and functions of the ovary, oocytes, and their mitochondria

We previously reported that superoxide generated in the ovary induces apoptosis of granulosa cells to break down follicular walls, thereby supporting ovulation in rodents, and suggested that oxidative stress underlies the mechanism of ovarian aging. To test this hypothesis, we successfully induced ovulation repeatedly in mice by sequentially administrating pregnant mare serum gonadotropin, human chorionic gonadotropin, and prostaglandin F2α. Kinetic analysis revealed that the number of ovulated oocytes decreased significantly with repeated cycles of ovulation with a concomitant decrease in the gene expression of mitochondrial transcription factor A and nuclear respiratory factor 1 and an increase in oocytes having abnormally distributed mitochondria. Repeated ovulation decreased the amounts of mitochondrial DNA and increased 8-hydroxydeoxyguanosine in oocytes. Cell culture analysis of the in vivo fertilized oocytes revealed that their maturation from two cells to blastocyst was inhibited significantly by repeated ovulation. All these events induced by repeated ovulation were suppressed by oral administration of L-carnitine. These results suggest that oxidative stress associated with ovulation underlies the mechanism of ovarian aging and that L-carnitine may have therapeutic potential in patients with infertility and increased incidence of aneuploidy and to suppress impaired maturation of zygotes frequently observed in childbearing at an advanced age.

Paternal inheritance of mitochondria in Chlamydomonas

To analyze mitochondrial DNA (mtDNA)inheritance, differences in mtDNA between Chlamydomonas reinhardtii and Chlamydomonas smithii, respiration deficiency and antibiotic resistance were used to distinguish mtDNA origins. The analyses indicated paternal inheritance. However, these experiments raised questions regarding whether paternal inheritance occurred normally.Mitochondrial nucleoids were observed in living zygotes from mating until 3 days after mating and then until progeny formation. However, selective disappearance of nucleoids was not observed. Subsequently, experimental serial backcrosses between the two strains demonstrated strict paternal inheritance. The fate of mt+ and mt- mtDNA was followed using the differences in mtDNA between the two strains. The slow elimination of mt+ mtDNA through zygote maturation in darkness was observed, and later the disappearance of mt+ mtDNA was observed at the beginning of meiosis. To explain the different fates of mtDNA, methylation status was investigated; however, no methylation was detected. Variously constructed diploid cells showed biparental inheritance. Thus, when the mating process occurs normally, paternal inheritance occurs. Mutations disrupting mtDNA inheritance have not yet been isolated. Mutations that disrupt maternal inheritance of chloroplast DNA (cpDNA) do not disrupt inheritance of mtDNA. The genes responsible for mtDNA inheritance are different from those of chloroplasts.

338. METHYL BINDING DOMAIN PROTEIN (MBD1) IN THE NUCLEUS OF THE MOUSE ZYGOTE

MBD1 is one of five proteins which bind methylated DNA and regulate gene transcription. The binding of these proteins, particularly MBD1, is commonly used as a proxy measurement of global CpG methylation. Since methylation is reported to be highly dynamic during the first cell-cycle, with reported asymmetric global demethylation of the paternal and maternal genomes by the time of syngamy, we were interested to assess the pattern of staining of the MBD1 during this stage of development. A specific antibody to MBD1 was shown by Western analysis to detect in zygotes a protein of predicted mass. Using immunolocalization, however, we found no staining in pronuclei. Brief acid treatment (10min, 4M HCl) followed by immunolabelling revealed strong pronuclear MBD1 staining throughout the maturation of the zygote and on metaphase chromosomes, indicative of epitope masking under normal staining conditions. Upon unmasking by acid treatment zygotes collected fresh from the oviduct did not show consistent differences in MBD1 staining between the maternal or paternal chromosomes or pronuclei, but for those embryos produced by IVF we found more MBD1 staining in the male paternal pronucleus. Brief treatment with trypsin caused a marked loss of MBD1 staining and this treatment increased the extent of staining of 5-methylcytosine. These results show that MBD1 antigen persists on DNA after treatments normally used for the detection of 5-methylcytosine. MBD1 at least partially masks methylcytosine from immunological detection and the results therefore raise the possibility that the reported changes in genome methylation in the zygote are a consequence of the binding of MBD1. If MBD1 binding is truly a proxy for methylation, the persistence of high levels of MBD1 throughout the first cell-cycle questions the current paradigm of global demethylation during zygote maturation.

The dynamic behaviour of mitochondria in living zygotes during maturation and meiosis in Chlamydomonas reinhardtii

To understand fully the function of mitochondria during the development of cells and organs, it is important to elucidate the dynamics of their morphology. However, the detailed morphology of mitochondria during meiosis has not yet been studied in algae. We examined the mitochondrial morphology of Chlamydomonas reinhardtii and classified zygotes into seven types by mitochondrial morphology in order to analyse the morphological change in mature and meiotic zygotes. We also investigated the oxygen consumption of living zygotes and the effects of tubulin and actin polymerization inhibitors on mitochondria, using fluorescence microscopy and oxygen electrodes. During zygote maturation, mitochondria fragmented into small particles, with a large decrease in oxygen consumption. When mature zygotes were exposed to light, mitochondria became tubular and formed a network, and oxygen consumption gradually recovered. At the same time, particle-like mitochondrial nucleoids became stringy and produced new nucleoid particles. Tubular mitochondria accumulated around the cell nucleus and then spread throughout the cell. Cell division followed (first and second rounds), and the resultant daughter cells had tubular mitochondria in a mesh-like arrangement. An inhibitor of tubulin polymerization, demecolcine, inhibited the assembly of mitochondria around the cell nucleus, whereas an inhibitor of actin polymerization, latrunculin B, inhibited the formation of tubular mitochondria. These results suggest that microtubules are probably involved in mitochondrial accumulation around the cell nucleus, whereas microfilaments may maintain the tubular network of mitochondria.

In vitro maturation and fertilization of bovine oocytes and in vitro culture of presumptive zygotes in the presence of bovine pestivirus

A pathogen which has been shown to commonly contaminate in vitro bovine embryo production system is bovine pestivirus (bovine viral diarrhea virus). Three experiments were designed to evaluate the in vitro maturation (experiment I), fertilization (experiment II) and embryo development (experiment III) of immature oocytes, inseminated oocytes and presumptive zygotes in the presence of a bovine pestivirus (non-cytopathic, nCP type 1). The virus inoculum used was derived from a persistently infected cow. In experiment I, follicular oocytes ( n=1257) recovered from slaughterhouse derived ovaries were randomly assigned to either a control group ( n=578) which did not become exposed to bovine pestivirus and a treatment group ( n=679) which was inoculated with bovine pestivirus (2.20–3.69 log 10 TCID 50/50 μl) at the time of commencement of in vitro maturation. Overall, there was no significant difference between the control and pestivirus inoculated oocytes in either the cumulus cell expansion rate (79±7.5% versus 74±10.7%) or the nuclear maturation rate (89±4.8% versus 85±7.4%), respectively. In experiment II, in vitro matured oocytes ( n=607) were inseminated either in the absence (control; n=301) or the presence of bovine pestivirus (4–4.6 log 10 TCID 50/50 μl; n=306). A significant ( P<0.01) reduction in the overall number of fertilized oocytes with two well formed male and female pronuclei was observed in the treatment group compared to the control group (58.5±5.8% versus 73.3±3.6%, respectively). In experiment III, after in vitro maturation and fertilization, presumptive zygotes were randomly assigned to either a control group ( n=139) which was not exposed to bovine pestivirus or a treatment group which was inoculated with bovine pestivirus (2.97–4.47 log 10 TCID 50/30 μl; n=139). The zygotes were then cultured under mineral oil in an atmosphere of 88% N 2, 7% O 2 and 5% CO 2 at 39 °C. The morphologic appearance of the embryos was assessed 48 h after the commencement of culture, and then every 48 h up to days 7–8 after insemination. The 22% (31/139) and 3.6% (5/139) of the presumptive zygotes developed to the morula or blastocyst stage in the control and the bovine pestivirus inoculated groups, respectively ( P<0.001). This study demonstrates that bovine pestivirus has a significant detrimental effect on in vitro fertilization and early in vitro embryo development.

Chloroplast DNA methylation and inheritance in Chlamydomonas.

When Chlamydomonas reinhardtii cells mate, a zygotic maturation program is activated, part of which leads to destruction of chloroplast DNA (cpDNA) from the mating type minus (mt-) parent, and, therefore, to uniparental inheritance of mating type plus (mt+) cpDNA. A long-standing model that explains the selective destruction of mt(-) cpDNA in zygotes invokes a methylation-restriction system. We tested this model by using the potent methylation inhibitor 5-aza-2'-deoxycytidine (5adc) to hypomethylate parental cpDNA and found that the pattern of cpDNA inheritance is altered by 5adc in a manner that is consistent with the model. Surprisingly, however, hypomethylated mt+ cpDNA is not destroyed in zygotes as the methylation-restriction model predicts it should be. Destruction of mt- cpDNA is also unaffected when the parental mt+ cpDNA is hypomethylated. Instead, loss of methylation affects the relative rates of replication of residual mt- cpDNA and mt+ cpDNA in germinating zygotes. The mode of action for 5adc on cpDNA replication in germinating zygotes may be via hypomethylation of mt+ cpDNA, but is also consistent with its action as a DNA-damaging agent. Interestingly, 5adc causes reduced cpDNA replication only in germinating zygotes, not in vegetatively grown cells, indicating that cpDNA replication is qualitatively different in these two stages of the life cycle. Our results demonstrate that methylation is not necessary for protection of the mt+ cpDNA in early zygotes and uncover a novel stage of the Chlamydomonas life cycle when replication of cpDNA is highly susceptible to perturbation. Our data support a model in which differential cpDNA replication in germinating zygotes is used as a mechanism to selectively amplify intact and properly methylated cpDNA molecules.

THE PRESENCE OF CALLOSE IN THE PRIMARY ZYGOTE WALL OF CHLAMYDOMONAS MONOICA AND THE EFFECTS OF ITS DEGRADATION ON ZYGOTE DEVELOPMENT

Chlamydomonas monoica constructs a temporary primary wall around its developing zygotes. This study aimed to confirm callose as a component of the primary wall, as well as to note the effects of primary wall degradation on zygote development. Glucanase, specific for the β‐1,3 glycosidic bonds comprising callose, was added to mating media at concentrations ranging from 5 to 1 mg ml−1 and light microscope observations were made as the zygotes developed. The overall health of the zygotes was assessed by comparing their ability to germinate after exposure to chloroform vapors. The bright staining of the primary wall with aniline blue, specific for β‐1,3 polysaccharides, suggested the presence of callose. This was further supported by the adverse effects of glucanase on zygote development. After mating, declining levels of intact zygotes were found as their maturation continued, and dead immature zygotes accumulated in the treated cultures. Twelve days after mating, when the zygotes were plated for germination, fully mature zygotes were identified in only the lowest of the six enzyme concentrations. In addition, germinating zygotes from the treated cultures showed increased sensitivity to killing by chloroform vapors relative to untreated zygotes. These results suggest that callose is a key component in the primary zygote wall, and that its degradation negatively affects zygote maturation. Electron microscopy will be used to help determine whether structural defects in the primary wall occur as a result of glucanase treatment, and whether such defects affect secondary zygospore wall assembly.

Disintegration of Chloroplasts during Zygote Maturation inClosterium ehrenbergii(Zygnematales, Chlorophyta)

The disintegration of chloroplasts during zygote maturation in Closterium ehrenbergii Meneghini ex Ralfs was examined mainly by electron microscopy. The young zygotes each contained four chloroplasts derived from the plus and the minus gametes. Ca. 7 d after gamete union, two of the four chloroplasts within a zygote began to disintegrate. The disintegrating chloroplasts turned yellow and rapidly lost starch grains. Subsequently, they became fragmented, each fragment having many plastoglobuli and residual thylakoid membranes. At this stage, vacuolar structures of various sizes in section appeared within the cytoplasm. Each of them was limited by a single membrane and contained small vesicles and electron‐translucent materials. The chloroplast fragments seemed to be separated from the ground cytoplasm by their fusion with the vacuolar structures, and they were broken down into aggregations of plastoglobuli and residual membrane pieces by 2 wk after plasmogamy. Dictyosomes sometimes lay close to the vacuolar...

Dynamics of Nuclear DNA Quantities during Zygote Development in Barley.

Quantities of DNA were estimated in the nuclei of mechanically isolated egg and zygote protoplasts in two cultivars of barley using 4[prime],6-diamidino-2-phenylindole staining and microfluorometry. Unlike many previous studies on DNA amounts within the sex cells of flowering plants, we obtained consistent and unambiguous results indicating that the egg and sperm nuclei are at the 1C DNA level (basic haploid amount) at the time of karyogamy. Karyogamy was initiated within 60 min postpollination, and the male chromatin became completely integrated into the egg nucleus within 6 to 7 hr postpollination (hpp). Zygotic nuclear DNA levels began to increase at ~9 to 12 hpp in cultivar Alexis and at 12 to 15 hpp in cultivar Igri. The 4C DNA complement was reached in most zygotes by 22 to 26 hpp in cultivar Alexis and by 23 to 29 hpp in cultivar Igri. These data are fundamental to a better understanding of fertilization and zygote maturation in flowering plants. They are also relevant to studies in which the timing of zygotic DNA replication is of interest, such as ongoing investigations on genetic transformations in barley using the microinjection technique.

The fate of mitochondrial DNAs of mt + and mt ? origin in gametes and zygotes of Chlamydomonas

In order to study the mechanism responsible for the uniparental transmission of the mitochondrial genome in crosses between Chlamydomonas reinhardtii and C. smithii, we have analyzed the fate of mitochondrial DNA during gametogenesis, zygospore differentiation and sporulation by hybridization experiments. Both mt+ and mt- gametes contain the same amount of mitochondrial DNA and the two parental genomes persist for several days in the zygotes. The DNA of mt+ origin is slowly eliminated during the period of zygote maturation. Light is required for total elimination of mt+ mitochondrial DNA in the zygospores. Using appropriate restriction enzymes, we have been unable to detect methylation of the mitochondrial DNA during gametogenesis or zygospore formation. The possibility that the mt+ mitochondria themselves are specifically eliminated in the course of zygote maturation is discussed.

Identification of novel genes specifically expressed in Chlamydomonas reinhardtii zygotes

The maturation of zygotes formed by the fusion of two gametes is the essential part of the diploid phase of the Chlamydomonas reinhardtii sexual life cycle and results in mature zygotes competent to germinate. To understand the molecular mechanisms underlying zygote maturation and the attainment of competence for germination we isolated genomic clones representing three different genes that are specifically expressed in Chlamydomonas reinhardtii zygotes. Accumulation of the RNAs started more than 24 h after mating, setting these genes apart from genes expressed in young zygotes. Upon light-induced germination of zygotes, the mRNAs disappeared. The patterns of RNA accumulation and disappearance were gene-specific and suggested a function of these genes in maturation and/or in initial steps of germination.

Loss of mt +-derived zygotic chloropiast DNA is associated with a lethal allele in Chlamydomonas monoica

The Chlamydomonas monoica mutant allele mtl-1, is associated with the formation of nonviable zygospores following “self-mating” of the mutant strain. Furthermore, mtl-1 heterozygote populations show a 50% reduction in germination frequency and no transmission of a chloroplast antibiotic resistance marker carried by the mtl-1 parent. To determine whether the effects on zygospore viability and chloroplast gene transmission resulted from the direct involvement of the mtl-1 locus in the control of mt+-directed uniparental inheritance of chloroplast DNA (cpDNA), we have used the DNA-specific fluorochrome DAPI to follow the fate of cpDNA during the maturation of zygotes. Throughout the first few hours after the initial fusion of gametes, the young zygotes show DAPI-fluorescent nucleoids distributed symmetrically around the region of nuclear fusion, and presumably located within both of the parental chloroplasts. Wild-type and mtl-1 mutant zygotes show similar early staining patterns. As the zygotes age, the staining patterns become asymmetric for the wildtype population, with all of the visible cytoplasmic nucleoids restricted to one “side” of the zygote. In contrast, mtl-1 homozygotes appear to lose cytoplasmic nucleoids from both “sides” of the zygote simultaneously and within 24 h are apparently devoid of cpDNA. By introducing a mutation which arrests cell fusion (and prevents plastid fusion), we can show that (1) the asymmetric nucleoid distribution in wildtype zygotes results from the loss of nucleoids from one gamete in each mating event, and (2) the additional loss of cpDNA in mtl-1 homozygotes does not require contact between parental plastids (thus the nuclease responsible for cpDNA degradation is not sequestered within the chloroplast of one gamete). We propose that the mtl-1 mutant strain is defective for a process which normally protects cpDNA of mt+ origin.