Unlike somatic cells mitosis, germ cell meiosis consists of two consecutive rounds of divisions that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric divisions. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it was localized to meiotic spindles, and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration; and symmetric division or large polar body emission. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.

Live offspring is obtained from in vitro production of porcine embryos, but the procedure is still associated with great inefficiencies. In mammalian oocytes, acquisition of meiotic competence coincides with a decrease in general transcriptional activity at the end of the oocyte growth phase. In this study, we investigated the expression and sub-cellular localization of positive transcription elongation factor P-TEFb (CDK9/Cyclin T1), a RNA polymerase II CTD kinase during pig oocyte growth and early embryonic development. Localization and expression of components involved in mRNA and rRNA transcription were assessed by immunocytochemistry in growing and fully-grown oocytes. In addition, meiotic resumption, germinal vesicle breakdown, nuclear transcription and embryonic genome activation (EGA) were analyzed in oocytes and embryos cultured in presence of a potent CDK9 inhibitor, flavopiridol. Our analyses, demonstrated that CDK9 became co- localized partially with phosphorylated Pol II CTD and mRNA splicing complexes. Surprisingly, CDK9 was co-localized with Pol I-specific transcription factor, UBF, and gradually localized in nucleolar peripheries at the final steps of oocyte growth. Later, CDK9 became associated with nucleolar structures at 4-cell stage. Treatment with flavopiridol resulted in arrest in meiotic resumption, germinal vesicle breakdown as well as a decline in global transcription. Flavopiridol also inhibited embryo development beyond EGA. All together, these data suggest that CDK9 has a dual role in both Pol I- and Pol II-dependent transcription in pig oocyte growth and embryonic development.

In vitro maturation of porcine immature cumulus-enclosed oocytes can be enhanced by co-incubation with spermatozoa even before fertilization. The aim of this study was to determine whether the addition of spermatozoa into the culture medium can stimulate the meiosis resumption of porcine cumulus-enclosed oocytes arrested at germinal vesicle (GV). Cumulus-enclosed oocytes (CEOs) were collected from follicles of 3 to 5mm diameter. Porcine CEOs were cultured in tissue culture medium containing various meiosis inhibitors and spermatozoa. Oocytes were examined for evidence of GV and GV breakdown after 24 h culture. After 24 h culture 43.8% of oocytes cultured in only TCM 199 remained at GV stage whereas 56.2% of oocytes were able to resume meiosis. When porcine CEOs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 90% of oocytes were not able to resume meiosis. However, co-culture of porcine CEOs with spermatozoa was able to overcome the inhibitory effect of dbcAMP and Fo. Irrespective of the presence of 3-isobutyl-1-methylxanthine (IBMX), no difference was observed in the proportion of oocyte reached germinal vesicle breakdown (GVBD). The present study suggests that dbcAMP and Fo prevent the spontaneous maturation of competent oocyte in culture after isolation from follicles and that mammalian spermatozoa contain a substance(s) that improves meiosis resumption in vitro of porcine cumulus-enclosed oocytes.

Accurate chromosome segregation is critical to ensure genomic integrity during cell division. This process is facilitated by the kinetochore, a multiprotein structure that is assembled on centromeric regions of chromosomes. The kinetochore establishes a mechanical link between the chromosomes and spindle microtubules and modulates cell cycle progression by regulating spindle assembly checkpoint (SAC). Defects in this process result in an aneuploidy, leading to miscarriages, infertility and various genetic disorder such as Down’s syndrome. Although the numerous kinetochore proteins have been identified and studied, the mechanisms that engaged in kinetochore assembly and chromosome segregation are poorly understood. Here we investigated the function of kinetochore protein Zwint-1 on homologous chromosome segregation during oocyte meiotic maturation. We found that Zwint-1 was localized at the kinetochore during meiotic maturation. Knockdown of Zwint-1 caused premature polar body extrusion, indicating acceleration of meiosis I. Interestingly, Zwint-1 knockdown impaired the recruitment of Mad2 at the kinetochores. However, BubR1 localization at the kinetochores was not affected by Zwint-1 knockdown, suggesting that Zwint-1 selectively regulates the recruitment of SAC components into the kinetochores. We also found that Zwint-1 knockdown abrogated chromosome alignment and segregation, thereby resulting in a high incidence of aneuploidy. These chromosomal defects were mostly due to the abnormal kinetochore-microtubule (kMT) attachments. Intriguingly, chromosome misalignment mediated by SAC inactivation was repaired, when anaphase onset was delayed by treating oocytes with proteasome inhibitor MG132. However, surprisingly, chromosomal defects following Zwint-1 knockdown were not restored by delaying anaphase onset. This result suggests that chromosomal defects induced by Zwint-1 knockdown are less likely associated with the failure of SAC activation. In addition, we observed that Aurora B/C kinase activity was not affected by Zwint-1 knockdown. Nevertheless, the meiotic defects induced by Zwint-1 knockdown were similar to those observed in Aurora B/C inhibition, suggesting that Zwint-1 is a downstream effector of Aurora B/C kinase during meiosis. Consistent with this, in Zwint-1 knockdown oocytes chromosomal defects following Aurora B/C inhibition were not restored when Aurora B/C inhibitor was removed, whereas the defects were well rescued in control oocytes after removing Aurora B/C inhibitor. This result suggests that the role of Aurora B/C kinases that correct erroneous kMT attachment is primarily regulated by Zwint-1. Collectively, our results demonstrated for the first time that Zwint-1 is an essential downstream effector of Aurora B/C kinase that corrects erroneous kMT attachment and regulates SAC activity, which ensures accurate homologous chromosome segregation during oocyte meiosis.

The correct development of male gametophytes (pollen grains) in flowering plants is essential for proliferate in gamete production. Here we have taken a map-based cloning approach using Arabidopsis male gametophytic mutant, named gemini pollen3 (gem3) to identify and characterize key gene that is expressed gametophytically for the completion of microgametogenesis focusing on genes which control cell division and cell fate determination. Previously reported gem1 and gem2 mutants with similar characteristics to gem3 that are disturbed at asymmetric division and cytokinesis at pollen mitosis I (PMI) in Arabidopsis. However, gem3 was mapped to a different genetic locus, and pollen developmental analysis revealed that gem3 exert an effect at meiosis and mitosis causing complete sterility. We also discovered that gem3 homozygous lines produce aberrant pollen grains, arising from incomplete cytokinesis during male meiosis with sporophytic phenotypes of twisted-shape leaves, large flowers. This mutation shows reduced genetic transmission of gem3 allele through male gametophyte. In previous results, the gem3 locus was confirmed by mapping to the region located on chromosome 5. To further confirm strong candidate gene, we performed sequencing and genetic complementation analysis. Currently, we are performing functional studies of the gem3 gene for the better understanding of molecular mechanisms that control asymmetric division at meiosis and mitosis during pollen development.

xBrassicoraphanus, a new synthetic intergeneric hybrid between Brassica rapa L. ssp. pekinensis and Raphanus sativus L., also locally known as ‘Baemoochae’, is an interesting subject for studying polyploidy and genome plasticity in the family Brassicaceae, but very few genomic and cytogenetic information. Here, we analysed the chromosome complements and pairing of the most fertile lines, BB1 and BB5, using dual-color fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) to check their chromosomal segregation stability. The somatic chromosome complement of B. rapa was confirmed to be 2n=20 (2.8~4.8μm), of R.sativus, 2n=18 (2.0~3.3μm), and of xBrassicoraphanus, 2n=38 (2.2~5.0μm). There were eight, eight, and seventeen metacentric pairs and two, one, and two submetacentric pairs in B. rapa, R. sativus, and xBrassicoraphanus, respectively. Additionally, three, two, and five pairs of 5S rDNA and five, three, and eight pairs of 45S rDNA were observed in B. rapa, R. sativus, and xBrassicoraphanus, respectively. This suggests that both B. rapa (AA) and R. sativus (RR) genomes, particularly the rDNA arrays, co-exist in xBrassicoraphanus (AARR) genome. In meiosis I, nineteen bivalents were most frequent, and GISH analysis showed ten bivalents from the A genome. This study would provide a useful information for further genomic study of xBrassicoraphanus and its improvement as a new promising breeding variety.

Reverse breeding is a new plant breeding strategy based on crossover suppression during meiosis. This brings forth unprecedented possibilities like the almost instantaneous generation of homozygous parents for a chosen heterozygote. As a proof of concept, an Arabidopsis (Columbia-Landsberg) heterozygote was created that carried a RNAi:DMC1 construct stopping crossover formation. Gametes of this heterozygote were grown directly into doubled haploid offspring. These offspring show different combinations of (non-recombinant) Columbia and Landsberg chromosomes. Among these doubled haploids we retrieved the original Columbia parent and a complete set of chromosome substitution lines. From among these we could easily select two so called “complementing DHs” from which the Col-Ler hybrid could be re-created. Essentially, breeders can now bring single choice uncharacterized heterozygotes into a hybrid breeding program by creating parental lines for them. Reverse breeding superficially resembles apomixis (clonal reproduction through seeds) since both allow the preservation of heterozygous genotypes. Reverse breeding, however, has very different uses because it generates homozygous breeding lines. It thus allows for the improvement of the starting heterozygote because new traits can be introgressed into its newly produced parental lines. Reverse breeding is thought to be suitable for crops with smaller chromosome numbers (x ≤ 12). It will be discussed how reverse breeding could be developed for such crops, and it will be shown how reverse breeding presents very interesting new possibilities studying epistasis and heterosis through chromosome substitution lines. Further experiments with reverse breeding lines allow testing of a variety of intriguing breeding questions like to what extent a (heterozygous) genome actually determines a plants phenotype.

Chromosome analysis using mitosis, meiosis and bicolor FISH were carried out in Bupleurum latissimum Nakai, which is one of the endemic plants in Ulleung island of korea. The somatic methaphase chromosomes number of this plant was 2n = 2x = 16 and the chromosome complements consisted of six pairs of metacentrics and two pairs of submetacentrics. The size of chromosomes ranged 2.40~4.20 μm and NOR (nucleolus organizer region) chromosome did not observed using conventional staining. In meiosis chromosomes, metaphase-I and anaphase-I were observed. Metaphase-I anaphase-I showed 8 bivalents and chromosomes migration to make two daughter cells. Using bicolor FISH, one pair of 5S and 45S rDNA signals were detected on the centromeric region of chromosome 3 and the end of short of chromosome 2,respectively. We also observed the NOR using 45S rDNA probe.

Hybries which was made up by chromosome of L. longiflorum and L. x elegans, using root-tip individual which was obtained through ovary slice culture, and root-tip of these parents, with hoirugen staining, gimsa staining and Q-H staining inaccordance with the location and the existence of secondary construction which waslocating near short arm centromere of No, 1,2,6,9. In metaphase of meiosis ofhybrid which was made up by univalent from 2 individuals to 10 individuals wasobserved, and nuclear plate which was having abnormal type's synthesis amounted to91% of all cells whieh were observed. This result showed the fact that someobstacle arose annormal progress of the divission after that time. 63% of the cellshad micronucleus from 1 individlial to 4 individuals in tetrad phase of meiosisdivision. The peroxidase and α -estelase zymogram phenotypes of parents andhybrids were determined using agarlose IEF gel. Crosses were performed betweenparents bearing dissimilar allelomorphs in orther to discern the genetic control ofthe resolved enzymes. Genetic variation of hybrids were detected at all but 2 plant progenies.