Optimization of ultraviolet irradiation of Clarias gariepinus (Burchell, 1822) eggs for androgen production

Summary The optimum distance and duration of ultraviolet (UV) irradiation for the complete inactivation of African catfish Clarias gariepinus egg nucleus was investigated in this study. The UV light was suspended above the unfertilized eggs at four distances (5, 10, 20 and 30 cm) and for five durations (1, 2, 3, 4 and 5 min). Then, the irradiated eggs were activated with sperm from diploid C. gariepinus and cold shocked at 5°C for 5 min just moments before cell cleavage. Ploidy analysis was performed using karyotype chromosome counting. The results obtained suggested that the further the distance, the better the hatchability rate, however prolonged duration seemed to significantly reduced hatchability. All treatments with surviving progenies at the end of the study showed evidence of successfully diploid gynogen (2n = 56) induction at different percentages. However, the optimal protocol that gave a moderately high hatchability/survival rate and completely induced gynogens was exposure of the eggs to UV irradiation at 20 cm for 1 min. It was concluded that the distance and duration of UV irradiation affects gynogenetic induction in African catfish C. gariepinus.

Production of triploid, doubled haploid (DH) and meiogynogenetic brook trout (Salvelinus fontinalis) – efficiency and development of body deformities

In the present research we produced triploid, mitogynogenetic (doubled haploid; DH) and meiogynogenetic brook trout (Salvelinus fontinalis) to examine efficiency of these technologies and potential susceptibility of chromosome set manipulated individuals for the spinal disorders. Triploidy was induced by shocking (High Hydrostatic Pressure – HHP) of fertilized eggs 30 min. after insemination. In turn, gynogenetic development was induced by activation of eggs with UV-irradiated sperm. Activated eggs were then exposed to HHP shock applied 30 and 420 minutes after insemination to provide meiogynogenotes and gynogenetic DHs, respectively. When compared to non-manipulated diploids, the highest survival rates was observed among triploid brook trout while DHs showed the highest mortality. Malformation rates in the diploid larvae from the control groups did not exceed 7.0% while percentage of malformed triploid individuals equaled 19.1%. Drastically increased number of deformed larvae (> 30%) was observed in both, DH and meiogynogenetic individuals. Intensification of kyphosis and scoliosis was clearly demonstrated in the gynogenetic and triploid brook trout. Genetic factors such as increased number of sets of chromosomes in triploids and expression of lethal alleles in the gynogenetic fish plus side effects of HHP shock utilized for retention of the second polar body or inhibition of the first cell cleavage when induced triploid and gynogenetic development have been discussed to affect survival rates and prevalence for the skeletal deformities in the chromosome set manipulated brook trout.

Dietary Micronutrient Supplementation for 12 Days in Obese Male Mice Restores Sperm Oxidative Stress

Male obesity, which often co-presents with micronutrient deficiencies, is associated with sub-fertility. Here we investigate whether short-term dietary supplementation of micronutrients (zinc, selenium, lycopene, vitamins E and C, folic acid, and green tea extract) to obese mice for 12 days (designed to span the epididymal transit) could improve sperm quality and fetal outcomes. Five-week-old C57BL6 males were fed a control diet (CD, n = 24) or high fat diet (HFD, n = 24) for 10 weeks before allocation to the 12-day intervention of maintaining their original diets (CD, n = 12, HFD n = 12) or with micronutrient supplementation (CD + S, n = 12, HFD + S, n = 12). Measures of sperm quality (motility, morphology, capacitation, binding), sperm oxidative stress (DCFDA, MSR, and 8OHdG), early embryo development (2-cell cleavage, 8OHdG), and fetal outcomes were assessed. HFD + S males had reduced sperm intracellular reactive oxygen species (ROS) concentrations and 8OHdG lesions, which resulted in reduced 8OHdG lesions in the male pronucleus, increased 2-cell cleavage rates, and partial restoration of fetal weight similar to controls. Sub-fertility associated with male obesity may be restored with very short-term micronutrient supplementation that targets the timing of the transit of sperm through the epididymis, which is the developmental window where sperm are the most susceptible to oxidative damage.

Zscan4c activates endogenous retrovirus MERVL and cleavage embryo genes

Endogenous retroviruses (ERVs) contribute to ∼10 percent of the mouse genome. They are often silenced in differentiated somatic cells but differentially expressed at various embryonic developmental stages. A minority of mouse embryonic stem cells (ESCs), like 2-cell cleavage embryos, highly express ERV MERVL. However, the role of ERVs and mechanism of their activation in these cells are still poorly understood. In this study, we investigated the regulation and function of the stage-specific expressed ERVs, with a particular focus on the totipotency marker MT2/MERVL. We show that the transcription factor Zscan4c functions as an activator of MT2/MERVL and 2-cell/4-cell embryo genes. Zinc finger domains of Zscan4c play an important role in this process. In addition, Zscan4c interacts with MT2 and regulates MT2-nearby 2-cell/4-cell genes through promoting enhancer activity of MT2. Furthermore, MT2 activation is accompanied by enhanced H3K4me1, H3K27ac, and H3K14ac deposition on MT2. Zscan4c also interacts with GBAF chromatin remodelling complex through SCAN domain to further activate MT2 enhancer activity. Taken together, we delineate a previously unrecognized regulatory axis that Zscan4c interacts with and activates MT2/MERVL loci and their nearby genes through epigenetic regulation.

Peculiarities of the molecular composition of heterochromatin associated with pronucleoli in mouse embryos

The nucleus of pre-implantation mammalian embryos is characterized by peculiar structural organization. At the initial stages of cleavage, the nucleus of the embryo contains the so-called nucleolus precursor bodies (NPBs) or pronucleoli rather than functionally active nucleoli. The NPBs are fibrillar electron-dense structures inactive in RNA synthesis. The vast majority of NPBs are surrounded by a ring-shaped zone of transcriptionally inactive heterochromatin. Intriguingly, these zones contain not only tri-methylated histone Н3K9me3 as an epigenetic mark of repressed chromatin but also acetylated histone H4K5ac, a well-known marker of active chromatin. Immunocytochemical data suggest that the molecular composition of this ‘ring heterochromatin’ in mouse embryos changes during the realization of embryonic genome activation events, as well as during artificial suppression of transcription. In zygotes, some factors of mRNA biogenesis including splicing factor SC35 (SRSF2) and basal transcription factor TFIID are detectable in the ring chromatin. At later stages of development, other nuclear proteins such as Y14, a core component of the exon-exon junction complex (EJC), as well as the proteins involved in chromatin remodeling (ATRX, Daxx) are also detectable in this area. A typical component of the ‘ring heterochromatin’ is actin. Anti-actin immunocytochemical labeling is most expressed at the two-cell cleavage stage after activation of the embryonic genome. Indicatively, the molecular composition of the ‘ring heterochromatin’ associated with different NPBs may differ significantly even in the same nucleus. This seems to reflect the functional heterogeneity of morphologically similar NPBs according to their competence to the process of nucleologenesis. Here, we discuss briefly some peculiarities of the molecular composition and possible functions of the NPB-associated heterochromatin in mouse early embryos.

Optogenetic dissection of mitotic spindle positioning in vivo

The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα∙GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα∙GDP, Gα∙GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP–GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.

Optogenetic dissection of mitotic spindle positioning in vivo

ABSTRACTThe position of the mitotic spindle determines the plane of cell cleavage, and thereby the location, size, and content of daughter cells. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules. This process requires an evolutionarily conserved complex of Gα-GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. It remains unknown whether this complex merely forms a membrane anchor for dynein, or whether the individual components have additional functions, for instance through Gα-GTP or dynein activation. To functionally dissect this system, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. Controlled germline expression and membrane recruitment of the Gα regulators RIC-8Ric-8A and RGS-7Loco/RGS3, and replacement of Gα with a light-inducible membrane anchor demonstrated that Gα-GTP signaling is dispensable for pulling force generation. In the absence of Gα, cortical recruitment of GPR-1/2 or LIN-5, but not dynein itself, induced high pulling forces. Local recruitment of LIN-5 overruled normal cell-cycle and polarity regulation, and provided experimental control over the spindle and cell cleavage plane. Our results define Gα∙GDP–GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle positioning forces. This study also highlights the possibilities for optogenetic control of endogenous proteins within an animal system.