Tools to Image Germplasm Dynamics During Early Zebrafish Development

During the first day of zebrafish development, ribonucleoprotein (RNP) complexes called germplasm form large aggregates that initially segregate asymmetrically during cleavage stages. After zygotic genome activation, the granules break into smaller fragments that associate with the nuclear membrane as perinuclear (germ) granules toward the end of gastrulation. The mechanisms underlying the highly dynamic behavior of germ granules are not well studied but thought to be facilitated by the cytoskeleton. Here, we present efficient mounting strategies using 3d-printed tools that generate wells on agarose-coated sample holders to allow high-resolution imaging of multiplexed embryos that are less than one day post-fertilization (dpf) on inverted (spinning disk confocal) as well as upright (lattice light-sheet and diSPIM) microscopes. In particular, our tools and methodology allow water dipping lenses to have direct access to mounted embryos, with no obstructions to the light path (e.g., through low melting agarose or methyl cellulose). Moreover, the multiplexed tight arrays of wells generated by our tools facilitate efficient mounting of early embryos (including cleavage stages) for live imaging. These methods and tools, together with new transgenic reporter lines, can facilitate the study of germ granule dynamics throughout their lifetime in detail, at high resolution and throughput, using live imaging technologies.

Zygotic contractility awakening during mouse preimplantation development

Actomyosin contractility is a major engine of preimplantation morphogenesis, which starts at the 8-cell stage during mouse embryonic development. Contractility becomes first visible with the appearance of periodic cortical waves of contraction (PeCoWaCo), which travel around blastomeres in an oscillatory fashion. How contractility of the mouse embryo becomes active remains unknown. We have taken advantage of PeCoWaCo to study the awakening of contractility during preimplantation development. We find that PeCoWaCo become detectable in most embryos only after the 2nd cleavage and gradually increase their oscillation frequency with each successive cleavage. To test the influence of cell size reduction during cleavage divisions, we use cell fusion and fragmentation to manipulate cell size across a 20-60 μm range. We find that the stepwise reduction in cell size caused by cleavage divisions does not explain the presence of PeCoWaCo or their accelerating rhythm. Instead, we discover that blastomeres gradually decrease their surface tensions until the 8-cell stage and that artificially softening cells enhances PeCoWaCo prematurely. Therefore, during cleavage stages, cortical softening awakens zygotic contractility before preimplantation morphogenesis.

ATP signaling in the integrative neural center of Aplysia californica

ATP and its ionotropic P2X receptors are components of the most ancient signaling system. However, little is known about the distribution and function of purinergic transmission in invertebrates. Here, we cloned, expressed, and pharmacologically characterized the P2X receptors in the sea slug Aplysia californica—a prominent neuroscience model. AcP2X receptors were successfully expressed in Xenopus oocytes and displayed activation by ATP with two-phased kinetics and Na+-dependence. Pharmacologically, they were different from other P2X receptors. The ATP analog, Bz-ATP, was a less effective agonist than ATP, and PPADS was a more potent inhibitor of the AcP2X receptors than the suramin. AcP2X were uniquely expressed within the cerebral F-cluster, the multifunctional integrative neurosecretory center. AcP2X receptors were also detected in the chemosensory structures and the early cleavage stages. Therefore, in molluscs, rapid ATP-dependent signaling can be implicated both in development and diverse homeostatic functions. Furthermore, this study illuminates novel cellular and systemic features of P2X-type ligand-gated ion channels for deciphering the evolution of neurotransmitters.

Role of BMP signaling during early development of the annelid Capitella teleta

The mechanisms regulating nervous system development are still unknown for a wide variety of taxa. In insects and vertebrates, bone morphogenetic protein (BMP) signaling is known to play a key role in both neural specification and dorsal-ventral (D-V) axis formation, leading to speculation about the conserved evolution of nervous systems. Studies outside insects and vertebrates show a more diverse picture of what, if any role, BMP signaling plays in neural development across Bilateria. This is especially true in the morphologically diverse Spiralia (~Lophotrochozoa). Despite several studies of D-V axis formation and neural induction in spiralians, there is no consensus for how these two processes are related, or whether BMP signaling may have played an ancestral role in either process. Here we incubated larvae of the sedentary annelid Capitella teleta in BMP4 protein at various cleavage stages to determine the role of BMP signaling during early development. Adding exogenous BMP protein to early-cleaving C. teleta embryos had a striking effect on formation of the brain, eyes, and foregut in a time-dependent manner. However, adding BMP did not block neural specification of the brain or VNC or block formation of the D-V axis. We identified three key time windows of BMP activity, and hypothesize that BMP may cause trans-fate switching of blastomere quadrant identities in at least one time window. 1. Early treatment around 2q caused the loss of the eyes, radialization of the brain, and a reduction of the foregut, which we interpret as a loss of A-, B- and C-quadrant identities with a possible trans-fate switch to a D-quadrant identity. 2. Treatment after 4q induced formation of a third ectopic brain lobe, eye, and foregut lobe, which we interpret as a trans-fate switch of B-quadrant micromeres to a C-quadrant identity. 3. Continuous BMP treatment from early cleavage through mid-larval stages resulted in a modest expansion of Ct-chrdl expression in the dorsal ectoderm and a concomitant loss of the ventral midline (neurotroch ciliary band). Loss of the ventral midline was accompanied by a collapse of the bilaterally-symmetric VNC although the total amount of neural tissue did not appear to be greatly affected. Our results compared to those from other annelids and molluscs suggest that BMP signaling was not ancestrally involved in delimiting neural tissue or establishing the D-V axis in the last common ancestor of annelids. However, the effects of ectopic BMP on quadrant-identity during cleavage stages may represent a very early ‘organizing’ function in the context of spiralian development. Ultimately, studies on a wider range of spiralian taxa are needed to determine if the ability of BMP signaling to block neural induction and help establish the D-V axis was lost within Annelida or if BMP signaling gained these functions multiple times across Bilateria. Ultimately, these comparisons will give us insight into the evolutionary origins of centralized nervous systems and body plans.

Spawning and developmental biology of endangered Pabdah catfish, Ompok pabda (Hamilton, 1822)

Pabdah catfish, Ompokpabdawas collected from the Old Brahmaputra river and successfully induced to breed by injecting pituitary gland extract to evaluate spawning performances and developmental stages under a photo microscope from February 2018 to August 2018. In this study, absolute fecundity of the O. pabdafemales varied from 6,676 to 20,410 eggs per fish. The GSI value of male and female was lower during September and gradually attained its maximum level in June with the highest average value 6.75±0.2 and 1.053±0.2 in female and male, respectively. The average fertilization and hatching rate of eggs were found 78.67±1.33% and 70.21±0.73%, respectively at 24.5±1.0°C. Fertilized eggs were brownish in color, very transparent and adhesive in nature with 1.0 to 1.2 mm in diameter. Cleavage stages were completed at 1.50 h of post fertilization and it eventually took 23.00 h for hatching. Larva was appeared with prominent three pairs of barbells after 12.00 h of hatching. It took 3 d post hatching to absorb their yolk sac and feeding exogenously. The present work supplemented to the deficient information on detailed spawning and developmental biology of O. pabdain order to save this species from extinction. Asian J. Med. Biol. Res. September 2020, 6(3): 449-459

The impact of male factor infertility on early and late morphokinetic parameters: a retrospective analysis of 4126 time-lapse monitored embryos

STUDY QUESTION Is there an effect of male factor infertility (MFI) on either early or late morphokinetic parameters obtained during embryonic culture to blastocyst stage in a time-lapse imaging (TLI) incubator? SUMMARY ANSWER Neither mild nor severe MFI had an impact on overall time to blastocyst or duration of individual cleavage stages in the total embryo population. WHAT IS KNOWN ALREADY Prior studies have suggested that paternal DNA and sperm quality affect embryo morphokinetic parameters, but the impact of MFI is not fully understood. STUDY DESIGN, SIZE, DURATION This retrospective cohort study, at a major academic fertility centre, included 536 couples (women, ≤44 years of age) undergoing IVF between September 2013 and September 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS Data from 4126 embryos cultured to the blastocyst stage in a TLI-monitored incubator were retrospectively reviewed. Embryos derived from the sperm of men with MFI were compared with those derived from patients with other infertility diagnoses. Generalized fixed and random effects models, t-test and χ2 were used as appropriate. MAIN RESULTS AND THE ROLE OF CHANCE Couples with MFI had a higher rate of ICSI utilization and fewer usable embryos on average, and the men were older compared with couples with other diagnoses. Additionally, the women in MFI couples were younger and had higher antral follicle counts (AFCs) and higher anti-Müllerian hormone (AMH) levels compared with the other women undergoing IVF. When controlling for maternal and paternal ages, AMH and fertilization method (conventional IVF versus ICSI), neither mild nor severe MFI affected duration of individual cleavage stages or overall time to the blastocyst stage, when all or only usable embryos were examined (coefficient 0.44 hours in all embryos, P = 0.57; coefficient 0.39 hours in usable embryos, P = 0.60). Whether the sperm was surgically extracted similarly had no significant effect on embryo morphokinetic parameters. When the fertilization method was assessed independently, ICSI lengthened the overall time to blastocyst stage by 1.66 hours (P = 0.03) on average, primarily due to an increase in duration of the time from 5-cell embryo stage to early blastulation (P5SB). LIMITATIONS, REASONS FOR CAUTION This large cohort study avoided embryo selection bias due to random assignment of embryos to the TLI incubators. However, our findings may not be generalizable to groups under-represented in our clinic population. Future studies should also evaluate the impact of male hormonal status and detailed sperm morphology, such as head versus flagellum defects, on embryo morphokinetic development. WIDER IMPLICATIONS OF THE FINDINGS Our findings suggest that the fertilization method rather than MFI per se impacts time to early blastulation. The clinical implications of this effect on embryo development warrant further investigation. STUDY FUNDING/COMPETING INTEREST(S) There were no sources of funding for this study. There are no competing interests. TRIAL REGISTRATION NUMBER N/A

73 APPLICATION OF THE HOLLOW FIBER VITRIFICATION METHOD TO THE CRYOPRESERVATION OF HIGHLY CRYOSENSITIVE EMBRYOS

We recently demonstrated that the hollow fibre vitrification (HFV) method (Matsunari et al. 2012) could effectively be applied to the cryopreservation of embryos from diverse species. In this study, we applied the HFV method to the cryopreservation of highly cryosensitive specimens, such as in vitro matured (IVM)/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, as well as IVM/IVF-derived cattle embryos at early cleavage stages. Porcine parthenogenetic morulae (d-4) derived from IVM oocytes were treated with 0.25% pronase to remove zona pellucidae. The resulting blastomeres were isolated from the zona-free morulae by a decompaction treatment followed by gentle pipetting. Bovine IVM-IVF embryos at the 2 to 4 cell (d-1), 8 to 16 cell (d-3), and morula stages (d-5) were then subjected to vitrification. The HFV procedure was performed as described previously using 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M trehalose as cryoprotectants. Four to twenty embryos, or all of the blastomeres isolated from a single morula, were individually loaded into a cellulose acetate hollow fibre (25 mm long, 185 μm φ, 15 μm membrane thickness) and vitrified. Survival of the vitrified embryos was assessed by in vitro development to blastocysts. Blastomeres recovered after vitrification were aggregated in micro-wells to examine their ability to form blastocysts. The HFV method was demonstrated to be effective for cryopreserving zona-free in vitro-produced porcine morulae and the blastomeres isolated from them (Table 1), as well as bovine IVM-IVF embryos at early cleavage stages. These data demonstrate that the HFV method is effective for highly cryosensitive specimens, such as IVM/IVF-derived porcine zona-free morulae and blastomeres isolated from those morulae, and IVM/IVF-derived cattle embryos at early cleavage stages. These achievements may expand the technological options in the production of cloned and genetically modified pigs that are useful for biomedical research. Table 1.Survival of zona-free porcine morulae and isolated blastomeres after vitrification (top) and blastocyst formation rates in bovine early-stage in vitro matured-IVF embryos after vitrification (bottom) This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, and MUIIBR.