Acrosome components after intracytoplasmic sperm injection: the decondensation frontier

http://www.sciencedirect.com/science/article/B6T6K-43CBFGC-14/1/c122d3f8e7188ef9ec4a133a8068995

The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes

Mapping Primary Gyrogenesis During Fetal Development in Primate Brains: High-Resolution in Utero Structural MRI of Fetal Brain Development in Pregnant Baboons

The global and regional changes in the fetal cerebral cortex in primates were mapped during primary gyrification (PG; weeks 17–25 of 26 weeks total gestation). Studying pregnant baboons using high-resolution MRI in utero, measurements included cerebral volume, cortical surface area, gyrification index and length and depth of 10 primary cortical sulci. Seven normally developing fetuses were imaged in two animals longitudinally and sequentially. We compared these results to those on PG that from the ferret studies and analyzed them in the context of our recent studies of phylogenetics of cerebral gyrification. We observed that in both primates and non-primates, the cerebrum undergoes a very rapid transformation into the gyrencephalic state, subsequently accompanied by an accelerated growth in brain volume and cortical surface area. However, PG trends in baboons exhibited some critical differences from those observed in ferrets. For example, in baboons, the growth along the long (length) axis of cortical sulci was unrelated to the growth along the short (depth) axis and far outpaced it. Additionally, the correlation between the rate of growth along the short sulcal axis and heritability of sulcal depth was negative and approached significance (r = −0.60; p < 0.10), while the same trend for long axis was positive and not significant (p = 0.3; p = 0.40). These findings, in an animal that shares a highly orchestrated pattern of PG with humans, suggest that ontogenic processes that influence changes in sulcal length and depth are diverse and possibly driven by different factors in primates than in non-primates

Central spindle self-organization and cytokinesis in artificially activated sea urchin eggs

Author Posting. © Marine Biological Laboratory, 2016. This article is posted here by permission of Marine Biological Laboratory for personal use, not for redistribution. The definitive version was published in Biological Bulletin 230, no.2 (2016): 85-95.The ability of microtubules of the mitotic apparatus to control the positioning and initiation of the cleavage furrow during cytokinesis was first established from studies on early echinoderm embryos. However, the identity of the microtubule population that imparts cytokinetic signaling is unclear. The two main––and not necessarily mutually exclusive–– candidates are the central spindle and the astral rays. In the present study, we examined cytokinesis in ammonia-activated sea urchin eggs, which lack paternally derived centrosomes and undergo mitosis mediated by unusual anastral, bipolar mini-spindles. Live cell imaging and immunolabeling for microtubules and the centralspindlin constituent and kinesin-related protein, MKLP1, demonstrated that furrowing in ammonia-activated eggs was associated with aligned arrays of centralspindlin-linked, opposed bundles of antiparallel microtubules. These autonomous, zipper- like arrays were not associated with a mitotic apparatus, but did possess characteristics similar to the central spindle region of control, fertilized embryos. Our results highlight the self-organizing nature of the central spindle region and its ability to induce cytokinesis-like furrowing, even in the absence of a complete mitotic apparatus.This research was supported by student/faculty summer research grants from the Dickinson College Research and Development Committee to JHH; Laura and Arthur Colwin Summer Research Fellowships from the MBL to JHH and CBS; a National Science Foundation Major Research Instrumentation grant to JHH (MRI-0320606); and a NSF collaborative research grant to JHH (MCB-1412688) and to CBS (MCB- 1412734)

Corrigendum to “Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction” [Dev. Biol. 276 (2004) 237–252]

AbstractTherapeutic cloning or nuclear transfer for stem cells (NTSC) seeks to overcome immune rejection through the development of embryonic stem cells (ES cells) derived from cloned blastocysts. The successful derivation of a human embryonic stem cell (hESC) line from blastocysts generated by somatic cell nuclear transfer (SCNT) provides proof-of-principle for “therapeutic cloning,” though immune matching of the differentiated NT-hES remains to be established. Here, in nonhuman primates (NHPs; rhesus and cynomologus macaques), the strategies used with human SCNT improve NHP-SCNT development significantly. Protocol improvements include the following: enucleation just prior to metaphase-II arrest; extrusion rather than extraction of the meiotic spindle-chromosome complex (SCC); nuclear transfer by electrofusion with simultaneous cytoplast activation; and sequential media. Embryo transfers (ET) of 135 SCNT-NHP into 25 staged surrogates did not result in convincing evidence of pregnancies after 30 days post-ET. These results demonstrate that (i) protocols optimized in humans generate preimplantation embryos in nonhuman primates; (ii) some, though perhaps not yet all, hurdles in deriving NT-nhpES cells from cloned macaque embryos (therapeutic cloning) have been overcome; (iii) reproductive cloning with SCNT-NHP embryos appears significantly less efficient than with fertilized embryos; (iv) therapeutic cloning with matured metaphase-II oocytes, aged oocytes, or “fertilization failures” might remain difficult since enucleation is optimally performed prior to metaphase-II arrest; and (v) challenges remain for producing reproductive successes since NT embryos appear inferior to fertilized ones due to spindle defects resulting from centrosome and motor deficiencies that produce aneuploid preimplantation embryos, among other anomalies including genomic imprinting, mitochondrial and cytoplasmic heterogeneities, cell cycle asynchronies, and improper nuclear reprogramming

Sperm Chromatin-Induced Ectopic Polar Body Extrusion in Mouse Eggs after ICSI and Delayed Egg Activation

Meiotic chromosomes in an oocyte are not only a maternal genome carrier but also provide a positional signal to induce cortical polarization and define asymmetric meiotic division of the oocyte, resulting in polar body extrusion and haploidization of the maternal genome. The meiotic chromosomes play dual function in determination of meiosis: 1) organizing a bipolar spindle formation and 2) inducing cortical polarization and assembly of a distinct cortical cytoskeleton structure in the overlying cortex for polar body extrusion. At fertilization, a sperm brings exogenous paternal chromatin into the egg, which induces ectopic cortical polarization at the sperm entry site and leads to a cone formation, known as fertilization cone. Here we show that the sperm chromatin-induced fertilization cone formation is an abortive polar body extrusion due to lack of spindle induction by the sperm chromatin during fertilization. If experimentally manipulating the fertilization process to allow sperm chromatin to induce both cortical polarization and spindle formation, the fertilization cone can be converted into polar body extrusion. This suggests that sperm chromatin is also able to induce polar body extrusion, like its maternal counterpart. The usually observed cone formation instead of ectopic polar body extrusion induced by sperm chromatin during fertilization is due to special sperm chromatin compaction which restrains it from rapid spindle induction and therefore provides a protective mechanism to prevent a possible paternal genome loss during ectopic polar body extrusion

Freeze-dried primate sperm retains early reproductive potential after intracytoplasmic sperm injection

Oocytes inseminated by intracytoplasmic sperm injection using fresh ejaculated or freeze-dried rhesus macaque sperm showed similar activation, sperm aster assembly, and male-female pronuclear apposition rates.http://www.sciencedirect.com/science/article/B6T6K-4NY4Y3G-3/1/b88ff573caaf542e4e022e698231d4b