Yasutaka Kakiuchi

Most fertilizing mouse spermatozoa begin their acrosome reaction before contact with the zona pellucida during in vitro fertilization

To fuse with oocytes, spermatozoa of eutherian mammals must pass through extracellular coats, the cumulus cell layer, and the zona pellucida (ZP). It is generally believed that the acrosome reaction (AR) of spermatozoa, essential for zona penetration and fusion with oocytes, is triggered by sperm contact with the zona pellucida. Therefore, in most previous studies of sperm–oocyte interactions in the mouse, the cumulus has been removed before insemination to facilitate the examination of sperm–zona interactions. We used transgenic mouse spermatozoa, which enabled us to detect the onset of the acrosome reaction using fluorescence microscopy. We found that the spermatozoa that began the acrosome reaction before reaching the zona were able to penetrate the zona and fused with the oocytes plasma membrane. In fact, most fertilizing spermatozoa underwent the acrosome reaction before reaching the zona pellucida of cumulus-enclosed oocytes, at least under the experimental conditions we used. The incidence of in vitro fertilization of cumulus-free oocytes was increased by coincubating oocytes with cumulus cells, suggesting an important role for cumulus cells and their matrix in natural fertilization.

Why small males have big sperm: dimorphic squid sperm linked to alternative mating behaviours

BackgroundSperm cells are the target of strong sexual selection that may drive changes in sperm structure and function to maximize fertilisation success. Sperm evolution is regarded to be one of the major consequences of sperm competition in polyandrous species, however it can also be driven by adaptation to the environmental conditions at the site of fertilization. Strong stabilizing selection limits intra-specific variation, and therefore polymorphism, among fertile sperm (eusperm). Here we analyzed reproductive morphology differences among males employing characteristic alternative mating behaviours, and so potentially different conditions of sperm competition and fertilization environment, in the squid Loligo bleekeri.ResultsLarge consort males transfer smaller (average total length = 73 μm) sperm to a females internal sperm storage location, inside the oviduct; whereas small sneaker males transfer larger (99 μm) sperm to an external location around the seminal receptacle near the mouth. No significant difference in swimming speed was observed between consort and sneaker sperm. Furthermore, sperm precedence in the seminal receptacle was not biased toward longer sperm, suggesting no evidence for large sperm being favoured in competition for space in the sperm storage organ among sneaker males.ConclusionsHere we report the first case, in the squid Loligo bleekeri, where distinctly dimorphic eusperm are produced by different sized males that employ alternative mating behaviours. Our results found no evidence that the distinct sperm dimorphism was driven by between- and within-tactic sperm competition. We propose that presence of alternative fertilization environments with distinct characteristics (i.e. internal or external), whether or not in combination with the effects of sperm competition, can drive the disruptive evolution of sperm size.

Sperm from Sneaker Male Squids Exhibit Chemotactic Swarming to CO2

Behavioral traits of sperm are adapted to the reproductive strategy that each species employs. In polyandrous species, spermatozoa often form motile clusters, which might be advantageous for competing with sperm from other males. Despite this presumed advantage for reproductive success, little is known about how sperm form such functional assemblies. Previously, we reported that males of the coastal squid Loligo bleekeri produce two morphologically different euspermatozoa that are linked to distinctly different mating behaviors. Consort and sneaker males use two distinct insemination sites, one inside and one outside the females body, respectively. Here, we show that sperm release a self-attracting molecule that causes only sneaker sperm to swarm. We identified CO2 as the sperm chemoattractant and membrane-bound flagellar carbonic anhydrase as its sensor. Downstream signaling results from the generation of extracellular H(+), intracellular acidosis, and recovery from acidosis. These signaling events elicit Ca(2+)-dependent turning behavior, resulting in chemotactic swarming. These results illuminate the bifurcating evolution of sperm underlying the distinct fertilization strategies of this species.

Reduction of polar auxin transport in tobacco by the tumorigenic Agrobacterium tumefaciens AK-6b gene

The plant-tumorigenic 6b (AK-6b) gene of Agrobacterium tumefaciens strain AKE10 induces morphological alterations to tobacco plants, Nicotiana tabacum. To investigate the molecular mechanisms underlying these processes, we generated transgenic tobacco harboring the AK-6b gene under the control of a dexamethazone-inducible promoter. Upon induction, transgenic tobacco seedlings exhibited distinct classes of aberrant morphologies, most notably adventitious outgrowths and stunted epicotyls. Histological analysis revealed massive proliferation and altered venation in the newly established outgrowths. Prominent vascular development suggested that auxin metabolism or signaling had been altered. Indeed, basipetal auxin transport in the hypocotyls of the transgenic seedlings was reduced by 50–80%, whereas intracellular auxin contents were only slightly reduced. Analysis of cell extracts by HPLC revealed a large accumulation of phenolic compounds, including the flavonoid kaempferol-3-rutinoside, in transgenic plants compared with wild-type seedlings. As some naturally occurring flavonoids have been shown to affect auxin transport, we suggest that the AK-6b gene expression impairs auxin transport via modulation of phenylpropanoid metabolism, and ultimately results in the observed morphological alterations.

STAT3 and PKC differentially regulate telomerase activity during megakaryocytic differentiation of K562 cells.

Telomerase is active in immature somatic cells, but not in differentiated cells. However, the regulation during cell differentiation is not well understood. In this study, a human chronic myelogenous leukemia cell line (K562) was induced to differentiate into megakaryocytes by TPA, and erythroid by STI571. A human acute myeloblastic leukemia cell line (HL60) was also induced to differentiate into monocytes by TPA and VD3, and granulocyte by ATRA. TPA induced transient increase of telomerase activity (mainly nuclear fraction) during megakaryocytic differentiation, while the expression of hTERT decreased gradually throughout the same period. Pretreatment with PKC inhibitors inhibited the megakaryocytic differentiation, transient increase of telomerase activity, while recombinant PKC increased telomerase activity. ChIP assay resulted STAT3 and STAT5 dissociated from the hTERT promoter, indicating that STAT3 and STAT5 are one of the transcriptional regulators. These results suggest that telomerase activity is regulated by two mechanisms during megakaryocytic differentiation.

Antinociceptive effect of cyclic phosphatidic acid and its derivative on animal models of acute and chronic pain

1. AbstractBackgroundCyclic phosphatidic acid (cPA) is a structural analog of lysophosphatidic acid (LPA), but possesses different biological functions, such as the inhibition of autotaxin (ATX), an LPA-synthesizing enzyme. As LPA is a signaling molecule involved in nociception in the peripheral and central systems, cPA is expected to possess analgesic activity. We characterized the effects of cPA and 2-carba-cPA (2ccPA), a chemically stable cPA analog, on acute and chronic pain.Results(1) The systemic injection of 2ccPA significantly inhibited somato-cardiac and somato-somatic C-reflexes but not the corresponding A-reflexes in anesthetized rats. (2) 2ccPA reduced sensitivity measured as the paw withdrawal response to electrical stimulation applied to the hind paws of mice through the C-fiber, but not Aδ or Aβ. (3) In mice, pretreatment with 2ccPA dose-dependently inhibited the second phase of formalin-induced licking and biting responses. (4) In mice, pretreatment and repeated post-treatments with 2ccPA significantly attenuated thermal hyperalgesia and mechanical allodynia following partial ligation of the sciatic nerve. (5) In rats, repeated post-treatments with 2ccPA also significantly attenuated thermal hyperalgesia and mechanical allodynia following chronic sciatic nerve constriction.ConclusionsOur results suggest that cPA and its stable analog 2ccPA inhibit chronic and acute inflammation-induced C-fiber stimulation, and that the central effects of 2ccPA following repeated treatments attenuate neuropathic pain.

Akt and PKC are involved not only in upregulation of telomerase activity but also in cell differentiation-related function via mTORC2 in leukemia cells

We have shown previously that PI3K/Akt pathway is active after cell differentiation in HL60 cells. In the present study, we have investigated whether additional molecules, such as protein kinase C (PKC), are involved in the regulation, not only of telomerase, but also of leukemia cell differentiation. We show that PKC activates telomerase and is, itself, activated following VD3- or ATRA-induced differentiation of HL60 cells, as was observed for PI3K/Akt. To clarify the significance of PI3K/Akt and PKC pathway activation in leukemia cell differentiation, we examined the active proteins in either the downstream or upstream regulation of these pathways. In conjunction with the activation of Akt or PKC, mTOR and S6K were phosphorylated and the protein expression levels of Rictor were increased, compared with Raptor, following cell differentiation. Silencing by Rictor siRNA resulted in the attenuation of Akt phosphorylation on Ser473 and PKCα/βII phosphorylation, as well as the inhibition of Rictor itself, suggesting that Rictor is an upstream regulator of both Akt and PKC. In addition, in cells induced to differentiate by ATRA or VD3, Nitroblue-tetrazolium (NBT) reduction and esterase activity, were blocked either by LY294002, a PI3K inhibitor, or by BIM, a PKC inhibitor, without affecting cell surface markers such as CD11b or CD14. Intriguingly, the silencing of Rictor by its siRNA also suppressed the reducing ability of NBT following VD3-induced cell differentiation. Taken together, our results show that Rictor associated with mTOR (mTORC2) regulates the activity of both Akt and PKC that are involved in cell functions such as NBT reduction and esterase activity induced by leukemia cell differentiation.

Modulation of the venation pattern of cotyledons of transgenic tobacco for the tumorigenic 6b gene of Agrobacterium tumefaciens AKE10

Neoplastic plant-tissue formation, termed crown gall disease, is induced on infection with Agrobacterium tumefaciens. The tumorous tissues develop an extensive vascular system, with a venation pattern distinct from that of native host plants. We report here that the plant-tumorigenic 6b gene of the A. tumefaciens strain AKE10 is capable of inducing extensive vein formation in transgenic tobacco seedlings with distinct pattern formation. Unlike the wild-type cotyledons, transgenic cotyledons had wavy and striate veins depending on the extent of severity of leaf morphology. Graph analysis of the transgenic cotyledonous vein patterns revealed an increase in the number of branch points of veins, end-points of veins, and areas surrounded by the veins. Histological analysis showed abnormal tissue growth on the abaxial side of the cotyledon blades and continual formation of adventitious veins. These adventitiously formed veins included inverted dorso-ventrality and formation of a radial axis.

Multiple oscillations in changing cell shape by the plasmodium of Physarum polycephalum: general formula governing oscillatory phenomena by the Physarum plasmodium

Abstract The long-term dynamics of an amoeboid cell shape were studied using Physarum polycephalum plasmodia with various sizes. Cell shape varied oscillatorily in a multiple periodic manner. The organism periodically elongated with period of T 7 = 10 h, branched with T 6 = 4 h, became uneven with T 5 = 30 min and T 4 = 10 min, and blew up with T 3 = 1.5 min. Tiny plasmodia changed shape much faster with T 3 = 1.3 min, T 2 = 24 s and T 1 = 3.3 s simultaneously. The plasmodial cytoskeleton also showed periodic pattern formation with T 6, T 5 and T 3. Periods of all known oscillatory phenomena in this organism correspond to some of the periods for the above seven rhythms, and the following geometric progression holds among the periods: Ti + 1/Ti = 7 and Ti + 2/Ti + 1 = 3, where i = 1, 3, 5. Thus, multiple oscillations in the plasmodium are organized globally.

MOV10 as a novel telomerase-associated protein

A novel telomerase-associated protein was isolated from porcine testis. The 115-kDa protein, purified with telomerase activity, was molecular cloned using human cDNA library, and identified as MOV10. The expression levels of both MOV10 mRNA and MOV10 protein in cancer cells were 2-3 times higher than that of the normal cells, and MOV10 mRNA was highly expressed in human testis and ovary. The anti-MOV10 antibody precipitated the telomerase activity from cancer cell extracts, and inhibited the telomerase activity in vitro. Sf9-expressed MOV10 protein bound to G-rich strand of both single- and double-stranded telomere-sequenced DNA, but not to single C-rich strand. ChIP assay showed the binding of MOV10 to telomere region in vivo. These data suggest that MOV10 is involved in the progression of telomerase-catalyzing reaction via the interaction of telomerase protein and telomere DNA.