Several proteins present on the sperm membranes serve as signal transducers facilitating sperm maturation, capacitation, acrosome reaction and fertilization.Among them, guanylate cyclase (GC), the inositol trisphosphate receptor (IP 3 R) and calmodulin have been reported rather extensively.Although biochemical data clearly suggest their roles in the fertilization process, the precise location of these proteins in the sperm membranes have not been adequately investigated and need clarification.Using immunolocalization procedures we report that guanylate cyclase receptors (GCR) are localized on the apical region of the acrosome and on the postacrosomal plasmalemma, while IP 3 Rs are present in the neck region of the sperm and at lower density along the axonemal membrane.In contrast, calmodulin is restricted to the postacrosomal, basal nuclear membrane and the neck region that corresponds to the redundant nuclear envelope and ER vesicles, with some reaction along the middle piece and tail membranes.Colocalization of these proteins on the sperm membrane together with biochemical data as reported previously provide further insight into their role in the spermatozoan motility and possibly the fertilization process.

Boar sperm fixed in paraformaldehyde, pemeabilized in cold acetone and incubated in the presence of DAPI and Rhodamine-conjugated phalloidin or with fluorescein-conjugated cytochalasin D show brilliant blue staining of the nucleus and brilliant red or green fluorescence respectively in the middle piece and tail regions of the sperm. The results suggest the presence of F-actin in the boar sperm middle piece. Immunocytochemical studies also show the presence of tropomyosin in the middle piece region of boar sperm. Western Blot studies, using lysates of isolated boar sperm middle pieces and tails, indicate prominent sperm-actin bands with similar electrophoretic patterns of rat cardiac and striated muscle. The results strongly suggest that a 41 kDa actin, similar to cardiac or striated muscle actin, is present in sperm middle pieces and corroborates the phalloidin and cytochalasin D localization by fluorescence microscopy. We suggest that the simple sliding filament mechanism of the axoneme may not, by itself, impose the propulsive forces needed to propel large spermatozoa through the viscous prefertilization milieu. We further propose that an actomyosin system associated with the microtubule/dynein complex might play a role in sustained motility of mammalian spermatozoa.

Cytosolic casein kinase type II activity has been identified in MCF-7 and MDA-MB-231 human breast cancer cells heterotransplanted into athymic nude mice. Sephacryl S-300 chromatography of MCF-7 and MDA-MB-231 tumor cytosols revealed a major peak of casein kinase activity with an estimated molecular weight of 150,000. This peak was further characterized and optimal conditions for breast tumor casein kinase activity were established. Polylysine (10 micrograms) acted as a potent stimulator with casein as the phosphate acceptor protein. This enzyme used both ATP and GTP as phosphate donors and the Km for GTP was 10 microM. The rate of phosphorylation with increasing concentrations of [gamma-32p]GTP revealed typical Michaelis-Menten kinetics and Vmax was approached at a concentration of 30 microM GTP. MgCl2 stimulated enzyme activity at concentrations between 10-20 mM. Quercetin, a bioflavonoid, inhibited casein kinase type II activity in a dose dependent manner. MCF-7 (hormone-dependent) human breast cancer cells (2-3 X 10(6)) were inoculated into the mammary fat pads of nude mice, supplemented with a 0.5 mg estradiol pellet. To determine the influence of various regulatory agents on casein kinase activity in vivo, tumor-bearing mice were treated for five days with estradiol, progesterone, dexamethasone or tamoxifen. Casein kinase type II was partially purified by gel filtration on a Sephacryl S-300 column and assayed in the presence of polylysine and casein. Dexamethasone treatment significantly decreased casein kinase II activity in MCF-7 tumors, which are receptor-positive for estrogen, androgen and glucocorticoid receptors.

Intracellular calcium release from the endoplasmic reticulum is a hallmark at egg activation of both vertebrates and invertebrates. This fertilization‐associated calcium release results from generation of the second messenger inositol 1,4,5‐trisphosphate (IP3) by one or more phospholipases C (PLC). We characterized Chaetopterus PLCβ and γ by reverse transcription/degenerate oligonucleotide primed PCR and rapid amplification of cDNA end PCR. Phylogenetic analyses suggested that the deduced PLCβ protein shared the greatest homology with mammalian PLCβ4; the deduced PLCγ protein shared the greatest homology with starfish PLCγ and diverged from mammalian PLCγ before mammalian the PLCγ1 and γ2 isoforms diverged. Western blot analyses with specific anti‐PLCβ and γ antibodies, respectively, revealed that 135 and 150 kDa proteins were expressed in eggs. The general PLC antagonist U‐73122 blocked fertilization‐induced egg activation; however, the inactive analog, U‐73343, had no effect on egg activation. We further tested whether egg activation was G protein‐PLCβ and/or protein tyrosine kinase‐PLCγ dependent. Cholera and pertussis toxins, well‐known effectors of G proteins, had no effect on egg activation; while two antagonists of PTK, genistein and tyrphostin B42, inhibited both fertilization‐induced and artificial egg activation. Taken together, our studies suggested that PLC activity from eggs contributes to Chaetopterus egg activation and PLCγ might play an important role during this biological process. Mol. Reprod. Dev. 76: 460–470, 2009. © 2008 Wiley‐Liss, Inc.

Phospholipase C (PLC) is a critical enzyme that is involved in generation of the second messenger inositol-1,4,5-trisphosphate (IP3) for the calcium release that brings about egg activation in vertebrates and invertebrates. In the present study, we for the first time identified a novel PLC isoform, termed cp-PLCγ, in Chaetopterus eggs by reverse transcription/degenerate oligonucleotide primered polymerase chain reaction (RT/DOP-PCR) and rapid amplification of cDNA end polymerase chain reaction (RACE PCR) strategies. Cp-PLCγ is expressed as a 150kDa protein in unfertilized and fertilized Chaetopterus eggs. The general PLC antagonist U-73122 blocked egg activation in response to fertilization. However, the inactive analog, U-73343, had no effect on early embryonic development. We further determined that protein tyrosine kinase (PTK) antagonists, genistein and tyrophostein B42, inhibit both normal and parthenogenetic egg activation in a dose-dependent manner. These data indicates that PLC activity from the eggs is required for egg activation and that PLCγ may play an important role by PTK-PLCγ signal cascade. This research was supported by the NSF HRD-0401697

Melanoma Inhibitory Activity (MIA) is a small, secreted protein normally expressed in chondrocytes and appears to be a pivotal player in their differentiation. Initially identified as a tumor suppressor, MIA is now a major metastatic marker for melanoma and has been identified as a key molecule in melanoma tumor progression. Melanoma patients expressing high levels of serum MIA have a poor prognosis and exhibit a reduced response to various chemotherapeutic agents. This observation prompted us to look for a correlation between MIA levels and metastatic potential in breast cancer cell lines. The cell lines chosen ranged from minimally to highly metastatic. We examined the expression and localization of MIA by western blot, RT-PCR and immunofluorescence. Western blot and RT-PCR detected MIA in the MDA-MB 435, MDA-MB 231, MCF-7, SKBR3, T-47 D and SKMEL5 (melanoma control) cell lines. RT-PCR and western blot analysis revealed relatively high MIA mRNA and protein levels respectively in MDA MB 435 and MDA MB 231, the two most metastatic cell lines. MIA was detected as diffuse staining throughout the cytoplasm of the breast cancer cells. Analyses of preliminary data suggest a positive correlation between MIA expression and tumor progression in these cell lines as in melanoma and some other cancer cells. Hence, MIA has the potential of being a predictive marker for patients’ outcomes in breast cancer. Supported by: NSF HRD-0401697

Ecological developmental biology (Eco-Devo) involves the study of development in its natural environmental context as opposed to the laboratory setting. Ernest E. Just was an early 20th century African-American embryologist who devoted his career to studying the early development of marine invertebrates in the United States and abroad. Through detailed study of the fertilization process, he came to see the cell cortex as playing a central role in development, inheritance, and evolution. This paper, after presenting some of Just's scientific and philosophical contributions, argues that Just was an Eco-Devo biologist. Three lines of evidence are given. First, Just believed that intimate knowledge of the natural history of the marine animal under study – hence, the natural setting in which fertilization occurs – was essential. Second, he stressed the importance of the egg's “normality”—how well its condition in the laboratory corresponds to the natural, fertilizable state. Finally, Just was an organicist, believing that organisms are holistic systems with emergent properties that arise from their organization and complexity. Although other scientists may stand out more clearly as founding architects of Eco-Devo, E. E. Just, with his unwavering insistence on the normality and holistic integrity of the egg cell, was one of its purest adherents.

We report that protein kinase C (PKC) plays a regulatory role in early cleavage in Chaetopterus eggs. Using Western blotting, we assayed the expression patterns of conventional PKCs (cPKC), novel PKCs (nPKC), and atypical PKCs (aPKC). During early development after fertilization, PKC protein levels varied independently by isoform. PKC protein expression during differentiation, without cleavage and after parthenogenetic activation, was very similar to that during normal development indicating that PKC gene expression does not require cellularization. Since PKC has been shown to regulate meiosis in this organism, we also assayed the membrane association of these isoforms as an indicator of their activation during meiosis and early cleavage. PKC‐γ transiently associated with membranes and therefore became activated before meiotic division and cleavage, whereas PKC‐α and ‐β transiently dissociated from membranes and therefore became inactivated at these times. Inhibition of these PKC isoforms by bisindolylmaleimide I had no effect on cleavage or early development to the trochophore larva, indicating that PKC‐γ activation is not essential for cleavage or early development. However, their persistent activation by thymeleatoxin blocked cleavage. The results indicate that the dissociation of PKC‐α and/or ‐β from the membrane fraction, and therefore their inactivation, is essential for normal cleavage. Elevated PKC activity is essential for nuclear envelope breakdown and spindle formation at meiosis I. By contrast, down‐regulation of this activity is essential for cleavage after fertilization. Mol. Reprod. Dev. 69: 308–315, 2004. © 2004 Wiley‐Liss, Inc.

Changes in protein tyrosine phosphorylation are an essential aspect of egg activation after fertilization. Such changes result from the net contributions of both tyrosine kinases and phosphatases (PTP). This study was conducted to determine what role(s) PTP may have in egg activation. We identified four novel PTP in Chaetopterus pergamentaceus oocytes, cpPTPNT6, cpPTPNT7, cpPTPR2B, and cpPTPR2A, that have significant homology to, respectively, human PTPσ, ‐ρ, ‐D2 and ‐BAS. The first two are cytosolic and the latter two are transmembrane. Several PTP inhibitors were tested to see if they would affect Chaetopterus pergamentaceus fertilization. Eggs treated with β‐bromo‐4‐hydroxyacetophenone (PTP inhibitor 1) exhibited microvillar elongation, which is a sign of cortical changes resulting from activation. Those treated with Na3VO4 underwent full parthenogenetic activation, including polar body formation and pseudocleavage and did so independently of extracellular Ca2+, which is required for the Ca2+ oscillations that initiate development after fertilization. Fluorescence microscopy identified phosphotyrosine‐containing proteins in the cortex and around the nucleus of vanadate‐activated eggs, whereas in fertilized eggs they were concentrated only in the cortex. Immunoblots of vanadate‐activated and fertilized eggs showed tyrosine hyperphosphorylation of approximately140 kDa protein. These results suggest that PTP most likely maintain the egg in an inactive state by dephosphorylation of proteins independent of the Ca2+ oscillations in the activation process.

Egg activation in all animals evidently requires the synthesis of inositol 1,4,5-trisphosphate (InsP(3)) from phosphatidylinositol 4,5-bisphosphate (PIP(2)) by phospholipase C (PLC). Depending on the organism, InsP(3) elicits either calcium oscillations or a single wave, which in turn initiates development. A soluble component in boar sperm that activates mammalian eggs has been suggested to be a PLC isoform. We tested this hypothesis in vitro using egg microsomes of Chaetopterus. Boar sperm factor elicited Ca(2+) release from the microsomes by an InsP(3)-dependent mechanism. The PLC inhibitor U-73122, but not its inactive analog U-73343, blocked the response to sperm factor but not to InsP(3). U-73122 also inhibited the activation of fertilized and parthenogenetic eggs. Chaetopterus sperm also contained a similar activity. These results strongly support the hypothesis that sperm PLCs are ubiquitous mediators of egg activation at fertilization.

We report cellular hypertrophy, mitochondrial proliferation and differentiation, myofibrillogenesis, and junctional maturation in cardiac progenitor cells between the 8 and 26-somite stages of the zebrafish, Danio rerio. However, coordinated contraction in embryonic cardiomyocytes did not occur until 26-somite stage when 'developed' intercalated discs, rooted sarcomeres, and a well-established sarcoplasmic reticulum had differentiated.

The luminal fluid of estrogen or DES-stimulated uterus of immature rats contains 10-12 isoforms of peroxidase between pI 4.5-6.0. N-terminal amino acid sequencing of diaminobenzidine-peroxidase bands eluted from IEF and SDS-PAGE gels showed the presence of cathepsin B and the complement family of proteins as the major comigrants. Sequential treatment of uterine fluid by cation, anion, and size exclusion chromatography resulted in a five-fold purification of peroxidase having a specific activity of 273 units/mg. Mass spectrometric studies of bands isolated from SDS-PAGE gels from the size-exclusion purified peroxidase fraction showed the presence of complement C3 along with novel previously uncharacterized proteins. Two dimensional electrophoresis followed by N-terminal amino acid sequencing confirmed the presence of cathepsin B isoforms and isoforms of a novel protein at approximately 87 kDa. Identification by mass spectrometry from the database for this novel protein was inconclusive but could most likely be a candidate for estrogen-induced peroxidase. Results conclusively prove that cathepsin B and complement C3 are major proteins in the estrogen-induced peroxidase fraction of uterine fluid.

Increases in the intracellular free calcium concentration are of great importance to the initiation of development in deuterostomes. Their involvement has not yet been clearly defined in protostomes. We used endogenous ligands (IP3, cADPR, ryanodine and NAADP) and pharmacological agents (thapsigargin [Tg], thimerosal, caffeine and heparin) to study smooth endoplasmic reticulum Ca2+ pump and release mechanisms in eggs of an annelid, Chaetopterus. Oocyte homogenates effectively sequestered Ca2+ and released it in response to IP3 in a concentration-dependent manner. Repeated additions of IP3 were unable to cause further release. Heparin inhibited Ca2+ release in response to IP3. The homogenates also released Ca2+ in response to thimerosal, and this release was sensitive to heparin. Two antibodies to IP3 receptors recognized an appropriate band in Chaetopterus egg lysates. These results indicate that the oocytes possess type-1 IP3-gated Ca2+ channels. Neither calcium itself, nor strontium, cADPR, ryanodine, caffeine nor NAADP released appreciable Ca2+. At low concentrations, Tg caused a slow release of Ca2+; at higher concentrations, it elicited a rapid release. Release of Ca2+ by Tg activated development. Since one theory of fertilization invokes the introduction of a Ca2+ releasing soluble protein into the egg upon sperm-egg fusion, we also tested whether soluble extracts of Chaetopterus sperm could stimulate Ca2+ release in Chaetopterus egg homogenates. There was no Ca2+ release when the sperm extract was added to the homogenate; however, homogenates exposed to sperm extract became refractory to IP3. Thus, Ca2+ release at fertilization in these oocytes occurs through IP3-gated channels.

Intracellular pH (pHi) measurements were performed in surf clam (Spisula solidissima) oocytes before and after artificial activation or fertilization [evidenced by germinal vesicle breakdown (GVBD)] by the dimethyloxazolidinedione (DMO) and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) methods. Results using both methods showed increases of pHi of 0.3 pH unit after activation by excess K+. Using BCECF, we found an increase of similar magnitude after fertilization or after the addition of serotonin. By contrast, GVBD did not occur when the pHi was increased to similar or even higher levels by exposing the oocytes to ammonia. In sodium-free seawater, excess K+ induced GVBD but the pHi of K+-activated oocytes decreased significantly below the resting level of unactivated oocytes. The pHi increases in K+-activated oocytes were otherwise proportional to the external Na+ concentration. The amiloride derivatives dimethylamiloride and hexamethylene amiloride (at 10-50 microM) efficiently inhibited the K+-induced increase of pHi but did not block GVBD. These two derivatives were able, however, to retard K+-induced GVBD, hexamethylene amiloride being the more efficient. This retardation of K+-induced GVBD could be abolished by the simultaneous addition of ammonia. Taken altogether, these results show that a pHi increase, driven by a typical Na+/H+ exchanger, follows activation of surf clam oocytes but that this pHi increase is neither sufficient nor required for GVBD, though it does allow its progression at an optimal rate.

In frog oocytes, activation of mitogen-activated protein kinase (MAPK, ERK) leads to activation of cdc2 and germinal vesicle breakdown (GVBD). By contrast, in starfish, MAPK is activated after GVBD. Here we have examined the relative involvements of MAPK and cdc2 in GVBD ofChaetopterusoocytes. MAPK was rapidly tyrosine-phosphorylated and activated (within 1–2 min) in response to exposure of the oocytes either to natural seawater (the normal trigger of GVBD in this organism) or to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which can also elicit GVBD. This response preceded the tyrosine dephosphorylation and activation of cdc2 by several minutes. MAPK phosphorylation and activation were transient, lasting only until GVBD occurred and the spindle migrated to the cortex. The enzyme was not phosphorylated again as a result of egg activation. These results are consistent with the hypothesis that the activation of MAPK has a role in GVBD. However, PD 98059, a potent and selective inhibitor of MEK, the protein kinase that phosphorylates and activates MAPK, blocked the phosphorylation of MAPK but did not block GVBD, the dephosphorylation and activation of cdc2, or spindle formation and migration. Oocytes that underwent GVBD in PD 98059 could be fertilized and cleaved normally. Ionophore A23187, although it caused germinal vesicles to disappear and caused transient phosphorylation of MAPK, did not cause dephosphorylation of cdc2, and therefore this disappearance is artifactual. These results suggest that MAPK activation is neither obligatory nor sufficient for either GVBD or meiotic metaphase arrest inChaetopterusand that activation of MAPK and cdc2 occur on independent, parallel pathways.

Binding of estrogen to its receptor (ER) activates early genes that drive responsive cells through the proliferative phase. Earlier studies to evaluate the expression of protooncogenes, growth factors, growth factor receptor and steroid hormone receptor gene activities in the rat uterine system indicated complex pathways that involve significant 'crosstalk' between ER-systems and signal transduction pathways (Bhattacharyya et al., 1994). To analyze the interactions between these factors, we examined two well characterized estrogen dependent (MCF-7) and estrogen independent (MDA-MB-231) human breast cancer cell lines. Antibodies to estrogen receptor, epidermal growth factor receptor, c-Fos, c-Jun, and Ras proteins, protein kinases involved in receptor tyrosine kinase signal transduction pathway, MEK1 and phosphotyrosine were utilized in immunocytochemical localization experiments to evaluate temporal expression of these factors in response to estrogen treatment. ER, which was diminished in MCF-7 cells grown in estrogen-stripped medium, increased 9-fold in estrogen-reconstituted medium by 120 min. Fos and Jun appeared at nuclear and perinuclear cytoplasmic sites within 60 min after estrogen treatment in MCF-7 cells. Fos/Jun proteins were prominent in MDA-MB-231 cells, especially in association with actin filaments. Immunolabeling studies revealed no EGF-r in MCF-7 cells, while MDA-MB-231 cells contained intense EGF-r labeling in the plasma membrane. Ras protein was prominent in the cytoplasm and at the cell surface within 60 min after treatment of MCF-7 cells with estrogen. Ras was intense in MDA cells. Similarly, MCF-7 and MDA cells contained high concentrations of MEK1 and phosphotyrosine (pTyr) containing proteins in their cytoplasm and immunolabeling remained high as long as MCF-7 cells were grown in medium containing estrogen. It is speculated that MEK1 (cytoplasmic) functioning through Fos/Jun or Myc/Max (nuclear) may regulate the activity of AP-1 transcription factor. In all cases however, MEK1 and pTyr protein labeling was more intense in the highly metastatic and hormone independent MDA-MB-231 breast cancer cells. Results revealed signal transduction pathway proteins in ER+ estrogen dependent cells suggesting possible crosstalk between both receptor pathways during the proliferative phase of MCF-7 cells.

Previous articleNext article No AccessFERTILIZATION AND DEVELOPMENTAL BIOLOGYIntracellular pH Measurements During Fertilization of Surf Clam (Spisula solidissima) OocytesF. Dube and W. R. EckbergF. Dube Search for more articles by this author and W. R. Eckberg Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 191, Number 2October 1996 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.1086/BBLv191n2p279 Views: 19Total views on this site Copyright © 1996 by Marine Biological LaboratoryPDF download Crossref reports no articles citing this article.

Previous articleNext article No AccessFERTILIZATION AND DEVELOPMENTAL BIOLOGYEffects of Altering pHi and pHo on the Activation of Chaetopterus EggsW. R. Eckberg and F. DubeW. R. Eckberg Search for more articles by this author and F. Dube Search for more articles by this author PDFPDF PLUS Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Biological Bulletin Volume 191, Number 2October 1996 Published in association with the Marine Biological Laboratory Article DOIhttps://doi.org/10.1086/BBLv191n2p280 Views: 10Total views on this site Copyright © 1996 by Marine Biological LaboratoryPDF download Crossref reports no articles citing this article.