Transcriptomic analysis of granulosa cell populations proximal and distal to the germinal disc of chicken preovulatory follicles.

Within the oocytes of chicken preovulatory follicles, the engulfed yolk constitutes 99% of the oocyte content, while the small germinal disc (GD) (which contains the nucleus and 99% ooplasm) occupies only less than 1%. Relative to the position of the GD, the single granulosa cell layer surrounding the oocyte can be sub-divided into two sub-populations: granulosa cells proximal (named Gp cells) and distal (Gd cells) to the GD. It was reported that Gp cells and Gd cells differ in their morphology, proliferative rate and steroidogenic capacity, however, the underlying mechanism controlling granulosa cell heterogeneity remains unclear. Here we analyzed the transcriptomes of Gd and Gp cells of preovulatory (F5 and F1) follicles in chicken ovaries. We found that: (1) genes associated with cell cycle and DNA replication (CDK1, CCNB3 etc.) have comparatively higher expression levels in Gp cells than in Gd cells, while genes associated with steroidogenesis (CYP51A1, DHCR24) are highly expressed in Gd cells, indicating that Gp cells are likely more mitotic and less steroidogenic than Gd cells; (2) genes associated with extracellular matrix remodeling, cell adhesion and sperm binding (ZP3, ZP2) are differentially expressed in Gp and Gd cells; (3) Furthermore, signaling molecules (WNT4/IHH) and receptors for NGF (NGFR), epidermal growth factor (EGFR), gonadotropins (FSHR/LHR) and prostaglandin (PTGER3) are abundantly but differentially expressed in Gp and Gd cells. Taken together, our data strongly supports the notion that Gp and Gd cells of preovulatory follicles differ in their proliferation rate, steroidogenic activity, ECM organization and sperm binding capacity, which are likely controlled by gonadotropins and local ovarian factors, such as GD-derived factors.

Androgen and mineralocorticoid receptors are present on the germinal disc region in laying hens: Potential mediators of sex ratio adjustment in birds?

Female birds skew offspring sex ratios based on environmental and social stimuli; however, the mechanism mediating this phenomenon remains unknown. Growing evidence suggests that testosterone and corticosterone may influence meiosis, as they skew sex ratios when given immediately before chromosomal segregation. It is unclear if these hormones act on the germinal disc (GD) or through a downstream mediator. It is also unknown whether the GD contains receptors for these hormones. If testosterone and/or corticosterone act on the GD to skew sex ratios, then the GD should have receptors for them and that receptor levels should be higher in the GD regions compared to other follicular regions. Furthermore, fluctuations of receptor levels should occur near meiotic segregation. We collected ovarian follicles at 5 h pre-ovulation (just before meiotic segregation) and 20 h pre-ovulation (when sex chromosomes are arrested), and measured androgen receptor (AR) and mineralocorticoid receptor (MR) protein levels via Western blot. ARs and MRs were on the follicle in the GD and non-GD regions, and at 5 h and 20 h pre-ovulation. Both AR and MR protein levels were higher in the GD region than the non-GD region at both time points, but did not differ between time points. These results suggest that hen ovarian follicles have receptors for testosterone and corticosterone, and that the ability for testosterone to respond may be specifically higher in the GD-region, providing further support for the role of testosterone in the alteration of meiotic segregation.

Modulation of F-actin dynamics by maternal Mid1ip1L controls germ plasm aggregation and furrow recruitment in the zebrafish embryo.

During the early embryonic cell cycles, zebrafish germ plasm ribonucleoparticles (RNPs) gradually multimerize and become recruited to the forming furrows. RNPs multimerization occurs prior to and during furrow initiation, as forming aggregates move outward through their association with the tips of growing interphase astral microtubules. Germ plasm RNPs are also associated with short cortical F-actin. We show that, in embryos mutant for the cytoskeletal regulator mid1ip1l, germ plasm RNPs fail to become recruited to the furrow, accumulating instead at the periphery of the blastodisc. RNP aggregates are associated with zones of mid1ip1l-dependent cyclical local cortical F-actin network enrichments, as well as contractions at both the cortex and the contractile ring. F-actin inhibition in wild-type embryos mimics the RNP peripheral accumulation defect of mid1ip1l mutants. Our studies suggest that a common mechanism underlies distinct steps of germ plasm RNP segregation. At the cortex, this process attenuates microtubule-dependent outward RNP movement to retain RNPs in the blastodisc cortex and allow their recruitment to the furrows. F-actin network contraction likely also facilitates higher-order germ plasm RNP multimerization.

Localization of epithelial sodium channel (ENaC) and CFTR in the germinal epithelium of the testis, Sertoli cells, and spermatozoa.

Spermatogenesis starts within the seminiferous tubules of the testis by mitotic division of spermatogonia that produces spermatocytes. Meiotic division of these spermatocytes produces haploid spermatids that differentiate into spermatozoa. In this study, we examined the expression of ENaC and CFTR (a Cl- channel) in rat testicular sections using confocal microscopic immunofluorescence. The structural integrity of the seminiferous tubule sections was verified by precise phalloidin staining of the actin fibers located abundantly at both basal and adluminal tight junctions. The acrosome forming regions in the round spermatids were stained using an FITC coupled lectin (wheat germ agglutinin). In all phases of the germ cells (spermatogonia, spermatocytes, and spermatids) ENaC was localized in cytoplasmic pools. Prior to spermiation, ENaC immunofluorescence appeared along the tails of the spermatids. In spermatozoa isolated from the epididymis, ENaC was localized at the acrosome and a central region of the sperm flagellum. The mature sperm are transcriptionally silent. Hence, we suggest that ENaC subunits in cytoplasmic pools in germ cells serve as the source of ENaC subunits located along the tail of spermatozoa. The locations of ENaC is compatible with a possible role in the acrosomal reaction and sperm mobility. In contrast to ENaC, CFTR immunofluorescence was most strongly observed specifically within the Sertoli cell nuclei. Based on the nuclear localization of CFTR we suggest that, in addition to its role as an ion channel, CFTR may have an independent role in gene regulation within the nuclei.

Germinal Center B-Cell-Associated Nuclear Protein (GANP) Involved in RNA Metabolism for B Cell Maturation.

Germinal center B-cell-associated nuclear protein (GANP) is upregulated in germinal center B cells against T-cell-dependent antigens in mice and humans. In mice, GANP depletion in B cells impairs antibody affinity maturation. Conversely, its transgenic overexpression augments the generation of high-affinity antigen-specific B cells. GANP associates with AID in the cytoplasm, shepherds AID into the nucleus, and augments its access to the rearranged immunoglobulin (Ig) variable (V) region of the genome in B cells, thereby precipitating the somatic hypermutation of V region genes. GANP is also upregulated in human CD4(+) T cells and is associated with APOBEC3G (A3G). GANP interacts with A3G and escorts it to the virion cores to potentiate its antiretroviral activity by inactivating HIV-1 genomic cDNA. Thus, GANP is characterized as a cofactor associated with AID/APOBEC cytidine deaminase family molecules in generating diversity of the IgV region of the genome and genetic alterations of exogenously introduced viral targets. GANP, encoded by human chromosome 21, as well as its mouse equivalent on chromosome 10, contains a region homologous to Saccharomyces Sac3 that was characterized as a component of the transcription/export 2 (TREX-2) complex and was predicted to be involved in RNA export and metabolism in mammalian cells. The metabolism of RNA during its maturation, from the transcription site at the chromosome within the nucleus to the cytoplasmic translation apparatus, needs to be elaborated with regard to acquired and innate immunity. In this review, we summarize the current knowledge on GANP as a component of TREX-2 in mammalian cells.

Observations of turkey eggs stored up to 27 days and incubated for 8 days: embryo developmental stage and weight differences and the differentiation of fertilized from unfertilized germinal discs.

For logistical reasons, egg storage prior to incubation is a growing practice in the commercial turkey industry. Yet the consequence of increasing egg storage over 7 d is a progressive increase in embryo mortality. The objective of this study was to provide the information necessary to differentiate an early dead embryo from an unfertilized egg after 8 days of incubation (DOI). Five groups of eggs each from inseminated and virgin hens were stored for progressively increasing periods of time (5-d or less, 6 to 10 d, 11 to 15 d, 16 to 20 d, and 21 to 27 d) and incubated. At 8 DOI, eggs were examined and the stage of development (Hamburger and Hamilton, 1951) and embryo weights in normally developed eggs were determined. There was a significant negative correlation between the stage of development and embryo weight with increasing storage periods. All remaining eggs from the inseminated and virgin hens were broken-out and the appearance of the yolk and the fertilized and unfertilized germinal discs examined. The yolks of both hen groups with unfertilized ova maintained a homogeneous uniform yellow-orange color. In contrast, yolks of ova that had been fertilized, with or without early-dead embryos, and yolks from virgin hens that showed evidence of parthenogenetic development (3%) had a heterogeneous appearance. Using fluorescence microscopy, the heterogeneous appearance was due to sheets of aberrant cells and less frequently dispersed cells and folds of the perivitelline layer. It was concluded that clear egg breakouts need to be performed to more accurately assess the impact of egg storage on embryonic mortality. Furthermore, such breakouts should be performed with a high intensity light directed across the surface of the germinal disc to clearly differentiate the subtle differences between an early-dead embryo and an unfertilized germinal disc.

Association of Egg Mass and Egg Sex: Gene Expression Analysis from Maternal RNA in the Germinal Disc Region of Layer Hens (Gallus gallus).

Female birds have been shown to manipulate offspring sex ratio. However, mechanisms of sex ratio bias are not well understood. Reduced feed availability and change in body condition can affect the mass of eggs in birds that could lead to a skew in sex ratio. We employed feed restriction in laying chickens (Gallus gallus) to induce a decrease in body condition and egg mass using 45 chicken hens in treatment and control groups. Feed restriction led to an overall decline of egg mass. In the second period of treatment (Days 9-18) with more severe feed restriction and a steeper decline of egg mass, the sex ratio per hen (proportion of male eggs) had a significant negative association with mean egg mass per hen. Based on this association, two groups of hens were selected from feed restriction group, that is, hens producing male bias with low egg mass and hens producing female bias with high egg mass with overall sex ratios of 0.71 and 0.44 respectively. Genomewide transcriptome analysis on the germinal disks of F1 preovulatory follicles collected at the time of occurrence of meiosis-I was performed. We did not find significantly differentially expressed genes in these two groups of hens. However, gene set enrichment analysis showed that a number of cellular processes related to cell cycle progression, mitotic/meiotic apparatus, and chromosomal movement were enriched in female-biased hens or high mean egg mass as compared with male-biased hens or low mean egg mass. The differentially expressed gene sets may be involved in meiotic drive regulating sex ratio in the chicken.

La imagenologia médica en el laboratorio de morfología: iconografía / Medical imaging laboratory Morphology: iconography of bicéfalo fetus / A imagiologia médica no laboratório de morfologia: iconografia do feto bicéfalo

El nacimiento de gemelos siameses es un fenómeno extraño en las salas de parto de todo el mundo, son producto de un mismo óvulo que por alguna extraña razón no llegó a dividirse; se presenta 1/50.000 o 1/120.000 nacimientos; se estima que el 50% nace muerto y el 35% muere el primer día de vida; y el 70% son de génerofemenino. Un feto bicéfalo es un producto desarrollado con dos cabezas que se unen entre sí por medio de sus porciones temporo-parietales mediales, estando conectados vascularmente y compartiendo o no órganos entre sí; son de tipo monocigóticos y producidos por la separación anormal o incompleta de la porción axial del disco germinativo bilaminar en etapas avanzadas del desarrollo embrionario. En el presente trabajo se expone un caso de siameses bicéfalo de sexo femenino, cuyo espécimen hacía parte de las colecciones de embriología del Laboratorio de Morfología de la Universidad Tecnológica de Pereira, el cual por registro de Historia Clínica y Consentimiento Informado de la madre, quien lo entregó a la Universidad, fue sometido a descripción anatomopatológica con el fin de exponer aspectos relacionados con las posibles causas que los originaron, así como el diagnóstico prenatal de estos defectos congénitos y su diagnóstico Imagineológico postmortem.

The birth of conjoined twins is a strange phenomenon in the delivery rooms around the world, are the product of a single egg that for some reason did not divide; is presenta1 / 50,000 or 1 / 120,000 births; is estimated that 50% stillborn and 35% die on the first day of life; and 70% are female. A two-headed fetus is a product developed with two heads that are joined together through their temporoparietal medial portions being connected vascularized organs and sharing with each other or not; are monozygotic type and produced by abnormal or incomplete separation of the axial portion of the bilaminar germ disc in advanced stages of embryonic development. In this paper a case of two-headed Siamese female, whose specimen was part of the collections of Morphology Embryology Laboratory of the Technological University of Pereira, who for record medical history and informed consent of the mother who is exposed delivered to the University underwent pathological description to explain aspects of the possible causes of these accidents and the prenatal diagnosis of these congenital defects and postmortem imaging diagnosis.

O nascimento de gêmeos siameses é um fenômeno estranho nas salas de parto de todo o mundo. São produto de um mesmo óvulo, que por alguma estranha razão não chegou a dividir-se; se apresenta em 1/50.000 ou 1/120.000 nascimentos. Estima-se que 50% nascem mortos e 35% morre ao primeiro dia de vida; e 70% são do gênero feminino.Um feto bicéfalo é um produto desenvolvido com duas cabeças que se unem entre si por meio de suas porções temporão-parietais mediais, estando conectados vascular-mente e compartilham ou não órgãos entre si; são de tipo monozigóticos e produzi-dos pela separação anormal ou incompleta da porção axial do disco germinativo bi laminar em etapas avançadas do desenvolvimento embrionário. Neste trabalho se expõe o caso de siamesas bicéfalas do sexo feminino, cujo espéci-men fazem parte das coleções de embriologia do Laboratório de Morfologia da Uni-versidade Tecnológica de Pereira, que por registro de Historia Clínica e Consenti-mento Informado da mãe, que o entregou à Universidade, foi submetido a descrição anatomopatológica com o fim de expor aspectos relacionados às possíveis causas que os originaram, assim como o diagnóstico pré-natal destes defeitos congênitos e seu diagnóstico Imagiológico post-mortem.

Glycosylated chicken ZP2 accumulates in the egg coat of immature oocytes and remains localized to the germinal disc region of mature eggs.

Vertebrate eggs are surrounded by an egg coat, which is a specific extracellular egg matrix consisting of several glycoproteins with a conserved zona pellucida (ZP) domain. Two mammalian egg coat subunits, ZP2 and ZP3, have been suggested to act as sperm receptors. In bird eggs, however, ZP2 has never been identified in the egg coat of mature oocytes and ovulated eggs. Here we report that chicken ZP2 is expressed in immature small follicles and remains as an egg-coat component locally in the germinal disc region of mature eggs. RT-PCR analysis indicated marked expression of the ZP2 and ZP4 genes in the granulosa cells of immature white follicles, whereas the ZP3 and ZPD genes showed marked expression in the cells of maturing yellow follicles. ZP2 was identified in the egg coat isolated from immature follicles as a heavily N-glycosylated glycoprotein of ∼200 kDa, which was enzymatically converted to a 70-kDa deglycosylated form. Immunoblotting and immunohistological analyses showed that ZP2 was localized around the germinal disc region of mature follicles. ZP2 was accumulated in the egg coat of immature white follicles at the earlier stages of oocyte development and became a minor component in the egg coat of maturing yellow follicles, except for the germinal disc region. Localization of ZP2 in the germinal disc region of mature eggs, where sperm bind to the egg coat at high density, suggests some role for ZP2 in the preferential binding and penetration of sperm in the germinal disc region of bird eggs.

HIRA is essential for the development of gibel carp.

HIRA is one of the chaperones of histone H3.3. Mutation of Hira results in embryonic lethality in mice, suggesting a critical role in embryogenesis. However, Hira-mutated Drosophila may survive to adults, indicating that it is dispensable in Drosophila development. The role of Hira in fish development is unknown. In this study we first investigated the expression of Hira during embryogenesis of gibel carp (Carassius auratus gibelio) by whole-mount in situ hybridization. We found that Hira signal appeared ubiquitously in the early embryos. After gastrulation, it appeared mainly along the anterior-posterior axis, including the tail bud. In hatching period, the signal was detected in head, heart, and the endoderm region on the back of yolk. Then by microinjection with morpholino-HIRA at the beginning of development, we observed delayed gastrulation and abnormal somitogenesis in gibel carp embryos. The HIRA morphants exhibited short trunk, limited yolk extension, and twisted tail. Most of the mutants died during embryogenesis or shortly after hatching. The rest of the HIRA morphants could survive to larvae but with severe defects in organogenesis. These data suggest that HIRA may be essential for the development of gibel carp, and this function is conserved in vertebrates.

A survey of 6,300 genomic fragments for cis-regulatory activity in the imaginal discs of Drosophila melanogaster.

Over 6,000 fragments from the genome of Drosophila melanogaster were analyzed for their ability to drive expression of GAL4 reporter genes in the third-instar larval imaginal discs. About 1,200 reporter genes drove expression in the eye, antenna, leg, wing, haltere, or genital imaginal discs. The patterns ranged from large regions to individual cells. About 75% of the active fragments drove expression in multiple discs; 20% were expressed in ventral, but not dorsal, discs (legs, genital, and antenna), whereas ∼23% were expressed in dorsal but not ventral discs (wing, haltere, and eye). Several patterns, for example, within the leg chordotonal organ, appeared a surprisingly large number of times. Unbiased searches for DNA sequence motifs suggest candidate transcription factors that may regulate enhancers with shared activities. Together, these expression patterns provide a valuable resource to the community and offer a broad overview of how transcriptional regulatory information is distributed in the Drosophila genome.

The subcellular localization of cyclin B2 is required for bipolar spindle formation during Xenopus oocyte maturation.

Cyclins B1 and B2 are subtypes of cyclin B, a regulatory subunit of a maturation/M-phase promoting factor, and they are also highly conserved in many vertebrate species. Cyclin B1 is essential for mitosis, whereas cyclin B2 is regarded as dispensable. However, the overexpression of the cyclin B2 N-terminus containing the cytoplasmic retention signal, but not cyclin B1, inhibits bipolar spindle formation in Xenopus oocytes and embryos. Here we show that endogenous cyclin B2 was localized in and around the germinal vesicle. The perinuclear localization of cyclin B2 was perturbed by the overexpression of its N-terminus containing the cytoplasmic retention signal, which resulted in a spindle defect. This spindle defect was rescued by the overexpression of bipolar kinesin Eg5, which is located at the perinuclear region in the proximity of endogenous cyclin B2. These results demonstrate that the proper localization of cyclin B2 is essential for bipolar spindle formation in Xenopus oocytes.

A reliable method for sexing unincubated bird eggs for studying primary sex ratio.

In birds, offspring sex ratio manipulation by mothers is now well established with potentially important consequences for evolution and animal breeding. In most studies on primary sex ratio of birds, eggs are sexed after incubation by the use of PCR methods targeted to the sex-linked CHD1 genes. Sexing of unincubated eggs would be preferred, but as fertile and infertile blastodiscs cannot be distinguished macroscopically, errors could arise from PCR amplifications of parental DNA associated with the vitelline membrane of infertile eggs. In this study, we stained blastodiscs without the vitelline membrane with Hoechst 33342. This allowed unequivocal distinction between fertile and infertile blastodiscs. Fertile blastodiscs contained thousands of fluorescent nuclei, whereas no nuclei were seen in infertile eggs. In addition, after nucleic acid analysis, fertile blastodiscs yielded much stronger chromosomal DNA and CHD1-targeted PCR bands on agarose gels compared with infertile blastodiscs. These findings indicate that fertile blastodiscs contain much more embryonic DNA than parental DNA, allowing reliable sexing of the fertile eggs. The differences between fertile and infertile blastodiscs in chromosomal DNA and CHD1 PCR banding intensities alone could also be used to distinguish fertile from infertile eggs without using Hoechst staining. We conclude that identifying fertile blastodiscs either by Hoechst staining or by analyzing the yield of chromosomal DNA and CHD1-PCR products, combined with CHD1-targeted PCR amplification, presents an easy and reliable method to sex unincubated eggs.

A marked animal-vegetal polarity in the localization of Na(+),K(+) -ATPase activity and its down-regulation following progesterone-induced maturation.

The stage-VI Xenopus oocyte has a very distinct animal-vegetal polarity with structural and functional asymmetry. In this study, we show the expression and distribution pattern of Na(+),K(+) -ATPase in stage-VI oocytes, and its changes following progesterone-induced maturation. Using enzyme-specific electron microscopy phosphatase histochemistry, [(3) H]-ouabain autoradiography, and immunofluorescence cytochemistry at light microscopic level, we find that Na(+),K(+) -ATPase activity is mainly confined to the animal hemisphere. Electron microscopy histochemical results also suggest that polarized distribution of Na(+),K(+) -ATPase activity persists following progesterone-induced maturation, and it becomes gradually more polarized towards the animal pole. The time course following progesterone-induced maturation suggests that there is an initial up-regulation and then gradual down-regulation of Na(+),K(+) -ATPase activity leading to germinal vesicle breakdown (GVBD). By GVBD, the Na(+),K(+) -ATPase activity is completely down-regulated due to endocytotic removal of pump molecules from the plasma membrane into the sub-cortical region of the oocyte. This study provides the first direct evidence for a marked asymmetric localization of Na(+),K(+) -ATPase activity in any vertebrate oocyte. Here, we propose that such asymmetry in Na(+),K(+) -ATPase activity in stage-VI oocytes, and their down-regulation following progesterone-induced maturation, is likely to have a role in the active state of the germinal vesicle in stage-VI oocytes and chromosomal condensation after GVBD.

Inhibition of germinal vesicle breakdown by antioxidants and the roles of signaling pathways related to nitric oxide and cGMP during meiotic resumption in oocytes of a marine worm.

In mammalian oocytes, cAMP elevations prevent the resumption of meiotic maturation and thereby block nuclear disassembly (germinal vesicle breakdown (GVBD)), whereas nitric oxide (NO) and its downstream mediator cGMP can either inhibit or induce GVBD. Alternatively, some invertebrate oocytes use cAMP to stimulate, rather than inhibit, GVBD, and in such cases, the effects of NO/cGMP signaling on GVBD remain unknown. Moreover, potential interactions between NO/cGMP and AMP-activated kinase (AMPK) have not been assessed during GVBD. Thus, this study analyzed intraoocytic signaling pathways related to NO/cGMP in a marine nemertean worm that uses cAMP to induce GVBD. For such tests, follicle-free nemertean oocytes were stimulated to mature by seawater (SW) and cAMP elevators. Based on immunoblots and NO assays of maturing oocytes, SW triggered AMPK deactivation, NO synthase (NOS) phosphorylation, and an NO elevation. Accordingly, SW-induced GVBD was blocked by treatments involving the AMPK agonist AICAR, antioxidants, the NO scavenger carboxy-PTIO, NOS inhibitors, and cGMP antagonists that target the NO-stimulated enzyme, soluble guanylate cyclase (sGC). Conversely, SW solutions combining NO/cGMP antagonists with a cAMP elevator restored GVBD. Similarly, AICAR plus a cAMP-elevating drug reestablished GVBD while deactivating AMPK and phosphorylating NOS. Furthermore, sGC stimulators and 8-Br-cGMP triggered GVBD. Such novel results indicate that NO/cGMP signaling can upregulate SW-induced GVBD and that cAMP-elevating drugs restore GVBD by overriding the inhibition of various NO/cGMP downregulators, including AMPK. Moreover, considering the opposite effects of intraoocytic cAMP in nemerteans vs mammals, these data coincide with previous reports that NO/cGMP signaling blocks GVBD in rats.

Influence of storage on the position of the germinal disc in the fertilized unincubated chicken egg.

The breeding of male layer chickens is currently considered to be highly uneconomical. In Germany alone, 40 to 50 million newly hatched male chickens were killed annually immediately after hatching. Therefore, it is necessary to find a method for sexing chickens early in the embryonic development, preferably before incubation. The genotypic sex of an egg can be determined using information found in the germinal disc, so knowledge of the exact position of the germinal disc is essential for further sexing, or for other actions such as the in ovo injection of agents. Previous studies have shown that the germinal disc is located somewhere on top of the yolk. However, no studies have yet been performed that investigate the influence of time spent in horizontal storage on the position of the germinal disc. Magnetic resonance imaging was chosen to determine this influence on the position of the germinal disc. It was found that eggs placed horizontally for long periods of time before scanning had significant changes in the positions of their germinal discs compared with those of eggs scanned minutes after positioning. The position of the germinal disc in eggs, minutes after horizontal positioning, deviated 14.7 ± 0.6 mm from the maximum vertical plane of the egg (zero position) in the z-direction; eggs scanned after 96 h of horizontal positioning showed a deviation of only 4.9 ± 1.6 mm. The x-axis also exhibited changes in the position of the germinal disc over time. Immediately after horizontal positioning, the eggs showed a deviation of 0.4 ± 0.4 mm in the x-direction, whereas the deviation after 96 h was 2.9 ± 0.5 mm. These results show that horizontal positioning of the egg hours before the measurement is necessary.

Signaling pathways in ascidian oocyte maturation: the roles of cAMP/Epac, intracellular calcium levels, and calmodulin kinase in regulating GVBD.

Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10 µM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10 µM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis.

Comparative biology of cAMP-induced germinal vesicle breakdown in marine invertebrate oocytes.

During maturation, oocytes must undergo a process of nuclear disassembly, or "germinal vesicle breakdown" (GVBD), that is regulated by signaling pathways involving cyclic AMP (cAMP). In vertebrate and starfish oocytes, cAMP elevation typically prevents GVBD. Alternatively, increased concentrations of intra-oocytic cAMP trigger, rather than inhibit, GVBD in several groups of marine invertebrates. To integrate what is known about the stimulation of GVBD by intra-oocytic cAMP, this article reviews published data for ascidian, bivalve, brittle star, jellyfish, and nemertean oocytes. The bulk of the review concentrates on the three most intensively analyzed groups known to display cAMP-induced GVBD-nemerteans, ascidians, and jellyfish. In addition, this synopsis also presents some previously unpublished findings regarding the stimulatory effects of intra-oocytic cAMP on GVBD in jellyfish and the annelid worm Pseudopotamilla occelata. Finally, factors that may account for the currently known distribution of cAMP-induced GVBD across animal groups are discussed.

Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes - a method for quantitative transcriptional reprogramming.

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis. Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein.