A new vision of the origin and the oocyte development in the Ostariophysi applied to Gymnotus sylvius (Teleostei: Gymnotiformes)

Tendo por base os novos conhecimentos oriundos de recentes estudos com Perciformes marinho, a origem e o desenvolvimento dos oócitos no Ostariophysi Gymnotus sylvius são aqui descritos. da mesma maneira que ocorre nos Perciformes, em Gymnotus sylvius as oogônias são encontradas no epitélio germinativo que margeia as lamelas ovígeras. No início da foliculogênese, a proliferação das oogônias e sua entrada em meiose dão origem a ninhos de células germinativas que se projetam em direção ao estroma ovariano, a partir do epitélio germinativo. Os ninhos e o epitélio germinativo são suportados pela mesma membrana basal que os separa do estroma. Coincidindo com a paralisação da meiose os oócitos, presentes nos ninhos, são separados uns dos outros por processos citoplasmáticos das células pré-foliculares. As células pré-foliculares derivam do epitélio germinativo sendo, portanto, inicialmente células epiteliais. Durante a foliculogênese, ao mesmo tempo em que envolvem os oócitos individualizando-os, as células pré-foliculares sintetizam a membrana basal ao seu redor. Os oócitos entram em crescimento primário ainda dentro dos ninhos. Ao término da foliculogênese, o oócito e as células foliculares que compõem o folículo são circundados pela membrana basal. O folículo permanece conectado ao epitélio germinativo uma vez que ambos compartilham uma porção comum da membrana basal. Células oriundas do estroma circundam o folículo ovariano exceto na região de compartilhamento da membrana basal formando a teca. O folículo, a membrana basal e a teca formam o complexo folicular. O desenvolvimento do oócito ocorre dentro do complexo folicular e compreende os estágios de crescimento primário e secundário, maturação e ovulação. Os alvéolos corticais surgem no ooplasma momentos antes do início do crescimento secundário ou estágio vitelogênico que tem início com a deposição de vitelo, progride até o oócito esteja completamente desenvolvido e o ooplasma preenchido pelos glóbulos de vitelo. A maturação é caracterizada pela migração do núcleo ou vesícula germinativa, pela quebra da vesícula germinativa, ou seja, pela fragmentação do envoltório nuclear e, retomada da meiose. Na ovulação o ovo é liberado do complexo folicular para o lúmen ovariano. em comparação com os Perciformes marinhos com ovos pelágicos, o desenvolvimento oocitário em Gymnotus sylvius tem menos etapas dentro dos estágios de desenvolvimento, sendo as duas mais notáveis delas as ausências da formação das gotas de lipídio durante os crescimentos primário e secundário (e a consequente fusão das gotas para formar um único glóbulo de lipídio durante a maturação) e, a hidrólise do vitelo antecedendo a ovulação.

Role of roscovitine and IBMX on kinetics of nuclear and cytoplasmic maturation of bovine oocytes in vitro

The 3-isobutyl-1-methylxanthine (IBMX) is able to prevent resumption of meiosis by maintaining elevated cyclic AMP (cAMP) concentrations in the oocyte, and roscovitine, a purine known to specifically inhibit MPF kinase activity, maintains bovine oocytes at the germinal vesicle (GV) stage. The present study was conducted to analyze whether cytoplasmic maturation (examined by the pattern of cortical granule (CG) distribution) of bovine oocytes is improved during meiotic arrest with IBMX and roscovitine. Oocytes were matured in vitro in a 10% Knockout(SR) supplemented TCM-199 medium (Control) with either 0.5 mM IBMX or 25 mu M roscovitine (ROSC). Oocytes were stained with fluorescein isothiocyanate conjugated Lens culinaris agglutinin (FITC-LCA) for CG evaluation and with Hoechst 33342 for nuclear stage assessment. At 16 h of culture, the percentage of oocytes remaining in the GV stage was higher (P < 0.05) in the ROSC group (32.41%) compared with the Control and IBMX groups (8.61% and 9.73%, respectively). At 24h of culture, progression of meiosis to M II stage was retarded (P < 0.05) in the ROSC group (24.05%) compared to the Control (60.20%), whereas the IBMX group (33.88%) showed no significant difference to the other two groups. At 16h of maturation, the proportion of oocytes with CG in clusters (immature cytoplasm) was similar between the groups, as was the percentage of peripheral CG (mature) at 24h of maturation. The results of the present study demonstrated that the meiotic inhibitors IBMX and roscovitine delay the progression of nuclear maturation without affecting cytoplasmic maturation, assessed by the analysis of CG repositioning. (c) 2006 Elsevier B.V. All rights reserved.

Effects of FGF10 on bovine oocyte meiosis progression, apoptosis, embryo development and relative abundance of developmentally important genes in vitro

Contents Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion-related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes (COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22h in TCM-199 supplemented with 0, 2.5, 10 or 50ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5ng/ml FGF10 increased (p<0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real-time RT-PCR showed a tendency of 50ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes.

Ooplast-mediated developmental rescue of bovine oocytes exposed to ethidium bromide

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The effect of interaction between macromolecule supplement and oxygen tension on bovine oocytes and embryos cultured in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of fetuin on zona pellucida hardening, fertilization and embryo development in cattle

Mammalian oocytes can undergo spontaneous meiotic maturation when they are liberated from their follicles and cultured in vitro; however, the zona pellucida (ZP) becomes resistant to chymotrypsin digestion, or hardens, when spontaneous maturation occurs in serum-free medium. Schroeder et al. [Biol. Reprod. 43 (1990) 891] described that fetuin, a component of fetal calf serum (FCS), inhibits ZP hardening during oocyte maturation. The aim of this experiment was to study the effect of the presence of cumulus cells and addition of hormones to maturation media on bovine zona hardening and embryo development in medium with and without fetuin. In Experiment 1, different concentrations of fetuin were added to the maturation medium. The time necessary for digestion of 50% of the ZP (d50) was not different when oocytes were matured in presence of 10% FCS, 1 mg/ml polyvinyl alcohol (PVA), or 4, 1 and 0.25 mg/ml of fetuin; cleavage rates were also similar. However, significantly more blastocysts (P < 0.05) were formed when FCS was used compared to PVA and 0.25 mg/ml of fetuin. In Experiment 11, we examined the influence of the presence of cumulus cells and hormones during the maturation of oocytes in media with PVA, BSA, FCS and fetuin. The d50 was significantly higher (P < 0.05) when oocytes were matured in presence of cumulus cells. The cleavage rate of cumulus-intact oocytes was similar for all groups. However, when oocytes were partially stripped before maturation, the cleavage rate was significantly higher (P < 0.05) when FCS or fetuin was used. In both stripped and non-stripped groups, significantly more blastocysts (P < 0.05) were formed when oocytes were matured with FCS compared to BSA and PVA. These results indicate that zona hardening, as described for mouse and human oocytes, does not have a large effect on bovine cumulus-intact oocytes. Apparently fetuin can be used as a substitute for FCS during bovine oocyte maturation, since it leads to similar developmental rates as FCS in intact and partially stripped oocytes. (C) 2002 Published by Elsevier B.V. B.V.

Comparison of efficiency between two artificial insemination methods using frozen-thawed semen in domestic cat (Felis catus) Artificial insemination in domestic cats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Superovulation in mares: Limitations and perspectives

Superovulation is an important tool for routine use in equine embryo transfer (ET) in order to reduce the costs and to enhance the efficiency of ET programs. Satisfactory superovulatory answers (2-7 ovulations) have been reported in mares treated with Equine Pituitary Extract (EPE) and more recently using a commercial Equine FSH. However, embryo recovery rates have been inconsistent and below expectations (20-50% embryos/ovulation). Recent studies have shown that superovulatory treatment leads to disturbances in oocyte maturation and transport, especially in mares with a high ovarian response. Higher and more consistent embryo recovery rates per ovulation have been observed in mares treated with lower doses of EPE. This paper presents a review of recent studies related to superovulation in mares.

Caracterização estrutural do fator de crescimento de fibroblasto-10 (FGF-10) e seu papel na fisiologia folicular ovariana

Background: Interest in folliculogenesis has grown extensively in recent years. Nevertheless, several aspects of follicular activity are still poorly understood. Thus, in vitro culture of ovarian follicles using new substances has been established as a very viable model, enhancing the prospects for a better understanding of follicular activity. Among the family members of the fibroblast growth factor (FGFs), FGF-10 has received recent attention for its ability to regulate the development of ovarian follicles and oocyte maturation. Given the relevance of FGF-10 in the folliculogenesis process, this review aimed to describe the structural features, expression and the main biological effects of FGF-10 on the development of ovarian follicles in mammals.Review: Along this work, it was shown aspects related to structural characterization of FGF-10 and its receptors, as well as FGF-10 expression in different cell types, emphasizing its importance to follicular development. FGF-10 is a paracrine member of the family of FGFs, and is characterized by promoting biological responses via cell surface receptors (FGFRs) of tyrosine kinase-type. of these receptors, FGFR-1, FGFR-2 and FGFR-3 may undergo alternative transcriptional arrangements, enabling the formation of two isoforms (b and c) that have varying degrees of affinity for the various FGFs. Thus, seven FGFR proteins (FGFRs 1b, 1c, 2b, 2c, 3b, 3c and 4) with different binding specificities are generated from the four FGFR genes. The FGFRs transmit intracellular signals after binding with the ligand through the phosphorylation of tyrosine, which activates various transduction patterns in the cytoplasm. The signal transduction of FGF-10 may occur through three main pathways: protein of rat sarcoma (Ras)/MAPK, PLC gamma/Ca(2+) and phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt), which are involved in the transmission of biological signals, leading to cellular proliferation and differentiation. FGF-10 mRNA expression was detected in the ovarian stroma, oocyte and theca cells of preantral and antral follicles. on the other hand, the expression of mRNA for FGF-10 receptors was found in, granulosa cells, theca cells, cumulus cells and oocytes. Although FGFs are widely distributed in different tissues and cell types, the importance and function of FGFs in the ovary are still poorly documented. FGF-10 has been shown to be an important mediator of mesenchymal and epithelial cell interactions during follicle development, promoting follicular survival, activation and growth. Besides the action on folliculogenesis, FGF-10 was recently identified as a growth factor able to improve oocyte competence. However, in antral follicles, the presence of FGF-10 is associated with increased follicular atresia, which matches its anti-estrogenic action.Discussion: From this review, we can conclude that FGF-10 is an important regulator of female reproduction. This growth factor acts in follicle survival, oocyte maturation, expansion of cumulus cells and proliferation of granulosa/theca cellsthrough direct and/or indirect actions in the control of folliculogenesis. Furthermore, FGF-10 seemed to have different effects throughout the follicular development. However, it is necessary to perform additional studies that may provide a better understanding about the importance of FGF-10 during folicullogenesis.

Copulation is necessary for the completion of a gonotrophic cycle in the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphological aspects of cell reabsorption in laying queens and workers of Apis mellifera (Hymenoptera, Apidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influência do fator de crescimento semelhante à insulina I (IGF-I) adicionado ao meio fluido sintético de tuba uterina (SOF) sobre a maturação in vitro de oócitos caninos (Canis familiaris)

The follicular growth and oocyte maturation knowledge are very important to the development and improvement of new biotechnologies such as in vitro fertilization and somatic cell nuclear transfer. In order to the necessity of clarify the basic mechanisms related to canine oocyte maturation, this investigation focuses on the evaluation of the effect of insulin-like growth factor-i (IGF-I), added to synthetic oviductal fluid medium (SOF) on the in vitro maturation of domestic dog oocytes. Thirty-seven bitches undergoing ovariohysterectomy for castration or due to pathological conditions of the uterus were selected as oocytes' donors (n=875). The oocytes were allocated in the following groups: MO (stained in the collection's time), Control (72h in SOF) and Experimental (72h in SOF plus 100 ng IGF-I). After 72 hours of maturation the oocytes' nuclear status were assessed by Hoechst 33342 dye. The best results in terms of oocyte harvest were observed in those juvenile donors,, females in estrus, nuliparous and pure breeds. No significant differences were observed between treatments control (SOF) or experimental (IGF-I).

Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zona-removed equine oocytes

Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/ mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 μM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/ BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm. © 2003 American Society of Animal Science. All rights reserved.

Uso da suplementação de LH recombinante na estimulação ovariana com protocolos de FSH recombinante e agonistas do GnRH: Uma revisão da literatura

The purpose of this review was to assess the efficacy of recombinant LH (r-LH) supplementation for controlled ovarian stimulation in recombinant FSH (r-FSH) and GnRH-agonist (GnRH-a) protocol for IVF/ICSI cycles. Search strategies included on-line surveys of databases from 1990 to 2006. Four trials fulfilled the inclusion criteria (Lisi et al. 2002, Humaidan et al. 2004, Marrs et al. 2004, Tarlatzis et al. 2006). When the review was carried out advantages were observed for the r-LH supplementation protocol with respect to a fewer days of stimulation, a fewer total amount of r-FSH administered and a higher serum estradiol levels on the day of hCG administration. However, these differences were not observed in number of oocyte retrieved, number of mature oocytes, clinical pregnancy per oocyte retrieval, implantation and miscarriage rates. Nevertheless, more randomized controlled trials are necessary before evidence-based recommendations regarding exogenous r-LH supplementation in ovarian stimulation protocols with r-FSH and GnRH-a for assisted reproduction treatment can be provided.