Essential role of germinal vesicle material in the meiotic cell cycle of Xenopus oocytes

In almost all animal species, immature oocytes are arrested naturally in the first meiotic prophase, with a large nucleus called the germinal vesicle. A number of previous studies showed that both activation of maturation/M phase-promoting factor (MPF) (assayed by semiquantitative cytological methods) and some other maturational events occur essentially normally in enucleated oocytes from many amphibian species and mice. Hence, for nearly three decades, it has generally been believed that nuclear material is dispensable for MPF activation and the meiotic cell cycle in vertebrate oocytes. Here, we have challenged this view by examining the histone H1 kinase activities and the molecular forms of MPF in experimentally manipulated Xenopus oocytes. We show that oocytes injected with nuclear material undergo much more rapid MPF activation and maturation than uninjected control oocytes. Conversely, enucleated oocytes, unlike nucleated counterparts, undergo only weak MPF activation in meiosis I and no detectable MPF reactivation in meiosis II, the latter accompanying inhibitory tyrosine phosphorylation of cdc2 kinase, the catalytic subunit of MPF. These results argue strongly that nuclear material is indispensable for the meiotic cell cycle, particularly MPF reactivation (or cdc2 tyrosine dephosphorylation) on entry into meiosis II, in Xenopus oocytes. The classical and general view may thus need reconsideration.

The Stem-Loop Binding Protein (SLBP1) Is Present in Coiled Bodies of the Xenopus Germinal Vesicle

The stem-loop binding protein (SLBP1) binds the 3′ stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the germinal vesicle of Xenopus laevis oocytes. In spread preparations of germinal vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection of myc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 3′ end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

Synchronous oogenesis during the semilunar spawning cycle of the tropical abalone Haliotis asinina

On the southern Great Barrier Reef, Haliotis asinina (Vetigastropoda: Pleurotomarioidea) synchronously spawn every 2 wk in a predictable fashion. allowing detailed analysis of reproduction, gametogenesis, and gonad development. Histological examination of the ovaries of members of the Heron Reef population during this semilunar cycle reveals that oogenesis is also synchronous and predictable, and requires more than two spawning cycles (i.e. >28 days) to complete. Shortly after a spawning event the ovary comprises two cohorts of primary oocytes, one of which will be released at the next spawning event, and clusters of oogonia. At this time there is a rapid proliferation and expansion of trabeculae, germinal epithelial, and oogonia, and a dramatic increase in the size of the vitellogenic oocytes to be: spawned at the next spawning event. Within 4 days these oocytes have filled the ovary. On the day of the next spawning a lumen forms in the ovary as a result of localized degradation of trabeculae. The large primary oocytes dissociate from the receding trabeculae. initiate maturation, and accumulate in the lumen; these oocytes become embedded in a jelly coat layer. The next cohort of oocytes remain attached to the trabeculae. The jelly coat appears to be completely dissolved within 30 min of spawning. Comparison of the oogenesis and ovary development in II. asinina with other abalone species indicates that these processes are very similar in tropical and temperate abalone. This suggests that insights into the regulation of reproduction and spawning in H. asinina are likely to be applicable to other haliotids.

Supplemented tissue culture medium 199 is a better medium for in vitro maturation of oocytes from women with polycystic ovary syndrome women than human tubal fluid

Objective: To compare oocyte maturation, fertilization and cleavage rates, and embryonic developmental quality after culture of human immature oocytes from polycystic ovary syndrome (PCOS) patients in human tubal fluid (HTF) or tissue culture medium (TCM) 199. Design: Prospective, randomized, controlled trial. Setting: University hospital. Patient(s): Thirteen women undergoing 23 in vitro maturation cycles, from whom 119 oocytes were retrieved. Intervention(s): Cumulus-enclosed germinal vesicle-stage oocytes matured in TCM-199-supplemented or HTF-supplemented media. Main Outcome Measure(s): Oocyte maturation and fertilization rates, embryonic developmental quality. Result(s): Significant differences were observed between TCM 199 and HTF regarding maturation rate (82% vs. 56.9%), fertilization rate (70% vs. 39.4%), and embryo quality (81.3% vs. 41.7%). Conclusion(S): Human tubal fluid medium, although widely used for embryo fertilization and maintenance in IVF techniques, is not,in appropriate medium for the maturation of oocytes obtained from PCOS patients in nonstimulated cycles. (Fertil Steril(R) 2009;91:509-13. (C)2009 by American Society for Reproductive Medicine.)

Bovine oocyte vitrification: Effect of ethylene glycol concentrations and meiotic stages

Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0-10, 10-14, and 18-24 h of INK respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 It of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-I or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification. (C) 2007 Elsevier B.V. All rights reserved.

Effects of gonadotropin-exposed medium with high concentrations of progesterone and estradiol-17 beta on in vitro maturation of canine oocytes

During the process of maturation in the oviduct, canine oocytes in the germinal vesicle stage are exposed to decreasing levels of estradiol-17 beta and increasing levels of progesterone. However, hormone concentrations in the microenvironments in which they act are higher than serum concentrations. Therefore, the aim of the present study was to compare the meiotic competence of canine oocytes harvested from anestrous bitches in culture medium containing high concentrations (20 mu g ml(-1)) of estradiol-17 beta and/or progesterone in association to gonadotropins (luteinizing hormone and follicle-stimulating hormone) using three different maturation periods (48, 72, and 96 h). Oocytes were cultured in tissue culture medium (TCM-199) and arranged in four experimental groups: group control, group E2 (estradiol-17 beta), group P4 (progesterone), and group E2 + P4. Regardless of the maturation period, groups P4 and E2 + P4 presented statistically higher rate of germinal vesicle breakdown oocytes compared to the group control and group E2. There were no significant differences among groups on germinal vesicle, metaphase I, metaphase II, and degenerated or unidentifiable oocytes rates. The mean percentage of metaphase II oocytes was higher at 96 h when compared to 72 h. Results of the present research indicate no influence of estradiol-17 beta supplementation, unless in association with progesterone. There is an evidence of the positive effect of progesterone on germinal vesicle breakdown. Results also showed that extended periods of in vitro maturation affect positively maturation rates to metaphase II of low competent oocytes harvested from anestrous bitches, independent of the maturation media. In conclusion, high concentrations of steroids, especially progesterone, have positive effect on in vitro oocyte maturation when the oocytes are derived from the anestrous status.

Bacterial lipopolysaccharide induces apoptosis in the trout ovary

BACKGROUND: In mammals it is well known that infections can lead to alterations in reproductive function. As part of the innate immune response, a number of cytokines and other immune factors is produced during bacterial infection or after treatment with lipopolysaccharide (LPS) and acts on the reproductive system. In fish, LPS can also induce an innate immune response but little is known about the activation of the immune system by LPS on reproduction in fish. Therefore, we conducted studies to examine the in vivo and in vitro effects of lipopolysaccharide (LPS) on the reproductive function of sexually mature female trout. METHODS: In saline- and LPS -injected brook trout, we measured the concentration of plasma steroids as well as the in vitro steroidogenic response (testosterone and 17alpha-hydroxyprogesterone) of ovarian follicles to luteinizing hormone (LH), the ability of 17alpha,20beta-dihydroxy-4-pregnen-3-one to induce germinal vesicle breakdown (GVBD) in vitro, and that of epinephrine to stimulate follicular contraction in vitro. We also examined the direct effects of LPS in vitro on steroid production, GVBD and contraction in brook trout ovarian follicles. The incidence of apoptosis was evaluated by TUNEL analysis. Furthermore, we examined the gene expression pattern in the ovary of saline- and LPS-injected rainbow trout by microarray analysis. RESULTS: LPS treatment in vivo did not affect plasma testosterone concentration or the basal in vitro production of steroids, although a small but significant potentiation of the effects of LH on testosterone production in vitro was observed in ovarian follicles from LPS-treated fish. In addition, LPS increased the plasma concentration of cortisol. LPS treatment in vitro did not affect the basal or LH-stimulated steroid production in brook trout ovarian follicles. In addition, we did not observe any effects of LPS in vivo or in vitro on GVBD or follicular contraction. Therefore, LPS did not appear to impair ovarian steroid production, oocyte final maturation or follicular contraction under the present experimental conditions. Interestingly, LPS administration in vivo induced apoptosis in follicular cells, an observation that correlated with changes in the expression of genes involved in apoptosis, as evidenced by microarray analysis. CONCLUSION: These results indicate that female trout are particularly resistant to an acute administration of LPS in terms of ovarian hormone responsiveness. However, LPS caused a marked increase in apoptosis in follicular cells, suggesting that the trout ovary could be sensitive to the pro-apoptotic effects of LPS-induced inflammatory cytokines.

Effect of conditioned medium of mesenchymal stem cells on the in vitro maturation and subsequent development of mouse oocyte

Mesenchymal stem cells (MSCs) secrete a variety of cytokines and growth factors in addition to self-renewal and multiple forms of differentiation. Some of these secreted bioactive factors could improve meiotic maturation in vitro and subsequent embryo developmental potential. The aim of the present study was to determine whether in vitro maturation (IVM) of mouse oocyte with or without cumulus cells could be improved by contact with conditioned medium (CM) of MSCs as well as the efficiency of CM to support follicular growth and oocyte maturation in the ovarian organ of mice cultured on soft agar. The developmental potential of matured oocyte was assessed by blastocyst formation after in vitro fertilization (IVF). Germinal vesicle stage oocytes with or without cumulus cells were subjected to IVM in either CM, Dulbecco's modified Eagle's medium (DMEM), α-minimum essential medium (α-MEM) or human tubal fluid (HTF). Approximately 120 oocytes were studied for each medium. CM produced a higher maturation rate (91.2%) than DMEM (54.7%), α-MEM (63.5%) and HTF (27.1%). Moreover, CM improved embryo development to blastocyst stage significantly more than DMEM and HTF (85 vs 7% and 41.7%, respectively) but there was no significant difference compared with α-MEM (85 vs 80.3%). The behavior of cortical granules of IVM oocytes cultured in CM revealed cytoplasmic maturation. Moreover, CM also supported preantral follicles growth well in organotypic culture on soft agar resulting in the maturation of 60% of them to developmentally competent oocytes. The production of estrogen progressively increased approximately 1-fold every other day during organ culture, while a dramatic 10-fold increase in progesterone was observed 17 h after human chorionic gonadotropin stimulus at the end of culture. Thus, CM is an effective medium for preantral follicle growth, oocyte maturation, and sequential embryo development.

Heteroplasmy in mammal mitochondrial deoxyribonucleic acid

La nature a développé diverses stratégies afin d’assurer le commencement de la vie dans des conditions d’homoplasmie, c’est-à-dire des conditions telles que les cellules sont dotées du même ADN mitochondrial. Toutefois, des nouveaux haplotypes de l’acide désoxyribonucléique mitochondrial (ADNmt) peuvent apparaitre et croître de plusieurs façons tout au long de la durée d’une vie menant à l’hétéroplasmie. Par exemple, l’hétéroplasmie de l’ADNmt peut être créée artificiellement par des technologies reproductives assistées, ainsi que naturellement par le processus de vieillissement. De ce fait, la thèse de ce doctorat fut divisée en deux principaux objectifs. Le premier étant celui d’analyser les changements survenus dans l’hétéroplasmie de l’ADNmt produit par le transfert nucléaire des cellules somatiques (SCNT) lors du développement de l’embryon jusqu’au fœtus et aux tissus adultes de bovins clonés. En ce qui concerne le second objectif, il s’agit d’analyser les changements survenus dans l’hétéroplasmie de l’ADNmt causés par le vieillissement dans une cellule somatique adulte et dans des tissus germinaux durant l’ovogénèse, ainsi qu’au début de l’embryogenèse et dans la procédure de culture in vitro sur des souris. Dans la première série d’expériences sur des bovins, des fibroblastes fœtaux transportant une mutation d’ADNmt (insertion de 66 pb) furent fusionnés avec des ovocytes receveurs transportant l’ADNmt du type sauvage. La présence d’ADNmt venant de la cellule donneuse a été analysée à différents stades de développement, soit sur des embryons âgés de 17 jours (n=17), des fœtus âgés de 40 jours (n=3), des fœtus âgés de 60 jours (n=3), un fœtus âgé de 240 jours et 3 clones post-nataux âgés de 18 à 24 mois. Chaque individu s’est avéré être hétéroplasmique et 99 % (103/104) des échantillons de tissus analysés étaient également hétéroplasmiques. Cependant, l’ovaire venant du fœtus de 240 jours fut le seul à être homoplasmique pour l’ADNmt de l’ovocyte receveur. Dans la plupart des échantillons analysés (95,2 %, soit 99/104) la moyenne d’hétéroplasmie était de 1,46 %. Par contre, un fœtus âgé de 40 jours a présenté un niveau élevé d’hétéroplasmie (20,9 %), indiquant ainsi que des évènements rares d’augmentation de l’ADNmt des cellules donneuses peuvent survenir. Étant donné que la majorité des clones SCNT montrait de l’hétéroplasmie de l’ADNmt à des proportions comparables à celles des cellules donneuses au moment de la reconstruction de l’embryon, on a pu conclure que l’hétéroplasmie produite par des techniques de transfert nucléaire utilisant des cellules somatiques est due à une ségrégation neutre de l’ADNmt. Dans la seconde série d’expériences sur des souris, des femelles de différents âges, c.à.d. jeunes (0 – 8 mois), moyennes (8 – 16 mois) et vieilles (16 – 24 mois), ont été synchronisées (gonadotrophines) et sacrifiées dans le but d’obtenir des ovocytes au stade de vésicule germinal, et des ovocytes au stade métaphase-II produits in vivo et in vitro. De plus, des embryons in vivo et in vitro au stade de deux-cellules et des embryons au stade de blastocystes ont été obtenus de femelles jeunes. Différents tissus somatiques, venant de femelles des trois stades d’âge ont été obtenus : cerveau, foie, muscle et du cumulus ovocytaire. De plus, l’effet du vieillissement a été mesuré selon la fertilité de la femelle. En effet, les effets sur l’hétéroplasmie du vieillissement, du stade de développement et de la culture in vitro ont été mesurés dans des ovocytes et dans des embryons. Les effets du vieillissement sur les mitochondries ont été mesurés par rapport au nombre total de copies de l’ADNmt, au pourcentage des délétions communes et sur l’expression de trois gènes : Ndufs4, Mt-nd2 and Mt-nd4. Il a été possible d’observer que la fertilité des femelles dans la colonie de souris diminuait avec l’âge. En fait, le vieillissement affectait l’ADNmt dans les tissus somatiques, cependant il n’avait pas d’effet sur le cumulus, les ovocytes et les embryons. Le nombre de délétions de l’ADNmt augmentait pendant la reprise de la méiose et celui-ci diminuait au début du développement embryonnaire. La culture in vitro n’affectait pas la quantité d’ADNmt dans la plupart des tissus germinaux. Puisque nous n’avons pas trouvé d’effet de l’âge dans la majorité des paramètres mitochondriaux analysés dans les ovocytes et les embryons, il est suggéré que la délétion commune de l’ADNmt dans les tissus germinaux est davantage reliée au statut cellulaire de la production d’énergie qu’au processus de vieillissement. Deux sources différentes de mutations de l’ADNmt produites dans les ovocytes normaux ou reconstitués ont produit différents résultats d’hétéroplasmie au début de l’embryogénèse. Chez les bovins, l’hétéroplasmie artificielle impliquant une petite insertion (66 pb) dans la région non codante (D-loop) de l’ADNmt a été vraisemblablement non nocive pour l’embryon, tolérant la persistance de l’ADNmt étranger pendant les différents stades du développement des clones. Chez les souris, l’hétéroplasmie naturelle produite par une grande délétion (4974 pb délétion commune) dans la région codante de l’ADNmt a été vraisemblablement nocive pour l’embryon et par conséquent éliminée pour assurer l’homoplasmie au début du développement embryonnaire.

The orphan nuclear receptor NR5A2 regulates peri-ovulatory events and their consequent luteinization in mice

Le récepteur nucléaire Nr5a2, également connu sous le nom de liver receptor homolog-1 (Lrh-1), est exprimé au niveau de l’ovaire chez la souris, exclusivement dans les cellules lutéales et de la granulosa. La perturbation de Nr5a2, spécifique aux cellules de la granulosa chez la souris à partir des follicules primaires dans la trajectoire du développement folliculaire a démontré que Nr5a2 est un régulateur clé de l’ovulation et de la fertilité chez la femelle. Notre hypothèse veut que Nr5a2 régule les évènements péri- et post-ovulatoires dans une séquence temporelle lors de la folliculogénèse. Afin d'étudier l’implication de Nr5a2 lors de l’ovulation et de la lutéinisation à différents stades du développement folliculaire, nous avons généré deux modèles de souris knockout spécifiques aux cellules de la granulosa pour Nr5a2: 1) Nr5a2Amhr2-/-, avec une réduction de Nr5a2 à partir des follicules primaires et subséquents; 2) Nr5a2Cyp19-/-, avec une réduction de Nr5a2 débutant au stade antral de développement en progressant. L’absence de Nr5a2 à partir des follicules antraux a résulté en une infertilité chez les femelles Nr5a2Cyp19-/-, de même qu’en des structures non-fonctionnelles similaires aux structures lutéales au niveau des ovaires, en une réduction des niveaux de progestérone synthétisée ainsi qu’en un échec dans le support d’une pseudo-gestation. La synthèse de progestérone a été entravée suite à l’absence de Nr5a2 par l’entremise d’une régulation à la baisse des gènes reliés au transport du cholestérol, Scarb1, StAR et Ldlr, démontré par qPCR. Les complexes cumulus-oocytes des femelles Nr5a2Cyp19-/- immatures super-stimulées ont subi une expansion in vivo, mais l’ovulation a été perturbée, possiblement par une régulation à la baisse du gène du récepteur de la progestérone (Pgr). Un essai d’expansion du cumulus in vitro a démontré une expansion défectueuse du cumulus chez les Nr5a2Amhr2-/-, associée à un dérèglement de la protéine des jonctions communicantes (Gja1; Cx43). Cependant, l’expansion du cumulus chez les Nr5a2Cyp19-/- n’a pas été autant affectée. Des résultats obtenus par qPCR ont démontré une régulation à la baisse dans l’expression des gènes Areg, Ereg, Btc et Tnfaip6 chez les deux modèles de cellules ovariennes knockout à 2h et 4h post hCG. Nous avons observé que 85% des oocytes, chez les deux génotypes mutants, peuvent subir une rupture de la vésicule germinative, confirmant leur capacité de maturation in vivo. La technique d’injection intra-cytoplasmique de spermatozoïdes a prouvé que les oocytes des deux génotypes mutants sont fertilisables et que 70% des embryons résultants ont poursuivi leur développement vers le stade de blastocyste, et ce, indépendamment du génotype. En conclusion, Nr5a2 régule la fertilité chez les femelles tout au long du processus du développement folliculaire. Il a été démontré que Nr5a2 est essentiel à la lutéinisation et que sa perturbation dans les cellules somatiques ovariennes ne compromet pas la capacité des oocytes à être fertilisés. En vue d’ensemble, nous avons fourni une investigation inédite et complète, utilisant de multiples modèles et techniques afin de déterminer les mécanismes par lesquels Nr5a2 régule les importants processus que sont l’expansion du cumulus, l’ovulation ainsi que la formation du corps jaune.

Cytoplasmic maturation of bovine oocytes: Structural and biochemical modifications and acquisition of developmental competence

Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3` terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development. (c) 2009 Elsevier Inc. All rights reserved.

Induced spawning of hatchery-raised Brazilian catfish, cachara Pseudoplatystoma fasciatum (Linnaeus, 1766)

In the months of January 2001 and 2002, female cachara Pseudoplatystoma fasciatum were selected during their first and second gonadal maturation (2 years and 7 months old and 3 years and 7 months old, respectively) with an of oocyte diameter of 937.5 mum (82.5% with central nuclei and 17.5% with peripheral nuclei). Nine females in first maturation received two doses of carp pituitary extract (CPE), 0.5 mg/kg and 5.0 mg/kg; seven received two doses of human chorionic gonadotropin (hCG), 5 and 10 IU/g; five received doses of 0.5 CPE mg/kg and 5 hCG IU/g (CPE+hCG); and four received 0.9% saline (saline). Nine females from CPE and seven from hCG presented oocytes with the same diameter at the moment of oocyte release (100% with germinal vesicle breakdown and fertilization rate of 53.44 +/- 18.3 and 54.81 +/- 11.8%; larvae number of 165,330 +/- 94.1 and 158,570 +/- 20.6, respectively). The five females from CPE+hCG did not respond to the hormonal treatment. The four females from the saline group did not ovulate. In January 2002, 6 of 15 selected females that were going through the second reproductive cycle received CPE (five received hCG and four received saline), showing oocyte diameters similar to the ones in the first maturation. At stripping, CPE females had an oocyte diameter of 1062.5 mum (the hCG females had oocyte diameters ranging from 937.5 to 1125.0 mum; fertilization rates of 56.08 +/- 30.9 and 81.90 +/- 17.3%; 364,547 +/- 244 and 633,129 +/- 190, larvae, respectively). The fertilization rates and larvae number were higher in the second gonad maturation, both for CPE and hCG. (C) 2004 Elsevier B.V. All rights reserved.

The administration of exogenous prostaglandin may improve ovulation in pacu (Piaractus mesopotamicus)

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Effect of Vitrification of Feline Ovarian Cortex on Follicular and Oocyte Quality and Competence

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.52 mm3) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.