El consumo semicrónico moderado de alcohol altera la foliculogénesis y la calidad núcleo-citoplasmática oocitaria, en el ratón / Moderate semi-chronic alcohol consumption alters folliculogenesis and oocyte nuclear-cytoplasmic quality in mice

Resumen El consumo crónico de alcohol es un problema de salud mundial que afecta particularmente a la población femenina. Sin embargo, los efectos de la ingesta semicrónica en cantidades moderadas a bajas en el ovario y el oocito son poco conocidos. En un modelo murino, se administró etanol al 10% en agua de bebida (hembras tratadas) o agua (hembras control) por 15 días, y luego de la superovulación o no (ovulación espontánea), se analizó el ciclo estral y la calidad ovárico-gamética. En las hembras tratadas, la frecuencia y duración del diestro aumentó, y las frecuencias de folículos y cuerpos lúteos disminuyeron vs hembras controles, valores que se restauraron luego de la superovulación. Sin embargo, en las hembras tratadas, la tasa de proliferación celular folicular y el desbalance de la expresión ovárica de VEGF (factor de crecimiento endotelial) persistieron luego de la superovulación. El número de ovocitos ovulados con metafase II anormal, fragmentados y activados partenogenéticamente fue mayor en las hembras tratadas respecto las controles. En conclusión, el consumo semicrónico moderado de alcohol produce anestro, ciclo estral irregular, foliculogénesis deficiente y anomalías núcleo-citoplasmáticas en los oocitos ovulados. Estas alteraciones podrían constituirse en un factor etiológico de pérdida gestacional temprana y desarrollo embrionario anormal luego del consumo de alcohol.

Abstract Chronic alcohol consumption is a global health problem that particularly affects the female population. However, the ef-fects of semi-chronic ethanol intake in low-moderate amounts on the ovary and oocyte are poorly understood. In a mouse model, 10% ethanol was administered in drinking water (treated females) or water (control females) for 15 days, and after superovulation or not (spontaneous ovulation), the estrous cycle and ovarian-gametic quality were analyzed. In treated females, the frequency and duration of the diestrus increased, and the frequencies of follicles and corpus luteum decreased vs control females, values that restored after superovulation. However, in treated females, the follicular cell proliferation rate and the imbalance in ovarian expression of VEGF (endothelial growth factor) persisted after superovulation. The number of ovulated oocytes with abnormal metaphase II, fragmented and parthenogenetically activated was higher in treated females than in control ones. In conclusion, moderate semi-chronic alcohol consumption produces anestrum, irregular estrous cycle, poor folliculogenesis, and nuclear-cytoplasmic abnormalities in ovulated oocytes. These alterations could constitute an etiological factor of early gestational loss and abnormal embryonic development after alcohol consumption.

Melatonin effects on ovarian follicular cells: a systematic review

SUMMARY Melatonin is known for its effects on both the sleep and reproductive system of mammals. The latter has melatonin receptors type 1 and 2, which act to regulate, among other things, cyclic AMP. Notwithstanding all the literature data, there is still no sound knowledge or a clear understanding of the hormone's action on the physiology of ovarian follicular cells. OBJECTIVE To review and evaluate studies about melatonin action on the ovarian granulosa/theca interna cells from the literature. METHODS The systematic review was carried out according to the PRISMA recommendations. The MEDLINE and Cochrane primary databases were consulted with the use of specific terms. There was no limitation on language or publication year. RESULTS Seven papers about melatonin action on granulosa cells were selected. The following can be attributed to the hormone's effects: a) progesterone increase in culture medium; b) increased estrogen production; c) antagonistic action on estrogen; d) improvement in cell quality resulting in improved embryo and higher pregnancy rates; e) improved cell proliferation via MAPK; f) reduction of free radicals. Nevertheless, there are contrarian papers reporting a reduction in progesterone production. CONCLUSION Melatonin interferes in sex steroid production, boosting progesterone output. Such action may help improve oocyte quality.

RESUMO A melatonina é conhecida por seus efeitos no sono e no sistema reprodutivo dos mamíferos. Este último tem receptores de melatonina tipos 1 e 2, que atuam para regular, entre outras coisas, o AMP cíclico. Apesar de todos os dados da literatura, ainda não há um conhecimento sólido ou uma compreensão clara da ação do hormônio na fisiologia das células foliculares ovarianas. OBJETIVO Revisar e avaliar estudos da ação da melatonina na literatura sobre as células internas da granulosa/teca ovariana. MÉTODOS A revisão sistemática foi realizada de acordo com as recomendações do Prisma. As bases de dados primárias Medline e Cochrane foram consultadas com o uso de termos específicos. Não houve bar na língua ou ano de publicação. RESULTADOS Sete artigos sobre a ação da melatonina nas células da granulosa foram selecionados. O que se segue pode ser atribuído aos efeitos do hormônio: a) aumento de progesterona no meio de cultura; b) aumento da produção de estrogênio; c) ação antagônica no estrogênio; d) melhoria na qualidade celular, resultando em melhor embrião e maiores taxas de gravidez; e) melhor proliferação celular via MAPK; f) redução de radicais livres. No entanto, existem artigos controversos relatando redução na produção de progesterona. CONCLUSÃO A melatonina interfere na produção de esteroides sexuais, aumentando a produção de progesterona. Tal ação pode ajudar a melhorar a qualidade do oócito.

Involvement of K+ATP and Ca2+ channels in hydrogen sulfide-suppressed ageing of porcine oocytes

BACKGROUND: Hydrogen sulfide has been shown to improve the quality of oocytes destined for in vitro fertilization. Although hydrogen sulfide is capable of modulating ion channel activity in somatic cells, the role of hydrogen sulfide in gametes and embryos remains unknown. Our observations confirmed the hypothesis that the KATP and L-type Ca2+ ion channels play roles in porcine oocyte ageing and revealed a plausible contribution of hydrogen sulfide to the modulation of ion channel activity. RESULTS: We confirmed the benefits of the activation and suppression of the KATP and L-type Ca2+ ion channels, respectively, for the preservation of oocyte quality. CONCLUSIONS: Our experiments identified hydrogen sulfide as promoting the desired ion channel activity, with the capacity to protect porcine oocytes against cell death. Further experiments are needed to determine the exact mechanism of hydrogen sulfide in gametes and embryos.

Effects of Anethum graveolens L. [dill] on oocyte and fertility of adult female rats

Our previous studies revealed Anethum graveolens L. caused some changes in female reproductive system that induced infertility. Therefore, in this study, oocyte changes as one of probable reasons of infertility were investigated. In this study, 59 adult female rats were divided into 3 groups of control, low dose [0.5 g/kg] and high dose [5 g/kg] of dill seed aqueous extract [LDE and HDE] treated groups that were gavaged with 1 ml of each dose for 10 days [2 estrous cycles]. Vaginal smears were prepared daily. Oocytes of superovulated animals were extracted and their morphometrical changes were measured [n=5]. Oocyte cell membrane glycoconjugates were stained with UEA, PNA, and DBA-FITC lectins [n=5]. Ultrastructural studies of oocytes were performed using TEM [n=5]. The number, weight, and crown-rump length of newborns were examined in three groups after mating with untreated males [n=5]. Data were analyzed using SPSS software. Results demonstrated that the duration of the estrous cycle, the diestrus phase and progesterone concentration in the experimental groups increased significantly compared to the control group [p<0.05]. Granulosa cells of corpus luteum in HDE-treated group were larger and clearer. The intensity reactions of galactose/N-acetylgalactoseamine terminal sugar of oocyte decreased insignificantly in experimental groups compared to the control group p>0.05. Duration of mating to pregnancy increased and the weight and crown-rump length of newborns decreased in experimental groups significantly [p<0.05]. Dill seed aqueous extract can induce infertility without any effect on oocyte structure

Melatonin impact on in vitro development of mouse preantral follicles and oocyte maturation

Melatonin acts as an indirect antioxidant and is a powerful direct free radical scavenger and direct responses to melatonin in the gonads are detected. This study aims to investigate the influence of different doses of melatonin on preantral follicle development and oogenesis of in vitro cultured mouse ovarian follicles. Preantral follicles with diameters of 150- 175 micro m were mechanically isolated from NMRI mouse ovaries. Follicles were cultured in droplets of alpha-minimal essential medium [alpha-MEM] supplemented with 5% FBS, 100 mIU/ml rhFSH, 1%ITS, 100 IU/ml penicillin and 100 micro g/ml streptomycin in conjunction with varying doses of melatonin [0, 1, 10, 100 nM and 100, 500 pM] for six days. On day six, in vitro ovulation was induced by the addition of hCG/rEGF to the culture medium and after 16-20 h the maturation state of the oocytes was assessed. There was a significant [P<0.05] decrease in the number of surviving follicles in the groups that received 10, 100 nM and 500 pM melatonin compared to the other groups. After induction of in vitro ovulation, follicles in groups that received 1, 10, and 100 nM melatonin had higher ovulation rates [P<0.05] compared with the other groups. Oocyte maturation capacity was adversely influenced by five concentrations of melatonin and GV arrest was significantly higher compared to the control group [P<0.01]. Our data indicates that a dose of 100 pM melatonin has no toxic effects on follicular development and can be used to reduce oxidative stress in follicle culture systems

In vitro assessment of reproductive toxicity of cigarette smoke and deleterious consequences of maternal exposure to its constituents

Cigarette smoke is known to be a serious health risk factor and considered reproductively toxic. In the current study, we investigated whether constituents of cigarette smoke, pyrazine, 2-ethylpyridine, and 3-ethylpyridine, adversely affect reproductive functioning such as oocyte maturation and sperm capacitation. Our findings indicated that three smoke components were involved in retardation of oocyte maturation in a dose-dependent manner and the lowest-observed-adverse-effect level (LOAEL) was determined to be 10-10M. However, individual smoke components administrated at the LOAEL did not attenuate oocyte maturation, demonstrating that all three toxicants were equally required for the observed growth impairment. When exposed to all three components at 10-10M during in vitro capacitation, murine sperm lost forward progression and were unable to show adequate hyperactivation, which is indicative of the incompletion of the capacitation process. Only sperm administrated with 3-ethylpyridine alone showed significant reduction in capacitation status, suggesting the chemical is the one responsible for disrupting sperm capacitation. Taken together, this is the first report that documents the effect of cigarette smoke components on oocyte maturation and sperm capacitation. The present findings demonstrate the adverse effects of smoke constituents of mammalian reproduction and the differences in sensitivity to smoke components between male and female gametes. Since both processes take place in the female reproductive system, our data provide new insights into deleterious consequences of maternal exposure to cigarette smoke.

Effect of calcium ionophore on unfertilized oocytes after ICSI cycles

Fertilization failure is one of the most problems in assisted reproduction technology [ART]. The aim of this study was the evaluation of oocytes activation by addition of calcium ionophore in unfertilized oocytes in ICSI cycles. This study was done on 15 ICSI cycles [stimulated with standard long protocol]. Mature retrieved oocytes with normal morphology that had no evidence of fertilization 24 hours after ICSI were included in the study. The oocytes with fertilization and unfertilized oocytes with degeneration were excluded from the study. The unfertilized oocytes were washed with GIVF medium and were transferred to GIVF medium that contained 5 micro mol of calcium ionophore and were incubated for 10 minutes. Then again oocytes were washed with GIVF medium and consequently were transferred to GIVF medium and were incubated at 37°C in 6% CO[2]. After 18 hours, the oocytes were examined and activated oocytes were defined with observation of at least one pronucleus or cleaved oocytes. After ovarian stimulation and oocytes retrieval, 175 mature oocytes were obtained and injection of sperm was done for all of them. 114 of 175 oocytes [66%] showed evidence of fertilization after 24 hours. A total of 61 oocytes [34%] showed no evidence of fertilization and 10 oocytes were degenerated and were excluded from the study. Only 51 unfertilized oocytes with normal morphology were selected and were exposed to calcium ionophore. 37 [72.5%] of treated oocytes were fertilized [2PN] and 32 [62.7%] of them showed evidence of cleavage. 6 [11.8%] embryos had good quality. According to our results, oocytes activation with calcium ionophore had an acceptable fertilization rate, however high quality embryos remained low. We propose future studies to evaluate embryo quality

Effect of various concentrations of minimal essential medium vitamins [MEM vitamins] on development of sheep oocytes during in-vitro maturation

Improvements in culture media formulations have led to an increase in the ability of sheep embryo in culture throughout the preimplantation period. This study was carried out to evaluate the effects of various concentrations of MEM vitamins during in vitro maturation of sheep oocytes and subsequent embryo development. Sheep ovaries were collected from a slaughterhouse and transported to the laboratory. Oocytes were matured in SOF medium supplemented with, eCG, hCG and EGF in various concentrations of MEM vitamins [control, 0.5, 1 and 1.5x] for 24h. The cumulus oocyte compelex [COCs] were co-incubated with epididymal spermatozoa of post mortem rams in synthetic oviduct fluid fertilization [SOFF] medium with 10% heat inactivated estrous sheep serum for 18h. Embryos were cultured in synthetic oviduct fluid culture 1 [SOFC1] medium for 48h followed by cultured in synthetic oviduct fluid culture 2 [SOFC2] medium for six days. Addition of 0.5 and 1 x MEM vitamins significantly increased [P< 0.05] overall blastocyst development [21.62% and 22.33%; respectively] compared with 1.5x MEM vitamins [15.59%], but there was no difference between control, 0.5 and 1x MEM vitamins in the percentage of embryos successfully developing to the blastocyst stage [19.50%, 21.62% and 22.33% respectively]. It seems that addition of 1.5x of MEM vitamins has detrimental effect on blastocyst rate

Evaluation of effect of silymarin on granulosa cell apoptosis and follicular development in patients undergoing in vitro fertilization

To investigate the effects of silymarin on follicular development, we enrolled 40 healthy women undergoing in vitro fertilization [IVF] due to male factor infertility in this trial. They underwent ovulation induction and on a random and blind basis, patients were assigned to receive silymarin [70 mg X 3/day] or placebo from the beginning of the induction cycle. The number and quality of oocytes retrieved were evaluated and apoptosis of granolusa cells was studied. There was no significant difference between the groups for mean number of follicles >/= 18 mm [P = 0.131], mean number of oocytes retrieved [P = 0.209] or endometrial thickness [P = 0.673]. However, the proportion of total apoptosis in the study group was significantly lower than in the placebo group [P = 0.032]. These data suggest that administration of silymarin in IVF patients concomitantly with gonadotropin results in reduction of granolusa cell apoptosis but does not have any effect in promotion of follicular development, oocyte retrieval or endometrial thickness

Effect of different types of cryoprotectants on developmental capacity of vitrified-thawed immature buffalo oocytes

The study aimed to determine the effects of different types of cryoprotectants on developmental capacity of vitrified-thawed immature buffalo oocytes. The vitrification solution [VS] consisted of Dulbecco's phosphate buffered saline [DPBS] supplemented with 0.5 M sucrose, 0.4% bovine serum albumin [BSA] and different types of molar [M] concentrations of the cryoprotectants which composed of either glycerol [G], ethylene glycol [EG] or dimesthyl sulfoxide [DMSO] in order to determine the best type of vitrification cryoprotectants. The concentrations tested were 4 M, 7 M and 7M concentration of G, EG and DMSO, respectively. Cumulus oocyte complexes [COCs] were obtained from slaughterhouse ovaries. Oocytes were vitrified immediately after collection .The COCs were pre-equilibrated in 50% of the VS for 3-5 min, then kept in VS for 1 min and loaded in pre-sterilized 0.25 ml straws for 7-10 days of storage in liquid nitrogen. The straws were thawed in warm water at 37°C for 10 seconds and COCs were evaluated for morphological damage. Morphologically normal COCs were cultured in vitro and evaluated for maturation. Oocytes were fertilized with frozen-thawed semen capacitated in Brackett and Oliphant [BO] medium contained heparin and caffeine and evaluated for cleavage and embryonic development. The results revealed that the proportion of buffalo oocytes found to be morphologically normal was significantly [p<0.05] higher in EG and DMSO than those obtained in G [85.0 and 83.33 vs 65.0%, respectively]. Among the damaged oocytes, cracking of zona pellucida was the most frequent abnormality observed. A significantly higher [p<0.05] percentage of maturation and cleavage rates derived from vitrified -thawed immature oocyte in EG and DMSO than those obtained in G [47.05, 46.67%; 28.57, 25.71 vs 30.76% and 10.0%, respectively]. A similar trend was observed in blastocyst stage produced in vitro. However, in vitro developmental competence was higher for vitrified-thawed fresh oocytes [control] than those obtained from all groups of cryoprotectants. that 7M solution of EG or DMSO could be used for vitrification of immature buffalo oocytes for their subsequent utilization in the in vitro maturation, fertilization and embryo production

Effects of the conditioned medium of mesenchymal stem cells on mouse oocyte activation and development

Mesenchymal stem cells (MSCs) have been reported to secrete a variety of cytokines and growth factors acting as trophic suppliers, but little is known regarding the effects of conditioned medium (CM) of MSCs isolated from femurs and tibias of mouse on the artificial activation of mouse oocytes and on the developmental competence of the parthenotes. In the current study, we investigated the effect of CM on the events of mouse oocyte activation, namely oscillations of cytosolic calcium concentration ([Ca²+]i), meiosis resumption, pronucleus formation, and parthenogenetic development. The surface markers of MSCs were identified with a fluorescence-activated cell sorter. The dynamic changes of the spindle and formation of pronuclei were examined by laser-scanning confocal microscopy. Exposure of cumulus-oocyte complexes to CM for 40 min was optimal for inducing oocyte parthenogenetic activation and evoking [Ca²+]i oscillations similar to those evoked by sperm (95 vs 100 percent; P > 0.05). Parthenogenetically activated oocytes immediately treated with 7.5 µg/mL cytochalasin B (CB), which inhibited spindle rotation and second polar body extrusion, were mostly diploid (93 vs 6 percent, P < 0.01) while CB-untreated oocytes were mostly haploid (5 vs 83 percent, P < 0.01). Consequently, the blastocyst rate was higher in the CB-treated than in the CB-untreated oocytes. There was no significant difference in developmental rate between oocytes activated with CM and 7 percent ethanol (62 vs 62 percent, P > 0.05), but the developmental competence of the fertilized oocytes was superior to that of the parthenotes (88 vs 62 percent, P < 0.05). The present results demonstrate that CM can effectively activate mouse oocytes, as judged by the generation of [Ca²+]i oscillations, completion of meiosis and parthenogenetic development.

effect of progesterone on the in vitro maturation and developmental competence of mouse germinal vesicle oocytes

The aim of the present study was to investigate the role of progesterone on the developmental competence of cumulus-oocyte complexes [COCs] and cumulus-denuded oocytes [CDOs] at germinal vesicle [GV] stage. GV oocytes of pregnant mare's serum gonadotropin [PMSG]-primed prepubertal mice were divided into two groups: CDOs and COCs. The oocytes were cultured in TCM199 with different concentrations of progesterone [10, 38, 50 and 100 micro M] and without progesterone [controls]. The number of oocytes at the GV, germinal vesicle breakdown [GVBD] and metaphase II [MII] stages were counted. In vitro fertilization [IVF] of MII oocytes and their development to the blastocyst stage were evaluated. Significantly different MII rates were observed between the COCs [85%] and CDOs [68%] control groups. The MII rates of 83%, 48%, 14% and 0% for COCs and 65%, 53%, 20% and 0% for CDOs were obtained in TCM199 that contained 10, 38, 50 and 100 micro M progesterone concentrations, respectively. These MII rates were lower [p<0.05] in both COCs and CDOs as compared to their respective control groups, except for 10 micro M. The fertilization and blastocyst rates of COCs [83% and 35%, respectively] were higher [p <0.05] than those of the CDOs [51% and 5%, respectively] control groups. The fertilization and blastocyst rates in the presence of 10 micro M [81% and 36%, respectively] and 38 micro M [85% and 30%, respectively] progesterone in COCs and CDOs [52% and 4% for 10 micro M; 56% and 4% for 38 micro M] were similar to their respective control groups. Adding progesterone to the medium could not improve maturation of mouse GV oocytes and their development to the blastocyst stage

Nitric oxide acts through different signaling pathways in maturation of cumulus cell-enclosed mouse oocytes

Nitric oxide [NO] have a dual action in mouse oocyte meiotic maturation which depends on its concentration, but the mechanisms by which it influences oocyte maturation has not been exactly clarified. In this study different signaling mechanisms which exist for in vitro maturation of meiosis was examined in cumulus cell-enclosed oocytes [CEOs] after injection of pregnant mare's serum gonadotropin [PMSG] to immature female mice. The CEOs were cultured in spontaneous maturation and hypoxanthine [HX] arrested model. Sodium nitroprusside [SNP, an NO donor, 10mM] delayed germinal vesicle breakdown [GVBD] significantly during the first 5 hrs of incubation and inhibited the formation of first polar body [PB1] at the end of 24 hrs of incubation. SNP [10-5M] stimulated the meiotic maturation of oocytes significantly by overcoming the inhibition of HX. Sildenafil [a cGMP stimulator, 100 nM], had a significant inhibitory effects on both spontaneous meiotic maturation and HX-arrested meiotic maturation. Forskolin [an adenylate cyclase stimulator, 6 micro M] and SNP [10mM] had the same effects on GVBD. Forskolin reversed the SNP [10-5M] stimulated meiotic maturation. These results suggest that differences in pathways are present between SNP-inhibited spontaneous meiotic maturation and SNP-stimulated meiotic maturation in mouse oocytes

influence of meiotic spindle configuration by cysteamine during in vitro maturation of mouse oocytes

The aim of this study was to assess effects of cysteamine as an anti-oxidant on rate of in vitro maturation of oocyte and determination of its effects on spindle size and shape. Pre-mature mice were primed with pregnant mare stimulating gonadotrophin [PMSG] and germinal vesicle [GV] stage oocytes were obtained 48 h after. The oocytes were cultured in tissue culture medium [TCM 199] with 50, 100, 200 and 500 microM cysteamine. Experiments also included a group of ovulated oocytes [in vivo matured] after priming with PMSG and human chorionic gonadotrophin. For spindle immuno-cytochemistry of metaphase II [MII], oocytes alpha and beta tubulin antibody were performed. Chromosome staining was performed with Hoechst. Our results showed that the rate of GV breakdown [GVBD] and MII increased in all cysteamine groups except in group cultured with 500 microM cysteamine. Immuno-cytochemistry analysis showed that spindle area in all in vitro Maturation oocytes increased when compared to in vivo oocytes. Spindle shape and size [spindle area] in 100 microM cysteamine in comparison to in vivo group was insignificant [P>0.05]. Our results revealed that cysteamine improved IVM rate in a dose dependant manner. The size and shape of spindle in the presence of given concentration of cysteamine was similar to in vivo. Therefore, cysteamine improved microtubule organization, rate of GVBD and MIl development

[Effects of different doses of LIF on IVM rate and cumulus expansion in mouse oocytes]

The aim of this research was to study the effects of different doses of LIF on GVBD and MII development rate and cumulus expansion. Immature mice superovulated with HMG and GV oocytes obtained from ovary 48 hours later. The GV oocytes were cultured in TCM199 with 100, 500 and 1000 RI /ml LIF. Cumulus expansions were evaluated with two examiners and numbers of MII oocytes were recorded. For denuding the oocytes hyaloronidase was used. Our results showed that the rate of GVBD and MII development increased in -groups with LIF compared with control group. Rate of MII development with 1000 IU/ml LIF was significantly higher than that of control group [P<0.05]. Cumulus expansion in group with 1000 IU/ml LIF improved significantly compared with control group [p<0.05]. Our results showed that LIF could improve IVM rate in dose dependant. Also cumulus expansion improved in group with LIF and increased oocyte quality

[Effect of fibroblastic growth factor on resumption of meiosis, in vitro maturation and embryo development of immature mouse oocytes]

The purpose of this study was to evaluate the effect of fibroblastic growth factor on resumption of meiosis, in vitro maturation of immature mouse oocytes and resulting embryo development with and without basic fibroblastic growth factor-4 [bFGF-4].Cumulus - oocyte complex [COCs] and germinal vesicle [GV] were obtained from female NMRI mice 46-48 hours after administration of an intra-peritoneal injection of 5 IU PMSG. COCs were cultured in TCM199 supplemented with different dosages of bFGF-4. After 24 hours, metaphase II [MII] oocytes were co-incubated with sperms for 4-6 hours in 16 medium. For all groups, the rate of cleaved embryos was assessed in the T6 medium until blastocyst stage. In all compared groups, the percentage of matured MII oocytes in the 10 ng/ml [%94.4] and 20 ng/ml [%92.5] of bFGF-4 treatment groups, was significantly higher [P<0.05] than those of the control group but the percentage of embryos that developed to blastocyst in 20 ng/ml bFGF-4 treatment group was significantly higher than those of the control group [P<0.05]. Exogenous bFGF-4 improved the oocyte maturation and embryo development

Regulation of Antiarrhythmic Drug Propafenone Effects on the C-type KV1.4 Potassium Channel by PHo and K+

The effects of the antiarrhythmic drug propafenone at c-type kv1.4 channels in Xenopus laevis oocytes were studied with the two-electrode voltage-clamp techinique. Defolliculated oocytes (stage V-VI) were injected with transcribed cRNAs of ferret Kv1.4 delta N channels. During recording, oocytes were continuously perfused with control solution or propafenone. Propafenone decreased the currents during voltage steps. The block was voltage-, use-, and concentration- dependent manners. The block was increased with positive going potentials. The voltage dependence of block could be fitted with the sum of monoexponential and a linear function. Propafenone accelerated the inactivate of current during the voltage step. The concentration of half-maximal block (IC(50)) was 121 micrometer/L. With high, normal, and low extracellular potassium concentrations, the changes of IC(50) value had no significant statistical differences. The block of propafenone was PH- dependent in high-, normal- and low- extracellular potassium concentrations. Acidification of the extracellular solution to PH 6.0 increased the IC50 values to 463 micrometer/L, alkalization to PH 8.0 reduced it to 58 micrometer/L. The results suggest that propafenone blocks the kv1.4 delta N channel in the open state and give some hints for an intracellular site of action.

[ effect of HCG on in vitro maturation of human immature oocytes and rate of 8 cell embryos mediated by ICSI]

The infertility treatment by using in vitro matured oocyte has many potential applications. It minimizes the risk of ovarian hyper stimulation and is an alternative treatment for women with polycystic ovarian syndrome who may have problems regarding stimulation for IVF. This method may prove important for subjects in need of fertility preservation and also provides information about the final stage of oocyte maturation. The aim of this study was to determine the effect of HCG on the rate of in vitro matured oocyte and 8 cell embryos mediated by ICSI. This experimental study was done in Montaserieh IVF center and Islamic Azad University at Mashhad. One hundred sixty eight immature oocytes aspirated from women undergoing IVF cycles were divided into 3 equal groups and allocated for maturation in 3 medium cultures. All of 3 culture groups contained G1 medium supplemented with HSA 10% [Human Serum Albumin]. Control group had no gonadotropin, but the first experimental group contained HCG 10 ILJ/ml and the second experimental group contained HCG 5 ILJ/ml. Matured oocytes were fertilized by ICSI and numbers of 8-cell embryos were investigated 72 hours after fertilization by invert microscope. The results showed that both of the HCG concentrations significantly increased the rate of matured oocytes and also the rate of 8-cell embryos with respect to control group [p<0.05]. No significant differences were found in rate of matured oocytes between the two experimental groups [p>0.05] while the number of 8-cell embryos was significantly higher in HCG 5IU/ml with respect to HCG 10 IU/ml and control group [p<0.05]. The results suggests that both of the HCG concentrations increase the rate of maturation of immature oocytes and the rate of 8-cell embryos but HCG [5IU/ml] has more effect on the number of 8-cell embryos mediated by ICSI

[Effect of demecolicne treatment on the developmental competency of in vitro matured bovine oocytes]

The aim of this study was to investigate whether demecolicne treatment of matured bovine oocytes adversely affects the process of in vitro fertilization and embryo development. Bovine Cumulus Oocyte Complexes [COC's] were matured in vitro and then were randomly allocated to two treatment groups of common concentrations of demecolicne [0.05 and 0.4 micro g/ml for 30 min] and a control group. COC's were then fertilized and cultured in vitro for up to 9 days when the ratios of in vitro embryo development and the viability of the hatched blastocysts were assessed and compared with the control group [p<0.05]. The ratios of the cleavage and blastocyst formation of demecolicne treated groups [0.4 and 0.05 micro g/ml] were 68.6, 63.5% and 23.3, 32.8%, which were not significantly different from the control group [73.3, 29.0%], respectively. The results of cell-viability were also not significantly different between the control vs. treatment groups. Since the overall indices of in vitro embryo development revealed no significant difference between the demecolicne treated compared to control bovine oocytes, it seems that demecolicne treatment of matured bovine oocytes may not compromise their potency for further in vitro development

effect of Endosulfan on the ovary of Bluegill sunfish: a histopathological study [Lepomis macrochirus]

The effects of pesticide endosulfan [an organochlorine compound], on the ovaries of bluegill fish [Lepomis macrochirus] were studied. Exposures for 24 hs with histological preparations at 25% [0.25 micro g/L], 75% [0.75 micro g/L], and 100% [1 micro g/L] sub lethal concentrations were examined. The control contained an abundance of the different stages of oocytes [Oocytes I, II, III, and IV] and had an intact ovigerous lamellae and follicular lining. The control also contained a thick and complete ovarian wall with evident provitelline and euvitelline nucleoli. After 24 hr exposure to a 25% concentration, many Oocyte II and III cells had damaged stroma and cytoplasmic and nuclear retraction. Adhesion is pronounced at the 25% concentration, but is even more profound at the 75% concentration. Empty follicles and a unique cytoplasmic clumping can be observed in Oocyte III and IV cells in the 75% concentration. The ovaries of fish exposed to a 100% concentration display an immense amount of empty follicles along with necrosis of nuclei and expelled nuclei. As the concentration increased, the amount of atretic cells increased, and the ovarian wall became more thinned and lifted. Macrophages were more evident as the concentration increased and the sizes of the different stages of oocytes, exposed to the different concentrations became smaller as well. This study showed that there is a clear correlation between the amount of damage seen and the amount of endosulfan