Is the effect of melatonin on vascular endothelial growth factor receptor-2 associated with angiogenesis in the rat ovary?

OBJECTIVES Vascular endothelial growth factor (VEGF) and its receptors play important roles in angiogenesis. Melatonin plays an important role in gonadal development; thus, its effect on the reproductive system is evident. We investigated the influence of melatonin on the expression of VEGF, vascular endothelial growth factor receptor-1 (VEGFR1) and vascular endothelial growth factor receptor-2 (VEGFR2), as well as on changes in oxidative stress markers and follicle numbers in rat ovaries. METHODS For this purpose, 45 Wistar rats were separated into the following groups: Group 1, control; Group 2, vehicle; and Group 3, melatonin. Rats in Group 3 were treated with melatonin at 50 mg/kg/day for 30 days. The effects of melatonin on the expression of VEGF, VEGFR1 and VEGFR2 were established by immunohistochemistry analysis. The effects of melatonin on antioxidant enzyme activities were demonstrated by spectrophotometric analysis. RESULTS Based on immunohistochemistry analysis, VEGFR2 was predominantly localized to theca cells in the ovary. Our data indicate that melatonin treatment can significantly increase VEGF and VEGFR1 expression in the ovary ( p <0.05). Additionally, the number of degenerated follicles significantly decreased with melatonin treatment ( p <0.05). Melatonin administration also led to significant increases in antioxidant enzyme levels in the ovary. CONCLUSION Melatonin treatment exerts protective effects on follicles against increased lipid peroxidation through modulating tissue antioxidant enzyme levels. These effects may be related to angiogenesis and antioxidant activities.

The effects of an experimentally induced unilateral varicose ovarian vein on the activities of anti-oxidant enzymes in an adult rat ovary / Efectos de la inducción experimental unilateral de una vena ovárica varicosa sobre las actividades de las enzimas anti-oxidantes en ovarios de ratas adultas

In this study, we aim to examine effects of an experimentally induced unilateral varicose ovarian vein on the activities of anti-oxidant enzymes in an adult rat ovary. In this experimental study, a total of 30 adult female Wistar albino rats were divided into three groups. 10 rats in group 1 as the varicocele group, 10 rats in group 2 as the control group and 10 rats in group 3 as the sham group, that underwent a sham operation and. Anti-oxidant assays were assessed via specific assay kits. Statistical analysis was performed using the one way ANOVA and Tukey's tests were used for post hoc multiple comparisons, P<0.05 was considered statistically significant. The effects of the unilateral varicosity was more evident on the left side when compared to the right side as all activities of the anti-oxidant assayed were significantly reduced, P 0.05 when compared to the right side. Also, in this present study, the effect of the unilateral varicose vein was bilateral as there were no significant differences recorded between the two sides. Finally the result of this study shows that varicocele may lead to female infertility through various factors that includes reduction in the activities of anti-oxidant enzymes.

En este estudio, nuestro objetivo fue examinar los efectos de la inducción experimental unilateral de una vena ovárica varicosa en la actividad de enzimas antioxidantes en un ovario de rata adulta. Un total de 30 ratas albinas Wistar, hembras adultas, se dividieron en tres grupos. Diez ratas en el grupo 1 (grupo varicocele), diez ratas en el grupo 2 (grupo de control) y diez ratas en el grupo 3 (grupo de tratamiento simulado), que se sometió a una operación simulada. Ensayos con anti-oxidantes se evaluaron a través de kits de ensayo específicos. El análisis estadístico se realizó mediante ANOVA de una vía y las pruebas de Tukey fueron utilizadas para comparaciones múltiples Post Hoc, siendo el P<0,05 considerado como estadísticamente significativo. Los efectos de la varicosidad unilateral fue más evidente en el lado izquierdo cuando fue comparada con el lado derecho en todas las actividades del ensayo con anti-oxidante que se redujeron significativamente, el P 0,05 cuando se compara con el lado derecho. Asimismo, en el presente estudio, el efecto de la vena varicosa unilateral fue bilateral ya que no hubo diferencias significativas registradas entre las dos partes. Por último, el resultado de este estudio muestra que el varicocele puede conducir a la infertilidad femenina a través de diversos factores que incluye la reducción en la actividad de las enzimas antioxidantes.

Immunohistochemical evaluation of proliferation, apoptosis and steroidogenic enzymes in the ovary of rats with polycystic ovary / Avaliação imunoistoquímica de proliferação, apoptose e enzimas esteroidogênicas no ovário de ratas com síndrome dos ovários policísticos

Objective: to evaluate the immunohistochemical expression of proliferative, apoptotic and steroidogenic enzyme markers in the ovaries of rats with polycystic ovary syndrome (PCOS). Methods: twenty rats were divided into two groups: GCtrl - estrous phase, and PCOS - with polycystic ovaries. The GCtrl animals were subjected to a lighting period from 7 am to 7 pm, while the animals with PCOS group remained with continuous lighting for 60 days. Subsequently, the animals were anesthetized, the ovaries were removed and fixed in 10% formaldehyde, prior to paraffin embedding. Sections were stained using H.E. or subjected to immunohistochemical methods for the detection of Ki-67, cleaved caspase-3, CYP11A1, CYP17A1 and CYP19A1. The results were analyzed using Student's t-test (p < 0,05). Results: morphological results showed evidence of interstitial cells originating from the inner theca cells of degenerating ovarian cysts in PCOS. Immunoexpression of Ki-67 was higher in the granulosa cells in GCtrl, and the theca interna cells in PCOS, while cleaved caspase-3 was higher in granulosa cells of ovarian cysts from PCOS and in the theca interna cells of GCtrl. Immunoreactivity of CYP11A1 in the theca interna, granulosa and interstitial cells was similar between the two groups, while CYP17A1 and CYP19A1 were higher in the granulosa and interstitial cells in the PCOS group. Conclusion: the results indicate that the interstitial cells are derived from the theca interna and that enzymatic changes occur in the theca interna and interstitial cells in ovaries of rats with PCOS, responsible for the high levels of androgens and estradiol. .

Objetivo: avaliar a expressão imunoistoquímica de marcadores de proliferação, apoptose e enzimas esteroidogênicas nos ovários de ratas com síndrome dos ovários policísticos (SOP). Métodos: vinte ratas foram divididas em dois grupos: controle (GCtrl), na fase de estro, e com síndrome dos ovários policísticos (GSOP). Os animais do GCtrl permaneceram com período de luz das 7 às 19 horas, e os do GSOP com iluminação contínua, durante 60 dias. Posteriormente, os animais foram anestesiados, os ovários removidos e fixados em formol a 10% para inclusão em parafina. Cortes histológicos foram corados pelo H.E. e outros submetidos a métodos imunoistoquímicos para detecção de Ki-67, caspase 3 clivada, CYP11A1, CYP17A1 e CYP19A1. Os resultados foram submetidos ao teste t de Student (p < 0,05). Resultados: a morfologia mostrou evidências da origem das células intersticiais a partir das células da teca interna dos cistos ovarianos em degeneração no GSOP. A imunoexpressão do Ki-67 mostrou-se aumentada nas células da granulosa no GCtrl e na teca interna do GSOP, enquanto a caspase 3 clivada se mostrou aumentada nas células da granulosa dos cistos ovarianos do GSOP e na teca interna do GCtrl. A imunorreatividade da CYP11A1 nas células da teca interna, bem como da granulosa e intersticiais, mostrou-se semelhante entre os dois grupos. As CYP17A1 e CYP19A1 apresentaram-se aumentadas nas células da granulosa e intersticiais no grupo SOP. Conclusão: os resultados indicam que as células intersticiais são oriundas da teca interna e que ocorrem alterações enzimáticas nas células da teca interna e intersticiais do ovário de ratas com SOP, responsáveis pelos altos níveis de androgênios e de estradiol. .

Aromatase activity in brain and ovary: Seasonal variations correlated with circannual gonadal cycle in the catfish, Heteropneustes fossilis.

Seasonal variations in the aromatase activity in H. fossilis estimated by a microassay were correlated with the sex steroids, vitellogenin in and ovarian weight during circannual reproductive cycle. In the female catfish, aromatase activity was detectable in the hypothalamus throughout the year whereas in ovary only during active vitellogenesis. In the catfish, hypothalamic aromatase levels increased two times during annual gonadal cycle, once in a fully gravid fish and then in a reproductively quiescent fish. On the other hand, increase in the ovarian aromatase activity was observed only during vitellogenesis, which showed a direct correlation with plasma levels of sex steroids. Further, plasma levels of testosterone and estradiol suggested a precursor-product relationship. At the completion of vitellogenesis, ovarian aromatase activity declined sharply resulting in elevation of plasma testosterone levels, which in turn could be utilized as substrate by the hypothalamic aromatase whose activity was the highest in the postvitellogenic catfish. At least two isoforms of gene, cyp19a and cyp19b, coding for aromatase in ovary and brain respectively were expressed in the catfish. Aromatase activity was more concentrated in those areas of catfish brain, which have been implicated in the control of reproduction.

Long-term melatonin treatment reduces ovarian mass and enhances tissue antioxidant defenses during ovulation in the rat

Melatonin regulates the reproductive cycle, energy metabolism and may also act as a potential antioxidant indoleamine. The present study was undertaken to investigate whether long-term melatonin treatment can induce reproductive alterations and if it can protect ovarian tissue against lipid peroxidation during ovulation. Twenty-four adult female Wistar rats, 60 days old (± 250-260 g), were randomly divided into two equal groups. The control group received 0.3 mL 0.9 percent NaCl + 0.04 mL 95 percent ethanol as vehicle, and the melatonin-treated group received vehicle + melatonin (100 µg·100 g body weight-1·day-1) both intraperitoneally daily for 60 days. All animals were killed by decapitation during the morning estrus at 4:00 am. Body weight gain and body mass index were reduced by melatonin after 10 days of treatment (P < 0.05). Also, a marked loss of appetite was observed with a fall in food intake, energy intake (melatonin 51.41 ± 1.28 vs control 57.35 ± 1.34 kcal/day) and glucose levels (melatonin 80.3 ± 4.49 vs control 103.5 ± 5.47 mg/dL) towards the end of treatment. Melatonin itself and changes in energy balance promoted reductions in ovarian mass (20.2 percent) and estrous cycle remained extensive (26.7 percent), arresting at diestrus. Regarding the oxidative profile, lipid hydroperoxide levels decreased after melatonin treatment (6.9 percent) and total antioxidant substances were enhanced within the ovaries (23.9 percent). Additionally, melatonin increased superoxide dismutase (21.3 percent), catalase (23.6 percent) and glutathione-reductase (14.8 percent) activities and the reducing power (10.2 percent GSH/GSSG ratio). We suggest that melatonin alters ovarian mass and estrous cyclicity and protects the ovaries by increasing superoxide dismutase, catalase and glutathione-reductase activities.

Correlation between function-morphology of effect of the stress and flunitrazepam administered during gestation in adult mouse ovary / Correlación entre la función y la morfología de los efectos de estrés y flunitrazepam administrado durante la gestación en ovario de ratón adulto

Previously we report long lasting effects on ovary of mice prenatally exposed to flunitrazepam (FNZ), a benzodiazepine with tranquilized action. In this work we find that the FNZ don't prevent the effects on ovary prenatally exposure to stress in mice. We studied adult females born from mothers that had been stressed by immobilization on day 6 ofgestation (GD-6) or group S, and from mothers stressed also by immobilization at GD-6, but which received a single oral dose of FNZ immediately after the stress group FNZS. The control groups were the SS that received the GD-6 saline solution and the group NT non-treated. Their ovaries were extracted for histology studies and to observe the activity of 3b hydroxysteroid dehydrogenase/isomerase (3 b-HSD). The histological analysis revealed high staining affinity ovarian cell of S and FNZS. Double oocytes and apoptotic bodies were found in the secondary atretic follicles, as well as abnormal primordial, primary and secondary follicle populations, as compared to SS and NT groups. The primordial, primary, and secondary follicles were significantly reduced in the experimental groups. But the primary and secondary atretic follicles were higher in both groups, and the number of corpora lutea was lower in both groups. The activity of 3 b-HSD was abnormally increased in both FNZS- and S-groups. These findings suggest that FNZ did not counteract the impairing effects of prenatal stress on adult offspring ovarian follicles, and could rather be responsible for long lasting changes occurring during embryonic programming.

Previamente comprobamos efectos de larga duración sobre el ovario de ratones expuestos prenatalmente a flunitrazepam (FNZ), una benzodiazepina con acción tranquilizante. En este trabajo encontramos que el FNZ, no revierte los efectos producidos por la exposición prenatal a estrés. Estudiamos hembras adultas nacidas de madres que se estresaron por inmovilización el día 6 de la gestación (DG-6) o grupo S, y de madres estresadas también por inmovilización el DG-6, las que recibieron una sola dosis de FNZ inmediatamente después del estrés (grupo FNZS). Los grupos de control fueron el SS al que se le administró solución salina y el NT no tratado. Se extrajeron sus ovarios para su estudio histológico y para observar la actividad de delta 3b-deshidroxiesteroide dehidrogenasa/isomerasa (3 b-HSD). El análisis histológico reveló una gran afinidad tintoreal en los ovarios de los grupos S y FNZS. En los ovarios de los ratones del grupo FNZS se encontraron en los folículos secundarios atrésicos ovocitos dobles y cuerpos apoptóticos así como una población mayor de folículos anormales primordiales, primarios y secundarios en comparación con los grupos SS y NT. Los folículos primarios y secundarios tuvieron una reducción significativa en los grupos experimentales pero los folículos atrésicos primarios y secundarios fueron más en ambos grupos y el número de cuerpos lúteos fue menor en ambos grupos. La actividad de 3 b-HSD aumentó de manera anormal tanto en los grupos FNZ y S. Estos hallazgos sugieren que el FNZ no contrarresta los efectos negativos del estrés prenatal sobre los folículos ováricos de las crías adultas, y podría ser responsable de los cambios largo plazo que ocurren a durante la programación embrionaria.

comparitive study of mouse ovarian alkaline phosphatase activity in normal and pseudopregnancies

The purpose of this study was to determine alkaline phosphatase [ALP] activity of ovary after ovarian induction using pregnant mare serum gonadotropin [PMSG] and human chorionic gonadotropin [hCG] during implantation periods. A total of 240 female NMRI mice aged 6-10 weeks were selected and divided into control and hyperstimulated. These mice were rendered normal or pseudopregnant. Five mice per each group were sacrificed by cervical dislocation at the first to sixth day of natural or pseudopregnancy. For biochemical assay the samples were obtained from the ovary, then were hemogenated using Tris HCl buffered salin [pH=8.3] and centrifuged with 14000 g. The activity of enzyme was determined using paranitrophenyle phosphate as substrate. Then specific activity of enzyme was calculated according to the total protein. The data were evaluated with Mann whitney test. For histochemistry the samples were cryosectioned [5mm thickness] and the ALP activity was determined by azo-coupling technique using alphanaphtole phosphate as substrate. The pattern of ALP activity in the biochemical and hisotchemical study was the same in each group. The activity of the ovarian ALP was increased during early pregnancy in the control and hyperstimulated natural pregnant groups. There were significant differences between these groups in every days except on the first and fourth day of pregnancy [p<0.05]. The ovarian enzyme activity was increased in pseudopregnancy control until 4th day and in the pseudopregnant hyperstimulation groups until 2nd day of pseudopregnancy then it was decreased. The daily patterny of these alterations were significantly different [p<0.05] comparing the above-mentioned groups. ALP activity was increased in every day of pregnancy [p<0.05] in normal pregnant hyperstimulated group in comparison with the psuedopregnant hyperstimulated group. Thus ovarion hyprstimulation alters the ovarian ALP activity during early pregnancy. These alteration may be due to esteroidogenesis activity of ovarian cells. However more investigation with complementary technique is needed

Histochemical localization of enzymes and lipids in the ovary of a vespertilionid bat, Scotophilus heathi, during the reproductive cycle

O presente estudo descreve as mudanças sazonais na D5 3b hidroxiesteróide desidrogenase (3b-HSD), glicose-6 fosfato desidrogenase (G-6-PD), e lipídios no ovário de um morcego vespertilionidae, Scotophilus heathi. As atividades totais dos lipídios e do 3b-HSD estão restritas às células tecais e intersticiais do ovário. Os lipídios, 3b-HSD e G-6-PD totais, aumentaram significantemente durante a recrudescência, e permaneceram elevados durante a dormência de inverno e o período de acasalamento, quando comparados a outras fases reprodutivas. A alta incidência de atividade de lipídios e enzimas nas células intersticiais durante o período de acasalamento e durante o período de ovulação sugere claramente que estas células estão ativamente envolvidas na esteroidogênese. O declínio da atividade dos lipídios e enzimas durante a dormência de inverno, o qual se correlaciona com os níveis decrescentes da esteroidogênese, podem ser o fator responsável pela longa sobrevivência do folículo de De Graaf no ovário do S. heathi.

Response related enzymatic changes in ovaries of superovulated mice.

Adult female mice were superovulated with PMSG followed by HCG and 140 blastocysts and 69 morulae were recovered from 24 mice. On the basis of the response, mice were divided into six groups; non responders, 1-5, 6-10, 11-20, 21-30 and >30 embryos. The ovaries of the animals were pooled group wise, homogenized in PBS (pH 7.4) and after centrifugation for 10-15 minutes, the supernatant was analyzed for the enzymes, guanine oxaloacetate transaminase (GOT), guanine pymvate transaminase (GPT), acid phosphatases (ACP) and alkaline phosphatases (AKP). Acid and alkaline phosphatase activities did not show any variation in relation to response to superovulation but GOT and GPT showed significantly increased activity in response to induction of superovulation. A statistically significant positive correlation was found between GOT and GPT activities and the superovulatory response in mice.

Cell nucleus activity during post-embryonic development of Apis mellifera L. (Hymenoptera: Apidae). Intranuclear acid phosphatase

We report nuclear acid phosphatase activity in the somatic (intra-ovariolar and stromatic) and germ cells of differentiating honey bee worker ovaries, as well as in the midgut cells of metamorphosing bees. There was heterogeneity in the intensity and distribution of electron dense deposits of lead phosphate, indicative of acid phosphatase activity in the nuclei of these tissues, during different phases of post-embryonic bee development. This heterogeneity was interpreted as a variation of the nuclear functional state, related to the cell functions in these tissues

Activity of glutathione related enzymes and ovarian steroid hormones in different sizes of follicles from goat and sheep ovary of different reproductive stages.

The investigations on enzymes related to glutathione like glutathione-S-transferase (GST) and glutathione peroxidase (GSH-Px) have been carried out mostly in human and rat ovaries, however the studies on these enzymes in ruminants are relatively absent. In the present study the changes in the activity of these enzymes, in different sizes of follicles from goat and sheep ovaries of different reproductive stages, were investigated. The results demonstrated that the activity of the enzyme GST increased with the increase in size of the follicles from small to large follicles of follicular phase ovary and from small to medium follicles of luteal phase ovary in both the species, thereafter it decreased in large follicles of luteal phase ovary. There was increasing pattern in the activity of GSH-Px in the follicular phase follicles and a decreasing pattern in the luteal phase follicles from both the species. Thus the changes in the activity of glutathione related enzymes namely GST and GSH-Px in different size follicles from both the species during different reproductive phases are evident from the results. It is reasonable, therefore, to assume that these enzymes may have functional role in the steroid hormone metabolism in ruminant ovary as reported in human ovary.

Host-mediated juvenoids affect development and metamorphosis of endoparasitic wasp, Chelonus blackburni (Hymenoptera: Braconidae) and consequent morphogenetic derangements in its reproductive system.

Juvenoids, applied topically on larvae of Corcyra cephalonica harbouring the larvae of the parasitoid, produce various types of developmental derangements in parasitoid C. blackburni. The deformed morphs, e.g., apparently normal adults, adultoids and prolonged larvae were developed at different rates by different doses of juvenoids. Larval and pupal mortality rates of the parasite were 6-13 and 4-8% respectively in different treatments. Deformities in reproductive system of the parasitoid were increased or decreased ovariole number, development of compound egg chamber and ill-developed ovariole.

Changes in catalase activity and hydrogen peroxide level in rat ovary during estrous cycle and induction of catalase in rat ovary by estradiol-17 beta.

Catalase activity in the whole ovary homogenate and hydrogen peroxide level in the differentially centrifuged fractions of the ovary homogenate during each stage of estrous cycle were measured. The highest catalase activity was observed in the metestrous which declined in the estrous and proestrous and was lowest in the diestrous. An inverse relationship was found between catalase activity and hydrogen peroxide production. Treatment of immature (28-29 days old) female rats with estradiol-17 beta (5 micrograms in 0.2 ml oil/animal/day for consecutive 3 days, s.c.) increased the ovarian catalase activity. The findings indicate that the free radical-scavanger system may have functional role in the ovary.

Subcellular distribution of oxygen free radical scavenging enzymes in goat ovary.

Oxygen free radical scavenging enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase were heterogeneously distributed in goat ovary. Activities of SOD and catalase were predominantly located in cytosolic fractions compared to mitochondrial and microsomal fractions. Most of the peroxidase activity was observed in microsomal fractions with little activity in cytosolic and mitochondrial fractions. Higher activities of all these enzymes were in luteal phase as compared to follicular phase. Most of the activities of these enzymes in luteal phase were restricted to luteal cells while in follicular phase these were mainly present in the follicles of the ovary.

Changes in catalase activity during follicular growth, atresia and luteinization in rat ovary.

Quantitative changes have been observed in the catalase activity during follicular growth, induced atresia and in corpora lutea of cycle and pregnancy. Large growing and preovulatory follicles showed higher enzyme activity as compared to the smaller follicles; the activity was mainly present in the thecal layer of the preovulatory follicle. After the blockade of ovulation with barbiturate, the activity increases significantly in the whole follicle and also in the thecal layers till third day of ovulation and afterward it declines on 5th day, suggesting that rise in catalase activity may exert a protective function against lytic actions of peroxide which is known to be produced in the ovary during several metabolic and steroidogenic events. The corpora lutea of the cycle showed significantly less enzyme activity than the corpora lutea of pregnancy. The significance of catalase activity during follicular and corpus luteum degeneration is discussed.

Effect of steroids on ovarian neutral proteinases during ovulation in cyclic albino rats.

Progesterone (0.5 mg/rat) and estradiol-17 beta (10 micrograms/rat) injections (im) on the morning of proestrous in cyclic female rats enhanced the ovulation rate (number of ova shed). These steroids also significantly (P less than 0.05) increased the activity of ovarian neutral proteinases, observed on the morning of estrus as compared to those in control and vehicle treated animals. Nonsteroidal estrogen antagonist, tamoxifen suppressed ovulation rate and ovarian neutral proteinase activity as compared to control, its vehicle and steroidal treatment. The results demonstrate a stimulatory effect of progesterone and estradiol 17 beta on ovulation and ovarian neutral proteinases.