201 ROLE OF THE NITRIC OXIDE SYSTEM IN EFFECTS OF PROLACTIN AND GROWTH HORMONE ON METAPHASE-II CHROMOSOMES IN BOVINE OOCYTES AGING IN VITRO

2016 ◽  
Vol 28 (2) ◽  
pp. 231
Author(s):  
I. Lebedeva ◽  
G. Singina ◽  
E. Shedova ◽  
A. Lopukhov ◽  
N. Zinovieva
Keyword(s):  
Nitric Oxide ◽  
Growth Hormone ◽  
Cumulus Cells ◽  
Total Activity ◽  
Inhibitory Effect ◽  
Pituitary Hormones ◽  
Oxide System ◽  
Nos Inhibitors ◽  
Bovine Oocytes

Aging of mammalian oocytes is the time-dependent process of cytological and molecular transformations leading to a decline in the ovum quality and developmental capacity. We have previously shown that 2 related pituitary hormones, prolactin (PRL) and growth hormone (GH), may decelerate abnormal changes in the morphology of metaphase II (MII) chromosomes in bovine cumulus-enclosed oocytes (CEO) aging in vitro. The goal of the present research was to examine the involvement of different isoforms of nitric oxide synthase (NOS) in the actions of PRL and GH on MII chromosomes in aging bovine oocytes. Bovine CEO were matured for 20 h in TCM 199 containing 10% FCS, 10 μg mL–1 porcine FSH, and 10 μg mL–1 ovine LH. After IVM, CEO or denuded oocytes (DO) were cultured for 24 h in the aging medium of TCM 199 supplemented with 10% FCS (control). In experimental groups, the medium contained either 50 ng mL–1 bovine PRL or 10 ng mL–1 bovine GH and/or NOS inhibitors. The following inhibitors were applied: (1) N-propyl-l-arginine (NPLA; an inhibitor of neuronal NOS (nNOS), 5 μM) and (2) L-NAME (an effective inhibitor of both endothelial NOS (eNOS) and nNOS, 20 μM). Destructive changes of MII chromosomes in oocytes were assessed by the following morphological signs: decondensation, partial adherence, chromosome clumping into a single mass, and fragmentation. The total activity of NOS in oocytes was determined by NADPH-diaphorase staining. The data from 4–5 replicates were analysed by ANOVA. During CEO aging in the control medium, the rate of MII oocytes with destructive changes of chromosomes rose from 16.8 ± 2.1% to 58.5 ± 1.4% (P < 0.001), whereas both PRL and GH reduced this rate up to 42.0 ± 1.3% and 46.5 ± 1.6%, respectively (P < 0.001). The nNOS inhibitor NPLA abolished (P < 0.001) the inhibitory effect of PRL on abnormal modifications of chromosomes in CEO but did not affect the frequency of these modifications in the control or GH-treated groups. In the absence of the hormones, L-NAME (the eNOS+nNOS inhibitor) decreased the rate of aging CEOs with chromosome abnormalities from 58.5 ± 1.4% to 41.2 ± 2.5% (P < 0.001), acting unidirectionally with PRL and GH. Meanwhile, L-NAME enhanced (P < 0.05) the suppressing effect of PRL on destructive changes of MII chromosomes but did not influence the similar effect of GH. At the same time the chromosome morphology in senescent DOs was unaffected by the hormones or NOS inhibitors. Furthermore, the total activity of NOS in oocytes separated of cumulus after 24 h of aging was similar in the control and experimental groups. Thus, the inhibitory effect of GH on abnormal modifications of MII chromosomes in aging bovine oocytes may be related to a reduction of the eNOS activity in cumulus cells, whereas the respective effect of PRL is likely to be achieved by both inactivation of eNOS and activation of nNOS. This research was supported by RFBR (No. 13–04–01888).

164 EFFECTS OF CUMULUS CELLS AND PITUITARY HORMONES ON AGE-ASSOCIATED CELLULAR CHANGES DURING THE PROLONGED CULTURE OF BOVINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 196
Author(s):  
I. Lebedeva ◽  
G. Singina ◽  
A. Lopukhov ◽  
N. Zinovieva
Keyword(s):  
Fetal Calf Serum ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Meiotic Arrest ◽  
Parthenogenetic Activation ◽  
Cellular Changes ◽  
Prolonged Culture ◽  
Bovine Oocytes

In vivo and in vitro aging of mature mammalian oocytes heavily reduces their quality and developmental capacity. Therefore, the knowledge of physiological factors modulating the speed of oocyte aging is of great importance for successful reproduction. The goal of the present research was to study effects of cumulus cells (CC) and two related pituitary hormones, prolactin (PRL) and growth hormone (GH), on the dynamics of age-associated cellular changes during the prolonged culture of bovine oocytes in vitro. Bovine cumulus–oocyte complexes (COC) were cultured for 20 h in the following maturation medium: TCM 199 containing 10% fetal calf serum, 10 μg mL–1 porcine FSH, and 5 μg mL–1 ovine LH. After IVM, COC were transferred to the aging medium consisting of TCM-199 supplemented with 10% fetal calf serum and cultured for 0, 12, 24, or 36 h in the absence (Control) or presence of 50 ng mL–1 bovine PRL (Research Center for Endocrinology, Moscow, Russia) or 10 ng mL–1 recombinant bovine GH (Monsanto, St. Louis, MO, USA). A portion of in vitro-matured oocytes were denuded of their CC and cultured in the control aging medium. At the end of culture, the state of the nuclear material in oocytes and embryos was evaluated by Tarkowski's cytogenetic method. The number of oocytes undergoing spontaneous parthenogenetic activation in the respective groups was determined by summarising the numbers of embryos cleaved and oocytes reaching anaphase-II to telophase-II stages or containing a pronucleus. Destructive changes in CC were assessed using the morphological signs of apoptosis. The data from 3 to 5 replicates were analysed by ANOVA. In the control group of COC, a rise in the rate of metaphase-II (M-II) oocytes with abnormal chromosome configurations occurred by 12 h of aging [31.8 ± 4.6% (12 h) v. 17.5 ± 2.6% (0 h); P < 0.05] and persisted up to 36 h (70.4 ± 2.0%; P < 0.001). At the same time, the frequency of oocyte parthenogenetic activation markedly increased only between 0 and 36 h of aging (from 0% to 20.7 ± 3.4%; P < 0.001). The addition of PRL or GH to the aging medium or removal of CCs resulted in a decline in the rate of M-II oocytes with degenerative changes of chromosomes throughout the culture period (at least P < 0.05). Furthermore, PRL and GH reduced the frequency of the oocyte activation at 36 h of the prolonged culture (up to 5.4 ± 2.5 and 1.7 ± 1.7%, respectively; P < 0.01), although CC did not influence meiotic arrest at M-II. Meanwhile, the rate of degenerated CC steadily increased as the culture time increased from 0 h (10.3 ± 1.1%) to 36 h (22.7 ± 2.2%; P < 0.001) and was unaffected by both hormones. The data suggest that, in bovine COC, CC accelerate abnormal changes in the chromosomal structure of aging M-II oocytes, whereas PRL and GH may decelerate these changes and support meiotic arrest during the prolonged culture of in vitro-matured oocytes. This research was partially supported by RFBR (project no. 13-04-01888).

Hydrogen sulfide downregulates the aortic l-arginine/nitric oxide pathway in rats

2007 ◽  
Vol 293 (4) ◽  
pp. R1608-R1618 ◽  
Author(s):  
Bin Geng ◽  
Yuying Cui ◽  
Jing Zhao ◽  
Fang Yu ◽  
Yi Zhu ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Hydrogen Sulfide ◽  
Umbilical Vein ◽  
Transcript Abundance ◽  
Inhibitory Effect ◽  
Akt Phosphorylation ◽  
Enos Activity ◽  
Nos Inhibitors

The aim of the present study was to investigate the effect of hydrogen sulfide (H2S) signaling by nitric oxide (NO) in isolated rat aortas and cultured human umbilical vein endothelial cells (HUVECs). Both administration of H2S and NaHS, as well as endogenous H2S, reduced NO formation, endothelial nitric oxide synthase (eNOS) activity, eNOS transcript abundance, and l-arginine (l-Arg) transport (all P < 0.01). The kinetics analysis of eNOS activity and l-Arg transport showed that H2S reduced Vmax values (all P < 0.01) without modifying Km parameters. Use of selective NOS inhibitors verified that eNOS [vs. inducible NOS (iNOS) and neuronal NOS (nNOS)] was the specific target of H2S regulation. H2S treatment (100 μmol/l) reduced Akt phosphorylation and decreased eNOS phosphorylation at Ser1177. H2S reduced l-Arg uptake by inhibition of a system y+ transporter and decreased the CAT-1 transcript. H2S treatment reduced protein expression of eNOS but not of nNOS and iNOS. Pinacidil (KATP channel opener) exhibited the similar inhibitory effects on the l-Arg/NOS/NO pathway. Glibenclamide (KATP channel inhibitor) partly blocked the inhibitory effect of H2S and pinacidil. An in vivo experiment revealed that H2S downregulated the vascular l-Arg/eNOS/NO pathway after intraperitoneal injection of NaHS (14 μmol/kg) in rats. Taken together, our findings suggest that H2S downregulates the vascular l-Arg/NOS/NO pathway in vitro and in vivo, and the KATP channel could be involved in the regulatory mechanism of H2S.

230 ROLE OF PITUITARY HORMONES AND CUMULUS CELLS IN MODULATING THE DEVELOPMENTAL CAPACITY OF AGING BOVINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 204
Author(s):  
G. Singina ◽  
I. Lebedeva ◽  
T. Taradajnic ◽  
N. Zinovieva
Keyword(s):  
Embryonic Development ◽  
Tyrosine Kinases ◽  
Cumulus Cells ◽  
Pituitary Hormones ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Prolonged Culture ◽  
Bovine Oocytes

The competence for embryonic development acquired during the oocyte maturation attenuates during the subsequent oocyte aging both in vivo and in vitro. Thus, the successful control of the female fertility requires information regarding factors responsible for the oocyte protection from early aging. The aim of the present research was to study the pattern and pathways of actions of two closely related pituitary hormones, prolactin (PRL), and growth hormone (GH), on the developmental potential of bovine oocytes during their aging in vitro. Therefore, we analysed (1) effects of PRL and GH during the prolonged culture of bovine oocytes on their subsequent development up to the blastocyst stage and (2) the role of cumulus cells (CC) and tyrosine kinases, the well-known mediators of PRL and GH signalling, in these effects. Bovine cumulus-enclosed oocytes (CEO) were cultured for 22 h in the following maturation medium: TCM 199 containing 10% fetal calf serum (FCS), 10 μg mL–1 of porcine FSH, and 10 μg mL–1 of ovine LH. After IVM, CEO or denuded oocytes (DO) were transferred to the aging medium consisting of TCM 199 supplemented with 10% FCS and cultured for 10 h in the absence (Control) or presence of 50 ng mL–1 bovine PRL or 10 ng mL–1 recombinant bovine GH and/or 10 μg mL–1 genistein (a non-selective inhibitor of tyrosine kinases). Genistein was not applied in the case of aging DO, since their developmental potential was not affected by both hormones. Following the prolonged culture, oocytes underwent IVF and IVC. Embryos were cultured in CR1aa medium until Day 5 post-insemination and then transferred to the same medium supplemented with 5% FCS and cultured up to Day 8. The embryo development was evaluated at Days 2 and 8 for cleavage and blastocyst formation. The data from 5 to 6 replicates using 135–184 oocytes per treatment were analysed by ANOVA. Aging of oocytes in the control medium had no effect on the cleavage rate, but caused the blastocyst yield to decline (P < 0.001) from 31.1 ± 2.3% (CEO fertilized immediately after maturation) to 10.5 ± 2.4% (aged CEO) and 7.9 ± 1.9% (aged DO). Cleavage rates of aging CEO and DO were unaffected by both PRL and GH. In the case of CEO, the addition of PRL (but not GH) to the aging medium raised the blastocyst yield from 8.2 ± 0.9% to 15.2 ± 2.1% (P < 0.05), whereas the removal of CC abolished this effect, reducing the yield up to 9.1 ± 2.7% (P < 0.05). At the same time, genistein did not influence the blastocyst yield in the PRL-treated group. The findings demonstrate that PRL can inhibit the attenuation of the developmental competence of bovine oocytes aging in vitro, with this effect being achieved via cumulus cells. Tyrosine kinases are unlikely to mediate the beneficial action of PRL on the CEO capacity for embryonic development. Meanwhile, closely related GH does not affect the developmental competence of aging bovine oocytes.This research was supported by RFBR (project No. 13-04-01888).

SENSITIVITY OF THE RAT DIAPHRAGM TO GROWTH HORMONE.

1967 ◽  
Vol 54 (4) ◽  
pp. 645-662 ◽  
Author(s):  
Å. Hjalmarson ◽  
K. Ahrén
Keyword(s):  
Growth Hormone ◽  
Inhibitory Effect ◽  
Rat Diaphragm ◽  
Intracellular Accumulation ◽  
Slight Effect ◽  
Kidney Capsule ◽  
Muscle Tissues ◽  
Hypophysectomized Rats ◽  
Isolated Rat

ABSTRACT The effect of growth hormone (GH) in vitro on the rate of intracellular accumulation of the non-utilizable amino acid α-aminoisobutyric acid (AIB) was studied in the intact rat diaphragm preparation. Bovine or ovine GH (25 μg/ml incubation medium) markedly stimulated the accumulation of AIB-14C by diaphragms from hypophysectomized rats, while there was no or only a very slight effect on diaphragms from normal rats. In diaphragms from rats with the pituitary gland autotransplanted to the kidney capsule GH in vitro stimulated the accumulation of AIB-14C significantly more than in diaphragms from normal rats but significantly less than in diaphragms from hypophysectomized rats. Injections of GH intramuscularly for 4 days to hypophysectomized rats made the diaphragms from these rats less sensitive or completely insensitive to GH in vitro. These results indicate strongly that the relative insensitivity to GH in vitro of diaphragms from normal rats is due to the fact that the muscle tissues from these rats has been exposed to the endogenously secreted GH. The results show that GH can influence the accumulation of AIB-14C in the isolated rat diaphragm in two different ways giving an acute or »stimulatory« effect and a late or »inhibitory« effect, and that it seems to be a time-relationship between these two effects of the hormone.

ANALYSIS OF THE BIPHASIC ACTION OF GROWTH HORMONE IN VITRO ON THE RAT DIAPHRAGM

1968 ◽  
Vol 57 (3_Suppl) ◽  
pp. S19-S35 ◽  
Author(s):  
Å. Hjalmarson
Keyword(s):  
Growth Hormone ◽  
Actinomycin D ◽  
Accumulation Rate ◽  
Inhibitory Effect ◽  
Bovine Growth Hormone ◽  
Rat Diaphragm ◽  
Dual Nature ◽  
Hypophysectomized Rats ◽  
D 20

ABSTRACT In vitro addition of bovine growth hormone (GH) to intact hemidiaphragms from hypophysectomized rats has previously been found to produce both an early stimulatory effect lasting for 2—3 hours and a subsequent late inhibitory effect during which the muscle is insensitive to further addition of GH (Hjalmarson 1968). These effects on the accumulation rate of α-aminoisobutyric acid (AIB) and D-xylose have been further studied. In presence of actinomycin D (20 μg/ml) or puromycin (100 μg/ml) the duration of the stimulatory effect of GH (25 μg/ml) was prolonged to last for at least 4—5 hours and the late inhibitory effect was prevented. Similar results were obtained when glucose-free incubation medium was used. Preincubation of the diaphragm at different glucose concentrations (0—5 mg/ml) for 3 hours did not change the GH sensitivity. Addition of insulin at start of incubation could not prevent GH from inducing its late inhibitory effect, while dexamethasone seemed to potentiate this effect of GH. Furthermore, adrenaline was found to decrease the uptake of AIB-14C and D-xylose-14C in the diaphragm, but not to change the sensitivity of the muscle to GH. Preincubation of the diaphragm for 3 hours with puromycin in a concentration of 200 μg/ml markedly decreased the subsequent basal uptake of both AIB-14C and D-xylose-14C, in the presence of puromycin, and abolished the stimulatory effect of GH on the accumulation of AIB-14C. However, the effect of GH on the accumulation of D-xylose-14C was unchanged. The present observations are discussed and evaluated in relation to various mechanisms of GH action proposed to explain the dual nature of the hormone.

scholarly journals Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1794
Author(s):  
Konstantina Stamperna ◽  
Themistoklis Giannoulis ◽  
Eleni Dovolou ◽  
Maria Kalemkeridou ◽  
Ioannis Nanas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Cumulus Cells ◽  
Intramolecular Interactions ◽  
Developmental Competence ◽  
Embryo Yield ◽  
Bovine Oocytes

Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.

Mechanism of the adverse effect of hyaluronidase used for oocyte denudation on early development of bovine embryos

Zygote ◽  
2021 ◽  
pp. 1-5
Author(s):  
Shiori Ashibe ◽  
Kanade Irisawa ◽  
Ken Yokawa ◽  
Yoshikazu Nagao
Keyword(s):  
Treatment Group ◽  
Assisted Reproductive Technologies ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Control Group ◽  
Free Calcium ◽  
Parthenogenetic Development ◽  
Early Embryos ◽  
Bovine Oocytes

Summary Hyaluronidase is widely used in animal and human assisted reproductive technologies (ARTs) to remove cumulus cells around oocytes. However, adverse effects of hyaluronidase treatment, such as increased rates of degeneration and parthenogenesis, have been found after treatment of human and mouse oocytes. Currently, the mechanism(s) of the detrimental effects are unclear. The present study was initiated to identify the mechanism of adverse responses to hyaluronidase treatment in bovine oocytes and early embryos. Cumulus cells were removed from cumulus–oocyte complexes (COCs) with or without hyaluronidase and the oocytes were subjected to intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). Significantly lower rates of blastocyst formation were obtained in the hyaluronidase treatment group after ICSI (22.4%) and IVF (21.2%) compared with the non-hyaluronidase control groups: 36.1% after ICSI and 30.4% after IVF. Next, we examined the effect of hyaluronidase on parthenogenetic development rates and on the cytoplasmic levels of free calcium ions (Ca2+), reactive oxygen species (ROS) and reduced glutathione (GSH). No differences in parthenogenesis rates were found between treated and untreated groups. Ca2+ levels in oocytes from the hyaluronidase treatment group indicated using mean fluorescence intensity were significantly higher (68.8 ± 5.3) compared with in the control group (45.0 ± 2.5). No differences were found in the levels of ROS or GSH between the treated and untreated groups. We conclude that hyaluronidase might trigger an increase in Ca2+ levels in oocytes, resulting in a decreased potential for normal embryonic development.

Failure of Human Growth Hormone and Human Placental Lactogen to Affect Glucose Consumption by Human Erythrocytes and Leucocytes In Vitro

1972 ◽  
Vol 50 (10) ◽  
pp. 1014-1017
Author(s):  
Catherine L. Tanser ◽  
Nannie K. M. de Leeuw
Keyword(s):  
Growth Hormone ◽  
Glucose Oxidase ◽  
Human Growth Hormone ◽  
Glucose Utilization ◽  
Human Growth ◽  
Glucose Consumption ◽  
Inhibitory Effect ◽  
Placental Lactogen ◽  
Human Placental Lactogen

The effect of human growth hormone (HGH) and human placental lactogen (HPL) on glucose consumption by erythrocytes and leucocytes in vitro was investigated. Glucose consumption was measured by determining glucose utilization during 3 h incubation at 37 °C, using the glucose oxidase method.HGH and HPL showed no effect on glucose consumption by erythrocytes, and HPL showed no effect on glucose consumption by leucocytes in vitro. Our results do not confirm previous reports of an inhibitory effect of HGH on glucose consumption by erythrocytes in vitro.

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