Radioprotective effect of ethanolic extract of Alocasia indica on γ-irradiation-induced reproductive alterations in ovary and uterus.

Evaluation of the modulatory effect of ethanolic extract of Alocasia indica tuber (EEAIT) against γ-irradiation induced ovarian and uterine toxicity. Extract preparation was done by 80% hydro-ethanol using Soxhlet apparatus. EEAIT was administered to female Swiss albino mice (n = 5) daily (200 and 400 mg/kg body weight/d) for 7 days before γ-irradiation exposure (2.9 Gy). FSH, LH, estrogen, progesterone, cytokine levels, and oxidative stress parameters were measured after 24 hours of γ-irradiation. Histology, folliculogenesis, viability of granulosa cells, ROS measurement by flow cytometry, western blot of P450scc, P45017A1, 3ß HSD and SF 1 were also performed. In addition, fertility status was assessed by fecundability and fecundity. The results showed that EEAIT exhibit a strong radioprotective activity by reducing the oxidative stress and thereby restored the ovarian and uterine alterations. EEAIT also improved the abnormality in follicle development, restored altered gonadal hormones and cytokines levels, increase the fertility status, reducing ROS level of granulosa cells with increasing granulosa cells viability and steroidogenic enzyme activity as compared to control. So EEAIT showed a radioprotective effect on γ-irradiation induced ovarian and uterine damage. Our results suggested that Alocasia indica tuber can be a potential radioprotector to prevent female infertility.

Effect of Mobile Phone Radiation on Cardiovascular Development of Chick Embryo.

The biological effects on cardiovascular development of chicken embryos were examined after radiation exposure using mobile phone (900 MHz; specific absorption rate˜1.07 W/kg) intermittently 3 h per day during incubation. Samples were selected by morphological and histological methods. The results showed the rate of embryonic mortality and cardiac deformity increased significantly in exposed group (P < 0.05). No any histological pathological changes were observed on Day 5-7 (D5-D7) of incubation. A higher distribution of lipid droplets was unexpectedly present in myocardial tissue from the exposure groups on D10-D13. Soon afterwards, myofilament disruption, atrioventricular valve focal necrosis, mitochondria vacuolization and atrial natriuretic peptide (ANP) decrease appeared on D15-D21 of incubation. Comet assay data showed the haemocyte mean tail in the exposed group was significantly larger than that of the control (P < 0.01). The arterial vascular wall of exposed group was thicker (P < 0.05) than that of the control on D13, which was reversed to normal in later stages. Our findings suggest that long-term exposure of MPR may induce myocardium pathological changes, DNA damage and increased mortality; however, there was little effect on vascular development.

GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro.

STUDY QUESTION: Is there any in vitro evidence for or against ovarian protection by co-administration of a GnRH agonist with chemotherapy in human? SUMMARY ANSWER: The co-administration of GnRH agonist leuprolide acetate with cytotoxic chemotherapy agents does not preserve ovarian reserve in vitro. WHAT IS KNOWN ALREADY: Randomized controlled trials of the co-administration of gonadotrophin-releasing hormone (GnRH) agonists with adjuvant chemotherapy to preserve ovarian function have shown contradictory results. This fact, together with the lack of a proven molecular mechanism of action for ovarian protection with GnRH agonist (GnRHa) places this approach as a fertility preservation strategy under scrutiny. We therefore aimed in this study to provide in vitro evidence for or against the role of GnRHa in the prevention of chemotherapy-induced damage in human ovary. STUDY DESIGN, SETTINGS, SIZE AND DURATION: This translational research study of ex vivo and in vitro models of human ovary and granulosa cells was conducted in a university hospital between 2013 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian cortical pieces (n = 15, age 14-37) and mitotic non-luteinized (COV434 and HGrC1) and non-mitotic luteinized human granulosa cells (HLGC) expressing GnRH receptor were used for the experiments. The samples were treated with cyclophosphamide, cisplatin, paclitaxel, 5-FU, or TAC combination regimen (docetaxel, adriamycin and cyclophosphamide) with and without GnRHa leuprolide acetate for 24 h. DNA damage, apoptosis, follicle reserve, hormone markers of ovarian function and reserve (estradiol (E2), progesterone (P) and anti-mullerian hormone (AMH)) and the expression of anti-apoptotic genes (bcl-2, bcl-xL, bcl-2L2, Mcl-1, BIRC-2 and XIAP) were compared among control, chemotherapy and chemotherapy + GnRHa groups. MAIN RESULTS AND THE ROLE OF CHANCE: The greatest magnitude of cytotoxicity was observed in the samples treated with cyclophosphamide, cisplatin and TAC regimen. Exposure to these drugs resulted in DNA damage, apoptosis and massive follicle loss along with a concurrent decline in the steroidogenic activity of the samples. GnRHa co-administered with chemotherapy agents stimulated its receptors and raised intracellular cAMP levels. But it neither activated anti-apoptotic pathways nor prevented follicle loss, DNA damage and apoptosis induced by these drugs. LIMITATIONS, REASONS FOR CAUTION: Our findings do not conclusively rule out the possibility that GnRHa may offer protection, if any, through some other mechanisms in vivo. WIDER IMPLICATIONS OF THE FINDINGS: GnRH agonist treatment with chemotherapy does not prevent or ameliorate ovarian damage and follicle loss in vitro. These data can be useful when consulting a young patient who may wish to receive GnRH treatment with chemotherapy to protect her ovaries from chemotherapy-induced damage.

Photobiological effect of low-level laser irradiation in bovine embryo production system.

The objectives of this study were to evaluate the effect of low-level laser irradiation (LLLI) on bovine oocyte and granulosa cells metabolism during in vitro maturation (IVM) and further embryo development. Cumulus-oocytes complexes (COCs) were subjected (experimental group) or not (control group) to irradiation with LLLI in a 633-nm wavelength and 1 J/cm2 fluency. The COCs were evaluated after 30 min, 8, 16, and 24 h of IVM. Cumulus cells were evaluated for cell cycle status, mitochondrial activity, and viability (flow cytometry). Oocytes were assessed for meiotic progression status (nuclear staining), cell cycle genes content [real-time polymerase chain reaction (PCR)], and signal transduction status (western blot). The COCs were also in vitro fertilized, and the cleavage and blastocyst rates were assessed. Comparisons among groups were statistically performed with 5% significance level. For cumulus cells, a significant increase in mitochondrial membrane potential and the number of cells progressing through the cycle could be observed. Significant increases on cyclin B and cyclin-dependent kinase (CDK4) levels were also observed. Concerning the oocytes, a significantly higher amount of total mitogen-activated protein kinase was found after 8 h of irradiation, followed by a decrease in all cell cycle genes transcripts, exception made for the CDK4. However, no differences were observed in meiotic progression or embryo production. In conclusion, LLLI is an efficient tool to modulate the granulosa cells and oocyte metabolism.

Challenging cell phone impact on reproduction: a review.

PURPOSE: The radiofrequency electromagnetic radiation (RF-EMR) produced by cell phones can enhance the excitability of the brain and has recently been classified as carcinogenic. The suggested use of hands-free kits lowers the exposure to the brain, but it might theoretically increase exposure to the reproductive organs. This report summarizes the potential effects of RF-EMR on reproductive potentials in both males and females. METHODS: A critical review of the literature pertaining to the impact of cell phone RF-EMR on reproduction in male and female animals and humans was performed, with a focus on gonad metabolism, apoptosis of reproductive cells, fertility status, and serum reproductive hormones. RESULTS: While some animal and human studies revealed alterations in reproductive physiology in both males and females, others did not report any association. The in vitro and in vivo studies to date are highly diverse, very inconsistent in conduct and, in many cases, report different primary outcomes. CONCLUSION: The increasing use of cell phone warrants well-designed studies to ascertain the effect of their RF-EMR on reproduction.

Antiapoptotic and proliferative activity of curcumin on ovarian follicles in mice exposed to whole body ionizing radiation.

The aim of this study was to evaluate the antiapoptotic and proliferative activity of curcumin (Cur) on the ovarian follicles in mice exposed to whole body ionizing radiation (Rd). The mice were exposed to 8.3 gray whole body Rd, and Cur groups were given as a daily dose of 100 mg/kg of Cur for 10 days (10 days before Rd). The ovaries were collected 3 and 12 h after irradiation. To date, no such studies have been performed on antiapoptotic and proliferative activity of Cur on the ovarian follicles in mice exposed to whole body Rd. Analysis of mice ovary after exposure to Rd by terminal-deoxynucleotidyl-transferase-mediated dUTP nick end labeling showed that there were apoptotic cells both in the follicular wall and the antrum, and that the number of follicles showing early atresic features was high 3 h after Rd. On the other hand, analysis of mice ovary 12 h after exposure to Rd showed that the number of follicles containing apoptotic cells with advanced atresic features was significantly higher when compared to the 3-h Rd exposure group. The proliferating cell nuclear antigen -positive granulosa cells were decreased in association with follicular atresia. The groups given treatment were observed to have some benefit from Cur against the damage caused by Rd. The results of this study demonstrate that Cur prevents follicular atresia in Rd-induced apoptosis in ovarian follicles.

Gamma-radiation-induced follicular degeneration in the prepubertal mouse ovary.

Prepubertal mice were whole-body irradiated with a mean lethal dose (LD50) of gamma-radiation using a 60Co source with a total dose of 7.2 Gy and a dose rate of 12.0 cGy/min. At day 0 before the irradiation and at day 1, 2, and 3 after the irradiation, the ovaries were collected and the morphological changes were assessed. The ratios (%) of atretic or polymorphonuclear leukocytes (neutrophil)-infiltrated follicles in the largest cross sections were calculated. In the early atretic follicle of the control mouse ovary, both apoptotic and mitotic cells were observed and occasionally neutrophils were infiltrated into the follicle cavity. However, in the atretic follicles 2 days post-irradiation, numerous cell fragments, apoptotic cells and bodies, and especially, a number of neutrophils were observed. In the non-irradiated control, the ratios of atretic follicles were 58.0+/-8.6 and 27.3+/-11.2 (mean+/-S.E.M.) in antral and preantral follicles, respectively. The ratios of the number of antral and preantral follicles with one or more neutrophils to the total number of atretic follicles were 29.3+/-12.0. At 2 days post-irradiation, the ratios of atretic follicles were increased to 94.0+/-3.4 and 86.9+/-7.6 in antral and preantral follicles, respectively. The ratios of neutrophil-containing follicles among the atretic one were increased to 65.9+/-11.5 and 57.8+/-15.4 at 2 and 3 days after the irradiation, respectively. Taken together, the present results show that gamma-radiation induces apoptotic and inflammatory degeneration of mouse ovarian follicles. Besides, neutrophils may be involved in the acute atretic degeneration in gamma-irradiated mouse ovarian follicles.

Cell type-specific genotoxic effects of intermittent extremely low-frequency electromagnetic fields.

The issue of adverse health effects of extremely low-frequency electromagnetic fields (ELF-EMFs) is highly controversial. Contradictory results regarding the genotoxic potential of ELF-EMF have been reported in the literature. To test whether this controversy might reflect differences between the cellular targets examined we exposed cultured cells derived from different tissues to an intermittent ELF-EMF (50 Hz sinusoidal, 1 mT) for 1-24h. The alkaline and neutral comet assays were used to assess ELF-EMF-induced DNA strand breaks. We could identify three responder (human fibroblasts, human melanocytes, rat granulosa cells) and three non-responder cell types (human lymphocytes, human monocytes, human skeletal muscle cells), which points to the significance of the cell system used when investigating genotoxic effects of ELF-EMF.

Amifostine has an inhibitory effect on the radiation-induced p53-branched cascade in the immature mouse ovary.

The organic thiophosphate, amifostine, is a promising pharmacological compound showing selective protection in many tissues against the toxic side-effects of radiation and cytotoxic drugs. The aim of the present study was to assess the radioprotective effects of amifostine on ovarian follicles. Three-week-old female mice, with or without pretreatment with amifostine, were irradiated with 6.42 Gy of gamma-ray. Reduced proliferation of granulosa cells was verified with BrdU staining and the incidences of follicular degeneration increased in ovarian follicles in the gamma-ray-irradiated mice compared to that of the control or amifostine-treated group. Biochemical changes caused by gamma-irradiation provoked a rise of p53 and Bax protein and a decline of the inactive form in caspase-3 and PARP protein. Caspase-3 and PARP cleaved into active peptides during apoptosis. This process was confirmed by the result of this study, which was that the amount of the stable form decreased immediately after irradiation. In the amifostine treatment group before irradiation, the increased rate of p53 and Bax was suppressed, particularly in the LDs-treated group. The relationship between PARP and caspase-3 levels showed the effect of amifostine exposure before irradiation. In conclusion, amifostine had an inhibitory effect on ovarian programmed cell death induced by gamma-ray, affecting the expression of apoptotic signaling molecules and the level of proliferation of the granulosa cells.

Non-thermal DNA breakage by mobile-phone radiation (1800 MHz) in human fibroblasts and in transformed GFSH-R17 rat granulosa cells in vitro.

Cultured human diploid fibroblasts and cultured rat granulosa cells were exposed to intermittent and continuous radiofrequency electromagnetic fields (RF-EMF) used in mobile phones, with different specific absorption rates (SAR) and different mobile-phone modulations. DNA strand breaks were determined by means of the alkaline and neutral comet assay. RF-EMF exposure (1800 MHz; SAR 1.2 or 2 W/kg; different modulations; during 4, 16 and 24h; intermittent 5 min on/10 min off or continuous wave) induced DNA single- and double-strand breaks. Effects occurred after 16 h exposure in both cell types and after different mobile-phone modulations. The intermittent exposure showed a stronger effect in the comet assay than continuous exposure. Therefore we conclude that the induced DNA damage cannot be based on thermal effects.

Relationship between oocyte apoptosis and ovarian tumours induced by high and low LET radiations in mice.

PURPOSE: To investigate the biological effectiveness of neutrons at the energy below 1 MeV on apoptosis and carcinogenesis in the mouse ovary. MATERIALS AND METHODS: Female mice were exposed to 1.0 Gy monoenergetic neutrons (0.317, 0.525 and 1.026 MeV), (252)Cf fission neutron (2.13 MeV) or (137)Cs gamma-rays at 7 days of age. Apoptosis of the oocyte and pregranulosa cells, and ovarian carcinogenesis were compared between the radiations. The efficiency of gamma-rays for granulosa cell tumorigenesis was tested by transplantation of the irradiated ovaries into non-irradiated mice. RESULTS: The cumulative apoptotic index of oocytes was 77.9%, 65.6% and 41.6% for the 0.525 MeV neutron, 2.13 MeV neutron and gamma-rays, respectively. Follicles with apoptotic pregranulosa cells were 53.0%, 18.3% and 22.8% of cumulative index for the three groups. Tubular adenomas developed in the groups of monoenergetic neutrons (26.1%) and gamma-ray (35.5%), whereas granulosa cell tumours developed only in the gamma-ray groups (3.2% for 1.0 Gy and 15.6% for 3.0 Gy). Partial-body irradiation with 3 Gy gamma-rays to the ovaries induced granulosa cell tumours with an incidence of 27.3%. CONCLUSION: Effectiveness of neutrons to cause apoptosis was higher for 0.525 MeV than for 2.13 MeV. The pregranulosa cell apoptosis occurred in an oocyte-prone manner. The higher effectiveness of neutrons than gamma-rays to induce oocyte and pregranulosa cell apoptosis correlates with the inhibition of granulosa cell tumour development.

Natural and radiation-induced degeneration of primordial and primary follicles in mouse ovary.

The present study deals with the morphological changes of the degenerating primordial and primary follicles induced by gamma-radiation. Prepubertal female mice of 3 weeks old ICR strain were gamma-irradiated with the dose of LD(80(30)) (8.3 Gy). The ovaries were collected at 3, 6 and 12 h after irradiation. The largest cross-sections were prepared by histological semithin sections for microscopical observations. The ratio (%) of normal to atretic follicles decreased with time after the irradiation in primordial follicles and in primary follicles as well. At 6 h after irradiation, the number of degenerated primordial follicles increased. Germinal vesicles disappeared and lipid droplets increased in number. Granulosa cells became round in shape and apoptotic cells started to appear. The ooplasmic membrane was not recognizable. The ratio of normal to atretic primordial follicles in the control group was 62.5. Then it became lower with time after the irradiation. It went down to 51.6, 49.0, 11.1 and 7.1 at 0, 3, 6 and 12 h, respectively. The ratio of normal to atretic primary follicles in the control mouse ovary was 81.3. It was 80.0, 75.0, 45.5 and 33. 3 at 0, 3, 6 and 12 h after irradiation, respectively. It is concluded that the ionizing radiation acutely induces the degeneration of primordial and primary follicles. The pattern of degeneration is one of the following: (1) apoptosis of one or more granulosa cells with a relatively intact oocyte, (2) apoptosis of an oocyte with intact follicle cells, or (3) apoptotic degenerations of both kinds of cells. These results can provide morphological clues for the identification of the degenerating primordial and primary follicles in normal and irradiated mouse ovaries.

Morphology and steroidogenesis of cultured granulosa cells obtained from ovaries of women treated with ionizing radiation.

The object of the study was the morphology and steroidogenesis of cultured granulosa cells obtained from 6 women aged 28-39 years who, because of Ib cervix carcinoma, were treated with ionizing radiation and later underwent surgery. It was observed that the granulosa cells were viable, had strong proliferative ability, and formed a monolayer on day 2 of culture. Contrary to our expectations, these cells produced larger amounts of steroids in culture than the control cells harvested from normal ovaries in late follicular phase. It was also found that the cells treated with ionizing radiation responded to exogenous gonadotropins with higher production of progesterone and estradiol than the controls. It is concluded that the increase in metabolic activity by granulosa cells from ovaries which had been indirectly affected by ionizing radiation is manifested by the stimulating influence of radiation on steroidogenesis.

Induction of apoptotic cell death in hen granulosa cells by ceramide.

Recent studies have demonstrated that ovarian follicle atresia occurs extensively before follicle selection into the avian preovulatory hierarchy, and that this process is mediated via granulosa cell apoptosis. Subsequent to follicle selection, granulosa cells are inherently resistant to apoptosis, and such resistance is correlated with increased expression of death suppressor genes such as bcl-xlong. In the present studies we used this avian ovary model system to 1) identify cellular characteristics and mechanisms related to apoptotic cell death of granulosa cells in vitro, and 2) further characterize functional differences between apoptosis-susceptible (4- to 8-mm follicle) and apoptosis-resistant (preovulatory follicle) granulosa cells. Treatment of granulosa cells from the largest preovulatory follicle with N-octanoylsphingosine (C8-ceramide) results in pronounced oligonucleosome formation, a hallmark of apoptosis. That this is indicative of programmed cell death is supported by an increased incidence of pyknotic nuclei and apoptotic bodies in C8-ceramide-treated samples compared to that in control cultured cells. Tumor necrosis factor-alpha, a stimulator of ceramide production, actively promotes oligonucleosome formation in apoptosis-susceptible, but not in apoptosis-resistant, granulosa cells. Induction of apoptosis is also observed after exposure of apoptosis-resistant granulosa cells to sphingomyelinase treatment and UV irradiation, which are known to stimulate endogenous ceramide production, and to the anticancer drug, daunorubicin, which initiates de novo ceramide biosynthesis via activation of ceramide synthase. Although treatment of granulosa cells with fumonisin B1, a specific ceramide synthase inhibitor, blocks daunorubicin-stimulated oligonucleosome formation, UV-induced cell death is unaffected. Taken together, these results demonstrate that pharmacological factors known to mimic the actions of ceramide or stimulate ceramide production can induce oligonucleosome formation and programmed cell death in granulosa cells. More importantly, however, the ability of a physiologically relevant initiator of ceramide biosynthesis, tumor necrosis factor-alpha, to promote cell death is evident only in apoptosis-susceptible granulosa cells collected from atresia-prone prehierarchal follicles. These data provide support for ceramide as an important intracellular signaling mechanism, mediating granulosa cell apoptosis and follicle atresia.

Evidence for a granulosa cell site of dihydrostestosterone metabolism in the adult rat ovary.

The ovarian enzyme 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) converts dihydrotestosterone (DHT) to 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol), a reduced androgen that does not bind to the granulosa cell androgen receptor. To determine the relative contribution of the granulosa cells to total ovarian 3 alpha-HSD activity, adult rats treated with either medroxyprogesterone acetate (MPA) or vehicle underwent ovarian microdissection. 3 alpha-Hydroxysteroid dehydrogenase is primarily located in excised follicles and corpora lutea, and is inhibited in the follicles but not corpora lutea by MPA (P less than 0.05). Elimination of healthy granulosa cells while maintaining healthy theca cells by irradiation of the exteriorized ovaries with 6000 rads resulted in a marked reduction in 3 alpha-HSD to 19% of control levels on a per-organ basis (P less than 0.01). The granulosa cell is the major ovarian site for 3 alpha-hydroxylation of ring A-reduced C19 steroids in the adult rat.

Effect of low intensity laser beam on steroid dehydrogenase activity and steroid hormone production in cultured porcine granulosa cells.

Effect of the beam emitted by a helium-neon (He-Ne) laser of a low power (2.8 mW) on the activity of delta 5, 3 beta-hydroxysteroid dehydrogenase (delta 5, 3 beta-HSD), as well as on the estradiol and progesterone production was studied in cultured granulosa cells obtained from porcine ovary. It has been found that the laser beam stimulates the activity of steroid dehydrogenase, increases estrogen levels and exerts a variable influence on the progesterone level. In control cultures the highest level of both hormones occurred simultaneously, while in cultures exposed to the laser beam progesterone level rose at a slower rate and reached maximum when the estrogen level dropped. Hyperplasia and epithelisation were observed only in the experimental cultures. The most intense cytochemical reaction for delta 5, 3 beta-HSD, as well as the most pronounced increase in estrogen and progesterone level occurred in cells exposed to a pulsating beam for 60 sec.

Participation of granulosa cell populations in radiation response of the follicular apparatus in the ovary of mice.

The aim of this paper was to investigate the fates of granulosa cells in three different types of the follicles during 10 hours after single irradiation with 100 resp. 200 R. Using flash labelling with 3H-thymidine, observations of the ovarian anatomical structures during 4--5 estruses after 150 R were made. It has been found, that the basic radiobiological event, influencing further fates of the follicles, is delay of granulosa cell proliferation. Consequently, the further growth and maturation of the follicles is retarded and a reduction of ovulation, a lengthening and an irregularity of estrus take place. The killing effect within granulosa cells is not significant and is of secondary importance.