Effects of Dextran-Coated Superparamagnetic Iron Oxide Nanoparticles on Mouse Embryo Development, Antioxidant Enzymes and Apoptosis Genes Expression, and Ultrastructure of Sperm, Oocytes and Granulosa Cells.

BACKGROUND: Although application of superparamagnetic iron oxide nanoparticles (SPIONs) in industry and medicine has increased, their potential toxicity in reproductive cells remains a controversial issue. This study was undertaken to address the response of sperm, oocyte, and resultant blastocyst to dextran-coated SPIONs (D-SPIONs) treatment during murine in vitro fertilization (IVF). MATERIALS AND METHODS: In this experimental study, murine mature oocytes were randomly divided into three groups: control, and low- and high-dose groups in which fertilization medium was mixed with 0, 50 and 250 µg/ml of DSPIONs, respectively. Sperm and/or cumulus oocyte complexes (COCs) were cultured for 4 h in this medium for electron microscopic analysis of sperm and COCs, and assessment of developmental competence and genes expression of Gpx1, Sod1, catalase, Bcl2l1 and Bax in the resultant blastocysts. RESULTS: Ultrastructural study of sperm, oocyte, and granulosa showed destructed mitochondria and membranes in spermatozoa, vacuolated mitochondria and distorted cristae in oocytes, and disrupted nuclei and disorganized cell membranes in granulosa in a dose-dependent manner. Data showed that cleavage and blastocyst rates in the 250 µg/ml of D-SPIONs were significantly lower than in the control group (P<0.05). Gene expression of GPx1, Sod1, catalase, Bcl2l1 and Bax in resultant blastocysts of the high-dose group and catalase and Bax in resultant blastocysts of the low-dose group, was higher than the controls. CONCLUSION: There is considerable concern regarding D-SPIONs toxic effects on IVF, and mitochondrial and cell membrane damage in mouse spermatozoa and oocytes, which may be related to oxidative stress and apoptotic events.

The effects of metformin on stereological and ultrastructural features of the ovary in streptozotocin -induced diabetes adult rats: An experimental study.

BACKGROUND: Diabetes is a chronic disease that can affect almost all of the body organs, including male and female reproductive systems. OBJECTIVE: This study was designed to investigate the preventive effects of metformin on stereological and ultrastructure characteristics of the ovary in the streptozotocin-induced diabetes adult female rats. MATERIALS AND METHODS: Seventy adult (8-10 wk) female Sprague-Dawley rats (180-200 gr) were equally divided, as follows: (n = 10/each) control; STZ-induced diabetes (single dose of 65 mg/kg STZ, IP); metformin-treated (50 mg/100 gr of body weight, orally); diabetic-metformin-treated; sham 1, (single dose of sodium citrate); sham 2, (0.5 ml of daily oral distilled water); and sham 3, (sodium citrate + distilled water treated). The body mass index, ovarian weight, blood sugar level, cholesterol, and triglyceride were measured. The stereological and ultrastructural features of ovary were assessed. RESULTS: The blood sugar of induced-diabetic rats was increased (p < 0.01). The BMI (p < 0.01), number of granulosa cells (p = 0.04), primordial, primary and secondary follicles (p = 0.03), total volume of ovary (p < 0.01) and cortex, nucleus diameter ratio to the ooplasm were decreased. The number of atretic follicles in the diabetic and diabetic + metformin-treated rats were increased (p < 0.01). The ultrastructural characteristics of ovary were more damaged in diabetic rats. CONCLUSION: Diabetes has destructive effects on ovarian follicles and causes follicular atresia. Also, the size of oocytes, numbers of granulosa cells and ooplasmic organelles, which are involved in the folliculogenesis are affected by diabetes and metformin has no preventive effects.

Apoptosis, Autophagy, and Necrosis in Murine Embryonic Gonadal Ridges and Neonatal Ovaries: An Animal Model.

BACKGROUND: In mammalian ovaries, loss of over two-thirds of germ cells happens due to cell death. Nonetheless, the exact mechanism of cell death has yet to be determined. The present basic practical study was designed to detect 3 types of programmed cell death, namely apoptosis, autophagy, and necrosis, in murine embryonic gonadal ridges and neonatal ovaries. METHODS: Twenty gonadal ridges and ovaries from female mouse embryos 13.5 days post coitum and newborn mice 1 day postnatal were collected. The TUNEL assay was performed to evaluate apoptosis. The interplay of autophagy was evaluated by immunohistochemistry for beclin-1. Necrotic cell death was analyzed by propidium iodide (PI) staining. The count and percentage of the labeled oocytes in the gonadal ridges and ovaries were evaluated and compared using the independent t test and one-way ANOVA. A P value less than 0.05 was considered statistically significant. RESULTS: We detected TUNEL-positive reaction in the embryonic germ cells and in the small and large oocytes of the neonatal ovaries. The germ cells and small oocytes reacted to beclin-1. PI absorption was detected in the embryonic germ cells and the large oocytes of the neonatal ovaries, but not in the small oocytes. The percentage of the TUNEL-positive and PI-labeled oocytes in the gonadal ridges was significantly higher than that in the neonatal ovaries (P<0.01 and P=0.01). In the neonatal ovaries, the percentage of the beclin-1-labeled oocytes was significantly higher than that in the embryonic phase (P<0.01). CONCLUSION: We showed that all 3 types of programmed cell death, namely apoptosis, autophagy, and necrosis, accounted for embryonic and neonatal germ-cell loss. Our observations demonstrated a potential role for necrosis, particularly in the embryonic gonadal ridge in comparison to the neonatal ovary, in mice.

Effects of pentoxifylline on the histological and ultra-structural features of vitrified mouse ovarian tissue: An experimental study.

BACKGROUND: Vitrification is a process that can be used to preserve gonads in the healthy and natural status. Oxidative stress is one of the disadvantages of vitrification. Pentoxifylline (PTX) is an antioxidant that can reduce reactive oxidative stress effects. OBJECTIVE: We aimed to investigate the effects of PTX on histological and ultra-structural features of vitrified and non-vitrified mouse ovarian tissue. MATERIALS AND METHODS: Twenty-five adult female Balb-C mice were randomly and equally divided into control group: the ovaries did not receive any treatment; experimental 1 and 2: the vitrified ovaries were incubated in phosphate buffer solution and bovine serum albumin without and with PTX, respectively, for 30 min; sham 1 and 2: the non-vitrified ovaries were incubated in phosphate buffer solution and bovine serum albumin and were incubated without and with PTX, respectively for 30 min. The right and left ovaries in all of the groups were evaluated using light and transmission electron microscopy, respectively. RESULTS: The histological and ultra-structural features of vitrified ovaries were seriously damaged. There was non-uniformed germinal epithelium and tunica albuginea, degenerated granulosa cells and stromal cells, puffy basement membrane and irregular thickness of zona pellucida, as well as a pyknotic nucleus and bubbly and segmented ooplasmic in the follicles. Also, ovarian tissues were damaged by the PTX in the non-vitrified ovaries. CONCLUSION: Vitrification can damage the histological and ultra-structural features of the ovary in mouse models. PTX as an antioxidant, with concentration of 1.8 mM could not prevent and restore these damages and had no adequate effects on the vitrified ovarian tissues.

The non-preventive effects of human menopausal gonadotropins on ovarian tissues in Nandrolone decanoate-treated female rats: A histochemical and ultra-structural study.

BACKGROUND: The follicular growth and development may be affected by abused drugs. Nandrolone decanoate (ND) as an anabolic androgenic steroid can damage the morphological and functional features of the ovary and may lead to reproductive failure. OBJECTIVE: This study was designed to evaluate the effects of synchronized and non-synchronized administration of Human Menopausal Gonadotropins (hMG) with ND on ovarian tissue and level of sex hormones in the adult female rat. MATERIALS AND METHODS: Forty adult female Sprague Dawley rats were divided into eight groups. The five experimental groups received 3 and/or 10 mg/kg of ND synchronized and non-synchronized with 10 IU of hMG and hMG alone. The two shams and control groups received solvents of ND and hMG. The animals' serum levels of Follicle-stimulating hormone, Luteinizing hormone, progesterone and estrogen and the weight, volume and dimensions of the ovaries were measured. The ovaries were prepared for apoptosis assessment and morphological study. RESULTS: The ovarian volume and sex hormones in the experimental groups were decreased, but ovarian weight and dimensions didn't change. The rate of apoptosis was increased in the experimental groups as follows; a low and high dose of ND synchronized with hMG 48.80±18.70 and 65.20±14.20 respectively vs. Sham 1, 33.20±17.80, a low and high dose of ND non-synchronized with hMD 55.80±17.20 and 75.20±14.30 respectively vs. Sham 2, 31.60±32.40 groups, p˂0.01. Follicular and stromal cells were damaged in the experimental groups except for the hMG group. CONCLUSION: Administration of ND decreased the serum level of Luteinizing hormone, Follicle-stimulating hormone, progesterone and estrogen and damaged ovarian tissue irreversibly and irreparably and hMG cannot prevent the destruction of the follicles in the adult female rats. This can be a serious warning to women who abuse ND.

The effects of activated omental extract on nuclear and cytoplasmic in vitro maturation of rat oocytes.

OBJECTIVE: The role of growth factors, including vascular endothelial growth factor of activated omentum on mitosis is clearly known, though not on all the aspects of in vitro oocyte maturation. This study was designed to assess the effect of activated-omental extract (AOE) on in vitro maturation (IVM) of rat cumulus-oocyte complexes (COCs). MATERIALS AND METHODS: In this experimental study, the COCs were incubated in Ham's F-10 supplemented with either 20% AOE, 20% fetal bovine serum (FBS) or serum-free media. Post-culture COCs were studied according to the cumulus cells (CCs) expansion, nuclear maturation and cytoplasmic maturation. Cumuli expansion was evaluated by inverted microscope without staining; nuclear maturation was assessed by aceto-orcein staining (light microscope) and cytoplasmic maturation was also observed by TEM. RESULTS: Expansion of CCs and nuclear maturation of the oocytes in in vitro for 24 hr was significantly higher in AOE- and FBS-supplemented groups (P=0.000 and 0.013) and (P=0.004 and 0.014), respectively, compared to serum-free group. At ultra-structural level, after 24 hr, both FBS and AOE-supplemented media showed uniformly wide perivitelline space (PVS). After 12 hr, the cortical granules were found in the oocytes cultured in FBS and AOE-supplemented media. Within 24 hr, both granules and mitochondria were large without any detectable topographic tendency across the ooplasm. In AOE and FBS-supplemented oocytes, the number and size of microvilli were more than those in serum-free one. CONCLUSION: Although AOE supplementation induced a higher rate of the CCs expansion, and resuming meiosis, it was not as potent as FBS to provide cytoplasmic maturation of rat oocytes.

Does metformin improve in vitro maturation and ultrastructure of oocytes retrieved from estradiol valerate polycystic ovary syndrome-induced rats.

BACKGROUND: Metformin decreases polycystic ovary syndrome (PCOS) symptoms, induces ovulation, and may improve developmental competence of in vitro oocyte maturation. This study was designed to define the effects of metformin on the characteristics of in vitro oocyte maturation in estradiol valerate (EV) PCOS-induced rats. METHODS: Forty-five adult female Sprague-Dawley rats were randomly divided into control; sham and PCOS-induced (treated by a single dose of estradiol valerate, 4 mg/rat, IM) groups. The body weight was measured weekly for 12 weeks. At the end of week 12, the serum levels of testosterone, estrogen, progesterone, LH, and FSH and blood glucose of all the rats were measured. About 380 cumulus oocyte complexes (control, 125; sham, 122; PCOS-induced rats, 133) were incubated in Ham's F10 in the absence and/or presence of metformin (M 5(-10)) for 12, 24, 36, and 48 h. The cumulus cells expansion and nuclear and cytoplasmic maturation of the oocytes was evaluated using 1 % aceto-orcein staining, and transmission electron microscopy (TEM). RESULTS: No significant differences were observed in the body weight of the rats. The serum level of testosterone was reduced, and progesterone and LH were significantly increased in the PCOS-induced rats (p < 0.05). However, no significant differences were observed in the serum levels of estrogen and FSH among the groups. Blood glucose level was higher in the PCOS-induced rats than control, (p < 0.01). The expansion of cumulus cells was observed in the metformin-treated oocytes. The oocytes retrieved from PCOS-induced rats show a stage of meiotic division (GVBD, MI, A-T, and MII) in 57.12 % of metformin-untreated and fairly significantly increased to 64.28 % in metformin-treated oocytes, (p < 0.05), but no differences were observed in the MII stage within groups. The redistribution of some cytoplasmic organelles throughout the ooplasm, particularly the peripheral cortical granules, was defined in the metformin-treated oocytes. CONCLUSIONS: Single dose of EV can creates a reversible PCO adult rat model. Metformin enhances the COCs to initiate meiotic resumption at the first 6 h of IVM. In our study the metformin inability to show all aspects of in vitro oocyte maturation and may be resulted from deficiency of EV to induce PCOS.

Modulatory effect of gonadotropins on rats' ovaries after nandrolone decanoate administration: a stereological study.

BACKGROUND: Nandrolone decanoate (ND) is an anabolic androgenic steroid (AAS) which influences the ovarian structure and function. We assessed the effects of ND on the ovarian volume, number of primordial follicles, and level of hormones and also evaluated the modulatory effects of gonadotropins on the histopathological changes imposed by the administration of ND. METHODS: Six groups of Sprague-Dawley adult female rats (n=30) were used. The experimental rats were injected intraperitoneally with 3 and 10 mg/kg ND with or without human menopausal gonadotropin (hMG), 10 IU weekly for one month. The vehicle and control rats were administered olive oil and saline, respectively, for the same period of time. The ovarian volume and number of primordial follicles were estimated by stereological methods. RESULTS: The results showed a decrease in the ovarian volume, number of primordial follicles, and level of gonadotropins in the ND-treated animals compared with the vehicle groups. In the rats treated with 3 mg/kg of ND with hMG, an increase in the ovarian volume and number of primordial follicles was shown as compared to the rats treated with the same dose of ND without hMG. CONCLUSION: ND exerted detrimental effects on the dimensions of the ovary, number of follicles, and level of sex hormones. However, hMG, prevented the harmful effects of ND (at least in a low dose) on the ovarian follicles.