Katalin Paksy

Effect of cadmium on morphology and steroidogenesis of cultured human ovarian granulosa cells

Cadmium (Cd) is able to decrease preovulatory luteinizing hormone (LH) levels in blood and inhibit ovulation in rats. In this study the direct effects of Cd on steroidogenesis in granulosa cells were investigated. The cells obtained from ovarian follicular aspirates of 41 women undergoing in vitro fertilization (IVF) were cultured. Cadmium‐induced alterations in the cellular morphology and in the production of progesterone by the cells was determined after exposure to concentrations of 8, 16, 32 and 64 μM CdCl2 for 2, 4, 8, 24 and 48 h. Progesterone secretion by granulosa cells could be stimulated with increasing concentrations of follicle‐stimulating hormone (FSH). Combined effects of Cd and FSH were also studied.

Age dependent accumulation of cadmium in the human ovary

Cadmium (Cd) was determined by atomic absorption spectrophotometry in small pieces (< 1 g) of healthy human ovaries excised for histologic examination. Cd levels in the ovary increased linearly between 30 and 65 years of age. Below 30 years, there was no age dependent increase and over 65 a tendency was observed for ovarian Cd levels to decrease. There was no difference in the Cd content of fresh luteal and nonluteal tissue taken from regularly cycling ovaries. In smokers, the amount of Cd in the ovaries was elevated compared to nonsmokers. In multiparous women (more than 3 children) a tendency of decreased Cd ovarian levels was observed. There was no difference between ovarian Cd content of physical and mental workers. It can be proposed that Cd may be a risk factor for conception and pregnancy in women in their forties.

Acute effects of cadmium on preovulatory serum FSH, LH, and prolactin levels and on ovulation and ovarian hormone secretion in estrous rats

On the day of diestrus II CFY rats were given 5, 10, or 15 mg/kg cadmium chloride (CdCl2) or 1.0 mL/kg of 0.9% NaCl. On the next day a group of animals was anesthetized with pentobarbital and blood was collected from the aorta at 13:00, 15:00, 16:30, or 18:00 h. for FSH, LH, prolactin (PRL), progesterone (P), and estradiol-17 beta (E2) determination. On the day of the expected estrus, the second group of animals was anesthetized with pentobarbital and cannulas were inserted in one of the femoral arteries and veins, and in one of the utero-ovarian veins. Five-minute blood fractions were collected from the ovary for 40 min, and following the first blood samples, 10 IU hCG was injected iv. Ovarian venous outflow and blood pressure were continuously recorded. From the blood fractions, P and E2 were determined, and their secretion rates were calculated. In a third group of treated animals, the ovaries were excised for histological examination, and oviducts were flushed for counting oocytes. CdCl2 in the dose of 10 or 15 mg/kg increased the PRL serum levels at 13:00 h; it diminished FSH serum levels in the dose of 10 mg/kg and LH serum levels in the doses of 10 and 15 mg/kg at 15:00 h. The decrease in LH levels continued until 16:30 h in the dose of 10 mg/kg CdCl2. In estrous animals, CdCl2 did not influence the blood pressure and ovarian blood flow. In animals receiving 10 or 15 mg/kg CdCl2, a decrease in basal secretion of P occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Lead accumulation in human ovarian follicular fluid, and in vitro effect of lead on progesterone production by cultured human ovarian granulosa cells

Lead content of ovarian follicular fluid obtained from 23 women was determined by atomic absorption spectrophotometry. In an in vitro experiment the direct effect of lead on the morphology and on progesterone (P) production by cultured granulosa cells of six women was investigated. Follicular fluid and granulosa cells were obtained from follicular aspirates of women undergoing in vitro fertilization (IVF) and embryo transfer (ET). Granulosa cells were cultured for 48 h to form monolayers in the presence or absence of lead acetate (100-1600 µ M). The effect of the metal proved to be concentration dependent. While 100-400 µ M lead had no effect on the integrity of the monolayer, concentrations as high as 800 µ M or higher inhibited cell adhesion and induced detachment of cells. The lead levels found in follicular fluid were 11.29 - 1.38 µg/L (0.056 - 0.007 µ M). With lead in vitro at 1600 µ M (331.5 mg/L) there resulted a significant decrease in P production by granulosa cells. This concentration is very much higher than that measured in follicular fluid of IVF/ET patients, specifically nonexposed to lead, and even higher than mean blood levels reported by others in high exposure groups. In conclusion, lead seems not to exert a specific effect on the steroidogenesis by cultured human granulosa cells. Therefore, the lead levels measured in the ovarian follicular fluid seem not to pose a hazard with respect to progesterone secretion by the ovary.

Effect of sinusoidal 50 Hz magnetic field on the testosterone production of mouse primary Leydig cell culture

This study evaluated the effect of sinusoidal 50 Hz magnetic field on the basal and human chorionic gonadotropin (hCG)-stimulated testosterone (T) production of 48-h mouse Leydig cell culture. The luteinizing hormone (LH) analog hCG was used to check the T response of the controls and to evaluate the possible effect of the applied magnetic field on the steroidogenic capacity of the exposed cells. Leydig cells were obtained from the testes of 35- to 45-g CFLP mice and isolated by mechanical dissociation without enzyme treatment. The cell cultures were exposed to sinusoidal 50 Hz 100 microT (root mean square) AC magnetic field during the entire time of a 48-h incubation. Testosterone content of the culture media was measured by radioimmunoassay. In cultures exposed to the magnetic field, a marked increase of basal T production was found (P < .05), compared with the unexposed controls, whereas no significant difference was seen between the exposed or unexposed cultures in the presence of maximally stimulating concentration of hCG. These findings demonstrate that sinusoidal 50 Hz 100 microT magnetic fields are able to stimulate the basal T production of primary mouse Leydig cell culture, leaving the steroidogenic responsiveness to hCG unaltered.

Toxic effects of cadmium on LHRH-induced LH release and ovulation in rats

CFY rats were given 5 or 10 mg/kg bw cadmium chloride (CdCl2) or 0.9% NaCl solution (1 mL/kg) subcutaneously on the day of diestrus II. Six days later (proestrus) at 1200 h they were anesthetized with pentobarbital, 0.5 or 2 micrograms/kg LHRH was injected intravenously at 1400 h, and blood was collected for LH determination. A second group of animals pretreated with 10 mg/kg bw CdCl2 and treated with 2 or 4 micrograms/kg LHRH was allowed to recover from the anesthesia and checked for ovulation the next day (estrus). In rats treated with 10 mg/kg of CdCl2, the LH content of pituitary gland diminished, but no significant difference was found in the LH response to LHRH. In controls (ovulation blocked by anesthesia) 2 as well as 4 micrograms/kg of LHRH completely restored ovulation, while after Cd pretreatment, ovulation recovered depending on the dose of LHRH. It is concluded that Cd-induced anovulation is related to altered function of the pituitary gland and ovary, which can be restored by excess LHRH.

Zinc protection against cadmium-induced infertility in female rats. Effect of zinc and cadmium on the progesterone production of cultured granulosa cells

Adult female rats were treated subcutaneously (sc) with zinc chloride (ZnCl, 10 or 20 mg kg body weight, bw) four times during two ovarian cycles. The third injection was accompanied by cadmium chloride (CdCl) administration sc (2.5, 5 and 10 mg kg bw). The fourth zinc (Zn) treatment was followed by mating.ZnCl (20 mg kg) itself impaired fertility by 20%, while CdCl dose-dependently blocked the receptivity of female rats. In combination with 2.5 and 5 mg kg CdCl the metal salts decreased fertility in an additive fashion, whereas at the highest CdCl dose (10 mg kg) a marked ameliorating effect of ZnCl (10 and 20 mg kg) on cadmium (Cd)-caused sterility was observed.In the pregnant animals apart from the higher Cd-induced blood progesterone levels and reduced body weight gain of dams, no significant treatment-related maternal and fetal effects could be observed. ZnCl (10 to 80 μm) and CdCl (10 to 80 μm) were added to the culture medium of ovarian granulosa cells. CdCl suppressed follicle-stimulating-hormone- (FSH-) and cAMP-stimulated progesterone accumulation. No protective effect of Zn against Cd-induced drop in progesterone production could be seen, while Zn by itself induced a significant increase in FSH-supported progesterone synthesis.In conclusion, while Zn protected against Cd-induced sterility in vivo, it failed to counteract the direct effect of Cd on steroid biosynthesis. The data indicate that Zn protection does not take place at the level of ovary. Moreover, Zn and Cd seem to affect FSH-stimulated progesterone production by different mechanisms.

Cadmium interferes with steroid biosynthesis in rat granulosa and luteal cellsin vitro

Recently, cadmium has been described to disturb ovarian function in rats. In this paper the direct influence of cadmium on steroid production of ovarian cellsin vitro has been studied. Granulosa and luteal cells were obtained from proestrous and pregnant rats, and incubated with 0, 5, 10, 20 or 40 μg ml−1 CdCl2 in the presence or absence of 0.1–1000 ng ml−1 follicle stimulating hormone (FSH) or luteinizing hormone (LH) for 24 or 48 h. Production of progesterone (P) and 17β-estradiol (E2) by granulosa and that of P by luteal cells were measured by radioimmunoassay. In FSH-stimulated granulosa cell cultures, 5 and 40 μg ml−1 CdCl2 suppressed P accumulation to 65 and 10%, respectively; accumulation of E2 (at 5 μg ml−1 CdCl2) decreased to 44%. P production of LH-supported luteal cells dropped to 86 and 66%, respectively, when 5 and 40 μg ml−1 CdCl2 was added to the medium. No alteration in basal P accumulation occurred in granulosa and luteal cell cultures following incubations with 20 and 40 μg ml−1 CdCl2, whereas basal E2 production of granulosa cells was markedly diminished. It is concluded that CdCl2 suppressing steroid synthesisin vitro exerts a direct influence on granulosa and luteal cell function.

Uptake and distribution of Cd in the ovaries, adrenals, and pituitary in pseudopregnant rats: effect of Cd on progesterone serum levels.

Pseudopregnant (PSP) rats were treated with 3.5 or 7.0 mg/kg body wt of CdCl2 on Day 1 of PSP sc. In the lower dose Cd content of the ovaries (luteal and nonluteal tissues), adrenals, pituitary, and blood on Days 1, 2, 5, 8, 10, and 12, and in the higher dose that of luteal and nonluteal tissue on Days 2 and 5 of PSP were determined with atomic absorption spectrophotometry. A rapid incorporation into the corpora lutea was measured on Day 1 and Day 2 of PSP followed by a decrease of Cd content toward the end of PSP whereas the nonluteal tissue, adrenals, and pituitary accumulated Cd gradually until the fifth to 10th day, respectively. Progesterone (P) serum levels were measured with RIA in the blood collected daily from the jugular vein following administration of 3.5 to 7.0 mg/kg body wt of CdCl2 sc on Day 1 or Day 8 of PSP. The serum levels of P remained unchanged when CdCl2 was administered on Day 1 of PSP; however, 7.0 mg/kg body wt CdCl2 given on Day 8 of PSP induced a significant decrease in serum levels of P. It is supposed that the regressing luteal tissue is more sensitive to the toxic effects of Cd than the developing one.

Effect of Ni2+ on the testosterone production of mouse primary Leydig cell culture

This study evaluated the effects of Ni2 on testosterone (T) production of mouse Leydig cells in vitro following an in vivo or in vitro exposure. CFLP mice were subjected to repeated exposure (4 treatments, subcutaneously, every 3 d) to 10, 20 or 40 mg/kg body weight of NiSO4 or 1.0 ml of 0.9% NaCl solution. Depressed human chorionic gonadotropin (hCG)-stimulated T response was seen over a 48-h culture of testicular interstitial cells obtained from the animals exposed to 20 mg/kg or higher dose of NiSO4, while the basal T production remained unaltered. There were no Ni2+-related changes in the body weights or in the weights of testes, epididymides, adrenals, and kidneys. No histopathological alteration was found in the examined organs of NiSO4-treated groups except the dose-dependent tubular lesions in kidney as a result of a specific rather than a general cytotoxic action. To assess the direct effect of Ni2+ on Leydig-cell T production, testicular interstitial cells were cultured with Ni2+ (62.5 to 1000 microM) for 48 h in the presence or absence of maximally stimulating concentration of hCG. Dose-dependent depression in hCG-stimulated T production was seen at 125 microM or higher dose of Ni2+, while basal T production was unaffected. In order to evaluate the time dependency of this effect the cells were cultured for various times in the presence or absence of 250 and 1000 microM Ni2+. Decreased hCG-stimulated T production was found in the cultures maintained at least for 4 h in the presence of 1000 microM Ni2+, whereas at 250 microM at least 16 h was required to elicit the depression. Cell viability was assessed by a metabolic activity (MTT) assay. The viability of cells was unaltered by 250 microM Ni2+, and only a slight decrease was found even at the end of the 48-h culture period in the presence of 1000 microM Ni2+. Our results show a dose-related depression in stimulated T production of mouse Leydig cells in culture following either in vivo or in vitro Ni2+ treatment at a dose that does not induce any general toxic or significant cytotoxic action. The data of the time-course study indicate that the effect of Ni2+ on Leydig-cell T production is both time and concentration dependent, and not due to cytotoxicity.