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 (100-1,600 microM). The effect of the metal proved to be concentration dependent. While 100-400 microM lead had no effect on the integrity of the monolayer, concentrations as high as 800 microM or higher inhibited cell adhesion and induced detachment of cells. The lead levels found in follicular fluid were 11.29 +/- 1.38 microg/L (0.056 +/- 0.007 microM). With lead in vitro at 1,600 microM (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.

In vitro comparative effect of Cd2+, Ni2+, and Co2+ on mouse postblastocyst development.

Postblastocyst development of mouse preembryos was studied in vitro in order to determine direct effect of Cd2+, Ni2+, and Co2+ ions on embryogenesis during the peri-implantation stage. Uterine horns were flushed on Day 4 of pregnancy and expanded blastocysts were cultured for 4 days in the presence of micromolar Cd2+ (1.1-26.4), Ni2+ (0. 1-500) or Co2+ (1-200). Area of trophoblast outgrowth was measured and used as a quantitative toxicological endpoint. Hatching, attachment, outgrowth, and formation of inner cells mass were also registered. Significant adverse effect on the development stages were observed at 2.2 microM (Cd2+), at 10 microM (Ni2+), and at 100 microM (Co2+). Cd2+ and Co2+ decreased the area of trophoblast markedly at concentrations of 1.1 and 10 microM, respectively. Ni2+ exposure resulted in a slight increase at 10 microM followed by a marked reduction in the trophoblast area at 250 microM. Reduced proliferative ability of trophoblast cells may point to compromised invasiveness of the embryo. The lowest Cd2+ concentration (1.1 microM=0.25 microg/ml) significantly deteriorating trophoblast development was found to be lower than Cd levels ranging up to 0.512 microg/g, reported in clinical ovarian samples of occupationally nonexposed women. The morphological alteration and loss of cellular contacts in blastocysts induced by exposure to Cd2+, Ni2+, or Co2+ may adversely influence adhesion and recognition events and may disturb aggregation of mononuclear trophoblastic cells to multinucleated cells in the course of peri-implantation in vivo as well.

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.

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.

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 microM 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. Cadmium diminished progesterone production in unstimulated and FSH-supported cells depending on its concentration and the exposure time. Follicle-stimulating hormone (100 ng ml[-1]) protected against Cd-induced suppression of progesterone production. Cadmium interfered with cell-cell junctions and the adherence of cells. No protective effect of FSH on Cd-induced alteration in cell morphology could be observed. Retraction of cytoplasmic extensions occurred at a lower dose and within a shorter exposure than a decrease in progesterone production. In conclusion, Cd exerted a direct effect on both granulosa cell morphology and on steroid biosynthesis. The lowest Cd concentration (16 microM) that was able to reduce progesterone production was about 3.5 times higher than levels reported in the ovary of a female smoker. The presented data can help to define environmental, occupational and life-style (smoking) risk factors in gonadal function during the preconception period of the female reproductive lifespan.

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 (s.c.) with zinc chloride (ZnCl2, 10 or 20 mg kg-1 body weight, bw) four times during two ovarian cycles. The third injection was accompanied by cadmium chloride (CdCl2) administration sc (2.5, 5 and 10 mg kg-1 bw). The fourth zinc (Zn) treatment was followed by mating. ZnCl2 (20 mg kg-1) itself impaired fertility by 20%, while CdCl2 dose-dependently blocked the receptivity of female rats. In combination with 2.5 and 5 mg kg-1 CdCl2 the metal salts decreased fertility in an additive fashion, whereas at the highest CdCl2 dose (10 mg kg-1) a marked ameliorating effect of ZnCl2 (10 and 20 mg kg-1) 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. ZnCl2 (10 to 80 microM) and CdCl2 (10 to 80 microM) were added to the culture medium of ovarian granulosa cells. CdCl2 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.

Effect of cadmium on female fertility, pregnancy and postnatal development in the rat.

Adult female rats having regular ovarian cycles were treated with 2.5, 5 or 10 mg/kg cadmium chloride (CdCl2) during estrus or diestrus and mated 32, 80 or 132 h post-treatment. Sperm positivity was checked next day on the predicted estrus. Maternal effects during pregnancy, fetal outcome on day 10 or at term as well as postnatal development of the F1 generation were recorded. CdCl2 caused sterility in 40 or 87% of animals at doses 5 and 10 mg/kg, respectively. Influence of Cd on fertility depended on the day of the cycle, and on the time elapsed between treatment and mating. The Cd-caused overt toxicity in fertile female rats was expressed by dose-dependent decrease in maternal body weight gain and increased progesterone blood levels. No treatment-related alteration in number and weight of conception day 10 of pregnancy or in weight and size of litters, rate of males and females at term and during the 21-day post-parturition study could be seen. It is concluded that Cd given prior mating may lead to sterility in a dose-dependent fashion. This is suggested to be caused by anovulation resulting from reversible pituitary disfunction. Animals proving fertile in spite of Cd-treatment have developed tolerance against Cd in terms of fetal outcome and postnatal development.

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.

Altered ovarian progesterone secretion induced by cadmium fails to interfere with embryo transport in the oviduct of the rat.

The effect of Cadmium (Cd) on embryo transport through the oviduct and on ovarian progesterone (P) secretion were studied in the rat. Animals were given 2.5, 5, 10 mg/kg CdCl2 or 1.0 mL/kg NaCl sc on day 1 of pregnancy. On days 1, 2, 3, 4, and 5, they were anesthetized with pentobarbital, cannulae were inserted in one of the utero-ovarian veins, and 5-minute blood samples were taken from the ovary. Ovarian venous outflow was recorded, P was determined from the blood fractions, and secretion rates were calculated. P levels were determined in peripheral blood. Body weights and the wet weight of adrenals, ovaries, and oviducts were checked; oviducts and uterine horns were flushed; and number, location, and developmental stage of embryos were observed. Cd content of the oviducts was measured. Cd accumulated dose and time dependently in oviducts and induced a dose-dependent depression and delay in the rise of ovarian P secretion during days 1 through 5 of pregnancy. In the peripheral blood, P levels also failed to rise until day 4 of pregnancy in Cd-treated rats. In embryo transfer, however, no alteration could be observed. It is hypothesized that lack of vascular contact in the oviduct makes it possible for the preimplantation embryos to escape toxic effects of Cd.

Cadmium interferes with steroid biosynthesis in rat granulosa and luteal cells in 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 cells in vitro has been studied. Granulosa and luteal cells were obtained from proestrous and pregnant rats, and incubated with 0, 5, 10, 20 or 40 micrograms 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 17 beta-estradiol (E2) by granulosa and that of P by luteal cells were measured by radioimmunoassay. In FSH-stimulated granulosa cell cultures, 5 and 40 micrograms ml-1 CdCl2 suppressed P accumulation to 65 and 10%, respectively; accumulation of E2 (at 5 micrograms ml-1 CdCl2) decreased to 44%. P production of LH-supported luteal cells dropped to 86 and 66%, respectively, when 5 and 40 micrograms 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 micrograms ml-1 CdCl2, whereas basal E2 production of granulosa cells was markedly diminished. It is concluded that CdCl2 suppressing steroid synthesis in vitro exerts a direct influence on granulosa and luteal cell function.

Distribution of cadmium in ovaries, adrenals and pituitary gland after chronic administration in rats.

Wistar rats were given 0.25, 0.5 or 1.0 mg/kg/week CdCl2 for 14, 18 or 22 weeks and the body weight, Cd content of ovaries, adrenals, pituitary gland, furthermore the progesterone and oestradiol-17 beta secretion of ovary were checked. Cd treatment caused a slight decrease in the body weight, but failed to alter the weight of endocrine organs. CdCl2 in a dose of 0.25 mg/kg/week resulted in almost the same Cd content in all the three organs. Rising the amount of Cd administered the pituitary gland accumulated more Cd than the adrenals, and the lowest levels were found in the ovary. CdCl2 even in the dose of 1.0 mg/kg/week failed to alter the ovarian cycle, progesterone and oestradiol-17 beta production of ovary. The data point also to a developing tolerance to Cd as the cumulative dose of CdCl2 lies close to the LD50 levels.

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.

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.

Long-term effects of a single cadmium chloride injection on the ovulation, ovarian progesterone and estradiol-17 beta secretion in rats.

On the day of dioestrus II rats were given 5, 10 or 15 mg/kg of cadmium chloride (CdCl2), or 1, 0 ml/kg of 0.9% NaCl solution. Then ovarian cycle was checked daily for a period of 12 cycle length. On the day of oestrus or expected oestrus in the 13th cycle the animals were anaesthetized 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 for 40 minutes and following the first blood samples 10 IU of hCG were injected iv. Ovarian venous outflow and blood pressure were continuously recorded. From the blood fractions progesterone (P) and oestradiol-17 beta (E2) were determined with RIA and the P and E2 secretion rates of ovary were calculated. Ovaries were excised and oviducts were flushed for counting oocytes. CdCl2 shortly after its administration induced a (dose-dependent) anoestrous period which turned into regular or irregular cycles depending on the dose. Part (28-32%) of the oestrous animals (14% that of the controls) remained unovulatory, when ovulation occurred normal number of ova was found. None of the doses of CdCl2 has influenced the blood pressure of animals and blood flow of the ovary. The basal secretion rate of P and E2 was not changed in the ovary compared to the controls. The hCG induced rise of P secretion, however, in the animals treated with 5 and 10 mg/kg bw CdCl2 was diminished and delayed, while in the animals treated with the 15 mg/kg Cd dose a complete lack of response was observed.

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)

Changes in the rat's motor behaviour during 4-hr inhalation exposure to prenarcotic concentrations of benzene and its derivatives.

Group motility was recorded continuously in male rats during the inhalation of benzene, toluene, ethylbenzene, o-, m- and p-xylene vapours. The solvents were applied in at least six concentrations, up to those inducing anaesthesia. Minimum narcotic concentrations (ppm) were: 5940 (benzene), 3590 (toluene), 2180 (ethyl-benzene), 2180 (0-xylene), 2100 (m-xylene), and 1940 (p-xylene). The results indicate that prenarcotic concentrations of these structurally related aromatic hydrocarbons and also the xylene isomers elicit qualitatively and quantitatively different acute behavioral effects. Except o-xylene which caused depression only the agents produced bell-shaped concentration-action curves characteristic of the biphasic effect, i.e., activation at lower and depression at higher concentrations. The curves differed in form and magnitude depending on the stimulatory potency and on the range of effective concentrations. Based on arbitrary assessment of central excitation, the five aromatics may be ranked as follows: benzene and toluene (striking activation), p-xylene (marked activation), ethylbenzene (moderate activation), m-xylene (slight activation). At the same time, high degree of motor incoordination, and in the case of benzene and p-xylene, also marked tremor could be seen.

Comparative study on the acute effects of benzene, toluene and m-xylene in the rat.

Solvents were tested for their influence on motivational state, as well as for their sedative effects, after intraperitoneal administration. The following tests were used: open field (exploratory activity, locomotion), activity wheel (running activity), tube test and tilted plane (muscle weakness, incoordination). The agents proved to be similar in causing muscle weakness and ataxia already at low dose levels. Benzene was the most potent in this respect. The three solvents, however, contrasted strongly in influencing motor activity. While benzene and especially toluene exerted CNS stimulation, no such effect could be detected following m-xylene treatment. On the basis of human experiences with these substances it may be suggested that the screening procedure applied in rats might be useful for predicting acute adverse effects in man.