Synergistic Action of Virgin Coconut Oil and Clomiphene in Reversing Endocrine Dysregulation in Letrozole-Model of Polycystic Ovarian Syndrome in Rats: Role of Nrf2/HMOX-1 Pathway.

Polycystic ovarian syndrome (PCOS) is an endocrine disorder in women's reproductive age. Currently, the pathophysiology of PCOS is unclear, and the limited treatment options are unsatisfactory. Virgin coconut oil (VCO) is functional food oil associated with pharmacological effects in reproductive disorders. Therefore, we aimed to evaluate whether VCO could enhance clomiphene (CLO) therapy against PCOS in female rats. Rats were randomly divided: (1) Control, (2) PCOS model, (3) PCOS + CLO, (4) PCOS + VCO, and (5) PCOS + CLO + VCO. The PCOS was induced via daily letrozole (1 mg/kg, orally) administration for 21 days. After the PCOS induction, CLO, VCO, and CLO + VCO were administered from days 22 to 36. Serum levels of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, estrogen, progesterone, and prolactin were estimated. Polymerase chain reaction gene expression for nuclear factor-erythroid-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), catalase (CAT), glutathione reductase (GSR), LH receptor (LHr), androgen receptor (AR), tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), and caspase-3 were analyzed. The letrozole-induced PCOS caused considerable increases in GnRH, LH, prolactin, estrogen, and testosterone, whereas FSH decreased significantly compared to the control. The gene expression of Nrf2, HO-1, CAT, and GSR were markedly diminished, while IL-1ß, TNF-α, caspase-3, AR, and LHr prominently increased compared to control. Interestingly, the CLO and VCO separately exerted anti-inflammatory and endocrine balance effects. However, VCO-enhanced CLO effect in LH, prolactin and testosterone, Nrf2, HO-1, CAT, GSR, and AR. VCO may synergize with CLO to depress hyperandrogenism and oxidative inflammation in PCOS.

Clomiphene citrate induces nuclear translocation of the TFEB transcription factor and triggers apoptosis by enhancing lysosomal membrane permeabilization.

The autophagy-lysosome pathway plays a central role in cellular homeostasis by regulating the cellular degradative machinery. The transcription factor EB (TFEB) regulates the biogenesis and function of both lysosomes and autophagosomes, and enhancement of TFEB function has emerged as an attractive therapeutic strategy for lysosome-related disorders. However, little is known about the role of TFEB activation in regulating the cellular fate. Here, we describe that clomiphene citrate (CC), a selective estrogen receptor modulator, promotes nuclear translocation of TFEB and increases lysosomal biogenesis in HeLa and MDA-MB-231 cells. Treatment with CC inhibits cell viability and causes apoptosis by increasing the release of proteases cathepsin B (CatB) and cathepsin D (CatD) from lysosomes into the cytosol. In contrast, knockdown of TFEB rescues the cells from CC-induced cell death. Furthermore, CC-induced TFEB activation also enhances the autophagy flux in HeLa cells. Knockdown of autophagy-related gene 7 (ATG7) significantly decreases the CC-induced CatB and CatD release and cell death, suggesting that autophagy contributes to the lysosomal membrane permeabilization (LMP) caused by CC. Altogether, these findings have broad implications for our understanding of TFEB function and provide new insights into CC pharmacological therapy.

Induction of sister chromatid exchanges and cell division delays by clomiphene citrate in human lymphocytes.

OBJECTIVE: Clomiphene citrate (CC) is a selective estrogen receptor modulator and is used for the treatment of in vitro fertilization, intracytoplasmic sperm injection, intrauterine insemination, and so on. In this study, sister chromatid exchanges (SCEs) and cell cycle delays were analyzed to investigate genotoxicity and cytotoxicity of CC in peripheral blood lymphocytes of healthy women. METHODS: Human peripheral blood lymphocytes obtained from two donors were used to detect genotoxicity and cytotoxicity of CC. Lymphocytes were treated with various concentrations (0.40, 0.80, 1.60, and 3.20 µg/ml) of CC. A negative (distilled water) and a positive control (mitomycin-C = 0.20 µg/ml) were also used simultaneously with test substance-treated cultures. SCEs and cell division delays were measured from 25 cells and 100 cells perdonor, respectively. RESULTS: CC significantly increased the mean SCE value at all concentrations compared with the negative control. This increase was found to be dose dependent (r = 0.83) and at the highest concentration, nearly two times higher increase was observed than the negative control. However, replication index was not affected by the CC treatment. CONCLUSION: The present study shows that CC is genotoxic for human lymphocytes in vitro. Further investigations, especially in vivo are now needed in different test organisms to clarify the genotoxic activity of CC, which should also help to better understand genotoxic mechanism of this ovulation-stimulating drug.

Evaluation of the genotoxicity of clomiphene citrate.

Clomiphene citrate (CC) is a selective estrogen-receptor modulator that is primarily used to enhance follicular development in women receiving in vitro fertilization (IVF) treatment. Although some studies suggested large increases in ovarian cancer risk related to fertility medications, this association has not been confirmed in other studies. Whether there could be a residual, small risk is still an open question. It is known that genomic instability and multiple genetic changes may be required in carcinogenesis. Genomic instability such as single-base changes, chromosomal rearrangements or aneuploidy may accelerate this process. Genomic instability is not only central to carcinogenesis, but it is also a factor in some neurodegenerative diseases such as amyotrophic lateral sclerosis or the neuromuscular disease myotonic dystrophy. For these reasons, this study was planned to examine genotoxic effects of CC in human lymphocytes by use of the chromosome aberration (CA) assay, the micronucleus (MN) test, the comet assay, and the test for bacterial mutagenicity in Salmonella typhimurium strains TA98 and TA100 (Ames test). Concentrations of 0.40, 0.80, 1.60, and 3.20µg/ml of CC significantly increased the frequency of chromosomal aberrations (p<0.01 and p<0.001) and micronuclei (p<0.05, p<0.01 and p<0.001) in cultured human lymphocytes, and of DNA damage (tail length, p<0.05, except 0.80µg/ml) in isolated lymphocytes compared with their respective controls. The highest CC concentration at 24h and highest two concentrations after the 48-h treatment significantly decreased the mitotic index. The Ames test showed that the concentrations of CC used in this study induced neither base-pair substitutions nor frame-shift mutations in S. typhimurium strains TA98 and TA100.

Tubo-ovarian dysplasia in relationship with ovulation induction in rats.

OBJECTIVE: To assess tubo-ovarian dysplasia via morphologic and immunohistochemical study of rats exposed to ovulation stimulation protocols. DESIGN: Animal experimental study. SETTING: Academic research hospital. ANIMAL(S): 72 female Wistar rats divided into three groups. INTERVENTION(S): Stimulation protocols using follicle-stimulating hormone (FSH) or clomiphene citrate for 3, 6, or 12 cycles, after which the animals were killed. MAIN OUTCOME MEASURE(S): Ovarian and tubal dysplasia score and immunohistochemical assessment using p53 and Ki67. RESULT(S): The ovarian dysplasia score was statistically significantly higher after 12 stimulation cycles in the groups receiving FSH (group B) or clomiphene citrate (group C) compared with control (group A). The tubal dysplasia score was statistically significantly increased after only three stimulation cycles in groups B and C. The Ki67 proliferation marker was statistically significantly expressed in the ovaries from group C, and in the fallopian tubes from groups B and C. P53 was constantly low in all three groups. CONCLUSION(S): Ovulation stimulation may induce tubal and ovarian histopathologic and immunohistochemical abnormalities with a dose effect. The role of the fallopian tubes and their interaction with the ovaries require further study.

In vivo evaluation of the genotoxic effects of clomiphene citrate on rat reticulocytes: a micronucleus genotoxicity.

OBJECTIVE: To determine the genotoxic effects of clomiphene citrate (CC) on rat reticulocytesin vivo. METHODS: In this prospective, randomized, controlled study, rats were each assigned randomly to the CC 50, CC 100, CC 200, or control group and were given repeat doses of 0.16, 0.32 or 0.64 mg CC, or normal saline, respectively. Each study group received its CC dose in 2 ml of saline intraperitoneally for 5 days, while the control group received only 2 ml of saline. Each treatment cycle was repeated six times. Six months later, the rats were euthanized. Bone marrow tissues were removed, and pluripotent reticulocyte cells with micronuclei, nuclear buds, and binuclear abnormalities were analyzed using an in situmicronuclei assay under light microscopy. The proportion of micronucleated erythrocytes was measured. RESULTS: Fewer cells with nuclear buds and binuclear abnormalities were detected in the CC 50 group and controls. The CC 100 and 200 groups had significantly (p < 0.05) more nuclear buds and binuclear abnormalities compared with the CC 50 group and controls in the cytogenetic analysis of bone marrow stem cells. CONCLUSION: In rats, the micronucleus genotoxicity assay suggests a dose-dependent CC effect on genomic instability in bone marrow stem cells in vivo.

Estradiol protects clomiphene citrate-induced apoptosis in ovarian follicular cells and ovulated cumulus-oocyte complexes.

OBJECTIVE: To determine whether clomiphene citrate (CC) reduces E(2) level in the ovary and circulation and induces apoptosis in ovarian follicular cells and ovulated cumulus-oocyte complexes (COCs). If yes, to determine whether E(2) coadministration could protect against these adverse effects of CC. DESIGN: A controlled prospective study. SETTING: Laboratory research setting. ANIMAL(S): Ninety sexually immature female rats that were 24-25 days of age. INTERVENTION(S): The immature female rats were injected with a single dose of 10 IU of pregnant mare serum gonadotropin. After 48 hours, 10 IU of hCG along with 10 mg of CC per kilogram of body weight, with or without 2.0 mg of E2 per kilogram of body weight were coadministered. After 16 hrs, the rats were killed; COCs were collected from oviduct and ovaries were isolated. MAIN OUTCOME MEASURE(S): Number of superovulated COCs, oocyte morphology, E2 level in ovary and serum, histology of ovary, DNA fragmentation, and bax protein expression in ovary and COCs. RESULT(S): The number of COCs and E2 level in ovary and serum were reduced, whereas membrane blebbing in oocytes, bax protein expression, and DNA fragmentation in ovarian follicular cells and ovulated COCs were induced after CC treatment. These adverse effects of CC were protected against if animals were coadministered with E2. CONCLUSION(S): Clomiphene citrate-induced apoptosis in ovarian follicular cells (probably granulosa cells), thereby reducing E2 level in ovary and circulation that might have resulted in poor development and maturation of oocytes leading to reduced ovulation.

Oral administration of clomiphene to neonatal rats causes reproductive tract abnormalities.

Oral administration of clomiphene at 2, 4, or 8 mg/kg to 4-day-old rats caused multiple histopathological abnormalities of the reproductive tract in both male and female animals. No histopathological abnormalities were observed in 30-day-old male rats at any dose examined. In contrast, 30-day-old females showed hypertrophy of the myometrium at all doses examined, and hypertrophy of the luminal or glandular epithelium, and dilatation of the uterine lumen were observed in the highest dose group. In post-pubertal rats, histopathologically marked changes were observed in the testes and epididymides in males, and in the ovaries and uterus in females in the highest dose group. In addition, relative weight of male reproductive organs in the highest dose group was decreased as compared with that in the controls. These results suggested that early neonatal exposure to clomiphene induced marked reproductive tract abnormalities in males after puberty, as well as in females.

Effects of clomiphene citrate on neonatal rat skin.

BACKGROUND: Clomiphene citrate is chemically related to non-steroidal estrogens, and has antiestrogenic properties. It is used in the treatment of anovulatory female infertility and its therapeutic effect mainly depends on inhibiting the negative feedback effects of endogenous estrogen by stimulating the gonadotropin releasing hormone. Today, it is also used in the treatment of male infertility. OBJECTIVES: In this study the effects of clomiphene citrate on skin maturation in neonatal rats were investigated. METHODS: Forty Spraque-Dawley female newborn rats were separated into two control and two experimental groups (n = 10). One day after birth. experimental newborn rats were given clomphene citrate subcutaneously in a dosage of 100 mg/kg/day for five days. The first experimental group of rats were anesthetised at 21 days whereas the second experimental group of rats were then anestetised on day 28. Biopsies were taken immediately from the perineal skin. Histopathological assessments were made and compared with their control groups. RESULTS: In both the experimental groups of newborn rats, increased keratinization and irregular hypertrophy were observed in the epidermal cells. Disorganization of the basal layer cells and hyperplasia were found to be more prominent in the first experimental group and dermal fibrosis and lymphohistiocytic inflammatory cell infiltration were especially prominent around the sebaceous glands in the second experimental group. CONCLUSION: The administration of clomiphene citrate in newborn rats showed impaired skin maturation.

Does maternal drug ingestion cause megacystis microcolon intestinal hypoperistalsis syndrome? I. Clomiphene trial.

PURPOSE: Megacystis Microcolon Intestinal Hypoperistalsis Syndrome (MMIHS) is a congenital and lethal disease, and the etiology of the disease is not clear. It is speculated that maternal ingestion of some drugs during pregnancy may be an etiologic factor. In this study we aimed to investigate the effect of maternal ingestion of clomiphene on fetal bladder and colon in pregnant rats. METHODS: We separated animals into a control group including 14 rats and a clomiphene group with 30 rats. Nothing was given to the control group during pregnancy. Two mg/kg/day clomiphene intraperitoneally was given to the study group from the 6th to 12th day of pregnancy. All of them were sacrificed on the 20th day of pregnancy. Histopathological examination of the fetal colon and bladder was performed. RESULTS: In the clomiphene group a significant decrease in the thickness of the bladder wall, an increase in bladder epithelium, an increase in muscle atrophy of the colon and bladder wall, an increase in vacuoler degeneration of the muscles of the bladder and colon wall, a decrease in ganglion cell numbers in the myenteric plexus of the bladder and a decrease in the thickness of the bladder tunica muscularis were determined. CONCLUSION: In our rat model we found histological structural changes in the rats' colon and bladder walls as a result of using clomiphene on days 6-12 of pregnancy; a similar pathological finding to those found in some of the MMIHS patients' colons and bladders.

Clastogenic and aneugenic effects of tamoxifen and some of its analogues in hepatocytes from dosed rats and in human lymphoblastoid cells transfected with human P450 cDNAs (MCL-5 cells).

Tamoxifen and its analogues 4-hydroxytamoxifen, toremifene, 4-hydroxytoremifene, clomifene and droloxifene were tested for clastogenic effects in a human lymphoblastoid cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase and in a cell line containing only the viral vector (Ho1). MCL-5 or Ho1 cells were incubated with 4-hydroxytamoxifen, 4-hydroxytoremifene, clomifene or droloxifene and the incidence of micronuclei estimated. With MCL-5 cells there was an increase in micronuclei with 4-hydroxytamoxifen, 4-hydroxytoremifene and clomifene but not with droloxifene. With Ho1 cells only 4-hydroxytamoxifen and 4-hydroxytoremifene caused an increase in micronuclei. MCL-5 cells were incubated with tamoxifen, 4-hydroxytamoxifen, toremifene, droloxifene, clomifene or diethylstilbestrol (0.25-10 microg/ml) for 48 h and subjected to 3 h treatment with vinblastine (0.25 microg/ml) to arrest cells in metaphase. The incidence of cells with chromosomal numerical aberrations (aneuploidy) was increased in cells treated with tamoxifen, 4-hydroxytamoxifen, toremifene, clomifene and diethylstilbestrol but not droloxifene. The frequency of cells with structural abnormalities (excluding gaps) was increased in cells treated with tamoxifen and toremifene but not 4-hydroxytamoxifen, clomifene, droloxifene or diethylstilbestrol. The clastogenic activities of tamoxifen (35 mg/kg), toremifene (36.3 mg/kg), droloxifene (35.2 mg/kg) and diethylstilbestrol (25 mg/kg) were compared in groups of four female Wistar rats. Each chemical was dissolved in glycerol formal, administered as a single dose by gavage and hepatocytes isolated by collagenase perfusion 24 h later. The cells were cultured in the presence of epidermal growth factor (40 ng/ml) for 48 h, colchicine (10 microg/ml) being added for the final 3 h of incubation. At least 100 chromosomal spreads were examined from each animal for the presence of numerical and structural abnormalities. The incidences of aneuploidy following treatment were: tamoxifen 81%, toremifene 46%, droloxifene 9.6%, diethylstilbestrol 45.7%, vehicle control 5.3%. The incidences of chromosomal structural abnormalities excluding gaps were: tamoxifen 4.3%, toremifene 0.8%, droloxifene 0.5%, diethylstilbestrol 0.8%, control 0.5%. The incidence of chromosomal structural aberrations excluding gaps in the treated animals was not statistically significantly different from controls except in the tamoxifen-treated group. Tamoxifen (35 mg/kg per os) and toremifene (36.3 mg/kg per os) were dosed to rats for 4 weeks and chromosomal spreads made from hepatocytes. The incidences of aneuploidy were: tamoxifen 94%, toremifene 57%, control 6.5%. The incidences of chromosomal aberrations excluding gaps were: tamoxifen 12%, toremifene 1%, control 0.5%. The incidence of tamoxifen-induced chromosomal structural abnormalities was significantly elevated compared with control levels. The results demonstrate that tamoxifen and toremifene are the only two drugs tested in the study that cause chromosomal structural and numerical aberrations in vitro and tamoxifen is the only drug that induces both these effects in rat liver cells stimulated to divide in culture following oral dosing. Since chromosomal mutations require cell division for their manifestation and tamoxifen is the only compound of those tested that causes hyperplasia in the rat liver, chromosomal aberrations and aneuploidy in the rat liver would only be expected to occur following treatment with tamoxifen alone, although aneuploidy could be induced by toremifene in conjunction with a promoter such as phenobarbitone.

[Assessment of genetic toxicity of the exposure to clomiphene citrate, with various bacterial test systems]. / Valoración de riesgo gentóxico de exposición a citrato de clomifeno, con diferentes sistemas de prueba bacterianos.

Clomiphene citrate (CC) induces framshift mutations on the Salmonella typhimurium Ames strains TA1538, TA97 and TA100 employing in vitro metabolic activation with S9 aerochlor 1254 induced rat livers, but not base pair substitution mutations with neither the standard or the preincubation method. CC induced genolethal DNA damages on the Escherichia coli PolA-/PolA+ with S9 on the preincubation method or without S9 on the disk diffusion one. The severe primary DNA damages produced b CC was verified by the SOS induction on the lysogenic lambda phage induction with De Marini (1988) method and the induction of colicin E1 plasmid on E. coli. These results are suggestive that CC may be an adduct forming compound which is able to inhibit replication if the cell lacks DNA polymerase, or it may produce framshift mutations after replications. CC induced damages could be large lesions conducing to unicatenary DNA strains, that are able to induce the lexA regulated genes. So, the use of this ovulation inductor is a risk of genotoxic damage and it is advisable to do a risk-benefit evaluation in any particular case before its prescription.

Preovulatory administration of clomiphene citrate to mice causes fetal growth retardation and neural tube defects (exencephaly) by an indirect maternal effect.

Clomiphene citrate was administered to female mice at different doses and different times prior to ovulation, in the preimplantation period after ovulation, and after implantation. Pregnancy outcome was determined on day 15 of gestation, when the number of implantations and resorptions were calculated relative to the number of ovulations, and fetuses were assessed for size and stage of development and morphological abnormalities. Preovulatory administration of clomiphene citrate caused decreased implantation rates and growth retardation of surviving fetuses, the degree of the effect being dependent on the dose and the time of drug injection relative to ovulation. The implantation rate was lowest, and the degree of fetal growth retardation highest, when clomiphene citrate was administered immediately before ovulation. An increased incidence of exencephaly was found in the fetuses of females injected with clomiphene citrate prior to ovulation. Transfer of blastocysts from treated mice to untreated fosters showed the effect of clomiphene citrate on implantation and fetal growth to be predominantly mediated through the female reproductive tract, rather than a direct effect on the embryo itself. Administration of clomiphene citrate in the preimplantational period resulted in complete inhibition of implantation, while the only effect when administration was after implantation was a slight reduction in fetal weight. These results indicate that preovulatory clomiphene citrate impairs uterine function, which has an indirect effect on the growth and development of the postimplantation embryo.

Effects of antiestrogens on adult and neonatal mouse reproductive organs.

Estrogenic potencies of various antiestrogens, including keoxifene (Kx) and trifluoperazine (Tfp), on reproductive tracts of ovariectomized adult mice, and effects of neonatal Kx and Tfp on reproductive organs were studied in C57BL/Tw mice. In adult ovariectomized mice, weight, DNA, and protein of the uterus and vagina were increased by 3 daily injections of 100 micrograms clomiphene, tamoxifen (Tx), and nafoxidine, and of 1 microgram estradiol-17 beta (E), but not by Kx. Antiestrogenic potency of Kx was studied in adult mice given injections of E. Kx significantly suppressed the E-induced increase in weight, DNA, and protein in the uterus and vagina. Tfp (20 micrograms), known as a tranquilizer and an antiestrogen, had no estrogenic effect on either organ. Male and female mice given 5 daily injections of Kx or Tfp from the day of birth were examined at 30, 40, and 60 days of age. Weights of testis, epididymis, and seminal vesicle in neonatally Kx-treated mice were significantly lower than in controls at 30 and 40 days. Spermatozoa were not formed in the seminiferous tubules of Kx-treated mice, although spermatogenesis occurred at 60 days. In neonatally Kx-treated females, weight of the uterus at 60 days and of the vagina at 40 and 60 days was significantly lower than in controls. Corpora lutea were absent from the ovaries of Kx-treated females. In neonatally Tfp-treated mice of both sexes at all ages examined, no differences were found in organ weights or histology, other than lower spermatogenic indices at 40 and 60 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of clomiphene citrate on early pregnancy in guinea-pigs.

Clomiphene citrate (2 mg/kg body wt) given on the day of mating can block or interrupt pregnancy in guinea-pigs. Corpus luteum function, uterine histology, implantation and embryo development were studied in clomiphene-treated and control animals on Days 5, 9 and 20 of pregnancy. Following treatment, only 25% of the females were regularly pregnant, presenting large and healthy foetuses. The other females examined showed either pregnancy with embryos undergoing resorption or no sign of pregnancy. In these females, corpus luteum size was reduced, progesterone concentrations were very low and the endometrial glands and the epithelium were often altered. It is concluded that clomiphene causes a reduction in fertility by altering the uterus and, by directly or indirectly inducing luteolysis, causes later pregnancy loss.

Validation of protocols for assessing early pregnancy failure in the rat: clomiphene citrate.

A battery of protocols for the assessment of maternally mediated toxicity during early pregnancy in the rat is being evaluated for its utility in detecting and defining mechanisms of early pregnancy failure. In this report, clomiphene citrate (CC), an estrogen agonist/antagonist, was used as a model compound in this evaluation process. The protocols involve dosing rats with CC during, and evaluation of multiple endpoints following, (a) the first 8 days of pregnancy; (b) early pseudopregnancy, accompanied by decidual induction; and (c) the pre- and postimplantation intervals of early pregnancy. In addition, the effect of CC on embryo transport rate was assessed. Eight days of dosing with CC during early pregnancy produced a dose-dependent reduction in the number of implantation sites seen on Day 9, concomitant with alterations in maternal hormonal parameters. No effect on the decidual cell response was found, indicating that the mechanism of fertility reduction was not mediated via compromised uterine decidualization. The preimplantation period was highly vulnerable to the toxic effects of CC while no postimplantation resorptions were seen. When embryo transport rate was measured, a statistically significant embryo transport rate acceleration was detected, but this was insufficient to account for all of the observed preimplantation loss. The data suggest an effect of CC on embryo viability, on the embryo's ability to implant, or on the reproductive tract resulting in compromised embryo survival as the potential mechanism(s) mediating CC-induced early pregnancy failure. The battery of protocols was thus successful in identifying CC as a reproductive toxicant and in elucidating the causes of the observed early pregnancy failure.

Critical period of induction by tamoxifen of genital organ abnormalities in female mice.

Genital organs of C57BL/Tw female mice given 5 daily injections of 100 micrograms tamoxifen (Tx) from the day of birth (day 0) were examined at 5, 10, 15, 20, 30 and 60 days of age. The incidence of polyovular follicles (PF) in Tx mice was higher, but the development of uterine glands and tunica muscularis uteri in Tx mice was lower than in the age-matched controls. Uteri of 15-day-old Tx mice underwent a weight increase resulting from edematous change in the stromal tissue lacking type I collagen and fibronectin. Adenosis-like lesions were found in the vaginae of 5- to 30-day-old Tx mice. Mice given neonatal injections of 100 micrograms clomiphene (Clm) and nafoxidine (Naf) were also examined at 60 days. Tx caused much greater damage to the ovary and uterus than did Clm and Naf. In order to examine the critical period of induction by Tx of female genital organ abnormalities, mice were given 5 daily injections of Tx starting at different early postnatal ages. Tx injections starting within 5 days caused a high incidence of PF in the ovary and aplasia of tunica muscularis in the uterus. The Tx treatment also induced atrophy of the uterine luminal epithelium when started within 7 days. The present study suggests, therefore, that the postnatal limit of the critical period for the female genital organs lies within 7 days after birth.

Pituitary-related weight changes affecting the liver, uterus and adrenal glands of rats treated with hexoestrol and clomiphene in high doses.

The synthetic oestrogen hexoestrol, administered at 60 mg/kg/day for 4 days to female rats in 3 studies, caused the following mean changes in the relative weights of some of the principal organs: liver (+37%), spleen (-11%), adrenals (+43%), kidneys (+3%), pituitary (+23%), uterus (+49%), and ovaries (+13%). The heart weights showed no consistent changes. The mean relative organ weights of hypophysectomized, hexoestrol-treated rats did not differ significantly from those of untreated hypophysectomized controls. The latter animals had lower organ weights than sham-operated controls. Pretreatment with clomiphene citrate at dose levels of 20-60 mg/kg/day prevented in a dose-dependent manner most of the organ weight changes induced by hexoestrol. The exception was the adrenal weight, which was increased. Compared with controls, the mean relative organ weights of rats receiving clomiphene alone at 60 mg/kg/day differed as follows: liver (-18%), spleen (-17%), heart (-16%), adrenals (+7%), kidneys (-11%), pituitary (-13%), uterus (-25%), and ovaries (-4%). The changes affecting the liver, spleen, heart, kidneys and uterus were significant. Rats immunised with a monkey antiserum to rat growth hormone and treated with hexoestrol had significantly lower relative liver weights than did animals treated with hexoestrol alone. No other significant differences were observed. It is tentatively concluded that there is a mediating or co-operative pituitary influence involved in the significant hepatic, uterine and adrenal weight gains caused by hexoestrol. Clomiphene may act centrally to affect these organ weight changes and, indeed, may act at this level to (mainly) anti-organotrophic effect even in the absence of exogenous oestrogen. In the case of the hexoestrol-induced liver enlargement, the role of pituitary growth hormone is worth further investigation.

Use of human cumulus granulosa cells for in vitro screening of reproductive toxicants.

We investigated the possibility that human granulosa cells from the cumulus mass obtained during human in vitro fertilization/embryo transfer (IVF/ET) might be useful for screening of potential reproductive toxicants in vitro. The cumulus granulosa cells detached from the zona pellucida after fertilization were allowed to spontaneously adhere to the incubation dish following transfer (removal) of the embryo. These cumulus cells survived in culture for at least four additional days, appeared on simple inspection to be morphologically normal luteinized granulosa cells, and produced large amounts of progesterone (P) over the culture interval. Production gradually declined during culture in the absence of human chorionic gonadotropin (hCG); however, inclusion of hCG (100 ng/mL) in the medium maintained P production at control (day 1) levels. Introduction of estrogenic agents previously shown to suppress P production in porcine or human culture systems using mural granulosa cells showed comparable effects in this human cumulus cell system. 17 beta-estradiol (10(-5) M), clomiphene citrate (10(-5) M), and o,p-DDT (10(-5)) significantly inhibited hCG-supported P production by human cumulus cells in vitro. This system has the advantages that (1) human cumulus granulosa cells are readily available from IVF/ET programs, (2) the techniques for maintaining the cells in culture are extremely simple, (3) a marker of highly differentiated granulosa cell function (P production) can be reliably measured, and (4) the cells respond predictably like other comparable granulosa cell systems. We conclude that human cumulus cells are a readily available and useful resource for in vitro screening of potential female reproductive toxicants.