Noninvasive sample collection for the genotyping of neonatal rats using adhesive tape.

To develop a noninvasive sample collection method for genotyping, we compared PCR products from samples collected from neonates using five different brands of adhesive tape. Next, the youngest application age to distinguish genotypes was established. The tapes were applied on the backs of rats on postnatal day (PND) 10. DNA extracts from two brands provided clear PCR products that enabled genotype identification. The youngest age for distinguishing genotypes was PND 5; however, the youngest age that provided accurate results was PND 7. Thus, the present method allows for genotyping during the neonatal period without invasive burden and may improve animal welfare by refining.

Dose-dependent acceleration in the delayed effects of neonatal oral exposure to low-dose 17α-ethynylestradiol on reproductive functions in female Sprague-Dawley rats.

Xenoestrogen exposure during the critical period of sexual differentiation of the brain causes delayed effects on female reproduction. We investigated the internal dose of orally administered ethynylestradiol (EE) during the critical period and its delayed effects by administering 0 (vehicle control), 0.4, or 2 µg/kg EE to female Sprague-Dawley rats for 5 days from postnatal day (PND) 1. Determination of serum EE level 24 hr after the initial dosing and 6 and 24 hr after the final dosing of 2 µg/kg indicated that the administered EE entered the circulation and cleared after every administration. Although the treatment did not affect physical development, including growth, eyelid opening, and vaginal opening, the estrous cycle was arrested from postnatal week (PNW) 12 even with 0.4 µg/kg EE, with an inverse correlation between doses and arresting ages. Although ovarian morphology at PNW 22-23 indicated that the treatment caused long-term anovulation and cystic follicle formation, the number of primordial follicles at PNW 22-23 was similar among the groups. Because this number was lower than that at PND 10 in all groups, primordial follicles may have been consumed under long-term anovulation. The treatment also caused other abnormalities, including mammary gland hyperplasia, increase in pituitary and liver weights, and decrease in the uterine weight. Because the highest circulating EE level in the 2 µg/kg-treated neonates is considered to be comparable to the physiological range of estradiol-17ß, we concluded that a slight increase in the circulating estrogens during the neonatal period exerts irreversible delayed effects.

[Basic principles for setting acute reference dose, ARfD in Japan].

Basic principles for simulation of acute reference dose (ARfD) setting were defined based on the work of Solecki et al. (2005). The principles are: (1) Appearance of acute toxicity within 24 h after oral administration. (2) Rationale for setting toxicity that appears or could appear after single oral administration. (3) ARfD setting is assumed to be necessary for all pesticides. (4) ARfD setting is not necessary when the value is at or above the cutoff level. (5) The setting basically applies to the general population. (6) ARfD is set based on the lowest NOAEL among all the available study data concerning endpoints for acute effects. (7) Effects of exposure during critical periods should be considered as endpoints for ARfD setting. (8) The approach for the safety coefficient is the same as that for acceptable daily intake. (9) If available, human data are acceptable as an endpoint for ARfD setting.

Membranous Glomerulonephropathy in a Hatano Low-avoidance Rat.

Membranous glomerulonephropathy can be experimentally induced in rats, but spontaneous cases have been rarely reported. In this report, we present a typical case of spontaneous membranous glomerulonephropathy in a rat. A male Hatano low-avoidance (LAA) strain rat had a tumor mass on the right auricle, and was sacrificed at 41 weeks of age. Urinary screening by reagent strips revealed intense proteinuria. Histological tests revealed frequent presence of irregularly sized eosinophilic hyaline materials on the capillary wall and in the mesangium of renal glomeruli. Immunofluorescence revealed granular deposits of IgG, IgM, and C3 in the glomeruli. Subepithelial dense deposits were observed by electron microscopy accompanied by podocyte foot process effacement and occasional irregular thickening of the glomerular basement membrane. The rat also developed chronic lymphocytic pancreatitis, and the tumor mass on the right auricle was diagnosed as a fibrosarcoma. Screening tests for antibodies against major infectious agents and antinuclear antibody were negative. Western blot and indirect immunofluorescence analyses suggested the presence of an autoantibody against the pancreatic component. The glomerulopathy was considered an early stage of membranous glomerulonephropathy.

Simulation of acute reference dose (ARfD) settings for pesticides in Japan.

In order to develop guidelines for setting acute reference doses (ARfDs) for pesticides in Japan, we conducted simulations of ARfD settings based on evaluation reports for 201 pesticides assessed by the Food Safety Commission (FSC) in Japan over the last 8 years. Our conceptual principles were based on the concepts written by Solecki et al. (2005) and were adapted for toxicological data required in Japan. Through this process, we were able to set the ARfDs for over 90% of the 201 pesticides tested. The studies that provided the rationale for ARfD setting were primarily reproductive and developmental toxicity studies, acute neurotoxicity studies, and pharmacology studies. For approximately 30% of the pesticides simulated in the present study, it was not necessary to establish ARfDs. Some of the simulated ARfDs resulting from their endpoints may be conservative estimates, because the evaluation reports were written for acceptable daily intake settings. Thus, it was sometimes difficult to distinguish acute toxic alerts from repeated toxicities. We were unable to set an ARfD for 14 pesticides because of insufficient data on acute toxicities. This could be improved by more complete recordkeeping. Furthermore, we categorized the 201 pesticides by mechanism of action or chemical structure. Our simulation indicates that the conceptual framework presented here can be used as a basis for the development of guidelines on ARfD settings for pesticides in Japan.

Vascular hamartoma in the uterus of a female Sprague-Dawley rat with an episode of vaginal bleeding.

An annular, reddened lesion with mild serosal hemorrhage and no tumorous mass formation was detected in the right uterine horn of a 37-week-old female Sprague-Dawley rat that had postpubertal vaginal bleeding. Histological examination revealed prominent proliferation of the endometrium, which occupied the uterine lumen. There were numerous aberrant vascular spaces filled with erythrocytes, proliferation of stromal cells, and inflammatory infiltrates including hemosiderin-laden macrophages in the endometrium. These vasculatures extended into the myometrium, and in a transverse section of the lesion, they were mostly distributed throughout the circumference of the uterus. They were irregular in shape and interconnected, forming a large vascular sinus and anastomosing reticular channels. In the area with serosal hemorrhage, the muscular layer covering the large irregular vascular space had undergone degeneration and necrosis. The lining cells of the vasculatures were often plump, and they protruded into the lumen and were arranged in a tombstone or hobnail manner. Immunostaining revealed that these cells were positive for von Willebrand factor and CD34. The aberrant vasculatures were not accompanied by pericytes or muscular layer, although a discontinuous muscular wall was present around some of them. From these results, the uterine lesion was diagnosed as a vascular hamartoma.

Cutaneous hybrid cyst in a sprague-dawley rat.

This report describes a spontaneous hybrid cyst in a Sprague-Dawley (SD) rat. A 52-week-old, male SD rat had a cutaneous cyst on the left mystacial pad. Histologically, the cyst wall showed infundibular differentiation with keratohyalin granules in the granular layer and matrical differentiation comprising basaloid epithelial cells with trichohyalin granules. The cyst cavity was filled with lamellar, flaky keratin and aggregates of shadow cells. Immunohistochemically, the infundibular-type epithelium was positive for cytokeratin (CK) AE1/AE3, CK KL1 and CK14 but negative for CK15, whereas the matrical-type epithelium was negative for all four CK isoforms examined. These immunohistochemical properties of the infundibular- and matrical-type epithelia were similar to those of the infundibulum and inferior segment of normal hair follicles, respectively. Based on these findings, the cyst was diagnosed as a hybrid cyst, comprising more than one type of cyst arising from various parts of the pilosebaceous unit.

Endocrinological differences between Hatano high- and low-avoidance rats during early two-way avoidance acquisition.

Hatano high (HAA)- and low (LAA)-avoidance rats were selected from Sprague-Dawley rats genetically on the basis of their active avoidance behavior in a shuttle-box test. The purpose of this study was to investigate stress-related alterations of hormones corticotropin-releasing hormone (CRH), arginine-vasopressin (AVP), prolactin, and adrenocorticotropin (ACTH) in the brain and blood during early avoidance acquisition using two lines of Hatano rats. In paraventricular nucleus (PVN) of the hypothalamus, the CRH levels in HAA rats were significantly increased after shuttle-box tasks compared with before the tasks, whereas the CRH levels in LAA rats significantly decreased after shuttle-box tasks compared with before the tasks. In the HAA rats, the CRH and AVP levels in the median eminence decreased after shuttle-box tasks, whereas there were no significant differences in the levels between before and after shuttle-box tasks in LAA rats. The plasma concentrations of ACTH were significantly higher in HAA rats than in LAA rats after shuttle-box tasks. These results show that the response of CRH-ACTH was higher in HAA rats than in LAA rats. This phenotype may be an important reason for the high avoidance rates of shuttle-box tasks in HAA rats. These endocrine differences in early avoidance acquisition may be involved in regulation of their avoidance responses in the shuttle-box task.

Endocrine mechanisms responsible for different follicular development during the estrous cycle in Hatano high- and low-avoidance rats.

Hatano high- and low-avoidance rats (HAA and LAA strains, respectively) were selected and bred according to the avoidance rate in a shuttle-box task. Although they have clear strain differences in ovarian function, their endocrine mechanisms still remain to be clarified. Differences in female reproductive endocrinology between the strains were investigated by means of measuring the plasma concentration of reproductive hormones during the estrous cycle. LAA rats showed approximately threefold lower basal and surge levels of LH, a more than fourfold lower level of FSH surges and higher levels of inhibin A and inhibin B during the estrous cycle compared with the levels seen in HAA rats. The concentration of estradiol-17ß in the proestrous stage was significantly lower in LAA rats than in HAA rats. Additionally, LH and FSH secretions from primary cultured anterior pituitary cells with or without in vitro GnRH stimulation were lower in the cells derived from LAA rats and, in terms of FSH secretion, were unresponsive to GnRH in contrast to cells derived from HAA rats. Although an increased number of preantral follicles in diestrus were observed in LAA rats, number of hCG-induced ovulation was lower in LAA rats. LAA rats may have much more follicle growth during the early stage of folliculogenesis, but most follicles might not grow into mature follicles. These results strongly suggest that the strain difference in ovarian function of these two Hatano rats is due to the difference in the regulation of hypothalamo-hypophyseal system for gonadotropins secretion.

Comparison of aryl hydrocarbon receptor gene expression in laser dissected granulosa cell layers of immature rat ovaries.

In order to understand ovarian toxicity of aryl hydrocarbon receptor (AhR) agonists, in situ gene expression of the AhR was examined during follicle development in immature rats. In situ hybridization on frozen sections of ovaries from 24-day-old Sprague-Dawley rats showed that the AhR mRNA was localized in the granulosa cells and occasionally in the theca cells of the follicles irrespective of the developmental stage. In situ gene quantification on granulosa cell layers collected by laser microdissection further revealed that the granulosa cells expressed less AhR mRNA according to development of belonging follicles, but more ß-subunit of inhibin A mRNA, a quality control gene. These results may help to elucidate vulnerable developmental stages of follicles to toxicities of the AhR agonists.

Effects of transmaternal exposure to genistein in Hatano high- and low-avoidance rats.

Hatano high- and low-avoidance (HAA and LAA) rats are separated by breeding from Sprague-Dawley rats by high versus low rates of avoidance responses in a shuttle-box task. In addition, compared to HAA rats, LAA rats show lower running-wheel activity, later sexual maturation, 5-day estrous cycling, lower sperm motility, more pronounced immunological reactions, and are generally less reactive to stress. The present study was designed to compare the effects of transmaternal exposure to genistein on these characteristics between HAA and LAA rats. To this aim, litters from both strains were fostered onto Sprague-Dawley rats receiving genistein by gavage with 5 mg/animal/day from day 17 of pregnancy through day 21 of lactation. Inhibited growth after weaning and reduced uterine weight at weaning were observed in the LAA offspring reared by genistein-treated dams. IgM antibody production in response to sheep red blood cells was significantly decreased in the HAA offspring reared by genistein-treated dams. During restraint stress, the plasma concentration of corticosterone was significantly lower in the LAA offspring reared by genistein-treated dams. Strain-related differences were detected in shuttle-box avoidance performance, running-wheel activity, estrous cycling, and sperm motility. The results demonstrate that transmaternal exposure to genistein potentially affects the immunological and stress responses as well as the post-weaning growth of the offspring. It suggests that a comparative study using Hatano rats would be useful for studying the influence of endocrine active chemicals on the whole body systems.

Differences in adrenocortical secretory and gene expression responses to stimulation in vitro by ACTH or prolactin between high- and low-avoidance Hatano rats.

Rats of the Hatano high-avoidance (HAA) and low-avoidance (LAA) strains have been genetically selected on the basis of their two-way active avoidance behavior, and have different endocrine responses to stress. The present study focused on the adrenal steroid hormone responses of the Hatano strains and identifies differences in regulation of the adrenal cortex in vitro of HAA and LAA rats. Although incubation with prolactin (PRL) and/or adrenocorticotrophic hormone (ACTH) resulted in a dose-dependent increase of corticosterone and progesterone release by adrenal cells from both HAA and LAA male rats, the responses were markedly increased for adrenal cells from LAA rats as compared with HAA rats. This finding suggested that adrenal glands of HAA rats are less sensitive to PRL and/or ACTH than adrenals from LAA rats. Several possible intra-adrenal regulators were investigated. The basal level of expression of steroidogenic acute regulatory protein (StAR) and the long form of the PRL receptor (PRLR-L) mRNAs was higher in adrenals of LAA rats. ACTH treatment of adrenal cells from HAA rats resulted in statistically significant increases in melanocortin receptor 2 (MC2R) mRNA expression, while neither ACTH nor PRL altered MC2R mRNA expression in adrenal cells of LAA rats. Conversely, the increase in PRLR-L mRNA expression induced by PRL was observed only in adrenal cells from LAA rats. Treatment of adrenal cells with PRL and/or ACTH increased the expression of StAR and CYP11A1 mRNAs for both Hatano strains. However, the induction of StAR mRNA expression was higher in LAA rats, but the CYP11A1 response was lower. These findings indicate that adrenal cells of the LAA strain have higher sensitivity to secretagogues than those of the HAA strain. These results suggest that PRL may also be important in stimulating secretion of adrenal steroid hormones.

Screening of toxicological properties of 4-methylbenzoic acid by oral administration to rats.

Oral toxicity of 4-methylbenzoic acid in male and female Sprague-Dawley rats was profiled through a twenty-eight-day repeated dose toxicity study (the 28-day study) and a screening test for reproductive/developmental toxicities (the reproduction/developmental study) conducted under Organisation for Economic Co-operation and Development (OECD) test guidelines. Daily administration of 4-methylbenzoic acid, at a dose level of 0, 100, 300 or 1,000 mg/kg, did not show any adverse effect on reproductive organs of animals in the 28-day study. In the reproductive/developmental study, however, 1,000 mg/kg/day of the compound reduced epididymal weights and increased incidence of cauda epididymal oligo/azoospermia. While the compound did not affect estrous cycle or mating performances, 1,000 mg/kg of the compound reduced fertility. Furthermore, 300 mg/kg or more of the compound increased pre-implantation loss, which resulted in a decrease in the number of offspring, and reduced body weight gain of the dams during the latter period of gestation. From these results, the no-observed-effect-level (NOEL) for reproductive/developmental toxicities is considered to be 100 mg/kg, whereas 1,000 mg/kg did not show any effect on neonates. In the 28-day study, NOEL is considered to be 300 mg/kg for male and female rats, since 1,000 mg/kg of the compound caused, in both sexes, a few minor changes, such as temporal salivation, a slight increase in food consumption and a moderate increase in blood aspartate aminotransferase (AST) activity. Thus, 4-methylbenzoic acid has the potential for reproductive toxicity, with diverse adverse effects on the epididymis, after repeated administration, observed in the two studies.

Effects of vertically transferred 3,3',4,4',5-pentachlorobiphenyl on gene expression in the ovaries of immature Sprague-Dawley rats.

We have previously shown that 3,3',4,4',5-pentachlorobiphenyl (PCB-126) vertically transferred from dams potentially exerts a direct effect on the ovaries of offspring and adversely affects female puberty. To investigate its toxicological targets in ovarian tissues, mRNAs encoding representative peptides that regulate follicular development in granulosa cells, theca cells, and oocytes were quantified using ovaries collected on postnatal days (PND) 5, 15, and 24 from the offspring of dams administered oral doses of 0, 1 or 3 microg/kg PCB-126 starting 2 weeks prior to mating and continuing until 20 days after delivery. Quantification using the real-time RT-PCR method revealed that PCB-126 lowered the amounts of mRNAs that encoded the inhibin alpha- and inhibin/activin beta A-subunits from PND 15 onwards; the amounts of mRNAs for inhibin/activin beta B-subunit, follicle-stimulating hormone (FSH) receptor, and aromatase on PND 15; and the amounts of luteinizing hormone receptor mRNA on PND 24 compared with those of the age-matched controls. In contrast, no differences were noted for mRNAs encoding c-kit, growth differentiation factor-9, bone morphogenetic protein-15, or kit ligand for any of the age groups examined. The serum FSH level on PND 24 was higher than that in the control. Since the earliest effects on the mRNAs in the rat ovaries were observed in those expressed in the granulosa cells of the growing follicles after the antral follicles had developed, molecules in granulosa cells but not in oocytes during the early stages of the antral follicles might be the primary targets of vertically transferred PCB-126.

Internal dose-effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in gonadotropin-primed weanling rat model.

Single sc injection of 5 IU equine chorionic gonadotropin (eCG) induces ovulation in weanling female rats 3 days later. It has been shown that treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 24 h before eCG injection reduces eCG-stimulated ovarian hypertrophy and inhibits ovulation. The present study intended to compare internal dose-effects of TCDD between these endpoints and representative endpoints for TCDD toxicity, such as weights of the liver and thymus, in weanling female rats given orally 0, 1, 4 or 16 microg/kg TCDD 24 h before eCG injection on postnatal day 25. Measurement of plasma TCDD concentrations by ELISA at 6, 72 and 96 h after TCDD revealed that significant levels of TCDD were maintained in systemic circulation until 96 h (on the day of induced ovulation) with the highest level at 6 h after TCDD treatment. Ovarian TCDD concentrations varied similarly and tended to be higher than those in the thymus at all time points, whereas hepatic concentrations of TCDD were the highest among the tissues. Although > or = 4 microg/kg TCDD affected the weights of the thymus and liver, no differences were observed in ovarian weights at any time point or in ovulation between corn oil-treated and TCDD-treated groups. Furthermore, ovarian levels of representative mRNAs in follicles were not affected by TCDD treatment. Since TCDD increased the amount of cytochrome P450 1A1 mRNA in the ovary, the administered TCDD stimulated the aryl hydrocarbon receptor-signaling pathway. From these results, we concluded that thymus weights of weanling female rats responded to TCDD at a lower internal dose as compared with that ovarian hypertrophy and follicular growth from early antral stage to ovulation would respond to.

Effects of vertically transferred 3,3',4,4',5-pentachlorobiphenyl (PCB-126) on the reproductive development of female rats.

In order to study the effects of vertically transferred coplanar polychlorinated biphenyls on female reproductive development, female rat offspring from dams of Sprague-Dawley strain, which received daily oral administration of vehicle (corn oil) or 1 or 3 microg/kg of 3,3',4,4',5-pentachlorobiphenyl (PCB-126) from 2 weeks prior to mating with intact males until 20 days after delivery were examined from birth until puberty. Hepatic expression of the aryl hydrocarbon receptor (AhR)-inducible enzyme cytochrome P450 1A1 (CYP1A1) was detected in all offspring from PCB-126-exposed dams, indicating vertical transfer of PCB-126. Furthermore, quantification of ovarian mRNAs encoding CYP1A1, AhR and ARNT demonstrated that the ovary equipped the AhR-signaling system through which transcription of the CYP1A1 gene was enhanced in a dose-dependent manner. Exposure to PCB-126 retarded the growth of offspring in both exposed groups, while the viability of the neonates of the exposed groups was comparable to that of the oil-exposed controls. The exposure to 3 mug/kg/day reduced the ovarian weight on postnatal day (PND) 24, with atresia of most of the antral follicles and delayed vaginal opening. Exposure to 1 microg/kg/day did not produce such effects; however, both doses of PCB-126 induced external urogenital anomalies, such as vaginal thread and hypospadias, in all of the PCB-126-exposed female offspring. These results indicate that vertically transferred PCB-126 is potent enough to exert a direct effect on the ovary and adversely affect female puberty by altering the morphological and functional development of the female reproductive system.

Immunological characteristics of Hatano high-and low-avoidance rats.

Hatano high- and low-avoidance (HAA and LAA) rats have been genetically selected on the basis of their two-way active avoidance behavior, and have been shown to differ in other behavioral and hormonal parameters. Since close interconnections among the nervous, endocrine and immune systems have been well documented, these two strains might possess differences in aspects of immunological action. In Experiment 1, plasma levels of IgG, IgM, complement 3 (C3), classical pathway hemolytic complement (CH50) and beta(2)-microglobulin were compared between males of the two strains at 5 and 24 weeks of age. Plasma levels of IgG and CH50 were lower in LAA than HAA rats at 5 weeks of age, whereas those differences disappeared at 24 weeks of age. There were no differences between the two strains in plasma levels of IgM, C3 and beta(2)-microglobulin. In Experiment 2, antibody production to sheep red blood cells (SRBC) and mitogen-induced lymphocyte proliferation were compared between 12-week-old males of the two strains. Antibody responses in the PFC assay, plasma anti-SRBC-IgM levels and spleen weights were higher in LAA than HAA rats. LPS-induced lymphocyte proliferation was greater in LAA than HAA rats. It was concluded that HAA rats show earlier development of immunological development, but that antibody production and mitotic response of B lymphocytes may be more pronounced in adult LAA than HAA rats. The strain differences observed in the immunological response may indicate the usefulness of using Hatano rats in studies of behavioral-immunological relationships.

New approach to in situ quantification of ovarian gene expression in rat using a laser microdissection technique: relationship between follicle types and regulation of inhibin-alpha and cytochrome P450aromatase genes in the rat ovary.

The recently developed laser microdissection (LMD) technique makes it possible to quantify local gene expression in the target cells of various tissues. Using the LMD technique, this study aimed at comparing the amounts of mRNAs encoding the inhibin-alpha subunit and cytochrome P450 aromatase (P450(arom)) in granulosa cells between preantral and antral follicles in immature rat ovaries. Serial frozen sections of the ovaries from 24-day-old female Wistar rats were made and 30 healthy preantral (100-200 microm maximum diameter) and ten healthy antral ( > 300 microm maximum diameter) follicles were selected in each ovary based on morphological examinations, including immunohistochemistry for inhibin-alpha, in sections adjacent to those used for LMD. The amounts of mRNAs encoding inhibin-alpha subunit and P450(arom) were quantified by real-time polymerase chain reaction (PCR). While the amount of P450(arom) mRNA in the granulosa cell layers from the antral follicles was about 12-times higher than that in the preantral follicles, no difference in the amount of inhibin-alpha mRNA was found between these two types of follicles. Thus, the LMD technique allowed the in situ quantification of gene expression in the ovary and revealed that each granulosa cell expresses a stable amount of inhibin-alpha subunit mRNA independently of antral formation in immature rat ovaries.

Gastric ulceration and expression of prolactin receptor in the brain in Hatano high- and low-avoidance rats.

Recently, prolactin was shown to inhibit the development of stress-induced ulcers. However, the mechanism for suppression of gastric ulcers by prolactin has not been clarified. Hatano high-avoidance (HAA) and low-avoidance (LAA) strains of rats were originally selected and bred from Sprague-Dawley rats based on shuttle-box tasks. The present study focused on the relationships among gastric ulceration and endocrine response with special reference to prolactin secretion and restraint stress in water of HAA and LAA rats. The restraint stress induced an elevation of plasma concentrations of ACTH, corticosterone, and prolactin. Peak levels of plasma ACTH during stressful condition were significantly higher in HAA rats than in LAA rats, while peak levels of prolactin were significantly lower in HAA rats than in LAA rats. The gastric erosion index was significantly higher in HAA rats than in LAA rats 7 h after restraint stress in water. The numbers of prolactin- receptor-positive cells determined by immunohistochemistry in the paraventricular nucleus was significantly increased in LAA rats than in HAA rats 7 h after restraint stress in water. These results indicate that HAA rats were more sensitive than LAA rats to restraint stress in water. The strain differences in gastric ulceration under stress may be involved in peripheral prolactin secretion and central prolactin receptor expression. The expression of prolactin receptor in the paraventricular nucleus may be important in suppressing gastric ulceration.

Reduction of primordial follicles caused by maternal treatment with busulfan promotes endometrial adenocarcinoma development in donryu rats.

Ovarian dysfunction leading to hormonal imbalance plays a crucial role in uterine carcinogenesis in rats as well as women. However, the effects of a reduction in primordial follicles at birth on uterine adenocarcinoma development have hitherto not been determined. The present study was therefore conducted using female Donryu rats, a high incidence rat strain of uterine adenocarcinoma. The animals were maternally exposed to 2.5 or 5.0 mg/kg of busulfan on gestation day 14 to reduce primordial follicles, and were then initiated by intrauterine treatment with N-ethyl-N'-nitro-N-nitrosoguanidine at 11 weeks of age. Both busulfan treatment doses caused earlier occurrence of persistent estrus, with dose-dependence as compared to controls. At 15 months of age, the rats were euthanized. The incidence of uterine adenocarcinomas and multiplicity of uterine neoplastic lesions were significantly increased by the 5.0 mg/kg, but not the 2.5 mg/kg busulfan treatment. Morphologically, the ovaries exposed to busulfan treatment exhibited severe atrophy, with few or no follicles and corpus lutea. Serum 17beta-estradiol (E2), progesterone, and inhibin levels were significantly decreased in the busulfan treatment groups, with a clear dose-relation. Interestingly, only the 5.0 mg/kg busulfan treatment elevated the E2/progesterone ratio. These results provide evidence that the reduction of primordial follicles promotes uterine adenocarcinoma development in rats in association with an earlier occurrence of the persistent estrus status.