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.

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.

Collaborative work on evaluation of ovarian toxicity. 5) Two- or four-week repeated-dose studies and fertility study of busulfan in female rats.

Busulfan, an antineoplastic agent that targets small follicles (primordial and primary follicles), was given orally to female Sprague-Dawley rats (0, 0.1, 0.5, or 1.5 mg/kg/day; n = 10 in each group) for 2 or 4 weeks to assess the optimal administration period for detection of the toxic effects on ovarian morphology. Isolated ovaries were used for histopathological analysis and follicle counts. In addition, a female fertility study was conducted by giving the same dose levels of busulfan from 2 weeks before mating to day 7 of pregnancy to determine the non-observed-adverse-effect-level (NOAEL) for female reproduction. In the 2-week study, all rats treated with busulfan showed normal estrous cyclicity and no toxicological changes in weight or histopathology of the ovaries. In the 4-week study, a decrease in small follicles was found histopathologically in 1 rat, even at 0.5 mg/kg, and in 4 rats at 1.5 mg/kg. Proliferating cell nuclear antigen immunohistochemistry of the follicles confirmed the above decrease in number of small follicles at 1.5 mg/kg. In the female fertility study, increases in dead embryos and post-implantation loss were found in rats at 1.5 mg/kg. Taken together, the NOAELs were 1.5 mg/kg for reproductive performance and 0.5 mg/kg for early embryonic development. In conclusion, the present study indicates that a 4-week administration period and appropriate assessment, including careful histopathological analysis of stage-based follicles are needed to detect small follicle depletion in a general toxicity study used as a first-titer screen.

Distribution of VEGF and flt-1 in the normal dog tissues.

Angiogenesis is essential for tumor progression and is regulated by several angiogenic factors such as vascular endothelial growth factor (VEGF). We investigated the expression and distribution of VEGF and its receptor flt-1 in twelve normal canine tissues in six beagle dogs using immunohistochemistry, RT-PCR and real-time RT-PCR. Immunochemical staining showed that both VEGF and flt-1 were expressed in many tissues and their mRNAs were detected in all organs examined by RT-PCR. Levels of VEGF164 and flt-1 mRNA expression were high in tissues containing many intensely immunopositive cells. The expression levels of VEGF164 and flt-1 mRNA tended to be similar. These results indicated that VEGF and flt-1 are closely associated in canine, as in human tissues, and quantifying their mRNAs might be helpful in evaluating angiogenesis.

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.

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.

Detection of gene expression of vascular endothelial growth factor and flk-1 in the renal glomeruli of the normal rat kidney using the laser microdissection system.

The recent development of the laser microdissection (LMD) technique enables one to target particular tissues or cells for gene or protein analyses. The purpose of this study was to detect local mRNA expression of vascular endothelial growth factor (VEGF) and its receptor, flk-1, in the glomeruli of normal rat kidneys using the LMD system. Frozen sections of the kidney of 8-week-old male Wistar rats were made. The glomeruli were dissected from the frozen sections with the LMD system, and total RNA was extracted from 200 glomeruli in each kidney. Reverse-transcription polymerase chain reaction (RT-PCR) revealed the local mRNA expression of three isoforms of VEGF, flk-1 and GAPDH in the glomeruli. Moreover, the real-time PCR was performed to evaluate the experimental condition for quantification of VEGF and flk-1 mRNA expression using this system, and the results showed that at least 10 glomeruli might be needed for quantifying local VEGF mRNA expression. However, cDNA from 200 glomeruli was not enough for quantitative evaluation of flk-1 mRNA with this system. These results demonstrate the reproducibility of the analysis of mRNA expression in the renal glomeruli using the LMD system and also suggest that the application of the LMD technique will provide information to further our understanding of the mechanisms involved in kidney diseases.