In vitro and in vivo effect of flutamide on steroid hormone secretion in canine and human inflammatory breast cancer cell lines.

The aim was to study the effects of flutamide on cell proliferation, in vivo tumour growth and steroid production in canine and human IBC cell lines. IPC-366 and SUM149 cell cultures were exposed to flutamide concentrations for 72 hours. Additionally, IPC-366 and SUM149 xenotransplanted mice were treated subcutaneously with flutamide 3 times a week for 2 weeks. Steroid hormones determination in culture media, serum and tumour homogenates (pregnenolone, progesterone, androstenedione, testosterone, dihydrotestosterone, 17ß-oestradiol and oestrone sulphate) were assayed by EIA. in vitro cell proliferation percentages showed a decrease in all flutamide dosages in IPC-366 and SUM149. in vivo flutamide reduced tumour size by 55% to 65%, and metastasis rates decreased. In treated groups, androgen levels in culture media, serum and tumour homogenates were increased as oestrogen levels decreased. These results suggest that flutamide treatment inhibits cell proliferation and promotes tumour reduction by increasing androgen levels and also support future therapy approaches.

Morfologia e dinâmica testicular em cutias (Dasyprocta prymnolopha) adultas / Testicular morphology and dynamic in adult agoutis (Dasyprocta prymnolopha)

Este trabalho objetivou apresentar a caracterização da morfologia do testículo de cutia (Dasyproctaprymnolopha) macho, com o intuito de colaborar com o conhecimento da morfofisiologia reprodutiva da espécie. Foram utilizados testículos de 47 animais, com idade entre um e dois anos, pesos homogêneos (2,08 ± 0,23kg), oriundos do Núcleo de Estudos e Preservação de Animais Silvestres do Centro de Ciências Agrárias da Universidade Federal do Piauí. As estruturas foram dissecadas, descritas, e fragmentos foram processados para a microscopia de luz, sendo, posteriormente avaliada a atividade gonadal. Observou-se que os testículos são órgãos elipsoides alongados, podendo ser encontrados na região inguinal ou na cavidade abdominal, não apresentando um escroto bem delimitado. Verificou-se também parênquima com característica histológica padrão para o órgão em mamíferos, com a identificação de oito tipos de associações celulares, caracterizando os estádios do ciclo do epitélio seminífero, com menor e maior frequência dos estádios 3 e 5, respectivamente.(AU)

This study meant to characterize the morphology of the testicle from (Dasyprocta prymnolopha) agouti males, in order to collaborate with the knowledge of reproductive morphophysiology of the specie. Testicles were used from 47 animals aged between 1 and 2 years, homogeneous weight (2.08±0.23kg), coming from the Centre for the Study and Conservation of Wild Animals of Agricultural Sciences Center of the Federal University of Piauí. The structures were dissected, described and fragments were processed for light microscopy, and, subsequently, gonadal activity was evaluated. Testes were observed to be elongated ellipsoidal bodies that can be found in the groin or in the abdominal cavity, not having a clearly defined scrotum. We also could see parenchymal with standard histological characteristic for the mammalian body, with the identification of eight types of cell associations, characterized epithelium Seminiferous stages of the cycle, with lower and higher frequency of stages 3 and 5, respectively.(AU)

Canine cell line, IPC-366, as a good model for the study of inflammatory breast cancer.

Inflammatory breast cancer (IBC) is an aggressive type of cancer with poor survival in women. Inflammatory mammary cancer (IMC) in dogs is very similar to human IBC and it has been proposed as a good surrogate model for study the human disease. The aim was to determine if IPC-366 shared characteristics with the IBC cell line SUM149. The comparison was conducted in terms of ability to grow (adherent and nonadherent conditions), stem cell markers expression using flow cytometry, protein production using western blot and tumorigenic capacity. Our results revealed that both are capable of forming long-term mammospheres with a grape-like morphology. Adherent and nonadherent cultures exhibited fast growth in vivo. Stem cell markers expressions showed that IPC-366 and SUM149 in adherent and nonadherent conditions has mesenchymal-like characteristics, E-cadherin and N-cadherin, was higher in adherent than in nonadherent cultures. Therefore, this study determines that both cell lines are similar and IPC-366 is a good model for the human and canine disease.

Isoflavones and their effects on the onset of puberty in male Wistar rats.

This study was performed to determine how two of the most important isoflavones, genistein and daidzein, affect the gonadal axis in male prepuberal rats. One hundred and seventy-five prepuberal male Wistar rats were allocated into seven groups: one control group and six experimental groups that were orally administered a high or low dose of genistein, daidzein or a mixture of both. Testosterone determination was assayed by EIA. The testes and body weights were measured, and the histology of the epididymis with the sperm content and epididymal sperm count were evaluated. In the control group, we observed an increase in the serum testosterone levels (>2.5 ng ml(-1) ) at the third week (52 days), which corresponded to the onset of puberty in these rats. The same increase in serum testosterone levels was observed at the fourth week in rats that received low doses of isoflavones; therefore, we concluded that the onset of puberty was delayed. At high doses, there was no significant increase in testosterone levels, which could be related to the fact that these male rats did not reach puberty. These findings were supported by the results obtained from the analysis of the epididymal content as well as the testes/body weight ratio.

The effects of isoflavones on androgens and glucocorticoids during puberty on male Wistar rats.

Isoflavones are the most common phytoestrogens found in human diets. However, it is still not clear whether isoflavones have effects on the reproductive and the endocrine systems under normal dietary intake and overdose. The aim of this study was to determine how the most important isoflavones, genistein and daidzein, affect androgen and glucorticoid levels on male prepuberal rats. A hundred and seventy-five 30-day-old male Wistar rats were dosed orally by stomach tube every day for 35 days, with saline solution, low and high doses of genistein, daidzein and a mixture of both. Serum samples were analysed by an enzyme immunoassay for hormone determinations. In control group, there was a peak of testosterone (T) and dihydrotestosterone levels associated to the onset of puberty, at the third week. However, in low-dose groups, the same peak was found at the fourth week (p < 0.05), indicating a delay in the onset of puberty in these groups. Moreover, high doses groups serum androgen levels were significantly lower (p < 0.05) than the control group from the first week until fifth week. This fact was supported by a epididymal histological analysis that indicate in low doses there were several content of spermatozoa at fourth week and in high doses there were few content of spermatozoa. Besides, corticosterone levels followed the same pattern of androgens in all groups. We can conclude that oral administration of isoflavones in male rats decreased the secretion of androgens and glucocorticoids causing a delay in the onset of puberty and may cause physiological and developmental problems.

Domestic carnivore's development: detection of Oct-4, a pluripotency marker, in pharyngeal arches.

Very few carnivore's embryology is reported mainly restricted to old literature without new technique analyses. Also, their development focuses on pharyngeal arches and stem cell sources and the high capacity for differentiation from those cells to generate embryonic tissue. We aimed to use immunohistochemistry to prove the potentiality of these stem cell niches. The results were to highlight the timetable for the development of dogs and cats, the proper formation of pharyngeal arches and the description of these cells on first and second arches since 17-25 days of pregnancy. After that, the differentiation process is reduced.

Collection and evaluation of epididymal sperm in captive agoutis (Dasyprocta aguti).

The objective was to establish a protocol for the collection and evaluation of epididymal sperm in agoutis. Eight males (1-2 y old) underwent left orchidectomy and epididymal sperma were collected by retrograde flush. Average values were flush volume 32 µL, pH 6.9, sperm concentration 748 x 10(6) sperm/mL, with motility 86.5% and vigor 4.6. Viable sperm were present in all flush samples; 66% of sperm were alive, and 41.9% of sperm responded positively to the hypoosmotic test (using distilled water). There were 21.1% morphologically abnormal sperm, of which 2.0 and 19.1% were primary and secondary defects, respectively. The acrosome was intact in 99.5% of sperm. The sperm head was 4.89 ± 0.41 µm long and 3.13 ± 0.35 µm wide, with an area of 13.01 ± 2.01 µm(2). Midpieces were 5.33 ± 0.44 µm long and 0.98 ± 0.13 wide, sperm tails were 29.91 ± 2.29 µm, and overall sperm length was 40.12 ± 2.44 µm. In conclusion, epididymal sperm collection from agoutis was satisfactory; the collected sperm has the potential to be stored, facilitating development of other reproductive biotechnologies for this species.

Quantification of sexual steroid hormones in faeces of Iberian wolf (Canis lupus signatus): a non-invasive sex typing method.

The determination of gender in wild animals is essential for behavioural and ecological studies, and also for conservation. The objectives of this study were (i) the determination of gender in faecal samples of Iberian wolf based on the differential concentrations of sexual steroid hormones (SSH) and (ii) to analyse the profiles of SSH in males and females (considering the gender determination carried out previously) during the non-reproductive and reproductive periods. The quantification of androgens (testosterone, T), progestin (progesterone, P) and oestrogen (oestradiol, E) was conducted by means of enzyme immunoassay. The k-means conglomerate analysis showed that the 59 faecal samples grouped into three different conglomerates, considering SSH levels. Groups 1 and 2 showed higher levels of T than group 3. Therefore, the faecal samples included in groups 1 and 2 (17 samples) corresponded to males and those of group 3 (42 samples) to females. The levels of T + P + E and T/P were higher in the group of males than in the group of females. The results of this study also showed that levels of T in males were higher during the reproductive period than in the non-reproductive period. However, the concentrations of P and E turned out to be higher during the non-reproductive season. In females, the levels of the three hormones (T, P and E) were higher during the reproductive period.

The effect of long-term exposure to combinations of growth promoters in Long Evans rats: part 2. Adrenal morphology (histopathology and immunochemical studies).

The aim of the study was to investigate the effects of long-term exposure (45 days) to growth promoters: clenbuterol (CB: 1 mg kg(-1) bw) and/or dexamethasone (DEX: 0.1 mg kg(-1) bw), in adrenal gland morphology, and the possibility of recovery after the withdrawal of drug treatment. Animals were sacrificed at different days of withdrawal (W0, W5, W10, W15 and W20), and adrenal glands processed for histopathology and immunohistochemistry. Adrenals of CB treatment showed typical features of long-term administration of beta-agonists at W0 such as capillary dilatation in the fasciculata-reticularis zone, and this feature was also presented at W20. Adrenals of CB+DEX treatments showed the same results of CB treatment at days W0 and W20. However, DEX treatment presented the typical results of the exposure to corticoids with the atrophy of adrenal cortex. Immunohistochemistry of adrenal cortex steroidogenic enzymes (P450: scc, 3beta-HSD, aromatase) denoted that neither positive staining nor localization was affected by treatments. Aromatase enzyme was immunolocalized in adrenal medulla cells in controls as well as in treated groups. The immunolocalization of glucocorticoid receptors showed an increase in CB (+++) and CB+DEX (++) treatments compared to the control group (0) and DEX treatment (0). Histopathological and immunohistochemical results are closely related to those found for adrenal endocrine function. We can conclude that chronic administration of growth promoters influence adrenal morphology and glucocorticoid receptor expression.

Amplified androstenedione enzymeimmunoassay for the diagnosis of cryptorchidism in the male horse: comparison with testosterone and estrone sulphate methods.

An amplified enzymeimmunoassay (EIA) was validated for androstenedione in the serum of male horses. We will use the assay as a tool for the diagnosis of equine cryptorchidism. We will compare androstenedione EIA to the currently used methods (testosterone and estrone sulphate determinations). The study was conducted on 115 horses of pure Spanish and Arabian breeds, that included 30 geldings, 60 bilateral cryptorchids and 25 stallions. Androstenedione standard curve covered a range between 0 and 1 ng per well. Low detection limit was 1.54 pg/ml. Intra- and inter-assay coefficients of variation (CV%) were <8.2 and <9.3, respectively (n=10). Recovery rate of known androstenedione concentrations averaged from 96.62+/-2.69 to 97.63+/-1.87%. Androstenedione mean+/-S.E. serum concentrations were 10.52+/-1.36 ng/ml in stallions (n=25), 0.51+/-0.04 ng/ml in cryptorchids (n=60), and 0.03+/-0.01 ng/ml in geldings (n=30). Diagnostic validation parameters in basal samples showed for estrone sulphate the lower positive predictive value (0.85) with the higher number of false positives, and lower specificity (0.84). Testosterone showed the higher number of false negatives with a negative predictive value of 0.85, and lower sensitivity (0.85). Among the three hormones evaluated, androstenedione presented the best results with the smaller number of horses diagnosed as false positives (0.93) or negatives (0.91). This technique also resulted in higher sensitivity, specificity and efficiency over the other two methods assayed. We concluded that our amplified EIA is a highly sensitive and specific assay that provides a rapid, simple, and inexpensive alternative to other methods.

Effects of the anaesthetic/tranquillizer treatments on selected plasma biochemical parameters in NZW rabbits.

In order to assess the response of plasma biochemical parameters to anaesthesia, 40 New Zealand White (NZW) rabbits were assigned to four treatment groups (n = 10): control (1 ml i.v. saline solution), fentanyl-droperidol (FD) (0.4 ml/kg s.c. of 'thalamonal' solution; 2.5 mg/ml droperidol, 0.05 mg/ml fentanyl), ketamine (K) (10 mg/kg i.v.) with either xylazine (X) (3 mg/kg i.v.) or diazepam (D) (2 mg/kg i.v.). Blood samples were obtained from the central ear artery at six time points: before injection, and at 10, 30, 60, 120 min and 24 h after injection of the anaesthetics/saline. Plasma ALT, AST, ALP, GGT, BUN, creatinine, phosphate and potassium levels were measured by the Hitachi 747 autoanalyser. The administration of K-X increased (P < 0.05) plasma ALT (from 11.4 +/- 0.9 to 20.2 +/- 1.7 IU/l, at 10 min), AST (from 10.5 +/- 3.3 to 34 +/- 2.1 IU/l, at 120 min), BUN (from 17.2 +/- 0.9 to 25.8 +/- 1.8 mg/dl, at 60 min) and creatinine concentrations (from 1 +/- 0.1 to 1.6 +/- 0.2 mg/dl, at 10 min). After K-D administration, we observed an increase (P < 0.05) in plasma ALT (from 11.4 +/- 0.9 to 20.2 +/- 1.1 IU/l, at 10 min), AST (from 11.4 +/- 1.6 to 28 +/- 3.7 IU/l, at 10 min), BUN (from 15.8 +/- 0.8 to 30 +/- 1.5 mg/dl, at 10 min) and creatinine levels (from 1 +/- 0.08 to 2.2 +/- 0.2 mg/dl, at 120 min). No significant changes were seen in the FD group. We conclude that K-X and K-D may affect plasma concentration of select serum enzymes and biochemical parameters. These results should be taken into account when blood samples are evaluated in treated rabbits.

Influence of epidermal growth factor on mammalian oocyte maturation via tyrosine-kinase pathway.

Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture with EGF and two specific tyrosine-kinase inhibitors (TKIs, tyrphostins A-23 y A-47). Maturation was determined as the percentage of oocytes at metaphase II stage after culture. Treatments with EGF significantly increased incidences of metaphase II stage compared to controls (86.2% vs. 55% and 70.4% vs. 22.5% in ovine and equine oocytes, respectively). Tyrphostins A-23 and A-47 were effective in suppressing EGF-effect on oocytes. EGF-receptor was localized in follicles, being more prominent in cumulus and granulosa cells. These results confirm that EGF has a physiological role in the regulation of oocyte maturation via tyrosine-kinase pathway.

Influence of epidermal growth factor on mammalian oocyte maturation via tyrosine-kinase pathway.

Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture with EGF and two specific tyrosine-kinase inhibitors (TKIs, tyrphostins A-23 y A-47). Maturation was determined as the percentage of oocytes at metaphase II stage after culture. Treatments with EGF significantly increased incidences of metaphase II stage compared to controls (86.2% vs. 55% and 70.4% vs. 22.5% in ovine and equine oocytes, respectively). Tyrphostins A-23 and A-47 were effective in suppressing EGF-effect on oocytes. EGF-receptor was localized in follicles, being more prominent in cumulus and granulosa cells. These results confirm that EGF has a physiological role in the regulation of oocyte maturation via tyrosine-kinase pathway.

Measurement of serum and peritoneal fluid LH concentrations as a diagnostic tool for human endometriosis.

A rapid, sensitive enzymeimmunoassay for the measurement of LH concentrations in serum and peritoneal fluid samples of healthy women and women with endometriosis is reported. The ligand (LH) was captured by a readily available, widely used and well-characterized monoclonal antibody (mAb, 518B7) generated against the beta subunit of bovine LH. This mAb, although specific for LH, shows very little species specificity and detects LH by radioimmunoassay in humans. A polyclonal antiserum raised in rabbits against hCG was conjugated to horseradish peroxidase and was used as the second antibody signal. This anti-hCG antiserum crossreacts with LH. The enzymeimmunoassay uses the standard human LH (hLH) preparations (NIADDK-hLH-I-3, AFP-827OB) and results are based on the relative concentrations of LH in serum and peritoneal fluid. Total assay time was < 3 h. The range of the standard curve was 0.002-0.500 ng LH per well and the lowest concentration of hLH that could be distinguished from zero concentration was 0.15 +/- 0.02 ng ml(-1) serum and 0.058 +/- 0.021 ng ml(-1) peritoneal fluid. Clinical diagnostic parameters for the LH enzymeimmunoassay showed a sensitivity of 85.71%, specificity 92.50%, efficiency 88.54%, positive predictive value 94.11% and negative predictive value 82.22%. The study was retrospective. Serum LH concentrations of women with endometriosis were 13.67 +/- 7.21 ng ml(-1), whereas serum LH concentrations of women in the control group were 4.52 +/- 2.03 ng ml(-1). One-way ANOVA showed significant differences (P < 0.001) between women with endometriosis and control groups. Women in the control group had peritoneal fluid LH values of 5.65 +/- 2.43 ng ml(-1), whereas peritoneal fluid LH values of 64.06 +/- 16.44 ng ml(-1) were obtained in women with endometriosis (P < 0.001). A cycle-dependent pattern of serum and peritoneal fluid LH concentration was observed in women in the control group, which was not observed in the peritoneal fluid of the group with endometriosis. The application of this assay to serum or peritoneal fluid samples provides the attractive possibility that it could be included in the panel of markers used for diagnosis of endometriosis.

Effect of peritoneal fluid from women with endometriosis on implantation in the mouse model.

OBJECTIVE: To investigate the potential role of peritoneal fluid (PF) from women with or without endometriosis in implantation in mice with use of the delayed implantation model. DESIGN: A murine experimental model with markers of uterine receptivity and prospective comparison of the effects of human PF on implantation. SETTING: Academic university and hospital program. INTERVENTION(S): PF collected from women with and without endometriosis was injected intraperitoneally into recently mated mice. MAIN OUTCOME MEASURE(S): Implantation sites were counted in treated and untreated animals, and the alphavbeta3 integrin was measured in the pregnant mouse uterus by immunohistochemistry with in situ hybridization. Leukemia inhibitory factor and the beta3 subunit of alphavbeta3 were measured by Northern blot during early pregnancy and after injections of PF. RESULT(S): Animals receiving PF from infertile women with endometriosis had a reduction in the number of implantation sites compared with animals that received PF from fertile women or from patients with recently treated endometriosis. In the mouse, expression of alphavbeta3 and leukemia inhibitory factor peaked at the time of implantation and was reduced by injections of human PF from infertile patients with endometriosis. CONCLUSION(S): Leukemia inhibitory factor and alphavbeta3 are coexpressed at the time of implantation in the mouse. PF from women with endometriosis has a detrimental effect on embryo implantation, perhaps by adversely affecting uterine receptivity.

Development of a long-term serum-free culture system for immature granulosa cells from diethylstilboestrol-treated prepubertal rabbits: influence of androstenedione and fibronectin on FSH-induced cytodifferentiation.

Granulosa cells from diethylstilboestrol-treated prepubertal rabbits were cultured for 6 days in M199 with FSH (1-100 ng ml(-1)) in uncoated or fibronectin-coated plates with or without androstenedione to define the time course profile of oestradiol and progesterone secretion, and the possible modulator role of androstenedione and fibronectin during FSH-induced rabbit granulosa cell differentiation. Every 48 h, cultures were photographed and samples of medium were collected and assayed by ELISA for oestradiol and progesterone. FSH increased oestradiol secretion in a dose-dependent manner. Androstenedione augmented FSH-stimulated oestradiol secretion, and led to a decrease in secretion of oestradiol with time in culture. FSH stimulated progesterone secretion in a dose-dependent manner. This was increased by androstenedione with 10 ng FSH ml(-1) (0-96 h) and 1 ng FSH ml(-1) (96-144 h). FSH-stimulated (100 ng ml(-1)) progesterone secretion decreased at 48-96 h. Fibronectin prevented this decrease, without affecting oestradiol or progesterone secretion at other time points. FSH caused cell reaggregation at 48 h. In conclusion, this serum-free culture system is appropriate for the study of mechanisms of rabbit granulosa cell differentiation. FSH induced cytodifferentiation and reaggregation of granulosa cells. Androstenedione appeared to act synergistically with FSH to promote steroidogenesis. Fibronectin sustained progesterone secretion during differentiation.

Blockade of the alpha(v)beta(3) integrin adversely affects implantation in the mouse.

The role of endometrial and embryonic integrins during implantation remains unresolved although work in animal models and in humans supports their involvement in this process. Temporal and spatial distribution of the alpha(v)beta(3) integrin on both embryo and endometrium in women and mice coincides with the time of initial attachment during implantation. In mice, the endometrial and embryonic alpha(v)beta(3) integrin is present at the time of implantation, as shown by reverse transcription-polymerase chain reaction and immunohistochemistry. In situ hybridization demonstrates the presence of the alpha(v)beta(3) integrin on the subluminal stromal cells of the uterus. Functional blockade of this integrin on the day of implantation by intrauterine injection of neutralizing monoclonal antibodies against alpha(v) or beta(3) integrin subunits, arg-gly-asp (RGD)-containing peptides, or of the disintegrin echistatin, reduced the number of implantation sites compared to controls receiving BSA. These studies demonstrate that, like the human, the murine alpha(v)beta(3) integrin is expressed at the time of implantation in the endometrium and on the blastocyst, and may play a critical role in the cascade of events leading to successful implantation.

The effects of different anaesthetic treatments on the adreno-cortical functions and glucose levels in NZW rabbits.

The effects of five anaesthetics on the corticosterone, cortisol and glucose concentrations were investigated in the NZW rabbit. Sixty animals were assigned to 6 treatment groups (n= 10 per group): control ( iv saline solution injection), ketamine (10 mg/kg iv) with either xylazine (3 mg/kg iv) or diazepam (2 mg/kg iv), pentobarbitone (30 mg/kg iv), thiopentone (20 mg/kg iv) and fentanyl/droperidol (1 mg/kg sc). Plasma glucocorticoids were measured by competitive enzymeimmunoassay EIA and glucose by an autoanalyzer, previously validated for this species in both cases. Blood samples were obtained at 6 time-points: before injection, at 10, 30, 60, 120 min and 24 h after injection of the anaesthetics/saline. A significant decrease of plasma glucocorticoids at 10-60 min was observed in the pentobarbitone and fentanyl/ droperidol groups, whereas the administration of ketamine/diazepam or thiopentone stimulated plasma glucocorticoid release, principally in the recovery period. However, in the ketamine/xylazine group no changes were observed in the glucocorticoid levels, except for a significative increase of cortisol at 60-120 min. Glucose levels significantly increased after ketamine/diazepam administration and principally, after ketamine/xylazine treatment. The present data suggest that ketamine/xylazine has little effect on glucocorticoid levels and provides an adequate level of surgical anaesthesia, hence it would be the anaesthetic of choice, although the hyperglycaemic effect after injection has to be considered for any experimental procedures in rabbits.