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.

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.

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.

Developmental competence of immature pig oocytes under the influence of EGF, IGF-I, follicular fluid and gonadotropins during IVM-IVF processes.

Epidermal growth factor (EGF) and insulin like growth factor-I (IGF-I) were evaluated for their effects on in vitro maturation and fertilization in presence or absence of gonadotropin and porcine follicular fluid. Four groups were made with the addition of growth factors: none (control), EGF, IGF-I or EGF+IGF-I. Each group underwent four predefined treatments with gonadotropin (FSH and LH), follicular fluid, a combination of both, or none (as control). Porcine cumulus-oocyte complexes (COCs) were matured in media containing the above-mentioned treatments for 42-44 h prior to fertilization with fresh sperm capacitated for 2.5 h. At the end of the fertilization period, the presumable embryos were fixed, stained and examined as whole-mounts to ascertain their nuclear status. The addition of EGF alone or in combination with IGF-I, significantly increased the proportion of monospermic oocytes forming 2 normal pronuclei. Also, supplementation with both growth factors together enhanced the percentages of pronucleus formation and total penetration. In addition, treatments with EGF+IGF-I significantly decreased (P<0.01) the incidence of degeneration in fertilized oocytes. However, no significant differences in the proportions of COCs undergoing polyspermy were observed among all treatments. These results suggest a stimulatory effect of tested growth factors in maturation and fertilization of pig oocytes. Furthermore, gonadotropins and follicular fluid can be replaced by the addition of EGF and IGF-I to the maturation media with positive effects on fertilization rate.

Steroid-level response to insulin-like growth factor-1 in oocytes matured in vitro.

The objective was to establish the influence of insulin-like growth factor-1 (IGF-1) on steroid production and nuclear maturation during oocyte in vitro maturation (IVM). Immature-selected rabbit follicular oocytes, divided as cumulus-oocyte complexes (COC) and denuded oocytes (DO), were cultured in Brackett's medium with different concentrations of IGF-1 at 0, 50, 100 and 200 ng/ml. After 8 and 16 h of culture, the oocytes were assessed for nuclear maturation by acetic-orcein stain, and media were analyzed by enzyme-immunoassay (EIA) for 17 beta-estradiol (E), progesterone (P), androstenedione (A) and testosterone (T) content. After culture treatments with IGF-1 significantly increased (P < 0.01) the incidence of nuclear activation (germinal vesicle breakdown stage, GVBD) and nuclear maturation (metaphase II stage); maximum stimulation occurred at 100 ng IGF-1/ml (86.9 vs. 49.3% in control). Compared to controls, the presence of IGF-1 in cultures was associated with a significant increase of E and A production by COCs (P < 0.01). However, P and T levels were not significantly influenced by the IGF-1. In addition, positive correlations between E/T and E/A ratios and nuclear maturation rates were only found in the IGF-1 treatments. Regarding the DOs, neither positive effects in nuclear maturation rates nor increase of steroid levels in culture were observed for any treatment. These results suggest that: (1) IGF-1 had a significant effect on E and A production during oocyte maturation; (2) the addition of IGF-1 enhanced nuclear maturation significantly in rabbit oocytes; and (3) all these effects are only possible in oocytes surrounded by cumulus cells.

Enzyme immunoassay for testosterone and androstenedione in culture medium from rabbit oocytes matured in vitro.

Two enzyme immunoassays (EIAs) were validated to determine testosterone and androstenedione levels in culture medium (Brackett's medium with or without the addition of IGF-I, hormone and serum-free), without previous extraction, from rabbit oocytes matured in vitro. Polyclonal testosterone (C917), and androstenedione (C9111) antibodies were raised in rabbits using testosterone 3-carboxymethyloxime:BSA, and androstenedione 3-carboxymethyloxime:BSA. Horseradish peroxidase was used as label, conjugated to testosterone 3-carboxymethyloxime, and to androstenedione 6-hemisuccinate. Standard dose response curves covered a range between 0 and 1 ng/well. The low detection limits of the technique were 11.43 pg/ml for testosterone, and 2.32 pg/ml for androstenedione. Intra- and inter-assay coefficient of variation percentages were < 6.4 and < 7.1 for testosterone, and < 5.1 and < 6.3 for androstenedione, respectively (n= 10). The recovery rate of known testosterone or androstenedione concentrations added to pools of culture maturation medium samples averaged 97.58 +/- 2.11%, and 95.73 +/- 1.59%, respectively. Compared with RIA, EIA values were in close agreement for testosterone (n= 15, r= 0.96, P< 0.001), and androstenedione (n= 15, r= 0.94, P< 0.001). Culture medium samples were obtained at the end of oocyte in vitro maturation (14-16 h). Mean +/- SE culture maturation medium concentrations (ng/ml) were 1.80 +/- 0.09 and 0.52 +/- 0.01 for testosterone, and 1.70 +/- 0.04 and 0.24 +/- 0.01 for androstenedione in both the oocytes with and without cumulus cells, respectively. We concluded that our EIA is a highly sensitive and specific assay that provides a rapid, simple, inexpensive and nonradiometric alternative to RIA for determining testosterone and androstenedione concentrations in oocyte maturation culture medium.

A sensitive EIA for 17 beta-estradiol and progesterone in culture medium for oocyte in vitro maturation procedures.

A sensitive heterologous enzyme immunoassay (EIA) was validated to determine 17 beta-estradiol (E2) and progesterone levels, without previous extraction, in culture medium from rabbit oocytes matured in vitro with and without the addition of IGF-I. Polyclonal E2 (C902), and progesterone (C914) antibodies were raised in rabbits using 6-keto-17 beta-estradiol 6-carboxymethyloxime:BSA, and 11 alpha-hydroxyprogesterone 11 alpha-hemisuccinate:BSA. Horseradish peroxidase was used as label, conjugated to 17 beta-estradiol 3-hemisuccinate, and to progesterone 3-carboxymethyloxime. Standard dose response curves covered a range between 0 and 1 ng/well (100 microliters). The low detection limits of the technique were 1.99 pg/well for E2, and 13.21 pg/well for progesterone. Intra- and interassay coefficient of variation percentage (% CV) were < 6.3 and < 7.8 for E2 and progesterone, respectively (n = 10). The recovery rate of known E2 or progesterone concentrations added to a pool of culture maturation medium averaged 96.39%, and 98.65%, respectively. Compared with RIA, EIA values were in close agreement for E2 (n = 15, R = 0.96, P < 0.001), and progesterone (n = 15, R = 0.99, P < 0.001). Medium samples were obtained after oocyte maturation in vitro for 16 h. Use of IGF-I significantly elevated steroids production in the oocyte surrounded cumulus cells. The EIA described here is highly sensitive and specific assay, and provides a rapid, simple, inexpensive, and non-radiometric alternative to RIA for determining E2 and progesterone levels in oocyte culture medium.

[3H]heparin binding in boar spermatozoa: characterization and correlation with routine semen quality parameters.

A simple technique has been established for the purpose of characterizing heparin binding to boar sperm. Binding experiments were performed using [3H]heparin and extended boar semen. [3H]Heparin binding to boar sperm was effectively displaced by increasing concentrations of heparin. [3H]Heparin binding was linear at least between 50,000 and 10(6) spermatozoa and was stable for at least 120 min. Binding was sensitive to fucoidan, chondroitin sulfate B, chondroitin sulfate C, and chondroitin sulfate A, while keratan sulfate had only a marginal effect on binding. The [3H]heparin binding was saturable. Assuming a 13,000 M(r) for the [3H]heparin used, binding to boar spermatozoa showed an apparent equilibrium dissociation constant (Kd) of 23.6 +/- 2.5 nM and a maximum binding capacity (Bmax) of 6.0 +/- 1.1 pmol/10(6) spermatozoa (average values from 6 boars, means +/- SEM). Interejaculate variations in binding parameters were dependent on the male. Thus, with respect to Bmax variation, 4 of 6 boars studied exhibited an interejaculate coefficient of variation of less than 0.33 (0.09, 0.11, 0.11, and 0.29, respectively, for 3 consecutive ejaculates), while in the case of Kd interejaculate variation, only 2 of the 6 boars studied showed acceptable variation coefficients (0.16 and 0.28). No seasonal effect was observed in either of the binding parameters, with the variations following boar-specific patterns. Kd and Bmax intermean differences for different boars during the course of the study period (April-June) were not significant (p > 0.05). Correlations of mean boar binding parameters (Kd and Bmax) with conventional semen quality parameters showed a correlation between Bmax values and those of the osmotic resistance test (r = 0.8990, p < 0.01), normal acrosomes (r = 0.7946, p < 0.05), and bended tails (r = -0.8632, p < 0.02). Kd values were correlated with cytoplasmic distal droplet (r = -0.7992, p < 0.05). The glycosaminoglycans and polysulfated boar sperm binding sites reported in the present work should be regarded as binding sites until further studies elucidate their physiological role. The results obtained suggest that this technique be given a role as a research tool.