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The present study was designed to (1) assess the role of triiodothyronine (T(3)) with regard to in vitro steroid hormone secretion by chicken ovarian follicles; (2) determine whether T(3) influences the in vivo function of the pituitary-ovarian axis in the hen; and (3) detect expression of thyroid hormone receptor (TR) mRNA in chicken ovarian follicles. In the first experiment, laying hens were decapitated 22.5h before ovulation. White prehierarchical follicles (1-8mm) and fragments of theca and granulosa layers of the 3 largest yellow preovulatory follicles F3-F1 (22-35mm) were incubated in a medium supplemented with T(3) (0, 0.1, 1, 10, 100, or 1000ng/mL) or ovine luteinizing hormone (LH) (10ng/mL) in combination with doses of T(3) (1, 10, and 100ng/mL). Triiodothyronine decreased basal and LH-stimulated estradiol secretion by white follicles and the theca layer of all preovulatory follicles. On the other hand, it increased progesterone secretion by F2 and F1 follicles. In the second experiment, hens were injected 1h after ovulation with saline (control) or T(3) (10microg/100g body weight, intraperitoneally). Results indicated that exogenous T(3) decreased plasma concentrations of LH and estradiol and increased plasma concentrations of progesterone. In the third experiment, using reverse transcription polymerase chain reaction (RT-PCR) analysis, expression of thyroid hormone receptor (TRalpha and TRbeta0), mRNA was detected in all of the ovarian compartments. The expression of TRalpha mRNA was relatively greater in comparison with TRbeta0. There were no differences between white ovarian follicles in the expression of TRalpha and TRbeta0 mRNA. A considerably higher TRalpha and lower TRbeta0 expression was detected in the granulosa layer of preovulatory follicles in comparison with the theca layer. In conclusion, the data indicate that thyroid hormones acting via nuclear receptors are involved in regulation of the pituitary-ovarian axis and processes associated with follicle growth and maturation.

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The study was performed to determine the hormonal status of mature germline chimeras obtained by blastodermal cell transfer from chicken embryos of a donor breed [Green-legged Partridgelike breed (GP) x Araucana (AR)] to those of a recipient breed [White Leghorn (WL)] being at the same stage of embryonic development. The egg-laying chimeras and WL hens (control) of the same age were used in the experiment. At first, blood samples were taken from each bird at 0.5, 5, 12.5 and 18.5 h following oviposition. Subsequently, the chimeras and the WL hens were decapitated 1-2 h after ovulation. A stroma and the following follicles were isolated from the ovary: white normal (1-4, 4-6 and 6-8 mm), white atretic and yellow preovulatory follicles (F4-F1). Sex hormones, progesterone (P4), testosterone (T) and oestradiol (E2) in blood plasma and ovarian follicles were determined radioimmunologically. The activity of the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in the granulosa and theca layers of the follicles was analysed histochemically. In chimeric chickens, a higher level of T in blood plasma during the ovulatory cycle was noticed. However, in the stroma, white prehierarchical and medium-size preovulatory ovarian follicles the level of T was significantly lower. With respect to E2, its elevated levels were found both in blood and in the ovarian follicles. There were no significant differences in P4 concentrations in blood plasma while in ovarian follicles a higher level was observed only in white 6-8 mm follicles. 3beta-HSD activity in granulosa and theca layers of the ovarian follicles in chimeras was not different from that in the WL hens. In conclusion, the results obtained indicate that germline chimeras exhibit significant alterations in sex hormone levels in the ovary and blood plasma, which in turn may affect their reproductive abilities.

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The effect of tamoxifen (TMX), an anti-estrogen compound, on immunoglobulins (Ig) level in blood plasma of laying hens was investigated. TMX (4 mg/hen/day) was given per os for seven consecutive days; control hens received placebo. Blood samples were collected from the wing vein every day before TMX treatment, and plasma Ig levels were measured by means of Rlebodzinski's test. TMX significantly decreased plasma Ig levels, maximally by 51% on day 2 of the experiment. The observed reduction in Ig level was accompanied by the significant, 37% decrease in the ratio of Ig/total protein (Tp). From the third day of TMX treatment, level of Ig and the ratio of Ig/Tp gradually increased and on the day 5 of the experiment no difference between control and experimental group was found. In non-immunoglobular (Tp-Ig) fractions of plasma proteins no significant alterations after TMX treatment were observed. Therefore, treatment of laying hens with TMX transiently decreased plasma Ig levels. Most likely the effect of TMX is associated with the antagonistic properties of TMX toward estrogen receptors. On the contrary, the transient decrease in plasma Ig levels of TMX-treated hens followed by the gradual increase suggests adaptation of the immunological system to treatment with the anti-estrogen preparation.

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HyLine Brown laying hens at 30 weeks of age were treated twice daily with Fadrozole, a non-steroidal aromatase inhibitor (AI; 1 mg/kg body weight; i.m.) for six consecutive days; control hens received saline. Blood was collected every day 0.5 h after oviposition, i.e. just before AI treatment. Ovarian steroids: progesterone (P4), testosterone (T) and estradiol (E2), and iodothyronines: thyroxine (T4), triiodothyronine (T3) and reverse-triiodothyronine (rT3) were measured in blood plasma by radioimmunoassay methods. In AI-treated hens a gradual delay in oviposition time was observed. AI significantly decreased P4 and E2 levels, maximally by 43% on day 4 and by 74% on day 5, respectively, and elevated T level, maximally by 248% on day 4. Simultaneously, the increases in T4 and T3 levels with no changes in rT3 levels were observed. The maximal effect of AI on T4 and T3 levels was found on day 4 (60% increase) and day 5 (312% increase), respectively. Moreover, statistically significant, negative coefficient of correlation between E2 and T3 (r = -0.51), and positive coefficient of correlation between T and T3 (r = 0.42) in AI-treated hens were found. The results obtained indicate that in mature laying hens there is a strong relationship between ovarian steroids and thyroid hormones, and suppression of E2 synthesis not only disrupts ovarian function but also affects the activity of the thyroid gland and peripheral metabolism of thyroid hormones.

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This study was conducted to investigate the interactions between growth hormone (GH) and insulin-like growth factor-I (IGF-I) on progesterone (P4) secretion by porcine luteal cells cultured in vitro. Cells isolated from corpora lutea (CL) collected at three different periods of the luteal phase (CL1--early luteal phase; CL2--middle luteal phase and CL3--late luteal phase) were incubated with different doses of GH (10, 100 or 200 ng/ml). After 48 h cultures were terminated and the media were frozen until further P4 concentration analysis. GH (100 ng/ml) increased P4 secretion by CL1 and CL2 and had no effect on CL3. In separate studies these cells were treated for 48 h with IGF-I alone or with GH combined with IGF-I. IGF-I alone increased basal P4 secretion only by cells collected from CL1 while concurrent treatment with GH had no effect on P4 secretion by any type of CL. To investigate the possible mechanism of GH and IGF-I mediated induction of P4 secretion, an inhibitory study was conducted. In this experiment, luteal cells collected from CL1 were cultured in the absence or presence of cycloheximide (an inhibitor of protein synthesis) or actinomycin D (an inhibitor of DNA transcription). Cycloheximide or actinomycin D completely blocked the stimulatory effect of both GH and IGF-I on P4 production but did not reduce basal progesterone secretion suggesting involvement of gene transcription and translation in the GH and IGF-I action on luteal cells. Additionally, the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) under the influence of GH added alone or together with IGF was measured by the conversion of pregnenolone to progesterone. Stimulation of P4 secretion in P5-treated cells in GH-stimulated cultures was not observed, however, high stimulatory effect was noted in IGF-I treated cultures. In conclusion, the present studies indicate that there is direct and cycle stage dependent influence of GH and IGF-I on steroidogenesis in procine luteal cells. It is suggested that both IGF and GH may exert some regulatory action during CL development in the pig.

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Thirty-four-week-old laying hens received injections of recombinant chicken leptin to assess the role of leptin in avian ovarian function. In the first experiment, the hens (n=60) were divided into three groups: (i). fed ad libitum; (ii). fasted; and (iii). fasted + leptin. Hens were fasted for 5 days and those treated with leptin received 250 microg leptin kg-1 body weight twice a day, i.p. In the second experiment, the hens (n=72) were divided into four groups: (i). fed ad libitum; (ii). fasted; (iii). fasted + leptin given only during fasting (5 days); or (iv). fasted and leptin given during both fasting and 5 days of re-feeding (10 days). LH was measured in blood plasma, and progesterone and oestradiol were measured in blood plasma and the ovary by radioimmunoassay. Apoptosis was examined in the walls of the three largest yellow hierarchical follicles (F3-F1; F38-12 mm), and the granulosa layer of F3 follicles. The expression of leptin receptor in the granulosa layer of F2 and F1 follicles was barely detectable. This was in contrast to a much higher expression of leptin receptor maintained in the theca layer of F3-F1 follicles. The present results indicate that in chickens leptin might be involved in the adaptation to starvation due to attenuation of follicular apoptosis. The presence of leptin receptors in the ovary indicates the possibility of a peripheral effect of the hormone.

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The deleted in colorectal cancer (DCC) protein is important in the pathway guidance of cells and cell processes during neural development, and DCC has also been implicated in the aberrant cellular migrations of neuroblastoma dissemination. We attempted to further define DCC protein function by the overexpression of full-length and truncated DCC constructs in a human neuroblastoma cell line. Overexpression of the truncated DCC protein resulted in a less epithelioid morphology. This was accompanied by decreases in expression of N-cadherin and alpha- and beta-catenin by immunoblot and Northern blot analysis. Levels of desmoglein were relatively less affected, whereas endogenous DCC protein levels were increased in the truncated transfectants. N-cadherin immunofluorescence was consistent with the immunoblot studies and localized the protein to the cytoplasm and sites of cell-cell contact. Cell aggregation studies demonstrated diminished calcium-dependent aggregation in the truncated transfectants. In conclusion, overexpression of a truncated DCC protein in neuroblastoma cells resulted in the loss of an epithelioid morphology, diminished expression of N-cadherin and alpha- and beta-catenin, and diminished calcium-dependent cell adhesion. These studies provide the first evidence of an apparent functional link between DCC and N-cadherin/catenin-dependent cell adhesion.

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The purpose of the present study was: (1) to demonstrate immunocytochemically the localization of histamine in the wall of four chicken oviductal parts, i.e. infundibulum, magnum, isthmus, and shell gland, (2) to identify the presence of mast cells in chicken oviduct, and (3) to determine histamine concentration in oviductal tissue by the spectrofluorometric method. Experiments were carried out on Isa Brown laying hens decapitated just after oviposition. The specific immuno-reactivity for histamine and the presence of mast cells were found in the wall of all the examined oviductal parts. The immuno-reactive histamine was localized in epithelium, tubular glands, connective tissue layer, circular and longitudinal muscles, and endothelium and muscles of blood vessels. The intensity of immuno-positive reaction was as follows: infundibulum > shell gland > magnum = isthmus and correlated with quantitatively determined histamine level and tissue density of mast cells. It is suggested that mast cells are the main source of histamine in the chicken oviduct.

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Little is known about the physiological events occurring in the chicken ovary during a pause in laying, therefore the aim of the present study was to examine changes in sex steroid concentration in the follicle wall and blood plasma during cessation of egg laying. The experiment was performed on laying Isa Brown hens. Control hens were fed ad libitum whereas the experimental ones were subjected to a pause in laying by complete food deprivation for 5 days and water deprivation on 3 day followed by feeding every second day up to 9 day and then ad libitum. Blood samples were taken from the wing vein each day. The hens were decapitated on day 3, 6, 9, and 16. The ovary was isolated and the following follicles were dissected: white (1-2; 2-4; 4-6; 6-8 mm) and yellow preovulatory ones (F1-F3). Progesterone and estradiol were measured in the follicle wall and blood plasma by RIA methods. The hens stopped egg laying on day 4 and began egg restoration on day 14 of the experiment. Cessation of egg laying was preceded by a decrease in estradiol and progesterone levels in the ovary as well as in the blood plasma. The plasma level of these steroids began to increase 7 days before the start of egg restoration. Autopsy of the ovary showed that the atrophy of the chicken yellow preovulatory follicles during the pause in laying was accompanied by a significant increase in the total number of white follicles.

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The concentrations of ovarian steroids (estradiol--E2, progesterone--P4 and testosterone--T) and thyroid hormones (thyroxine--T4 and triiodothyronine--T3) were determined in blood plasma of the domestic hen during sexual maturation and the initial period of egg lay. Blood samples were collected from Hy-Line pullets at 3 day intervals from days 87 to 144 day of life, i.e. 42 days before and 14 days after the onset of egg lay (OEL). Ovarian and thyroid hormones were measured by RIA methods. During sexual maturation an increase in ovarian steroids in the blood plasma was observed. The maximum E2 and P4 levels were recorded on day 6 and day 3 prior to OEL, respectively. In the case of plasma T level, an increase from 42 to 18 days before OEL followed by a decrease and a renewed increase from day 9 till OEL was observed. The relatively unchanged plasma level of T4 until day 9 before OEL decreased significantly just before the first oviposition while the T3 level gradually decreased between day 42 and day 9 before OEL, and then increased and again decreased from day 3 before till day 3 after OEL. During sexual maturation the following statistically significant coefficients of correlation between ovarian steroids and T3 were found: E2 vs. T3-->r = -0.551 and P4 vs. T3-->r = -0.373. There was no significant correlation between T and T3 or between the examined steroids and T4. The data obtained indicate that during sexual maturation of the domestic hen there is a negative relationship between the ovary and the thyroid gland.

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In a laying hen, histamine was found to be present in all compartments of the ovary, i.e. stroma with follicles < 1 mm, small white (1-4 mm), large white (4-8 mm), atretic white, yellow preovulatory (8-35 mm) and postovulatory follicles. Stroma containing non-yolky follicles exhibited the highest histamine concentration (6080 +/- 331 ng/g wet wt. tissue) which differed significantly (P < 0.01) from histamine levels observed in all examined classes of ovarian follicles. High histamine concentration was found in small, large and atretic white follicles as well as in older postovulatory follicles whereas low levels of histamine contained yellow preovulatory and younger postovulatory follicles. Population of yolky white follicles presented significant (P < 0.01) differences in histamine level among small (4280 +/- 333), atretic (2940 +/- 193) and large (2010 +/- 110 ng/g) follicles. Within hierarchy of yellow preovulatory (F7-F1) follicles initial decrease in histamine concentration, from 859.3 +/- 51.5 ng/g in F7 follicle to 363.9 +/- 28.3 ng/g in F4 follicle, was followed by the increase as follicle matured, reaching the highest level in F1 follicle (711.4 +/- 35.9 ng/g). In postovulatory (P1-P5) follicles histamine concentration gradually increased as they were getting older, from 604.3 +/- 49.3 ng/g in P1 follicle to 2253 +/- 197 ng/g in P5 follicle. Determination of histamine in relation to ovulation revealed significant (P < 0.01) difference both in histamine concentration and content between the largest preovulatory F1 follicle and the largest postovulatory P1 follicle, being 0.5 h before and 0.5 h after ovulation, respectively. It is suggested that in chicken, ovarian histamine may play a role in the follicular development and/or the ovulatory process.

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This study was undertaken to determine histamine concentration in chicken oviductal parts (infundibulum, magnum, isthmus and shell gland) in relation to the egg location within the oviduct and ovulation. The experiment was performed on Hisex Brown laying hens with regular sequences of at least four eggs. Ovulation occurred within 5-15 min of oviposition of the previous egg in the series. Histamine was determined spectrofluorometrically in the following stages of the egg-laying cycle: during c2 oviposition; 0.5 h, 6.5 h, 12.5 h and 18.5 h after c2 oviposition; and during c3 oviposition. Irrespective of the egg formation stage histamine concentration in the examined oviductal parts was arranged in the following order: infundibulum > magnum > isthmus > shell gland. During the egg-laying cycle histamine concentration significantly changed. During oviposition, i.e. just before ovulation of the next egg in the series, histamine concentration significantly increased in the infundibulum while 6.5 h after oviposition, i.e. about 1.5 h of the egg stay in the shell gland, there was a significant increase in histamine concentration both in the infundibulum and the shell gland. In the magnum histamine concentration was elevated when the ovum entered the segment, i.e. 0.5 h after oviposition. There were no changes in histamine concentration in the isthmus. It is suggested that histamine participates in the local events taking place in the hen oviduct during the egg formation cycle.

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Two follicular compartments, granulosa (G) and theca interna (T) cells isolated from porcine ovaries were cultured alone or in co-culture (GT). Cells were grown as monolayers in a control medium without hormone and in a media supplemented with arginine-vasotocin (AVT) at a concentration of either 10(-7)M or 2 x 10(-7)M. Progesterone (P4), estrogen (E2) and androgen (A) concentrations in the culture media were taken as measures of the effect of AVT on the function of follicular cells. Steroids were analysed by radioimmunoassay. AVT action in this culture system was expressed as a decrease in progesterone secretion by cultures of granulosa cells alone, and especially as a change in the pattern of estradiol and androgen secretion by co-cultures. Control T and G cells cultured alone secreted small amounts of A (238.0pg/10(5) cells, 27.3pg/10(5) cells, respectively), and E2 (272.5pg/10(5) cells, 10.6pg/10(5) cells, respectively) while in co-culture these two cell types interacted and the result of this positive interaction was a significant increase in secretion of these two steroids (941.0pg/10(5) cells androgen secretion and 854.1pg/10(5) cells estradiol secretion). This phenomenon is similar to that observed in the intact follicle in vivo. AVT introduced to the culture medium impaired the effect of this positive interaction of mixed G and T cells on the production of high levels of E2 and A by untreated co-cultures.

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The purpose of the present study was to examine the effect of methallibure (MLB), a non-steroid inhibitor of pituitary gonadotrophic activity on serotonin (5-HT) levels in the wall of preovulatory follicles (F1-F4) in the domestic hen. 5-HT was determined spectrofluorometrically. Hens were treated with MLB (10 mg/hen per os) twice a day for 3 successive days. 5-HT was determined in F1-F4 (F1 greater than F2 greater than F3 greater than F4) follicles of the control group, on the next day (MLB-1 group) and 6 days following cessation of MLB administration (MLB-6 group). During MLB treatment egg production was inhibited in all hens. In the MLB-6 group, five hens out of seven took up egg laying on the sixth day after MLB administration. Within each examined group there were no significant differences in 5-HT concentration between F1-F4 follicles. In comparison to the control, MLB caused a significant (P less than 0.01-0.05) increase of 5-HT concentration in F1 (MLB-1 group) and F4 (both MLB-groups). The results obtained indicate that there is a relationship between pituitary activity and 5-HT levels in the preovulatory follicles of the domestic hen.

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The purpose of the present study was to demonstrate visually and localize the presence of serotonin (5-HT) in the ovary and oviduct of the domestic hen using a histochemical Falck-Hillarp method. Experiments were carried out on White Leghorn laying hens with no egg in the shell gland. The specific yellow fluorescence, indicating the presence of 5-HT, was found both in the ovary and all examined oviductal parts. The strongest fluorescence was present in the ovarian stroma containing small follicles with a diameter under 4 mm. In the wall of the largest preovulatory follicle a very strong fluorescence was located mainly in the theca layer. In the oviductal parts, the intensity of 5-HT fluorescence in the infundibulum and magnum was fairly strong, whereas in the isthmus and shell gland it was weak. Fluorescence seen in the infundibulum, magnum, and isthmus was primarily localized along the luminal borders of the fold surface epithelium. In the shell gland 5-HT fluorescence was found within the uterine folds, especially in the tubular glands. Moreover, the presence of an egg in the definite oviductal segment (infundibulum or isthmus) increased the intensity of yellow fluorescence in this part.

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The interrelationship between prostaglandins (PG) and vasotocin (AVT) in the oviposition of the domestic hen was investigated. Single or combined injections of indomethacin (IND), an inhibitor of PG synthesis, and AVT gave delay or induction of oviposition. Injection (i.m.) of IND (5 mg/kg) 5 h before oviposition resulted in 15.1 h (+/- 0.93) delay of oviposition. Injection (i.v.) of AVT (0.1 microgram/kg) 2.5 h before oviposition caused premature oviposition within a few minutes (3.1 +/- 0.2). Combined injection of IND and AVT at 5 h and 2.5 h, respectively, before oviposition caused the delay of oviposition (15.8 h +/- 0.8). The results indicate that IND blocked the induction of oviposition by AVT.

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Porcine luteal cells were obtained from the corpora lutea on the 13th day of the estrous cycle. The cells were digested with 0.25% trypsin and suspended in the Medium 199 with an addition of 10% calf serum, at a concentration of 5 X 10(4) cells/ml. The cells were incubated with or without 4 and 40 mi.u./ml of oxytocin, 10 and 100 ng/ml of arginine-vasotocin, 1 microgram LH and 50 U/ml hCG. Levels of progesterone, testosterone and estradiol 17 beta were determined with the radioimmunological method, following 6 h incubation. It was found that progesterone secretion under the influence of oxytocine (4 mi.u./ml) was less than in the control group and in the group with LH. Similarly, arginine-vasotocin at a dose of 10 ng/ml inhibited progesterone secretion (P less than 0.05). Higher doses of these peptides had no suppressive effect on the luteal cells. Oxytocin and arginine-vasotocin had no influence on testosterone secretion by luteal cells. However, these cells produced less (P less than 0.05) estradiol 17 beta under the influence of oxytocine than of hCG. The results point to a direct effect of oxytocin and arginine-vasotocin on steroidogenesis in the corpora lutea of cyclic pigs.

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