Effect of growth hormone on basal and LH-stimulated steroid secretion by chicken yellow ovarian follicles. An in vitro study.

The aim of the present study was to determine the effect of growth hormone (GH) on basal and LH-regulated steroid secretion by yellow hierarchical follicles before and after maturation, and the granulosa and theca layers of the largest preovulatory follicles during the ovulatory cycle in the chicken. In the first experiment, whole yellow follicles (8-12 mm, 12-18 mm, 18-24 mm and 24-30 mm) isolated from 15 and 17-18 week-old chickens were used. In the second experiment, the granulosa and theca layers of the 3 largest yellow preovulatory follicles (F3

Independent, non-IGF-I mediated, GH action on estradiol secretion by prehierarchical ovarian follicles in chicken. In vitro study.

Information concerning the role of growth hormone (GH) in the local regulation of ovarian activity in birds is limited. Therefore, the aim of the present study was to determine whether in the domestic hen GH influences in vitro estradiol secretion by prehierarchical ovarian follicles. Moreover, the interaction between GH and IGF-I on estradiol secretion was examined. Small white (1-4 mm), large white (4-6 mm) and yellowish (6-8 mm) ovarian follicles were isolated at the stage of 2 h after ovulation. In the first experiment (n = 8 hens), whole follicles (small white, n = 6/dose/ovary; large white, n = 1/dose/ovary and yellowish, n = 1/dose/ovary) were ipcubated for 24 h at 38 degrees C in a medium supplemented with 0 (control), 1, 10 or 100 ng/ml of chicken GH (cGH). In the second experiment (n = 6 hens), follicles were incubated in the same way in a medium with 0 (control), 10 ng/ml cGH, 25 ng/ml human IGF-I or cGH+hIGF-I (10 ng/ml+25 ng/ml). Following incubation the estradiol concentration was determined in media (RIA) and protein in the tissues of the follicular wall (Lowry). The secretion of estradiol was expressed per milligram of protein. The experiments revealed that both cGH and hIGF-I stimulated estradiol secretion by examined chicken ovarian follicles. The simultaneous addition of cGH and hIGF-I increased estradiol secretion by ovarian follicles as compare to the control. These hormones added together did not have an additive effect when compared to their separate actions. The results obtained suggest that both GH and IGF-I are important stimulators of estradiol production in chicken nonhierarchical ovarian follicles. We propose independent, non-IGF-I-mediated GH action on estradiol secretion.

Effect of growth hormone on steroid content, proliferation and apoptosis in the chicken ovary during sexual maturation.

The present study was undertaken to examine in vivo the effect of growth hormone (GH) on progesterone and estradiol levels and on cell proliferation and apoptosis in the chicken ovary during sexual maturation. Hy-Line chickens (10 weeks old) were injected three times a week with 200 µg recombinant chicken GH (cGH) per kilogram body weight until sexual maturity. GH treatment significantly increased ovarian weight at 16 weeks of age, i.e., ~1 week before onset of egg laying. The progesterone content in the ovary just before and at the time of sexual maturity and the estradiol content before onset of egg laying were also elevated after cGH injections. The highest number of proliferating (positive for proliferating cell nuclear antigen) and apoptotic (positive for terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labeling) cells was found in the ovarian stroma and white follicles (>1-4 mm diameter), whereas the lowest number of these cells was detected in yellow (>8-30 mm) follicles. Administration of cGH significantly stimulated cell proliferation and inhibited cell apoptosis in the ovarian stroma and small ovarian follicles. The number of ovarian follicles and the weight of the ovary prior to the first oviposition were also higher in cGH-injected hens. Thus, prior to and after the onset of egg laying, GH participates in the growth, maturation and hormonal activity of ovarian follicles in the chicken, via the regulation of steroidogenesis, proliferation and apoptosis processes.

The expression of pituitary FSHbeta and LHbeta mRNA and gonadal FSH and LH receptor mRNA in the chicken embryo.

In avian species, synthesis of sex steroids by embryonic gonads is regulated by luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In order to elucidate the role of the two gonadotropins in gonadal axis development during the second half of chicken embryogenesis, pituitary expression of LH beta subunit (LHbeta) and FSH beta subunit (FSHbeta) mRNAs as well as gonadal expression of LH and FSH receptor (LHR and FSHR) mRNAs were determined on days 11 (E11) and 17 (E17) of embryonic development and after hatching (D1). In the pituitary of the female embryo, the gene expression of FSHbeta was the lowest on E11 and increased on E17. In the male pituitary, the expression of FSHbeta did not differ among the studied days. The FSHbeta mRNA expression on E11 was higher in the male than in the female pituitary gland. The expression of LHbeta mRNA in the female pituitary increased on D1 in comparison to E11. In the male pituitary gland, the expression of LHbeta gene was relatively constant. The expression of mRNA encoding FSHR in the ovary increased on E17, while in testes it did not differ among the studied days. There were no significant alterations in LHR gene expression in the ovary or in the testes in the examined period however, the gene expression on E17 was higher in the ovary than in the testes. We observed positive correlations between the pituitary FSHbeta mRNA expression and ovarian expression of FSHR mRNA (r = 0.63; p<0.01) as well as between LHbeta mRNA and LHR mRNA in the testes (r=0.65; p<0.01). The reported alterations in gene expression of FSHbeta, LHbeta and their receptors between sexes and among the stages of embryonic development indicate time- and sex-dependent action of gonadotropins in gonads of chicken embryos.

Effect of tamoxifen on sex steroid concentrations in chicken ovarian follicles.

The aim of the present investigation was to study the effect of tamoxifen (TAM), an oestrogen receptor antagonist, on the concentrations of sex hormones in chicken ovarian follicles. The experiment was carried out on Hy-line hens which were randomly divided into two groups (control and experimental). TAM was given at a dose of 4 mg/hen (per os) at first once a day for 7 consecutive days, and subsequently four times a day for the next 6 days. Control hens received placebo. Birds were killed on the day after the last TAM treatment. From the dissected ovaries the following compartments were isolated: stroma with follicles < 1 mm, white non-hierarchical (1-4 mm and 4-8 mm) and yellow hierarchical follicles (F6-F1; 18-35 mm). The concentrations of the sex steroids progesterone (P4), testosterone (T) and oestradiol (E2) in the ovarian follicles were determined by radioimmunoassay. In the TAM-treated group, a gradual decrease in egg-laying rate was observed from the 4th day of the experiment. Eventually, egg laying stopped entirely on the 12th day of the experiment. TAM significantly decreased the weight of the ovary and affected the sex hormone concentrations in the ovarian follicles. Following TAM treatment (1) a significant increase in E2 and T concentrations in the stroma, white follicles and the F4 and F1 follicles, (2) a significant decrease in E2 and T concentrations in the F2 follicle, and (3) a significant decline of P4 in the F4 to F1 follicles were observed. The results indicate that the blockade of oestrogen receptors by TAM significantly modulates the process of chicken ovarian steroidogenesis.

Effect of 9-cis retinoic acid (RA) on progesterone and estradiol secretion and RA receptor expression in the chicken ovarian follicles.

Several lines of evidence indicate that retinoids, derivates of vitamin A, affect reproductive function in birds, however, the mechanism of their action in the ovary is still unknown. Therefore, the present study was designed (i) to show whether in the domestic hen 9-cis retinoic acid (9-cis RA), one of the retinoids, influences steroid secretion in vitro by white and yellow chicken ovarian follicles, and (ii) to detect expression of retinoic acid RXR receptor mRNA in these follicles. The white follicles (small: 1-4 mm, medium: 4-6 mm and large 6-8 mm in diameter) and the three largest yellow preovulatory follicles (F3-F1; 25-37 mm) were isolated from the ovary 3 h before ovulation. The granulosa layer was separated from the theca layer in the preovulatory follicles, which were subsequently divided into 4 equal pieces. The isolated whole white follicles or parts of the granulosa or theca layers were incubated for 24 h at 38 degrees C in Eagle's medium in the following 4 groups: control, ovine LH (oLH; 10 ng/ml), 9-cis RA (100 ng/ml) and 9-cis RA + oLH. After incubation, the medium was collected for estradiol (E2) and progesterone (P4) determination while tissues were saved for protein assay. It was found that 9-cis RA affects steroid secretion from chicken ovarian follicles. It decreased E2 secretion from white follicles and from the theca layer of the two largest (F2 and F1) preovulatory follicles. 9-cis RA had no effect on oLH-stimulated E2 secretion by the white follicles and yellow F2 and F1 follicles, but it diminished E2 secretion by F3 follicles. As regards P4, the effect of 9-cis RA was opposite; it increased P4 secretion from the granulosa layer of all preovulatory follicles. 9-cis RA did not change oLH-stimulated P4 secretion by granulosa layers ofF3 and F2 follicles, however, it inhibited oLH-enhanced P4 secretion from the F1 granulosa layer. In a separate experiment, the presence of mRNA encoding RXR was found in the stroma and all follicles of the chicken ovary by means of the RT-PCR technique. The results indicate that retinoids, acting by specific nuclear receptors, are modulators of follicular steroidogenesis in the chicken ovary.

MRNA expression and immunocytochemical localization of leptin receptor in the oviduct of the laying hen (Gallus domesticus).

The role of leptin in female reproduction is fairly well established in mammals, whereas reports concerning leptin action in birds are scarce. The aim of the present study was to detect leptin receptor (LEP-R) mRNA and to localize the leptin receptor protein in the oviduct of laying hens 2h after ovulation by the RT-PCR method and immunocytochemical staining. The RT-PCR reaction demonstrated expression of the long form ofleptin receptor mRNA in all examined oviductal parts (infundibulum, magnum, isthmus and shell gland) and the weakest level was found in the isthmus. The expression of the short isoform was lower than the long form in all examined tissue samples and no differences between oviductal parts were observed. Immunostaining specific for leptin receptor was found in the walls of all examined oviductal parts. The intensity of the immunopositive reaction was the strongest in the epithelium of all examined parts of the oviduct and in the endothelium and muscles of blood vessels. The weakest immunopositive reaction was observed in tubular glands, the connective tissue layer and in circular and longitudinal muscles. The results obtained in this experiment suggest that the oviduct may be a target tissue for leptin, where this polypeptide hormon may participate in egg formation and/or its transport through the oviduct of the domestic hen.

Expression of alpha and beta estrogen receptors in the chicken ovary.

The role of estrogens in hen reproduction is well established. However, the distribution of estrogen receptors in the chicken ovary is unknown. Therefore, the mRNA expression of alpha (ERaalpha) and beta (ERbeta) estrogen receptors was examined within the ovaries of laying hens. Expression of ERs was determined by RT-PCR analysis. The presence of ERalpha and ERbeta mRNAs was found in the ovarian stroma and white, yellowish, small yellow and the largest preovulatory (F3-F1) follicles. ERalpha and ERbeta mRNAs were detected in the granulosa and theca layers of the walls of preovulatory follicles. The expression of ERalpha mRNA was markedly higher than ERbeta mRNA in all examined ovarian compartments. Within the ovary, the relative expression of both ER mRNAs depends on the follicular diameter and the layer of the follicular wall. The results demonstrate the expression of both ERalpha mRNA and ERbeta mRNA in all compartments of the chicken ovary, suggesting different pathways of estrogen action in the avian ovary. Much higher expression of ERalpha mRNA indicates that this form of estrogen receptor is predominant in the chicken ovary. The clarification of the mechanism of ERalpha and ERbeta participation in the ovarian functions of birds necessitates further experiments examining ERs at the protein level.

Expression and localization of growth hormone and its receptors in the chicken ovary during sexual maturation.

Roles of pituitary growth hormone (GH) in female reproduction are well established. Autocrine and/or paracrine actions of GH in the mammalian ovary have additionally been proposed, although whether the ovary is an extra-pituitary site of GH expression in the laying hen is uncertain. This possibility has therefore been assessed in the ovaries of Hy-Line hens before (between 10-16 weeks of age) and after (week 17) the onset of egg laying. Reverse transcription/polymerase chain reaction (RT-PCR) analysis has consistently detected a full-length (690 bp) pituitary GH cDNA in ovarian stroma from 10 weeks of age, although GH expression is far lower than that in the pituitary gland or hypothalamus. GH mRNA is also present in small (>1-4 mm diameter) follicles after their ontogenetic appearance at 14 weeks of age and in all other developing follicles after 16 weeks of age (>4-30 mm diameter). Immunoreactivity for GH is similarly present in the ovarian stroma from 10 weeks of age and in small (<4 mm diameter) and large (>4-30 mm) follicles from 14 and 16 weeks of age, respectively. The relative intensity of GH staining in the ovarian follicles is consistently greater in the granulosa cells than in the thecal cells and is comparable with that in the follicular epithelium. A 321-bp fragment of GH receptor (GHR) cDNA, coding for the intracellular domain of the receptor, has also been detected by RT-PCR in the ovary and is present in stromal tissue by 10 weeks of age, in small follicles (<4 mm diameter) by 14 weeks of age, and in larger follicles (>4-30 mm diameter) from 16 weeks. GHR immunoreactivity has similarly been detected, like GH, in the developing ovary and in all follicles and is more intense in granulosa cells than in the theca interna or externa. The expression and location of the GH gene therefore parallels that of the GHR gene during ovarian development in the laying hen, as does the appearance of GH and GHR immunoreactivity. These results support the possibility that GH has autocrine and/or paracrine actions in ovarian function prior to and after the onset of lay in hens.

Generation of cloned and chimeric embryos/offspring using the new methods of animal biotechnology.

The article summarizes results of studies concerning: 1/ qualitative evaluation of pig nuclear donor cells to somatic cell cloning, 2/ developmental potency of sheep somatic cells to create chimera, 3/ efficient production of chicken chimera. The quality of nuclear donor cells is one of the most important factors to determine the efficiency of somatic cell cloning. Morphological criteria commonly used for qualitative evaluation of somatic cells may be insufficient for practical application in the cloning. Therefore, different types of somatic cells being the source of genomic DNA in the cloning procedure were analyzed on apoptosis with the use of live-DNA or plasma membrane fluorescent markers. It has been found that morphological criteria are a sufficient selection factor for qualitative evaluation of nuclear donor cells to somatic cell cloning. Developmental potencies of sheep somatic cells in embryos and chimeric animals were studied using blastocyst complementation test. Fetal fibroblasts stained with vital fluorescent dye and microsurgically placed in morulae or blastocysts were later identified in embryos cultured in vitro. Transfer of Polish merino blastocysts harbouring Heatherhead fibroblasts to recipient ewes brought about normal births at term. Newly-born animals were of merino appearance with dark patches on their noses, near the mouth and on their clovens. This overt chimerism shows that fetal fibroblasts introduced to sheep morulae/blastocysts revealed full developmental plasticity. To achieve the efficient production of chicken chimeras, the blastodermal cells from embryos of the donor breeds, (Green-legged Partridgelike breed or GPxAraucana) were transferred into the embryos of the recipient breed (White Leghorn), and the effect of chimerism on the selected reproductive and physiological traits of recipients was examined. Using the model which allowed identification of the chimerism at many loci, it has been found that 93.9% of the examined birds were chimeras. The effect of donor cells on the reproduction and physiology of the recipients was evident.

Exogenous leptin advances puberty in domestic hen.

The present study was undertaken to examine the effect of recombinant chicken leptin administered to fed ad libitum and feed-restricted immature chickens of a layer strain on ovarian development and the timing of sexual maturity. In the first experiment 11-week-old pullets (77 days of age) fed ad libitum were injected daily with leptin at four dose levels (4, 16, 64 and 256 microg/kg body weight) until sexual maturity (lay of the first egg). Leptin treatment at the highest dose significantly (P<0.05) advanced the onset of puberty (day 116.3+/-1.0) in comparison to controls (day 121.3+/-1.2). The rises of luteinizing hormone, estradiol and progesterone in blood plasma were also advanced by leptin treatment. In the second experiment, both full-fed and feed-restricted pullets (79 days of age) were injected daily with leptin (256 microg/kg body weight). In birds fed ad libitum, exogenous leptin again significantly (P<0.05) advanced first ovipostion (day 118.4+/-1.4 versus day 124.4+/-1.7), while abolishing the significant (P<0.05) delay caused by feed restriction (day 131.5+/-1.6) and restoring the normal onset of sexual maturity (day 125.7+/-1.6). Analysis of the ovaries in 106-day-old pullets revealed that leptin injections advanced follicular development, particularly in birds fed ad libitum, and significantly (P<0.01) reduced follicular apoptosis both in full-fed and feed-restricted birds. In conclusion, we have shown that in female chickens exogenous leptin advances the onset of puberty by attenuation of ovarian apoptosis and enhancement of folliculogenesis.

Effect of prolonged progesterone treatment on the proenkephalin mRNA gene expression and enkephalins concentration in the sheep brain.

The opioids modulate reproduction in sheep mostly by inhibiting the activity of the hypothalamo-pituitary-gonadal axis. However, the mechanism by which the negative feedback control systems regulate opioid synthesis and secretion in sheep is still not recognized. As a part of a research dealing with interaction between opioids and steroids, the effect of prolonged administration of progesterone (P4) and opioid receptor agonist or antagonist on the Met-enkephalin synthesis and concentration was examined in sheep brain. Long term P4 treatment significantly decreased the synthesis and the concentration of the opioid peptide in the hypothalamus and pituitary, however, the effect was more pronounced in the hypothalamus. Injections of Met-enkephalin completely or partially reversed the effect of P4. Naltrexone given together with opioid peptide modulated the response to the opioid agonist. The results show that there is an interaction between P4 and endogenous opioids in the central nervous system of cyclic sheep.

Steroidogenic activity of chicken ovary during pause in egg laying.

The present study was designed (i) to assess the changes in the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and cytochrome P450 aromatase (P450arom) in the ovaries of hens which are subjected to a pause in egg laying by fasting, and (ii) relate these changes with progesterone (P(4)) and estradiol (E(2)) production in the ovary. Hy-Line Brown laying hens (n=90) were fasted for 5 days with water deprivation only on day 3 and subsequently fed every second day up to day 13 and then ad libitum. Birds were euthanized (n=18) on day 0, 3, 6, 9 and 16 of the experiment. The activities of 3beta-HSD and P450arom were evaluated in stroma with cortical follicles (<1mm) and in the wall of white non-hierarchical (1-8 mm) and yellow hierarchical follicles (>8 mm) by histochemical and immunohistochemical method, respectively. Ovarian P(4) and E(2) were measured radioimmunologically. Hens stopped egg laying on day 4 of the experiment and pause in egg laying lasted up to day 12. The hens then began to gradually resume egg laying and on day 16 all hens laid eggs. It was found that during the pause in egg laying: (i) the activity of 3beta-HSD in stroma and normal white follicles was slightly decreased while P450arom activity was significantly increased; (ii) in yellow hierarchical follicles which became atretic and regressed, activity of both enzymes were markedly decreased; (iii) ovarian P(4) production dramatically decreased, whereas ovarian E(2) production after an initial decrease significantly increased. In white atretic follicles the activity of 3beta-HSD and P450arom was very weak during the whole experiment. In conclusion, the present results indicate that during a pause in egg laying white follicles become resistant to atresia.

Histamine affects blood flow through the reproductive organs of the domestic hen (Gallus domesticus).

The study was undertaken to determine the effect of histamine on blood flow to the ovary and oviduct in the domestic hen (Gallus domesticus). Cardiac output and blood flow were measured with 86RbCl through the ovarian stroma, white ovarian follicles, yellow preovulatory follicles, postovulatory follicles and four oviductal parts: infundibulum, magnum, isthmus and shell gland 1 min and 5 min after histamine treatment. In comparison with control hens which received 0.9% NaCl, histamine significantly increased (by 21.4%) cardiac output exclusively 5 min after its treatment. Blood flow (ml/min/g tissue) through the stroma, the infundibulum and the shell gland was significantly elevated both 1 min (54.3%, 84.3% and 64.2%, respectively) and 5 min (87.1%, 111.5% and 70.4%, respectively) after histamine administration and through the ovarian follicles (29.3%-61.9%) exclusively 5 min after histamine treatment. The increase in blood flow through the ovarian stroma, follicles and the oviductal parts following the administration of histamine was not the result of increased cardiac output but the consequence of local histamine action on blood flow through the ovary and oviduct. The results of the present study indicate that histamine, by influencing the hemodynamics of blood vessels and in consequence changing the blood flow through the reproductive organs, participates in the processes taking place in the ovary during growth, maturation and regression of the follicles, and in the oviduct during formation of the egg.

Effect of prolactin on estradiol and progesterone secretion by isolated chicken ovarian follicles.

In nonbroody birds, participation of prolactin in the reproductive functions is still unknown and its role in the local regulation of ovarian activity has had little attention. Therefore, the aim of the present study was to determine whether in the domestic hen prolactin influences in vitro steroid secretion by white and yellow chicken ovarian follicles. Small white (1-4 mm), medium white (4-6 mm), large white (6-8 mm) and 3 largest yellow preovulatory follicles (F3-F1; F3

Synergistic action of growth hormone and insulin-like growth factor I (IGF-I) on proliferation and estradiol secretion in porcine granulosa and theca cells cultured alone or in coculture.

The effect of growth hormone (GH) on insulin-like growth factor I (IGF-I) secretion and the effects of GH and IGF-I on [(3)H] thymidine incorporation and estradiol (E2) secretion by theca interna (Tc) and granulosa cells (Gc) cultured alone and in coculture were studied in cultured porcine follicular cells, prepared from small (SF), medium (MF) and large (LF) preovulatory follicles. We demonstrated that both Tc and Gc secrete IGF-I and that GH had no effect on IGF-I secretion by Tc but, increased IGF-I secretion by Gc isolated from SF and cultured alone or in coculture. IGF-I stimulated secretion of E2 by all cells, except in Tc derived from SF in which the effect was not statistically significant. The only stimulatory effect of concurrent treatment with GH on E2 secretion was noted in Tc derived from MF. IGF-I increased the [(3)H] thymidine incorporation in all Tc cells but GH did not augment this effect. In Gc, IGF-I stimulated [(3)H] thymidine incorporation in cells derived from SF and MF but not from LF. GH had no stimulatory effect except on Gc derived from LF and grown alone. The highest stimulatory effect was observed in SF. This was smaller in MF and no effect was noted in LF. In conclusion, our work shows that both Gc and Tc are sites of IGF-I production and for the first time shows the stimulatory role of IGF-I in proliferation of Tc cells derived from all types of follicles and augmentation of E2 secretion in Tc derived from MF and LF. The promotion of the mitogenic activity in Tc by IGF-I during all stages of follicular development suggests an important role for theca cells in follicular growth.