Is membrane androgen and estrogen receptor signaling imperative in the governing function of the adrenal cortex in the Eurasian beaver (Castor fiber L.)?

There is a need to fully know the physiology of Eurasian beaver due to its essential role in environmental homeostasis. However, a "human factor" impacts this, including stress conditions and environmental pollution. Adrenal glands protect these all. The regulation of endocrine processes by nonclassical androgen and estrogen signaling, the first and fastest control, is still a matter of research. The specific analyses performed here in mature female and male beaver adrenals contained: anatomical and histological examinations, expression and localization of membrane androgen receptor (zinc transporter, Zinc- and Iron-like protein 9; ZIP9) and membrane estrogen receptor coupled with G protein (GPER), and measurement of zinc (Zn2+) and copper (Ca2+) ion levels and corticosterone levels. We revealed normal anatomical localization, size, and tissue histology in female and male beavers, respectively. Equally, ZIP9 and GPER were localized in the membrane of all adrenal cortex cells. The protein expression of these receptors was higher (p < 0.001) in male than female adrenal cortex cells. Similarly, Zn2+ and Ca2+ ion levels were higher (p < 0.05, p < 0.01) in male than female adrenal cortex. The increased corticosterone levels (p < 0.001) were detected in the adrenal cortex of females when compared to males. The present study is the first to report the presence of nonclassical androgen and estrogen signaling and its possible regulatory function in the adrenal cortex of Eurasian beavers. We assume that this first-activated and fast-transmitted regulation can be important in the context of the effect of environmental physical and chemical stressors especially on adrenal cortex cells. The beaver adrenals may constitute an additional supplementary model for searching for universal mechanisms of adrenal cortex physiology and diseases.

Nitrophenols suppress steroidogenesis in prehierarchical chicken ovarian follicles by targeting STAR, HSD3B1, and CYP19A1 and downregulating LH and estrogen receptor expression.

To assess the effects of 4-nitrophenol (PNP) and 3-methyl-4-nitrophenol (PNMC) on steroidogenesis in the chicken ovary, white (WF, 1-4 mm) and yellowish (YF, 4-8 mm) prehierarchical follicles were incubated in a medium supplemented with PNP or PNMC (10-8-10-4 M), ovine LH (oLH; 10 ng/mL), and combinations of oLH with PNP or PNMC (10-6 M). Testosterone (T) and estradiol (E2) concentrations in media and mRNA expression for steroidogenic proteins (STAR, HSD3B1, and CYP19A1), and LH receptors (LHR), estrogen receptor α (ESR1) and ß (ESR2) in follicles were determined by RIA and real-time qPCR, respectively. PNP and PNMC decreased T and E2 secretion by the WF and YF, and oLH-stimulated T secretion from these follicles. PNP decreased basal STAR and HSD3B1 mRNA levels both in the WF and YF, and CYP19A1 mRNAs in the WF. PNP reduced oLH-affected mRNA expression of these genes in the YF. PNMC inhibited basal STAR, HSD3B1, and CYP19A1 mRNA expression in the WF, but not in the YF. PNMC reduced oLH-stimulated STAR and CYP19A1 expression in the YF and WF, respectively. PNP decreased basal mRNA expression of LHR, ESR1, and ESR2 in the WF, but it increased ESR1 and ESR2 mRNA levels in the YF. PNMC reduced both basal and oLH-affected LHR, ESR1, and ESR2 mRNA expression in the WF; however, it did not influence expression of these genes in the YF. We suggest that nitrophenols by influencing sex steroid synthesis and transcription of LH and estrogen receptors in prehierarchical ovarian follicles may impair their development and selection to the preovulatory hierarchy.

In vitro effects of PNP and PNMC on apoptosis and proliferation in the hen ovarian stroma and prehierarchal follicles.

This study aimed to examine the mRNA expression, activity, and immunolocalisation of apoptosis/proliferation regulating factors following in vitro exposure of the stroma, white (WFs), and yellowish (YFs) follicles of the chicken ovary to 4-nitrophenol (PNP) or 3-methyl-4-nitrophenol (PNMC). PNMC increased the mRNA expression of caspase-3, -8, Apaf-1, and cytochrome c in the ovarian stroma. The activity of caspase-3, -8, and -9 decreased in WFs in both nitrophenol-treated groups. PNP reduced the number of caspase-3-positive cells in the stromal connective tissue (CT) and the theca interna and externa layers of WFs. In the stroma, the proliferating index decreased in the wall of primary follicles in both nitrophenol-treated groups, however, in the CT, the effect of PNMC was opposite. In the theca interna of WFs, PNP diminished the proliferating index. These results suggest that nitrophenols might impact the development of chicken ovarian follicles by affecting cell death and proliferation.

Response of the chicken ovary to GH treatment during a pause in laying induced by fasting.

This study was undertaken to examine the effect of GH treatment during a pause in laying on (1) ovarian follicle formation, growth (folliculogenesis), and atresia; (2) follicle cell proliferation and apoptosis; and (3) mRNA expression of selected yolk-specific proteins in the chicken liver. A pause in egg laying was induced by food deprivation for 5 d, followed by feeding every other day, and then feeding daily from day 10 onward. Birds were divided into 3 groups: control (n = 18) fed ad libitum, subjected to a pause in laying (n = 18), and subjected to a pause in laying and injected every day with 200 µg/kg BW of chicken GH (chGH; n = 18). The liver, ovarian stroma, and follicles were isolated from the hens of each group on days 6 (ovary regression), 13 (ovary recrudescence), and 17 or 20 (ovary rejuvenated) of the experiment. The results showed that injection of chGH during fasting (1) increased the number of follicles <1 mm and proliferating cell nuclear antigen (PCNA)-positive (proliferating) cells in these follicles; (2) attenuated the expression of PCNA and survivin mRNA in the white follicles and the activity of caspases 3, 8, and 9 in the stroma and white follicles; (3) intensified the atresia of yellow hierarchical follicles; and (4) deepened the effect of starvation on egg yolk gene expression concomitantly with considerably increased IGF-1 transcription levels in the liver (P < 0.05 to P < 0.001). Prolongation of chGH injections into the refeeding period did not exert pronounced effects on the examined parameters. In summary, the results provide evidence that GH promotes the formation and development of prehierarchical follicles in the hen ovary during a pause in laying by regulating cell proliferation and apoptosis. Alterations in cell proliferation- and apoptosis-related gene expression or enzyme activity in ovarian follicles as well as the expression of egg yolk proteins in the liver after chGH treatment strongly suggest that this hormone is involved in determining the rate of regression and rejuvenation of the chicken ovary during a pause in laying.

Expression of aquaporin 4 in the chicken ovary in relation to follicle development.

In the mammalian ovary, aquaporins (AQPs) are thought to be involved in the regulation of fluid transport within the follicular wall and antrum formation. Data concerning the AQPs in the avian ovary is very limited. Therefore, the present study was designed to examine whether the AQP4 is present in the chicken ovary, and if so, what is its distribution in the ovarian compartment of the laying hen. Localization of AQP4 in the ovarian follicles at different stage of development was also investigated. After decapitation of hens the stroma with primordial follicles and white (1-4 mm), yellowish (4-8 mm), small yellow and the three largest yellow pre-ovulatory follicles F3-F1 (F3 < F2 < F1; 20-36 mm) were isolated from the ovary. The granulosa and theca layers were separated from the pre-ovulatory follicles. The AQP4 mRNA and protein were detected in all examined ovarian compartments by the real-time PCR and Western blot analyses, respectively. The relative expression of AQP4 was depended on follicular size and the layer of follicular wall. It was the lowest in the granulosa layer of pre-ovulatory follicles and the highest in the ovarian stroma as well as white and yellowish follicles. Along with approaching of the largest follicle to ovulation the gradual decrease in AQP4 protein level in the granulosa layer was observed. Immunoreactivity for AQP4 was present in the granulosa and theca cells (theca interna ≥ theca externa > granulosa). The obtained results suggest that AQP4 may take part in the regulation of water transport required for follicle development in the chicken ovary.

Effect of growth hormone on steroid concentrations and mRNA expression of their receptor, and selected egg-specific protein genes in the chicken oviduct during pause in laying induced by fasting.

This study was undertaken to examine the effect of growth hormone (GH) treatment during pause in laying on (1) the concentration of steroids in blood plasma and oviduct tissues, (2) the expression of mRNA of steroid receptors, and (3) the mRNA expression of selected egg-specific proteins in the chicken oviduct. A pause in egg laying was induced by food deprivation for 5 d, followed by feeding every other day, and then feeding daily from Day 10 onward. Birds were divided into three groups: control (n = 18) fed ad libitum, subjected to pause in laying (n = 18), and subjected to pause in laying and injected every day with 200 µg/kg BW of chicken GH (chGH; n = 18). The oviduct was isolated from hens of each group on Days 6 (when the oviduct was regressed), 13 (during oviduct recrudescence), and 17 or 20 (rejuvenated oviduct) of the experiment. Fasting caused a decrease in plasma concentrations of progesterone (P4), testosterone, and estradiol on Day 6 and a reduction in tissue concentrations of these steroids on Days 6 and 13. Fasting also caused an increased relative expression of estrogen receptor α and ß (ERα, ERß) and progesterone receptor (PR) in the magnum and shell gland on Day 6, increased ERα and PR in the magnum on Days 13 and 17 or 20, and increased androgen receptor (AR) mRNA in the magnum on Days 6 and 13 and in the shell gland on Day 13. A fasting-induced elevation in ovocalyxin-36 mRNA expression on Day 6 and a decrease in avidin mRNA on Days 6 and 13 and in ovocleidin-116 on Day 13 were also observed (P < 0.05 to P < 0.001). Administration of chGH abolished the fasting-induced decrease in the concentration of steroids in plasma and tissues. Furthermore, chGH enhanced the effect of fasting on mRNA expression of PR, ERα, and avidin in the magnum on Day 6, and ERα in the shell gland on Day 13. The gene expression of ovalbumin on Days 6 and 13, ovocalyxin-36 and ovocleidin-116 on Day 6 was decreased in chGH-treated chickens. In contrast, the expression of ovalbumin on Day 17 or 20 was increased (P < 0.05 to P < 0.001). The results obtained indicate that, by alterations in the concentration of steroid hormones and their receptor expression in the chicken oviduct, GH determines the rate of regression and rejuvenation of this organ during molting. Moreover, changes in the expression of selected egg proteins indicate that GH might be the regulator of the secretory activity of the hen oviduct.

Effect of 3,3',5-triiodothyronine and 3,5-diiodothyronine on progesterone production, cAMP synthesis, and mRNA expression of STAR, CYP11A1, and HSD3B genes in granulosa layer of chicken preovulatory follicles.

In vitro studies were performed to assess whether stimulatory effects of triiodothyronine (T3) on progesterone (P4) production in a granulosa layer (GL) of chicken preovulatory follicles are associated with 3',5'-cyclic adenosine monophosphate (cAMP) synthesis and mRNA expression of STAR protein, CYP11A1, and HSD3B. Effects of 3,5-diiodothyronine (3,5-T2) on steroidogenic function in these follicles were also investigated. The GL of F3 to F1 follicles was incubated in medium supplemented with T3 or 3,5-T2, LH, or forskolin (F), and a combination of each iodothyronine with LH or F. Levels of P4 and cAMP in culture media were determined by RIA. Expression of genes involved in P4 synthesis (ie, STAR protein, CYP11A1, and HSD3B) in the GL of F3 to F1 follicles incubated in medium with T3 or 3,5-T2 and their combination with LH was performed by real-time PCR. Triiodothyronine increased basal and LH- and F-stimulated P4 secretion by preovulatory follicles. The 3,5-T2 elevated P4 synthesis by F3, had no effect on F2 follicles, and diminished P4 production by the GL of F1 follicles. It had no effect on LH-stimulated P4 production; however, it augmented F-stimulated P4 production by F2 and F1 follicles. Although T3 did not affect basal and F-stimulated cAMP synthesis by the GL of preovulatory follicles, it increased LH-stimulated synthesis of this nucleotide. However, 3,5-T2 elevated F-stimulated cAMP synthesis in F3 and F2 follicles; it did not change basal and LH-stimulated cAMP production. Triiodothyronine decreased basal STAR and CYP11A1 mRNAs in F3 follicles, increased them in F1 follicles, and elevated HSD3B mRNA levels in F1 follicles. Triiodothyronine augmented LH-stimulated STAR, CYP11A1, and HSD3B mRNA levels in F2 and CYP11A1 in F1 follicles. However, T3 decreased LH-stimulated STAR and HSD3B mRNA levels in F1 follicles. The 3,5-T2 did not affect basal STAR and CYP11A1 mRNA expression in all investigated follicles; however, it decreased LH-stimulated STAR expression in F2 and F1 ones. The effects of 3,5-T2 caused elevated basal but diminished LH-stimulated HSD3B mRNA levels. In conclusion, data indicate that both iodothyronines are involved in P4 production in the GL of chicken preovulatory follicles acting alone and additively with LH. Effects of iodothyronines depend on follicle maturation and are associated with modulation of cAMP synthesis and STAR, CYP11A1, and HSD3B mRNA expression. We suggest that iodothyronines participate in maturation and ovulation of chicken follicles.

Sex steroids level in blood plasma and ovarian follicles of the chimeric chicken.

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.

Tamoxifen decreases level of immunoglobulins in blood of the hen (Gallus domesticus) without alteration in non-immunoglobular fractions of plasma proteins.

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.

Attenuation by leptin of the effects of fasting on ovarian function in hens (Gallus domesticus).

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.

Presence of histamine and mast cells in chicken oviduct.

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.

Simultaneous determination of plasma ovarian and thyroid hormones during sexual maturation of the hen (Gallus domesticus).

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.