Ovaries Of Laying Hens Research Articles

Tamoxifen-induced alterations in the expression of connexin 43 in the chicken ovary

Connexin 43 (Cx43) is a gap junction protein that participates in small molecule exchange between adjacent cells. It is a predominant Cx within the mammalian ovary, where is associated with proper follicle development. The expression and regulation of Cx43 in the chicken ovary is largely unknown. The aim of the present study was to examine the expression of the Cx43 gene (GJA1) and protein as well as the immunolocalization of Cx43 in the laying hen ovary in relation to follicle development, and to examine how tamoxifen (TMX; an estrogen receptor modulator) treatment affects these factors. qRT-PCR and western blotting demonstrated differences in Cx43 mRNA transcript and protein abundances in ovarian white follicles, yellowish follicles, small yellow follicles, and the largest yellow preovulatory follicles (F3–F1). In general, Cx43 was more abundant in hierarchical than prehierarchical follicles and in granulosa cells compared with theca cells. Further, the response to TMX treatment depended on the stage of follicle development and the layer of the follicular wall. Ovarian regression following TMX treatment was accompanied by an increase in Cx43 expression in most ovarian tissues, which may impact the formation and function of Cx43 hemichannels. Overall, our results showed, for the first time, the differences in Cx43 mRNA and protein levels between ovarian follicles, suggesting the potential involvement of this gap junction protein in the regulation of ovarian follicle development and function. In addition, the results indicate a possible role for estradiol in regulation of Cx43 transcription and/or translation in the chicken ovary. Understanding the contribution of Cx43 in mechanisms underlying ovarian follicle development may be of considerable importance for poultry egg production.

Tamoxifen-induced alterations in the expression of selected matrix metalloproteinases (MMP-2, -9, -10, and −13) and their tissue inhibitors (TIMP-2 and -3) in the chicken ovary

Matrix metalloproteinases (MMPs) are a family of peptidases that disintegrate extracellular matrix (ECM) molecules associated with tissue remodeling, including reproductive tissues. Their actions are largely controlled by specific tissue inhibitors of MMPs (TIMPs). The role and regulation of MMPs in the chicken ovary is largely unknown. The aim of the present study was to examine the effect of tamoxifen (TMX; estrogen receptor modulator) treatment on the expression of selected members of the MMP system in the laying hen ovary. The activity of MMP-2 and -9 was also examined. Real-time polymerase chain reaction and western blot analyses revealed changes in mRNA and/or protein expression of MMP-2, -9, -10, −13, TIMP-2, and TIMP-3 in the following ovarian follicles after TMX treatment: white (WF), yellowish (YF), small yellow (SYF), and the largest yellow preovulatory (F3–F1). The response to TMX depended on the stage of follicle development and the layer of follicular wall. Moreover, ovarian regression following TMX treatment was accompanied by both an increase in total activity of MMP-2 in the theca layer of F3–F2 and granulosa layer of F2, and a decrease in total activity of MMP-2 in the WF, YF, and SYF, and MMP-9 in theca of F3–F1. In conclusion, the TMX-induced changes in MMP-2, -9, -10, and -13, and TIMP-2 and -3 mRNA expression, as well as MMP-2 and -9 activity, were dependent on tissue and the stage of follicular maturation. Our findings strongly suggests a role for estrogen in regulating the transcription, translation, and/or posttranslational activity of members of the MMP system. Further, these components may be involved in the orchestration of ECM turnover and cellular functions during ovary regression, which occur under conditions of reduced estrogenic activity.

Expression of gelatinases (MMP-2 and MMP-9) and tissue inhibitors of metalloproteinases (TIMP-2 and TIMP-3) in the chicken ovary in relation to follicle development and atresia

Matrix metalloproteinases (MMPs) are a family of peptidases that possess the ability to break down extracellular matrix macromolecules associated with tissue turnover in various physiological and pathological conditions. Their activity is largely regulated by specific tissue inhibitors of MMPs (TIMPs). Information concerning the role of MMPs in the chicken ovary is very limited. The aim of the present study was to determine the expression and localization of selected members of the MMP system in different compartments of the laying hen ovary and to investigate whether their expression changes at different stages of the ovulatory cycle. MMP-2 and -9 activity was also examined. Expression of MMP-2, -9 and tissue inhibitors of MMPs (TIMP-2 and -3) in the ovarian follicles was examined 22 h and 3 h before F1 ovulation. Real-time polymerase chain reaction and western blot revealed differential mRNA and protein expression of MMP-2, MMP-9, TIMP-2, and TIMP-3 in the ovarian follicles: white, yellowish, small yellow, the largest preovulatory (F3-F1), and white atretic. Within the ovary, the relative expression of MMP and TIMP mRNA depended on follicle development, the layer of follicular wall, and ovulation stage. The relatively higher expression of MMP-2 and MMP-9 mRNA in the ovarian follicles 3 h compared to 22 h before ovulation was found. As follicle development progressed toward ovulation, elevated MMP-2 and -9 activity was noted. Atresia of white follicles was accompanied by an increase in gelatinase activities. Immunohistochemistry demonstrated tissue- and follicle-dependent immunoreactivity of the examined MMPs and TIMPs. In summary, the results show tissue- and stage of the ovulatory cycle-dependent differences in MMP and TIMP expression, as well as MMP-2 and -9 activity. Findings that suggest these molecules might significantly participate in the complex remodeling of extracellular matrix required for follicle development, ovulation, and atresia in the chicken ovary.

Histology of the Ovary of the Laying Hen (Gallus domesticus).

The laying hen (Gallus domesticus) is a robust animal model for epithelial ovarian cancer. The use of animal models is critical in identifying early disease markers and developing and testing chemotherapies. We describe the microscopic characteristics of the normally functioning laying hen ovary and proximal oviduct to establish baselines from which lesions associated with ovarian cancer can be more readily identified. Ovaries and oviducts were collected from 18-month-old laying hens (n = 18) and fixed in 10% neutral buffered formalin. Hematoxylin- and eosin-stained sections were examined by light microscopy. Both post-ovulatory follicular regression and atresia of small follicles produce remnant clusters of vacuolated cells with no histological evidence that scar tissue persists. Infiltrates of heterophils are associated with atresia of small follicles, a relationship not previously documented in laying hen ovaries. Because these tissues can be mistaken for cancerous lesions, we present a detailed histological description of remnant Wolffian tissues in the laying hen ovary. Immunohistochemical staining for pancytokeratin produced a positive response in ovarian surface epithelium and staining for vimentin produced a positive response in granulosa cells of follicles. Epithelial cells lining glands of the remnant epoöphoron had a positive response to both pancytokeratin and vimentin, a result also observed in women.

Ethylene Glycol and Dimethyl Sulfoxide Combination Reduces Cryoinjuries and Apoptotic Gene Expression in Vitrified Laying Hen Ovary.

Successful cryopreservation of avian gonads is important not only for avian breeding but is also crucial for preservation of species, especially of endangered birds. The aim of this study was to evaluate the effect of vitrification by several cryoprotectants on the ovarian tissues of laying hens. Ovarian tissues were randomly divided into six groups: control (nonvitrified: C), dehydrated using ethylene glycol (EG), dehydrated with propylene glycol (PROH), dehydrated using dimethyl sulfoxide (DMSO), and two combined groups, EG+DMSO and EG+PROH. The composition of vitrification solutions was as follows: EG group: V1 = 7.5% EG and V2 = 15% EG +0.5 M sucrose, DMSO group: V1 = 7.5% DMSO and V2 = 15% DMSO +0.5 M sucrose, PROH group: V1 = 7.5% PROH and V2 = 15% PROH +0.5 M sucrose, EG+DMSO group: V1 = 7.5% EG +7.5% DMSO and V2 = 15% EG +15% DMSO +0.5 M sucrose and EG+PROH group: V1 = 7.5% EG +7.5% PROH and V2 = 15% EG +15% PROH +0.5 M sucrose. Ovarian tissues of each group were dehydrated for 10 minutes with V1 solution and 2 minutes with V2. Among the vitrified groups, intact primordial and primary follicles showed significant increase in EG+DMSO, but follicular attrition had the highest rate in the PROH group (p < 0.05). Immunohistochemical analysis showed that the percentage of active caspase 3-positive cells was lower (p < 0.05) when using EG+DMSO versus PROH. Further gene expression of caspase 3, 8, and 9 was highest in the PROH group (p < 0.05). Vitrification of ovaries of laying hens using EG+DMSO can afford effective protection of primordial and primary follicles during preservation and may therefore be successfully used for storing avian gonadal tissues.

Granulosa cell responsiveness to follicle stimulating hormone during early growth of hen ovarian follicles

In the laying hen ovary, the cyclic recruitment of a follicle represents a process in which a single follicle is selected to enter the rapid growth phase and undergo final maturation prior to ovulation. Published data support the proposal that final differentiation of the granulosa cell (GC) layer commences at the time of follicle selection. This process is characterized by the enhanced capacity for FSH-induced cell signaling via the protein kinase A/cyclic adenosine monophosphate (cAMP) pathway. One consequence of such signaling within the GC layer is the initial capacity for steroidogenesis (predominantly progesterone production) mediated by increased expression of mRNA encoding steroidogenic acute regulatory protein (STAR) and the cholesterol side-chain cleavage enzyme (CYP11A). Prior to selection, the GC layer remains minimally responsive to a 3 h challenge with FSH (10 ng/mL), in vitro, compared to that from the most recently selected 9- to 12-mm follicle. By comparison, when the duration of the cell culture prior to FSH challenge is increased to 18 h, GCs collected from 1- to 2-mm, 3- to 5-mm, and 6- to 8-mm follicles respond to a 3 h FSH challenge by increasing STAR expression and progesterone production, with the greatest response from GCs collected from 6- to 8-mm follicles. Culture with Bone Morphogenetic Protein 6 (BMP6) enhances both CYP11A expression and FSH responsiveness at each stage of development, with the greatest response again occurring in GCs from 6- to 8-mm follicles. Significantly, factors that activate mitogen activated protein kinase (MAPK) or protein kinase C (PKC) signaling prevent the ability of prolonged culture or culture with BMP6 to induce FSH-responsiveness and the initiation of GC differentiation at each stage of development. Collectively, these results provide further support for the hypothesis that prior to follicle selection, inhibitory cell signaling (e.g., MAPK, PKC) maintains the GC layer in an undifferentiated state in follicles of all sizes, even in the presence of a differentiation-promoting signal (BMP6). The process by which the GC layer from the single 6- to 8-mm follicle selected each ovulatory cycle ultimately escapes inhibitory signaling to initiate FSH-responsiveness remains to be established.

Expression of Matrix Metalloproteinase-2 mRNA in the Chicken Ovary in Relation to Follicle Remodelling

In the mammalian ovary, matrix metalloproteinase-2 (MMP-2) is thought to be involved in the regulated turnover of the extracellular matrix during the process of follicle development, ovulation and regression. Information concerning the role of MMP-2 in the chicken ovary is scarce. The present investigation was undertaken to determine the distribution of MMP-2 mRNA within the laying hen ovary as well as to define whether the expression of MMP-2 mRNA changes during the ovulatory cycle. Expression of MMP-2 mRNA in ovarian compartments was determined by RT-PCR analysis at two stages of the ovulatory cycle, i.e. at 22 h and 3 h before ovulation. MMP-2 mRNA was detected in the ovarian stroma and follicles: white, yellowish, small yellow, the largest preovulatory (F3-F1) and postovulatory (P1-P5). In the wall of preovulatory follicles MMP-2 mRNA was found in the theca and granulosa layers. Within the ovary the relative expression of MMP-2 mRNA depended on follicular size, the layer of the follicular wall, the stage of the ovulatory cycle and the degree of regression in the postovulatory follicles. The results obtained indicate that in the chicken ovary MMP-2 may take part in the extracellular matrix remodelling required for follicle growth, development and regression.

Differential expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and activin type-I and type-II receptors in preovulatory and prehierarchical follicles of the laying hen ovary

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGFbeta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (approximately 40 mm). Ovaries were collected (n = 16) from mid-sequence hens maintained on a long-day photoschedule (16 h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of mRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0.05) in 8-9.9 mm follicles, whereas ActRIIA rose significantly from 6-7.9 mm to 8-9.9 mm, before falling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan mRNA expression rose 3-fold from 4-5.9 mm to 8-9.9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4-7.9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0.05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 mm to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActRIIA increased 2-fold from F2 to F1 (P < 0.05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before falling approximately 50% in the F1. In all follicles studied expression of betaglycan and ActRI (granulosa: r = 0.65, P < 0.001, n = 144/group; theca: r = 0.49, P < 0.001, n = 144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI, ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11.4-fold; ActRIIB, 5.1-fold; ActRI, 3.8-fold; ActRIIA, 2.8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.

Distribution of histamine in laying hen ovary.

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.

Immunocytochemical localization of progesterone receptor in the chick ovary.

The distribution of progesterone receptor (PR) in the chick ovary was studied using light- and electron-microscopic immunocytochemical methods. With the light microscopic technique, PR was observed in the germinal epithelium cells of estrogen-treated and estrogen-untreated immature chicks. With the preembedding immunocytochemical technique, which proved to be more sensitive than immunohistochemistry using paraffin sections, the germinal epithelium and also part of the thecal and stromal cells were stained when immature chicks were not treated with estrogen. After estrogen treatment, the number of PR-positive stromal and thecal cells increased, as did the immunostaining intensity in their nuclei. The granulosa cells were PR-positive only after estrogen treatment. In the ovary of laying hens, the most intense staining for PR was localized in the germinal epithelium. PR was also present in the thecal, stromal, and granulosa cells. At the subcellular level, PR was detected only in the cell nuclei, indicating that ovarian PR is intranuclear independent of the presence of progesterone. In conclusion, immunocytochemical methods proved to be suitable for studying steroid hormone receptors in steroid-producing tissues (e.g. the ovary), because excess endogenous hormones do not affect detection of the receptor with the antibody as they do detection with labeled ligands. Immunocytochemically, the germinal epithelium, stromal, thecal, and granulosa cells of the laying hen ovary were shown to be target cells for progesterone. The inducibility of PR by estrogen in the thecal, stromal, and granulosa cells suggests that these cell types are also sensitive to estrogen.

IODINE ENVIRONMENT AND THYROID FUNCTION IN THE DEVELOPING CHICK EMBRYOS

To evaluate quantitatively and qualitatively iodine metabolism and thyroid function of the developing chick embryo, we measured total iodine contents and identified chemical nature of the main iodine components of the egg (white Leghorn) by an autoanalizer for PBI (Technicon) and other analytical methods.The iodine was predominantly distributed in the yolk (21 μg, 74%) at first, while a small portion (0.9 μg) was found in the white. The contents in the shell (6 pig) and shell membrane (0.4 μg) remained constant during incubation but the latter increased to 1.5 pig on Day 18. After Day 10, when vascularization of the yolk was well developed and the thyroid started to accumulate iodide, yolk iodine decreased abruptly to Day 12 (6.2 pig), then gradually to Day 18 (0.9 μig), while iodine contents in the amniotic and allantoic fluids increased inversely from Day 10 to Macimally Day 15 (14.2 and 8.4 pig, respectively). On Day 18, however, the iodine in the two compartments, particularly in the amnion was reduced (3.9 and 6.9 pig, respectively).In accordance with those changes, serum iodine in the embryo started to increase gradually from Day 10 (44 μg/ml), tliereafteir vigorously to Day 20 (1, 580 ng/ml). After hatching, serum iodine decreased dramatically probably via urine excretion.Thyroidal iodine also increased from Day 10 (34 ng/gland) and rapidly from Day 15 (560 ng/gland) to Day 18 (2, 341 ng/gland). Serum T4 level measured by RIA increased gradually to Day 19 (17 ng/ml), whereas T3 showed no marked change.We then extracted the iodine in the yolk, serum (Day 19) and the two fluids (Day 15) with CHCl3 and MeOH (2:1) and found the iodine more than 80% in MeOH-H2O layer and others were found as protein-bound and lipid-bound iodine. The iodine rich fraction was analyzed by five methods, i) Sephadex G 10 column, ii) Sephadex G 25 column, iii) thin-layer chromatography, iv) cation-exchange chromatography, and v) a fully automated amino acid analyzer. We identified two major components, as iodide and monoiodohistidine (MIH). Besides those, diiodotyrosine was also identified among other minor components. The two major components were identified in amniotic and allantoic fluids as well as in the yolk and serum.We concluded from the present findings that laying hen ovary concentrates an excess amount of iodine mainly as iodine and MIH into the yolk, and iodide as active source for thyroid hormone synthesis and MIH as an inert iodine compound circulating in compartments of the developing chick embryo. Thus, a role of MIH in the closed environment might be a detoxication mechanism which was subservient to avoid the Wolff-Chaik off efect caused by excess iodide to inhibit thyroid functions.

Antigenicity of Laying Hen Ovary

Antigenicity of Laying Hen Ovary