Theca Layer Research Articles

Expression of cocaine- and amphetamine-regulated transcript (CART) in hen ovary

BackgroundCocaine- and amphetamine-regulated transcript (CART), discovered initially by via differential display RT-PCR analysis of brains of rats administered cocaine, is expressed mainly in central nervous system or neuronal origin cells, and is involved in a wide range of behaviors, such as regulation of food intake, energy homeostasis, and reproduction. The hens egg-laying rate mainly depends on the developmental status of follicles, expression of CART have not been identified from hen follicles, the regulatory mechanisms of CART biological activities are still unknown. The objective of this study was to characterize the mRNA expression of CART in hen follicular granulosa cells and determine CART peptide localization and regulatory role during follicular development.MethodsSmall white follicles (1–2 mm in diameter) were treated for RNA isolation; Small white follicles (1–2 mm in diameter) and large white follicles (4–6 mm in diameter) were treated for immunohistochemical localization and large white follicles (4–6 mm in diameter), small yellow follicles (6–8 mm in diameter), large yellow follicles (9–12 mm in diameter), mature follicles (F5, F4, F3, F2, F1, >12 mm in diameter) were treated for RNA isolation and Real time PCR.ResultsThe results showed that full length of the CDS of hen CART was 336 bp encoding a 111 amino acid polypeptide. In the hen ovary, CART peptide was primarily localized to the theca layer, but not all, the oocyte and granulosa layer, with diffused, weaker staining than relative to the theca cell layer. Further, amount of CART mRNA was more (P < 0.05) in granulosa cells of 6–8 mm follicles compared with that in granulosa cells of other follicles. However, CART mRNA amount was greater in theca cells of 4–6 mm follicles relative to follicles of other sizes (P < 0.05).ConclusionsResults suggest that CART could play a potential role in developmental regulation of chicken follicles.

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 (1-4mm), yellowish (4-8mm), small yellow and the three largest yellow pre-ovulatory follicles F3-F1 (F3<F2<F1; 20-36mm) 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.

Expression of cytokine-induced neutrophil chemoattractant suppresses tumor necrosis factor alpha expression and thereby prevents the follicles from undergoing atresia and apoptosis.

AimCytokine‐induced neutrophil chemoattractant (CINC/gro) is a CXC family chemokine, similar to interleukin‐8 in rats, and is one of the factors that regulates ovulation. However, the mechanism that regulates atresia of the ovaries postovulation is not clearly defined.MethodsWhether antibody‐blocking of CINC/gro can alter the number of ovulated oocytes and modulate neutrophil infiltration was investigated. The effect of the antibody on the level of inflammatory cytokine production and follicular atresia was examined. Apoptosis was measured by the terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) method and via analysis of the messenger RNA expression of Bcl‐2 and Bcl2‐associated X (Bax).ResultsThe anti‐CINC/gro antibody treatment decreased the number of ovulated oocytes. The messenger RNA levels of cyclooxygenase‐2 and interleukin‐1 beta were decreased by the antibody treatment, whereas that of tumor necrosis factor (TNF) alpha was increased. The TUNEL analysis revealed a larger number of apoptotic cells in the antibody group, compared with those in the control group, as well as a significant increase in the Bax/Bcl‐2 ratio 24 hours after human chorionic gonadotropin administration.ConclusionThese findings suggest that ovulation is accelerated by neutrophil infiltration into the theca layer. The CINC/gro appears to synergize with interleukin‐1 beta for ovulation. By contrast, the data suggest that CINC/gro expression suppresses TNF alpha expression and that CINC/gro expression therefore prevents the follicles from undergoing atresia and apoptosis.

Time course effect of lipopolysaccharide on Toll-like receptors expression and steroidogenesis in the Chinese goose ovary.

The ovary of Chinese goose is easily infected by microorganisms because of the mating behaviour in water, which causes decreased laying performance. This study investigated the time course effect of lipopolysaccharide (LPS) on the steroidogenesis and mRNA expression of Toll-like receptors (TLRs), a class of key pattern recognition receptor, in the breeding goose ovary. The laying geese were treated intravenously with LPS for 0, 6, 12, 24 and 36 h, and all birds were slaughtered approximately 8 h after oviposition. The expression levels of TLRs in the white and yellowish follicles, and granulosa and theca layers of hierarchical follicles were examined by real-time PCR. All 10 members of avian TLR family were differentially expressed among the different follicular tissues. Moreover, at 24 and 36 h after LPS treatment, the hierarchical follicle morphological structure was altered, but the expression levels of TLRs were still higher than the control. Furthermore, during LPS treatment period, the expression pattern of TLRs 2A and 4 genes was similar to that of TLR15 in the white follicles, TLRs 1B, 5 and 15 in the yellowish follicles, TLRs 7 and 15 in the granulosa layer, and TLRs 1A, 2B, 3, 7 and 15 in the theca layer, which had a negative correlation with the kinetics of plasma P4 and E2 concentrations. In conclusion, the mechanism by which pathogen infection inhibited goose follicular growth and further decreased egg production may involve a gradually enhanced inflammatory response and reduced endocrine function. This may be due to stimulated TLRs in the ovary.

Regulatory Mechanisms Underlying the Expression of Prolactin Receptor in Chicken Granulosa Cells

Prolactin (PRL) has both pro- and anti-gonadal roles in the regulation of avian ovarian functions through its interaction with the receptor (PRLR). However, neither the pattern of expression of PRLR nor its regulatory mechanisms during follicle development have been clearly defined. The objective of the present study was to investigate mechanisms of PRLR expression in chicken granulosa cells. Levels of PRLR transcript were highest in the stroma and walls of follicles < 2 mm in diameter and progressively declined with the maturation of follicles. In preovulatory follicles, PRLR was expressed at higher levels in granulosa than theca layers. FSH exerted the greatest stimulatory effect on PRLR and StAR expression in cultured granulosa cells of the 6–8 mm follicles but this effect declined as follicles matured to F1. In contrast, LH did not alter the expression of PRLR in granulosa cells of all follicular classes but increased levels of StAR in F2 and F1 granulosa cells. Both non-glycosylated- (NG-) and glycosylated- (G-) PRL upregulated basal PRLR expression in granulosa cells of the 6–8 mm, F3 or F1 follicles but had little effect in F2 follicles. Furthermore, FSH-stimulated PRLR expression was reduced by the addition of either isoform of PRL especially in F2 granulosa cells. These results indicate that PRLR is differentially distributed and regulated by FSH or PRL variants independently or in combination in the follicular hierarchy. By using activators and inhibitors, we further demonstrated that multiple signaling pathways, including PKA, PKC, PI3K, mTOR and AMPK, are not only directly involved in, but they can also converge to modulate ERK2 activity to regulate FSH-mediated PRLR and StAR expression in undifferentiated granulosa cells. These data provide new insights into the regulatory mechanisms controlling the expression of PRLR in granulosa cells.

Lysophosphatidic acid expression in theca cells depends on the type of bovine ovarian follicle.

Lysophosphatidic acid (LPA) exerts various actions on the mammalian reproductive system. In cows, LPA stimulates the synthesis and secretion of luteotropic factors in the ovary, which affects the growth and development of ovarian follicles. The role of LPA in granulosa cells, oocyte and oocyte-cumulus complex (COC) has previously been investigated; but its role in the theca layer, which is an important structural and functional component of the ovarian follicle, is still unclear. The goal of this study was to investigate the expression of LPA in theca cells originating from different bovine ovarian follicle types. Theca cells were separated from healthy, transitional and atretic ovarian follicles, based on intrafollicular estradiol: progesterone ratios. LPA concentration in the follicular fluid (FF) in different follicle types was measured, and expression of the enzymes responsible for LPA synthesis (autotaxin [AX], phospholipase A2 [PLA2]) and receptors for LPA (LPAR1-4) were determined. The obtained results confirmed the follicle-type dependent presence of LPA in the FF of the bovine ovarian follicles. The highest concentration of LPA was detected in follicles classified as healthy and dominant. LPAR1-4, PLA2 and AX expression in theca cells in all of the types of follicles examined were detected at mRNA and protein level. These results suggest that theca cells can be a source of LPA synthesis other than granulosa cells and COCs, as well as the target for its action in the bovine ovarian follicle, with PLA2 and LPAR4 playing major roles in LPA synthesis and action.

The dynamics of trkA expression in the bovine ovary are associated with a luteotrophic effect of ovulation-inducing factor/nerve growth factor (OIF/NGF).

BackgroundOvulation-inducing factor in semen (OIF/NGF) influences ovulation and CL form and function in camelids and, remarkably, in cows. To test the hypothesis that the luteotrophic effect of OIF/NGF is mediated by an increase in trkA receptors in the ovulatory follicle and early CL, a study was designed to characterize the spatial and temporal distribution of trkA in ovarian follicles and CL at known stages of the bovine estrous cycle.MethodsSexually mature cattle (n = 14) were examined daily by transrectal ultrasonography to determine the day of ovulation (Day 0), and assigned randomly to be unilaterally ovariectomized on Day 2, 4, 6 or in the pre-ovulatory period just before or after exogenous LH treatment. After a complete interovulatory interval, the cows were re-assigned to a different day–group on which the remaining ovary was removed. Sections of ovarian tissue representing the dominant follicle, largest subordinate follicle, and the CL were processed for immunofluorescent detection and quantification of trkA receptor.ResultsTrkA immuno-fluorescence in ovarian tissues was restricted to follicles and the CL (no reaction in stroma or vessels), and was restricted to the cytoplasm (no nuclear staining). The trkA staining intensity, area of staining, and proportion of cells stained was greater in both theca and granulosa layers of dominant follicles than in that of subordinate follicles (P ≤ 0.05) in all day-groups except the Pre-LH group. Among dominant follicles, a progressive reduction in the immuno-positive reaction was detected from Day 2 to Day 6. Among subordinate follicles, immuno-reactivity remained low and unchanged except a rise in the Pre-LH group. The number of immuno-positive cells was greater in early developing CL (Days 2 and 4 combined) than in mature or regressing stage CL (Day 6, Pre- and Post-LH combined; P = 0.01). The intracellular distribution of trkA was more diffuse and widespread in dominant than subordinate follicles, particularly on Day 2 and Post-LH (P < 0.05).ConclusionsDistinct differences in trkA expression between dominant and subordinate follicles, particularly when circulating progesterone is minimal (early luteal development and after luteolysis) is consistent with a local role of OIF/NGF in follicle selection and early luteogenesis.

Ovarian follicular dynamics after aromatizable or non aromatizable neonatal androgenization.

The effects of neonatal testosterone or dihydrotestosterone exposure on ovarian follicular dynamics were analysed at prepubertal, pubertal or adult age in Wistar rats. Both androgens induced a transitory increase on follicular endowment that was partially corrected at puberty. At adult age testosterone prevented ovulation, without significant modifications on follicular dynamics. An increased number of cystic structures were observed from puberty to adult age. However, ovaries of rats treated with dihydrotestosterone showed follicles with evident morphological alterations in granulosa and thecal layers although several corpora lutea were observed. A significant increase in preantral follicles and few cystic structures were detected at advanced adulthood. The size of cyst increased with age. No immunohistochemical changes on growth factors or enzymes related to steroidogenesis in growing follicles were obvious in any group. In both androgenized groups, cysts shared immunohistochemical characteristics exhibited by preovulatory follicles but they were unable to ovulate spontaneously. Our results provide an insight into the role of different androgens in female reproductive system development, indicating a direct effect of dihydrotestosterone on ovarian tissues whereas a central effect would be the main feature of neonatal testosterone exposure. Heterogeneous clinical manifestations seen in pathologies such as polycystic ovary syndrome among women could be associated with subtle hormonal changes during follicular population development.

Apoptotic Cell Localization in Preantral and Antral Follicles in Relation to Non-cyclic and Cyclic Gilts.

The objective of this study was to determine apoptotic cell localization in preantral and antral follicles of porcine ovaries. Additionally, the proportion of cells undergoing apoptosis was also compared between delayed puberty gilts and normal cyclic gilts. Ovarian tissues were obtained from 34 culled gilts with age and weight of 270.1±3.9days and 143.8±2.4kg, respectively. The gilts were classified according to their ovarian appearance as 'non-cyclic' (n=7) and 'cyclic' (n=27) gilts. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay was used to determine apoptotic cell expression in different compartments of the ovarian tissue sections. All apparent preantral (n=110) and antral (n=262) follicles were evaluated using image analysis software. It was found that apoptotic cells were expressed in both granulosa (22.2%) and theca cell layers (21.3%) of the follicles in the porcine ovaries. The proportion of apoptotic cells in the granulosa layer in the follicles was positively correlated with that in the theca layer (r=0.90, p<0.001). Apoptosis did not differ significantly between preantral and antral follicles in either granulosa (27.8% and 26.4%, p>0.05) or theca cell layers (28.6% and 26.5%, p>0.05). The proportion of apoptotic cells in non-cyclic gilts was higher than cyclic gilts in both granulosa (31.7% and 22.6%, p<0.001) and theca cell layers (34.8% and 20.2%, p<0.001). This study indicated that apoptosis of the granulosa and theca cell layers in the follicles was more pronounced in the ovarian tissue of delayed puberty gilts than cyclic gilts. This implied that apoptosis could be used as a biologic marker for follicular development/function and also that apoptosis was significantly associated with anoestrus or delayed puberty in gilts, commonly observed in tropical climates.

Overexpression of Oct4 in porcine ovarian stem/stromal cells enhances differentiation of oocyte-like cells in vitro and ovarian follicular formation in vivo.

BackgroundRecent findings have revealed that the female gonad may have regenerative activity with having germ line stem cells in juveniles and adults. Application of these germ line stem cells could be an alternative therapy for reproductive disorders in regenerative medicine.MethodsTo enhance the potency of differentiation into oocyte-like cells (OLCs) and folliculogenesis, we overexpressed Oct4 in ovarian stem/stromal cell (OvSCs) and examined the cellular properties related to stemness and self-renewal ability and finally demonstrated the ability of in vitro differentiation and folliculogenesis.ResultsOvarian cortex included putative stem cells in terms of AP activity, cell cycle status, cell proliferation, expression of mesenchymal lineage surface markers and pluripotent transcriptional markers. Further, Oct4 transfected OvSCs (Oct4-OvSCs) were enhanced their AP activity and cell proliferation compared to OvSCs. The potential on in vitro differentiation into OLCs and in vivo folliculogenesis was also evaluated in OvSCs and Oct4-OvSCs, respectively. Oct4-OvSCs possessed higher oogenesis potential in vitro than OvSCs, in terms of expression of germ cell markers by RT-PCR and the number of OLCs. When OvSCs and Oct4-OvSCs were xeno-transplanted into infertile mice ovaries, the OvSCs transplantation induced new primary follicle formation and hormonal levels of estradiol and FSH remained similar to that of normal mice. However, Oct4-OvSCs possessed higher ability for folliculogenesis based on inducing developing follicles with thecal layer and granulosa cells and more similar estradiol level to normal mice.ConclusionsThese findings demonstrated that putative stem cells were present in ovarian cortex and exhibited differentiation ability into OLCs and folliculogenesis in vivo, and Oct4-overexpression enhanced these ability, suggesting their cellular models based on gene therapy in understanding the mechanisms of oogenesis and folliculogenesis, and finally in view of reproductive cell therapy.

Metabolic changes, ovarian function and growth related gene expression in goats given stair-step feeding during estrous cycle

The study was conducted to determine effects of the stair-step feeding during estrous cycle on metabolic changes, ovarian function, and growth related gene expression. Mature goats (n=48) that exhibited at least two normal, consecutive estrous cycles were randomly assigned to either the control or stair step fed group. In the control group, goats were fed at 100% of nutrient requirement (at a rate to maintain normal growth of 46–50g/day) throughout two consecutive estrous cycles. The goats in the stair-step group were fed 70 and 130% of the control for first estrous and second estrous cycles, respectively. Blood and ovaries samples were collected throughout the 6-week experiment to examine concentration of blood metabolites, progesterone (P4) and ovarian cells proliferation. Serum glucose, insulin concentrations and number of visible follicles were greater (P<0.05) in stair-step fed goats than in control goats. Serum P4 concentrations were not different (P>0.05) throughout the experiment. Percentages of good oocyte quality obtained from all sizes of follicle were better (P<0.05) in the stair-step fed goats than that of the control goats. Rates of cell proliferation in granulosa and thecal layers across all follicular size, as determined by labeling index, were greater (P<0.05) in stair-step fed goats than in control goats. The mRNA levels of BCL-2 gene in granulosa and thecal layers in all follicular size were similar between groups, except for granulosa layer of follicle size 1–3mm, expression of BCL-2 gene in the stair-step fed goats was greater than (P<0.05) that of the control group. These data demonstrate the stair-step feeding management affects metabolic, ovarian functions, and expression of BCL-2 gene in goats.

Vascular endothelial growth factor and angiopoietins during hen ovarian follicle development

Growth and maturation of ovarian follicles in the hen (Gallus gallus) requires a network of blood vessels that increases in complexity during development. The present studies investigate expression of vascular endothelial growth factor A (VEGF), angiopoietin1 (ANGPT1) and ANGPT2 mRNAs together with their associated receptors (VEGFR and TIE2, respectively) during maturation. Elevated expression of VEGF and its receptors is associated with healthy, compared to atretic, follicles. Levels of VEGF significantly increase, while antagonistic ANGPT2 decrease, in granulosa cells (GC) at follicle selection. By comparison, levels of VEGF, VEGFR1, VEGFR2, ANGPT1, ANGPT2 and TIE2 within the theca layer do not change (P>0.05) relative to developmental stages surrounding follicle selection (6–8mm versus 9–12mm follicles). Prior to selection, treatment with transforming growth factor β1 (TGFβ1) significantly increases levels of VEGF in undifferentiated GC from prehierarchal (6–8mm) follicles and actively differentiating GC from selected (9–12 and F4) follicles. Moreover, subsequent to selection follicle stimulating hormone (FSH) increases VEGF expression in GC from 9 to 12mm follicles, and eventually luteinizing hormone (LH) promotes VEGF expression in GC from more mature preovulatory follicles. It is concluded that prior to follicle selection VEGF expression is regulated by autocrine and paracrine actions of TGFβ1 (but not FSH), and that a comparatively limited extent of vasculature is sufficient to maintain prehierarchal follicles in a viable and undifferentiated state. At follicle selection, FSH- and subsequently LH-induced VEGF production within the GC layer enhance angiogenesis within the theca layer, which facilitates the rapid growth of preovulatory follicles via enhanced incorporation of yellow yolk.

Morphometric characteristics of preantral and antral follicles and expression of factors involved in folliculogenesis in ovaries of adult baboons (Papio anubis).

Baboons are commonly utilized as an animal model for studies of human reproduction. However, folliculogenesis in this species has not been fully documented. The aim of this study was to assess follicle morphometry and expression of essential proteins involved in folliculogenesis in baboons. Ovaries were recovered from four adult baboons and processed for histological evaluation and immunohistochemical analyses. Follicle proportion, follicle and oocyte diameter, theca layer thickness, number of granulosa cells, and follicle density were calculated. Immunohistochemical staining was also carried out for connexin 43 (Cx43), aromatase, and zona pellucida 3 (ZP3). A total of 2221 follicles were counted and measured. Proportions of primordial, primary, secondary, small antral, and large antral follicles were 49, 26, 23, 1, and 1%, respectively. The increase in follicle diameter was due not only to the increase in oocyte diameter but also to granulosa cell proliferation. Almost all antral follicles were positive for Cx43 (89.8%), aromatase (84.8%), and ZP3 (100%). Most secondary follicles were positive for Cx43 (65%) and ZP3 (64.5%), and some primary follicles were positive only for Cx43. No primordial follicles stained positive in any of these immunohistochemical analyses. Only antral follicles showed aromatase activity. On the basis of these results, we can conclude that folliculogenesis in baboons appears to be similar to that in humans, and this animal therefore constitutes a valuable model.

The effect of Non- ionizing electromagnetic field with a frequency of 50 Hz in Rat ovary: A transmission electron microscopy study

Recently, there are increasing concerns and interests about the potential effects of Electromagnetic Field (EMF) on both human and animal health. The goal of this study was to evaluate the harmful effects of 50 Hz non-ionizing EMF on rat oocytes. In this experimental study 30 rats were randomly taken from laboratory animals and their ags and weights were determined. These 3 month's old rats were randomly divided into 3 groups. The control group consisted of 10 rats without receiving any treatment and kept under normal conditions. Experimental group 1 (10 rats) received EMF for 8 weeks (3 weeks intrauterine +5 weeks after births) and experimental group 2 (10 rats) received EMF for 13 weeks (3 weeks intrauterine +10 weeks after birth). After removing the ovaries and isolating follicles, granulosa cells were fixed in glutaraldehyde and osmium tetroxide. Electron microscopy was used to investigate the traumatic effects of EMF on follicles. In control group nucleus membrane and mitochondria in follicle's cytoplasm seemed normal in appearance. Theca layer of primary follicles in experimental group was separated clearly, zona layer demonstrated trot with irregular thickness and ovarian stroma seemed isolated with dilated vessels showing infiltration. According to the results of this study, it can be concluded that EMF has harmful effects on the ovarian follicles.

Comparative histopathology of the estrous or menstrual cycle in laboratory animals.

Accurate analysis of female reproductive toxicity requires a thorough understanding the differences in and specifics of estrous or menstrual cycles between laboratory animals. There are some species differences such as the time of sex maturation, the length of the estrous or menstrual cycle, the length of the luteal phase, the number of dominant follicles or corpora lutea, the size of follicles, processes of luteinization, and hormonal changes during the estrous or menstrual cycle. Rodents have a short estrous cycle, and their ovarian cycling features are the same in both ovaries, which contain a large number of follicles and corpora lutea. The dog estrous cycle is much longer than those of other laboratory animals, and it includes a long anestrus phase. The duration of the menstrual cycle of monkeys is roughly 30 days, and their ovarian cycling features are different between the left and right ovaries. In both rodents and dogs, the theca cells invade the early luteum, mixing with granulosa cells during luteinization. However in monkeys, the theca layer dose not mix with the granulosa cells as it invaginates only slightly into the early luteum. In addition, we found that high progesterone levels after ovulation are sustained for a much shorter duration in rodents than in dogs and monkeys due to the comparatively rapid passage of the rodent luteal phase. Based on these species differences, animal species for use in ovarian toxicology studies need to be selected appropriately.

A comparative gametogenesis and biochemical differentiation of goatfish (Family: Mullidae) from the Red Sea

Comparative study between few species of goatfishes (Mullidae), locally called Sultan Ibrahim, has been investigated from Jeddah coast. The present study compares gonad structure, oogenesis and spermatogenesis in three species of goatfish. The various stages are similar in the different species, but differ in time of spawning. The reproductive biology of three goatfishes, Mulloidichthys flavolineatus (1 black blotch), Mulloidichthys flavolineatus (3 black blotches), Mulloidichthys vanicolensis, were examined. A macroscopic and histological examination of the gonads demonstrated that all the three species are typically gonochoristic. Species-specific reproductive parameters including male stages, changes in gonado-somatic index (GSI), classification of oocyte stages were analyzed for each of the three targeted species. All three species have asynchronous ovary. The spawning season for the three studied species differ from one species to another i.e., the spawning season for M. vanicolensis females and males from April to July; M. flavolineatus, (1 black blotches) for both sexes from May until December; for M. flavolineatus (3 black blotches) showed a peak in May to early October for both sexes. Ultrastructurally the mature spermatozoon’s head shape appears like a computermouse, the tiny mid-piece consist two mitochondria and a flagellum. The chromatin, within the nucleus is electron-dense and homogenous in M. flavolineatus (with one black blotch), while the chromatin is granular in (M. vanicolensis and M. flavolineatus (3 black blotches)). The posterior region of the nucleus is deeply penetrated by the nuclear fossa which contains, the basal part of flagellum and centriolar complex. The ultrastructure of the eggs of the three Mullidae species is circular in shape and contains a simple, circular micropyle. The ovarian follicular epithelium of the three Mullidae species consists of two layers, an outer theca layer and an inner granulosa layer with zona radiata under them. Zona radiata consists of 2 layer-interna and externa. Zona radiata interna is thicker than zona radiata externa. Differences in protein patterns of muscles and gonads were observed in the present study.

Localization of Vascular Endothelial Growth Factor and Fibroblast Growth Factor 2 in the Ovary of the Ostrich (Struthio camelus).

Vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2) play a paramount role in the regulation of normal and pathologic angiogenesis in the ovary of mammals. Very little is known on the expression of these two growth factors in the avian ovary. The aim of this study was to determine for the first time the localization of VEGF and FGF-2 in the ovary of the ostrich using immunohistochemical techniques to investigate the vascularization of the rapidly growing huge ostrich oocyte. At the oocyte periphery, distinct VEGF-positive granules are visible. In our opinion, the expression of VEGF in the growing oocytes, which does not occur in mammals such as bovines, does not significantly contribute to angiogenesis in the theca interna and externa, where all the original and developing vessels are located, but may contribute to the mitoses and survival of granulosa cells during folliculogenesis. A different immunostaining can be demonstrated for FGF-2: from late pre-vitellogenic follicles, FGF-2 immunopositivity can be observed at the inner perivitelline layer area. In the stroma, the smooth muscle cells of small arteries and the endothelial cells of venules and veins are positively stained for FGF-2. Another interesting finding of this study is the occurrence of a significant number of VEGF- and FGF-2 positive heterophilic granulocytes within the ovarian stroma, which migrate from the periphery of the ovary towards the growing follicles. We assume that the growth factors of the heterophilic granulocytes contribute significantly to the angiogenesis seen in both theca layers.

Histological and immunohistochemical study of the ovaries and oviducts of the juvenile female of Caiman latirostris (Crocodilia: Alligatoridae)

The purpose of this study was to examine the ovaries and the oviduct of juvenile females of the broad-snouted caiman, Caiman latirostris (Daudin, 1802), using light microscopy, histochemical and immunocytochemical techniques. The ovarian cortex was observed to contain groups of germinative cells and abundant previtellogenic follicles at different stages of development. In previtellogenic follicles, the oocyte was surrounded by a single cuboidal layer of granulosa cells, supported by the theca layer. The theca was formed by a concentric layer containing collage nous fibers, predominantly type I, and several smooth muscle fibers. The inner perivitelline layer appeared as a narrow basophilic region between the oocyte and the granulosa. The ovarian medullary region of loose connective tissue contained blood and lymphatic vessels, as well as numerous lacunae, being covered by a simple squamous epithelium, supported by a thin layer of connective tissue. The oviduct of C. latirostris showed five histologically distinct regions: infundibulum, magnum, isthmus, uterus and vagina. The infundibulum was composed of an irregularly folded mucosa covered by a simple columnar epithelium with the presence of ciliated cells, weakly reactive to PAS staining. In the magnum, the mucosa was highly folded, and pseudostratified columnar epithelium contains mucous cells, which reacted positively to PAS staining. The isthmus was lining by an epithelium of ciliated and non-ciliated secretory cells, but no gland was visualized in mucosa. In the uterus, the folded mucosa was composed of a simple epithelium of high cylindrical cells and the lamina propria, which was predominantly formed by strongly birefringent fibers, but yellow-green weakly birefringent fibers were also present. The epithelium of the vagina contained intensely ciliated and non-ciliated cells, both of which were positive to PAS staining and no gland was observed in this region. Although the ovary and the oviduct showed some morphological particularities, they are similar to other crocodilians.

The Effect of Green Tea Extract on Reproductive Improvement in Estradiol Valerate-Induced Polycystic Ovarian Syndrome in Rat.

Polycystic ovary syndrome (PCOS) is a reproductive and metabolic disorder in which the level of oxidative elements in blood rises. Green tea is a potent antioxidant since it contains catechins. In this study, the effect of hydro-alcoholic green tea extract on PCOS rats was examined. For PCOS induction, 96 mature Wistar rats were given estradiol valerate. After 60 days, the rats were divided into four groups including PCOS group and three experimental groups, which were given 50, 100 and 200 mg/Kg BW green tea extract 10 days, intraperitoneally. The serum concentration level of FSH, LH, testosterone and insulin were measured using ELISA method, while the serum concentration level of glucose was measured using glucose oxidase methods. Insulin resistance was calculated using HOMA-IR formulation. The data were analyzed using the one-Way ANOVA method considering P<0/05 level of significance. There were a significant reduction in LH serum level, body and ovarian weight between the green tea extract treated-groups compare to PCOS. Moreover, a significant reduction in insulin resistance index was seen in the treatment groups related to PCOS. Histomorphometric studies also showed the significant changes in the number of follicles and theca layer thickness. These changes demonstrated a marked improvement in the symptoms of PCOS which may be due to green tea effects on oxidative stress pathways. Green tea can be considered as a potentially effective drug for treatment of PCOS, Insulin resistance and Type II diabetes.

Effect of PCB 126 on aryl hydrocarbon receptor 1 (AHR1) and AHR1 nuclear translocator 1 (ARNT1) mRNA expression and CYP1 monooxygenase activity in chicken (Gallus domesticus) ovarian follicles.

The aim of the experiment was to study the in vitro effect of 3,3',4,4',5-pentachlorobiphenyl (PCB 126; a coplanar PCB congener) on aryl hydrocarbon receptor (AHR1) and AHR1 nuclear translocator (ARNT1) mRNA expression and the activity of CYP1 family monooxygenases in chicken ovarian follicles. White (1-4 mm) and yellowish (4-8 mm) prehierarchical follicles as well as fragments of the theca and granulosa layers of the 3 largest preovulatory follicles (F3-F1) were incubated in a medium supplemented with 0 (control group), 1, 10 or 100 nM PCB 126. The incubation was carried out for 6 h or 24 h for determination of mRNA expression of AHR1 and ARNT1 genes (real-time qPCR) and CYP1 monooxygenase activity (EROD and MROD fluorometric assays), respectively. It was found that chicken ovarian follicles express mRNA of AHR1 and ARNT1 genes. A modulatory effect of PCB 126 on AHR1 and ARNT1 expression depended not only on the biphenyl concentration but also on the follicular layer and the maturational state of the follicle. EROD and MROD activities appeared predominantly in the granulosa layer of the yellow preovulatory follicles. PCB 126 induced these activities in a dose-dependent manner in all ovarian follicles. The obtained results suggest that ovarian follicles, especially the granulosa layer, are involved in the detoxification process of PCBs in the laying hen. Taking this finding into consideration it can be suggested that the granulosa layer of the yellow hierarchical follicles plays a key role in the protective mechanism which reduces the amount of transferred dioxin-like compounds into the yolk of the oocyte.