Hypoxia-inducible factor-1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis.

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.

Changes in the expression of prostaglandin family members in bovine corpus luteum during the estrous cycle and pregnancy.

The aim of this study was to characterize certain prostaglandin family members in the bovine corpus luteum (CL) during the estrous cycle and pregnancy. The CL tissue was assigned to the stages 1-2, 3-4, 5-7, 8-12, 13-16 and >18 days (after regression) of the estrous cycle and 1-2, 3-4, 6-7, and >8 months of pregnancy. In these samples, we investigated prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, and PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES). The expression of messenger RNA (mRNA) was measured by reverse transcription quantitative polymerase chain reaction, hormones by enzyme immunoassay, and localization by immunohistochemistry. The mRNA expression of COX-2, PTGFS, and PTGES in CL during the early-luteal phase was high followed by a continuous and significant downregulation afterward, as well as during all phases of pregnancy. The concentration of PTGF in CL tissue was high during the early-luteal phase, decreased significantly in the mid-luteal phase, and increased again afterward. In contrast, the concentration of PTGE increased significantly during the late-luteal phase followed by a decrease during regression. The PTGE level increased again during late pregnancy. Immunohistochemically, the large granulose-luteal cells show strong staining for COX-2 and PTGES during the early-luteal stage followed by lower activity afterward. During pregnancy, most of the luteal cells were only weakly positive or negative. In conclusion, our results indicate that the examined prostaglandin family members are involved in the local mechanisms that regulate luteal function, specifically during CL formation, function, and regression and during pregnancy in the cow.

Functional Morphology of Thecal Glands in the Ovary of Japanese Quails (Coturnix japonica).

The role of thecal glands in the ovary of birds remains controversial. Using transmission electron microscopy and immunohistochemistry, immunohistochemical localisation of cyclooxygenase I and II (COX-1 and COX-2), oestrogen receptor α and ß (ER-α and ER-ß), androgen receptor (AR) and progesterone receptor (PR), a detailed analysis of the thecal glands was performed. Our ultrastructural studies revealed that the thecal glands of the quail ovary consist of 2 cell types, steroid-producing cells (SPCs) and enclosing cells (ENCs). The SPCs are large, light cells containing a varying number of lipid droplets. Their cytoplasm is characterised by a large amount of smooth endoplasmic reticulum. The ENCs are always located at the periphery of the gland. Some ENCs contain an abundant number of microfilaments, but lipid droplets and dense bodies were rare. Within 1 gland, SPCs with distinct COX-2 immunostaining were interspersed between usually larger numbers of moderately COX-2-positive cells. A completely different staining pattern was observed for COX-1, where the cytoplasm of the ENCs was distinctly immunopositive. The SPCs stained only weakly with antibodies to COX-1. The thecal glands showed distinct reactions for ER-ß but only a weak to negative one for ER-α, PR, and AR. Our immunohistochemical and ultrastructural data support our hypothesis that the thecal glands of the quail are involved in steroid hormone and prostaglandin synthesis. The prostaglandins secreted by the thecal glands probably contribute to the ovulation of the follicle first in the hierarchy.

Expression and localization of members of the thrombospondin family during final follicle maturation and corpus luteum formation and function in the bovine ovary.

The aim of this study was to characterize the expression patterns and localization of the thrombospondin family members (THBS1, THBS2) and their receptors (CD36 and CD47) in bovine ovaries. First, the antral follicles were classified into 5 groups based on the follicle size and estradiol-17beta (E2) concentration in the follicular fluid (< 0.5, 0.5-5, 5-40, 40-180 and >180 E2 ng/ml). Second, the corpus luteum (CL) was assigned to the following stages: days 1-2, 3-4, 5-7, 8-12, 13-16 and >18 of the estrous cycle and of pregnancy (month 1-2, 3-4, 6-7 and > 8). Third, the corpora lutea were collected by transvaginal ovariectomy before and 0.5, 2, 4, 12, 24, 48 and 64 h after inducing luteolysis by injecting a prostaglandin F2alpha analog. The mRNA expression of examined factors was measured by RT-qPCR, steroid hormone concentration by EIA, and localization by immunohistochemistry. The mRNA expression of THBS1, THBS2, CD36, and CD47 in the granulosa cells and theca interna was high in the small follicles and reduced in the preovulatory follicles. The mRNA expression of THBS1, THBS2, and CD47 in the CL during the estrous cycle was high, but decreased significantly during pregnancy. After induced luteolysis, thrombospondins increased significantly to reach the maximum level at 12 h for THBS1, 24 h for THBS2, and 48 h for CD36. The temporal expression and localization pattern of the thrombospondins and their specific receptors in the antral follicles and corpora lutea during the different physiological phases of the estrous cycle and induced luteolysis appear to be compatible with their inhibitory role in the control of ovarian angiogenesis.

Prostaglandins as local regulators of ovarian physiology in ruminants.

Prostaglandins are synthesized from arachidonic acid through the catalytic activities of cyclooxygenase, while the production of different prostaglandin types, prostaglandin F2 alpha (PGF) and prostaglandin E2 (PGE), are regulated by specific prostaglandin synthases (PGFS and PGES). Prostaglandin ligands (PGF and PGE) bind to specific high-affinity receptors and initiate biologically distinct signalling pathways. In the ovaries, prostaglandins are known to be important endocrine regulators of female reproduction, in addition to maintaining local function through autocrine and/or paracrine effect. Many research groups in different animal species have already identified a variety of factors and molecular mechanisms that are responsible for the regulation of prostaglandin functions. In addition, prostaglandins stimulate their intrafollicular and intraluteal production via the pathway of prostaglandin self-regulation in the ovary. Therefore, the objective of the review article is to discuss recent findings about local regulation patterns of prostaglandin ligands PGF and PGE during different physiological stages of ovarian function in domestic ruminants, especially in bovine. In conclusion, the discussed local regulation mechanisms of prostaglandins in the ovary may stimulate further research activities in different methodological approaches, especially during final follicle maturation and ovulation, as well as corpus luteum formation and function.

Effect of the gonadotropin surge on steroid receptor regulation in preovulatory follicles and newly formed corpora lutea in the cow.

The objective of the study was to characterize the mRNA expression patterns of specific steroid hormone receptors namely, estrogen receptors (ESRRA-estrogen related receptor alpha and ESRRB-estrogen related receptor beta) and progesterone receptors (PGR) in superovulation-induced bovine follicles during the periovulation and subsequent corpus luteum (CL) formation. The bovine ovaries (n = 5 cow / group), containing preovulatory follicles or early CL, were collected relative to injection of the gonadotropin-releasing hormone (GnRH) at (I) 0 h, (II) 4 h, (III) 10 h, (IV) 20 h, (V) 25 h (preovulatory follicles) and (VI) 60 h (CL, 2-3 days after induced ovulation). In this experiment, we analyzed the steroid receptor mRNA expression and their localization in the follicle and CL tissue. The high mRNA expression of ESRRA, ESRRB, and PGR analyzed in the follicles before ovulation is significantly reduced in the group of follicles during ovulation (25 h after GnRH), rising again significantly after ovulation in newly formed CL, only for ESRRA and PGR (P < 0.05). Immunohistochemically, the nuclei of antral follicles' granulosa cells showed a positive staining for ESRRA, followed by higher activity in the large luteal cells just after ovulation (early CL). In contrast, the lower PGR immunopresence in preovulatory follicles increased in both small and large luteal cell nuclei after follicle ovulation. Our results of steroid receptor mRNA expression in this experimentally induced gonadotropin surge provide insight into the molecular mechanisms of the effects of steroid hormones on follicular-luteal tissue in the period close to the ovulation and subsequent CL formation in the cow.

Expression and localization of nodal in bovine oviduct and uterus during different functional stages of oestrus cycle and pregnancy.

Members of TGF-ß superfamily play a major role in the endometrial changes involved in the establishment and maintenance of pregnancy. Their deregulated expression and action could lead to absolute or partial failure of embryo implantation. Nonetheless, the precise function and mechanism of many of these cytokines remain unclear. Nodal, a transforming growth factor beta (TGF-ß) superfamily member, was characterized in the human and rodent uterus and implicated in the tissue remodeling events during menstruation and embryo implantation. In order to study its possible role in the cattle reproductive process, we have analyzed Nodal expression pattern and localization in the oviduct and uterine horn during the oestrus cycle and early pregnancy (day 20). Nodal was detected both in oviduct and uterus during either the oestrus cycle or pregnancy; however, it shows a differential expression profile in the uterine horn at dioestrus and pregnancy, decreasing 1.5 and 1.4 folds in comparison with oestrus. Nodal immunostaining intensity was observed in stromal and in epithelial cells of the surface and the glandular epithelium. The staining pattern correlates with the RT-qPCR expression profile. This work is the first to evidence the presence of Nodal in the bovine reproductive tract; our data suggest that Nodal is a novel cytokine that would be involved in the remodelling occurring in the endometrium of cattle during the oestrus cycle and in the embryo implantation. The identification of new molecules that participate in endometrium cycling and/or pregnancy may be useful for predicting the ability of the uterine tissue to establish and maintain pregnancy or for detecting the infertility processes. These results highlight Nodal as a possible novel marker of the fertility process, nevertheless further studies should be done to determine its role in the reproductive system.

Expression of intermediate filaments in the Balbiani body and ovarian follicular wall of the Japanese quail (Coturnix japonica).

In the present study, we examined the distribution of 6 groups of intermediate filaments (IFs; cytokeratins, CKs, vimentin, synemin, desmin, glial fibrillary acidic protein and lamins) in oocytes and follicular walls of the Japanese quail (Coturnix japonica) during their development using immunohistochemical and ultrastructural techniques. A distinctly vimentin- and synemin-positive Balbiani body, which is a transient accumulation of organelles (mitochondria, Golgi complex and endoplasmic reticulum) that occurs in the oocytes of all vertebrates including birds, could be detected in the oocytes of primordial and early pre-vitellogenic follicles. In larger pre-vitellogenic follicles, the Balbiani body has dispersed and the positivity of the granulosa cells appeared to concentrate in the basal portion of their cytoplasm. Our ultrastructural data demonstrated that the matrix of the Bal-biani body consists of fine IFs, which may play a role in the formation and dispersion of the Balbiani body. Of the CKs studied (panCK, CK5, CK7, CK8, CK14, CK15, CK18 and CK19), only CK5 showed a slight positive staining in both the theca externa and the Balbiani bodies of pre-vitellogenic oocytes. In conclusion, our data, which describe the changes in avian IF protein expression during folliculogenesis, suggest that the functions of the IFs (vimentin and synemin) of oocytes and follicular walls are not primarily mechanical but may be involved in the transient tethering of mitochondria in the area of the Balbiani body and in the gain of endocrine competence during the differentiation of granulosa cells.

Assembly of the inner perivitelline layer, a homolog of the mammalian zona pellucida: an immunohistochemical and ultrastructural study.

The avian inner perivitelline layer (IPVL), a homologous structure to the mammalian zona pellucida, is deposited between the granulosa cells and the oocyte cell membrane during folliculogenesis. The glycoprotein meshwork of the IPVL forms a 3-dimensional matrix and possesses important functions in the fertilization process: it contributes to the binding of avian spermatozoa to the oocyte and induces acrosomal exocytosis. In contrast to the zona pellucida of mammals, the IPVL does not prevent the physiological polyspermy found in birds. Previous studies have shown that in the Japanese quail (Coturnix japonica) at least 5 glycoproteins are constituents of the IPVL (ZP1, ZP2, ZP3, ZP4, and ZPD). In this study, we investigated the spatiotemporal assembly pattern of the IPVL during folliculogenesis using immunohistochemical and ultrastructural methods. The obtained results clearly show that these glycoproteins are incorporated into the IPVL at distinct points during follicular development, supporting the hypothesis that ZP2 and ZP4 form a type of prematrix into which ZP1, ZP3, and ZPD are integrated at a later stage of development.

Biology and biotechnology of follicle development.

Growth and development of ovarian follicles require a series of coordinated events that induce morphological and functional changes within the follicle, leading to cell differentiation and oocyte development. The preantral early antral follicle transition is the stage of follicular development during which gonadotropin dependence is obtained and the progression into growing or atresia of the follicle is made. Follicular growth during this period is tightly regulated by oocyte-granulosatheca cell interactions. A cluster of early expressed genes is required for normal folliculogenesis. Granulosa cell factors stimulate the recruitment of theca cells from cortical stromal cells. Thecal factors promote granulosa cell proliferation and suppress granulosa cell apoptosis. Cell-cell and cell-extracellular matrix interactions influence the production of growth factors in the different follicular compartments (oocyte, granulosa, and theca cells). Several autocrine and paracrine factors are involved in follicular growth and differentiation; their activity is present even at the time of ovulation, decreasing the gap junction communication, and stimulating the theca cell proliferation. In addition, the identification of the factors that promote follicular growth from the preantral stage to the small antral stage may provide important information for the identification for assisted reproduction techniques.

Regulatory changes of local produced prostaglandins in corpus luteum after experimentally induced luteolysis in the cow.

The objective of the study was to evaluate the expression patterns of prostaglandin F2alpha (PGF), prostaglandin E2 (PGE), PGF receptor (FP), PGE receptors (EP2 and EP4), prostaglandin-endoperoxide synthase 2 (PTGS2) and prostaglandin synthases (PGFS and PGES) in corpora lutea (CL) during experimentally induced luteolysis in cow. The Fleckvieh cows in the mid-luteal phase (days 8-12, control group) were injected with cloprostenol (PGF analogue), and CL were collected by transvaginal ovariectomy before (days 8-12, control group) and at 0.5, 2, 4, 12, 24, 48 and 64 h after PGF application (n = 5 per group). The mRNA expression was determined by RT-qPCR, the hormone concentrations by enzyme immunoassay and localization by immunohistochemistry. PTGS2 gene expression increased significantly 2 h after PGF application, followed by continuous and significant downregulation afterwards. The PGF tissue concentration increased significantly just after PGF injection and again during structural luteolysis (after 12 h), whereas PGE concentration significantly decreased during structural luteolysis. The FP receptor mRNA decreased significantly at 2 h and again at 12 h after PGF. In contrast, EP4 receptor mRNA increased significantly just after the PGF application (0.5 h). The immunostaining of PGES and PTGS2 on day 15-17 shows numerous positive luteal cells, followed by lower activity afterwards on day 18 (luteolysis). In conclusion, the changes of examined prostaglandin family members in CL tissue after PGF application may be key components of the local mechanisms regulating the cascade of actions leading to functional and subsequent structural luteolysis in the bovine ovary.

Zona pellucida protein ZP2 is expressed in the oocyte of Japanese quail (Coturnix japonica).

The avian perivitelline layer (PL), a vestment homologous to the zona pellucida (ZP) of mammalian oocytes, is composed of at least three glycoproteins. Our previous studies have demonstrated that the matrix's components, ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is synthesized in the liver and is transported to the ovary by blood circulation. In this study, we report the isolation of cDNA encoding quail ZP2 and its expression in the female bird. By RNase protection assay and in situ hybridization, we demonstrate that ZP2 transcripts are restricted to the oocytes of small white follicles (SWF). The expression level of ZP2 decreased dramatically during follicular development, and the highest expression was observed in the SWF. Western blot and immunohistochemical analyses using the specific antibody against ZP2 indicate that the 80 kDa protein is the authentic ZP2, and the immunoreactive ZP2 protein is also present in the oocytes. Moreover, ultrastructural analysis demonstrated that the immunoreactive ZP2 localizes to the zona radiata, the perivitelline space, and the oocyte cytoplasm in the SWF. By means of western blot analysis and immunofluorescence microscopy, we detected a possible interaction of the recombinant ZP2 with ZP3 and that this interaction might lead to the formation of amorphous structure on the cell surface. These results demonstrate for the first time that the avian ZP gene is expressed in the oocyte, and that the ZP2 protein in the oocyte might play a role for the PL formation in the immature follicles of the ovary.

Prostaglandins in Superovulation Induced Bovine Follicles During the Preovulatory Period and Early Corpus Luteum.

The aim of this study was to characterize the regulation pattern of prostaglandin family members namely prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES) in the bovine follicles during preovulatory period and early corpus luteum (CL). Ovaries containing preovulatory follicles or CL were collected by transvaginal ovariectomy (n = 5 cows/group), and the follicles were classified: (I) before GnRH treatment; (II) 4 h after GnRH; (III) 10 h after GnRH; (IV) 20 h after GnRH; (V) 25 h after GnRH, and (VI) 60 h after GnRH (early CL). In these samples, the concentrations of progesterone (P4), estradiol (E2), PTGF and PTGE were investigated in the follicular fluid (FF) by validated EIA. Relative mRNA abundance of genes encoding for prostaglandin receptors (PTGFR, PTGER2, PTGER4), COX-2, PTGFS and PTGES were quantified by RT-qPCR. The localization of COX-2 and PTGES were investigated by established immunohistochemistry in fixed follicular and CL tissue samples. The high E2 concentration in the FF of the follicle group before GnRH treatment (495.8 ng/ml) and during luteinizing hormone (LH) surge (4 h after GnRH, 574.36 ng/ml), is followed by a significant (P<0.05) downregulation afterwards with the lowest level during ovulation (25 h after GnRH, 53.11 ng/ml). In contrast the concentration of P4 was very low before LH surge (50.64 mg/ml) followed by a significant upregulation (P < 0.05) during ovulation (537.18 ng/ml). The mRNA expression of COX-2 increased significantely (P < 0.05) 4 h after GnRH and again 20 h after GnRH, followed by a significant decrease (P < 0.05) after ovulation (early CL). The mRNA of PTGFS in follicles before GnRH was high followed by a continuous and significant downregulation (P < 0.05) afterwards. In contrast, PTGES mRNA abundance increased significantely (P < 0.05) in follicles 20 h after GnRH treatment and remained high afterwards. The mRNA abundance of PTGFR, PTGER2, and PTGER4 in follicles before GnRH was high, followed by a continuous and significant down regulation afterwards and significant increase (P < 0.05) only after ovulation (early CL). The low concentration of PTGF (0.04 ng/ml) and PTGE (0.15 ng/ml) in FF before GnRH, increased continuously in follicle groups before ovulation and displayed a further significant and dramatic increase (P < 0.05) around ovulation (101.01 ng/ml, respectively, 484.21 ng/ml). Immunohistochemically, the granulosa cells showed an intensive signal for COX-2 and PTGES in follicles during preovulation and in granulosa-luteal cells of the early CL. In conclusion, our results indicate that the examined bovine prostaglandin family members are involved in the local mechanisms regulating final follicle maturation and ovulation during the folliculo-luteal transition and CL formation.

Expression of prostaglandin synthesizing enzymes (cyclooxygenase 1 and cyclooxygenase 2) in the ovary of the ostrich (Struthio camelus).

Cyclooxygenase is the rate limiting enzyme in the production of prostaglandins. In birds two isoforms are present: cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2). Despite evidence implicating that cyclooxygenases and PGs are critical factors in female reproduction in birds, little is known about COX expression in the avian ovary. In birds, cyclooxygenases have been studied in very few species only. In this study we report on the expression of COX-1 and COX-2 in the ovary of the ostrich (Struthio camelus) using immunohistochemistry and non-radioactive in situ hybridization techniques. Our results demonstrate that COX-1 is strongly expressed in the cytoplasm of oocytes of previtellogenic follicles, whereas COX-2 shows the strongest immunostaining in the granulosa cells of previtellogenic follicles. The signals of both isoenzymes fade significantly with increasing diameter and finally nearly vanish in the vitellogenic follicles with a size >1.8 cm. This expression pattern in the ostrich (S. camelus) is, therefore, completely different from the localization of COX-1 and COX-2 in the hen (Gallus gallus), a finding which also suggests different functions of the cyclooxygenases in the ostrich species. Non-radioactive in situ hybridization confirmed that COX-1 is synthesized in the ooplasm and COX-2 in the granulosa layers of early previtellogenic follicles. According to the results of this study it appears unlikely that COX-1 or COX-2 play a major role in ovulation and oviposition in the ostrich.

Immunohistochemical and ultrastructural characterization of the ovarian surface epithelium of Japanese quail (Coturnix japonica).

Contrary to humans, most ovarian tumors in other species do not arise from the ovarian surface epithelium but are of follicular-, stromal- or germ-cell origin. One of the few species where ovarian cancer arises from the ovarian surface epithelium (OSE) is chicken (Gallus domesticus). Little is known about the morphology of the OSE in other avian species. In our study we analyzed the OSE morphology of Japanese quail (Coturnix japonica) using ultrastructural and histochemical techniques. Carbohydrate residues have been studied by using a panel of fluorescein isothiocyanate labeled lectins. Japanese quails are commonly used animal models in biomedical research as their housing is comparatively inexpensive and they show a short generation interval. Our ultrastructural and histochemical results demonstrate that the quail ovarian surface epithelium shows characteristic features which resemble the epithelia of both chicken and human. Additionally, the ovarian surface epithelium of the Japanese quail contains cytokeratin as well as vimentin intermediate filaments in their cytoplasm. Therewith and among other parts the quail OSE shows many characteristic features also seen in those of humans, which may qualify quail's ovary as a potential animal model for human ovarian carcinomas.

Oocytic expression of zona pellucida protein ZP4 in Japanese quail (Coturnix japonica).

The avian perivitelline layer, an extracellular matrix homologous to the zona pellucida (ZP) of mammalian oocytes, is composed mainly by zona pellucida gene family glycoproteins. Our previous studies in Japanese quail have demonstrated that the matrix's components, ZP3 and ZPD, are synthesized in ovarian granulosa cells. Another component, ZP1, is synthesized in the liver. Recently, we demonstrated that another minor constituent, ZP2 is produced in the oocytes of the immature follicles. In the present study, we report the isolation of complementary DNA encoding quail ZP4 and its expression and origin in the female birds. By ribonuclease protection assay and in situ hybridization, we demonstrated that ZP4 transcripts were transcribed in the oocytes of small white follicles. The expression level of ZP4 decreased dramatically during follicular development, and the highest expression was observed in the small white follicles. Western blot analysis using the specific antibody against ZP4 indicated that the immunoreactive 58.2 kDa protein was present in the lysates of the small white follicles. These results demonstrate for the first time that the avian ZP4 is expressed in the oocyte, and that the expression pattern of the gene is similar to that of ZP2.