Ultrastructural evidence of telocytes in the embryonic chick heart.

The cardiac telocyte (TC) is a novel interstitial cell type with a unique ultrastructure and great potential in therapy. The present study examined its presence in the heart of chicken embryos ageing 7-15 days old (Hamburger-Hamilton [HH] stages 31-41) using transmission electron microscopy. TCs were identified across all stages in the atrial and ventricular myocardium, close to maturing cardiomyocytes, blood vessels and lymphatics. Early-stage TCs have immature features resembling mesenchymal cells. Late-stage TCs were distinct, possessing the cytoplasmic prolongations termed telopodes (Tps), which are very long and thin, usually 1-3 in number, and display a moniliform appearance and have an average thickness below 0.2 µm. TCs residing in the epicardium and endocardium were also detected. In the subepicardium near developing coronary vessels, they were localized in the cardiac stem cell niches, coexisting with cardiac stem cells and cardiomyocyte progenitors. Electron-dense structures and the release of extracellular vesicles were observed between embryonic TCs and surrounding structures, suggesting roles in intercellular communication, cardiomyocyte differentiation and maturation, angiogenesis, and stem cell nursing and guidance.

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.

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.

Glycobiology of developing chicken kidney: Profiling the galectin family and selected ß-galactosides.

The concept of the sugar code interprets the cellular glycophenotype as a rich source of information read by glycan-lectin recognition in situ. This study's aim is the comprehensive characterization of galectin expression by immunohistochemistry during chicken nephrogenesis along with mapping binding sites by (ga)lectin histochemistry. Light and two-color fluorescence microscopy were used. First, six plant/fungal lectins that are specific for galectin-binding parts of N- and O-glycans were applied. The spatiotemporally regulated distributions of these glycans in meso- and metanephros equip cells with potential binding partners for the galectins. Complete galectin profiling from HH Stage 20 (about 70-72 hr) onward revealed cell-, galectin-, and stage-dependent expression patterns. Representatives of all three types of modular architecture of the galectin family are detectable, and overlaps of signal distribution in light and two-color fluorescence microscopy illustrate a possibility for functional cooperation among them. Performing systematic galectin histochemistry facilitated comparisons between staining profiles of plant lectins and galectins. They revealed several cases for differences so that tissue lectins appear to be selective among the ß-galactosides. Notably, selectivity is also disclosed in intrafamily comparison. Thus, combining experimental series with plant and tissue lectins is a means to characterize target populations of glycans presented by cellular glycoconjugates for individual galectins. Our results document the presence and sophisticated level of elaboration among ß-galactosides and among the members of the family of galectins during organogenesis, using chicken galectins and kidney as model. Thus, they provide a clear guideline for functional assays using supramolecular tools, cells, and organ cultures.

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.

Correction to: Influence of protein (human galectin-3) design on aspects of lectin activity.

In the original publication of the article, the "Result" section has been published.

Influence of protein (human galectin-3) design on aspects of lectin activity.

The concept of biomedical significance of the functional pairing between tissue lectins and their glycoconjugate counterreceptors has reached the mainstream of research on the flow of biological information. A major challenge now is to identify the principles of structure-activity relationships that underlie specificity of recognition and the ensuing post-binding processes. Toward this end, we focus on a distinct feature on the side of the lectin, i.e. its architecture to present the carbohydrate recognition domain (CRD). Working with a multifunctional human lectin, i.e. galectin-3, as model, its CRD is used in protein engineering to build variants with different modular assembly. Hereby, it becomes possible to compare activity features of the natural design, i.e. CRD attached to an N-terminal tail, with those of homo- and heterodimers and the tail-free protein. Thermodynamics of binding disaccharides proved full activity of all proteins at very similar affinity. The following glycan array testing revealed maintained preferential contact formation with N-acetyllactosamine oligomers and histo-blood group ABH epitopes irrespective of variant design. The study of carbohydrate-inhibitable binding of the test panel disclosed up to qualitative cell-type-dependent differences in sections of fixed murine epididymis and especially jejunum. By probing topological aspects of binding, the susceptibility to inhibition by a tetravalent glycocluster was markedly different for the wild-type vs the homodimeric variant proteins. The results teach the salient lesson that protein design matters: the type of CRD presentation can have a profound bearing on whether basically suited oligosaccharides, which for example tested positively in an array, will become binding partners in situ. When lectin-glycoconjugate aggregates (lattices) are formed, their structural organization will depend on this parameter. Further testing (ga)lectin variants will thus be instrumental (i) to define the full range of impact of altering protein assembly and (ii) to explain why certain types of design have been favored during the course of evolution, besides opening biomedical perspectives for potential applications of the novel galectin forms.

How galectins have become multifunctional proteins.

Having identified glycans of cellular glycoconjugates as versatile molecular messages, their recognition by sugar receptors (lectins) is a fundamental mechanism within the flow of biological information. This type of molecular interplay is increasingly revealed to be involved in a wide range of (patho)physiological processes. To do so, it is a vital prerequisite that a lectin (and its expression) can develop more than a single skill, that is the general ability to bind glycans. By studying the example of vertebrate galectins as a model, a total of five relevant characteristics is disclosed: i) access to intra- and extracellular sites, ii) fine-tuned gene regulation (with evidence for co-regulation of counterreceptors) including the existence of variants due to alternative splicing or single nucleotide polymorphisms, iii) specificity to distinct glycans from the glycome with different molecular meaning, iv) binding capacity also to peptide motifs at different sites on the protein and v) diversity of modular architecture. They combine to endow these lectins with the capacity to serve as multi-purpose tools. Underscoring the arising broad-scale significance of tissue lectins, their numbers in terms of known families and group members have steadily grown by respective research that therefore unveiled a well-stocked toolbox. The generation of a network of (ga)lectins by evolutionary diversification affords the opportunity for additive/synergistic or antagonistic interplay in situ, an emerging aspect of (ga)lectin functionality. It warrants close scrutiny. The realization of the enormous potential of combinatorial permutations using the five listed features gives further efforts to understand the rules of functional glycomics/lectinomics a clear direction.

Chicken lens development: complete signature of expression of galectins during embryogenesis and evidence for their complex formation with α-, ß-, δ-, and τ-crystallins, N-CAM, and N-cadherin obtained by affinity chromatography.

The emerging multifunctionality of galectins by specific protein-glycan/protein interactions explains the interest to determine their expression during embryogenesis. Complete network analysis of all seven chicken galectins (CGs) is presented in the course of differentiation of eye lens that originates from a single type of progenitor cell. It answers the questions on levels of expression and individual patterns of distribution. A qualitative difference occurs in the CG-1A/B paralogue pair, underscoring conspicuous divergence. Considering different cell phenotypes, lens fiber and also epithelial cells can both express the same CG, with developmental upregulation for CG-3 and CG-8. Except for expression of the lens-specific CG (C-GRIFIN), no other CG appeared to be controlled by the transcription factors L-Maf and Pax6. Studying presence and nature of binding partners for CGs, we tested labeled galectins in histochemistry and in ligand blotting. Mass spectrometric (glyco)protein identification after affinity chromatography prominently yielded four types of crystallins, N-CAM, and, in the cases of CG-3 and CG-8, N-cadherin. Should such pairing be functional in situ, it may be involved in tightly packing intracellular lens proteins and forming membrane contact as well as in gaining plasticity and stability of adhesion processes. The expression of CGs throughout embryogenesis is postulated to give meaning to spatiotemporal alterations in the local glycome.

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.

How altering the modular architecture affects aspects of lectin activity: case study on human galectin-1.

Discoveries on involvement of glycan-protein recognition in many (patho)physiological processes are directing attention to exploring the significance of a fundamental structural aspect of sugar receptors beyond glycan specificity, i.e., occurrence of distinct types of modular architecture. In order to trace clues for defining design-functionality relationships in human lectins, a lectin's structural unit has been used as source material for engineering custom-made variants of the wild-type protein. Their availability facilitates comparative analysis toward the stated aim. With adhesion/growth-regulatory human galectin-1 as example, the strategy of evaluating how changes of its design (here, from the homodimer of non-covalently associated domains to (i) linker-connected di- and tetramers and (ii) a galectin-3-like protein) affect activity is illustrated by using three assay systems of increasing degree of glycan complexity. Whereas calorimetry with two cognate disaccharides and array testing with 647 (glyco)compounds disclosed no major changes, galectin histochemical staining profiles of tissue sections that present natural glycome complexity revealed differences between wild-type and linker-connected homo-oligomers as well as between the galectin-3-like variant and wild-type galectin-3 for cell-type positivity, level of intensity at the same site and susceptibility for inhibition by a bivalent glycocompound. These results underscore the strength of the documented approach. Moreover, they give direction to proceed to (i) extending its application to other members of this lectin family, especially galectin-3 and (ii) then analyzing impact of architectural alterations on cell surface lattice formation and ensuing biosignaling systematically, considering the variants' potential for translational medicine.

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.

Galectin-related protein: An integral member of the network of chicken galectins: 2. From expression profiling to its immunocyto- and histochemical localization and application as tool for ligand detection.

BACKGROUND: Galectin-related protein (GRP), present in vertebrates, is special within this family of adhesion/growth-regulatory proteins due to its strong positive selection and loss of canonical lectin activity. METHODS: RT-PCR and Western blotting together with flow cytofluorimetry and immunocyto- and histochemistry monitor expression and localization of chicken GRP. The promoter sequence of the GRP gene is processed computationally to detect putative sites for binding transcription factors. The labeled protein is applied as probe to detect binding sites on cells and in sections, along with glycocompounds to test inhibition of the association. RESULTS: Expression of GRP in chicken is limited to bursa of Fabricius, immunohistochemically found in B cells, also in bursal epithelium and vessels. Presence in B cells is shared with only one canonical galectin, i.e. CG-8. Binding to a chicken lymphoma line was specific and saturable, not affected by lactose but completely blocked by heparin, as also seen in sections. CONCLUSIONS: Expression monitoring initiated for GRP reveals a distinct site of localization in chicken, much more restricted than for any of its canonical galectins.

Network monitoring of adhesion/growth-regulatory galectins: localization of the five canonical chicken proteins in embryonic and maturing bone and cartilage and their introduction as histochemical tools.

Divergence from an ancestral gene leads to a family of homologous proteins. Whether they are physiologically distinct, similar, or even redundant is an open question in each case. Defining profiles of tissue localization is a step toward giving diversity a functional meaning. Due to the significance of endogenous sugar receptors (lectins) as effectors for a wide range of cellular activities we have focused on galectins. The comparatively low level of network complexity constituted by only five canonical proteins makes chicken galectins (CGs) an attractive choice to perform comprehensive analysis, here studied on bone/cartilage as organ system. Galectin expression was monitored by Western blotting and immunohistochemistry using non-cross-reactive antibodies. Overall, three galectins (CG-1B, CG-3, CG-8) were present with individual expression patterns, one was found exclusively in the mesenchyme (CG-1A), the fifth (CG-2) not being detectable. The documented extents of separation are a sign for functional divergence; in cases with overlapping stainings, as for example in the osteoprogenitor layer or periosteum, cooperation may also be possible. Recombinant production enabled the introduction of the endogenous lectins as tools for binding-site localization. Their testing revealed developmental regulation and cell-type-specific staining. Of relevance for research on mammalian galectins, this study illustrates that certain cell types can express more than one galectin, letting functional interrelationships appear likely. Thus, complete network analysis irrespective of its degree of complexity is mandatory.

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.

Fetal Development of the Bovine Uterus: A Light Microscopy and Immunohistochemical Study.

Important steps during the prenatal development of the bovine uterus are described using conventional hematoxylin-eosin staining of fetuses from different developmental stages [crown-rump length (CRL) 9.2-94.0 cm]. Additionally, a number of intermediate filaments (keratin 7, 8, 14, 18, 19; and vimentin), the basement membrane protein laminin, smooth-muscle marker (SMA), and S100 were studied to further characterize certain differentiation processes. During early development, the uterine epithelium is simple or (pseudo)stratified with bud-like protrusions. Developing caruncles can be observed in the corpus uteri at a CRL of 15.8 cm onwards, showing a simple, keratin-positive epithelium. In contrast, the intercaruncular areas are characterized by a (pseudo)stratified epithelium, which also shows positive staining in a different manner for the investigated keratins. A differentiation of smooth muscle cell layers can be observed from a CRL of 24.4 cm onwards. Intense SMA-positive cells/fibers, arranged perpendicularly to the developing circular SMA-positive muscle cell layer, can be found preferentially located in the developing caruncles. Lymphocytes occur in the uterine epithelium and stroma in the corpora and cornua of fetuses with a CLR of 15.8 cm and higher.

Expression of Intermediate Filaments and Germ Cell Markers in the Developing Bovine Ovary: An Immunohistochemical and Laser-Assisted Microdissection Study.

In the present investigation, bovine ovary prenatal development was studied using immunohistochemistry and laser-assisted microdissection (LAM). A major aim of this study was to evaluate the protein expression pattern of intermediate filaments (IF) and distinguish S100 protein (S100 alpha and S100 beta protein) isoforms during prenatal follicle differentiation, subsequently correlating them with germ cell marker expression. A development-specific expression pattern of different keratins as well as vimentin was detected in the prenatal bovine ovary; K18-specific expression was found during all developmental stages (i.e. in surface epithelium, germ cell cord somatic cells, and follicle cells), and keratins 5, 7, 8, 14, and 19 and vimentin had a stage-specific expression pattern in the different cell populations of the prenatal ovaries. Additionally, our results represent new data on the expression pattern of germ cell markers during bovine ovary prenatal development. S100 alpha and beta protein was localized to oocyte cytoplasm of different follicle stages, and S100 alpha staining could be observed in granulosa cells. Furthermore, through isolation of characteristic ovary cell populations using LAM, specific confirmation of some genes of interest (KRT8, KRT18, S100 alpha, S100 beta, and OCT4, DDX4) could be obtained by RT-PCR in single cell groups of the developing bovine ovary.