Although many aspects of ovarian differentiation have been established, comparatively little is known about prenatal follicle formation and differentiation of bovine ovaries. The objective of this investigation was to study the role of the surface epithelium during the development of germ cell nests, germ cell cords and follicle formation in the fetal bovine ovary. Associated important proliferation and apoptotic features were further investigated. Additionally, the expression pattern of the S100 protein was detected. A strong increase of mitotic figures was detected in the surface epithelium, germ cell nests and germ cell cords of ovaries with a crown-rump length (CRL) of 13.0-58.0 cm. Oocytes were positively stained with S100 in bovine ovaries from fetuses with a CRL of 21.0 cm. The staining intensity enhanced parallel to increasing oocyte and follicle sizes during the ovary development. In later stages, a strong staining for S100 was observed in healthy oocytes in contradistinction to atretic oocytes where no expression of the S100 protein could be found. In conclusion, increasing mitosis index of surface epithelium cells, as well as oogonia directly beneath the surface epithelium, in combination with open surface connection during stages from a CRL of 11.0-94.0 cm of bovine fetal ovaries could play an important role in the period of time of ongoing folliculogenesis and derivation of granulosa cells. Additionally, S100-positive oocytes in primordial and later follicle stages joined by a high rate of Ki67-positive index in surrounding granulosa cells indicate that in the oocytes the S100 protein can perhaps be a useful marker for intact oocytes in bovine ovaries.
The mechanisms underlying detachment of foetal membranes after birth in cows are still unclear. To address this problem in a systematic manner, we performed the first holistic transcriptome study of bovine placentomes antepartum (AP; nZ4 cows) and intrapartum (IP; nZ4 cows) using Affymetrix GeneChip Bovine Genome Arrays. Three placentomes were extracted from each cow, and tissue samples from the contact zones of the placentomes (foeto-maternal units) were recovered by systematic random sampling and processed for RNA extraction and for stereological quantification of cellular composition. Statistical analysis of microarray data (false discovery rate 1%) revealed 759 mRNAs with at least twofold higher levels in the samples of the AP group, whereas 514 mRNAs showed higher levels in the IP group. The differentially expressed genes were classified according to biological processes and molecular functions using the Functional Annotation Clustering tool of the DAVID Bioinformatics Resources. Genes with higher mRNA levels in the AP group were nearly completely related to mitotic cell cycle and tissue differentiation. During parturition, a complete shift occurred because the genes with higher mRNA levels in IP were nearly all related to three different physiological processes/complexes: i) apoptosis, ii) degradation of extra cellular matrix and iii) innate immune response, which play a fundamental role in placental detachment. These results are an excellent basis for future studies investigating the molecular basis of retained foetal membranes.
Summary A histochemical study using conventional carbohydrate histochemistry (periodic‐acid staining including diastase controls, alcian blue staining at pH 1 and 2.5) as well as using a battery of 14 fluorescein isothiocyanate (FITC)‐labelled lectins to identify glycoconjugates present in 10 different areas of the skin of a catfish (Arius tenuispinis) was carried out. The lectins used were: mannose‐binding lectins (Con A, LCA and PSA), galactose‐binding lectins (PNA, RCA), N‐acetylgalactosamine‐binding lectins (DBA, SBA, SJA and GSL I), N‐acetylglucosamine‐binding lectins (WGA and WGAs), fucose‐binding lectins (UEA) and lectins which bind to complex carbohydrate configurations (PHA E, PHA L). Conventional glycoconjugate staining (PAS staining, alcian blue at pH 1 and 2.5) showed that the mucous goblet cells contain a considerable amount of glycoconjugates in all locations of the skin, whereas the other unicellular gland type, the club cells, lacked these glycoconjugates. The glycoproteins found in goblet cells are neutral and therefore stain magenta when subjected to PAS staining. Alcian blue staining indicating acid glycoproteins was distinctly positive at pH 1, but gave only a comparable staining at pH 2.5. The mucus of the goblet cells therefore also contains acid glycoproteins rich in sulphate groups. Using FITC‐labelled lectins, the carbohydrate composition of the glycoproteins of goblet cells could be more fully characterized. A distinct staining of the mucus of goblet cells was found with the mannose‐binding lectins LCA and PSA; the galactosamine‐binding lectins DBA, SBA and GLS I; the glucosamine‐binding lectin WGA; and PHA E which stains glycoproteins with complex carbohydrate configurations. No reaction occurred with the fucose‐binding lectin UEA and the sialic acid‐specific lectin SNA. In addition, the galactose‐binding lectins PNA and RCA showed only a weak or completely negative staining of the mucus in the goblet cells. The specificity of the lectin staining could be proved by inhibiting binding of the lectins by competitive inhibition with the corresponding sugars. From these data, we can conclude that the mucus produced by the epidermal goblet cells of A. tenuispinis is rich in mannose, N‐acetylgalactosamine and N‐acetylglucosamine residues.
Equine recurrent uveitis (ERU) causes painful inflammatory attacks and oftentimes blindness in the affected eyes. The disease is considered a late sequela of systemic leptospirosis. The most effective therapy is the surgical removal of the vitreous (vitrectomy), which is not only therapeutic, but provides vitreous material that can be assessed diagnostically. For example, the lipL32 gene, culturable Leptospira spp., and anti-Leptospira antibodies have all been detected in vitreous samples obtained from eyes with chronic ERU. Despite this clear evidence of leptospiral involvement, the systemic administration of antibiotics in infected horses is ineffective at resolving ERU. This syndrome of chronic recurrent inflammation, which is unresponsive to antibiotic therapy, combined with apparent bacteria evading the immune response, is consistent with a biofilm-associated infection. The purpose of this study, therefore, was to detect the in vivo biofilm formation of Leptospira spp. in vitreous samples collected during vitrectomy and examined using a Warthin-Starry silver stain and immunohistochemistry. All known steps of biofilm formation were visualized in these samples, including individual Leptospira spp., leptospiral microcolonies and dense roundish accumulations of Leptospira spp. In many instances spirochetes were surrounded by an extracellular substance. Taken together, data from the present study show that ERU is a biofilm-associated intraocular leptospiral infection, which best explains the typical clinical course.
The importance of using techniques that allow the study of pure populations of cells has been increasingly recognized. The authors used laser-assisted microdissection (LAM) in combination with quantitative real-time PCR (qPCR) to assess the relative expression of mRNAs encoding estrogen receptor α (ERα) and progesterone receptor (PR) in the different compartments of the bovine oviduct (epithelium, stroma, smooth muscle coat) during the follicular and mid-luteal phases of the estrus cycle. The localization of receptor mRNA was further studied using non-radioactive in situ hybridization (NISH). A special focus was on whether formalin fixation and paraffin embedding influence the quality and quantity of mRNA obtained from microdissected material. Distinct cyclic changes of the mRNA in the bovine oviduct were observed with elevated levels of PR mRNA transcripts in the epithelium and smooth muscle coat during the follicular phase. The expression of PR mRNA did not vary significantly in the stroma of the bovine oviduct during follicular and mid-luteal phases. In conclusion, the authors found that LAM with qPCR can precisely locate and accurately quantify mRNA expression in specific cell populations from formalin-fixed and paraffin-embedded oviductal tissue.
The cellular cytoskeleton is composed of three fibrillar systems, namely actin microfilaments, microtubules and intermediate filaments (IFs). It not only is a structural system, which mediates functional compartmentalization, but also contributes to many cellular processes such as transport, mitosis, secretion, formation of cell extensions, intercellular communication and apoptosis. In this study, we have examined the distribution of four groups of IFs [cytokeratins (CKs), vimentin, desmin and lamins] in the somatic and germinal cells of the bovine ovary using RT-PCR and immunohistochemical techniques. Using RT-PCR, specific transcripts for all intermediate proteins studied (CK8, CK18, desmin, vimentin, lamin A/C and lamin B1) were detected. A characteristic immunohistochemical staining pattern was observed for the different IFs within the ovary. In this study, we used antibodies against type I CK (acidic CKs: CK14, CK18 and CK19) and type II CK (basic CKs: CK5 and CK8). Among these, only antibodies against CK18 gave a characteristic pattern of immunostaining in the ovary, which included the surface epithelium, the follicle cells, the endothelium of blood vessels and rete ovarii. Antibodies against all other CKs resulted in a weak staining of a limited number of cellular structures (CK5 and CK19) or were completely negative (CK8 and CK14, apart from the surface epithelium). Vimentin antibodies resulted occasionally in a weak staining of the granulosa cells of primary and secondary follicles. In late secondary follicles, the basal and the most apical follicle cells contacting the zona pellucida usually showed a marked immunostaining for vimentin. In antral follicles, three different immunostaining patterns for vimentin were observed. Desmin immunostaining was confined to the smooth muscle cells of blood vessels. Although mRNA for lamin A/C and lamin B1 could be demonstrated using RT-PCR, no immunostaining was found for lamins, neither in the follicle cells nor in the oocytes.
The expression pattern of several intermediate-filament proteins (vimentin, cytokeratin 8, 18 and 19) and the basal lamina component laminin was investigated in the Wolffian and the Müllerian ducts of bovine embryos and fetuses. The material studied comprised sexually undifferentiated stages [crown-rump length (CRL) 0.9 cm/1.0 cm/1.2 cm/1.9 cm/2.5 cm] and female stages (CRL 3.0 cm/4.2 cm/5.1 cm). Laminin could be demonstrated in the basal lamina of the developing Wolffian and Müllerian duct as well as in the stroma surrounding the Müllerian duct. The intermediate-filament protein vimentin was expressed in the mesothelium of the funnel field and in the epithelium of the Müllerian duct in all studied specimens, whereas the epithelial cells of the Wolffian duct only showed vimentin expression from a CRL of 2.2 cm onwards. In the cranial part of the Müllerian ducts only a few cells stained with pan-cytokeratin antibodies, whereas mesothelium and epithelium of the Wolffian duct showed as distinct immunostaining in all investigated stages. Both genital ducts showed no immunostaining with the antibody against cytokeratin 19 at any time of development. We conclude from our immunohistochemical results that the epithelial cells of the Wollfian duct do not contribute cells to the developing Müllerian duct.
ABSTRACT:One of the many roles of tumor necrosis factor (TNF)-α is to control mammalian corpus luteum (CL) PG synthesis and apoptotic cell death. Here, the cellular localization of TNF-α and its type I (TNF-RI) and type II (TNF-RII) receptors in bovine luteal tissue were analyzed using in situ hybridization, immunohistochemistry, and quantitative realtime PCR. Transcripts for TNF-α were expressed in bovine CL throughout the estrous cycle, but were significantly more abundant (P < 0.01) at the regressed luteal stage than at the other stages. Localization of TNF-α transcripts and protein were observed in large and small bovine luteal cells, as well as in immune cells.Moreover, transcripts for TNF-RI and TNF-RII were expressed in bovine CL throughout the estrous cycle. The abundance of TNF-RII transcripts was greater (P < 0.01) at the regressed luteal stage than at the other stages, whereas TNF-RI transcript abundance did not significantly change. Expression of TNF-RI and TNF-RII transcripts and proteins were observed in both the large and small luteal cells, and the proteins were also expressed in the immune cells and vascular endothelial cells. These results suggest that TNF-α sources include immune cells, as well as large and small luteal cells, and that TNF-RI and TNF-RII are present in the luteal cells of the bovine CL.
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