Rebecca Anna-Maria Kenngott

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.

Summary: The aim of this investigation was to study the ultrastructural features during the development of fetal bovine ovaries (crown rump length ranging from 11.4 to 94.0 cm). An interesting observation was the occurrence of big elongated cells containing a variety of electron dense granules and light homogenous vacuoles/bodies. They were located between the stroma cells surrounding the germ cell cord ends, adjacent to the first formed primordial follicles, typically situated near blood vessels. ER alpha and ER beta receptor positive cells could be detected in the same regions by means of immunohistochemistry. Intercellular bridges linked the germ cells nests oogonia. Germ cell cords consisted of centrally located, large, pale oogonia, surrounded by elongated somatic cells with very long cytoplasm extensions. Primordial follicles with flat pale follicular cells could be observed on the inner end of the cords. Extrusions of the outer nuclear membrane could often been recognised in voluminous oocytes.

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.

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 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 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.

Summary: In this study, we investigated the ultrastructural changes during the prenatal differentiation of oviductal epithelium in 16 bovine embryos and fetuses from CRL of 18.0 cm to a CRL of 94.0 cm. Ciliated and secretory cells of bovine uterine tube, a derivative of the Müllerian duct, differentiate to distinct development stages in the prenatal period. The typical cellular pattern, which is generally characteristic for the adult bovine oviduct, is also obtained during fetal life. In the early stages (CRL 18.0/20.4 cm), the bovine oviductal epithelium appears mostly undifferentiated. The epithelial cells show only a few mitochondria, some cisternae of rough endoplasmic reticulum (rER) and a small Golgi‐complex. Most of the cytoplasm is filled with a large amount of glycogen, which decreases during later development. Interspersed between the undifferentiated epithelial cells, a few cells undergoing ciliogenesis can be observed. Ciliogenesis increased significantly during the later prenatal developmental stages. At a CRL of 55.0 cm, ciliated cells appear fully differentiated with mature cells covering their luminal surface. Formation of cilia usually use the acentriolar pathway. Fibrous granules occurred initially in association with the Golgi‐apparatus and r(ER) in the supranuclear cytoplasm. Fibrous granules later fuse with deuterosomes and give rise to procentrioles, which are translocated to the luminal plasma membrane. There they become arranged in a line just beneath the apical cell membrane and further differentiate to basal bodies from which the formation of cilia and striated rootlets take place. Clear signs of differentiation of secretory cells were first seen in our material in fetuses with a CRL of 51.0 cm and 64.0 cm. These cells contain a well developed rER and Golgi‐apparatus with dilated cisterns. In the supranuclear cytoplasm, the number of secretory granules continuously increases during later development and the cells adapt to the morphology of mature secretory cells at the CRL 94.0 cm.

Summary: The expression pattern of several intermediate‐filament proteins (vimentin, cytokeratin 8, 18 and19) 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.

Summary: In this study the development of the bovine Fallopian tube was investigated using light microscopic methods. Formation and differentiation of the Müllerian duct were studied in mesonephroi of 16 embryos and fetuses with a crown‐rump lengths (CRL) of 0.9–8.4 cm. The funnel field, the rostral beginning of the Müllerian duct was first observed at a CRL of 0.9 cm. It appears as a thickening of the mesothelium on the craniolateral side of the mesonephros. During later development the Müllerian duct emerges by caudal outgrowth from the funnel field. Formation of a common basal lamina surrounding the caudal tips of Müllerian and Wolffian ducts could be observed at all stages up to CRL of 2.7 cm. The mesothelium and the epithelium of the Wolffian duct adjacent to the Müllerian duct showed a modification of epithelium height in all examined stages. Probably the Wolffian duct influences the growth of Müllerian duct by epithelio‐mesenchymal interactions. Fetuses from a CRL of 12.0 to 94.0 cm were used for investigation of the prenatal differentiation of the oviductal mucosa. Folding of the oviductal mucosa started at a CRL of 29.0 cm and continued until birth. Individual primary, secondary and tertiary folds are formed in special proliferation zones and epithelium‐folding buds. The cellular differentiation of the oviductal epithelium involves the formation of ciliated and secretory cells during different times of prenatal development. Ciliogenesis was first detected at a CRL of 33.0 cm. Active secretory cells could be observed in the oviductal epithelium from a CRL of 64.0 cm onwards.