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B Berisha, D Schams, M Kosmann, W Amselgruber and R Einspanier

Locally produced growth factors may have important modulatory roles in final ovarian follicular growth. The aim of this study was to investigate the possible participation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF2) in bovine follicles during final growth. Ovaries were collected from a slaughterhouse within 10-20 min after exsanguination. A classification of follicles into five groups (<0.5; >0.5-5; >5-20; >20-180; >180 ng/ml) was performed according to the follicular fluid (FF) oestradiol-17 beta content. For a better characterisation of classes the mRNA expressions of FSH receptor, LH receptor and aromatase cytochrome P450 in theca interna (TI) and granulosa cells (GC) were determined. Analysis of VEGF transcript by RT-PCR showed that GC and theca cells express predominantly the smallest isoforms (VEGF(121) and VEGF(165)). VEGF mRNA expression in both tissues (TI and GC) and VEGF protein concentration in total follicle tissue increased significantly (and correlated) with developmental stages of follicle growth. The expression of mRNA for VEGF receptor (VEGFR)-1 and VEGFR-2 was very weak in GC, without any regulatory change during final follicle growth. In contrast, TI showed strong expression of mRNA for both receptors in all follicle classes examined. VEGF protein concentrations in FF increased significantly and continuously to maximum levels in preovulatory follicles. As shown by immunohistochemistry, VEGF protein was clearly localised in TI and GC of preovulatory follicles. FGF2 and FGF receptor (FGFR) mRNA expression in TI increased significantly during final growth of follicles. In contrast, the FGF2 and FGFR mRNA expression in GC was very weak and without any regulatory change during follicle growth. Histological observation revealed that FGF2 protein was localised in theca tissue (cytoplasm of endothelial cells and pericytes) but not in GC. Our results suggest that VEGF and FGF families are involved in the proliferation of capillaries that accompanies the selection of the preovulatory follicle resulting in an increased supply of nutrients and precursors, and therefore supporting the growth of the dominant follicle.

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A Plath, R Einspanier, F Peters, F Sinowatz and D Schams

It is now widely accepted that the mammary gland is under interconnected hormonal and local control. Growth factors are involved in the intercellular signalling of the gland. Our aim was the detection of transforming growth factors alpha (TGF-alpha) and beta 1 (TGF-beta 1) messenger RNA during mammogenesis, lactogenesis, galactopoiesis and involution in the bovine mammary gland (total n = 27). During these stages the RNA was assessed by means of ribonuclease protection assay and reverse transcription-polymerase chain reaction (RT-PCR). To study possible influences of oestrogen, progesterone and prolactin on growth factor expression, mammary RNA was obtained from heifers after induced mammogenesis and lactogenesis, with and without additional prolactin inhibition (total n = 20). Very low levels of TGF-alpha and TGF-beta 1 expression were detected during lactogenesis and galactopoiesis, increasing levels during mammogenesis of primigravid heifers, and highest levels during mammogenesis of virgin heifers and during involution. TGF-alpha expression after induced mammogenesis was greater than after induced lactogenesis or physiological mammogenesis during pregnancy. Furthermore, TGF-alpha mRNA contents increased after prolactin inhibition. TGF-beta 1 expression was almost equal after induced mammogenesis and lactogenesis, but greater than during the physiological mammogenesis and lactogenesis. In conclusion, it can be assumed that growth promoting TGF-alpha and growth inhibiting TGF-beta 1 are co-expressed in the bovine mammary gland. Higher mRNA contents of both factors during mammogenesis and involution may indicate autocrine or paracrine functions for these growth factors during proliferation and reorganisation of the mammary tissue.

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S E Ulbrich, S Rehfeld, S Bauersachs, E Wolf, R Rottmayer, S Hiendleder, M Vermehren, F Sinowatz, H H D Meyer and R Einspanier

Nitric oxide synthases (NOS) account for the endogenous production of nitric oxide (NO), a small and permeable bioreactive molecule. NO is known to act as a paracrine mediator during various processes associated with female reproduction. In the present study, the mRNA expression of the endothelial (eNOS) and inducible (iNOS) NO synthases were examined in bovine oviduct epithelial cells (BOEC) during the oestrous cycle. In addition, eNOS and iNOS mRNA and protein were localised by in situ hybridisation and immunocytochemistry respectively. Furthermore, the effects of exogenously applied oestradiol-17β and progesterone on NOS mRNA regulation were studied in a suspension culture of BOEC. The eNOS mRNA abundance was low around ovulation (day 0) and increased significantly until pro-oestrus (day 18) in the ampulla. Immunoreactive protein of eNOS was detected predominantly in endothelial cells as well as in secretory oviduct epithelial cells at pro-oestrus. The iNOS mRNA concentration was significantly reduced in the isthmus at pro-oestrus (day 18) and oestrus (day 0) compared with persistently high levels in the ampulla. By in situ hybridisation, specific iNOS transcripts were additionally demonstrated in the oviduct epithelium. Immunoreactive iNOS protein was localised in secretory epithelial cells as well as in the lamina muscularis. The in vitro stimulation showed that both NOS were stimulated by progesterone, but not by oestradiol-17β. The region-specific modulated expression of eNOS and iNOS provides evidence for an involvement of endogenously produced NO in the regulation of oviductal functions.