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.

To study the involvement of the IGFs in mammary development and lactation of the cow, the temporal expressions of IGF-I and -II, its receptor type 1 (IGFR-1), IGF-binding proteins (IGFBPs)-1 to -6 and GH receptor (GHR) mRNA were examined. This was carried out for different stages of mammogenesis, lactogenesis, galactopoiesis and involution in the bovine mammary gland of 26 animals. Furthermore, IGF-I was localised by immunohistochemistry. The highest mRNA concentrations for IGF-I were detected in the mammary tissue of late pregnant heifers (days 255-272) and significantly lower expression was detected during lactogenesis and galactopoiesis. Immunohistochemistry of IGF-I revealed only a weak staining in the epithelium of the ducts during mammogenesis. The epithelium of the alveoli were negative during mammogenesis, lactogenesis and galactopoiesis but displayed distinct IGF-I activity during involution. In the stroma a distinct staining of the cytoplasm of adipocytes and of vascular smooth muscle cells was observed. A certain percentage of fibroblasts (usually 20-30%) were also immunopositive. In contrast, highest expression for IGFR-1 was detected during galactopoiesis and involution. The lowest mRNA concentration for IGFR-1 was found during pregnancy (days 194-213). In general, the expression of IGF-II was not regulated during mammogenesis and lactation, but decreased during involution. The mRNA for the six binding proteins was detected in the bovine mammary gland. The dominant binding proteins were IGFBP-3 and -5. The highest expression of IGFBP-3 was observed during mid-pregnancy and the lowest during late lactation, involution and in non-pregnant heifers. The mRNA for IGFBP-5 increased during late mammogenesis and lactogenesis followed by a decrease thereafter. In general, the mRNA concentrations for IGFBP-2, -4 and -6 were barely detectable during all stages. In contrast, the expression for IGFBP-1 was upregulated in the mammary gland of virgin heifers and increased around the onset of lactation. mRNA for GHR was found during all stages examined without outstanding fluctuations. In conclusion, locally produced IGF-I and -II may mediate mammogenesis. The high mammary IGFR-1 mRNA during lactation suggests a role for peripheral IGF-I in maintenance of lactation. The role of IGFBPs in the mammary gland needs further evaluation.

The newly formed corpus luteum (CL) rapidly develops after ovulation and has the features of active vascularisation and mitosis of steroidogenic cells. These stage-specific mechanisms also may contribute to gain the function of prostaglandin F2 alpha (PGF2 alpha)-resistant CL at this stage. Recent studies suggest that the vasoactive peptide angiotensin II (Ang II) regulates luteal function. Thus, this study aimed to investigate (i) the expression of angiotensin-converting enzyme (ACE) mRNA by RT-PCR and the ACE protein expression by immunohistochemistry, (ii) the effects of angiogenic growth factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), on the secretion of Ang II, PGF2 alpha, progesterone and oxytocin (OT), and (iii) the effects of luteal vasoactive peptides (Ang II and endothelin-1 (ET-1)) or OT on the secretion of PGF2 alpha, progesterone and OT from bovine early CL (days 3--4 of the oestrous cycle), and evaluate a possible interaction of these substances with PGF2 alpha. The expression of mRNA for ACE was found in theca interna of mature follicle, early CL and endothelial cells from developing CL as well as pituitary and kidney, but granulosa cells of mature follicle were negative. The immunohistochemical analysis revealed that blood capillaries (endothelial cells) were stained for ACE, but luteal cells were negative in early CL. To examine the effects of substances on the secretory function of the CL, an in vitro microdialysis system was used as a model. The infusion of bFGF and VEGF stimulated Ang II and PGF2 alpha secretion as well as progesterone, but not OT secretion in early CL. The infusion of Ang II after PGF2 alpha infusion continued the stimulatory effect on progesterone and OT release within early CL until 3 h thereafter. However, the infusion of ET-1 alone had no effect on progesterone or OT release. The infusion of luteal peptides such as Ang II and OT stimulated PGF2 alpha secretion, whereas the infusion of ET-1 did not. In conclusion, the overall results of this study indicate that a functional angiotensin system exists on the endothelial cells of early CL, and that angiogenic factors bFGF and VEGF upregulate luteal Ang II and PGF2 alpha secretion, which fundamentally supports the mechanism of progesterone secretion in bovine early CL. This idea supports the concept that the local regulatory mechanism involved in active angiogenesis ensures the progesterone secretion in the developing CL in vivo.

It is now well established that oestrogen and progesterone are absolutely essential for mammary gland development. Lactation can be induced in non-pregnant animals by sex steroid hormone treatment. Most of the genomic actions of oestrogens are mediated by two oestrogen receptors (ER)-alpha and ERbeta, and for gestagens in ruminants by the progesterone receptor (PR). Our aim was the evaluation of mRNA expression and protein (localisation and Western blotting) during mammogenesis, lactogenesis, galactopoiesis (early, middle and late) and involution (8, 24, 28, 96-108 h and 14-28 days after the end of milking) in the bovine mammary gland (total no. 53). During these stages, the mRNA was assessed by means of real-time RT-PCR (LightCycler). The protein for ERalpha, ERbeta and PR was localised by immunohistochemistry and Western blotting. The mRNA expression results indicated the existence of ERalpha, ERbeta and PR in bovine mammary gland. Both ERalpha and PR are expressed in fg/ micro g total RNA range. The highest mRNA expression was found for ERalpha and PR in the tIssue of non-pregnant heifers, followed by a significant decrease to a lower level at the time of lactogenesis with low concentrations remaining during lactation and the first 4 weeks of involution. In contrast, the expression of ERbeta was about 1000-fold lower (ag/ micro g total RNA) and showed no clear difference during the stages examined, with a significant increase only 2-4 weeks after the end of milking. Immunolocalisation for ERalpha revealed a strong positive staining in nuclei of lactocytes in non-pregnant heifers, became undetectable during pregnancy, lactogenesis and lactation, and was again detectable 14-28 days after the end of milking. In contrast, PR was localised in the nuclei of epithelial cells in the mammary tIssue of non-pregnant heifers, in primigravid animals, and during late lactation and involution. During lactogenesis, peak and mid lactation, fewer nuclei of epithelial cells were positive, but increased staining of the cytoplasm of epithelial cells was obvious. ERalpha and ERbeta protein was found in all mammary gland stages examined by Western blotting. In contrast to mRNA expression, the protein signal for ERalpha was weaker in the tIssue of non-pregnant heifers and during involution (4 weeks). ERbeta protein showed a stronger signal (two isoform bands) in non-pregnant heifers and 4 weeks after the end of milking. This correlated with the mRNA expression data. Three isoforms of PR (A, B and C) were found by Western blotting in the tIssue of non-pregnant heifers, but only isoform B remained during the following stages (lactogenesis, galactopoiesis and involution). In conclusion, the mRNA expression and protein data for ER and PR showed clear regulatory changes, suggesting involvement of these receptors in bovine mammary gland development and involution.