IntroductionRapid induction of keratinocyte and fibroblast migration into wounds is necessary for tissue repair. Reepithelialization begins soon after injury as epidermal cells become migratory (1, 2). Because plasma fibronectin (pFn) circulates in the blood at 0.3-0.5 mg/mL (reviewed in ref.3) and binds to fibrin, it is a significant component of clot provisional matrix. If the basement membrane is destroyed, epidermal cells migrate over the pFnrich provisional matrix (4-6). The fibronectin of the provisional matrix is also known to promote the movement of fibroblasts, macrophages, and blood vessels into the wound space (1, 7). Keratinocytes, other epithelial cell types such as mammary and prostate epithelial cells, and fibroblasts express the α5β1 integrin fibronectin receptor (8-11). Although keratinocytes express less α5β1 than α3β1, α5β1 is more important for migration (8). Also, expression of α5β1 (12, 13) and specific metalloproteinases (14) are increased in motile keratinocytes at the leading edge of the new epithelium. Keratinocyte and fibroblast cell movement is supported by pFn (15,16). Fragments of pFn containing the cell-binding domain, which are found in wounds due to localized proteinase activity (17, 18), stimulate monocyte (7) and fibroblast chemotaxis through the dermis (19-21), as well as matrix metalloproteinase expression by fibroblasts (11). This interaction also stimulates cellular invasion of the provisional matrix and wound stroma (16; reviewed in ref. 22). The pFn cell-binding domain also induces monocytes to extravasate from proximal vessels, enabling them to enter the wound and differentiate into inflammatory macrophages (23), and can stimulate normal human endothelial cell motility in vitro (24). Thus, the invasive/migratory phenotype expressed by epithelial cells and fibroblasts after wounding may result from pFn fragmentation and α5β1 integrin-mediated signaling. Furthermore, this interaction promotes the immigration or motility of a number of cell types involved in healing through a variety of extracellular matrices, including the stroma as well as vascular basement membranes (23,24).To define the invasion-stimulatory sequences of pFn, the naturally serum-free basement membranes and associated extracellular matrices of sea urchin embryos (SU-ECM) were used as in vitro invasion substrates (25). We report that the PHSRN sequence of pFn stimulates SU-ECM invasion by keratinocytes and fibroblasts. Acetylated and amidated PHSRN peptide (Ac-PHSRN-NH 2 ) is greatly increased in activity, whereas a randomized sequence peptide, Ac-HSPNR-NH 2 , is inactive. In derivatives of the PHSRN peptide, substitution of arginine by alanine or glutamic acid also results in inactivity. The PHSRN sequence of the plasma fibronectin (pFn) cell-binding domain induces human keratinocytes and fibroblasts to invade the naturally serum-free extracellular matricies of sea urchin embryos. The potency of acetylated, amidated PHSRN (Ac-PHSRN-NH 2 ) is significantly increased, making it more active on a molar basis ...
To explain the high rate of blood flow in the corpus luteum, we hypothesize that luteal blood vessels offer minimal resistance to flow and are incapable of vasomotion. This hypothesis was tested in rabbits at mid-pseudopregnancy by measuring blood flow in the corpus luteum and ovarian stroma with tracer-labeled microspheres at three levels of arterial blood pressure, which was manipulated by constricting the aorta above the ovarian artery. In addition, the distribution of vascular smooth muscle in the ovary was evaluated with morphological and immunocytochemical techniques. Decreases in arterial pressure were paralleled by reductions in blood flow in the corpus luteum, whereas ovarian stromal blood flow was unchanged. Consistent with our hypothesis, there was no change in the low level of vascular resistance offered by blood vessels in the corpus luteum, supporting the view that they are maximally dilated and incapable of autoregulation. Morphologically, the vessels within the corpus luteum appeared as large sinusoidal capillaries without smooth muscle, providing an anatomical explanation for the lack of vasomotor control demonstrated physiologically. The absence of vascular smooth muscle was confirmed with immunocytochemistry using an antibody against the muscle-specific intermediate filament, desmin. The fluorescein-labeled antibody decorated arteries and arterioles within the ovarian stroma and near the capsule of the corpus luteum, but did not decorate vessels in the corpus luteum of pseudopregnancy, providing additional evidence that the vessels of the corpus luteum lack the smooth muscle investment necessary to change vascular caliber. From these findings, we have proposed a novel scheme to explain intraovarian blood flow regulation. Vascular resistance in the ovarian stroma, as in most tissues, is acutely regulated by dilation or constriction of intratissue arterioles. In contrast, vascular resistance within the corpus luteum is modeled as a relatively invariable parameter, fixed at a low level by the morphological characteristics of the luteal vasculature. Therefore, the corpus luteum operates on a linear (maximally "vasodilated") pressure-flow curve, does not actively regulate intratissue blood flow, and is subject to acute regulation of perfusion only through changes in extra-luteal vessels.
Using spectrofluorimetry and fluorescence microscopy, we analyzed the uptake and degradation of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-albumin) by the rat visceral yolk sac (VYS) during whole embryo culture. Rat conceptuses exposed continuously to FITC-albumin had linear increases of both acid-soluble and acid-insoluble FITC fluorescence in the VYS. Smaller amounts of FITC fluorescence that were nearly all acid soluble accumulated in the extraembryonic fluid, while the embryo proper did not accumulate a significant amount of fluorescence. During a chase period following a pulse exposure to FITC albumin, FITC fluorescence in the VYS decreased linearly, while that in the extraembryonic fluid and culture medium increased. Addition of proteinase inhibitors to the culture medium together with FITC-albumin increased acid-insoluble FITC-fluorescence in the VYS tissue but decreased acid-soluble fluorescent degradation products in the yolk sac, extraembryonic fluid, and the culture medium. Fluorescence microscopy of yolk sacs exposed to FITCalbumin revealed that the fluorescence was localized in apical vacuoles of the yolk sac epithelium and decreased substantially during a chase period. In conceptuses exposed to proteinase inhibitors, the yolk sac epithelium had enlarged vacuoles containing FITCfluorescence whose clearance in pulse-chase experiments was effectively blocked. Overall, these data suggest that FITC-albumin resembles 125 I-albumin in its processing by the VYS and that the fluorescent protein is an attractive alternative tracer molecule for studies of the effects of embryotoxicants on yolk sac function during whole embryo culture.
We investigated the physiological basis for the trophic effect of glucocorticoids in rat corpora lutea in the absence of pituitary gonadotropins. Immature (Day 29) Sprague-Dawley rats were given eCG and hCG to induce the development of corpora lutea and were hypophysectomized on Day 32. Beginning on Day 40, rats received twice-daily s.c. injections of either dexamethasone (dex; 200 microg/rat/day) or vehicle (controls) and then were killed on Day 44. Plasma 20alpha-dihydroprogesterone, a major steroid produced by the corpora lutea, was higher (p = 0.01) in dex-treated than in control rats (44.5 +/- 2.3 vs. 23.0 +/- 5.6 ng/ml). Dexamethasone treatment increased lipid droplets and lipid in the corpora lutea as revealed by electron microscopy and oil red O staining. Cholesterol esters were higher in corpora lutea of dex-treated rats compared to controls (14.8 +/- 1.1 vs. 2.2 +/- 0.5 microg/mg corpora lutea wet tissue, respectively; p = 0.05). Another group of hypophysectomized rats was treated with either a high or a lower dosage of corticosterone, both of which caused an elevation to > 2-fold of plasma 20alpha-dihydroprogesterone concentration compared to controls. Glucocorticoid receptor protein (about 92 kDa) was detected in both luteal and nonluteal ovarian tissues in this animal model. These effects of glucocorticoids and the presence of the glucocorticoid receptor raise the possibility of a physiological role for glucocorticoids in the rat corpus luteum.
The luteotropic roles of prolactin and testosterone (or estradiol formed in luteal tissue) were investigated in hypophysectomized rats with homografts of granulosa lutein tissue. Using this approach, we could determine the effects of prolactin independently of estrogen, since granulosa lutein tissue does not produce estrogen de novo under these conditions. Luteinizing granulosa cells were expressed from the ovaries of immature pregnant mare's serum gonadotropin-primed Fischer 344 rats 6 h after injection of human chorionic gonadotropin. The cells were transplanted beneath the kidney capsule of adult, hypophysectomized, ovariectomized Fischer 344 recipients, which were treated with hormones daily for 12 or 14 days. In rats without treatment (no hormones, n = 3) and in rats treated with only testosterone (Silastic capsule, n = 6), only small amounts of luteal tissue (less than 5 mg/rat) were found and serum progesterone remained at low concentrations (10 ng or less) throughout the experiment. In contrast, in rats treated either with ovine prolactin (300 micrograms/day, n = 10) or with the combination of prolactin and testosterone (n = 12), serum progesterone increased to 43 ng/ml by Day 8. Beyond Day 8, serum progesterone continued to rise in rats treated with the combination of prolactin and testosterone to reach a mean value of 87 ng/ml by Day 14, and mean homograft wet weight was 49 mg/rat; in rats treated with only prolactin, serum progesterone decreased to 25 ng/ml by Day 14 and homograft wet weight was lower (24 mg/rat). Prolactin and testosterone together stimulated more homograft aromatase activity in vivo than did prolactin alone, but the in vitro production of progesterone was not different.(ABSTRACT TRUNCATED AT 250 WORDS)
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