Requirement of Nck adaptors for actin dynamics and cell migration stimulated by platelet-derived growth factor B.

The Nck family of Src homology (SH) 2/SH3 domain adaptors functions to link tyrosine phosphorylation induced by extracellular signals with downstream regulators of actin dynamics. We investigated the role of mammalian Nck adaptors in signaling from the activated platelet-derived growth factor (PDGF) receptor (PDGFbetaR) to the actin cytoskeleton. We report here that Nck adaptors are required for cytoskeletal reorganization and chemotaxis stimulated by PDGF-B. Analysis of tyrosine-phosphorylated proteins demonstrated that Crk-associated substrate (p130(Cas)), not the activated PDGFbetaR itself, is the major Nck SH2 domain-binding protein in PDGF-B-stimulated cells. Both Nck- and p130(Cas)-deficient cells fail to display cytoskeletal rearrangements, including the formation of membrane ruffles and the disassembly of actin bundles, typically shown by their WT counterparts in response to PDGF-B. Furthermore, Nck and p130(Cas) colocalize in phosphotyrosine-enriched membrane ruffles induced by PDGF-B in NIH 3T3 cells. These results suggest that Nck adaptors play an essential role in linking the activated PDGFbetaR with actin dynamics through a pathway that involves p130(Cas).

Follicular development: the role of the follicular microenvironment in selection of the dominant follicle.

The importance of endocrine signals in the regulation of follicular development has long been recognized. However, the follicular microenvironment also plays a critical role in determining follicular fate. This review summarizes our studies on the role of the intrafollicular IGF system in selection of the dominant follicle (DF) in cattle. During the bovine estrous cycle, the largest antral follicles develop in two or three successive waves of follicular recruitment and selection of a DF. High concentrations of estradiol in the follicular fluid are the hallmark of dominant and preovulatory follicles and are associated with lower concentrations of low molecular weight (MW) insulin-like growth factor binding proteins (IGFBP-2, -4, and -5), which can prevent binding of IGF to its receptor. Our studies have shown that dominant and preovulatory follicles also have much higher levels of an IGFBP-4/-5 protease activity, which is the bovine equivalent of the human IGFBP-4 protease, pregnancy-associated plasma protein-A (PAPP-A). Studies of follicles isolated just after the emergence of the DF showed that PAPP-A is present in the follicular fluid of the DF as soon as it can be detected as morphologically dominant. To examine whether higher levels of PAPP-A in one follicle of the cohort (the future DF) precedes morphological dominance, the four largest follicles were isolated from pairs of bovine ovaries obtained before one follicle of the cohort was significantly larger the others, around the time that one follicle was first detected as morphologically dominant and after dominance was well established. Analysis of the temporal sequence of changes in estradiol, low MW IGFBPs, free IGF, and PAPP-A in the follicular fluid suggested that an increase in PAPP-A is the earliest biochemical difference yet detected in the future DF and that follicular selection is the result of a progressive series of changes beginning with the acquisition of PAPP-A, which leads to a decrease in IGFBP-4 and -5 and an increase in free IGF, which synergizes with FSH to increase estradiol production. Co-dominant follicles, induced by injection of small doses of recombinant bovine (rb) FSH, both had levels of PAPP-A similar to the single DF of control heifers, supporting the hypothesized role of FSH in the induction of PAPP-A in the DF. Taken together, these results suggest a critical role for FSH-induced PAPP-A, and thus for free IGF, in the selection of the DF. In contrast, other experiments provided evidence for a deleterious effect of IGF on the initiation of bovine follicular growth and the survival of primordial and primary follicles in vitro. These results underscore the importance of the follicular microenvironment in determining follicular fate and indicate that its effects can be stage-specific.

Proteolysis of insulin-like growth factor binding proteins -4 and -5 in bovine follicular fluid: implications for ovarian follicular selection and dominance.

Dominant follicles are characterized by low levels of low molecular weight IGF binding proteins (IGFBPs) and by proteolytic activity against IGFBP-4 and -5. To examine the hypothesis that proteolysis of IGFBP-4 and -5 plays a critical role in selection of the dominant follicle, we isolated follicles at various stages during the first wave of follicular development during the bovine estrous cycle, using ultrasonography to follow follicular growth. Ovariectomies were performed before divergence in follicular size (group 1; largest follicle, approximately 7 mm in diameter), at about the expected time of size divergence (group 2; largest follicle, approximately 8 mm) or after a dominant follicle was clearly present (group 3; largest follicle, > or =9 mm). The four largest follicles (F1-F4) were dissected and concentrations of steroids, IGFBPs and free IGF-I and levels of proteolytic activity for IGFBP-4 and -5 in the follicular fluid were determined. Follicles in group 1 did not differ significantly in size or estradiol concentrations, but levels of proteolytic activity against IGFBP-4 and -5 were higher in F1-F2 than in F3-F4. However, in group 2 the largest follicle (F1) had higher estradiol, free IGF-I, and IGFBP-4 and -5 proteolytic activity than F2-F4, whereas only slight (dissected) or no (ultrasound) differences in diameters could be detected. Differences between F1 and F2-F4 in diameter, estradiol, free IGF-I, and IGFBP-4 and -5 proteolytic activity were even greater in group 3. In addition, the hormonal regulation of IGFBP-4 and -5 proteolysis was evaluated in vivo by injecting heifers with small doses of recombinant bovine FSH to induce codominant follicles (group 4). The induced codominant follicles were larger and had higher IGFBP-4 and -5 proteolytic activity than subordinate follicles. The results suggest that follicular selection is a progression of changes starting with acquisition of an FSH-inducible IGFBP-4/-5 proteolytic activity, leading to an increase in intrafollicular concentration of free IGF-I that, in turn, synergizes with FSH to promote greater estradiol production by the follicle destined for dominance.

Selection of the dominant follicle and insulin-like growth factor (IGF)-binding proteins: evidence that pregnancy-associated plasma protein A contributes to proteolysis of IGF-binding protein 5 in bovine follicular fluid.

Development of a dominant follicle is associated with decreased intrafollicular low molecular weight IGF-binding proteins (namely IGFBP-2, -4, and -5) and increased proteolysis of IGFBP-4 by pregnancy-associated plasma protein A (PAPP-A). In addition to IGFBP-4 proteolytic activity, bovine follicular fluid contains strong proteolytic activity for IGFBP-5, but not for IGFBP-2. Here we show that the IGFBP-5 protease present in bovine follicular fluid is a neutral/basic pH-favoring, Zn(2+) metalloprotease very similar to the previously described IGFBP-4 protease. We hypothesized that immunoneutralization and immunoprecipitation with anti-PAPP-A antibodies would result in abrogation of the IGFBP-4, but not the IGFBP-5, proteolytic activity in follicular fluid. As expected, anti-PAPP-A antibodies were able to neutralize and precipitate the IGFBP-4, but not the IGFBP-5, proteolytic activity of human pregnancy serum, which was used as a positive control for PAPP-A. Surprisingly, immunoneutralization and immunoprecipitation of follicular fluid from bovine preovulatory follicles with anti-PAPP-A antibodies abrogated both IGFBP-4 and IGFBP-5 proteolysis. Quantitative results derived from phosphorimaging revealed a complete inhibition of both IGFBP-4 and -5 proteolysis by follicular fluid incubated for 2 or 5 h in the presence of anti-PAPP-A antibodies. After 18 h of incubation, anti-PAPP-A antibodies still inhibited IGFBP-5 degradation, although with an efficiency lower than that for IGFBP-4 degradation. Both proteolytic activities have identical electrophoretic mobility, and a single band ( approximately 400 kDa) was detected by Western immunoblotting of bovine follicular fluid with anti-PAPP-A antibodies. Proteolysis of IGFBP-5 was readily detectable in follicular fluid from dominant follicles and was negligible in subordinate follicles from the same cohort. These results suggest that an active intrafollicular IGFBP-4/-5 proteolytic system, in which PAPP-A is the major protease involved, is an important determinant of follicular fate.

Differentiation of dominant versus subordinate follicles in cattle.

Selection of a dominant follicle, capable of ovulating, from among a cohort of similarly sized follicles is a critical transition in follicular development. The mechanisms that regulate the selection of a species-specific number of dominant follicles for ovulation are not well understood. Cattle provide a very useful animal model for studies on follicular selection and dominance. During the bovine estrous cycle, two or three sequential waves of follicular development occur, each producing a dominant follicle capable of ovulating if luteal regression occurs. Follicles are large enough to allow analysis of multiple endpoints within a single follicle, and follicular development and regression can be followed via ultrasonographic imaging. Characteristics of recruited and selected follicles, obtained at various times during the first follicular wave, have been determined in some studies, whereas dominant and subordinate follicles have been compared around the time of selection in others. As follicular recruitment proceeds, mRNA for P450 aromatase increases. By the time of morphological selection, the dominant follicle has much higher concentrations of estradiol in follicular fluid, and its granulosa cells produce more estradiol in vitro than cells from subordinate follicles. Shortly after selection, dominant follicles have higher levels of mRNAs for gonadotropin receptors and steroidogenic enzymes. It has been hypothesized that granulosa cells of the selected follicle acquire LH receptors (LHr) to allow them to increase aromatization in response to LH, as well as FSH. However, LH does not appear to stimulate estradiol production by bovine granulosa cells, and the role of LHr acquisition remains to be determined. Recent evidence suggests a key role for changes in the intrafollicular insulin-like growth factor (IGF) system in selection of the dominant follicle. When follicular fluid was sampled in vivo before morphological selection, the lowest concentration of IGF binding protein-4 (IGFBP-4) was more predictive of future dominance than size or estradiol concentration. Consistent with this finding, dominant follicles acquire an FSH-induced IGFBP-4 protease activity. Thus, a decrease in IGFBP-4, which would make more IGF available to interact with its receptors and synergize with FSH to promote follicular growth and aromatization, appears to be a critical determinant of follicular selection for dominance.

A potential role for insulin-like growth factor binding protein-4 proteolysis in the establishment of ovarian follicular dominance in cattle.

A critical transition in ovarian follicular development is the selection of a dominant follicle, capable of ovulating, from a cohort of synchronously growing antral follicles. However, little is known about mechanisms and factors that regulate the selection and growth of dominant ovarian follicles. We have investigated whether a component of the insulin-like growth factor (IGF) system, namely IGFBP-4 protease, is associated with the establishment of follicular dominance in cattle. IGFBP proteases degrade IGFBPs, freeing IGFs to interact with their receptors. In experiment 1, follicular fluid from preovulatory follicles (n = 4) degraded about 80% of the added recombinant human (rh) IGFBP-4 within 18 h of incubation. The IGFBP-4 protease exhibited optimal activity at neutral/basic pH and its sensitivity to various protease inhibitors suggested a metalloprotease. The decline in the intensity of the band corresponding to intact rhIGFBP-4 was accompanied by the appearance of immunoreactive fragments of molecular weights approximately 18 and 14 kDa, which were not detectable by ligand blot analysis. In experiment 2, follicular fluid samples were collected from dominant and subordinate follicles on Day 2 or 3 of the first follicular wave, after ovariectomy (experiment 2a, n = 3/day) or by ultrasound-guided follicular aspiration (experiment 2b, n = 4-5/day). Estradiol concentrations in follicular fluid from dominant vs. subordinate follicles confirmed their identities and indicated that the dominant follicle had been selected by Day 2 of the follicular wave. In both experiments 2a and 2b, IGFBP-4 proteolytic activity was 2- to 3.5-fold (P < 0.05) and 5-fold (P < 0.01) higher in follicular fluid from dominant than subordinate follicles on Days 2 and 3 of the follicular wave, respectively. The finding that IGFBP-4 proteolytic activity is higher in dominant, estrogen-active follicles than in subordinate follicles of the same cohort, as early as Day 2 of the follicular wave, strongly suggests a role for IGFBP-4 protease in the establishment of ovarian follicular dominance.

Development of codominant follicles in cattle is associated with a follicle-stimulating hormone-dependent insulin-like growth factor binding protein-4 protease.

Low molecular weight insulin-like growth factor binding proteins (IGFBPs), particularly IGFBP-4, are believed to inhibit the actions of insulin-like growth factors (IGFs). We showed previously that ovarian follicular dominance in cattle is associated with the presence of a protease that degrades IGFBP-4. To test the hypothesis that specific IGFBP-4 proteolysis is associated with selection of the dominant follicle, we induced codominant follicles (co-DFs) during the first follicular wave of the estrous cycle. The ovaries of Holstein heifers were examined twice daily by ultrasonography; when the largest follicle reached 6 mm in diameter, saline (control, n = 5) or 2 mg of recombinant bovine (rb) FSH (FSH, n = 5) was injected i.m. every 12 h for 48 h. Follicular fluid was collected by aspiration from the two largest follicles/heifer 12 h after the last injection. IGFBPs in follicular fluid were quantified by Western ligand blotting/phosphorimaging. IGFBP-4 protease activity was measured by incubating follicular fluid with recombinant human (rh) IGFBP-4 substrate, followed by ligand blotting/phosphorimaging to quantify the percent of substrate loss and Western immunoblotting to detect specific proteolytic fragments. Co-DFs of FSH heifers did not differ (P > 0.05) from the single dominant follicle of controls in size, or in concentration of progesterone or level of IGFBP-4 in follicular fluid. In contrast, the largest subordinate follicle of control heifers was smaller, with lower progesterone and higher IGFBP-4 in the follicular fluid (P < 0.05). Concentrations of estradiol in follicular fluid were high in dominant follicles, intermediate in co-DFs, and low in subordinate follicles (P < 0.05). IGFBP-4 protease activity in co-DFs was similar (P > 0.05) to that of dominant follicles, but fourfold higher (P < 0.05) than that of subordinate follicles. The results strongly suggest that an FSH-dependent IGFBP-4 protease is associated with selection of the dominant follicle in cattle.

Ovarian follicular wave synchronization and induction of ovulation in postpartum beef cows.

An experiment was designed to evaluate a) the effect of a progesterone-estradiol combined treatment on ovarian follicular dynamics in postpartum beef cows, and b) ovulation and the subsequent luteal activity after short-term calf removal and GnRH agonist treatment. Multiparous Angus cows (25 to 40 d after calving) were assigned to the following treatments: untreated (Control, n = 9); short term calf removal (CR, n = 8); progesterone (CIDR, n = 9) and progesterone plus estradiol-17 beta (CIDR + E-17 beta, n = 9). Progesterone treatment (CIDR) lasted 8 d and the day of device insertion was considered as Day 0. Cows in the CIDR + E-17 beta group also received an i.m. injection of 5 mg of E-17 beta on Day 1. On Day 8, calves were removed for 48 h (CR, CIDR and CIDR + E-17 beta groups) and 6 h before the end of calf removal these cows also received an i.m. injection of 8 micrograms of Busereline (GnRH). Anestrus was confirmed in all cows by the absence of luteal tissue and progesterone concentrations below 1 ng ml-1 at the beginning of the experiment. Although mean (+/- SEM) interval from the beginning of the experiment (Day 0) to wave emergence did not differ (P > 0.05) among treatment groups (Control, 1.9 +/- 1.0, range -2 to 7 d; CR, 3.9 +/- 0.7, range 0 to 6 d; CIDR, 2.8 +/- 0.5, range 0 to 4 d and CIDR + E-17 beta, 4.1 +/- 0.2, range 3 to 5), the variability was less (P < 0.05) in the CIDR + E-17 beta group. The proportion of cows ovulating 24 to 48 h after GnRH administration tended (P = 0.08) to be higher in cows from CIDR + E-17 beta group (8/9) than in those of CR (5/8) or CIDR (6/9) groups, respectively and was associated with a higher proportion (P < 0.05) of CIDR + E-17 beta treated cows (9/9) that had a dominant follicle in the growing/early static phase at the time of GnRH treatment compared to the other GnRH treated groups (5/8, and 4/9 for CR and CIDR groups, respectively). Two CR cows ovulated 0-24 h after GnRH and only one Control cow ovulated the day before the time of GnRH administration. Cows pretreated with progesterone had longer (P < 0.05) luteal lifespan (CIDR, 14.5 +/- 0.7, CIDR + E-17 beta, 13.9 +/- 0.6 d) than those not treated with CIDR (Control, 5, CR, 4.0 +/- 0.4). We conclude that progesterone plus estradiol treatment results in tightly synchronized wave emergence and high GnRH-induced ovulation rate with normal luteal activity in postpartum beef cattle.

[Development and validation of a survey for evaluation of the satisfaction of patients with medical consultation in primary care clinics]. / Desarrollo y validación de una encuesta para evaluar la satisfacción de los pacientes con la consulta médica en consultorios de atención primaria.

The aim of this work was to devise, and assess the reliability, of an instrument to measure satisfaction with medical consultation of primary care urban patients at public outpatient clinics. A model was elaborated, based on data collected from the literature and from focal groups of patients and physicians, and an enquiry was developed and applied at four outpatient clinics. Fifty enquiries were obtained and analyzed to discard questions of difficult understanding. Next, a second self administered questionnaire and with fewer questions was devised. Its reliability was assessed in 53 enquiries, obtaining a Cronbach's a of 0.904. Questions that decreased consistency were discarded, finally obtaining a self administered instrument with 22 questions. This enquiry was used in 174 patients. Results were subjected to factorial analysis with varimax rotation, which separated three factors that explain 64% of the variance. It is concluded that a valid and reliable instrument was obtained.

Comparison of the activity of the cis and trans isomer of vitamin K1 in vitamin K-deficient and coumarin anticoagulant-pretreated rats.

The cis and trans isomers of vitamin K1 have been prepared at a purity of better than 99.5% and tested for effect on plasma level of factor VII in coumarin anticoagulant-pretreated and vitamin K-deficient rats. Both isomers show activity, but in coumarin anticoagulant-pretreated animals the cis isomer has approximately 10% and in vitamin K-deficient approximately 1% of the activity of the trans isomer. The cis isomer also shows slower onset and rate of increase of the response. Reduction of the 2',3'-double bond of the phytyl side chain of either isomer to the same 2',3'-dihydro derivative of vitamin K1 leaves the activity of the trans isomer unchanged but increases the activity of the cis isomer to that of the trans isomer. The results suggest that the phytyl side chain not only serves to increase lipid solubility, but may play a more direct functional role.