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The follicular phase of the ovine ovarian cycle demonstrates parallel increases in ovarian estrogens and uterine blood flow (UBF). Although estrogen and nitric oxide contribute to the rise in UBF, the signaling pathway remains unclear. We examined the relationship between the rise in UBF during the ovarian cycle of nonpregnant sheep and changes in the uterine vascular cGMP-dependent pathway and large-conductance Ca2+-activated K+ channels (BKca). Nonpregnant ewes (n = 19) were synchronized to either follicular or luteal phase using a vaginal progesterone-releasing device (CIDR), followed by intramuscular [PGF2.sub.[alpha]], CIDR removal, and treatment with pregnant mare serum gonadotropin. UBF was measured with flow probes before tissue collection, and second-generation uterine artery segments were collected from nine follicular and seven luteal phase ewes. The poreforming [alpha]- and regulatory [beta]-subunits that constitute the [BK.sub.Ca], soluble guanylyl cyclase (sGC), and cGMP-dependent protein kinase G (cPKG) isoforms ([cPKG.sub.1[alpha]] and [cPKG.sub.1[beta]]) were measured by Western analysis and cGMP levels by RIA. BKca subunits were localized by immunohistochemistry. UBF rose >3-fold (P < 0.04) in follicular phase ewes, paralleling a 2.3-fold rise in smooth muscle cGMP and 32% increase in [cPKG.sub.1[alpha]], (P < 0.05). sGC, [cPKG.sub.1[beta]], and the [BK.sub.Ca] [alpha]-subunit were unchanged. Notably, expression of 131- and [32-regulatory subunits rose 51 and 79% (P -< 0.05), respectively. Increases in endogenous ovarian estrogens in follicular-phase ewes result in increases in UBF associated with upregulation of the cGMP- and cPKG-dependent pathway and increased vascular [BK.sub.Ca] [beta]/[alpha]-subunit stoichiometry, suggesting enhanced [BK.sub.Ca] activation contributes to the follicular phase rise in UBF. estrogen; uterine blood flow; protein kinase G; guanylyl cyclase doi: 10.1152/ajpendo.00375.2009

Regulation of uteroplacental blood flow (UPBF) during pregnancy remains unclear. Large conductance, [Ca.sup.2+]-activated [K.sup.+] channels ([BK.sub.Ca]), consisting of [alpha]-and regulatory [beta]-subunits, are expressed in uterine vascular smooth muscle (UVSM) and contribute to the maintenance of UPBF in the last third of ovine pregnancy, but their expression pattern and activation pathways are unclear. We examined [BK.sub.Ca] subunit expression, the cGMP-dependent signaling pathway, and the functional role of [BK.sub.Ca] in uterine arteries (UA) from nonpregnant (n = 7), pregnant (n = 38; 56-145 days gestation; term, ~150 days), and postpartum (n = 15; 2-56 days) sheep. The a-subunit protein switched from 83-87 and 105 kDa forms in nonpregnant UVSM to 100 kDa throughout pregnancy, reversal occurring >30 days postpartum. The 39-kDa [[beta].sub.1]-subunit was the primary regulatory subunit. Levels of 100-kDa [alpha]-subunit rose ~70% during placentation (P < 0.05) and were unchanged in the last two-thirds of pregnancy; in contrast, [[beta].sub.1]-protein rose throughout pregnancy ([R.sup.2] = 0.996; P < 0.001; n = 13), increasing 50% during placentation and approximately twofold in the remainder of gestation. Although UVSM soluble guanylyl cyclase was unchanged, cGMP and protein kinase [G.sub.1[alpha]] increased (P < 0.02), paralleling the rise and fall in [[beta].sub.1]-protein during pregnancy and the puerperium. [BK.sub.Ca] inhibition not only decreased UA nitric oxide (NO)-induced relaxation but also enhanced [alpha]-agonist-induced vasoconstriction. UVSM [BK.sub.Ca] modify relaxation-contraction responses in the last two-thirds of ovine pregnancy, and this is associated with alterations in [alpha]-subunit composition, [alpha]:[[beta].sub.1]subunit stoichiometry, and upregulation of the cGMP-dependent pathway, suggesting that [BK.sub.Ca] activation via NO-cGMP and [[beta].sub.1] augmentation may contribute to the regulation of UPBF. uteroplacental blood flow; regulatory subunits; protein kinase G isoforms; cyclic guanosine 3',5'-monophosphate

Vascular smooth muscle (VSM) maturation is developmentally regulated and differs between vascular beds. The maturation and contribution of VSM function to tissue blood flow and blood pressure regulation during early gestation are unknown. The carotid artery (CA) contributes to fetal cerebral blood flow regulation and well being. We studied CA VSM contractility, protein contents, and phenotype beginning in the midthird of ovine development. CAs were collected from early (88-101 day of gestation) and late (138-150 day; term = day 150) fetal (n = 14), newborn (6-8 day old; n = 7), and adult (n = 5) sheep to measure forces in endothelium-denuded rings with KC1, phenylephrine, and ANG II; changes in cellular proteins, including total and soluble protein, actin and myosin, myosin heavy chain isoforms (MHC), filamin, and proliferating cell nuclear antigen; and vascular remodeling. KC1 and phenylephrine elicited age- and dose-dependent contraction responses (P < 0.001) at all ages except early fetal, which were unresponsive. In contrast, ANG II elicited dose responses only in adults, with contractility increasing greater than fivefold vs. that shown in fetal or neonatal animals (P < 0.001). Increased contractility paralleled age-dependent increases (P < 0.01) in soluble protein, actin and myosin, filamin, adult smooth muscle MHC-2 (SM2) and medial wall thickness and reciprocal decreases (P < 0.001) in nonmuscle MHC-B, proliferating cell nuclear antigen and medial cellular density. VSM nonreceptor- and receptor-mediated contractions are absent or markedly attenuated in midgestation and increase age dependently, paralleling the transition from synthetic to contractile VSM phenotype and, in the case of ANG II, paralleling the switch to the [AT.sub.1] receptor. The mechanisms regulating VSM maturation and thus blood pressure and tissue perfusion in early development remain to be determined. myosin heavy chain isoforms; nonmuscle myosin; fetal development; receptor and nonreceptor function; smooth muscle growth; angiotensin-II doi:10.1152/ajpregu.00851.2006

Daily estradiol-17[beta] ([E.sub.2][beta]) increases basal uterine blood flow (UBF) and enhances acute [E.sub.2][beta]-mediated increases in UBF in ovariectomized nonpregnant ewes. The acute [E.sub.2][beta]-mediated rise in UBF involves vascular smooth muscle (VSM) large-conductance [Ca.sup.2+]-activated [K.sup.+] channels (B[K.sub.Ca]). B[K.sub.Ca], consist of pore-forming [alpha]-subunits and regulatory [[beta].sub.1]-subunits that modulate channel function and [E.sub.2][beta] responsiveness. It is unclear whether [E.sub.2][beta] also alters subunit expression and thus channel density and/or function, thereby contributing to the rise in basal UBF and enhanced UBF responses that follow daily [E.sub.2][beta]. Therefore, we examined B[K.sub.Ca] subunit expression by using reverse transcription-PCR and immunoblot analysis of arterial VSM from reproductive and nonreproductive tissues and myometrium from ovariectomized nonpregnant ewes after daily [E.sub.2][beta] (1 [micro]g/kg iv) or vehicle without or with acute [E.sub.2][beta] (1 [micro]g]kg). Tissue distribution was determined by immunohistochemistry. Acute [E.sub.2][beta] did not alter [alpha]- or [[beta].sub.1]-subunit expression in any tissue (P > 0.1). Daily [E.sub.2][beta] also did not affect [alpha]-subunit mRNA or protein in any tissue (P > 0.1) or mesenteric arterial VSM [[beta].sub.1]-subunit. However, daily [E.sub.2][beta] increased uterine and mammary arterial VSM 131-subunit mRNA by 32% and 83% (P < 0.05), uterine VSM protein by 30%, and myometrial [[beta].sub.1]-subunit mRNA and protein by 74% (P [less than equal to] 0.005). Immunostaining of uterine arteries, myometrium, and intramyometrial arteries paralleled immunoblot analyses for both subunits. Although B[K.sub.Ca] density is unaffected by daily and acute [E.sub.2][beta], daily [E.sub.2][beta] increases [[beta].sub.1]-subunit in proximal and distal uterine arterial VSM. Thus prolonged [E.sub.2][beta] exposure may alter B[K.sub.Ca] function, estrogen responsiveness, and basal vascular tone and reactivity in reproductive arteries by modifying [alpha]:[[beta].sub.1] stoichiometry. myometrium; nonpregnant ewes; vasodilation; uterine blood flow; mesenteric artery; mammary artery

The follicutar phase of the ovine ovarian cycle demonstrates parallel increases in ovarian estrogens and uterine blood flow (UBF). Although estrogen and nitric oxide contribute to the rise in UBF, the signaling pathway remains unclear. We examined the relationship between the rise in UBF during the ovarian cycle of nonpregnant sheep and changes in the uterine vascular cGMP-dependent pathway and large-conductance Ca2+-activated K+ channels (BKCa). Nonpregnant ewes (n = 19) were synchronized to either follicular or luteal phase using a vaginal progesterone-releasing device (CIDR), followed by intramuscular PGF2α, CIDR removal, and treatment with pregnant mare serum gonadotropin. UBF was measured with flow probes before tissue collection, and second-generation uterine artery segments were collected from nine follicular and seven luteal phase ewes. The poreforming α-and regulatory β-subunits that constitute the BKCa, soluble guanylyl cyclase (sUC), and cGMP-dependent protein kinase G (cPKG) isoforms (cPKG1α and cPKG1β) were measured by Western analysis and cGMP levels by RIA. BKCa subunits were localized by immunohistochemistry. UBF rose >3-fold (P < 0.04) in follicular phase ewes, paralleling a 2.3-fold rise in smooth muscle cGMP and 32% increase in cPKG1α (P < 0.05). sGC, cPKG1β, and the BKCa α-subunit were unchanged. Notably, expression of β1- and β2-regulatory subunits rose 51 and 79% (P ⩽ 0.05), respectively. Increases in endogenous ovarian estrogens in follicular-phase ewes result in increases in UBF associated with upregulation of the cGMP- and cPKG-dependent pathway and increased vascular BKCa β/α-subunit stoichiometry, suggesting enhanced BKCa activation contributes to the follicular phase rise in UBF. [ABSTRACT FROM AUTHOR]

The intact ovine uterine vascular bed (UVB) is sensitive to α-agonists and refractory to angiotensin II (ANG II) during pregnancy; the converse occurs in the systemic circulation. The mechanism(s) responsible for these differences in uterine sensitivity are unclear and may reflect predominance of nonconstricting AT(2) receptors (AT(2)R) in uterine vascular smooth muscle (UVSM). The contribution of the placental vasculature also is unclear. Third generation and precaruncular/placental arteries from nonpregnant (n = 16) and term pregnant (n = 23) sheep were used to study contraction responses to KCl, norepinephrine (NE), and ANG II (with/without ATR specific inhibitors) and determine UVSM ATR subtype expression and contractile protein content. KCl and NE increased third generation and precaruncular/placental UVSM contractions in a dose- and pregnancy-dependent manner (P ≤ 0.001). ANG II only elicited modest contractions in third generation pregnant UVSM (P = 0.04) and none in precaruncular/placental UVSM. Moreover, compared with KCl and NE, ANG II contractions were diminished ≥ 5-fold. Whereas KCl and ANG II contracted third generation>>precaruncular/placental UVSM, NE-induced contractions were similar throughout the UVB. However, each agonist increased third generation contractions ≥ 2-fold at term, paralleling increased actin/myosin and cellular protein content (P ≤ 0.01). UVSM AT(1)R and AT(2)R expression was similar throughout the UVB and unchanged during pregnancy (P > 0.1). AT(1)R inhibition blocked ANG II-mediated contractions; AT(2)R blockade, however, did not enhance contractions. AT(2)R predominate throughout the UVB of nonpregnant and pregnant sheep, contributing to an inherent refractoriness to ANG II. In contrast, NE elicits enhanced contractility throughout the ovine UVB that exceeds ANG II and increases further at term pregnancy.

Intracellular regulation of oocyte meiosis is not completely understood. However, reversible phosphorylation, which involves serine/threonine protein kinases and phosphatases (PP), is an important mediator. Glycogen synthase kinase-3 (GSK-3) is a highly conserved serine/threonine protein kinase. Currently no reports exist on presence or function of GSK-3 in mammalian oocytes. The aim of this study was to determine GSK-3 presence/absence, transcript and protein expression, intracellular protein distribution, and to investigate the functional importance of GSK-3 in mouse oocyte meiosis. Germinal vesicle-intact (GVI) oocytes contained both GSK-3 transcript and protein. Although GSK-3 beta-isoform is the only transcript identifiable in GVI oocytes, both alpha- and beta-isoforms were recognized by Western blot analysis. In growing, meiotic-incompetent oocytes GSK-3 was present, diffusely located throughout the cytoplasm and absent in the nucleus, whereas in meiotic-competent oocytes this cytoplasmic GSK-3 displays a predominant peri-oolemma staining. Treatment of mouse GVI oocytes with lithium chloride (LiCl), which inhibits both inositol monophosphatase (IMPase) and GSK-3, had no significant influence on oocyte viability, morphology, or development to metaphase II (MII). However, LiCl caused abnormal spindle formation and significantly increased incidence of abnormal homologue segregation during the first meiotic division. L690,330, which is a specific IMPase inhibitor, had no significant effect on oocyte viability, morphology, MII development, or homologue segregation. This is the first report of GSK-3 in mammalian oocytes. LiCl inhibition of mouse oocyte GSK-3 modified organization of microtubules and/or function of meiotic spindles thus compromising segregation of condensed bivalent chromosomes., (Copyright 2003 Wiley-Liss, Inc.)