Modulation of P2Y2 receptors in bovine cumulus oocyte complexes: effects on intracellular calcium, zona hardening and developmental competence.

The improvement of cryopreserved oocyte survival is imperative for the preservation of female fertility. In this study, we investigate whether P2Y2 receptors (P2Y2R) can be directly implicated in calcium (Ca2+) homeostasis misbalances observed during the cryopreservation process of cumulus oocyte complexes (COC). Firstly, RNA was extracted from bovine immature and mature oocytes and cumulus cells and real-time PCR performed to identify P2Y2R transcripts (experiment 1). Changes in intracellular calcium concentration [Ca2+]i of mature COC and oocytes (experiment 2) were measured upon exposure to cryoprotectants (CPA), UTP (P2Y2R stimulator, 100 µM), and/or suramin (P2Y2R inhibitor, 100 and 300 µM). The functional role of P2Y2R was investigated by analyzing the effect on oocyte viability of its modulation prior and during oocyte exposure to CPA (experiment 3). Mature COC were randomly divided into groups, and exposed to CPA and different P2Y2 modulators. Oocytes' viability, cortical granules location, and competence for development were assessed. Results showed that P2Y2R mRNAs are expressed in both oocytes and cumulus cells. Stimulation with UTP and CPA led to [Ca2+]i increase, and this effect was totally or partially blocked by suramin (P2Y2R inhibitor). Oocyte exposure to CPA and UTP reduced embryo rates compared with control and suramin100µM (P ≤ 0.04). The observed enhanced premature zona hardening in oocytes exposed to CPA (P = 0.04) and UTP (P = 0.005) stimulus was inhibited by suramin 100 µM. In conclusion, inhibition of P2Y2R during cryoprotectant exposure reduces premature intracellular Ca2+ release and significantly improves the developmental competence of exposed bovine oocytes.

Bovine oocyte membrane permeability and cryosurvival: Effects of different cryoprotectants and calcium in the vitrification media.

The cryopreservation process must be improved to enhance oocyte cryosurvival and functionality. Two protocols with different cryoprotectants (CPAs), containing either ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose (EGDMSO) or 1,2-propanediol and sucrose (PrOH) were evaluated. In both protocols, calcium (Ca2+) free or -containing base media were tested. Oocytes were subjected to vitrification or only exposed to CPAs without immersion in liquid nitrogen. Oocyte's viability, cortical granules location and competence for development after fertilization were assessed. Finally, fatty acid composition and membrane permeability of oocytes exposed to CPAs were analyzed. Independently of Ca2+ concentration in the vitrification media, the development rates were higher in oocytes vitrified with EGDMSO protocols (p = 0.0005). After warming, higher cleavage rates were obtained in EGDMSO + Ca2+ compared to the PrOH without Ca2+ protocol (p = 0.02). Oocytes exposed to PrOH without Ca2+ presented lower cleavage rates compared to control (p = 0.04). An enhanced premature zona hardening in vitrified oocytes as well as lower concentrations of the fatty acids c11:18:1 and 20:4n-6 in cumulus oocyte complexes exposed to PrOH protocols were identified. The oocytes minimum volume and permeability were affected by the exposure to PrOH and Ca2+ (p ≤ 0.007). In conclusion, the most effective protocol for bovine oocytes cryopreservation combines EG and DMSO, independently of Ca2+ concentration in the media. A higher toxicity and an incomplete depletion of water during PrOH loading may hamper oocyte viability. The type of CPAs and Ca2+ interfered differentially on oocyte pathways to functionality, and this should be considered when choosing a cryopreservation protocol.

Role of cholinergic-activated KCa1.1 (BK), KCa3.1 (SK4) and KV7.1 (KCNQ1) channels in mouse colonic Cl- secretion.

AIM: Colonic crypts are the site of Cl- secretion. Basolateral K+ channels provide the driving force for luminal cystic fibrosis transmembrane regulator-mediated Cl- exit. Relevant colonic epithelial K+ channels are the intermediate conductance Ca2+-activated K(Ca)3.1 (SK4) channel and the cAMP-activated K(V)7.1 (KCNQ1) channel. In addition, big conductance Ca2+-activated K(Ca)1.1 (BK) channels may play a role in Ca2+-activated Cl- secretion. Here we use K(Ca)1.1 and K(Ca)3.1 knock-out mice, and the K(V)7.1 channel inhibitor 293B (10 microm) to investigate the role of K(Ca)1.1, K(Ca)3.1 and K(V)7.1 channels in cholinergic-stimulated Cl- secretion. METHODS: A Ussing chamber was used to quantify agonist-stimulated increases in short circuit current (Isc) in distal colon. Chloride secretion was activated by bl. forskolin (FSK, 2 microm) followed by bl. carbachol (CCH, 100 microm). Luminal Ba2+ (5 mm) was used to inhibit K(Ca)1.1 channels. RESULTS: K(Ca)1.1 WT and KO mice displayed identical FSK and CCH-stimulated Isc changes, indicating that K(Ca)1.1 channels are not involved in FSK- and cholinergic-stimulated Cl- secretion. CCH-stimulated DeltaIsc was significantly reduced in K(Ca)3.1 KO mice, underscoring the known relevance of this channel in the activation of Cl- secretion by an intracellular Ca2+ increasing agonist. The residual CCH effect observed in K(Ca)3.1 KO mice suggests that yet another K+ channel is driving the CCH-stimulated Cl- secretion. In the presence of the specific K(V)7.1 channel blocker 293B, the residual CCH effect was abolished. CONCLUSIONS: This demonstrates that both K(Ca)3.1 and K(V)7.1 channels are activated by cholinergic agonists and drive Cl- secretion. In contrast, K(Ca)1.1 channels are not involved in stimulated electrogenic Cl- secretion.

Distal colonic Na(+) absorption inhibited by luminal P2Y(2) receptors.

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na(+) channel (ENaC)-mediated Na(+) absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y(2) and a luminal P2Y(4) receptor, stimulate K(+) secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na(+) absorption in distal colonic mucosa of mice treated on a low Na(+) diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V (te)): -13.7 +/- 1.9 mV (lumen negative), transepithelial resistance (R (te)): 24.1 +/- 1.8 Omega cm(2), equivalent short circuit current (I (sc)): -563.9 +/- 63.8 microA/cm(2) (n = 21). Amiloride completely inhibited I (sc) to -0.5 +/- 8.5 microA/cm(2). Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I (sc) by 160.7 +/- 29.7 microA/cm(2) (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC(50) values of 10 microM and 3 microM respectively. In P2Y(2) knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na(+) absorption was absent. In contrast, in P2Y(4) KO mice the inhibitory effect of luminal UTP on Na(+) absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na(+) diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y(2) and P2Y(4) receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y(2) receptor mediates inhibition of electrogenic Na(+) absorption.

K+ secretion activated by luminal P2Y2 and P2Y4 receptors in mouse colon.

Extracellular nucleotides are important regulators of epithelial ion transport, frequently exerting their action from the luminal side. Luminal P2Y receptors have previously been identified in rat distal colonic mucosa. Their activation by UTP and ATP stimulates K+ secretion. The aim of this study was to clarify which of the P2Y receptor subtypes are responsible for the stimulated K+ secretion. To this end P2Y2 and P2Y4 knock-out mice were used to measure distal colonic ion transport in an Ussing chamber. In mouse (NMRI) distal colonic mucosa, luminal UTP and ATP with similar potency induced a rapid and transient increase of the transepithelial voltage (V(te)) (UTP: from -0.81 +/- 0.23 to 3.11 +/- 0.61 mV, n = 24), an increase of equivalent short circuit current (I(sc)) by 166.9 +/- 22.8 microA cm(-2) and a decrease of transepithelial resistance (R(te)) from 29.4 +/- 2.4 to 23.5 +/- 2.0 Omega cm2. This effect was completely inhibited by luminal Ba2+ (5 mm, n = 5) and iberiotoxin (240 nm, n = 6), indicating UTP/ATP-stimulated K+ secretion. RT-PCR analysis of isolated colonic crypts revealed P2Y2, P2Y4 and P2Y6 specific transcripts. The luminal UTP-stimulated K+ secretion was still present in P2Y2 receptor knock-out mice, but significantly reduced (DeltaV(te): 0.83 +/- 0.26 mV) compared to wild-type littermates (DeltaV(te): 2.08 +/- 0.52 mV, n = 9). In P2Y4 receptor knock-out mice the UTP-induced K+ secretion was similarly reduced. Luminal UTP-stimulated K+ secretion was completely absent in P2Y2/P2Y4 double receptor KO mice. Basolateral UTP showed no effect. In summary, these results indicate that both the P2Y2 and P2Y4 receptors are present in the luminal membrane of mouse distal colonic mucosa, and stimulation of these receptors leads to K+ secretion.

Lecithinase reaction of Staphylococcus aureus strains of different origin on Baird-Parker medium.

Staphylococcus aureus produces one or more enzymes with lipolytic activity, but differences between strains have been reported (Owens and John 1975; O'Toole 1987; Rollof et al. 1987). The biological and biochemical properties of these enzymes have been investigated and results were recently reviewed (Kötting et al. 1984). Baird-Parker medium (Baird-Parker 1962) is a selective medium commonly used for the isolation of Staph. aureus. The presence of egg yolk in this medium permits the detection of two reactions due to lipolytic activity of staphylococci: (1) Lecithinase reaction, a zone of precipitate in the medium surrounding the colonies; and (2) Lipase reaction or 'pearly layer', an iridescent film in and immediately surrounding colonies, visible by reflected light (iridescent sheen or 'oil in water'). In this study, human and bovine strains, previously biotyped according to the scheme of Devriese et al. (1984), were compared for production of a zone of precipitation, lecithinase reaction, on Baird-Parker medium. Bovine and human strains of Staph. aureus were compared for production of the egg yolk reaction (lecithinase reaction) on Baird-Parker medium and the results were related to their biotypes and site of origin of the sample. Human strains and strains biotyped as human biotypes had higher percentage of positive results than bovine isolates and/or biotypes. However, all strains isolated from body sites of heifers produced a positive reaction regardless of the biotype.

Isolation and identification of coagulase-negative Staphylococcus species from bovine body sites and streak canals of nulliparous heifers.

Heifers (n = 103) ranging in age from 1d to 2 yr were sampled to determine the coagulase-negative staphylococcal flora of haircoat, nares, vagina, teat skin, and streak canal. A total of 2706 staphylococal strains were identified from 3612 bacterial isolates. Other genera or groups identified included Bacillus, Micrococcus, Corynebacterium, and coliforms. Staphylococci were identified utilizing a simplified biochemical scheme. Staphylococcus xylosus, S. chromogenes, and S. warneri were the predominant species recovered from anatomic sites and streak canal. Staphylococcal strains identified from specific body sites (expressed as percentage of heifers harboring these species) were: nares 74% S. xylosus and 48% S. warneri; haircoat, 70% S. xylosus and 57% S. chromogenes; vagina, 60% S. chromogenes and 54% S. xylosus; teat skin 62% S. chromogenes and 61% S. warneri; streak canal 53% S. chromogenes, and 43% S. warneri. The prevalent staphylococcal strains identified differed from heifers in confined housing compared with heifers on pasture. Differences observed in distribution of Staphylococcus species among body sites, particularly those between teat skin and streak canal, suggest that establishment of staphylococcal microflora depends on the ability of a species to adapt to and colonize anatomic sites as well as on environmental conditions present.