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The roles of pH in regulation of uterine contraction in the laying hens
Animal Reproduction Science 118 (2010) 317–323
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
The roles of pH in regulation of uterine contraction in the laying hens S. Kupittayanant a,∗ , P. Kupittayanant b a b
School of Physiology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand School of Animal Production Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
a r t i c l e
i n f o
Article history: Received 27 February 2009 Received in revised form 1 July 2009 Accepted 7 July 2009 Available online 16 July 2009 Keywords: Laying hen pH Uterus Calcium
a b s t r a c t In the laying hens, the uterus (shell gland) plays essential roles in calcium transfer for calcification of the eggshell and expulsion of the egg through the vagina for oviposition. Much is known about the effects of pH changes on eggshell production of the uterus. However, very little is understood about the effects of pH changes on uterine contractility. We investigated the effects of pH changes on uterine contraction in the laying hens. The laying hens were humanely killed, and strips of uterine smooth muscles were isolated. Isometric force was measured and the effects of intracellular and extracellular pH changes studied. The results show that alterations of pH clearly have marked effects on force in the hen uterus. Both intracellular and extracellular acidifications significantly decreased uterine activity, whether it arises spontaneously or in the presence of agonists such as prostaglandin F2␣ and arachidonic acid. Alkalinization produced the opposite effects. Thus, changes in pH can regulate uterine contraction. This insight into pH regulation of the uterine activity provides a focus for egg production management directed at physiological and pathological oviposition in the laying hens. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The hen uterus is a myogenic organ. Without stimulation of nerves and hormones, it can produce spontaneously phasic contractions for several hours in the appropriate condition (Kupittayanant et al., 2008). Our previous work has shown that the frequency of spontaneous activity, in vivo, is around 18 contractions every 10 min (Kupittayanant et al., 2008). The major pathway that regulates such contractions depends on the calcium–calmodulin myosin light chain kinase (MLCK) pathway, as without either calcium or MLCK no force was produced (Kupittayanant et al., 2008). Agonist such as prostaglandin F2␣ (PGF2␣ ) can alter spontaneously contracting uterus not only by stimulating the calcium–calmodulin MLCK pathway but also by activating the non-calcium–calmodulin MLCK pathway (Kupittayanant et al., 2008). Thus, uterine contractility of
∗ Corresponding author. Tel.: +66 44224633; fax: +66 44224633. E-mail address: [email protected] (S. Kupittayanant). 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.07.001
the laying hens can be regulated and modulated via several pathways. It is well established that changes in intracellular and extracellular pH can modulate uterine contractility in the human and laboratory animals (Phoenix and Wray, 1993; Taggart and Wray, 1993; Parratt et al., 1995; Taggart et al., 1996; Larcombe-McDouall et al., 1998; Naderali and Wray, 1999; Pierce et al., 2003; Wray, 2007). Physiological changes in intracellular pH may occur for several reasons such as restricting blood flow and oxygen supply to the uterus, increased uterine contractility, and alteration of acid–base balance (Wray, 2007). When extracellular pH is altered, intracellular pH will change (Pierce et al., 2003). To the best of our knowledge, there is no data demonstrating the effects of pH changes on uterine contractility in the laying hens. However, the effects of pH changes on eggshell production of the uterus are well described (see below). Acidification, which occurs during normal process of egg formation, enhances eggshell calcification (Johnson, 2002), whereas alkalinization, occurred during heat stress, inhibits calcification causing soft-shelled eggs (El Hadi and
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Sykes, 1982; Tanor et al., 1984; Mashaly et al., 1986; Odom et al., 1986; Koelkebeck and Odom, 1994; Rozenboim et al., 2007). During the physiological process of egg formation, the uterus is the oviductal part that the egg spends the longest for calcification of the eggshell (18–20 h depending on cycle length) (Johnson, 2002). There, acidification occurs while calcium is deposited to form the eggshell (Simkiss, 1969). However, it is unclear whether the uterus is quiescent while the process of calcification takes place. Under pathological conditions, the heat stressed laying hens often lay eggs with thinner shells because of alkalinization as a result of elevating blood pH after excessive loss of CO2 from their lungs (El Hadi and Sykes, 1982; Tanor et al., 1984; Mashaly et al., 1986; Odom et al., 1986; Koelkebeck and Odom, 1994; Rozenboim et al., 2007). In addition, it is likely that the oviposition interval in the heat stressed laying hens is always shorter (< 24 h) compared with the unstressed laying hens (24–26 h) (Novero et al., 1991). These studies led us to hypothesize that changes in pH (both acidification and alkalinization) might affect uterine contraction. Thus, an inactive uterus might be required during acidification to complete the process of calcification, whereas alkalinization might activate the uterus that may lead to premature oviposition as occurs during heat stress. Furthermore, there are several hormones control oviposition including PGF2␣ . It is not known whether pH changes can occur in the presence of PGF2␣ . It was reported that the level of PGF2␣ increases sharply before oviposition to aid uterine contraction and an injection of its precursor, AA, leads to increases in intraluminal pressure of the uterus (Johnson, 2002). Thus, it is not clear whether acidification could affect either PGF2␣ - or AA-induced contraction. We therefore examined the effects of pH changes on the uterus. The experiments were designed to answer the questions: (1) How does the spontaneous contractility of the uterus change with acidification or alkalinization? (2) Do changes in pH alter uterine contractility when the agonists (PGF2␣ or arachidonic acid, AA) are present? (3) Are the effects of extracellular pH on contractility the same as that of the effects of intracellular pH? 2. Materials and methods This study was conducted in accordance with guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council of Thailand. The experiment protocol was approved by the Institutional Animal Ethics Committee, Suranaree University of Technology, Nakhon Ratchasima, Thailand. 2.1. Uterine samples and myometrial strip preparations Uterine samples and myometrial strip preparations were taken from the group of laying hens used previously by Kupittayanant et al. (2008). Briefly, myometrial tissues were obtained from crossbred laying hens (12–17 months old and weighing 1.8–2.3 kg). The hens were maintained in individual cages. The lighting regimen was 14 h L:10 h D (light, 06:00–20:00 h) and food and water were freely available. The oviposition time was recorded
daily for each hen so that the time of next oviposition could be predicted. The mean oviposition interval was 24.20 ± 0.05 h. Hens were killed by cervical dislocation 4 h before the onset of oviposition. The uteri were removed and immediately immersed in physiological saline solution. The samples were transported to the nearby laboratory for immediate uses or stored at 4 ◦ C, until dissection was performed. 2.2. Tension measurements As with previous work (Kupittayanant et al., 2008), strips of longitudinal fibres (8 mm × 2 mm × 1 mm) were dissected. The strips were mounted in a 25 mL temperature-controlled organ bath (Panlab s.l. for ADInstruments Pty Ltd., Spain). The myometrial strips were attached at each end to two metal hooks. One hook was attached to a fixed point, and the other hook was attached to a tension transducer (ADInstruments Pty Ltd., Spain). Passive resting tension of 1 g was given, which produced baseline tension. The tissue-bathing medium used was physiological solution maintained at pH of 7.4, temperature of 37 ◦ C, and gassed with 100% O2 . The strip was allowed to contract spontaneously and an equilibrium period of 30 min was given before the application of any chemical. The measurements were made whilst the strip was continually perfused with physiological solution (control) or physiological solution containing drugs and/or chemicals. The electrical signal from the transducer was amplified and converted to a digital signal and recorded on a computer using Chart software (ADInstruments Pty Ltd., Australia). 2.3. Solutions and chemicals Physiological salt solution (pH, 7.4) contained (in mM); NaCl (154), KCl (5.4), MgSO4 (1.2), glucose (12), CaCl2 (2), and HEPES (10). The agonists PGF2␣ and AA were used at concentrations of 1 M (Wechsung and Houvenaghel, 1981; Kupittayanant et al., 2008). All chemicals were obtained from Sigma Chemical Co. (St. Louis, USA). 2.4. Experimental design 2.4.1. Intracellular pH changes As with earlier study (Pierce et al., 2003), changes in intracellular pH at constant external pH were produced by iso-osmotic substitution of sodium chloride with 40 mM sodium butyrate (NaBu) or 40 mM ammonium chloride (NH4 Cl). Although intracellular pH was not measured directly in our study, previous work used the human myometrial tissue preparation had shown that similar applications of NaBu or NH4 Cl produced intracellular pH changes of approximately 0.14 pH units (Parratt et al., 1995). These changes occur within 1 min as NaBu and NH4 Cl cross the cell membranes and dissociate, after that intracellular pH will be restored gradually to normal value within 20 min by pH regulatory mechanisms (Parratt et al., 1995).
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2.4.2. Extracellular pH changes Changes in extracellular pH were made by the addition of hydrochloric acid or sodium hydroxide to the perfusing solution to produce acidification or alkalinization (Pierce et al., 2003). It has been reported that the mean value of the intracellular pH of the uterus during the period from 1 to 10 h of calcification is around 6.5, whereas the value for the last 8 h (i.e. from 10 to 18 h) is 6.9 (Simkiss, 1969). Thus, the extracellular acidification to pH 6.9 and alkalinization to pH 7.9 were used for the subsequent experiments. 2.5. Data presentation and statistical analysis The data were analyzed using Microcal Origin Software. Parameters that were measured include the contraction amplitude, the contraction integral, and the contraction frequency. The phasic contractions in weak acid or weak base were measured over 20 min from the start of their application. Results were expressed as percentages of control contractions (i.e. the control is 100%). To test the effects of weak acid or weak base on PGF2␣ or AA, contractions were compared for the 10 min in PGF2␣ or AA (i.e. 11–20 min after start of PGF2␣ or AA exposure), to the 11–20 min in PGF2␣ or AA with weak acid or weak base. In some experiments, PGF2␣ or AA was added after weak acid or weak base and the effects compared. Integrated force (area under the curve) was measured over a 10- or 20-min period as appropriate. Significance was tested using appropriate t tests and P values <0.05 taken to be significant. Data are given as mean ± S.E.M. and “n” represents the number of samples, each one from a different hen. 3. Results 3.1. Effects of intracellular pH changes on uterine contraction Under control conditions spontaneous contractions could be recorded for several hours with only small changes in amplitude and frequency. A typical recording of a control trace is shown in Fig. 1A. The direct effects of intracellular acidification on uterine contraction arising spontaneously were investigated. Force was recorded in isolated uterine strips in the presence and absence of a weak acid, NaBu. The addition of NaBu (40 mM) to spontaneous phasic activity of the hen myometrium (Fig. 1B) produced an initial increase in tension (approximately 3-min duration; position ii), followed by a decrease in tension (both the amplitude and the frequency of the contractions; position iii) and its baseline, in all preparations (n = 5; position iii). Removal of NaBu led to a rapid increase in contraction integral (position iv) as a result of rebound alkalinization and spontaneous phasic activity returning gradually in 20 min. The effects of NaBu on preparations in response to PGF2␣ were examined. PGF2␣ (1 M) increased contractile activity within the hen myometrium by stimulating phasic activity in all preparations (n = 5) (Fig. 1B; positions i and ii). The contractions in the presence of PGF2␣ were decreased by acidification (a typical example is shown in Fig. 1C; position iii). The mean decrease in contrac-
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tions was 74.2 ± 5.53% (P < 0.05). The contraction frequency decreased in a manner similar to that seen during acidification of a spontaneously active preparation (Fig. 1B; position iii). We next investigated whether the effect of NaBu on spontaneously phasic force could be overcome if the preparation was stimulated by PGF2␣ . After 20 min in NaBu, PGF2␣ was added in the continued presence of NaBu, but produced no significant increase in the contraction integral (Fig. 1D; position ii). As expected, under control conditions, AA (1 M) increased contractile activity compared to spontaneous activity (Fig. 1E; positions i and ii). Acidification by the addition of NaBu decreased AA-induced force (Fig. 1E; position iii). If AA was added after NaBu, it could not overcome the inhibitory effect of NaBu (Fig. 1F; position ii). Having established the effects of intracellular acidification on the hen uterus, we next investigated the effects of intracellular alkalinization. It is not known whether alkalinization occurred during heat stress could have an effect on contractile force and whether this could lead to premature oviposition resulting in soft-shelled eggs. As shown above, acidification inhibited uterine activity, irrespective of how the uterine force was produced. Thus, one would expect the opposite effect of alkalinization. We therefore tested the hypothesis. The application of a weak base, 40 mM NH4 Cl (n = 6) produced a large and significant rise in tension, such that the contractions almost coalesce to become tonic (Fig. 2A; position ii). After 5 min, the amplitude of tension had fallen to 84.88 ± 4.07% (position iii) compared with the first response. The removal of NH4 Cl decreased (position iv) or abolished (3/7 preparations) phasic activity as a result of a rebound acidification. Normal spontaneous activity returned 20 min after the removal of NH4 Cl. The effects of NH4 Cl were also investigated on preparations in response to PGF2␣ . Alkalinization by the addition of NH4 Cl (n = 5) in the continued presence of PGF2␣ led to a further increase in force (Fig. 2B; position iii). Tension development increased by 127 ± 8.67% (P < 0.05) compared with the preceding contractions induced by PGF2␣ alone (positions i and ii) over comparable 30-min periods. Similarly if PGF2␣ was added after NH4 Cl, it produced a further increase in force (Fig. 2C; position ii). Alkalinization by the addition of NH4 Cl also increased AA-induced force (Fig. 2D; positions i and ii). If AA was added after NH4 Cl, it produced a further increase in force in the continued present of NH4 Cl (Fig. 2E; position ii). In the laying hens, we observed that intracellular pH changes took place longer (>20 min) before the pH regulatory mechanisms come into play. It could be that the laying hen uterus has different pH regulatory mechanisms that help the uterus to survive in acidic condition for more than 20 h during eggshell formation. 3.2. Effects of extracellular pH changes on uterine contractility When extracellular pH is altered, intracellular pH will gradually follow (Pierce et al., 2003). Thus, to confirm the effects of intracellular pH we therefore examined the effects of extracellular pH changes on the uterine activity.
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Fig. 1. A representative trace illustrates the effects of intracellular pH changes (weak acid) on uterine contractility of the laying hens. A, a control trace. B, spontaneous contraction recorded in response to 40 mM NaBu before (i), during (ii and iii), and after (iv). C PGF2␣ -induced contraction in the absence (i and ii) and presence (iii) of NaBu. D, NaBu response in the absence (i) and presence of PGF2␣ (ii). E, AA-induced contraction in the absence (i and ii) and presence (iii) of NaBu. F, NaBu response in the absence (i) and presence of AA (ii). The scale on X and Y axis in this and subsequent figures indicates tension (g) and time (min) respectively. The up arrow in this and subsequent figures shows the periods when drugs are removed.
The immediate effects of extracellular acidification to pH 6.9 were to initially increase (Fig. 3A; position ii) followed by a decrease in the amplitude of contractions of the phasic contractions (position iii). The first contraction at pH 6.9 were significantly increased to 144 ± 13.88% (position, ii), when they were compared with the preceding control contraction (position i); after 5 min, the amplitude of tension had significantly fallen to 57.25 ± 6.51% (position iii) compared with control transients (n = 5; position ii). There was also a significant decrease in the frequency of the contractions to 68.66 ± 9.95% (position iii). As with internal acidification, external acidification caused a decrease in the baseline tension. A return of normal spontaneous contractions occurred 20 min after the removal of the acidic
solution. During extracellular acidification to pH 6.9, the contractions that were evoked by the agonist PGF2␣ showed a significant decrease in phasic contraction integral (Fig. 3B; position iii) to 73.25 ± 1.70% and frequency of contractions to 66 ± 1.90% of control values (Fig. 3B; position ii). If PGF2␣ was added after, it could not reverse the inhibitory effect of external acidification (Fig. 3C; position iii). As with PGF2␣ , external acidification (n = 5) produced a significant decrease in contractions stimulated by AA (Fig. 3D; position iii). This was also a case when AA was added after acidification (Fig. 3E; position iii). External alkalinization to pH 7.9 produced an immediate and significant increase in the amplitude of the spontaneous contractions to 128 ± 3.21% (Fig. 4A; positions ii and
Fig. 2. A representative trace illustrates the effects of intracellular pH changes (weak base) on uterine contractility of the laying hens. A, spontaneous contraction recorded in response to 40 mM NH4 Cl before (i), during (ii and iii), and after (iv). B, PGF2␣ -induced contraction in the absence (i and ii) and presence (iii) of NH4 Cl. C, NH4 Cl response in the absence (i) and presence of PGF2␣ (ii). D, AA-induced contraction in the absence (i and ii) and presence (iii) of NH4 Cl. E, NH4 Cl response in the absence (i) and presence of AA (ii).
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Fig. 3. A representative trace illustrates the effects of extracellular pH changes (pH 6.9) on uterine contractility of the laying hens. A, spontaneous contraction recorded in response to pH 6.9 before (i), during (ii and iii), and after (iv). B, PGF2␣ -induced contraction before (i and ii) and after (iii) acidification to pH 6.9. C, acidification to pH 6.9 before (i and ii) and after (iii) application of PGF2␣ . D, AA-induced contraction before (i and ii) and after (iii) acidification to pH 6.9. E, acidification to pH 6.9 before (i and ii) and after (iii) application of AA (ii).
iii) of control value (position, i). A significant increase in the frequency of the contractions to 130.33 ± 5.33% also occurred. Restoration of pH to 7.4 saw rapid (5 min) restoration to control level. The contractions that were evoked by the agonist PGF2␣ showed an increase (Fig. 4B; position iii) in amplitude to 117.75 ± 3.85% (P < 0.05) during alkalinization to pH 7.9, compared with preceding control transients (control was 100% (position ii); n = 5). During this phase, there was a significant increase in the frequency of the contractions to 132.75 ± 12.03% (Fig. 4B; position iii). If PGF2␣ was added after alkalinization to pH 7.9, force was also further increased (Fig. 4C; position iii). As with PGF2␣ , external alkalinization (n = 5) produced a significant increase in contractions stimulated by AA (Fig. 4D; posi-
tion iii). This was also the case when AA was added after alkalinization (Fig. 4E; position iii). 4. Discussion By investigating the effects of pH changes on the hen uterus, we found that acidification decreases the normal phasic activity, even if either PGF2␣ or AA is present, whereas alkalinization increases the normal phasic contraction and force is further potentiate when either PGF2␣ or AA is present. The induced pH changes in our study are the same as those occurring in normal oviposition (Mashaly et al., 1986), suggesting that either acidification or alkalinization can regulate and modulate uterine contraction that
Fig. 4. A representative trace illustrates the effects of extracellular pH changes (pH 7.9) on uterine contractility of the laying hens. A, spontaneous contraction recorded in response to pH 7.9 before (i), during (ii and iii), and after (iv). B, PGF2␣ -induced contraction before (i and ii) and after (iii) alkalinization to pH 7.9. C, alkalinization to pH 7.9 before (i and ii) and after (iii) application of PGF2␣ . D, AA-induced contraction before (i and ii) and after (iii) alkalinization to pH 7.9. E, alkalinization to pH 7.9 before (i and ii) and after (iii) application of AA (ii).
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Fig. 5. A proposed model for explaining the roles of pH in regulation and modulation of uterine contraction in the laying hens. A, a decrease in pH (either intracellular or extracellular pH) leads to an inhibition of uterine contraction resulting in the prevention of expulsion of the egg, but promotion calcification. B, an increase in pH produces the opposite effect.
may lead to a delayed or premature oviposition, respectively. A proposed model for explaining the roles of pH in regulation and modulation of uterine contraction in the laying hens is shown in Fig. 5. In the laying hens, both acidification and alkalinization either induced internally or externally can affect uterine activities. The effects on force, seen during acidification and alkalinization, are opposite suggesting that either acidification or alkalinization is acting at the same locus in the calcium–calmodulin MLCK pathway. An earlier study that used human myometrium and weak acids and weak bases had shown that intracellular acidification and alkalinization influence L-type calcium entry as changes in phasic activity were mirrored by changes in intracellular calcium concentration for both intracellular alkalinization- and acidification-induced changes (Pierce et al., 2003). There are data, in other smooth muscles, showing that intracellular acidification may also inhibit force production directly at the level of cross-bridge cycling as the reduction in force cannot be explained by a fall in intracellular calcium concentration (Peng et al., 1998; Smith et al., 1998). As changes in intracellular calcium concentration was not measured
directly in our study, it is unclear whether the effects of pH changes in the present study of hen myometrium were due to the alteration L-type calcium entry. In the hen myometrium, it has been shown that calcium source for spontaneous contraction predominantly comes from calcium entry through the voltage-dependent opening of L-type calcium channels across the plasma membrane (Kupittayanant et al., 2008). Mechanisms whereby uterine contractility is regulated in hen are almost resembled to those found in human myometrium (Kupittayanant et al., 2008). Thus, it seems reasonable to conclude that pH changes are, at least in part, acting on L-type calcium entry. A previous study that examined the uterine contraction frequency during ovulatory cycle had shown that low frequency of uterine contraction was found during the period when the egg is in the uterus and maximum frequency was found at expulsion of the egg from the uterus (Shimada and Asai, 1978). It was concluded that the frequency changes may be attributable to either or both actions of the posterior pituitary hormones and the postovulatory follicles and that the process of ovulation may be involved in the changes of frequency (Shimada and Asai, 1978). It is not clear what sets the contraction frequency in the hen uterus. However, when the egg is in the uterus or during calcification the mean value of the uterine pH was acidic and became alkaline when the egg is about to leave the uterus or at the end of calcification (Simkiss, 1969). In addition, our data also indicate that acidic pH decreased the uterine contraction frequency and alkalosis increased it. Taken together, in the laying hens, it is likely that pH has an essential role in regulation of the uterine contraction frequency. Our findings are the first to show the effects of pH changes on the uterus of the laying hens. Furthermore, our results suggest that the process of calcification and oviposition may be not only controlled and modulated by the hormones as previously demonstrated (Day and Nalbandov, 1977; Hertelendy and Biellier, 1978; Olson et al., 1978; Wechsung and Houvenaghel, 1978; Toth et al., 1979; Rzasa, 1984; De Saedeleer et al., 1989; Saito et al., 1990; Takahashi et al., 1992; Li et al., 1994), but pH changes also contribute to such process. This fundamental breakthrough in our understanding of the physiological basis by which uterine contraction is controlled by pH can be exploited for subsequent managing advantage in case of either delayed or premature oviposition. Acknowledgements We thank Prof. Susan Wray for discussions and Miss Chittawadee Suwannachat for technical help. Funding was provided by the National Research Council, Thailand. References Day, S.L., Nalbandov, A.V., 1977. Presence of prostaglandin F (PGF) in hen follicles and its physiological role in ovulation and oviposition. Biol. Reprod. 16, 486–494. De Saedeleer, V., Wechsung, E., Houvenaghel, A., 1989. Influence of substance P on in vitro motility of the oviduct and intestine in the domestic hen. Anim. Reprod. Sci. 21, 293–300. El Hadi, H., Sykes, A.H., 1982. Thermal panting and respiratory alkalosis in the laying hen. Br. Poult. Sci. 23, 49–57.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
The roles of pH in regulation of uterine contraction in the laying hens S. Kupittayanant a,∗ , P. Kupittayanant b a b
School of Physiology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand School of Animal Production Technology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand
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Article history: Received 27 February 2009 Received in revised form 1 July 2009 Accepted 7 July 2009 Available online 16 July 2009 Keywords: Laying hen pH Uterus Calcium
a b s t r a c t In the laying hens, the uterus (shell gland) plays essential roles in calcium transfer for calcification of the eggshell and expulsion of the egg through the vagina for oviposition. Much is known about the effects of pH changes on eggshell production of the uterus. However, very little is understood about the effects of pH changes on uterine contractility. We investigated the effects of pH changes on uterine contraction in the laying hens. The laying hens were humanely killed, and strips of uterine smooth muscles were isolated. Isometric force was measured and the effects of intracellular and extracellular pH changes studied. The results show that alterations of pH clearly have marked effects on force in the hen uterus. Both intracellular and extracellular acidifications significantly decreased uterine activity, whether it arises spontaneously or in the presence of agonists such as prostaglandin F2␣ and arachidonic acid. Alkalinization produced the opposite effects. Thus, changes in pH can regulate uterine contraction. This insight into pH regulation of the uterine activity provides a focus for egg production management directed at physiological and pathological oviposition in the laying hens. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The hen uterus is a myogenic organ. Without stimulation of nerves and hormones, it can produce spontaneously phasic contractions for several hours in the appropriate condition (Kupittayanant et al., 2008). Our previous work has shown that the frequency of spontaneous activity, in vivo, is around 18 contractions every 10 min (Kupittayanant et al., 2008). The major pathway that regulates such contractions depends on the calcium–calmodulin myosin light chain kinase (MLCK) pathway, as without either calcium or MLCK no force was produced (Kupittayanant et al., 2008). Agonist such as prostaglandin F2␣ (PGF2␣ ) can alter spontaneously contracting uterus not only by stimulating the calcium–calmodulin MLCK pathway but also by activating the non-calcium–calmodulin MLCK pathway (Kupittayanant et al., 2008). Thus, uterine contractility of
∗ Corresponding author. Tel.: +66 44224633; fax: +66 44224633. E-mail address: [email protected] (S. Kupittayanant). 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.07.001
the laying hens can be regulated and modulated via several pathways. It is well established that changes in intracellular and extracellular pH can modulate uterine contractility in the human and laboratory animals (Phoenix and Wray, 1993; Taggart and Wray, 1993; Parratt et al., 1995; Taggart et al., 1996; Larcombe-McDouall et al., 1998; Naderali and Wray, 1999; Pierce et al., 2003; Wray, 2007). Physiological changes in intracellular pH may occur for several reasons such as restricting blood flow and oxygen supply to the uterus, increased uterine contractility, and alteration of acid–base balance (Wray, 2007). When extracellular pH is altered, intracellular pH will change (Pierce et al., 2003). To the best of our knowledge, there is no data demonstrating the effects of pH changes on uterine contractility in the laying hens. However, the effects of pH changes on eggshell production of the uterus are well described (see below). Acidification, which occurs during normal process of egg formation, enhances eggshell calcification (Johnson, 2002), whereas alkalinization, occurred during heat stress, inhibits calcification causing soft-shelled eggs (El Hadi and
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Sykes, 1982; Tanor et al., 1984; Mashaly et al., 1986; Odom et al., 1986; Koelkebeck and Odom, 1994; Rozenboim et al., 2007). During the physiological process of egg formation, the uterus is the oviductal part that the egg spends the longest for calcification of the eggshell (18–20 h depending on cycle length) (Johnson, 2002). There, acidification occurs while calcium is deposited to form the eggshell (Simkiss, 1969). However, it is unclear whether the uterus is quiescent while the process of calcification takes place. Under pathological conditions, the heat stressed laying hens often lay eggs with thinner shells because of alkalinization as a result of elevating blood pH after excessive loss of CO2 from their lungs (El Hadi and Sykes, 1982; Tanor et al., 1984; Mashaly et al., 1986; Odom et al., 1986; Koelkebeck and Odom, 1994; Rozenboim et al., 2007). In addition, it is likely that the oviposition interval in the heat stressed laying hens is always shorter (< 24 h) compared with the unstressed laying hens (24–26 h) (Novero et al., 1991). These studies led us to hypothesize that changes in pH (both acidification and alkalinization) might affect uterine contraction. Thus, an inactive uterus might be required during acidification to complete the process of calcification, whereas alkalinization might activate the uterus that may lead to premature oviposition as occurs during heat stress. Furthermore, there are several hormones control oviposition including PGF2␣ . It is not known whether pH changes can occur in the presence of PGF2␣ . It was reported that the level of PGF2␣ increases sharply before oviposition to aid uterine contraction and an injection of its precursor, AA, leads to increases in intraluminal pressure of the uterus (Johnson, 2002). Thus, it is not clear whether acidification could affect either PGF2␣ - or AA-induced contraction. We therefore examined the effects of pH changes on the uterus. The experiments were designed to answer the questions: (1) How does the spontaneous contractility of the uterus change with acidification or alkalinization? (2) Do changes in pH alter uterine contractility when the agonists (PGF2␣ or arachidonic acid, AA) are present? (3) Are the effects of extracellular pH on contractility the same as that of the effects of intracellular pH? 2. Materials and methods This study was conducted in accordance with guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council of Thailand. The experiment protocol was approved by the Institutional Animal Ethics Committee, Suranaree University of Technology, Nakhon Ratchasima, Thailand. 2.1. Uterine samples and myometrial strip preparations Uterine samples and myometrial strip preparations were taken from the group of laying hens used previously by Kupittayanant et al. (2008). Briefly, myometrial tissues were obtained from crossbred laying hens (12–17 months old and weighing 1.8–2.3 kg). The hens were maintained in individual cages. The lighting regimen was 14 h L:10 h D (light, 06:00–20:00 h) and food and water were freely available. The oviposition time was recorded
daily for each hen so that the time of next oviposition could be predicted. The mean oviposition interval was 24.20 ± 0.05 h. Hens were killed by cervical dislocation 4 h before the onset of oviposition. The uteri were removed and immediately immersed in physiological saline solution. The samples were transported to the nearby laboratory for immediate uses or stored at 4 ◦ C, until dissection was performed. 2.2. Tension measurements As with previous work (Kupittayanant et al., 2008), strips of longitudinal fibres (8 mm × 2 mm × 1 mm) were dissected. The strips were mounted in a 25 mL temperature-controlled organ bath (Panlab s.l. for ADInstruments Pty Ltd., Spain). The myometrial strips were attached at each end to two metal hooks. One hook was attached to a fixed point, and the other hook was attached to a tension transducer (ADInstruments Pty Ltd., Spain). Passive resting tension of 1 g was given, which produced baseline tension. The tissue-bathing medium used was physiological solution maintained at pH of 7.4, temperature of 37 ◦ C, and gassed with 100% O2 . The strip was allowed to contract spontaneously and an equilibrium period of 30 min was given before the application of any chemical. The measurements were made whilst the strip was continually perfused with physiological solution (control) or physiological solution containing drugs and/or chemicals. The electrical signal from the transducer was amplified and converted to a digital signal and recorded on a computer using Chart software (ADInstruments Pty Ltd., Australia). 2.3. Solutions and chemicals Physiological salt solution (pH, 7.4) contained (in mM); NaCl (154), KCl (5.4), MgSO4 (1.2), glucose (12), CaCl2 (2), and HEPES (10). The agonists PGF2␣ and AA were used at concentrations of 1 M (Wechsung and Houvenaghel, 1981; Kupittayanant et al., 2008). All chemicals were obtained from Sigma Chemical Co. (St. Louis, USA). 2.4. Experimental design 2.4.1. Intracellular pH changes As with earlier study (Pierce et al., 2003), changes in intracellular pH at constant external pH were produced by iso-osmotic substitution of sodium chloride with 40 mM sodium butyrate (NaBu) or 40 mM ammonium chloride (NH4 Cl). Although intracellular pH was not measured directly in our study, previous work used the human myometrial tissue preparation had shown that similar applications of NaBu or NH4 Cl produced intracellular pH changes of approximately 0.14 pH units (Parratt et al., 1995). These changes occur within 1 min as NaBu and NH4 Cl cross the cell membranes and dissociate, after that intracellular pH will be restored gradually to normal value within 20 min by pH regulatory mechanisms (Parratt et al., 1995).
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2.4.2. Extracellular pH changes Changes in extracellular pH were made by the addition of hydrochloric acid or sodium hydroxide to the perfusing solution to produce acidification or alkalinization (Pierce et al., 2003). It has been reported that the mean value of the intracellular pH of the uterus during the period from 1 to 10 h of calcification is around 6.5, whereas the value for the last 8 h (i.e. from 10 to 18 h) is 6.9 (Simkiss, 1969). Thus, the extracellular acidification to pH 6.9 and alkalinization to pH 7.9 were used for the subsequent experiments. 2.5. Data presentation and statistical analysis The data were analyzed using Microcal Origin Software. Parameters that were measured include the contraction amplitude, the contraction integral, and the contraction frequency. The phasic contractions in weak acid or weak base were measured over 20 min from the start of their application. Results were expressed as percentages of control contractions (i.e. the control is 100%). To test the effects of weak acid or weak base on PGF2␣ or AA, contractions were compared for the 10 min in PGF2␣ or AA (i.e. 11–20 min after start of PGF2␣ or AA exposure), to the 11–20 min in PGF2␣ or AA with weak acid or weak base. In some experiments, PGF2␣ or AA was added after weak acid or weak base and the effects compared. Integrated force (area under the curve) was measured over a 10- or 20-min period as appropriate. Significance was tested using appropriate t tests and P values <0.05 taken to be significant. Data are given as mean ± S.E.M. and “n” represents the number of samples, each one from a different hen. 3. Results 3.1. Effects of intracellular pH changes on uterine contraction Under control conditions spontaneous contractions could be recorded for several hours with only small changes in amplitude and frequency. A typical recording of a control trace is shown in Fig. 1A. The direct effects of intracellular acidification on uterine contraction arising spontaneously were investigated. Force was recorded in isolated uterine strips in the presence and absence of a weak acid, NaBu. The addition of NaBu (40 mM) to spontaneous phasic activity of the hen myometrium (Fig. 1B) produced an initial increase in tension (approximately 3-min duration; position ii), followed by a decrease in tension (both the amplitude and the frequency of the contractions; position iii) and its baseline, in all preparations (n = 5; position iii). Removal of NaBu led to a rapid increase in contraction integral (position iv) as a result of rebound alkalinization and spontaneous phasic activity returning gradually in 20 min. The effects of NaBu on preparations in response to PGF2␣ were examined. PGF2␣ (1 M) increased contractile activity within the hen myometrium by stimulating phasic activity in all preparations (n = 5) (Fig. 1B; positions i and ii). The contractions in the presence of PGF2␣ were decreased by acidification (a typical example is shown in Fig. 1C; position iii). The mean decrease in contrac-
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tions was 74.2 ± 5.53% (P < 0.05). The contraction frequency decreased in a manner similar to that seen during acidification of a spontaneously active preparation (Fig. 1B; position iii). We next investigated whether the effect of NaBu on spontaneously phasic force could be overcome if the preparation was stimulated by PGF2␣ . After 20 min in NaBu, PGF2␣ was added in the continued presence of NaBu, but produced no significant increase in the contraction integral (Fig. 1D; position ii). As expected, under control conditions, AA (1 M) increased contractile activity compared to spontaneous activity (Fig. 1E; positions i and ii). Acidification by the addition of NaBu decreased AA-induced force (Fig. 1E; position iii). If AA was added after NaBu, it could not overcome the inhibitory effect of NaBu (Fig. 1F; position ii). Having established the effects of intracellular acidification on the hen uterus, we next investigated the effects of intracellular alkalinization. It is not known whether alkalinization occurred during heat stress could have an effect on contractile force and whether this could lead to premature oviposition resulting in soft-shelled eggs. As shown above, acidification inhibited uterine activity, irrespective of how the uterine force was produced. Thus, one would expect the opposite effect of alkalinization. We therefore tested the hypothesis. The application of a weak base, 40 mM NH4 Cl (n = 6) produced a large and significant rise in tension, such that the contractions almost coalesce to become tonic (Fig. 2A; position ii). After 5 min, the amplitude of tension had fallen to 84.88 ± 4.07% (position iii) compared with the first response. The removal of NH4 Cl decreased (position iv) or abolished (3/7 preparations) phasic activity as a result of a rebound acidification. Normal spontaneous activity returned 20 min after the removal of NH4 Cl. The effects of NH4 Cl were also investigated on preparations in response to PGF2␣ . Alkalinization by the addition of NH4 Cl (n = 5) in the continued presence of PGF2␣ led to a further increase in force (Fig. 2B; position iii). Tension development increased by 127 ± 8.67% (P < 0.05) compared with the preceding contractions induced by PGF2␣ alone (positions i and ii) over comparable 30-min periods. Similarly if PGF2␣ was added after NH4 Cl, it produced a further increase in force (Fig. 2C; position ii). Alkalinization by the addition of NH4 Cl also increased AA-induced force (Fig. 2D; positions i and ii). If AA was added after NH4 Cl, it produced a further increase in force in the continued present of NH4 Cl (Fig. 2E; position ii). In the laying hens, we observed that intracellular pH changes took place longer (>20 min) before the pH regulatory mechanisms come into play. It could be that the laying hen uterus has different pH regulatory mechanisms that help the uterus to survive in acidic condition for more than 20 h during eggshell formation. 3.2. Effects of extracellular pH changes on uterine contractility When extracellular pH is altered, intracellular pH will gradually follow (Pierce et al., 2003). Thus, to confirm the effects of intracellular pH we therefore examined the effects of extracellular pH changes on the uterine activity.
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Fig. 1. A representative trace illustrates the effects of intracellular pH changes (weak acid) on uterine contractility of the laying hens. A, a control trace. B, spontaneous contraction recorded in response to 40 mM NaBu before (i), during (ii and iii), and after (iv). C PGF2␣ -induced contraction in the absence (i and ii) and presence (iii) of NaBu. D, NaBu response in the absence (i) and presence of PGF2␣ (ii). E, AA-induced contraction in the absence (i and ii) and presence (iii) of NaBu. F, NaBu response in the absence (i) and presence of AA (ii). The scale on X and Y axis in this and subsequent figures indicates tension (g) and time (min) respectively. The up arrow in this and subsequent figures shows the periods when drugs are removed.
The immediate effects of extracellular acidification to pH 6.9 were to initially increase (Fig. 3A; position ii) followed by a decrease in the amplitude of contractions of the phasic contractions (position iii). The first contraction at pH 6.9 were significantly increased to 144 ± 13.88% (position, ii), when they were compared with the preceding control contraction (position i); after 5 min, the amplitude of tension had significantly fallen to 57.25 ± 6.51% (position iii) compared with control transients (n = 5; position ii). There was also a significant decrease in the frequency of the contractions to 68.66 ± 9.95% (position iii). As with internal acidification, external acidification caused a decrease in the baseline tension. A return of normal spontaneous contractions occurred 20 min after the removal of the acidic
solution. During extracellular acidification to pH 6.9, the contractions that were evoked by the agonist PGF2␣ showed a significant decrease in phasic contraction integral (Fig. 3B; position iii) to 73.25 ± 1.70% and frequency of contractions to 66 ± 1.90% of control values (Fig. 3B; position ii). If PGF2␣ was added after, it could not reverse the inhibitory effect of external acidification (Fig. 3C; position iii). As with PGF2␣ , external acidification (n = 5) produced a significant decrease in contractions stimulated by AA (Fig. 3D; position iii). This was also a case when AA was added after acidification (Fig. 3E; position iii). External alkalinization to pH 7.9 produced an immediate and significant increase in the amplitude of the spontaneous contractions to 128 ± 3.21% (Fig. 4A; positions ii and
Fig. 2. A representative trace illustrates the effects of intracellular pH changes (weak base) on uterine contractility of the laying hens. A, spontaneous contraction recorded in response to 40 mM NH4 Cl before (i), during (ii and iii), and after (iv). B, PGF2␣ -induced contraction in the absence (i and ii) and presence (iii) of NH4 Cl. C, NH4 Cl response in the absence (i) and presence of PGF2␣ (ii). D, AA-induced contraction in the absence (i and ii) and presence (iii) of NH4 Cl. E, NH4 Cl response in the absence (i) and presence of AA (ii).
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Fig. 3. A representative trace illustrates the effects of extracellular pH changes (pH 6.9) on uterine contractility of the laying hens. A, spontaneous contraction recorded in response to pH 6.9 before (i), during (ii and iii), and after (iv). B, PGF2␣ -induced contraction before (i and ii) and after (iii) acidification to pH 6.9. C, acidification to pH 6.9 before (i and ii) and after (iii) application of PGF2␣ . D, AA-induced contraction before (i and ii) and after (iii) acidification to pH 6.9. E, acidification to pH 6.9 before (i and ii) and after (iii) application of AA (ii).
iii) of control value (position, i). A significant increase in the frequency of the contractions to 130.33 ± 5.33% also occurred. Restoration of pH to 7.4 saw rapid (5 min) restoration to control level. The contractions that were evoked by the agonist PGF2␣ showed an increase (Fig. 4B; position iii) in amplitude to 117.75 ± 3.85% (P < 0.05) during alkalinization to pH 7.9, compared with preceding control transients (control was 100% (position ii); n = 5). During this phase, there was a significant increase in the frequency of the contractions to 132.75 ± 12.03% (Fig. 4B; position iii). If PGF2␣ was added after alkalinization to pH 7.9, force was also further increased (Fig. 4C; position iii). As with PGF2␣ , external alkalinization (n = 5) produced a significant increase in contractions stimulated by AA (Fig. 4D; posi-
tion iii). This was also the case when AA was added after alkalinization (Fig. 4E; position iii). 4. Discussion By investigating the effects of pH changes on the hen uterus, we found that acidification decreases the normal phasic activity, even if either PGF2␣ or AA is present, whereas alkalinization increases the normal phasic contraction and force is further potentiate when either PGF2␣ or AA is present. The induced pH changes in our study are the same as those occurring in normal oviposition (Mashaly et al., 1986), suggesting that either acidification or alkalinization can regulate and modulate uterine contraction that
Fig. 4. A representative trace illustrates the effects of extracellular pH changes (pH 7.9) on uterine contractility of the laying hens. A, spontaneous contraction recorded in response to pH 7.9 before (i), during (ii and iii), and after (iv). B, PGF2␣ -induced contraction before (i and ii) and after (iii) alkalinization to pH 7.9. C, alkalinization to pH 7.9 before (i and ii) and after (iii) application of PGF2␣ . D, AA-induced contraction before (i and ii) and after (iii) alkalinization to pH 7.9. E, alkalinization to pH 7.9 before (i and ii) and after (iii) application of AA (ii).
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Fig. 5. A proposed model for explaining the roles of pH in regulation and modulation of uterine contraction in the laying hens. A, a decrease in pH (either intracellular or extracellular pH) leads to an inhibition of uterine contraction resulting in the prevention of expulsion of the egg, but promotion calcification. B, an increase in pH produces the opposite effect.
may lead to a delayed or premature oviposition, respectively. A proposed model for explaining the roles of pH in regulation and modulation of uterine contraction in the laying hens is shown in Fig. 5. In the laying hens, both acidification and alkalinization either induced internally or externally can affect uterine activities. The effects on force, seen during acidification and alkalinization, are opposite suggesting that either acidification or alkalinization is acting at the same locus in the calcium–calmodulin MLCK pathway. An earlier study that used human myometrium and weak acids and weak bases had shown that intracellular acidification and alkalinization influence L-type calcium entry as changes in phasic activity were mirrored by changes in intracellular calcium concentration for both intracellular alkalinization- and acidification-induced changes (Pierce et al., 2003). There are data, in other smooth muscles, showing that intracellular acidification may also inhibit force production directly at the level of cross-bridge cycling as the reduction in force cannot be explained by a fall in intracellular calcium concentration (Peng et al., 1998; Smith et al., 1998). As changes in intracellular calcium concentration was not measured
directly in our study, it is unclear whether the effects of pH changes in the present study of hen myometrium were due to the alteration L-type calcium entry. In the hen myometrium, it has been shown that calcium source for spontaneous contraction predominantly comes from calcium entry through the voltage-dependent opening of L-type calcium channels across the plasma membrane (Kupittayanant et al., 2008). Mechanisms whereby uterine contractility is regulated in hen are almost resembled to those found in human myometrium (Kupittayanant et al., 2008). Thus, it seems reasonable to conclude that pH changes are, at least in part, acting on L-type calcium entry. A previous study that examined the uterine contraction frequency during ovulatory cycle had shown that low frequency of uterine contraction was found during the period when the egg is in the uterus and maximum frequency was found at expulsion of the egg from the uterus (Shimada and Asai, 1978). It was concluded that the frequency changes may be attributable to either or both actions of the posterior pituitary hormones and the postovulatory follicles and that the process of ovulation may be involved in the changes of frequency (Shimada and Asai, 1978). It is not clear what sets the contraction frequency in the hen uterus. However, when the egg is in the uterus or during calcification the mean value of the uterine pH was acidic and became alkaline when the egg is about to leave the uterus or at the end of calcification (Simkiss, 1969). In addition, our data also indicate that acidic pH decreased the uterine contraction frequency and alkalosis increased it. Taken together, in the laying hens, it is likely that pH has an essential role in regulation of the uterine contraction frequency. Our findings are the first to show the effects of pH changes on the uterus of the laying hens. Furthermore, our results suggest that the process of calcification and oviposition may be not only controlled and modulated by the hormones as previously demonstrated (Day and Nalbandov, 1977; Hertelendy and Biellier, 1978; Olson et al., 1978; Wechsung and Houvenaghel, 1978; Toth et al., 1979; Rzasa, 1984; De Saedeleer et al., 1989; Saito et al., 1990; Takahashi et al., 1992; Li et al., 1994), but pH changes also contribute to such process. This fundamental breakthrough in our understanding of the physiological basis by which uterine contraction is controlled by pH can be exploited for subsequent managing advantage in case of either delayed or premature oviposition. Acknowledgements We thank Prof. Susan Wray for discussions and Miss Chittawadee Suwannachat for technical help. Funding was provided by the National Research Council, Thailand. References Day, S.L., Nalbandov, A.V., 1977. Presence of prostaglandin F (PGF) in hen follicles and its physiological role in ovulation and oviposition. Biol. Reprod. 16, 486–494. De Saedeleer, V., Wechsung, E., Houvenaghel, A., 1989. Influence of substance P on in vitro motility of the oviduct and intestine in the domestic hen. Anim. Reprod. Sci. 21, 293–300. El Hadi, H., Sykes, A.H., 1982. Thermal panting and respiratory alkalosis in the laying hen. Br. Poult. Sci. 23, 49–57.
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