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Protein Synthesis in Tissues and in the Whole Body of Laying Hens During Egg Formation1
Protein Synthesis in Tissues and in the Whole Body of Laying Hens During Egg Formation1 K. HIRAMOTO, T. MURAMATSU,2 and J. OKUMURA Laboratory of Animal Nutrition, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan (Received for publication December 20, 1988) ABSTRACT The present study was conducted to investigate whether or not protein synthesis in tissues and in the whole body of laying hens would be affected by the position of an ovum passing along the oviduct during egg formation. Protein synthesis in tissues was measured in vivo by a primed-continuous infusion of [ "N]methionine for 3 h, finishing at the time when an ovum would have stayed at one of the segments within the oviduct, i.e., infundibulum, magnum, isthmus, uterus, or vagina. The dissection of the entire oviduct immediately after the isotope infusion confirmed whether or not the ovum was in the expected position. Whole-body protein synthesis, estimated from plateau enrichment of free [ 15N]methionine in plasma at the end of the infusion, was not significantly affected by the position of an ovum along the various segments of the oviduct. The highest values were observed in the entire oviduct when an ovum was in the magnum portion, both for protein synthesis and protein synthesis per unit of RNA. Protein synthesis per unit of RNA in the sum of tissues other than the liver and oviduct was highest when an ovum was in the isthmus. The synthesis of liver protein was relatively constant and was not significantly affected by the position of the ovum passing along the oviduct (Key words: egg formation, protein synthesis, magnum, oviduct, liver) 1990 Poultry Science 69:264-269 INTRODUCTION
In laying hens, the magnum (a portion of the oviduct) is generally known as the formation site for egg albumen protein (Smith et ai, 1957). Albumen protein is rapidly formed in the magnum during the intervals between the passage of successive ova (Conrad and Scott, 1942; Mandeles and Ducay, 1962). Smith et al. (1959) suggested that approximately 45% of egg albumen protein is made just as an ovum passes through the magnum, based on their measurements of the volume difference in the magnum observed from .5 to 4.0 h after ovulation. However, these changes may largely reflect die secretion of egg albumen protein into the lumen rather than its synthesis. Previously, protein synthesis in various tissues of laying hens during the egg-formation cycle has not been measured in vivo. Recendy, Muramatsu et al. (1987a,b) developed a method for measuring protein synthesis in vivo in the tissues and the whole body of laying hens by a primed-continuous infusion of stable isotopes in combination with subsequent
analyses of enrichment with a gas-chromatograph mass spectrometer. The method can be used to determine protein kinetics in tissue of interest in vivo. For laying hens, both the liver and oviduct had faster fractional rates of protein synthesis (percentage per day) man any other tissues measured (Hiramoto et al., unpublished data). However, because of the rapid formation of egg albumen, protein synthesis in tissue such as that in the oviduct might fluctuate substantially during the egg-formation cycle. On the other hand, the synthesis of liver protein might remain constant throughout the egg-formation cycle. Redshaw and Follett (1972) demonstrated that the concentration of lipovitellin, a major protein in the egg yolk, in plasma did not vary throughout the day. The present study was conducted to investigate whether or not protein synthesis in tissues and in the whole body of laying hens would be affected by the position of an ovum passing along the oviduct during egg formation. MATERIALS AND METHODS
'Financial support was provided by a grant-in-aid. No. 62480082, for Scientific Research from the Ministry of Education, Science, and Culture, Japan. ^To whom correspondence should be addressed.
Animals The laying hens used were Single Comb White Leghorns 12 mo of age and weighing 1.57
264
PROTEIN SYNTHESIS AND EGG FORMATION IN LAYING HENS
265
± .24 (SD) kg and having an egg-production rate (Muromachi Kagaku Kogyo Ltd., Tokyo, Jaof 90% on the average. The hens were housed pan). The effluent was dried under reduced individually in cages in an air-conditioned room pressure at 50 C. Free amino acids in plasma at 25 C with a lighting period of 14 h per day. were derivatized to form n-trifluoro-acetyl nThey were maintained on a commercial layer butyl amino acid (TFA) esters according to the diet (Marubeni Shiryo Ltd., Tokyo, Japan) with method of Gehrke et al. (1968). The samples of 15.5% CP and 2,900 kcal of ME/kg. amino acid in free and protein fractions were Before measuring protein synthesis, the derivatized in a similar fashion. The preparation oviposition times of individual hens were of amino acid samples in tissues for enrichment recorded for 2 wk so that the expected position determination has been described in detail by of the ovum within the oviduct could be Muramatsu et al. (1987b,c). estimated at the end of isotope infusion. The isotope abundance in the TFA esters thus Whether or not the ovum was in the expected obtained was analyzed by using a computerposition was confirmed by dissecting the entire controlled, selected-ion-monitoring, gas-chrooviduct immediately after the isotope infusion. matograph mass spectrometer (Model QPAt 3 h before the appropriate time when an 1,000, Shimadzu Corp., Kyoto, Japan). The ovum would have stayed approximately at the analytical conditions of the [15N]methionine mid-point in one of the oviduct segments, sterile were as follows: column, .5% ethylene glycol polyethylene cannulas were inserted into veins adipate on chromosorb W (AW 80-100 mesh) of both the left and right wings, followed by a packed in a glass column with an internal 3-h, primed-intravenous infusion of DL- diameter of 2.6 mm and 3,000 mm long; carrier [15N]methionine. Blood-sampling was done gas, helium at a flow rate of 30 mL/min; column each hour. The blood samples were centrifuged temperature, 170 C; separator temperature, 250 at 1,800 x g for 10 min in order to separate the C; ion-source temperature, 250 C; ionizing plasma fraction, which was subsequendy depro- voltage, 70 eV; mass numbers of fragment ions, teinized with 5 vol of 1% (wt/vol) of picric acid. m + z = 228 and 229. The deproteinized samples were stored at -20 C Tissue nitrogen was determined by the until analyzed. The details of die infusion Kjeldahl method (Hawk et al., 1954). Tissue method are described elsewhere (Muramatsu et protein was calculated as N times 6.25. For al., 1987a,b). At the end of die infusion period, tissue RNA analysis, modified Schmidt-Thaundie hen was killed; then die liver, the entire hauser method as described by Munro and Fleck oviduct, a piece of skin around the leg muscle (1969) was used. (Af. biceps femoris), the breast muscle (Af. pectoralis superficialis), the leg muscle (Af. biceps femoris), the gizzard plus the proventric- Calculation ulus, and the entire intestine were removed Methionine flux (milligrams per 3 h) was quickly, weighed, and frozen by plunging them calculated according to the equation of Conway into liquid N2. The tissue samples were stored at et al. (1980) with modifications as previously -20 C until analysis. described given by Muramatsu et al. (1987b). The values designated as skin covering the The methionine flux was converted into wholewhole body were extrapolated from the ratios of body protein flux (milligrams per day) based on a specific area to the whole body. Those ratios the assumption that methionine would account were determined in a preliminary experiment for 1.8% of the body protein in chickens (Scott et using laying hens with similar body weights. In al., 1982). The calculation for the fractional rate the same manner, the values designed as breast of protein synthesis was done as described or leg muscle were derived from the ratios of previously by Muramatsu et al. (1987b). corresponding, specific muscles to the entire Since the DL-enatiomer of [15N]methionine breast or leg muscle, ratios also determined in was used as a tracer, the values for protein the preliminary experiment. synthesis were underestimated; so adequate correction, varying from one tissue to another probably due to differences in the stereoselecChemical Analysis tion or oxidation of D-methionine, had to be The picric acid in the deproteinized plasma made (Hiramoto et al., 1989). The correction was removed by passing the samples through a factors derived from differences in fractional column of the CI" form of Dowex 2-X8 synthesis rates of tissue or whole-body protein
266
HTRAMOTO ET AL. TABLE 1. Changes in protein and RNA contents in various tissues with respect to the location of an ovum along the oviduct of laying hens Location of an ovum Infundibulum
Measurement, by tissue Protein, g/tissue Liver 6.4 Oviduct 10.0*" Proventriculus plus gizzard 4.6 Intestine 6.3 Skin 19.2 Breast muscle 35.9 Leg muscle 8.6 Sum, other than liver and oviduct 74.6 RNA, mg/tissue Uver 552 Oviduct 317c Proventriculus plus gizzard 15l" Intestine 354* Skin 442" Breast muscle 452 Leg muscle 100 Sum, other than Uver and oviduct 1,499
Magnum
Isthmus
6.6 10.3* 4.9 6.6 19.3 32.7 8.4 71.9
7.5 7.5b 4.6 6.4 22.2 35.1 7.8 76.2
547 623* 143" 321*" 447*" 396 76 1381
611 520*" 150" 227" 425" 393 102 1,295
Uterus 6.3 8.0*" 4.5 6.3 20.1 37.5 9.2 77.5 491 431 bc 127" 303*" 545* 480 103 1,557
Vagina 6.0 9.1*" 4.8 6.0 19.2 37.1 8.0 75.1 559 388"° 188* 376* 468*" 493 106 1,631
Pooled SEM1 1.0 .8 .4 .7 2.0 3.2 .8 5.8 47 45 10 32 31 50 12 102
a_c
Means with no common superscripts within a row differ significantly (P<.05). !n = 4.
(estimated by using L- and DL-[15N]methionine) were as follows: whole body, 1.44; liver, 1.12; oviduct, 1.06; proventriculus plus gizzard, 1.13; intestine, 1.07; skin, 1.56; breast muscle, 1.17; leg muscle, 1.11 (Hiramoto et al., 1989). Statistical Analysis The data were subjected to a one-way analysis of variance (Snedecor and Cochran, 1980). The significance of differences between means was assessed by using Duncan's multiple range test (Duncan, 1955). RESULTS
Changes in the protein and RNA contents of various tissues are given in Table 1. To see how the values in tissues excluding the liver and oviduct that are related to the formation of egg protein would fluctuate in relation to the location of the ovum along the oviduct, the sum for tissues other than the liver and oviduct (the proventriculus plus gizzard, intestine, skin, breast muscle, and leg muscle) was also determined. No significant difference in protein content was detected in any tissues or in the sum except for the oviduct in relation to the location of an ovum. Protein content in the
oviduct was significandy (P<.05) reduced when an ovum moved from the magnum to the isthmus. The RNA content in the oviduct was highest when an ovum existed in the magnum, gradually decreasing as an ovum moved from the isthmus to the vagina. No significant changes in RNA contents were observed for the liver, breast muscle, leg muscle, and the sum of tissues other than liver and oviduct. The RNA content in the proventriculus plus gizzard was highest when an ovum was in the vagina. The RNA in the intestine gradually decreased as an ovum passed from the infundibulum to die isthmus, and increased as the ovum passed from the isthmus to the vagina. The RNA in the skin was higher when an ovum was in the uterus than when an ovum was in the infundibulum or the isthmus. Table 2 shows die changes in protein synthesis and protein synthesis per unit of RNA in various tissues, in the sum of tissues other man the liver and oviduct, and in the whole body in relation to the location of an ovum. Whole-body protein synthesis showed no significant fluctuations in relation to the location of an ovum. In the oviduct, protein synthesis was highest when an ovum was in the magnum. In the skin, a significant increase in protein synthesis was detected when an ovum moved down the oviduct from the
267
PROTEIN SYNTHESIS AND EGG FORMATION IN LAYING HENS
TABLE 2. Changes in protein synthesis and in protein synthesis per unit of RNA for various tissues and in the whole body with respect to the location of an ovum along the oviduct of laying hens Location of an ovum Measurement, by tissue
Infundibulum
Magnum
Protein synthesis, mg/h per bird or per tissue Liver 243 247 207bc Oviduct 536" Proventriculus plus gizzard 163 117 Intestine 201 163 Skin 256b 221 b Breast muscle 327 290 Leg muscle 98 69 Sum, other than liver and oviduct 1,044 859 Whole body2 1,753 2,180 Protein synthesis per unit RNA, mg/h per mg RNA Liver .44 .44 Oviduct .65b .91a Proventriculus plus gizzard 1.07 .83 Intestine .57ab .51 ab b Skin .59 A& .74 .74 Breast muscle Leg muscle .99 1.05 b Sum, other than liver and oviduct .70 .62b
Isthmus
Uterus
258 285 b 122 172 386" 343 66 1,089 1,942
240 173c 128 190 309 ab 344 103 1,072 1,817
.43
.ss* .80 .77" .91" .87 .66 .84"
.50 .40° 1.01 .64* .56" .71 .97 .69 b
Vagina 230 240 bc 148 186 294ab
385 80 1,093 2,191 .42 .61*° .79 .49b .62b .77 .78 .67b
Pooled SEM1 36 31 16 26 38 49 12 93 260 .09 .07 .09 .08 .07 .06 .13 .04
a_c
Means with no common superscripts within a row differ significantly (P<.05). !n = 4. 2 Estimated from the plateau enrichment of free [15N]methionine in plasma.
infundibulum and magnum to the isthmus. In the liver and in the rest of tissues, protein synthesis was not significantly changed as an ovum passed along the oviduct. Protein synthesis per unit of RNA showed no significant fluctuations in the proventriculus plus gizzard or in the breast and leg muscles in relation to the location of an ovum (Table 2). In the oviduct, protein synthesis per unit of RNA followed a pattern similar to that for protein synthesis, being the highest when an ovum was in the magnum. The values for protein synthesis per unit of RNA in the skin and in the sum of the tissues (other than the liver and oviduct) were significantly higher when an ovum was in the isthmus compared with the rest of the segments. In the intestine, the value for protein synthesis per unit of RNA was significantly higher when an ovum was in the isthmus than when it was in the vagina. Protein synthesis in the liver per unit of RNA was fairly constant and was not significantly changed in relation to the location of an ovum. DISCUSSION
The results from the present study clearly showed that protein synthesis in the oviduct varied substantially as an ovum passed along
the oviduct: a sharp increase was found when the ovum was in the magnum portion, followed by an immediate decrease at the isthmus portion and by a further reduction to the initial level when an ovum was at the uterus and the vagina (Table 2). The increased protein synthesis when an ovum was in the magnum probably reflects the enhanced synthesis of egg albumen, the secreted protein. Among the possible factors responsible for the changes observed in protein synthesis, alterations of hormonal status would be important as well as direct mechanical stimulation caused by an ovum and reactions mediated by chemical activators from an ovum and by neurons. Estrogen increases the synthesis of ovalbumin, conalbumin, ovomucoid, and lysozyme (Palmiter, 1972a); progesterone induces avidin synthesis (O'Malley, 1967). Seaver et al. (1980) reported that both estrogen and progesterone induced an increase in the protein synthesis in egg whites. Detailed studies revealed that these steroid hormones increased the level of mRNA coding for the corresponding egg-white proteins (Palmiter, 1973) and enhanced the subsequent translation by increasing the rate of initiation (Palmiter, 1972b) or of elongation (Rhoads, 1975).
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HIRAMOTO ET AL.
The involvement of these steroid hormones in the observed alterations of protein synthesis in the oviduct may be supported by the fact that the plasma levels of estrogen and progesterone fluctuate within the ovulation cycle; after the surge of luteinizing hormone about 6 to 8 h before the ovulation, the level of plasma estrogen and progesterone increases, but with certain delays (Furr et al., 1973; Shodono et al., 1975; Johnson and van Tienhoven, 1980; Tanabe et al, 1980). The increase in the steroid hormone level in plasma just before ovulation might result in an elevated level of protein synthesis. Since a sharp induction of protein synthesis in the oviduct was detected when the ovum was in the magnum portion (Table 2), the corresponding time for induced protein synthesis could be within a few hours after ovulation. Therefore, the time lag between the elevation in the plasma level of steroid hormones and the enhanced protein synthesis in the oviduct might reflect the interval necessary for a series of biochemical reactions to lead to the induction of protein synthesis in egg albumen. By contrast with the oviduct, protein synthesis in liver, a major site of yolk-protein synthesis (Deeley et al., 1975), was not significantly affected by the position of an ovum along the oviduct, in spite of the fact that protein synthesis in the egg yolk is also under estrogen control (Shapiro et al, 1976; Ryffel, 1978). However, the consistency of protein synthesis in the liver observed in the present study agrees with the findings of Green and Tata (1976), who demonstrated that the synthesis of vitellogenin (the precursor of lipovitellin and phosvitin) was constant as long as the concentration of plasma estrogen was maintained above a certain level. Alternatively the contribution of yolk protein synthesis in the yolk to the total protein synthesis in the liver might be too small to show any variations. In the sum of tissues (other than liver and oviduct), protein synthesis per unit of RNA was reduced when an ovum was in the magnum, but immediately increased when an ovum passed from the magnum to isthmus (Table 2). No significant change in protein synthesis was detected in the sum of these tissues when synthesis was expressed per tissue. The reduced protein synthesis per unit of RNA in other tissues not related to the formation of egg protein might facilitate the increase in oviduct protein synthesis by sup-
plying more amino acids to the oviduct at this stage of egg formation. Thus, when oviduct protein synthesis increases, protein synthesis in other tissues except for the liver and oviduct may decrease, and vice versa. The data on protein synthesis per unit of RNA suggest that protein synthesis may be increased in other tissues, but that this was not the case when protein synthesis was expressed per tissue. Reversed interorgan changes in protein synthesis may account for the ovum position not affecting whole-body protein synthesis. In the present investigation, the position of an ovum within the oviduct was predicted by recording the time of oviposition for 2 wk prior to the infusion experiment and was confirmed after the infusion by dissecting the ovum. Although dissection clarified the location of the ovum, the exact time the ovum had been retained in a particular portion of the oviduct is not known. Since the transit time at the sites other than the uterus was short with respect to the infusion period of 3 h, the estimates for protein synthesis were subject to the error arising from not being able to predict the exact location. REFERENCES Conrad, R. M., and H. M. Scott, 1942. The accumulation of protein in the oviduct of the fowl. Poultry Sci. 21: 81-85. Conway, J. M , D. M. Bier, K. J. Motil, J. F. Burke, and V. R. Young, 1980. Whole-body lysine flux in young adult men: effects of reduced total protein and of lysine intake. Am. J. Physiol. 239:E192-E200. Deeley, R. G., K. P. Mullinix, W. Wetekam, H. M. Kronenberg, M. Meyers, J. D. Eldridge, and R. F. Goldberger, 1975. Vitellogenin synthesis in the avian liver. J. Biol. Chem. 250:9060-9066. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1-42. Furr, B.J.A., R. C. Bonney, R. J. England, and F. J. Cunningham, 1973. Luteinizing hormone and progesterone in peripheral blood during the ovulatory cycle of the hen, Gallus domesticus. J. Endocrinol. 57: 159-169. Gehrke, C. W., D. Roach, R. W. Zumwalt, L. D. Stalling, and L. L. Wall, 1968. Pages 27-44 in: Quantitative gasliquid chromatography of amino acids in proteins and biological substances. Analytical Biochemistry Laboratories, Inc., Columbia, MO. Green, C. D., and J. R. Tata, 1976. Direct induction by estradiol of vitellogenin synthesis in organ cultures of male Xenopus laevis liver. Cell 7:131-139. Hawk, P. B., B. L. Oser, and W. H. Summerson, 1954. Total nitrogen. Pages 874-882 in: Practical Physiological Chemistry. 13th ed. McGraw-Hill Book Co., New York, NY. Hiramoto, K., T. Muramatsu, and J. Okumura, 1989. Under-
PROTEIN SYNTHESIS AND EGG FORMATION IN LAYING HENS estimation of protein synthesis caused by using DLisomer of [13N]methionine in laying hens. Nutr. Rep. Int. 39:635-642. Johnson, A. L., and A. van Tienhoven, 1980. Plasma concentrations of six steroid and LH during the ovulatory cycle of the hen, Gallus domesticus. Biol. Reprod. 23:386-393. Mandeles, S., and E. D. Ducay, 1962. Site of egg white protein formation. J. Biol. Chem. 237:3196-3199. Munro, H. M., and A. Fleck, 1969. Analysis of tissues and body fluids for nitrogenous constituent. Pages 423-525 in: Mammalian Protein Metabolism. Vol. 3. Academic Press, New York, NY. Muramatsu, T., K. Hiramoto, I. Tasaki, and J. Okumura, 1987a. Whole body protein turnover in laying hens with special reference to protein depletion and repletion. Nutr. Rep. Int. 35:607-614. Muramatsu, T., K. Hiramoto, I. Tasaki, and J. Okumura, 1987b. Effect of protein starvation on protein turnover in liver, oviduct and whole body of laying hens. Comp. Biochem. Physiol. 87B:227-232. Muramatsu, T., S. Muramatsu, J. Okumura, and I. Tasaki, 1987c. Developmental fall in whole body protein turnover of chick embryos during incubation. Br. Poult. Sci. 28:165-172. O'Malley, B. W., 1967. In vitro hormonal induction of a specific protein (avidin) in chick oviduct. Biochemistry 6:2546-2551. Palmiter, R. D., 1972a. Regulation of protein synthesis in chick oviduct I. Independent regulation of ovalbumin, conalbumin, ovomucoid and lysozyme induction. J. Biol. Chem. 247:6450-6461. Palmiter, R. D., 1972b. Regulation of protein synthesis in chick oviduct II. Modulation of polypeptide elongation and initiation rates by estrogen and progesterone. J. Biol. Chem. 247:6770-6780. Palmiter, R. D., 1973. Rate of ovalbumin messenger ribonucleic acid synthesis in the oviduct of estrogen-primed chicks. J. Biol. Chem. 248:8260-8270. Redshaw, M. R., and B. K. Follett, 1972. The physiology of egg yolk production in the hen. Pages 3-21 in: Egg
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Formation and Production. B. M. Freeman and P. E. Lake, ed. British Poultry Science, Ltd., Edinburgh, Scotland, U.K. Rhoads, R. E., 1975. Ovalbumin messenger ribonucleic acid. Purification and fractionation on basis of polyadenylate content by thermal elution from oligodeoxythymidylate-cellulose. J. Biol. Chem. 250: 8088-8097. Ryffel, G. U., 1978. Synthesis of vitellogenin, an attractive model for investigating hormone-induced gene activation. Mol. Cell. Endocr. 12:237-246. Scott, M. L., M. C. Nesheim, and R. J. Young, 1982. Nutrition of the Chicken, 3rd ed. M. L. Scott and Associates Publishers, Ithaca, NY. Seaver, S. S., D. C. van Eys-Fuchs, J. F. Hoffmann, and P. B. Coulson, 1980. Ovalbumin messenger ribonucleic acid accumulation in the chick oviduct during secondary stimulation: influence of combinations of steroid hormones and circannual rhythms. Biochemistry 19: 1410-1416. Shapiro, D. J., H. J. Baker, and D. T. Stitt, 1976. In vitro translation and estradiol-17fJ induction of Xenopus laevis vitellogenin messenger RNA. J. Biol. Chem. 251:3105-3111. Shodono, M., T. Nakamura, Y. Tanabe, and K. Wakabayashi, 1975. Simultaneous determinations of oestradiol-17p, progesterone and luteinizing hormone in the plasma during the ovulatory cycle of the hen. Acta Endocrinol. 78:565-573. Smith. A. H., S. A. Court, and E. W. Martin, 1959. Formation of albumen precursors by the hen's oviduct. Am. J. Physiol. 197:1041-1044. Smith, A. H., C. M. Winget, and T. J. Hage, 1957. Effect of local X-irradiation on albumen formation by the hen's oviduct. Am. J. Physiol. 191:605-609. Snedecor, G. W., and W. G. Cochran, 1980. Statistical Methods, 7th ed. Iowa State Univ. Press, Ames, IA. Tanabe, Y., K. Tanaka, and T. Ookawa, 1980. Neural and endocrine aspects. Pages 169-172 in: Biological Rhythms in Birds. K. Tanaka and M. Kamiyoshi, ed. Japanese Scientific Societies Press, Tokyo, Japan.
In laying hens, the magnum (a portion of the oviduct) is generally known as the formation site for egg albumen protein (Smith et ai, 1957). Albumen protein is rapidly formed in the magnum during the intervals between the passage of successive ova (Conrad and Scott, 1942; Mandeles and Ducay, 1962). Smith et al. (1959) suggested that approximately 45% of egg albumen protein is made just as an ovum passes through the magnum, based on their measurements of the volume difference in the magnum observed from .5 to 4.0 h after ovulation. However, these changes may largely reflect die secretion of egg albumen protein into the lumen rather than its synthesis. Previously, protein synthesis in various tissues of laying hens during the egg-formation cycle has not been measured in vivo. Recendy, Muramatsu et al. (1987a,b) developed a method for measuring protein synthesis in vivo in the tissues and the whole body of laying hens by a primed-continuous infusion of stable isotopes in combination with subsequent
analyses of enrichment with a gas-chromatograph mass spectrometer. The method can be used to determine protein kinetics in tissue of interest in vivo. For laying hens, both the liver and oviduct had faster fractional rates of protein synthesis (percentage per day) man any other tissues measured (Hiramoto et al., unpublished data). However, because of the rapid formation of egg albumen, protein synthesis in tissue such as that in the oviduct might fluctuate substantially during the egg-formation cycle. On the other hand, the synthesis of liver protein might remain constant throughout the egg-formation cycle. Redshaw and Follett (1972) demonstrated that the concentration of lipovitellin, a major protein in the egg yolk, in plasma did not vary throughout the day. The present study was conducted to investigate whether or not protein synthesis in tissues and in the whole body of laying hens would be affected by the position of an ovum passing along the oviduct during egg formation. MATERIALS AND METHODS
'Financial support was provided by a grant-in-aid. No. 62480082, for Scientific Research from the Ministry of Education, Science, and Culture, Japan. ^To whom correspondence should be addressed.
Animals The laying hens used were Single Comb White Leghorns 12 mo of age and weighing 1.57
264
PROTEIN SYNTHESIS AND EGG FORMATION IN LAYING HENS
265
± .24 (SD) kg and having an egg-production rate (Muromachi Kagaku Kogyo Ltd., Tokyo, Jaof 90% on the average. The hens were housed pan). The effluent was dried under reduced individually in cages in an air-conditioned room pressure at 50 C. Free amino acids in plasma at 25 C with a lighting period of 14 h per day. were derivatized to form n-trifluoro-acetyl nThey were maintained on a commercial layer butyl amino acid (TFA) esters according to the diet (Marubeni Shiryo Ltd., Tokyo, Japan) with method of Gehrke et al. (1968). The samples of 15.5% CP and 2,900 kcal of ME/kg. amino acid in free and protein fractions were Before measuring protein synthesis, the derivatized in a similar fashion. The preparation oviposition times of individual hens were of amino acid samples in tissues for enrichment recorded for 2 wk so that the expected position determination has been described in detail by of the ovum within the oviduct could be Muramatsu et al. (1987b,c). estimated at the end of isotope infusion. The isotope abundance in the TFA esters thus Whether or not the ovum was in the expected obtained was analyzed by using a computerposition was confirmed by dissecting the entire controlled, selected-ion-monitoring, gas-chrooviduct immediately after the isotope infusion. matograph mass spectrometer (Model QPAt 3 h before the appropriate time when an 1,000, Shimadzu Corp., Kyoto, Japan). The ovum would have stayed approximately at the analytical conditions of the [15N]methionine mid-point in one of the oviduct segments, sterile were as follows: column, .5% ethylene glycol polyethylene cannulas were inserted into veins adipate on chromosorb W (AW 80-100 mesh) of both the left and right wings, followed by a packed in a glass column with an internal 3-h, primed-intravenous infusion of DL- diameter of 2.6 mm and 3,000 mm long; carrier [15N]methionine. Blood-sampling was done gas, helium at a flow rate of 30 mL/min; column each hour. The blood samples were centrifuged temperature, 170 C; separator temperature, 250 at 1,800 x g for 10 min in order to separate the C; ion-source temperature, 250 C; ionizing plasma fraction, which was subsequendy depro- voltage, 70 eV; mass numbers of fragment ions, teinized with 5 vol of 1% (wt/vol) of picric acid. m + z = 228 and 229. The deproteinized samples were stored at -20 C Tissue nitrogen was determined by the until analyzed. The details of die infusion Kjeldahl method (Hawk et al., 1954). Tissue method are described elsewhere (Muramatsu et protein was calculated as N times 6.25. For al., 1987a,b). At the end of die infusion period, tissue RNA analysis, modified Schmidt-Thaundie hen was killed; then die liver, the entire hauser method as described by Munro and Fleck oviduct, a piece of skin around the leg muscle (1969) was used. (Af. biceps femoris), the breast muscle (Af. pectoralis superficialis), the leg muscle (Af. biceps femoris), the gizzard plus the proventric- Calculation ulus, and the entire intestine were removed Methionine flux (milligrams per 3 h) was quickly, weighed, and frozen by plunging them calculated according to the equation of Conway into liquid N2. The tissue samples were stored at et al. (1980) with modifications as previously -20 C until analysis. described given by Muramatsu et al. (1987b). The values designated as skin covering the The methionine flux was converted into wholewhole body were extrapolated from the ratios of body protein flux (milligrams per day) based on a specific area to the whole body. Those ratios the assumption that methionine would account were determined in a preliminary experiment for 1.8% of the body protein in chickens (Scott et using laying hens with similar body weights. In al., 1982). The calculation for the fractional rate the same manner, the values designed as breast of protein synthesis was done as described or leg muscle were derived from the ratios of previously by Muramatsu et al. (1987b). corresponding, specific muscles to the entire Since the DL-enatiomer of [15N]methionine breast or leg muscle, ratios also determined in was used as a tracer, the values for protein the preliminary experiment. synthesis were underestimated; so adequate correction, varying from one tissue to another probably due to differences in the stereoselecChemical Analysis tion or oxidation of D-methionine, had to be The picric acid in the deproteinized plasma made (Hiramoto et al., 1989). The correction was removed by passing the samples through a factors derived from differences in fractional column of the CI" form of Dowex 2-X8 synthesis rates of tissue or whole-body protein
266
HTRAMOTO ET AL. TABLE 1. Changes in protein and RNA contents in various tissues with respect to the location of an ovum along the oviduct of laying hens Location of an ovum Infundibulum
Measurement, by tissue Protein, g/tissue Liver 6.4 Oviduct 10.0*" Proventriculus plus gizzard 4.6 Intestine 6.3 Skin 19.2 Breast muscle 35.9 Leg muscle 8.6 Sum, other than liver and oviduct 74.6 RNA, mg/tissue Uver 552 Oviduct 317c Proventriculus plus gizzard 15l" Intestine 354* Skin 442" Breast muscle 452 Leg muscle 100 Sum, other than Uver and oviduct 1,499
Magnum
Isthmus
6.6 10.3* 4.9 6.6 19.3 32.7 8.4 71.9
7.5 7.5b 4.6 6.4 22.2 35.1 7.8 76.2
547 623* 143" 321*" 447*" 396 76 1381
611 520*" 150" 227" 425" 393 102 1,295
Uterus 6.3 8.0*" 4.5 6.3 20.1 37.5 9.2 77.5 491 431 bc 127" 303*" 545* 480 103 1,557
Vagina 6.0 9.1*" 4.8 6.0 19.2 37.1 8.0 75.1 559 388"° 188* 376* 468*" 493 106 1,631
Pooled SEM1 1.0 .8 .4 .7 2.0 3.2 .8 5.8 47 45 10 32 31 50 12 102
a_c
Means with no common superscripts within a row differ significantly (P<.05). !n = 4.
(estimated by using L- and DL-[15N]methionine) were as follows: whole body, 1.44; liver, 1.12; oviduct, 1.06; proventriculus plus gizzard, 1.13; intestine, 1.07; skin, 1.56; breast muscle, 1.17; leg muscle, 1.11 (Hiramoto et al., 1989). Statistical Analysis The data were subjected to a one-way analysis of variance (Snedecor and Cochran, 1980). The significance of differences between means was assessed by using Duncan's multiple range test (Duncan, 1955). RESULTS
Changes in the protein and RNA contents of various tissues are given in Table 1. To see how the values in tissues excluding the liver and oviduct that are related to the formation of egg protein would fluctuate in relation to the location of the ovum along the oviduct, the sum for tissues other than the liver and oviduct (the proventriculus plus gizzard, intestine, skin, breast muscle, and leg muscle) was also determined. No significant difference in protein content was detected in any tissues or in the sum except for the oviduct in relation to the location of an ovum. Protein content in the
oviduct was significandy (P<.05) reduced when an ovum moved from the magnum to the isthmus. The RNA content in the oviduct was highest when an ovum existed in the magnum, gradually decreasing as an ovum moved from the isthmus to the vagina. No significant changes in RNA contents were observed for the liver, breast muscle, leg muscle, and the sum of tissues other than liver and oviduct. The RNA content in the proventriculus plus gizzard was highest when an ovum was in the vagina. The RNA in the intestine gradually decreased as an ovum passed from the infundibulum to die isthmus, and increased as the ovum passed from the isthmus to the vagina. The RNA in the skin was higher when an ovum was in the uterus than when an ovum was in the infundibulum or the isthmus. Table 2 shows die changes in protein synthesis and protein synthesis per unit of RNA in various tissues, in the sum of tissues other man the liver and oviduct, and in the whole body in relation to the location of an ovum. Whole-body protein synthesis showed no significant fluctuations in relation to the location of an ovum. In the oviduct, protein synthesis was highest when an ovum was in the magnum. In the skin, a significant increase in protein synthesis was detected when an ovum moved down the oviduct from the
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PROTEIN SYNTHESIS AND EGG FORMATION IN LAYING HENS
TABLE 2. Changes in protein synthesis and in protein synthesis per unit of RNA for various tissues and in the whole body with respect to the location of an ovum along the oviduct of laying hens Location of an ovum Measurement, by tissue
Infundibulum
Magnum
Protein synthesis, mg/h per bird or per tissue Liver 243 247 207bc Oviduct 536" Proventriculus plus gizzard 163 117 Intestine 201 163 Skin 256b 221 b Breast muscle 327 290 Leg muscle 98 69 Sum, other than liver and oviduct 1,044 859 Whole body2 1,753 2,180 Protein synthesis per unit RNA, mg/h per mg RNA Liver .44 .44 Oviduct .65b .91a Proventriculus plus gizzard 1.07 .83 Intestine .57ab .51 ab b Skin .59 A& .74 .74 Breast muscle Leg muscle .99 1.05 b Sum, other than liver and oviduct .70 .62b
Isthmus
Uterus
258 285 b 122 172 386" 343 66 1,089 1,942
240 173c 128 190 309 ab 344 103 1,072 1,817
.43
.ss* .80 .77" .91" .87 .66 .84"
.50 .40° 1.01 .64* .56" .71 .97 .69 b
Vagina 230 240 bc 148 186 294ab
385 80 1,093 2,191 .42 .61*° .79 .49b .62b .77 .78 .67b
Pooled SEM1 36 31 16 26 38 49 12 93 260 .09 .07 .09 .08 .07 .06 .13 .04
a_c
Means with no common superscripts within a row differ significantly (P<.05). !n = 4. 2 Estimated from the plateau enrichment of free [15N]methionine in plasma.
infundibulum and magnum to the isthmus. In the liver and in the rest of tissues, protein synthesis was not significantly changed as an ovum passed along the oviduct. Protein synthesis per unit of RNA showed no significant fluctuations in the proventriculus plus gizzard or in the breast and leg muscles in relation to the location of an ovum (Table 2). In the oviduct, protein synthesis per unit of RNA followed a pattern similar to that for protein synthesis, being the highest when an ovum was in the magnum. The values for protein synthesis per unit of RNA in the skin and in the sum of the tissues (other than the liver and oviduct) were significantly higher when an ovum was in the isthmus compared with the rest of the segments. In the intestine, the value for protein synthesis per unit of RNA was significantly higher when an ovum was in the isthmus than when it was in the vagina. Protein synthesis in the liver per unit of RNA was fairly constant and was not significantly changed in relation to the location of an ovum. DISCUSSION
The results from the present study clearly showed that protein synthesis in the oviduct varied substantially as an ovum passed along
the oviduct: a sharp increase was found when the ovum was in the magnum portion, followed by an immediate decrease at the isthmus portion and by a further reduction to the initial level when an ovum was at the uterus and the vagina (Table 2). The increased protein synthesis when an ovum was in the magnum probably reflects the enhanced synthesis of egg albumen, the secreted protein. Among the possible factors responsible for the changes observed in protein synthesis, alterations of hormonal status would be important as well as direct mechanical stimulation caused by an ovum and reactions mediated by chemical activators from an ovum and by neurons. Estrogen increases the synthesis of ovalbumin, conalbumin, ovomucoid, and lysozyme (Palmiter, 1972a); progesterone induces avidin synthesis (O'Malley, 1967). Seaver et al. (1980) reported that both estrogen and progesterone induced an increase in the protein synthesis in egg whites. Detailed studies revealed that these steroid hormones increased the level of mRNA coding for the corresponding egg-white proteins (Palmiter, 1973) and enhanced the subsequent translation by increasing the rate of initiation (Palmiter, 1972b) or of elongation (Rhoads, 1975).
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HIRAMOTO ET AL.
The involvement of these steroid hormones in the observed alterations of protein synthesis in the oviduct may be supported by the fact that the plasma levels of estrogen and progesterone fluctuate within the ovulation cycle; after the surge of luteinizing hormone about 6 to 8 h before the ovulation, the level of plasma estrogen and progesterone increases, but with certain delays (Furr et al., 1973; Shodono et al., 1975; Johnson and van Tienhoven, 1980; Tanabe et al, 1980). The increase in the steroid hormone level in plasma just before ovulation might result in an elevated level of protein synthesis. Since a sharp induction of protein synthesis in the oviduct was detected when the ovum was in the magnum portion (Table 2), the corresponding time for induced protein synthesis could be within a few hours after ovulation. Therefore, the time lag between the elevation in the plasma level of steroid hormones and the enhanced protein synthesis in the oviduct might reflect the interval necessary for a series of biochemical reactions to lead to the induction of protein synthesis in egg albumen. By contrast with the oviduct, protein synthesis in liver, a major site of yolk-protein synthesis (Deeley et al., 1975), was not significantly affected by the position of an ovum along the oviduct, in spite of the fact that protein synthesis in the egg yolk is also under estrogen control (Shapiro et al, 1976; Ryffel, 1978). However, the consistency of protein synthesis in the liver observed in the present study agrees with the findings of Green and Tata (1976), who demonstrated that the synthesis of vitellogenin (the precursor of lipovitellin and phosvitin) was constant as long as the concentration of plasma estrogen was maintained above a certain level. Alternatively the contribution of yolk protein synthesis in the yolk to the total protein synthesis in the liver might be too small to show any variations. In the sum of tissues (other than liver and oviduct), protein synthesis per unit of RNA was reduced when an ovum was in the magnum, but immediately increased when an ovum passed from the magnum to isthmus (Table 2). No significant change in protein synthesis was detected in the sum of these tissues when synthesis was expressed per tissue. The reduced protein synthesis per unit of RNA in other tissues not related to the formation of egg protein might facilitate the increase in oviduct protein synthesis by sup-
plying more amino acids to the oviduct at this stage of egg formation. Thus, when oviduct protein synthesis increases, protein synthesis in other tissues except for the liver and oviduct may decrease, and vice versa. The data on protein synthesis per unit of RNA suggest that protein synthesis may be increased in other tissues, but that this was not the case when protein synthesis was expressed per tissue. Reversed interorgan changes in protein synthesis may account for the ovum position not affecting whole-body protein synthesis. In the present investigation, the position of an ovum within the oviduct was predicted by recording the time of oviposition for 2 wk prior to the infusion experiment and was confirmed after the infusion by dissecting the ovum. Although dissection clarified the location of the ovum, the exact time the ovum had been retained in a particular portion of the oviduct is not known. Since the transit time at the sites other than the uterus was short with respect to the infusion period of 3 h, the estimates for protein synthesis were subject to the error arising from not being able to predict the exact location. REFERENCES Conrad, R. M., and H. M. Scott, 1942. The accumulation of protein in the oviduct of the fowl. Poultry Sci. 21: 81-85. Conway, J. M , D. M. Bier, K. J. Motil, J. F. Burke, and V. R. Young, 1980. Whole-body lysine flux in young adult men: effects of reduced total protein and of lysine intake. Am. J. Physiol. 239:E192-E200. Deeley, R. G., K. P. Mullinix, W. Wetekam, H. M. Kronenberg, M. Meyers, J. D. Eldridge, and R. F. Goldberger, 1975. Vitellogenin synthesis in the avian liver. J. Biol. Chem. 250:9060-9066. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics 11:1-42. Furr, B.J.A., R. C. Bonney, R. J. England, and F. J. Cunningham, 1973. Luteinizing hormone and progesterone in peripheral blood during the ovulatory cycle of the hen, Gallus domesticus. J. Endocrinol. 57: 159-169. Gehrke, C. W., D. Roach, R. W. Zumwalt, L. D. Stalling, and L. L. Wall, 1968. Pages 27-44 in: Quantitative gasliquid chromatography of amino acids in proteins and biological substances. Analytical Biochemistry Laboratories, Inc., Columbia, MO. Green, C. D., and J. R. Tata, 1976. Direct induction by estradiol of vitellogenin synthesis in organ cultures of male Xenopus laevis liver. Cell 7:131-139. Hawk, P. B., B. L. Oser, and W. H. Summerson, 1954. Total nitrogen. Pages 874-882 in: Practical Physiological Chemistry. 13th ed. McGraw-Hill Book Co., New York, NY. Hiramoto, K., T. Muramatsu, and J. Okumura, 1989. Under-
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