relations between maternal and fetal plasma ...

Relations between maternal and fetal plasma concentrations of placental lactogen and placental and fetal weights in well-fed ewes P. A. Schoknecht, S. N. Nobrega, J. A. Petterson, R. A. Ehrhardt, R. Slepetis and A. W. Bell J ANIM SCI 1991, 69:1059-1063.

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RELATIONS BETWEEN MATERNAL AND FETAL PLASMA CONCENTRATIONS OF PLACENTAL LACTOGEN AND PLACENTAL AND FETAL WEIGHTS IN WELL-FED EWES' P. A. Schoknecht, S. N. Nobrega, J. A. Petterson, R. A. Ehrhardt, R. Slepetis, and A. W. Bell2 Cornell University3, Ithaca, N Y 14853 ABSTRACT

The concentration of ovine placental lactogen (oPL) in maternal plasma varies with litter size and nutritional status, making it difficult to compare these concentrations across studies. In this study, 27 Dorset and Finn-Dorset crossbred ewes with litters of known size and gestational age were used to relate concentrations of oPL in maternal plasma to placental and fetal weights. Fetal oPL concentrations also were correlated to these variables in 12 chronically catheterized singleton fetuses. The concentration of oPL in maternal plasma increased with increasing placental weight across litter sizes ranging from 1 to 3 (r = .716). When expressed per gram of placenta, oPL was greater (P e .05) in those ewes carrying multiple fetuses. There was no correlation between maternal and fetal oPL in time-matched samples or in average values between individuals for ewes carrying singleton pregnancies. Within the singleton group, placental weight and fetal weight were well correlated (r = .761), as were the concentration of fetal plasma oPL and fetal weight (r = .699). Placental weight plus fetal oPL could explain 81% of the variation seen in fetal weight. These results imply that maternal and fetal oPL release are controlled independently and that fetal oPL affects fetal growth by a mechanism not directly related to placental size. Key Words: Placental Lactogen, Placenta, Fetus, Litter Size, Sheep J. Anim. Sci. 1991. 69:105%1063

increases circulating oPL (Brinsmead et al., 1981), but casein supplementation decreases Ovine placental lactogen (oPL), produced oPL concentration (Gluckman and Bany, by the binucleate cells of the chorion (Wood- 1988). Fetal concentrations of oPL in late ing, 1981), is released into both the fetal and gestation generally are 30 to 100 ng/ml maternal circulations. Its circulating concentra- (Gluckman et al.. 1979; Taylor et al., 1980), tion in maternal plasma during late pregnancy but they increase when feed intake is severely is highly variable (600 to 3,000 ng/ml) and restricted in the ewe (Brinsmead et al., 1981). increases with litter size Faylor et al., 1980; Because both placental size and nutritional Butler et al., 1981). Experimental reduction of status affect concentrations of oPL in maternal placental mass decreases maternal oPL concen- plasma, it is difficult to compare these trations (Taylor et al., 1983; Falconer et al., concentrations across studies. Therefore, to 1985; Newnham et al., 1986). Starvation better define and explain the relations between oPL concentration, placental weight, and fetal weight we studied these variables in well-fed ewes of hown gestational age and litter size. introduction

'This research was supported by funds from the New York Agric. Exp. Sta. %o whom correspondence should be addressed. 3~partmentof ~ n i m .~ c i . Received July 5, 1990. Accepted September 10, 1990.

Materials and Methods

Animals and Feeding. Twenty-seven multiparous Dorset and Finn-Dorset crossbred ewes

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from 3 to 6 yr of age were bred to a Dorset ram; the first day of pregnancy was considered the last day on which coitus was observed. Litter size was determined using ultrasound4 at approximately d 50 of pregnancy. Ewes were housed individually and fed a total mixed diet containing 2.5 Mcal ME/kg dry weight and 15% CP. Beginning on d 100 of pregnancy, ewes carrying single fetuses received 25 g/kg body weight, ewes with twins received 38 g/kg body weight, and those carrying triplets received 42 @g body weight to meet their predicted nutrient requirements (NRC, 1985). Prior to d 100, all ewes were group-fed and maintained at the Comell University Teaching and Research Center. Experimental Procedure. Maternal arterial blood samples were taken via a hindlimb iliac artery catheter from all ewes carrying singles and twins. Blood samples (3 ml) were collected by jugular venipuncture from the ewes carrying triplets; they also were control animals in a separate nutrition study. The constraints of that study did not allow place ment of a hindlimb catheter. For the litter size analysis, oPL concentrations from blood samples collected within 1 wk of slaughter were averaged for each individual ewe. For ewes carrying singles, this average was from two samples taken on 133 and 136 of gestation. For twins, eight samples taken on d 138 were averaged. Two samples were taken from ewes carrying triplets on d 129 and 136 of gestation; these were averaged for each ewe. Fetal arterial blood samples (3 ml) also were taken from only the singleton fetuses via an aortic catheter that was surgically implanted on d 115, using the procedure of Rudolph and Heymann (1980). Maternal and fetal samples were taken within 10 min of each other in all cases, on d 122, 123, 125, 127, 130, 133, and 136 of gestation. For the time-matched analysis, individual fetal and maternal samples were compared. For all other fetal and maternal comparisons, the mean of seven samples for each individual was used. All samples were collected into heparinized tubes and immediately centrifuged at 2,000 x g at 4'C; the

%xhnicare ZOODX, 3.5-MHz traosducer, Johnson and Johnson, East Brumwick, NJ. 'sigma Diagnostics, procedure *IO, sigma chemical Co., St. Louis. MO.

plasma was stored at -2O'C prior to analysis. Measurements. The concentration of plasma oPL was measured using a specific double antibody produced by W. B. Currie using oPL purified from ovine placentomes. The purification procedure and radioimmunoassay methodology are detailed in Bell et al. (1989). Intraand interassay CV were 4.2 and 5.3%, respectively. The concentration of plasma glucose was measured using an enzymatic kit assay5, with intra- and interassay CV of 2.7 and 3.9%. respectively. Ewes were stunned with a captive bolt pistol and exsanguinated at d 136 to 138 of gestation. The gravid uterus was removed and individual fetuses and placentas were weighed. Placental weight was the aggregate weight of all placentomes, dissected from the chorioallantoic membranes and endometrium on fetal and maternal surfaces, respectively. Statistical Analysis. All statistical tests were from Neter et al. (1985) and were perFormed using SAS (1985). Mean values in Table 1 were compared across litter size by one-way ANOVA and Tukey's test. Concentrations of plasma oPL for singleton fetuses were regressed across time using a simple linear regression. Correlation coefficients were calculated by simple linear regression and tested for their difference from zero. Multiple regressions and covariate analyses were run to predict fetal body weight from placental weight, fetal oPL, and maternal oPL. Results

Ewes. Live weight of ewes averaged 53.2 f 2.3 kg (mean f SE) at d 100 of pregnancy and 60.5 f 3.1 kg at d 136. Concentrations of plasma glucose ranged from 49.5 to 65.3 mg/ dl, with a mean of 53.2 f 2.3 mg/dl. Litter Size. Data for fetal weight, placental weight, concentration of oPL in maternal plasma, and plasma oPL/g placenta are shown in Table 1, according to litter size. Both total fetal and placental weights increased (P < .05) in a predictable manner according to litter size. Concentrations of oPL in maternal plasma also increased with increasing litter size (P -c .05), although the difference in means between twins and triplets was not statistically significant. The correlation coefficient for the relation between fetal weight and the concentration of oPL in maternal plasma was .707 (P < .01). Maternal oPL concentration per gram of


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PLACENTAL LACTOGEN AND CONCEF'IWS

TABLE 1. TOTAL FETAL AND PLACENTAL WEIGHTS AND MATERNAL PLASMA OVINE PLACENTAL LACTOGEN (OK) CONCENTRATIONS IN EWES OF DLPFERENT LlTIER SIZE ( M E A N S f SE) Litter Size

1 2 3

No. of ewes 12 12 3

Fetal wt,

Placental

oPL conc.,

&

mg

4.35 f .14" 7.12 f .43b 9.45 f 1.01'

440 f 22"

nghnl 341 f 28" 659 f 57b 975 f 351b

600 f Mb 692 f 137b

%bvcValueswithin columns with different superscripts are si@icantly

placental tissue also was greater (P < .05) in those ewes carrying multiple fetuses. The regression of the concentration of oPL in maternal plasma against placental weight by litter size is shown in Figure 1. The concentration of oPL in maternal plasma was significantly correlated to placental weight (r = .716; P < .01). Single Fetuses. The concentration of oPL in fetal plasma did not vary significantly within animals over the 2-wk sampling period, so values were averaged for each animal. The overall mean was 30.0 f 1.2 ng/ml. There was no relation between individual matched samples for fetuses and their dams (r = .141, n = 52), nor was there a relation when values were averaged across the sampling period for each individual (r = .224, n = 12). Correlation coefficients for relations be tween maternal and fetal oPL concentrations, placental weight, and fetal weight in singlepregnant ewes are shown in Table 2. Correlations between concentrations of oPL in maternal plasma and any other variable, including placental size, were not significant. However, the concentration of fetal plasma oPL was correlated positively (.699,P c .05) to fetal weight. This correlation was similar to that

TABLE 2. CORRELAnoN COEFFICIENTS FOR RELATIONS B E T " MATERNAL PLASMA OVINE PLACENTAL LACTOGEN (OPL) CONCENTRATION, FETAL PLASMA oPL CONCENTRATION, PLACENTAL WEIGHT, AND FETAL WEIGHT FOR SINGLE FETUSES AND THEIR DAMS Fetal

Item

OPL

OFT

wt

-.224 ,249 -47

-

-

.327 .699*

.761**

*P e .05; n = 12. **P < .01; n = 12.

different at P < .05.

DlSCuSSlOn

The concentration of maternal plasma oPL varies considerably during late pregnancy in ewes on an adequate, well-defiied plane of nutrition. Thus, we presume that energy status was not influencing oPL concentrations of our ewes, as has been of concern previously (Brinsmead et al., 1981). The weight gains

.

I 1500-

1Mx)-

400

Plecental

Fetal oPL Placentalwt Fetal wt

-

.8 f .la 1.1 f .lb 1.4 rt .3b

between placental weight and fetal weight (r = .761; P .01). Squared, these values mean that placental weight and fetal oPL could account for 58% and 49% of the variation in fetal weight, respectively. The concentration of fetal plasma oPL still had a significant effect when placental weight was used as a covariate. Placental weight and fetal oPL concentration together could explain 81% of the variation in fetal weight. No additional variation was explained by the addition of the concentration of maternal plasma oPL to the model.

m Maternal

oEX&lacenta, ngl(ml%)

m

800

lo00

Placenta!Weight

(gams)

Figure 1. Regression of maternal plasma ovine placental lactogen (oPL) Concentration and placental weight for ewes carrying singles (O), twins (e),or triplets (A). Hasma OK= 1.49 (placental weight) -248.68, r = .716.


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seen in these ewes, along with concentrations of plasma glucose within the normal euglycemic range, indicate that these ewes were well fed during pregnancy. Most of the variation in the concentration of maternal plasma oPL was due to litter size; the range in ewes carrying triplets was particularly large (456 to 1,645 ndml). Circulating levels of oPL expressed per gram of placenta (presumed to be an index of weight-specific placental production) increased with litter size, as did the correlation between circulating oPL and placental size within litters. These data imply that individual placentas respond to external signals, possibly of fetal origin. Such signals have been proposed previously by Bell et al. (1987). Circulating oPL values were relatively constant across time and between individuals for single fetuses. Lack of any association between maternal and fetal concentrations for matched samples suggests that release of oPL is controlled differently on the fetal and maternal sides of the placenta. Fetal oPL increased with placental size within single fetuses and could explain almost 50% of the variation in fetal weight, similar to that explained by placental weight alone. Multiple regression analysis showed that, together, these two variables could explain most (81%) of the variation in fetal weight. The proposed function of oPL in the ewe is to alter maternal metabolism to spare glucose for use by the growing conceptus (Grumbach et al., 1968). This idea is supported by the increase in circulating oPL with increasing litter size, because, even in well-fed ewes, the energetic demand on a ewe carrying multiple fetuses is large. However, conclusive evidence for this hypothesis has not yet been obtained in sheep or in any other species. Recent evidence has led some to theorize that oPL in the fetus is a regulator of fetal growth (Chene et al., 1988). Our data are consistent with this contention to the extent that the concentration of fetal plasma oPL and fetal weight were highly correlated. However, a large fetus could produce signals that result in increased oPL production. Although some of the apparent effect of oPL may simply be due to covariance with placental weight, oPL also seems to have an additional, separate, role.

lmpiicatlons

A strong correlation between litter size and the concentration of ovine placental lactogen in maternal plasma and the increasing concentration of ovine placental lactogen per gram of placenta with litter size suggest that signals from the fetus affect placental production of this hormone. This is consistent with the hypothesis that ovine placental lactogen alters maternal metabolism to increase the amount of glucose available to the fetuses. Our results also suggest a role for placental lactogen in the control of fetal growth, an idea that has been theorized but never tested. These data, along with recent in vitro evidence, support the need to determine the roles of placental lactogen in the pregnant animal. Literature Cited Bell, A. W.. D. E. Bauman and W. B. Currie. 1987. Regulation of nutrient paaitioning and metabolism duringpre-andpostnatalgrowth.J.Anim. Sci.65:186. Bell, A. W., B. W. McBride, R. Slepetis, R J. Early and W. B. Cunie. 1989. Chronic heat stress and prenatal development in sheep: I. Conceptus growth and maternal plasma hormones and metabolites. J. Anim. Sci. 67:3289. Brimmead, M. W., B. J. Bancroft, G. D. Thorburnand M.J. Waters. 1981. Fetal and maternal ovine placental lactogen during hyperglycaemia,hypoglycaemia, and fasting. J. E n d d o l . 90.337. Butler. W. R., S. M. Menkamp, L. A. Cappiello and S. Haudwerger. 1981. The relatiomhip between breed and litter size in sheep and maternal serum concentrations ofplacentallactogen, estradiol, and progesterone. J. Anim. Sci. 5331077. Chene, N., J. Martal and J. Charrier. 1988. Ovine chorionic somatomammol~opin and foetal growth. Reprod. Nutr. Dev. 28:1707. Falconer, J., J. Owens, E. AUotta and I. Robinson. 1985. Effect of restriction of placental growth on the concentrations of insulin, glucose and placental lactogen in the plasma of sheep. J. Ehdocrinol. 1W7. Gluckman, P. D. and T. N. Bany. 1988. Relationships between plasma concentrations of placental lactogen, insulin-likegrowth factors,metabolites and lamb size in late gestationewes subject to nutritionalsupplementation and in their lambs at birth. Domest. Anim. Endocrinol. 5209. Gluckman, P.D., S. L. Kaplan, A. M.Rudolph and M. M. Orumbach. 1979. Hormone ontogeny in the ovine fetus. II. Ovine chorionic somatomammotropin in midand late gestation in the fetal and m a t d circulations. Endocrinology 10431828. Orumbach, M. M., S. L. Kaplan, J. Sciama and I. Burr. 1968. Chorionic growth hormone-prolactin (CGP): Secretion, disposition, biological activity in maa, and postulated function as the growth hormone of the second half of pregnancy. AM. N. Y.Acad. Sci. 148: 501.


PLACENTAL LACTOGEN AND CONCEPTUS NRC. 1985. Nutrient Requirements of Sheep (6th Ed.). National Academy Press, Washington, DC. Neter, J., W. Wasserman and M.H. Kutner. 1985. Applied Linear Statistical Models (2nd Ed.). h i n , Homewood, E.

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Amsterdam. SAS. 1985. SAS User’s Guide: Statistics. SAS Inst., Inc., Gary, NC. Taylor, M.J., G. Jenkin,J. S. Robinsonand G. D. Thorburn. 1983. Effect of intrauterine death and fetectomy on Newnham,J.P.,R.W.Lam,C.J.Hobel,D.H.PolkandD. ovine placental lactogen production. Res. Vet. Sci. 35: A. Fisher. 1986. Blunted response of maternal ovine 22. placental lactogen levels to arginine stimulation after Taylor, M.J., G. Jenkin, J. S. Robinson, G. D. Thorbum, H. single umbilid artery ligation in pregnant sheep. Am. Friesen and J.S.D. Chaa 1980. Concentrations of J. Obstet. and Gynecol. 154663. placental lactogen in chronicallycatheterizedewes and Rudolph, A. M. and M.A. Heymann. 1980. Methods for fetuses in late pregnancy. J. Endocrinol. 85:27. studying the circulation of the fetus inutero. In: P. W. Wooding, F.B.P. 1981. Localization of ovine placental Nathanielsz (Ed.) Animal Models in Fetal Medicine. lactogen in sheep placentomes by electron microscope pp 1-57. ElsevierNorth-Holland Biomedical Ress, immunocytochemistry. J. Reprod. F d . 62:15.


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