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Effects of recombinant ovine placental lactogen and

recombinant ovine growth hormone on growth of lambs and

milk production of ewes

a a b c ,

_*

Haim Leibovich , Arieh Gertler , Fuller Bazer , Elisha Gootwine

Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel b

Center for Animal Biotechnology and Genomics, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, College Station, TX 77843-2471, USA c

Institute of Animal Science, ARO, The Volcani Center, P.O. Box 6, 50250 Bet Dagan, Israel Received 12 August 1999; received in revised form 20 December 1999; accepted 20 May 2000

Abstract

The somatogenic and galactopoietic effects of recombinant ovine placental lactogen (oPL) were compared with the effects of recombinant ovine growth hormone (oGH) in post-weaned growing lambs and in lactating ewes. In two experiments that each lasted 35 days, 2-month-old lambs were given daily subcutaneous injections (0.1 mg / kg live weight) of oPL or oGH, and their daily growth rates were compared with those of non-treated control lambs. Ovine GH and oPL had similar

profound (P,0.01) growth-stimulating effects, enhancing lamb growth by 10 to 25%. In two other experiments, lactating

ewes were injected with oGH or oPL (0.1 mg / kg live weight / day) for 14 days in mid-lactation. Treatment with oGH

increased (P,0.001) daily milk production by up to 55% over control ewes. Ovine PL increased (P,0.01) milk production

by up to 25%. In all experiments, treatment of lambs or lactating ewes with oGH, but not with oPL increased (P,0.05)

serum insulin-like growth factor-I concentrations. It is concluded that oPL and oGH have similar somatogenic effects in

lambs. Both hormones exhibited galactopoietic effects, but oGH was considerably more potent than oPL.  2001 Elsevier

Keywords: Placental lactogen; Growth hormone; Growth; Milk production; Sheep; IGF-I

1. Introduction ruminants synthesize and secrete unique proteins that

belong to the growth hormone / prolactin (GH / PRL) Similar to other mammalian species, placentae of family and are called placental lactogens (PLs) (Byatt et al., 1992b). Ovine (o) (Martal and Djiane, 1975; Warren et al., 1990) and bovine (b) (Murthy et

Contribution from the Agricultural Research Organization, _{al., 1982) PLs are 22 to 23 kDa proteins which are,} The Volcani Center, Bet Dagan, Israel. No. 337 / 99, 1999 series.

based on structural analysis and gene mapping, more *Corresponding author. Tel.: 1972-3-9683-752; fax:1

972-8-closely related to their respective PRL than GH 475-075.

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One unique property of ruminant PL which was 2. Materials and methods

observed early on is their ability to bind to both PRL

and GH receptors, including receptors for human The Volcani Center Animal Care Committee ap-(h)GH (Forsyth, 1986; Anthony et al., 1995a,b). This proved the experimental protocols used in this observation suggested that GH and PL may have research.

similar biological activities. However, attempts to

prove this hypothesis using in vitro experimental 2.1. Materials systems yielded contradictory results depending on

whether a homologous or a heterologous model Recombinant oPL and oGH were prepared as system was chosen. Both bPL and oPL were potent described previously (Sakal et al., 1997). Human agonists in FDC-11P cells stably transfected with insulin-like growth factor-I (IGF-I) and antiserum human or rabbit GH receptors, and their activities against IGF-I were obtained from Fugisawa Pharma-were comparable with those of hGH and oGH, ceutical (Osaka, Japan). Antiserum against oGH was respectively (Helman et al., 1997, 1998; Sakal et al., obtained from the National Hormone Pituitary Pro-1997). In contrast, in ‘‘293’’ cells transiently trans- gram (University of Maryland School of Medicine, fected with oGH or hGH receptors, ruminant PL MD, USA). Goat anti-rabbit IgG and normal rabbit were active only in cells transfected with hGH serum were purchased from Bio-Makor (Rehovot, receptors (Herman et al., 1999). In cells transfected Israel). Antiserum against oPL was prepared in

125 with oGH receptors, only oGH acted as an agonist, rabbits in our laboratory. Carrier-free Na[ I] was whereas ruminant PL, despite their ability to bind to purchased from New England Nuclear Corp. (Bos-those receptors, exhibited antagonistic activity. ton, MA, USA).

Therefore, it was concluded that ruminant PL can

bind to, but not homodimerize, oGH receptors 2.2. Animals, housing and feeding (Herman et al., 1999).

Results from in vivo comparisons of PL and GH The experiments were conducted with the Kat-bioactivities are also inconsistent. Whereas adminis- zenelbugen dairy flock, which includes 600 Assaf tration of both bPL or bGH increased growth and breeding ewes, at Moshav Talmai Elazer. This flock milk production in rats, only bGH affected milk is kept indoors all year round and the ewes are fed yield in dairy cows (Byatt et al., 1992a). Further- according to Israeli Ministry of Agriculture recom-more, oPL failed to show GH-like galactopoietic mendations (Landau and Leibovich, 1992). All the properties when injected into ewes (Min et al., lambs of this flock are routinely separated from their 1995). Bovine GH, but not bPL, had stimulatory dams on the day of lambing, and are moved to an effects on lamb growth rate in one study (McLaug- artificial rearing unit where they receive a commer-hlin et al., 1993) while, in another study, administra- cial milk replacer ad libitum until weaning at approx-tion of oPL to growing lambs significantly increased imately 25 days of age. Following weaning, lambs their growth rate, whereas bGH did not (Min et al., have free access to concentrates, hay and water. 1996). This latter result contradicts several other Ewes are milked at 08:00 and 16:00 daily from the studies where bGH had a growth-promoting effect in day of lambing until milk yield decreases to approxi-lambs (Wagner and Veenhuizen, 1978; Rosemberg et mately 0.5 l / day.

al., 1989).

Recombinant oPL (Colosi et al., 1989; Sakal et al., 2.3. Growth-promoting effects following 1997) and bPL (Krivi et al., 1989) have been administration of oPL and oGH

prepared, and were produced in our laboratory

(Gertler et al., 1992; Sakal et al., 1997) in amounts Sixty male lambs born in February 1995 and 36 sufficient for in vivo studies with a large number of male lambs born in March 1997 were used in two animals. Therefore, the present study compared the experiments. In each experiment, 2-month-old lambs effects of oPL and oGH in a homologous in vivo were allocated to experimental groups according to system, using two experimental models: growing their weight, post-weaning growth rate, litter size and lambs and lactating ewes. age. The lambs were fed concentrates (16% crude

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protein) ad libitum and hay at 0.1 kg / head / day. infrared analyzer (Milkoscan 134; N. Foss Electric, Lambs were treated daily at 10:00 for 35 days with Hillerod, Denmark).

saline solution containing 0.1 mg / kg body weight of

oPL (oPL group) or oGH (oGH group), or received 2.6. Radioimmunoassays saline only (control group). Lambs were weighed at

the beginning of the injection period and once a Serum concentrations of oGH (Peri et al., 1993), week thereafter until the end of the injection period. IGF-I (Breier et al., 1991) and oPL (Kann, 1971) Group feed consumption was monitored daily. Blood were determined as described previously. Iodination samples for hormone assays were collected from the of oGH and oPL was performed as described previ-jugular vein on the morning time of day 34 of the ously (Gertler et al., 1984). The inter- and intra-injections, prior to the daily hormone administration, assay coefficients of variation were 12.4% and 8.7% to obtain serum, which was stored at 2208C until for IGF-I, 10.4% and 6.2% for oGH, and 8.8% and assayed. 6.4% for oPL, respectively.

2.7. Data analyses 2.4. Galactopoietic effects following administration

of oPL and oGH

Lamb growth rates were calculated by fitting linear regressions to the body weight measurements Forty-five multiparous ewes that lambed during 2

and then subjecting them to analysis of variance. The weeks in October 1997 and 143 multiparous ewes

model included the main fixed effects of experiment that lambed during 2 weeks in October 1998 were

(1, 2) and experimental group (oPL, oGH and used in two experiments to determine daily milk

control). Lamb weight at the beginning of the production, recorded weekly from lambing. In each

experiment was used as a covariate. Results from experiment, at about 8 weeks after lambing and

each of the milk production experiments were ana-when all ewes had passed their peak of lactation,

lyzed separately. Data on milk production on each they were assigned to three groups. Distribution of

day of recording was analyzed by analysis of vari-ewes into the different groups was according to their

ance. The model included experimental group as the weight and their daily milk production averaged

main effect, and milk production on day 0, days from across the last three daily milk records. The ewes

lambing and ewe body weight at the beginning of the were treated daily at 17:00 for 14 days with saline

injection period were taken as covariates. Serum solution containing 0.1 mg / kg body weight of either

hormone concentrations and milk composition were oPL (oPL group) or oGH (oGH group), or received

subjected to analysis of variance to test for effects of saline only (control group). Milk production was

treatment within day of sampling. Statistical analyses monitored the day before the injections were begun

were conducted using the Statistical Analysis Insti-(day 0) and then every 3 to 5 days, for up to 28 days.

tute (SAS) computer package (1985). All data are Body weight was recorded for all ewes before and at

expressed as least squares means and standard errors. the end of the hormonal injection periods. Group

Differences of P,0.05 were considered significant. feed consumption was monitored daily in the first

experiment only. Blood samples were collected 10 to 11 days after initiation of hormone treatments at

3. Results

10:00 to obtain serum for assay for concentrations of IGF-I, oGH and oPL.

3.1. Comparison of oGH and oPL effects in growing lambs

2.5. Milk composition analysis

Least-squares means and levels of significance for Milk composition was evaluated on day 13 of the growth rates, and oGH, oPL and IGF-I serum injection period in the second experiment only. concentrations are presented in Table 1. Experimen-Concentrations of fat, protein, lactose and total solids tal group and initial weight of lambs had significant in milk were measured using a semi-automated effects on growth rate. Both oGH and oPL had

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Table 1

Growth performance, and oGH, oPL and IGF-I serum concentrations (LS mean6S.E.M.) of control lambs and lambs treated daily for 35 1

days with 0.1 mg / kg body weight of oPL or oGH

Experimental treatment

Control oPL oGH

Experiment1

No. of lambs 20 20 20

Initial body weight (kg) 23.560.6 23.260.6 24.160.6

a b b

Growth rate (kg / d) 0.4860.01 0.5360.01 0.5360.01

2 a a b

GH (ng / ml) 7.561.0 8.161.0 12.261.2

PL (ng / ml) N.D. 56.1612.3 N.D.

2 a a b

IGF-I (ng / ml) 304623 321625 466635

Experiment2

No. of lambs 12 12 12

Initial average body weight (kg) 25.060.9 24.460.9 24.660.9

a b b

Growth rate (kg / d) 0.3660.02 0.4560.02 0.4560.02

2 a a b

GH (ng / ml) 2.460.3 2.860.6 7.261.2

PL (ng / ml) N.D. 58.5615.6 N.D.

2 a a b

IGF-I (ng / ml) 310615 308620 365616

1 a,b

Within rows, means with different superscripts are different (P,0.05). 2

Measured 34 days after the beginning of the injection periods. N.D., Not detectable.

profound and similar growth-stimulating effects of 3.2. Comparison of oGH and oPL effects in approximately 10 and 25%, in experiments 1 (P, lactating ewes

0.01) and 2 (P,0.005), respectively. The relative

effects of the hormonal treatments on growth rates At 6 weeks after lambing, ewes reached their peak were inversely related to the average growth rates of lactation at an average of 2.4 and 2.1 kg / d milk yield the control groups, which were 480 and 360 g / d in in experiments 1 and 2, respectively. The experiment experiments 1 and 2, respectively. Feed intake for was started 8 weeks after lambing when the body control lambs averaged 1.51 and 1.41 kg DM / d in weight of ewes averaged 8062.6 kg. Daily milk experiments 1 and 2, respectively, and were numeri- production prior to and during the experimental cally higher (6%) for lambs treated with oGH or period is presented in Fig. 1. There were no pre-oPL. Ovine GH, but not oPL, increased (P,0.05) treatment differences in milk yield among the treat-IGF-I concentrations (Table 1). Concentrations of ment groups for ewes in either experiment. For oGH in serum prior to treatment in all lambs control ewes, milk production gradually decreased belonging to the control, oGH and oPL groups during the injection periods in both experiments. In averaged 6.9 and 2.3 ng / ml in experiments 1 and 2, contrast, ewes injected with oGH experienced a respectively. Ovine GH serum concentrations re- dramatic increase (P,0.001) in daily milk pro-mained similar to the pre-treatment concentrations in duction within 5 to 6 days after the first day of the control and the oPL groups, while treatment with injection. The maximum percentage increase in milk oGH increased (P,0.05) oGH serum concentrations production due to oGH was observed 10 days after at day 34 of the injection period to an average of the first injection, being 45 to 55% above controls. 12.2 and 7.2 ng / ml in experiments 1 and 2, respec- At that point, the absolute daily yield was higher tively (Table 1). Ovine PL was not detectable in than that at peak lactation prior to the oGH in-serum from lambs prior to or during the treatment jections. Injection with oPL also increased milk periods in lambs in control and oGH groups. In the yields in both experiments, but the effect was oPL group, oPL concentrations at day 34 of the moderate. The maximum percentage increase above injection period were 58615 ng / ml. control ewes was observed 11 days after the first

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Table 2

Milk yield and composition (LS mean6S.E.M.) on the 13th day of the injection period of ewes belonging to the control, oPL and

1 oGH groups

Experimental group

Control oPL oGH

No. of ewes 15 15 15

a a b

Milk yield (l) 1.9160.1 2.1460.1 2.9760.1 Total solids (%) 17.160.3 17.060.2 16.460.2

a b b

Lactose (%) 5.160.5 4.760.4 4.760.4

a a b

Protein (%) 5.660.1 5.660.1 5.160.1 Fat (%) 5.660.2 5.760.2 5.560.2

1 a,b

Within rows, means with different superscripts are different (P,0.05).

not detectable in control ewes or ewes treated with oGH, but averaged 73611 ng / ml in ewes treated with oPL. Feed intake for the oGH and oPL-treated ewes averaged 3.6 and 3.8 kg DM / d, respectively, numerically higher than the feed intake in control ewes (3.1 kg DM / d).

Fig. 1. Milk production in ewes treated daily for 14 days with 0.1

Results on milk composition are summarized in mg / kg body weight of oPL (triangles), oGH (open circles) or

saline (squares). The values are given as LS means6S.E.M. The Table 2. The increase (P,0.05) in milk yield in the numbers of ewes in each treatment were 15 in experiment 1 and _{oPL and the oGH groups did not affect total solids or} 47–48 in experiment 2.

fat concentration. However, percent protein was lower (P,0.05) in the oGH group and percent injection (P,0.01), being 25 and 20% in experi- lactose was lower (P,0.05) in both the oPL and ments 1 and 2, respectively. Upon cessation of the oGH groups.

hormonal treatments, there was a decrease in milk production within 10 to 14 days and daily milk

yields for ewes in the treatment groups were not 4. Discussion

different from those of the control ewes. IGF-I

concentrations in control ewes on days 10 to 11 of In this study, we have compared for the first time the injection periods were 88611 and 126610 ng / the in vivo somatogenic and lactogenic activities of ml in experiments 1 and 2, respectively. While oPL oGH and oPL in a homologous system. Our results administration did not significantly change serum indicate that the growth-promoting activity of oPL in concentrations of IGF-I (101611 and 12169 ng / ml lambs is similar to that of oGH. However, the in experiments 1 and 2, respectively), concentrations galactopoietic effects of oPL in ewes were sig-of IGF-I in oGH-treated groups increased (P,0.05) nificantly less than for oGH. The mechanism(s) to 265611 and 294614 ng / ml in experiments 1 and responsible for the stimulatory effects of oPL on 2, respectively. growth and milk production may differ from those of Ovine GH and oPL serum concentrations were oGH, as only oGH treatment resulted in an increase determined in ewes only in experiment 1, 10 to 11 in serum IGF-I concentrations.

days after the initiation of the injection periods. In In the present study, oPL and oGH were found to the control and oPL groups, concentrations of oGH have a similar growth-promoting effect. To our in serum were similar (2.460.18 and 2.460.28 ng / knowledge, a similar homologous comparison of the ml, respectively) and less (P,0.01) than in ewes somatogenic activity of bGH and bPL has not been injected with oGH (3.460.2 ng / ml). Ovine PL was reported. However, in a non-homologous system,

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bGH, but not bPL, stimulated growth of lambs genetic selection for high milk production as dairy (McLaughlin et al., 1993). The growth-promoting cows. Thus, the high response to the oGH in ewes effects of oPL in our study (10–25%), obtained by may reflect the difference between the actual levels treating 2-month-old lambs for 35 days, are similar of production and the physiological potential for to the 12% increase in growth rate of 3-day-old production. Interestingly, injection of bGH into lambs following treatment with oPL for 21 days lactating goats has also increased milk production by (Min et al., 1996). In contrast, Ogawa et al. (1995) 50–60% (Jammes et al., 1996).

saw no somatogenic and anticatabolic effects of oPL It should be emphasized that the in vivo following daily injection of 2-month-old lambs for 5 somatogenic and lactogenic effects of oPL in the days. Thus, collectively, these results indicate that present study occurred during physiological states of the somatogenic effect of PL (as compared with that growth and lactation when oPL is not naturally of GH) may depend on the age of the lambs, the present. Thus, our results advance our understanding duration of the injection period and the homology of of the biological potency of oPL, but its physiologi-the experimental system. cal role during pregnancy needs to be investigated

In two experiments conducted with lactating ewes further.

injected for 14 days, both oGH and oPL had In both the growth and milk production experi-galactopoietic activity (Fig. 1), with oGH being ments, administration of oGH but not oPL increased consistently much more potent. Similar results show- IGF-I serum concentrations. The fact that oPL ing positive but different galactopoietic effects of administration does not increase circulating blood bGH and bPL were also reported by Byatt et al. concentrations of IGF-I was also reported in other (1992a) in dairy cows. In addition, we recently studies (Ogawa et al., 1995; Min et al., 1996). compared in vivo effects of oGH and oPL and Collectively, these results raise the question as to the determined that both hormones exhibit mammogenic way oPL effect is transduced, as its appears to be activity in pseudo-pregnant ewes (Kann et al., 1999). different from that of oGH. One unique property of Our results concerning the galactopoietic activity ruminant PL, which was observed early on, is their of oPL contradicts the results of Min et al. (1995), ability to bind to both prolactin (PRL) and GH who reported no galactopoietic effect following five receptors (Forsyth, 1986; Anthony et al., 1995a,b). consecutive daily injections of lactating ewes with Thus, several alternatives concerning the mechanism oPL. The differences between the two studies may of oPL action may be considered: (a) the PL signal is be related to the short duration of the oPL treatment transduced through homologous PRL receptors; (b) in the Min et al. (1995) study. As shown in Fig. 1, PL is capable of heterodimerization with the homolo-the development of homolo-the oPL galactopoetic effect in gous GH receptor through site I and the PRL our study was relatively slow and, although evident receptor through site II; (c) a unique unidentified PL in both experiments after 5 days, it was only receptor exists but has not yet been identified; and significant in experiment 1. The effect of oPL (d) an unknown variant(s) of GH receptor, mutated at became significant only after 10 days in experiment the extracellular domain allows dimerization of GH 2. receptor by PL and this variant GH receptor is The strong galactopoietic effect of oGH in the expressed in specific tissues or under unique physio-present study (45–55% above control) resembles the logical conditions. Recently, we (Herman et al., effect of bGH in sheep (Fernandez et al., 1995; 2000) have shown that oPL heterodimerizes the Chiofalo et al., 1999) and was much higher than the extracellular domains (ECDs) of ruminant GH re-typical effects of bGH in cows (15 to 25%; Bauman ceptor (GHR) and PRL receptor (PRLR). We also and Vernon, 1993). While different biological poten- showed that PL or PL analogues that exhibit little or cies of the bGH and oGH preparations may account no activity in cells transfected with PRLR and no for some differences in biological effects, an alter- activity in cells transfected with oGHR, do exhibit native explanation may be related to the fact that largely enhanced activity in cells co-transfected with dairy sheep have not undergone the same extensive both PRLR and GHR. Those results may provide

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growth hormone, and placental lactogen. J. Dairy Sci. 69, new ideas on the oPL specific mechanism of action,

886–903. which could explain the similar but different oGH

Gertler, A., Ashkenazi, A., Madar, Z., 1984. Binding sites of and oPL activity in the present study. _{human growth hormone and ovine and bovine prolactins in the} mammary gland and the liver of lactating dairy cow. Mol. Cell. Endocrinol. 34, 51–57.

Gertler, A., Hauser, S.D., Sakal, E., Vashdi, D., Staten, N.R.,

Acknowledgements

Freeman, J.J., Krivi, G.G., 1992. Preparation, purification and determination of the biological activities of 12 N terminus-We acknowledge the technical help of Mr. Eyal

truncated recombinant analogues of bovine placental lactogen. Vardi in the preparation of recombinant oPL and _{J. Biol. Chem. 267, 12655–12659.}

oGH, Ms. Nava Chapnik-Cohen for her help in the _{Gootwine, E., Yossefi, S., 1998. Genetic mapping of the ovine} Nb2 bioassays and Mr. Moti Katzenelbugen for his placental lactogen gene to sheep chromosome 20. Anim. Genet.

27, 69. cooperation. We also thank the National Institute of

Helman, D., Staten, N.R., Byatt, J., Grosclaude, J., McKinnie, Health, Hormone Program for a gift of anti IGF-I

R.E., Djiane, J., Gertler, A., 1997. Site-directed mutagenesis of antibodies. This research was supported by Research

recombinant bovine placental lactogen at lysine-73 leads to Grant No. US-2643-95 from BARD, The United _{selective attenuation of its somatogenic activity. Endocrinology} States–Israel Binational Agricultural Research and 138, 4069–4080.

Development Fund. Helman, D., Staten, N.R., Grosclaude, J., Daniel, N., Nespoulous, C., Djiane, J., Gertler, A., 1998. Novel recombinant analogues of bovine placental lactogen. G133K and G133R provide a tool to understand the difference between the action of prolactin and

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protein) ad libitum and hay at 0.1 kg / head / day. infrared analyzer (Milkoscan 134; N. Foss Electric, Lambs were treated daily at 10:00 for 35 days with Hillerod, Denmark).

saline solution containing 0.1 mg / kg body weight of

oPL (oPL group) or oGH (oGH group), or received 2.6. Radioimmunoassays saline only (control group). Lambs were weighed at

assayed. 6.4% for oPL, respectively.