American Journal of Primatology 26:35-46 (1992)

Characterization of Prolactin and Growth Hormone Immuno- and Bioactivities in the Pituitary Gland and Serum of the Squirrel Monkey (Saimiri boliviensis boliviensis)

J.G. SCAMMELL,' R. VON HAVEN,' H.G. FRIESEN? L.B. WEAR,' M.E. THOMPSON,' A.G. BRADY,' L.E. WILLIAMS,' AND C.R. ABEE' 'Departments of Pharmacology and 2Comparatiue Medicine, University of South Alabama, Mobile, Alabama; 3Department of Physiology, University of Manitoba, Winnipeg, Canada

The immunological and biological activities of growth hormone (GH) and prolactin (PRL) in the pituitary gland and serum of the squirrel monkey (Saimiri boliviensis boliuiensis) have been studied. Proteins in pituitary homogenates were solubilized in 1% SDS, electrophoresed on 12% poly- acrylamide gels, and transferred to nitrocellulose. Squirrel monkey GH and PRL were identified by immunoblotting with anti-human GH anti- bodies and a monoclonal antibody to ovine PRL, 6Fl1, respectively. Squir- rel monkey GH appeared predominantly as two proteins of apparent mo- lecular weight 22 and 20 kD, corresponding to native and variant forms of human GH. Squirrel monkey PRL appeared as two proteins of apparent molecular weight 24 and 26.5 kD, which co-migrated with native and glycosylated forms of ovine PRL. The cross-reactivity of neutralizing an- tibodies to human GH and PRL with squirrel monkey GH and PRL were examined using the Nb2 lymphoma bioassay. One of three monoclonal antibodies to human GH (2A1) neutralized squirrel monkey GH with an apparent affinity for squirrel monkey GH = 70 ng IgG/ml) which was fourfold lower than for human GH (IC5,, = 15 ng IgG/ml). Both poly- clonal [AR38-5(1)1 and monoclonal (9C3) antibodies to human PRL inhib- ited the activity of squirrel monkey PRL, although their affinities for squirrel monkey PRL were four- and twentyfold lower than for human PRL. The activities of antibodies 2A1 and 9C3 on GH and PRL in squirrel monkey serum were also examined by the Nb2 bioassay. The anti-gluco- corticoid RU486 was used in all incubations with squirrel monkey serum to eliminate the effect of high glucocorticoid levels on Nb2 cell growth. The mitogenic activity of squirrel monkey serum in the Nb2 assay was com- pletely eliminated in the presence of 2A1 and 9C3. This study represents the first description of the biochemistry of GH and PRL in the squirrel monkey.

Key words: monoclonal antibody, Nb2 bioassay, RU486

Received for publication November 16, 1990; revision accepted February 18, 1991.

Address reprint requests to Dr. Jonathan G. Scammell, Department of Pharmacology, University of South Alabama, College of Medicine, MSB 3130, Mobile, AL 36688.

0 1992 Wiley-Liss, Inc.

36 / Scammell et al.

INTRODUCTION The biochemistry of the anterior pituitary peptides growth hormone (GH) and

prolactin (PRL) in many non-human primates is not completely understood. Yet GH and PRL play important roles in regulating complex physiological functions for which non-human primates are useful research models. Our long-term inter- ests lie in the characterization of GH and PRL forms in primates and the identi- fication of epitopes in GH and PRL which are conserved among species. These studies may lead to the development of immunoassays for GH and PRL for many different primates using antibodies to conserved epitopes and also may provide insight into structural components of the GH and PRL molecules which are bio- logically important.

Growth hormone and PRL have been shown to exist in multiple forms in many species. For example, while GH exists predominantly as a single 22,000 dalton form, a 20 kD variant has also been described in human and murine pituitary glands [Lewis et al., 1978; Sinha & Gilligan, 19841. Different isoforms of GH have also been isolated from human pituitary glands by isoelectric focusing [Hummel et al., 19751, and a 25 kD band was identified in human prolactinoma by Western blot analysis [Sinha et al., 19871. In addition to native PRL, polymorphic forms of PRL with different charges [Hummel et al., 1975; Oetting & Walker, 19851 or molecular sizes [Sinha et al., 1987; Sinha & Jacobsen, 19871 have been observed in the pituitary glands of many species. Glycosylated forms of PRL, which migrate in sodium dodecyl sulfate (SDS) gels more slowly than the principal form of PRL (molecular weight 23,000), have now been well characterized in ovine and human pituitary glands and serum [Lewis et al., 1984, 1985; Markoff & Lee, 19871.

Although a number of studies have described GH and PRL isoforms in hu- mans, GH and PRL in non-human primates have been less well characterized. GH and PRL from cynomolgus monkey pituitary gland have been shown to possess different charges [Hummel et al., 19751. Li and co-workers [1986] have purified and sequenced GH from rhesus monkey pituitary gland and have found it to differ from human GH by only four amino acids. Bethea and Papkoff [1986] have found that rhesus monkey PRL purified from pituitary cell culture medium migrated as a single band of approximate molecular weight 23,000 on SDS gels. In the present study, we have investigated the GH and PRL forms in the pituitary glands of the squirrel monkey (Saimiri boliviensis boliviensis). The squirrel monkey is a South American primate, which has been an important animal model in biomedical re- search [Abee, 19891. Further, we have examined the relative reactivities of anti- bodies generated to GH and PRL from different species with squirrel monkey GH and PRL from pituitary tissue.

METHODS Materials

Culture medium was obtained from Grand Island Biological Co. (Grand Island, NY), and horse and fetal bovine sera were purchased from Hyclone Laboratories (Logan, UT). Human serum pooled from normal, healthy subjects was kindly pro- vided by Dr. G. Longenecker (University of South Alabama). Baboon serum from a single animal was provided by Dr. P.S. Dannies (Yale University School of Medicine). Bovine serum albumin (BSA) was from Arne1 Products (New York, NY) and electrophoresis reagents were purchased from Bio-Rad (Richmond, CA). Re- combinant-derived human GH was obtained from Genentech, Inc. (South San Francisco, CAI, while 20 kD variant of human GH was provided by Dr. A.F. Parlow

PRL and GH in Squirrel Monkey / 37

and the NIDDK. Sheep pituitary glands were purchased from Pel-Freez Biologicals (Rogers, AR). RU486 was provided by Roussel-Uclaf (Romainville, France).

Subjects and Social Environments Subjects were adult squirrel monkeys (Saimiri boliviensis boliviensis) that

were housed indoors (22°C) in laboratory breeding groups of seven to nine females with one to two males. Animals were exposed to a 12h:12h 1ight:dark cycle except during the summer and fall when they had access to outdoors and were exposed to the natural 1ight:dark interval. Squirrel monkey pituitary glands were rapidly removed at necropsy and frozen in liquid N2 in the Squirrel Monkey Tissue Re- source of the University of South Alabama Primate Research Laboratory. Blood samples were obtained by femoral venipuncture within 3 minutes of capture of the animals. Serum was separated by centrifugation at 22°C for 20 minutes. Samples from at least six monkeys were pooled as a serum lot, aliquoted, and stored at -20°C until assayed. Serum samples were heated to 56°C for 20 minutes prior to assay to eliminate high cytolytic complement activity [Thompson et al., 19891.

Antibodies Monoclonal antibody 6 F l l was generated to a sequential inoculum of ovine

and human PRL [Scott et al., 19881. Rabbit and guinea pig anti-human GH anti- sera were obtained from Chemicon International, Inc. (El Segundo, CA) and Arne1 Products, respectively. The neutralizing activities of anti-human GH (3D5, previ- ously referred to as 5D5 [Binder et al., 19891) and anti-human PRL (9C3) mono- clonal antibodies have been described previously [Binder et al., 1989; McNeilly & Friesen, 19851. The activities of two additional anti-human GH monoclonal anti- bodies (2A1 and 3B1, referred to as 3A1 and 3B3, respectively [Binder et al., 1989; Gertler et al., 19861) and AR38-5(1), a polyclonal antibody generated to human PRL, were also examined. The concentrations of mouse IgG in each monoclonal antibody preparation were determined in the ascites fluid by single radial immu- nodiffusion using a Mouse IgG Quantitative Immunodiffusion Kit (ICN Immuno- biologicals, Lisle, IL). Peroxidase-labeled goat anti-mouse, anti-rabbit, and anti- guinea pig IgGs were purchased from Hyclone Laboratories.

Immunoblotting Pituitary glands from adult squirrel monkeys of both sexes were thawed and

individually homogenized [25 mg/ml (w/v)l in 0.3 M sucrose, 0.05 M Tris (pH 7.4) at 4°C. Aliquots were solubilized in an equal volume of 2 x concentrated solubili- zation buffer, containing 2% SDS, 0.004% bromophenol blue, 20% glycerol, 0.124 M Tris (pH 6.7) with 4% 2-mercaptoethanol, followed by heating at 100°C for 3 min- utes. Sheep pituitary glands were processed in the same manner. Following elec- trophoresis of solubilized pituitary homogenates or purified growth hormone prep- arations on 12% gels, proteins were transferred to nitrocellulose filter paper (Bio- Rad) as described by Burnette [1981]. Filter papers were incubated with 1:1,000 dilutions of either guinea-pig or rabbit anti-human GH antiserum, or 6Fl1, as described previously [Scammell et al., 1990aJ. Peroxidase-labeled second antibod- ies (each at a dilution of 1:1,000) and the Bio-Rad HRP Color Development Reagent were used to detect the binding of the primary antibodies. The peroxidase reaction products were quantitated using an LKB Ultroscan XL Laser Densitometer.

Cell Culture

Fischer’s medium supplemented with 10% horse serum, 5% fetal calf serum, Nb2 rat node lymphoma cells were maintained as suspension cultures in

38 / Scammell et al.

B C

66K

36K

24K 20K

14K

MW Fig. 1. A representative Western blot of growth hormone forms in squirrel monkey pituitary gland. Twenty micrograms of pituitary homogenate from a six-year-old male squirrel monkey (A), 100 ng of purified human GH (B), or 100 ng of human 20K variant GH (Cf were subjected to SDS-polyacrylamide gel electrophoresis, trans- ferred to nitrocellulose, and immunolabeled with anti-human GH antiserum (Chemicon). Reactivity was re- vealed with peroxidase-labeled goat anti-rabbit IgG and the Bio-Rad HRP Color Development Reagent.

M 2-mercaptoethanol, 50 U/ml penicillin and 50 kg/ml streptomycin [Tanaka et al., 19801. The effects of hormones, serum, and antibodies on Nb2 cell growth were determined in triplicate in Costar 22-mm 12-well tissue culture dishes (Costar, Cambridge, MA) at a starting density of lo5 cells/well in 1 ml of the above medium but without fetal calf serum. The cells were incubated for 72 hours, after which the cell number was determined using a Coulter Counter.

The data presented here are from single experiments. All experiments were performed at least twice with similar results.

RESULTS Variant Forms of Growth Hormone and Prolactin in the Squirrel Monkey Pituitary Gland

When squirrel monkey pituitary extracts were electrophoresed on SDS gels, transferred to nitrocellulose, and immunolabeled with anti-human GH antiserum, two predominant bands were recognized (Figure 1A). The major band (apparent molecular weight 22,000) migrated to the position of human GH (Figure lB), while another band (molecular weight 20,000) was observed that migrated with purified 20 kD variant of human GH (Figure 1C). Human GH antiserum cross-reacted with two additional proteins (approximate size 26 and 33 kD) in the squirrel monkey

PRL and GH in Squirrel Monkey I 39

A 8

6 6 K

3 6 K

24K 20K

MW Fig. 2. A representative Western blot of prolactin forms in squirrel monkey and sheep pituitary glands. Twenty micrograms of pituitary homogenate from an eight-year-old female monkey (A) and four micrograms of sheep pituitary homogenate (B) were subjected to SDS-polyacrylamide gel electrophoresis, transferred to nitrocellu- lose, and immunolabeled with anti-PRL antibody 6F11. Reactivity was revealed with peroxidase-labeled goat anti-mouse IgG and the Bio-Rad HRP Color Development Reagent.

pituitary gland, although these bands were minor components of the total GH immunoreactivity (Figure 1A). Similar results were obtained with the two com- mercially available anti-human GH antisera used in this study.

When pituitary extracts were immunolabeled with a monoclonal antibody which recognizes PRL from several species including squirrel monkey PRL [Scam- me11 et al., 1990b1, two bands were observed (Figure 2A). The major band (appar- ent molecular weight 24,000) migrated to the same position as the predominant PRL species in the sheep pituitary gland (Figure 2B). Another protein (apparent molecular weight 26,500) in squirrel monkey pituitary was also recognized by monoclonal antibody 6Fll. This protein migrated to the same position as an im- munoreactive protein in sheep pituitary, which has been demonstrated to be a glycosylated form of PRL [Lewis et al., 19841. Densitometric analysis of the blots of immunoreactive GH and PRL in squirrel monkey pituitary demonstrated that approximately 80% of the total immunoreactivity of each peptide was found in the native form of the peptides. We found no sex-related differences in the proportion of GH to 20 kD GH, or PRL to 26.5 kD PRL, when pituitary homogenates from a total of 3 female and 3 male squirrel monkeys were individually immunolabeled with anti-GH or anti-PRL antibodies.

40 / Scammell et al.

A

5

4

2A1 3D5

0 381

x -1

$ 2

3j -I -1 W 0

1 0 0.1 1 10 100 1000

B

0 0.1 1 10 100 1000

IgG CONCENTRATION (nglml )

Fig. 3. The effect of anti-human GH monoclonal antibodies on the growth of Nb2 cells induced by 250 pgiml hGH (A) or squirrel monkey GH (B). Squirrel monkey GH was derived from a pituitary homogenate (12 ngiml) incubated in the presence of antibody 9C3 (1/1,000), which inhibits the bioaetivity of squirrel monkey PRL (see Figure 4). Control dishes contained 1.1 x lo5 cellsidishes after 3 days. Each point represents the mean of triplicate dishes. Variability among triplicates was less than 5%.

Cross-Reactivity of Antibodies to Human Growth Hormone and Prolactin with Squirrel Monkey Growth Hormone and Prolactin

The cross-reactivity of monoclonal antibodies generated to human GH and PRL with squirrel monkey GH and PRL was also examined. Antibodies 2A1,3D5, and 3B1 are monoclonal antibodies which neutralize human GH, while AR38-5(1) and 9C3 are polyclonal and monoclonal antibodies, respectively, which neutralize hu- man PRL. Various concentrations of antibodies were incubated with squirrel mon- key pituitary homogenates in tissue culture with Nb2 lymphoma cells. The Nb2 cell line has been utilized as a specific and sensitive bioassay for lactogenic hor- mones, including human PRL and GH [Tanaka et al., 19801.

The activity of human GH on Nb2 mitogenesis was inhibited by all three antibodies to GH, although antibody 2A1 was less potent than either 3D5 or 3Bl (Figure 3A). The activity of GH in squirrel monkey pituitary homogenates was inhibited only by the 2A1 antibody (Figure 3B). The affinity of 2A1 for squirrel monkey GH was somewhat lower than that for human GH (apparent IC,o of 70 versus 15 ng IgG/ml, respectively). The bioactivities of both human PRL (Figure

PRL and GH in Squirrel Monkey / 41

A

1 k l I

3125 625 125 25 5

B

i l I

ANTIBODY DILUTION ( 1 I1 000) 3125 625 125 25 5

Fig. 4. The effect of anti-human PRL antibodies on the growth of Nb2 cells induced by 80 pglml human PRL (A) or squirrel monkey PRL (B). Squirrel monkey PRL was derived from a pituitary homogenate (12 ngiml) incubated in the presence of antibody 2A1 (2 p.g IgGiml), which inhibits the bioactivity of squirrel monkey GH (see Figure 3). Control dishes contained 1.1 x lo5 cells/dish after 3 days. Each point represents the mean of the triplicate dishes. Variability among triplicates was less than 5%.

4A) and squirrel monkey PRL (Figure 4B) were inhibited by anti-human PRL antibodies, AR38-5(1) and 9C3, although these antibodies had a greater affinity for human PRL than squirrel monkey PRL. Antibody AR38-5(1) was approxi- mately fourfold more potent, while the monoclonal antibody 9C3 was approxi- mately twentyfold more potent, in inhibiting human than squirrel monkey PRL.

The Effect of Anti-hGH and Anti-hPRL Antibodies on the Lactogenic Activity of Squirrel Monkey Serum

The activity of antibodies 2A1 and 9C3 on the lactogenic activity of squirrel monkey serum was next examined. First, as high concentrations of glucocorticoids have been shown to inhibit Nb2 cell growth [Thompson et al., 19891, we investi- gated whether the unusually high concentrations of cortisol reported in squirrel monkey serum [Brown et al., 19701 might interfere with the Nb2 assay. Nb2 cells were incubated with pooled lots of squirrel monkey serum with or without the anti-glucocorticoid RU486 [Baulieu, 19891. We found that RU486 potentiated in a dose-dependent manner Nb2 cell growth induced by squirrel monkey serum (Fig- ure 5). The effect of RU486 was most likely due to antagonism of the inhibitory action of glucocorticoids rather than progesterone, as we found that progesterone

42 / Scammell et al.

, 1

0 0.005 0.05 0.5 5 50

RU486 (UM)

Fig. 5. Potentiation of squirrel monkey serum-induced Nb2 cell growth by RU486. Two different lots of pooled squirrel monkey serum were heat-inactivated and incubated with Nb2 cells in the presence of RU486 for 3 days. RU486 significantly potentiated the growth of both lots of serum above control levels (0 F,,,,,, = 39.24, P < 0.001; 0 F,,,,,, = 34.46, P < 0.001). Individual comparisons were made by Duncan’s multiple range test: * = significantly different from control levels ( P cc 0.05). Control dishes contained 1.21 x lo5 cellsidish after 3 days. Each point represents the mean of triplicate dishes. Variability among triplicates was less than 5%.

M) had no effect on basal or PRL-stimulated Nb2 cell growth (data not shown). Concentrations of RU486 above 5 p M resulted in Nb2 cell growth below maximal levels (Figure 5). The vehicle, ethanol (highest final concentration 0.5%), was without effect on either basal or PRL-stimulated Nb2 cell growth (data not shown). RU486 was used at a concentration of 1 pM in all further experiments involving squirrel monkey serum.

The incubation of anti-hGH antibody 2A1 and anti-hPRL antibody 9C3 with squirrel monkey serum completely inhibited lactogenic activity (Table I), indicat- ing that, as observed for human serum [Tanaka et al., 19801, the mitogenic activity of squirrel monkey serum in the Nb2 assay was due to either GH or PRL. However, the relative activities of GH (2Al-inhibitable) and PRL (9C3-inhibitable) in squir- rel monkey serum differed from those found in human or baboon serum. While greater than 75% of the lactogenic activity of human or baboon serum could be attributed to PRL (derived from standard curves of Nb2 cell growth induced by purified human PRL or human GH), Nb2 cell growth was unaffected by the anti- human PRL antibody 9C3. Similar results were obtained with two other pooled lots of squirrel monkey serum and when AR38-5(1) was used instead of 9C3 (data not shown). These data suggest that the major circulating lactogenic hormone in se- rum obtained from unanesthetized squirrel monkeys under manual restraint is GH.

DISCUSSION The present study is the first characterization of GH and PRL in the squirrel

monkey, the most commonly used South American primate in biomedical research.

PRL and GH in Squirrel Monkey / 43

TABLE I. Effect of Neutralizing Antibodies to hGH (2Al), hPRL (9C3), or Both hGH and hPRL (2A1 + 9C3) on the Lactogenic Activity of Squirrel Monkey, Human, or Baboon Serum

Serum

No addition Control 2A1 9c3 2A1 + 9C3

Squirrel Monkey 1.10" 3.19 1.93b 3.19 1.05b Serum 5 0.06 f 0.05 f 0.07 f. 0.05 f. 0.05 Human 1.33 4.18 3.93 1.61b 1 .40b Serum f 0.03 f 0.23 f. 0.25 f 0.06 f 0.06 Baboon 1.30 4.12 3.5gb 2.48b 1.38' Serum k 0.06 f. 0.07 f 0.06 ? 0.07 f. 0.05

~~ ~~ ~

"Mean ? SEM cell number ( x lo@) of triplicate dishes of Nb2 cells. Nb2 cells were plated at 1 x lo5 cellddish and were incubated for three days. Pooled squirrel monkey serum was added in a volume of 100 p1, while 20 p1 of pooled human serum or 20 p1 of serum from a single baboon were used. RU486 (1 rM) was added to all wells containing squirrel monkey serum. Antibody 2A1 was used a t a concentration of 2 bg IgG/ml, while antibody 9C3 was used at a concentration of 1/2000. The data were analyzed by ANOVA (squirrel monkey serum F,,,,,,, = 674, P < 0.001; human serum F,,,,,, = 109, P < 0.001; baboon serum F,,,,,,, = 599, P < 0.001). Individual comparisons were made by Duncan's multiple range test. bSignificantly different from the control serum level for each species (P < 0.05).

We have demonstrated the existence of different forms of GH and PRL in the squirrel monkey pituitary gland and have defined common immunological fea- tures of squirrel monkey and human GH and PRL by screening monoclonal anti- bodies to human GH or PRL against the squirrel monkey peptides.

We found that the predominant form of GH in the squirrel monkey pituitary gland migrated to the same position on SDS gels as purified human 22 kD GH. We also demonstrated that the squirrel monkey pituitary resembles the human gland in that it contains a variant form of GH in addition to native 22 kD GH. Variant GH from squirrel monkey pituitary gland migrated to the same position as purified human 20 kD variant GH on SDS gels and was recognized by anti-human GH antiserum, suggesting that the variant GH from squirrel monkey and human share some physicochemical properties. The human 20 kD variant GH is identical to the major form of human GH except for the deletion of a &amino acid segment [Lewis et al., 19801.

The primary structures of squirrel monkey 20 or 22 kD GHs and their extent of homology with human GH forms are unknown. Although rhesus monkey GH differs from human GH in only four amino acids [Li et al., 19861, complement fixation studies have indicated that squirrel monkey and human GH are more distantly related [Tashjian et al., 19661. However, pituitary extracts from squirrel monkey showed parallelism with human GH standard in radioimmunoassays em- ploying polyclonal antiserum to human GH [Hummel et al., 19741, suggesting common epitopes. Further, an epitope recognized by a monoclonal antibody to human GH (2A1) is conserved in squirrel monkey, while two other epitopes in human GH recognized by antibodies 3D5 and 3B1 were not found in squirrel monkey GH. Antibody 2A1 likely recognizes an epitope that includes a portion of the amino-terminus of GH, as deletion of the N-terminal 13 amino acids prefer- entially affects the binding of 2A1 to the mutant GH [Gertler et al., 19861. This portion of the GH molecule has been postulated to be part of the binding domain of GH which interacts with the PRL receptor on Nb2 cells [Binder et al., 19891. As this antibody cross-reacts with a functionally important region of both squirrel monkey and human GH, the epitope recognized by 2A1 may prove to be conserved among other primate species.

44 / Scammell et al.

We found that PRL in the squirrel monkey pituitary gland also existed in two forms, which co-migrated on SDS gels with native and glycosylated ovine PRL, apparent molecular weights 24,000 and 26,500. Variant forms of PRL have been described extensively in many species, although there have been few reports of different PRL forms in non-human primates. Bethea and Papkoff [1986] isolated only a single form of PRL (molecular weight 23,000) from rhesus monkey pituitary cell cultures, although a preliminary report suggested that rhesus monkey serum contains a larger molecular weight PRL variant in addition to the native peptide [Thompson et al., 19881. Prolactin forms in the rhesus monkey pituitary gland have not yet been reported. We also found that both polyclonal and monoclonal antibodies generated to human PRL cross-reacted and neutralized squirrel mon- key PRL, although with lower affinity than for human PRL. This suggests that the epitopes recognized by these antibodies are biologically important and are at least partially conserved in human and squirrel monkey PRL. Thus, these antibodies may prove to be useful in the characterization of PRL forms and the development of immunoassays for PRL in other primate species.

The functional significance of different forms of GH and PRL is not completely understood. The 20 kD variant form of human GH has generally lower GH-like bioactivity when compared to human GH [Lewis, 19841. It is secreted into the circulation [Baumann et al., 19831, although the factors regulating the synthesis and secretion of 20 kD human GH are unknown. On the other hand, an emerging body of evidence suggests that the relative secretion of native and glycosylated PRLs may be regulated. The ratio of glycosy1ated:non-glycosylated PRL varies markedly in the serum during development in the pig [Sinha et al., 19881 and during pregnancy in humans [Markoff et al., 1988al. As glycosylated ovine PRL possesses only 20-80% of the bioactivity of ovine PRL [Markoff et al., 1988b], the level of circulating glycosylated PRL may be important in controlling the meta- bolic activity of PRL. The appearance of variant forms of GH or PRL in the serum may result from differential secretion of one form over another, as suggested by the study of Pellegrini and co-workers [Pellegrini et al., 19901. We did not investigate the forms of GH or PRL in the serum of the squirrel monkey in this study.

We were surprised to find that the predominant lactogen in the serum of the squirrel monkey was GH. This finding is in contrast to human serum or baboon serum, in which PRL-like activity predominates. However, the method of blood sampling may have contributed to the relatively high GH levels in the squirrel monkey. Blood samples were obtained from unanesthetized subjects in all groups in this study, and Brown et al. [1971] have shown that capture is consistently stressful to squirrel monkeys, resulting in a profound increase in serum GH levels. Nevertheless, we have demonstrated that the lactogenic activity of squirrel mon- key serum is attributable to only GH and PRL. Furthermore, we have demon- strated that if the Nb2 assay is to be utilized for the determination of GH and PRL levels in the serum of this species as we have proposed [Thompson et al., 19891, it is necessary to coincubate the cells with the anti-glucocorticoid RU486 to block the cytotoxic activity of glucocorticoids on Nb2 cells.

CONCLUSIONS 1. Growth hormone in squirrel monkey pituitary gland existed in two forms

which migrated to the same positions on SDS gels as native (molecular weight 22,000) and variant (molecular weight 20,000) forms of human growth hormones.

2. Prolactin in squirrel monkey pituitary gland existed in two forms which migrated to the same positions on SDS gels as native (molecular weight 24,000) and glycosylated (molecular weight 26,500) forms of ovine prolactin.

PRL and GH in Squirrel Monkey / 45

3. Antibodies were identified which cross-reacted a n d neutralized squirrel

4. The anti-glucocorticoid, RU486, potentiated the stimulatory effect of squir-

5. Growth hormone and prolactin can be individually determined in squirrel

monkey growth hormone a n d prolactin.

rel monkey serum in the Nb2 assay.

monkey pituitary gland a n d serum using the Nb2 bioassay.

ACKNOWLEDGMENTS The authors wish to thank Ms. J u d i Naylor for secretarial assistance and the

staff of the University of South Alabama Primate Research Laboratory for their technical help. This work was supported by NIH grants DK-40593 (J.G.S.), RR- 01254 (C.R.A.), and the MRC of Canada (H.G.F.). J.G.S. is a recipient of a Faculty Development Award from the Pharmaceutical Manufacturers’ Association Foun- dation, Inc. R.V.H. was supported by a Research Experiences for Undergraduates Award from the National Science Foundation (DIR-8900788). M.E.T. is a recipient of a Graduate Minority Fellowship from the National Science Foundation.

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TRY AND CYTOCHEMISTRY 38:117- INGS OF THE 17TH ANNUAL MEETING