BIOLOGY

OF

REPRODUCTION

44,

827-833

Zebra Chorionic

Gonadotropin:

R McFARIANE,3

JAMES

Hormone

(1991)

Research

NANCY

Institute,3

Zoological

Partial

M. CZEKALA,4

University

Society

Purification and

of Ca4fornia,

of San

Diego,4

HAROLD

San

San

and Characterization1 PAPKOFF23

Franasco,

Diego,

Cahfornia

Calfornia

94143

92112

ABSTRACT Six

of pregnant

samples

to have

than

rification

second

A yield

of

7%.

Its molecular

The

zCG

was

Lii. Comparisons because the and

zLH;

and

size In

were preparation FSH

(d)

for

UI

made

with

and

activity,

for

UI

is more

but

like

second

of

mare

(0.9-5.3

p.g/ml);

ml)

was

fractionated

to achieve

HPLC

Is smaller

chorionic

that

showed

not

analysis

than

FSH

receptors

dLH,

and

is bioactive

zLH-all

evidence

otropin

for the

in species

to primates

so

presence

studied

far

key. This

to the horse laboratory has had

chemical

and

of both the

the eLH

key, dCG

biological

appears

properties

gonadotropins. resulted in the

studies

preparation and chorionic gonadotropins

pituitary

and

The presence of associated rionic gonadotropin (hCG)

an extremely

low

There

three

dae,

are

each

and

sesses

in this laboracharacterization

species

have

different

terns,

and

is known

species

social vary

of the

to be

of zebra

as distinct

(mountain,

organization

in their reproductive

(z)

adaptability biology

in the as the

family horse

ceptor

assays

gether

with

highly

Equi-

26, 1990. 3, 1990. ‘These studies were supported 2Correspondence: Dr. Harold Box 0534, University of California, December

October

in part Papkoff,

by NIH, Hormone

San Francisco,

Francisco,

species,

Institute, CA 94143.

LH. for

assay

preliminary to eCG, ciii, dCG,

results,

have

eLH-which

high

levels

activity.

FSH

LII

and

FSH

activity

(RAAs), HPLC

purified

and

characterized in an earand indicated a slight with zLH. Thus it was

as does

eCG.

literature (zCG) in it posThe

avail-

a rat

as definitive studies.

filtration

testis

Leydig

cell

assay

to-

used to characterize the zCG preparation and to compare it to eCG insufficient serum was available to prepare material, the results obtained cannot be re-

further

gel

but they

MATERIALS Serum

apart

was

do provide

data

as a basis

for

HSW

and

AND

METHODS

Hormones

Serum samples (2 ml) from five pregnant Hartmann’s were obtained from the San Diego Zoo. The day of conception and thus the day of pregnancy of the samples was calculated from the day of parturition with the gestation period taken as 362 days [9]. The day of pregnancy of zebras

HD-05722. Research

San

The

similar

and

If any

garded

the

Received

eCG

cell

Leydig

or don-

and Grevy’s) systems and mating patto arid lands [8]. Little

from some observational data [9] and a study based on autopsies of pregnant females [10]. Both of these studies were on Hartmann’s zebras (Equus [Hppot:gr&s] zebra barimannae), a subspecies of the mountain zebra. Partially purified

Accepted

UI.

minimal

both

plains,

of these

zebra

immunologially

from

partially purified and dCG.Since

activity in human choreported [7], but is of

of zebra

and

about

ability of a limited number of pregnant zebra serum samples has allowed us to address this question. A combination of classical, affinity, and ion exchange chromatography procedures were used to concentrate the zCG. RIM radiore-

level.

considered

key. These

FSH-like has been

rat testis

to be as equine

same

of interest to us to confirm an early report in the [12] on the presence of a chorionic gonadotropin pregnant zebra serum and to determine whether

(CG) from horse (e) [1-3], and the donkey (d) [4, 5]. Unique to horse is the highly potent FSH activity exhibited by both and eCG in heterologous bioassays [3,6]. In the donhowever, the degree of FSH-like activity in dLI-I and is considerably less in comparison to the horse [4,5]. pituitary

have

the

equine

the

pu-

gonadotropln

of zCG

and

FSH,

as an UI;

content about

were the

for

chorlonic

LH, and

shown

cases

employed

zebra

the

by RIA and in most

zebra pituitary gonadotropins were lier study [11] from this laboratory intrinsic FSH activity to be associated

gonad-

to be restricted

of Equidae

Previous

and

therefore,

of which

and its close relative the donan ongoing interest in the bio-

and

chorionic tory have

of a chorionic

previously

indicated

and

It differs,

INTRODUCTION Clear

UI

levels

of the

ovine

than

for

(RItA)

analyzed

methods

material

gonadotropin,

zCG

first

a concentration

greater

were trimester

pregnancy

by

of the

eCG,

assays

donkey

dCG,

trimesters

the

(10

radloreceptor

equine and of high purity,

in RRA5

sera

obtained.

values eCG,

and of

gonadotropin

was

and

first

to that

of the

by ‘/,/VO

RIAs

the

chorlonic

of glycoprotein

is not

competes

A pool

donkey

as judged

from

comparable

levels.

1.0 mg

tested

and

of intrinsic

(e)

serum

levels

trimester

of equine

(zCG).

(z)

gonadotropln

chorionic

higher

zebra

1092,

FAX: 415-

731-3612.

827

samples

varied

from

55 to 168

days

(Table

1). Samples

obtained at two times (55 and 143 days) were available from one of the zebras

during pregnancy (HMZ-04). A small

sample maining

donkey serum used together

of lyophilized pooled pregnant from a previous study [4] was

lyophilized

pregnant

bian

for comparison

horses

serum

from

with

Thoroughbred

the zebra

and

serum.

rewith

Ma-

828

McFARI.ANE

TABLE donkey

1. The concentration of CG in the and zebra in eCG equivalents. g/ml

Sample

4.2 5.2 0.9 2.6 5.3 0.9 1.5 5.3 2.2

Arabian

Donkey HMZ-01 HMZ-04 HMZ-04

HMZ-09 HMZ-15 HMZ-19 *The

day

of pregnancy

tation length of 345 for the zebra.

date

horse

of parturition and

were

donkey

and

to be

iodinated

first first second first second second first first

performed

and

ges-

362

days

action reaction

used

with

phenylglycoiril;

first

61 121 on

Jodinations

horse,

Trimester

168

for the

AL.

Hormones

55-65 55-60 60-71 145 55 143

is based days

of the

Day of pregnancy*

eCG

Thoroughbred

plasma

ET

Pierce,

was added

IL). The

and

applied

same buffer, Arlington

in 50 glass 100 to

followed by 500 His., IL). The re-

for 10 mm and then the to 1 ml of PBS containing

to a small

desalting

BlO-RAD), and 1-ml fractions volume peak was collected and

Pac 1ODG,

void

were

dissolved X 75-mm

then rinsed with 0.05 M PBS and together with 5 pg of the protein

vessel was gently vortexed mixture was transferred BSA,

assays

iodinations

500 ng of lodo-Gen evaporated in a 12

be iodinated in 50 p.1 of the pCi (5 p.1) of 1251 (Amersham,

0.1%

in various

(1,3,4,6-tetrachloro-3,6-di-

Rockford,

as follows; p.1 of chloroform was

tube. The tube pJ of PBS was

as radioligands

lodo-Gen

column

were used

(Econo-

collected. The in subsequent

assays. The hormones radioligands were previously described

used in this study as standards or assay purified in this laboratory and have been as follows: eLH and eFSH [3], eCG [13],

ovine

oFSH

(o)

LII [14],

[15],

and

partially

A partially purified eCG preparation as a standard in all of the assays

except

cell

was

bioassay

where

purified

eCG

purified

(1700

zLH [11].

nJ/mg

was

for the

used

rat Leydig

The zebra pregnancy serum samples were pooled (about 10 ml) and applied to a 70-ml column of Affi-Gel Blue, 50100-mesh (BlO-RAD, Richmond, CA), that had been equilibrated with 0.05 M Tris/HCI (pH 8.0), 0.5 M NaCI, and 0.01% NaN3. The zCG fraction was eluted with 0.5 M NaSCN. This fraction was dialyzed and then lyophilized before dissolving in 0.9% NaC1 and (NH4)2S04 added to 4% saturation. The pH was lowered to 4.0 with HPO3, and the resulting precipitate was removed. The supernatant was on

taway,

lized.

NJ)

a column

of Sephadex

equilibrated

This fraction

with

then

was

G25

(Pharmacia,

0.05 M NH4HCO3 dissolved in starting

applied to a 20-mi column of concanavalin arose (BlO-RAD) in 20 mM Tris/HCI (pH

and

lyophilization

dissolved

in starting

Sephadex

C50

M ammonium

eluted material

Lyophilized

assays

(designated

with

the same

After fraction was then to a sulfopropyl

equilibrated

with

0.01

at pH 4.0. After the unbound proteins the column was washed with 0.02 M amand the bound zCG fraction was finally

with 0.2 M NH4HCO3 (designated zCG).

tographed on sorbed fraction

column

and lyophibuffer and

acetate

been eluted, monium acetate, had

buffer

(Pharmacia)

as above, this and applied

Pisca-

A (Con A) Seph7.4), 0.15 M NaCl,

1 mM CaC12. The adsorbed fraction was eluted buffer containing 0.2 M methyl-D-mannopyranoside. desalting

pregnant

(pH donkey

9.0), serum

Affi-Gel Blue as described was used as a comparative dCG).

and

HPLC

yielded was

also above. standard

1 mg

of

gel

filtration

characteristics eCG.

with with umn.

used.

Purification

desalted

HPLC was

used

compare

the

elution

with that of purified A Perkin-Elmer Corp. (Norwalk, CT) series 410 HPLC UV detector (LC-95) and integrator (LCI-100) was used a 300 X 7.5-mm TSK-250 (BlO-RAD) gel filtration colThe solvent was 0.1 M Na2SO4 0.1 M NaH2PO4 (pH at a flow rate of 1 mi/mm. UV absorption was mea-

6.8) sured at 225 nm, and samples onto the column. The column mixture of 5 molecular weight The

to

of the zCG preparation

MWs

and

Ve/Vo

values

of 10-20 pg was calibrated (MW) standards for

the

were injected by use of a (BlO-RAD).

standards

were

as fol-

lows: thyroglobulin (670 000, 1.01); bovine gamma globulin (158 000, 1.50), ovalbumin (44 000, 1.72), horse myoglobin (17 000, 2.06), and vitamin B12 (1 350, 2.45). Fractions (0.5 ml) were collected during the HPLC gel filtrations of the purified eCG and the zCG preparations. The fractions were then assayed for immunoactivity by using the LII RIA (described related with the absorbance

RIM

and The

below), and the chromatograms.

results

were

cor-

RRAs RIA employed

for measuring

LH utilized

a mono-

clonal antibody against bovine LH (bLH) and 1251-eLH as radioligand and has been described in detail [16]. The antibody cross-reacts with every mammalian species of LII thus far

tested

(>12).

An RRA for LH has

been

that uses rat testis membranes as the receptor 1251-eLH as radioligand. An RRA for FSH was

viously described membrane fractions

described

[13]

source and used as pre-

[5, 13]. This as receptor

An RIA for eCG

assay employed calf testis source and 1251-oFSH as rahas been described [2] that uses

chroma-

dioligand.

The adin the

‘251-eCG as radioligand. The RIAs and the RRA in duplicate occasions.

samples were assayed and on at least two

by the separate

ZEBRA

CHORIONIC

GONADOTROPIN

829

Bioassay

zCG: LH bioactivity

was

assay as described was carried out

in

Cambridge,

Approximately

MA).

bated in 500 Solution and

monitored

[17] with 48-well

p.1 of media 0.1% BSA)

State

assayed

100000

production obtained

Univ.,

Fort

cell

modifications. The assay culture plates (Costar, cells

were

incu-

Balanced Salt the doses of

The plates were incubated of CO2 and 02 mixture

testosterone an antibody

(Colorado

minor tissue

in a rat Leydig

(M-199 with Earle’s to which was added

standard or test material. h at 37#{176}C in the presence after which RIA utilizing

in vitro

for 2 (5:95)

was determined by an from Dr. G.D. Niswender

Collins,

CO).

The

samples

‘4-a I.-’

CsJ

cs.J I-i-,

cI)

iO

0

iO

.0

L ‘

0 tI

.0

were

in triplicate. %s_______

_____,.I

Statistical

Analysis

Relative dard

potencies

hormones

linear

of the

were

regression

formed software.

SAS

preparation from

Linear

regression

lines.

using

zCG

calculated

Institute

(SAS

and

the

Inc.,

the

stan-

ED50

of log-logit

analysis

was

Cary,

NC)

I

I

5

10

16

20

per-

statistical

eCG: E

:

RESULTS Preliminary

experiments

the

monoclonal

tect

the

showed

antibody

CG

in the

against

pregnant

from

two

first

samples

and

The estimates

previously

[4], are

interest the

dency for

levels

-15, -19)

of CG in the

considerably

trimester

lower

than

of pregnancy,

for the mare

purification

the

been new

first

noted

The

of CG (eCG

having

most

employed

used

Application

glycoprotein trated for

of the

zebra protein)

in testing

mare.

.0

Of

5

those

the

ten-

the

zCG

methods

that

had

purification

previously

dCG [4,6]. were adsorption

and

The

results eCG

of HPLC are

shown

Time

‘15

20

in Minutes

procedures

the zCG was various assays

to a pool

sufficiently employed.

gel

filtration

in Figure

for

1. The

the

concen-

zCG

and

chromatogram

FIG. 1. Gel filtration HPLC chromatograms of the zCG preparation and a purified eGG, on a TSK-250 column (330 x 7.5 mm), and the immunoactivity (LH RIA) of the 30-sec fractions collected.

of

Pregnant and zebra) the CG

HPLC rified

10

Two

serum samples (approximately 10 ml, yielded a final product of 1 mg of

which in the

I’-’

second

than

to concentrate

applied in the isolation of eCG steps that proved to be effective

of the various 650-750 mg

‘8

0

levels

the to

the CG on Affi-Gel Blue and on Con A Sepharose. serum from all three species (horse, donkey, behaved identically with respect to concentrating therein.

‘0 .0

[2].

procedures

part

‘0

as noted

in the

similar

I-’-)

a)

equiv-

as serum a pool of

donkey

c.’l 0

in Table 1. The values from 0.9-5.3 p.g/ml,

(IIMZ-04,

mare;

to de-

to monitor

is the fact that the zebra samples from (HMZ-01, -04, -09) tended to be lower

trimester from

samples

to the

cJ

utilizing

used

serum samples as well of pregnant horse and

breeds

trimester

comparable

RIA

be

pregnant donkey serum are shown of CG in the zebra samples ranged with

the

LII could

zebra

fractionation experiments. alents) in the six zebra samples

that

a pu-

for

the

zCG

material

show

that

this

preparation

contained

a

considerable amount of contaminant, compared with the purified eCG. The results of the LII RIA on the fractions collected indicated that the immunoactivity was correlated with the peak emerging from the column at a V/V0 of 1.60, which was somewhat retarded compared with the similar immunoactive peak from the eCG (Ve/Vo 1.42). Experiments with eLH and oLH under the same HPLC conditions gave Ve/Vo values of 1.66 and 1.92, respectively. These results suggest that zCG has a smaller molecular size than eCG and is similar in size to eLH, which in turn appears to be larger than oLH. The immunoreactive zCG peak con-

830

McFARLANE

tamed

7%

content

of the

of the

total

area,

preparation

and

suggests

to be of that

the order

ET AL.

zCG

maximal

100

of magnitude

as well. RIM assayed in two RIAs, one for an antibody against eCG. The re2 and 3 and Table 2. In the LH tested had similar (parallel) doseresponse characteristics. The zCG preparation was about 3% as potent as the eCG standard and about 38% as potent as the zLH. The most and least potent preparations, as expected, were the purified eLH and the crude dCG fraction. In the eCG RIA (Fig. 3 and Table 2), the zLH, zCG, and the dCG had similar slopes in their competition curves. The potencies of zLH (1.6%) and zCG (0.9%) were less than in the LH RIA, while that of dCG remained the same (0.1%). The

zCG

preparation

was

the other using suits are seen in Figures RIA, all the preparations LII,

The LH

and

ratios RIA

of the ED50 for the hormone preparations in the to the ED for the same preparations in the eCG

25

RIA are shown in Table 3. A comparison of the ratios show that both dCG and eCG had a lower ED50 in the eCG RIA than they did in the LII RIA. The reverse was seen for both

0

100

0.1

10.0

1.0

100.0

ng

1000.0

10000.0

100000.0

/ tube

FIG. 3. Competitive binding curves of zCG compared and purified eLH and eGG, zLH, and a crude dCG, in an RIA using a polyclonal antibody against eCG and ‘25l-eCG tracer. 75

zLH and the icantly pear

zCG,

zebra

suggesting

that

preparations

different

than

to behave

the

by these

those

epitopes

two

of the dCG

in a similar

recognized

antibodies

and

are

eCG,

in signif-

which

ap-

manner.

50

RRAS The results for the rat testis LII RRA (Fig. 4) showed that all of the materials tested (zCG, zLH, dCG, and eCG) were capable of competing with the radioligand for the LH receptors. Further, the slope of the displacement curves were all similar.

25

increased sults

TABLE relative

0 0.01

0.10

1.00

10.00

100.00

1000.00

10000.00

ng / tube FIG. 2. Competitive binding curves and eCG, zLH, and a crude CG, in an against bLH and tThleLH tracer.

of ZCG compared and RIA using a monoclonal

purified LH antibody

The

potencies

relative

obtained

of eLH,

potency

in both

(Table

of the

2. The percent (%) potency to purified eGG.

RIAs.

of the

Sample

LH AlA

LH RRA

eLH eGG dCG zLH zCG

2607.0 100.0 0.1 8.4 3.2

6568.0 100.0 0.7 14.9 2.3

zLH,

andd

2) compared The

dCG showed with the re-

potency

hormones

in various

LH Bioassay

of each

of

assays

eGG

RIA

1688.0 100.0 0.6 2.5 0.3

100.0 0.1 1.6 0.9

ZEBRA TABLE 3. The RIA ED to the for the hormone used

in this

CHORIONIC

GONADOTROPIN

831

ratio of the LH eCG RIA ED preparations VzLH

study.

zC0

LH AlA:

Sample

eCG

ED

RIA

ration 1.19

eLH eGG dCG zLH

1.84

1.78 0.36 0.52

zCG

-l

a,

a,

these preparations relative to eLH was greatly reduced because of the high activity exhibited by eLH in this system. The results of the calf testis membrane RRA for FSH suggested that zCG does not possess the significant FSH-like activity exhibited by eLH and eCG. We have found that an ED is obtained in this assay with about 1-4 ng of either 0FSH or eFSH, 18 ng eLH, or 1 000 ng of the eCG standard (1 700 lU/mg). A dose of 10 pg of zCG did not displace significant amounts of the radiolabeled oFSH.

C 0 C-

a, In

4,

0 4’

In a,

I-

0I C

100 0.01

0.10

1.00

10.00

ng FIG. 5. Dose-response zLH and a crude dCG. production was measured.

eCG,

75

100.00

1000.00

10000.00

/ tube

curves for zCG in a rat Leydig

compared cell assay

with purified eLH and in which testosterone

Bioassay The

results 5 and the anticipated Figure

0

50

to

the

eCG

of the rat Leydig cell assay for U-I shown in Table 2 demonstrate that the zCG possessed LII bioactivity, although low in comparison standard

(0.3%).

The

slopes

of

the

sponse curves for eCG, zCG, dCG, and zLH were were the maximal responses except for the crude high

maximal

of augmenting preparations.

tent

25

(0.6%)

dose-re-

similar dCG;

as this

response may have been due to the presence factors not present in the more purified Also of interest is that the dCG was more pothan the zCG (0.3%). DISCUSSION

This ployed

both 0 0.01

pituitary 0.10

1.00

10.00

ng

100.00

1000.00

10000.00

/ tube

FIG. 4. Competitive binding displacement curves on zCG pu rifled eLH and eCG, zLH, and a crude dCG, in an LH RRA membranes as receptor source and I-eLH as tracer.

compared

using

with

rat testis

high

work had its genesis with studies hormone-specific assays to show

on eCG that emthat it possesses

LII and

FSH activity [18,19]. Comparisons with equine LII and FSH [3] revealed tht eLH also possesses a intrinsic FSH activity. These observations were later

confirmed and extended by others [20,21]. Because of this unique feature of eCG and eLH, we thought it would be of interest to examine the properties of these hormones in

closely

related

species

(i.e. donkey,

zebra).

Our

studies

on

832

McFARLANE

ET AL.

[4,51

ploying

a polyclonal

showed significant differences. Thus, it was difficult to ascribe significant intrinsic FSH-like activity to the CILH, and in the dCG, the FSH-like activity was very low compared to

parison

of the

that found in CG. In a recent pituitary gonadotropins [101,

study on we found

were

had

intrinsic

purified

donkey

little,

present

chorionic

if any, study,

and

pituitary

significant

the

availability

gonadotropins

semipurified that zLH, FSH

of several

activity. small

zebra dLH,

like

In

of

pregnant zebra serum enabled us to complete this comparative approach with respect to the zebra. Analysis of the individual pregnant zebra samples by RIA (Table to be similar

1) indicated that the first trimester values of CG tended higher than second trimester levels and thus were to the many observations made in the horse. Far

more zebras

data is required, however, from individual pregnant throughout gestation before conclusive comparisons

dCG

very

UI

antiserum

to eCG

all the

different.

This

antibodies

sults

confirmed

tions

indicated

against

RIA ratios

to have very similar while that of the zCG may

clonal antibody recognizing preparations whereas the

the

samples

eCG and respectively),

rabbit

ED

the

zCG

of the

in that

they

displacing the radiolabeled eLH branes. It was thus not unexpected

purified of the

brane lated

the

three

pituitary

species

[1,4]. The behavior like eCG and dCG,

of CG share

chemical

of zCG onto Sepharose-linked been used for human CG

glycoproteins

glycoprotein,

[23],

as expected.

to be both useful and used to adsorb serum

implies The

use

interesting. albumin

to find that zCG would that eCG and oUI are about 10 ml of pregnant

that

the

This gel [24], and

zCG Blue

that

of oW,

and

purified material and not ascertain to what tide and carbohydrate purified gonadotropins. Analysis

of the

to the conclusion nologically and but biologically eCG

in that

it

to that

is also

a

proved

does

not

have

has been widely was surprising

and

hCG

against

as we had mammalian

would

that elephant [25,26]. The

Without

bioassay

do and zCG

a significant calf testis that the bLH

a highly

lead

previously species

so [16].

rhinoceros could also

More

degree

one

shown of UI recently

of FSH

membrane RRA for RIA employing the

would

calf

conclude, little, if any, testis mem-

is more

closely

re-

it

that it resembles eCG and dCG immuin its ability to compete for UI receptors, it appears more like dCG (and hCG) than

antibody

the zCG insofar twelve diverse

RRAs, and

We

We thank Dr. Donald L. Thompson, Jr. (Louisiana State University, Baton Rouge, provided samples of pregnant Arabian and Thoroughbred mare serum. We also thank Dr. Claudia Cabrera and Mr. Stephen Coulson who assisted with some of the assays.

REFERENCES 1. Schams serum

D, Papkoff

cross-react

with

that more than as well as eCG we

have

shown

UI also cross-react in the RIA be measured in an RIA em-

H. Chemical

and

Biochim

Biophys

gonadotropin.

immunological Acta

on pregnant

studies

1972;

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263:139-148.

SW, Papkoff H. Immunochemical studies with pregnant mare serum gonadotropin. Biol Reprod 1979; 21:425-431. 3. Licht P, Bona-Gallo A, Aggarwal BE, Farmer SW, Castelino is, Papkoff H. Biological and binding activities of equine pituitary gonadotrophins and pregnant mare serum gonadotrophin. J Endocrinol 1979; 83:311-322. 4. Aggarwal BE, Farmer SW, Papkoff H, Stewart F, Allen WE. Purification and characterization of donkey chorionic gonadotropin. J Endocrinol 1980; 85:449-455. 5. Roser JF, Papkoff H, Murthy HMS, Chang Y-S, Chioupek RC, Potes JAC. Chemical, 2. Farmer

biological

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donkey

(Equrt

7. Sins

1980;

BC,

Can

for

intrinsic gonadotropin and

Suppl 23:7-11. 9. Joubert E. Notes nae

in South

10. Westlin-van mann’s

KJ, Homer

organization

pools

from

the

Biol Reprod

caballus). properties

GE. Biochemical

K, Birken

and

of equine

mare

of pregnant

Equus

Africa. LM, van zebra

5, Canfield

hormone-like Endocrinology

reproduction

on the reproduction

West Aarde

zebra

H, Seidel

two different

follicle stimulating luteinizing hormone.

H. Social

8. Klingel

from

gonadotropins

(Equis

sera.

Biol

23:570-576.

ES, Nisla

dence

Papkoff

SW,

gonadotropin

Reprod

of pituitaly

properties

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asinus):

1984; 30:1253-1262. BB, Farmer

chorionic

by RIM,

of

L.A) who

6. Aggarwal

activity, as measured by the FSH. It was not unexpected monoclonal

of eLH.

capable

ACKNOWLEDGMENTS

a detailed chemical analysis, one canextent size is a reflection of the pepcontributions relative to other highly

zCG

and in this respect than to the horse.

also be adsorbed. We have found adsorbed as well. From the pool of zebra serum, a yield of 1.0 mg was

similar

RRA reprepara-

similar-

obtained. The HPLC analysis of this material (Fig. 1) suggested that the zCG content was about 7%, comparable to the partially purified eCG standard employed in the assays (1 700 lU/mg). Purified eCG has a specific activity of about 15000 lU/mg [17]. The HPLC results also suggested that the molecular size of the zCG is less than that of eCG, greater than

RRA for FSH, to the donkey

Con A, which [22] and bovine

of Affi-Gel

mono-

the rat testis memthe zCG and related

could not displace the 0FSH radioligand. therefore, that the zCG-like zLH-possesses intrinsic FSH activity as measured by the

ities. Adsorption had previously

The various

all were

from that

be made with the pregnant mare. The methods used to fractionate and partially purify the zCG were derived from previous studies in which highly

suggesting

of the

preparations were active in the rat Leydig cell bioassay for UI (Fig. 5, Table 2). Perhaps most interesting to us were the results of the calf testis RRA for FSH, which showed that even extremely high doses (10 p.g) of the zCG preparation

can

eCG and dCG were prepared zCG in fractionation was much

(1.78 and 1.84, and zLH (0.36)

antiserum.

nature

RIAs

[2]. Com3) showed

identical epitope in all the and zLH are not detected by

polyclonal

UI-like

by the

values (0.52)

be a reflection an

in the

eCG

(Table

in equids.

in Hartmann

Madoqua

1974;

Aarde

RJ, Skinner

bartmannae.

RE, Ross

GT. New

evi-

activity in human chorionic 1978; 102:1356-1361.

zebra

J Reprod Equus

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zebra

1975;

barnnan-

1:31-35.

JD.

J Reprod

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1988;

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Hart-

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BL, Papkoff H. Biological and immunological gonadotropins: Comparison with horse and donkey gonadotropins. Biol Reprod 1987; 36:1134-1141. 12. Bun WE. General discussion of PMSG. In: Wohlstenholme GEW, Knight J (eds), Gonadotropins: Physicochemical and Immunological Properties, Ciba Fdn Grp 22. Boston: Little Brown & Co., 1965: 64. 13. Maueri EL, Papkoff H, Murthy HMS, RoserJF, Chang Y-S. Comparison of the prop11. Matteri EL, Baldwin properties

of zebra

DM,

pituitaly

Lasley

ZEBRA erties

of highly

purified

cial concentrates docrinol 14. Papkoff

equine

of pregnant

1986;

chorionic mare serum

gonadotropin

isolated

and endometrial

cups.

CHORIONIC

from Dom

commerEn-

Anim

3:39-48.

H, Gospodarowicz

cell-stimulating

D, Candiotti

hormones

of ovine

A, Li CH. Preparation

in high yield. Arch

Biochem

Biophys

interstitial

1965;

111:431-

438. 15. Papkoff

H, Gospodarowicz

stimulating

16. Matteri

hormone.

EL, Roser

monoclonal

malian

JF, Baldwin

antibody

species.

D, Li CH. Purification Arch

Dom

which Anim

Biochem DM, detects Endocrinol

Biophys Lipovetsky

luteinizing

and 1967;

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120:434-439.

V, Papkoff

hormone

from

diverse

of a mam-

1987; 4:157-164.

J, Sairam MR. The effects of interstitial cell-stimulating hormone, its subunits and recombinations, on isolated rat Leydig cells. Arch Biochem Biophys 1975; 67:294-300. 18. Papkoff H. Chemical and biological properties of the subunits of pregnant mare serum gonadotropin. Biochem Biophys Rca Comm 1974; 58:397-404. 19. Papkoff H, Bewley TA, Ramachandran J. Physicochemical and biological characterization of pregnant mare serum gonadotropin and its subunits. Biochim Bio17.

Ramachandran

phys

Acts

1978; 532:185-194.

20. Combarnous

Y, Guillou

hormone

(FSH)

chorionic

gonadotropin)

F, Martinat in male

of in v,irO follicle-stimulating

N, Comparison

of equine

activity

gonadotropins

(luteinizing

and female

rats.

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Endocrinology

FSH,

1984;

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1827.

21. Bousfleld hormone

GE, Ward DN. receptor-binding

Biochim 22. Dufau

H. Characterization

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Biophys

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of intrinsic

follicle-stimulating

equine

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ML, Tsuruhara

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Direct

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concanavalin

hormones

A in the pu-

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J, Pannell R Selective removal of albumin Clin Chim Acts 1973; 49:49-52.

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JR, Cabrera

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