Partial Purification and Characterization of Rhinoceros Gonadotropins ...

BIOLOGY

OF

Partial

Purification

44, 94-101

REPRODUCTION

and and

JAMES

R

Prolactin:

Research

of Rhinoceros

Comparison

CLAUDIA

McFARLANE,

Hormone

(1991)

Characterization

M.

Institute,

with

CABRERA,

University

Gonadotropins,

the

STEPHEN

Horse

and

San

Hormone,

Sheep1

A. COULSON,

of California,

Growth

and

Francisco,

HAROLD

PAPKOFF

California

94143

ABSTRACT The

rhinoceros

is an

objectives

of this

assessment,

and

A single

of

LH,

LB was

the

low.

Black

are

growth

FSH

presence

and

(GH),

activity

FSH

could

fraction

(RIA:

and

PRL

assay

detected

in

be detected

White

FSH;

RRA;

Adsorption

with

0,8% and

of

and

interested tuitary

are endangered habitat [1]; their

in vitro

and

chorionic intrinsic

in vivo

gonadotropin FSH-like activity

assays

[3,4]

a Black Rhinoceros (Diceros in comparing the properties

Accepted

August

Received

July studies

Francisco,

X

an RIA using

pituitary equine

Western

lactin

could

be

for

used

their

LH, and

extracted

(pH

SP-C50)

to yield

partially

a monoclonal

testis

9.5),

then

purified

‘FSH”

antibody

radioreceptor

fraction,

but

assay

was

readily

to bovine (RRA)

was

detected

in

FSH), blots.

Results

A-Sepharose

showed

a single

indicated

that

and

with

their

equine

bicornic), of several

counterparts,

31,

immunoreactive some

of the

PR!.

GB bands

(eCG) in a

and

in part

by

NIH

grant

HD-05722

Dr. San

Francisco,

It. Papkoff,

HSW

1092,

Box

0534,

University

to study

the

preparations

and

growth

hormone

(GH)

RRAs, and preparawere adand

pro-

obtained.

AND

METHODS

Hormones

female

zoo

cause of chronic pituitary arrived temperature

in deter-

NSF

established

animals.

These

pituitaries

were

g)

obtained

for us by Dr. J.H. Olsen, Busch Gardens, Tampa, FL, and Dr. S.B. Citino, Metro Zoo, Miami, FL. Each animal was 28 yr old at death. The White Rhinoceros was euthanized be-

we were of the pi-

and

previously

A single pituitary each from a White Rhinoceros (1.3 a Black Rhinoceros (1.2 g) was obtained post-mortem

from

radiore-

tracted

1990. supported

(PRL)

techniques

other species. A variety of RIAs, used to characterize the various together with Western blotting

used

Pituitaries

severe in this

state.

It was

as described

below.

health problems. Regrettably, its laboratory in a thawed, ambient immediately The

Black

refrozen Rhinoceros

until

ex-

pituitary

arrived frozen. The hormones used in this study as standards or radioligands were purified in this laboratory and have previously described as follows: equine LH and equine

assay been FSH

(eLH, equine

from

eFSH [3]), ovine LH (oLH, [6]), ovine FSH (0FSH, PRL (ePRL, [8]), and equine GH (eGH, obtained

(7]),

C.H. Li, University of California, San Francisco, CA; deceased). Human FSH (hFSH) was obtained from the National Hormone and Pituitary Program of the NIDDK, NIH

9, 1990. were

to purification

MATERIALS

grant

87-10021. 2Correspondence:

San

Sephadex

RIA or a calf

ditionally

mining which assays could be used for physiological detection studies, and whether or not the rhinoceros LH also possessed the FSH activity displayed by eLH. To achieve these objectives, the rhinoceros pituitaries were

DCB

and

for equine and a bioassay were tions. SDS-PAGE

has had a major interest in the biochemproperties of gonadotropins from the species. Particularly interesting is that both

hormones

These

homogenized

The

endocrinology,

assays

equine

of

was

and

subjected

ceptor assays (RRAs) [3, 5]. So far, this characteristic has not been found in any other mammalian species. Thus, when we were offered pituitaries from a White (Ceratotherium

simum)

UI) an FSH

Concanavalin

literature reveals that little is known of the reendocrinology in these species. Rhinoceros are of the order Perissodactvla [2], which also comhorse family (Equidae: horse, donkey, zebra) and

LH (eLH) and equine to possess potent

number

activity

(DEAE

Rhinoceros

depends on intensive management of native popand maintenance of captive colonies. A survey of

tapirs (Tapiridae). This laboratory ical and biological horse and related equine appear

which

glycosylated.

All species of the family Rhinocerotidae and rapidly disappearing from their native

the recent productive members prises the

FSH

X equine

INTRODUCTION

survival ulations

determine

Rhinoceros

LB by either

by SDS-PAGE

bands.

PRL

the

of its reproductive

is known

(PRL).

(0.1-1%

in the

)C equine

determined

was

immunoreactive

intrinsic g)

chromatography

cell

0,2%

(1.2

Little

hormones,

the

a Black

prolactin

and

family,

horse

pituitary

LB possesses g) and

ion-exchange

and

hormone

activity

of GH

multiple

(1.3

to the

rhinoceros

rhinoceros

a White

by a rat Leydig

LB).

Rhinoceros

The band

No

purify

fractionation,

measured

equine

X

extremely

salt

FSH,

readily

(6-11%

to partially

from

related

species

whether

each

to pH and

fractions

were

to ascertain

pituitary

subjected

LB

study

endangered

of California,

(Baltimore,

CA 94143.

94

MD).

RHINOCEROS

PITUITARY

Purification Each pituitary was extracted and purified separately. The extraction and purification techniques employed have previously been described in detail [3,9, 10]. In brief, each pituitary was minced, homogenized, overnight at 4#{176}C. After removal GH-PRL fraction (White, precipitated with HPO3

of

1P, 89 mg; (pH 4.0),

and extracted (pH insoluble residue, Black, leaving

9.5) the

13P, 74 mg) was a glycoprotein 13S, 57 mg) purification

concentrate fraction (White iS, 113 mg; Black, in the supernatant fluid. Separation and further

of LH and FSH from the White Rhinoceros fraction (iS) proceeded as follows: fraction iS was chromatographed on a column of DEAE-cellulose (BIO-RAD, Richmond, CA) with 0.03

M NH4NCO3,

pH 9.0; the

LH) was applied C50; Pharmacia, was

to a column Piscataway,

adsorbed

and

eluted

2.6 mg). On the DEAE and eluted with 0.2 M chromatographed pH 4.0; after

serial

unabsorbed

fraction

(putative

of sulfopropyl-Sephadex NJ) with 0.03 M NH4HCO3; with

1 M NH4HCO3

column,

(fraction

Black

on SP-C50 with 0.03 M sodium elution with the pH 4.0 buffer

Rhinoceros

dled in a similar matography steps Rhinoceros (fraction prepared

glycoprotein

fashion except was reversed.

4B,

FSH is normally adsorbed pH 9.0. This fraction was

NH4HCO3,

acetate at and 0.03

M ammonium acetate, FSH is normally eluted with NH4HCO3 (fraction 5C, 0.1 mg). FSH activity was quently found to be absent in the White Rhinoceros tions. The

(SPLH

concentrate

0.03 M subsefracwas

han-

the order of the two chroThe procedure for the Black

yielded LH (fraction 14SB, 1.3 mg), and FSH 15D, 0.6 mg). For HPLC comparisons, an oLH was in a similar fashion to fractions 4B and I4SB.

Analysis

of fractions

1P and

13P

by

SDS-PAGE

showed

bands suggestive of GH and PRL in both (see Fig. 6). These hormones were further purified by subjecting the fractions to DEAE chromatography: GH and PRL were adsorbed at 0.03 M NH4HCO3, pH 9.0, and eluted with 0.2 M NH4HCO3, yielding fractions and PRL in 11BC Sepharose unabsorbed The Black

I 1BC (White) and was further purified

16B (Black). The by Concanavalin

(Con A; BlO-RAD) chromatography, fraction (12A) and an adsorbed Rhinoceros fraction 16B, which

GH A-

yielding an fraction (12B). contained little

PRL relative to GH, was put on a Sephadex G-100 (Pharmacia) column equilibrated in 0.05 M NH4HCO3. The GH was found in the peak eluting with a Ve/Vo of 2.2, and yielded fraction

17B

(5 mg).

95

HORMONES

be iodinated in 50 p.1 of the same buffer and 500 p.Ci (5 p.l) of 1251 (Amersham, Arlington Heights, IL). The reaction vessel was gently vortexed for 10 mm; then the reaction mixture was transferred to 1 ml of PBS containing 0.1%

BSA. This

mixture

was

applied

to a small

desalting

column

(Econo-Pac lected. The

1ODG, BlO-RAD), and 1-mi fractions were colvoid volume peak was collected and used in

subsequent

assays.

RIM

and

The clonal

RRAs

RIA employed antibody against

gand and has has cross-reacted

been with

for measuring LH utilized bovine LH and 125I-eLH

described every

in detail mammalian

a monoas radioli-

[ii]. The antibody species of LH thus

far tested (>12). An RRA for LH as been described uses rat testis membranes as the receptor source eLH as radioligand. rabbit polyclonal hFSH

as

FSH antibody

radioligand.

scribed [13] and many mammalian

was measured raised against

This

the

assay

has

[12] and

that 125I

in an RIA with a 0FSH, with 125I

been

previously

de-

antibody also cross-reacts with FSH from and nonmammalian species. An RRA for

FSH was used as previously described [9, 12]. This assay employed calf testis membrane fractions as receptor source and ‘251-oFSH as radioligand. PRL was measured with a previously described RIA [9] using a rabbit poiyclonal antibody raised

against

purified

The GH RIA used against rat GH; this [14]. with

ePRL,

The antiserum was eGH as radioligand.

RIAs and

the

with

a monkey assay has

‘251-ePRL

as radioligand.

polyclonal antiserum raised been characterized elsewhere

used at a final The samples

RRA in duplicate

and

dilution of 1:75 000, were assayed by the

on

at least

two

separate

occasions.

Bioassay LH bioactivity

was

monitored

in vitro

tions. plates were

The assay was carried out in 48-well tissue culture (Costar, Cambridge, MA). Approximately 100 000 cells incubated in 500 p.! of medium (M-199 with Earle’s and 0.1% BSA) or test material.

samples

were

with

minor

modifica-

to which was added the doses of stanThe plates were incubated for 2 h at

37#{176}C in the presence which testosterone The

[15],

cell

as previously

BSS dard

described

in a rat Leydig

assay

of CO2 production assayed

and 02 mixture was determined

(5:95), after by an RIA.

in triplicate.

HPLC lodinations Hormones were iodinated phenylglycolril;

to be with

used as radioligands in various Iodo-Gen (1 ,3,4,6-tetrachloro-3a,6a-di-

Pierce,

performed as follows: xl of chloroform was tube. The tube pA of PBS was

Rockford,

IL). The

500 ng of lodo-Gen evaporated in a 12

iodinations dissolved X 75-mm

was then rinsed with 0.05 M PBS, and added together with 5 .tg of the protein

assays were in 50 glass 100 to

HPLC gel characteristics

filtration of the

was used rhinoceros

to compare LH preparations

the

elution with that

of a similarly prepared oLH. A Perkin-Elmer (series 410) HPLC with UV detector (LC-95) and integrator (LCI-100) (Perkin-Elmer Corp., Norwalk, CT) was used with a 300 X 7.5-mm

TSK-250

vent

was

0.1

rate

of 1 ml/min.

(BIO-RAD)

M Na2SO4,

gel

filtration

column.

0.1 M NaH2PO4,

UV absorption

was

pH

measured

6.8,

The

sol-

at a flow at 225

nm,

96

MCFARLANE

and

samples

of

The

column

was

ular

mass standards values for each

Ve/Vo

roglobulin-67

10-20

p.g were

calibrated

kDa,

injected

using

(BIO-RAD). The of the standards i .01;

bovine

and

Western

Purification by SDS-PAGE gel

was

the

column.

of five

Ovine

molec-

molecular mass were as follows:

gamma

1.50; ovalbumin-44 kDa, 1.72; horse and vitamin B12-i.35 kDa, 2.45.

SDS-PAGE

onto

a mixture

ET AL.

globulin-i

Black

Rhinoceros

58 kDa,

myoglobin-17

kDa,

2.06; ‘0

Blotting

of rhinoceros GH and PRL was monitored as outlined by Laemm!i [i6]. A 4.5% stacking

used

with

kDa),

a 12.5%

soybean

sozyme (14.4 Separated

gel.

Samples

were

trypsin

kDa). proteins

inhibitor

reduced

were

(21.5

kDa),

transferred

to

(Amersham) sheets, and then probed with manner similar to that described by Towbin antibody probes used were rabbit antiserum

with

and

t2’4

iH2’4

Elution

t2’4

Time

(minI

ly-

Hybond-N

antibodies in a et al. [17]. The against ePRL

FIG. 1. Gel filtration HPLC chromatograms of 0LH and the LH preparations from the White (4B) and the Black Rhinoceros (14SB) on a TSK-250 column (330 x 7.5-mm). The column was equilibrated and the sample eluted with 0.1 M Na2SO4. 0.1 M NaH2PO4pH 6.8).

[9] and bovine GH (obtained from C.H. Li) at a dilution of 1:500. A peroxidase-conjugated second antibody (Boehringer-Mannheim Biochemicals, Indianapolis, IN) was applied and then developed with 4-chloro-i-napthol.

Statistical

Rhinoceros

and thy-

5% (3-mercaptoethanol (Sigma Chemical Company, St. Louis, MO) prior to electrophoresis. The following molecular mass standards (BLO-RAD) were used: phosphorylase-b (97.4 kDa), BSA (66.2 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (31.0

White

100

Analysis

Relative

potencies

of the

rhinoceros

standard hormones were calculated logit linear regression lines. Linear performed using SAS (SAS Institute software.

preparations

and

the

from the ED50 of logregression analysis was Inc., Cary, NC) statistical

75

RESULTS 0

Gonadotropins The

50

rhinoceros

amined

LH preparations

by HPLC

These eluted

and

compared

results are shown at about ii mm

integrated

area.

at 11 mm (data not

and comprise shown). The

(4B)

that

and

A more

of the

FSH preparations

were

not

expected

and

14SB)

to a similarly

were

ex-

prepared

oW.

in Figure 1. The major 0LH peak and comprised about 70% of the highly

purified

closer White

Black

were

oW

to 90-95% Rhinoceros

Rhinoceros

peak eluting at about 11 mm. was only 27% in 4B and 39% the

(4B

would

(14SB)

The percentage in 14SB. Because

available

to be as purified

also

elute

and as the

these

each

had

a

of this peak far less of preparations

0

LH preparations,

HPLC analysis was not performed with them. Three different assays were used to assess

0.1

LH activity

in

the rhinoceros preparations. The results for the RIA are shown in Figure 2 and Table 1, and it can be seen that although

the

final

LH preparations

(4B,

25

of the total area LH preparation

14SB)

were

by no

1.0

10.0

ng /

100.0

1000.0

Tube

FIG. 2. Competitive binding curves of rhinoceros pituitary tions compared with purified ovine and equine LH in an RIA using clonal antibody against bovine LH and #{176}l-eLHtracer.

preparaa mono-

RHINOCEROS

TABLE

1. The

potency

of the

purified

equine

(taken

as 100%)

LH

100.0 115.0 6.4 10.7

means

pure 7-fold)

to

activities

were

(see

Fig.

than

i), the

about

preparation (i5D) All the dose-response

cating a considerable rhinoceros and eW.

preparations

preparations

compared

in various

LH bioassay

100.0 29.1 0.6 7.2

to highly

assays. FSH RIA

FSH RRA

0.01