Measurement of ovine plasma androgens by a competitive protein binding method : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science

(1)

Copyright is owned by the Author of the thesis. Permission is given for

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(2)

Department of Veterinary Clinical ^Sciences Massey University PALMERSTON NORTH

MEASUREHENT OF OVINE PLASHA ANDROGENS BY A COMPETITIVE PROTEIN BINDING METHOD

A thesis presented in partial fulfilment of the requirements for the degree of Master of Science

by

�Jilliarn Jatnes TORREY

Massey University New Zealand

1971

(3)

ACKNOh'LEDGEHEHT

S

I wish to t

h a

nk my supervisor, Dr. :.�. Greenway, for provi�ing

this t

o

p

ic and for

�is help

and un�erstanding du

r

ing the

study.

Thanks are due

to the Biochemistry Department,

Hassey

University, for

allmvins

!'le to undertake this

t

opic; to His

s

H.

Chaoman f

^o

r collection of

^blood samples;

to

Dr. b.N. Jruere of the Veterinary Clinical Science

Department,

:nnsse-:,' University, for

providing

2dvice and

the experimental

aniw.al s >

to the. staff

at Palmerston N

o

r

th

Putlic Hospital

who pro,.ri6:.:oC: me with

late

preg::ar1cy plasma

sarr,ple.s;

and to Professor G.Co Liggir1s

and his

stuff, of the

Po�;tzr2du:=:te

School of

Obstetrics

<md Gynaecologys t.ucldand University, for

the h

^e^l

p f

^u

l 2dvicc t::::q have

^eiver.

me.

Special apprcci�tion is

extended to Mrs. J.

Horsfall

fo r

ty p

in_g and

aiding in the

p riⁿ

t

ⁱ

n

g of

this th

e

s is

^.

Final

tha:1�cs must

go to my parents and t ho se :::-.; <=>nds

clear to me,

who have

provided emotional and moral

support.

(4)

ABSTl:ACT

A method has been developed for measuring pl

a

s

m

a testosterone in sheep using competitive protein binding

(CPB)

techniques.

iii

The procedure requires column chromatography on I.3-20 Sephadex gel of a methylene chloride extrc::ct of plasma, and final determination of pl�sma testosterone by the

CPB

technique using salt precipitation of the protein bound fraction. The sensitivity of the assay has allm-ved. npplication to the mec:surement of plasma testosterone levels in nonnal male, norraal feaale, Klinefelter and freemartin sheep.

It

has

also enabled the monitoring of the effect of stimulation - with intravenous pregnnnt

mare

sermn gonadotrophin and human

chorionic gon2dotrophin

(HCG)

on the plasmG testosterone levels of norraal male and Klinefelter sheep. A modification to the method, borohydride reduction of the initial methylene chloride extract, has enabled the plasma androstenedione levels to be determined,

simultD.neously with plasma testosterone, in t-.;.To of the

HCG

stimulation studies.

(5)

iv

Trivial nrunes hnve been u sed throughout the text for mo s t steroids. These trivid nomes together �;rith the sys tematic nClllles according to the I nternat iond Union of Pure .::nd Appl ied Chemi s try - I. U. P ,/�.,c. see Biocheraistry

(

_\!ash,

)

_{8, 2227}

(

1969) are l isted in t he section on terminology ana abbreviat ions.

CPB

TLC PC cc TPNH

PHSG

HCG FSH 1., r.

17-ketosteroid CP!:l

(

cpm

)

l U ng

corepetitive protein binding

testos terone-�H, tritiated testos terone

thin

lc7er chromatography poper chromotography col umn chrom�tography

triphosphopyri dine nucleotide , reduced fonn pregnnnt mare serum gonadotrophin

human chorionic gonadotrophin fol l icle s t imulat ing hormone l uteinising hormone

steroid l>lith n ketone on carbon-17 count s per minute

international units nanogram

mi

c

rogr

am

(6)

Trivid �Jrune

Androst2nediol Androstenedione /mdrosterone Choleste:col

DehydroepianCrosterone (DHEA) Dihy

c

rot

e s

tosteron

e

Estrac.iol-17�

l7a-hyC:roxyprcgnenolonc Lanosterol

Pregnenolone Progesterone Sr;udene

TestosteYone

V

Systematic Nme

5a-Androstcm-W, 17

�

-diol

�-Ancrostcn-3, 17-dione 5a-Androstan-3a-ol-17-onc 5-Cholesten-3�-ol

W-Hydroxy-5-androsten-17-one 17�-Hydroxy-Sa-androstane-3-one

1,3,5(10)-cstratrien-3,17�-diol 3�, 17a-Dihycro��y-5-pregnen-20-one Sa-Lanost�-6,24-dien-3�-ol

3�-Hydroxy-5-pregnen-20-one 4-Pregnene-3,20-dione

2,6,10,15,19,23-Hexamethyl-2,6, 10,14,1e,22-tetracosehexane

(7)

Chapter

Abstract Nomenclature

Tf...BLE

^OF ^CONTENTS

Terminology and �b0reviations

vi

page

iii

iv

1 Introductory Reviet

1.1 Role of Androgens in Sheep

1.1.1 The structure and function of androgens 1.1.2 Androgen biosynthesis ancl secretion

1.1.3 Naturally occurring ovine nn

�

rog

e

n

i

c steroids 1.1.4 Physiologic2l role of androgens in sheep

1 2 2 3 6,.

1. 2 i.-ieasurement of £-m�rogens by Competitive Protein Binding 5 1.2.1 Theory of competitive protein binding essay 5

1.2.2

Testosterone binding globulin 7

1.2.3 Competitive protein binding

methods

for pla

s

Qn

testosterone 7

1.2.4 t ^..nclrostene-:::ione assu:'

D

1.

3

:.ction of Gonadotrophic Hormones on :mdrogen Secretion

9

1.

3.

¹ Prer.;nant r:-..:::.re scrura gono�c trophin 10

1.

3. 2

Hur:wn cho:.cionic gcnaJotrophin 10

1.4 Clinical Sienificance of Pathologic�l Entities Studie�

11

1.4.1 Klinefelter rams 12

1.4.2 Freemartin sheep 12

2 Materials and Experimental Methods 2.1 Faterinls

2.2

Experimental Sheep

2.2.1 Normal sheep 2.2.2 Klinefelter

raQs

2.2.3 F

r

e

mar

t

i

n

sheep

2.3 Collection a

n

d Preservation of

S�ples

2.3.1

Collection

2.3.2 Treatment

and storage

2.4

Purification of Binding Plasma

13

15 15 15

16

(8)

2.5

^Preparation of Bin,:int

C

^omplex Solution

2.6

^St�ncard

Curve

2.

7

^Extrac^t^{ion of} Ple.st;:J.C Neutral Steroi::.s

2.�

C�romatogrnphic Purification of

Testosterone 2.

9 :1ecovery of

Adcec1

^Tracer

2.10

^�^li^q^u^ots for the Stan�ard Curve

2.11

Jisplacenent c'.ue

to

^{the N}^oⁿ-specific 11:3lccnk"

2.12 Calculation

2.13

AnGrostenedione

�ssay

2.14

^Gonadotrophin Stimulation Studies

?..14.1

^P^r^ec;nant mare serum gon:-l,_:otrophin stiuulation

vii

16 16

17

13

18 18

19 19

of anJrogen secr2tion 19

2. �J+. 2

Ht.ll:lnn chorionic

gvnc�otrn;_:>hin stinulation

of ancrogen S(:Cretion

20

Di!:cus10ion of Lethoc Develonnent

3.1

^Sample

Tre2t�ent 21

3.2

^RcQson^s ^forPurification of 3in�ing Plasna

22 3.

3 Depencence of ].::mc,e of Stancknl Curve on Prepar:ltion

of 3inJing

Complex

^S

ol

^u

t

^ion

23

3.4 Factors !,ffecting

the Shape

^of ^the

Stc:nc2rd

^Curve

23 3.5

^Ja^c^kg^r^o^un^� ^to the ?inal Extraction

Method 24 3.6

Steroict Separation by

C

hromatogra

p

hy

25

3.6.1 Column chromatogrc::phy

3.6.2

Thin layer chromatogrnphy

3.7

^Reasons for the Recovery Determinations

3.8 Contributing Pactors in the Non-specific "Blank"

3.

9

Development

of

^the lmdrostenecione Assay

3.10 Determination of Dose Level and San�ling Time for

25

26 26

27

29

Gonadotrophin Stucies 29

3.10.1 Action of pregnant mare serum gonadotrophin 29

::;.10.2 l.ction of hllLwn chorionic gon�dotrophin

30

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4

Experimental

anJ Results

viii

4.1 C�1.::1racterizc:.tion

of

Conpetitive Protein Binding He

t

h^o

d

31

L;.1.1 Recove

r

y of

added

testosterone 31

1>.1.;.:

^{Check on}

al

i,-·:uot size

for

the standard curve

32

4.1.3

Reprocucecbility cf samples

32

4.1.4

Recovery

of added

androstenedione

33 4.2

Det.;minntion of PlasDa

Testost:2rone

/: ^.^•

� .1 Norr,wl eHes

/: ^{. •}

?.. 2 Kl inefcl

te!' rams

L:-.2.3

FreeraD.rtin

sheep

!, ^•�-. l, No nnal r ans

4. 3

Act

i

on

of: Precno.nt

l.i2re Serura GonL::-�otrophin t,.L,

Lktion of Hurac-.n

Chorionic Gona.JGtrophin

1.: ·'f.1

:C:ffcct

of hUTI<:n chorionic 20neJdotrophin

on

34-

34

3L:.

35 36

36 37

plasma testosterone

37

L:-.1+.2

Effect of

hurJ.on

chorionic gonac'otrophin

on

5 L'iscus sion

5.1 il.ssay Eetho:�

5 .1.1 ::3l.:m��

plcsm2 ancrostene�ione

5.1.2

P

recisio

n

and accurac7

5.1.S Sensitivity

5.1.1} Aliquot

^size

for the

standarc: curve 5.? Plasma

Testosterone Levels

5.3

Relation of Plasc2

Testosterone tc

the

Clinical

5.3.1 5.3.2

(' . uyntrome

Freemartin

sheep

Klincfelter

r��s

5.L� Comparison of

the Klinefelter Sync:ro!ne

in Htunan

and Ovine Species

5.4.1

Circulatine plasma

testosterone

5.4.2

Gonadotrophic stimulation

40

4l:.

Lr7 47 48

(10)

5.5 Response to Gonadotrophins 5.5.1

5.5.2 5.5.3 5.5.4

Summe�ry

Action of pregnant mare seru.rn gonadotrophin Action of human chorionic gonadotrophin on

plasma testosterone levels

Effect of human chorionic gonacotrophin on plasma androstenedione levels Comparison of PUSG and HCG action

Literature Cite�

Appenc1ix I

ix

49 50

51

52 53

55

57

60

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Figure

1.1

1.2

1.3 1.4

3.1

3.2

� � Jo...'

3.4

[�.

1

4.2 4.2

4. 3

5.1

I

X

LIST OF FIGURES

Page

S t ero id Structure

2a

Tes t o s t erone Biosynthesis

2b

S t andard Curve 6a

S t eroid Structures

7a

Infe r i or S t andard Curve 24a

Improved S tand.:1rd Curve

2

^4b

LH-20 Elu tion Profi l e-

OD 25a

LH-20

E l ut ion Profi l e-

')

T--'H

25b

PHSG Injection (Te.s tost erone) 38a HCG Injec t ion (Tes t o s t erone) 38a HCG Injection (Tes t o s t e rone) 392 HCG Injec t ion (Andros tene1ione)

39a

Plasma Tes tos terone 3asel ine Levels 43a

Standard Curve 61a

(12)

Tnble

T��le

::.1

L,

.1

L;. ^•

/:

I ^'•

.. � . .:;

5.1

LIST OF T!J3LES

'Techniques

for

co�.1p0titivc protein bincing

Lssc;

for plasma testosterone

f:.espo1:se

tc P' !.""'� .:.uu Stinul<;tion

Response to ·:lCC Stir;mlntion (testosterone)

�.esponse

to

^ECG Stinulc:tion (nn:rostenedione)

�}ec:n plasnc::. testosterone in arte:-ial olooJ

xi

7b

37

38

39