BIOLOGY

OF

REPRODUCTION

15,

141-146

Pituitary-Testicular

(1976)

Responses

Luteinizing

in Rams

J.

WILLIAM

J.

BREMNER,

B. HUDSON

Medical

K.

and

Research

Hormone FINDLAY,

D.

Center,

M.

Reproduction

Cameron Institute

of

I. A.

CUMMING,

1-lenry’s

Hospital,

3004,

Laboratory, of Experimental

University

Infusions

(LH RH)’

KRETSER

Research Section, University and Department of Agriculture,

S. S. Florey

Howard

de

Prince

Melbourne,

and

to Prolonged

Hormone-Releasing

of Melbourne,

Werribee, 3030, Physiology

of Melbourne,

and

Medicine,

3052

ABSTRACT Luteinizing hormone releasing hormone (LHRH) was administered to adult rams as constant intravenous infusions lasting 4 to 6 h. Dosages of 0.05, 0.1, 0.5 and 1.0 ug/min were used. All studies were undertaken during the period of decreasing day length (January to June). Plasma luteinizing hormone (LH) values during administration of the two highest dosages revealed a biphasic pattern of increase during the first 2 h of the infusion, as has been found in similar studies in humans. LH values were maximal at approximately 2#{189}h of the infusions, then decreased in spite of continued LHRH administration, thereby demonstrating the development of pituitary refractoriness during prolonged LHRH administration. A similar refractory period has not been noted during analogous studies in men. Plasma follicle stimulating hormone (FSH) values generally increased gradually during LHRH infusions, with no evidence of a biphasic pattern. No FSH increase was noted in some studies, even at the highest LHR1-I dosage used. Plasma testosterone (T) levels increased sharply 15-30 mm after the initial rise in LH values. Maximal levels of T obtained were 3- to 5-fold greater than basal values and were considerably higher than those found during similar studies in men. These results suggest that T production from the ram testis is more sensitive to grnadotrophin stimulation than is that from the human testis. In spite of the prolonged elevations in gonadotrophins and the marked increases in plasma T levels, plasma

estradiol

levels

were

consistently

close

to or below

INTRODUCTION The (LH)

patterns and

of

follicle

responses hormone

serum

levels

hormone

extensively

in

many

have shown

been in

infusions increase

progressive Paulsen, otrophin

less well humans of LHRH in

serum

LH

increase 1974; values

de

values,

in FSH Kretser

in men

levels et al.,

remain

LH

and

pattern

but

a

1975).

LHRH

infusions

are

terminated

h)

(Bremner

and

Paulsen,

1976).

al. (1972) have LHRH infusions

shown cause

values

than

of

animals. single

gradual,

(Bremner

until 19

to maximal which

pg/mi).

levels

Received ‘Portions 7th Annual Reproductive

in ewes, in serum

at approximately

a decline

and

serum Kley

Gonad-

at near-maximal

have

toward

basal

3 values

the

gonadotrophins,

et

T (Roth et al., 1973; al., 1974; Wollesen

quantitatively Bremner and

April 6, 1976. January 14, 1976. of these data have been presented at the Meeting of the Australian Society for Biology, 1975.

small Paulsen,

of single intravenous rams (Galloway et rapid 141

particularly

in

In men, it is controversial whether injections of LHRH lead to increases in

during prolonged LHRH rises in serum T in men Accepted

that, a rise

(up Cum-

occurs in spite of continued LHRH administration. Gonadal steroid production during LHRH administration has received much less attention

It has been intravenous

to a biphasic

(15

h, following

responses to of this hormone

characterized. that constant lead

luteinizing have been (Gual

sensitivity

ming et prolonged

(FSH)

species

Rosemberg, 1973). Gonadotrophin more prolonged administration

to

hormone

luteinizing

stimulating

to single injections of releasing hormone (LHRH)

studied

assay

and

quantitatively

Judd et al.,

et al., 1974).

1974; Even

administration, are very gradual

the and

(de Kretser et a!., 1976). One recent a!.,

injections 1975) has larger

1975; study

of LHRH in shown a more

T response

than

BREMNER

142

has

been

demonstrated

in

humans.

Three

The present study was undertaken terize LH, FSH, T and estradiol (E2) during

constant

into

normal

being

intravenous rams.

investigated

tern

of

LH

humans,

(2)

refractoriness the situation increase longed

a

develops in humans,

is seen endogenous

(1)

in rams

whether

in

of LHRH

particular,

were:

increase

to characresponses

infusions

In

the

whether

is biphasic period

points the

these animals gonadotrophin

(4) whether an be demonstrated,

increase since

that and a!.,

in other species E2 is under gonadotrophin 1971; Kelch et al.,

pat-

basal

of

pituitary

at

1 5-mm

intervals

were

RESULTS

to of T

during proelevation,

in plasma E2 levels it has been found

is formed in the testes control (Leonard et 1972; de Jong et al.,

1973). AND

samples

further sampling was at 15-mm intervals during the infusions and at 30-mm intervals for 2 h following the infusions. Five other rams received 4-h infusions of 0.5 ug per minute with an identical blood sampling schedule except that blood was obtained at 5-mm intervals during the first hour of the infusion.

In

METHODS

Seven Corriedale crossbred sexually-experienced rams, aged 3-4 years were used. All studies were undertaken during the period of decreasing day length (January to June). Animals were fed on pasture and brought into a closed shed 1-2 days prior to the studies. Synthetic LHRH (Hoechst Op 62) dissolved in 0.9 percent saline was administered by infusion pump (4.0 mI/h) into one jugular vein. Blood for hormone studies was obtained from an indwelling catheter in the opposite jugular vein. Plasma was separated and frozen at -20#{176}C until hormone assay. LH (Lee et al., 1976a), FSH (Salamonsen et aI., 1973), T(Wanget al., 1974) and E2 was measured by specific radioimmunoassays. The LH standard used was NIH-LH-S8 and the FSH standard was NIH-FSH-S6. E2 was measured by a modification of the method of Dufau Ct al. (1970), using an antiserum produced in rabbits following injections of estradiol 3-carboxymethoxine coupled with porcine thyroglobulin. Cross reactivity of this antiserum was: E2 100 percent, estrone 22 percent, estriol 2.6 percent and T 0.2 percent. 2 ml plasma samples were extracted with dichloromethane and chromatographed on Sephadex G-1 5 to separate estrone and E2. The gel was prepared in distilled water. Following application of plasma samples to the column, androgens were eluted with distilled water and estrogens with absolute methanol. Separation of bound and free hormone was achieved using ammonium sulphate precipitation. Recovery of E2 was 63.3 ± 6.5 percent and the blank was 1.46 ± 0.78 pg. Dilutions of plasma extracts produced displacement curves parallel to those of the standards. Accuracy studies included addition of 25, 50, 75 and 100 pg of E2 to charcoal-treated plasma. A linear relationship between E2 added and measured was obtained (y = 0.99X+1.51, r = 0.99). The sensitivity of the assay was 15 pg/mI, intraassay variability was 12 percent and interassay variability was 20 percent. Two rams received each of 4 different dosages of LHRH (0.05, 0.1, 0.5 and 1.0 ug/min) in 4-h infusions, separated by at least one week. One animal received a second 0.5 ug/min infusion lasting 6 h.

the

LHRH,

given

2 animals

plasma

infusions

levels

(Fig.

values

fall

1A).

following

During

ug/min),

A

in

second

reached.

During phase

reduced, mm sured

although

of

maximal

lowest

dosage LH

continued

tained

of

until

ug/min

began

at

45

values

were

infusions,

the

was levels

LHRH

of

(0.05

began

mm.

150

the

animals

markedly LH

at 150

the same as those meadosage infusions. At the

increase

during

increase

maximal

dosages

both

15 mm, then approximately

increase

were essentially during the high

gradual

in

phase

infusions.

higher

first until

0.1

decreased

the

two

values

the stable

LH

maximal

LH

of continued LHRH and continued to

until the of

all

mm

of

LH

continuing

early

mm,

at the

sharply essentially

mm.

mm,

in spite 240

during

reaching

150

infusions

1.0

increased remained 45

After

of

dosages

increased

termination

the and

4 different

of LH

at approximately

during all infusions administration until

(0.5

MATERIALS

blood

obtained;

as it is in

in rams in contrast (3) what pattern

and can

ET AL.

lowest

by

ug/min), 30

a

mm

Maximal values dosage infusions

and obwere

a S

TiI,#{128} IIORSi

FIG.

during

1. Mean

and after

serum LHRH

LH,

FSH

infusions

and

into

T values 2 rams.

before,

PROLONGED

markedly

less

higher LH at

dosage levels

sampling sions

confirmed

during

high

and the that

the

(Fig.

2).

LHRH

infusion

20-25

mm,

that

received

had

increase

The

first

begun

5

of after

and

which

fact

longed

administration

to LH on

that the LHRH

values

or

for decreas-

two

of 0.5 ug/min, other 360 mm.

shown

60 in

mm in spite of until 240 mm.

ram pituitary stimulation for

becomes during proclearly a single

until

increases but

no

animal

animal

mm

during

increase

during

one

360

did

LHRH

in

the

FSH

lowest

during

dosage

the

in ram

(Fig. in either

infusions

highest

dosage

or

baseline

or areas

under

closely

similar During

for

however,

the

increase 15-30 in LH (Fig. 1C). by 90-180 mm,

response

began. T above

curves

were

the three highest the 0.5 ug/min LHRH

the

in

infusions.

which a very gradual decline computed either as increases

infusions.

to a

infusions

levels began a dramatic following the initial rise Maximal levels were attained

sion,

lead

occurred

mm

following responses,

not

elevation of serum LH values. levels in general revealed slight,

T

the

levels were decreasing

is illustrated are

r*pid

continued

stable

were found after 180 LHRH administration

The refractory

studied

in

were found until approximately a second phase of rapid increase

LH levels continued

3.

LH

mm

infusion

1B),

occurs

143

RAMS

sustained Plasma FSH

gradual

in serum phase

IN

of the

LHRH

infusions

LH began that lasted until maximal reached at 120 to 165 mm. Again,

Fig.

the

5-mm blood of the infu-

mm

within

following

during

INFUSIONS

more

LURH

commenced

ing values mm when

found

underwent first 60

dosage

phases

increase

those

infusions. in the 5 rams

ug/min during

0.5

two

than

LHRI-I

T response

was

dosage infu-

much

small-

er. There were levels of E2

no detectable during LHRH

values

from

ranging

tion

of the

assay

below

(15

with LHRH infusions one lasting 240 mm and the It can be seen that prolongation

increases in administration, the

pg/ml)

limit

of

the

detec-

to 21 pg/mI.

occasions

DISCUSSI

The stant

present

dosage increase tory

demonstrate that conLHRH at an appropriate

of

to rams induces a biphasic in serum LU values. This is very

pattern

during

analogous

(Bremner 1975)

results

administration

ON

and except

similar

to

LHRH

Paulsen, that

that

de

second

in

phase somewhat (45-60

90

The

mechanisms

the

physiologic

biphasic

pattern

of

et

al.,

of increase

LH values begins it does in humans

mm).

men

Kretser

in serum rams than lying

described

infusions

1974;

the

pattern of early secre-

earlier in versus

mm

underincrease

are

C

.-.

4-lW

l,Wm

s+I_,

w,w,

-j

0

r

60

60

240

U4

0.5

FIG. 2. Serum ug/min LHRH

(

HOURS)

LH values before, infusions in 5 rams.

during

and

after

360

05,4/mm 1mw

TIME

300

1.1411

(mm)

FIG. 3. Serum LH values before, during and after 2 LHRH infusions of 0.5 ug/min to the same ram, one lasting 4 h and the other several weeks later, lasting 6 h.

BREMNER

144

unknown.

It has

Paulsen,

been

1974)

two pools of LH longer stimulation Other

protein

sky,

1972)

also

been

in the for

possible involved

to

patterns

pancreas and Chakraborty biphasic

(Grod-

1971) in

The

intracellular synthesis

and

hormone

pituitary. et

al.

of

into ewes, intravenously) dosage also did

A biphasic data from 0.025

(1974) increase

but

the

in the a biphasic

directly

into

et al., 1972). also explain

discrepancies

to

single (Galloway

bly related (100 ug).

to the Studies

kinson

et

al.,

values

occurring

LHRH cause

injection. a biphasic

mura similar

et

of

of

two

hr

of LHRH dosages

1974)

have

found 30

within

The higher pattern of

following

et al., infusions

because levels

were

falling in

LI-I

1974) in the

of persistent following during

spite

of

high the

h,

did

last

depletion portant, inhibitory

infusions.

No

values lowest

administration.

Similar

of pituitary reprolonged LHRH

results

ported in the ewe by Cumming Chakraborty et al. (1974) and

have et al. Rippell

been

re-

(1972), et al.

the

alone

of refractorithat continuous for as long state 1976).

difference

the

in

the The

in pituitary

most

increases

in

of

the

high effect rate of

LHRH

pattern

increases

either or

revealed

the

a biphasic No

infusions

dosage

cases

during

found.

seen

highest

as

of

LH stores may be immay exert a “short-loop” the pituitary (Motta et al.,

levels

was

were dosage

animal in

FSH

during

one

infusion,

of

in

animal

the during

emphasizing

the

inconstancy of FSH responsiveness to LHRH in rams. Similarly, Hopkinson et al. (1974) found FSH increases in one of five rams given 10 ug intravenous four given

rise

the development in the ram during

fact

but may be related to the a much more pronounced T

evidence

increase

40

T levels

results

the

depletion

to a refractory and Paulsen,

pituitary LH itself effect on

gradual

during

demonstrate fractoriness

LH

species

FSH

modest,

continued

These

this

1969). Plasma

The

infusions.

the

content

during

development

of

LH

4-h

not lead (Bremner

or

after

the

days, of

study,

developed

pituitary the

is unknown, rams have

mm

than

that

for

LHRH stimulation. Extending the administration of LHRU to a total of 6 h led to no greater release

present

state

10

state LU

increase during LHRH administration; levels of T may exert a negative feedback on the pituitary. Alternatively, the

FSH

dosages tend to LH increase (An-

LHRH

infusion

used (Hop-

maximal mm

Hopkinson during

presumably serum

the

in and for

large using

dosages smaller

19

function fact that re-

for

ness.

reason

in dosage apparent

as

LHRI-1

in the

explain

pituitary

injections of 1975) is proba-

dosage injections. Plasma LH levels mm

late

in

LH

of

the refractory While pituitary

infusion, in which much less LH than in the high dosage infusions,

suggest not

were unable to dempituitary LH content

in

In men, it has been shown infusions of LHRH, 0.2 ug/min

intravenous and Pelletier,

a!., 1972; that seen

study,

elevation

as

lowest dosage was released may

artery

of

refractory

would

carotid

reports

occur

a

measured

a

(0.038 to the

single injections of LHRH et a!., 1974; Galloway Particularly, the tendency

values

to

find

not

LURH

of the pituitary a single 50 ug

injections

was

at 2.3 ugfh state of the

a refractory state 96 h following

which time persisted.

that

a

to depletion of et al. (1974)

change

daily

demonstrated LH content

in part, Rippell

They

any

pituitary

the

be due, stores.

to

a!. pituitary

LHRH infusion the refractory

injection.

onstrate

with

be seen infusions,

Differences many of the

published

in

sponsiveness to rams (Hopkinson Pelletier, 1975). LH

the

LHRH

et in

h of that

may LH

during

present study LH increase.

pattern of LH rise can ewes that received LHRH

(Cumming probably

present

not

during

dosage used comparable

was

infusions not cause

ug/min

following LHRH

did

LH

in

Chakraborty decrease

demonstrated lasting up

sec-

mechanisms secretory

secretion

(1974). four-fold

pituitary pituitary

have

a biphasic

stimulation.

AL.

following 24 and suggested

of these hormones occurs in the case of LH. It is

of

pattern

infusions ug/min

90

requiring the other.

as insulin

released

to

formation, storage and transport (Tixiand Farquhar, 1975) may underlie the

biphasic

peak

be

that similar in hormone

granule er-Vidal

one

(lversen,

and

be due

than

such

constant release than

may

pituitary, release

glucagon

during

(Bremner

pattern

hormones, and

ond phase of much earlier

lowest which

this

found

pattern

speculated

that

ET

maximal

which shown

mm ram

injections of ug injections.

began the

T

was

higher values

there that following (Sanford

increase

of

quite dosage

by

75

and

within

commencement

in the

to

LHRH

15 the

steep, 180

of

to

LH

30 rise.

particularly

infusions, to

three

mm,

reaching following

was a gradual decline. It has been T also rise’s sharply within 15-30 spontaneous et a!., 1974)

LH and

increases following

in the LHRH

PROLONGED

administration

to

prepubertal

et

In

men,

aL,

1976b).

(Naftolin

et

LHRH

al.,

al., and

1973;

Lincoln,

(Kley

1974; de Paulsen,

increases

or

Similarly,

exogenous

no

intravenously no change (Marshall

et

lambs

(Lee

the

spikes

Australia Fund.

and

LU

3-4

1974)

and

1974;

Wol-

al., to

1975; small levels.

et lead

in

serum

T

UCG

when

given

h to

normal

men

cause

than is no

is

species

of

the

human

explanation

difference.

for

The

ram

testis.

this

testis

apparis some

ten times as heavy as the human testis (approximately 250 grams vs 25 grams). However, the concentration of T within the testis is some one tenth as great in the ram as in man (approximately 100 pg/mg vs 1000 pg/mg, Bremner et al., unpublished observations) and Leydig cells are more difficult to demonstrate histologically in

the

normal

(Fawcett

E2

levels

Serum

in

trophin

of

small below

the

and

T levels.

It

than

that

testicular

The

in et

other al.,

present of E2 extra-testicular

levels

T

of

was

E2

not

E2

levels

above

than

that

found (Baker

ACKNOWL

in the

et et that

great

enough

15 pg/mI, in et al.,

some

to a figure other

a!., a!., the

increase much species

1976).

EDGMENTS assistance of Mrs. Anne Mr. Peter Langdon and of Mrs. Jill Volfsbergs

the

illustrations of Ms. Anne Hayres. We are grateful to Dr. H. Papkoff for his gift of highly purified ovine FSH (49 X NII-I-FSH-S1) and ovine LH (2 X NIH-LH-S1), to Hoechst Pharmaceuticals for their gift of synthetic LHRH, and to the National Institutes of Health for their gift of the gonadotrophin standards. the National

This

work was supported by Health and Medical Research

Trust

A., Buckmaster, J. M., Cerini, J. C., E., Chamley, W. A., Findlay, J. K. and J. R. (1972). Effect of progesterone on

Cumming, Cerini, Goding,

I. M.

(1973).

grants from Council of

Effect

of

of oestradiol-17j3 J. Endocrinol.

a

E2 production gonadotrophin

demonstrate

We appreciate the technical Davies, Ms. Ausma Dulmanis, Mr. R. W. Baxter, the typing

and

measurements

Research

Chakraborty, P. K., Adams, T. E., Tarnavsky, G. K. and Reeves, J. J. (1974). Serum and pituitary LH concentrations in ewes infused with LHRHFSHRH.J. Animal Sci. 39, 1150-1157.

in that

production from the testis conversion of the high

plasma

man

increases

species (Leonard 1972; and de Jong

lower including

detectable

however,

Wool

the release of luteinizing hormone induced by a synthetic gonadotrophin-releasing factor in the ewe. Neuroendocrinology 10, 338-348. de Jong. F. H., Hey, A. H. and van der Molen, H. J.

occurred which was the assay used. It may

results

combination and from

human

gonado-

marked

be useful in detecting demonstrated during

stimulation 1971; Kelch 1973).

no

is possible,

vein

the

prolonged

the

rise in E2 production the sensitivity of

ram would as has been

in

revealed

spite

elevation

serum

testis

1973).

increases

be

ram

et al.,

Australian

REFERENCES

T production by the to gonadotrophin

that

obvious

145

Arimura, A., Debeljuk, L., Matsuo, H. and Schally, A. V. (1972). Release of luteinizing hormone by synthetic LH-releasing hormone in the ewe and ram. Proc. Soc. Exp. Biol. Med. 139, 851-854. Baker, H. W. G., Burger, H. G., de Kretser, D. M., Hudson, B., O’Connor, S., Wang, C., Mirovics, A., Court, J., Dunlop, M. and Rennie, G. C. (1976). Changes in the pituitary-testicular system with age. Clin. Endocrinol. (In press.) Bremner, W. J. and Paulsen, C. A. (1974). Two pools of luteinizing hormone in the human pituitary: evidence from constant administration of luteinizing hormone releasing hormone. J. Clin. Endocr. Metab. 39, 81 1-815. Bremner, W. J. and Paulsen, C. A. (1976). Prolonged intravenous infusions of LH-releasing hormone into normal men. Horm. Metab. Res. (In press).

or small increases in serum T levels et al., 1973; Maurer et al., 1973).

stimulation ent

RAMS

and

LH

These findings suggest that ram testis is more responsive There

IN

al.,

Kretser 1976)

change

over

INFUSIONS

spontaneous

administration

lesen et Bremner

tam

LHRH

de

gonadotrophmns and testosterone

on the by the

secretion rat testis.

57, 277-284.

Kretser, (1975).

D. M., Serum

Burger, LH and

infusions

of luteinizing

H. G. FSH

and Dumpys, responses in

hormone

releasing

R. 4-h

hormone

(LHRH) in normal men, Sertoli-cell only syndrome and Klinefelter’s syndrome. J. Clin. Endocr. Metab. 41, 876-886. Dufau, M. L., Dulmanis, A., Catt, K. J. and Hudson, B. (1970). Measurement of plasma oestradiol-17a by

competitive plasma.

Fawcett, (1973). lymphatics tissue 500-53 Galloway, (1975). terone releasing 1-9.

Galloway,

binding assay employing pregnancy J. Clin. Endocr. Metab. 30, 35 1-356. D. W., Neaves, W. B. and Flores, M. N.

Comparative observations on intertubular and the organization of the interstitial of the mammalian testis. Biol. Reprod. 9, 2. D. B., Cotta, V., Pelletier, J. and Terqui, M. Circulating luteinizing hormone and testosresponse in rams after luteinizing hormone hormone treatment. Acta Endocr. 77, D.

hormone

B. and release

Pelletier, in entire

following injection of mone releasing hormone

propionate Grodsky, G. hypothesis

mathematical

pretreatment. M. for

(1972). packet

modeling.

J. (1975). and

Luteinizing castrated rams

synthetic and effect

luteinizing horof testosterone

J. Endocrinol. A threshold storage of

J.

64,

7-16.

distribution insulin and

Clin.

Invest.

its 51,

2047-2059.

Gual, C. and Rosemberg, mic Hypophysiotropic

E. (eds.)

Hormones.

(1973).

Hypothala-

Excerpta

Mcdi-

146

BREMNER

ca, Amsterdam. Hopkinson, C. R. N., Pant, H. C. and Fitzpatrick, R. J. (1974). Release of LH and FSH in the normal intact ram by synthetic LH-RF and the effect of pretreatment with testosterone propionate. J. Reprod. Fert. 39, 135-1 39. lversen, J. (1971). Secretion of glucagon from the isolated, perfused canine pancreas. J. Clin. Invest. 50, 2123-21 36. Judd, H. L., Rebar, R., Vandenberg, G. and Yen, S. S. C. (1974). Effect of luteinizing hormone-releasing factor on Leydig cell function. J. Clin. Endocr. Metab. 38, 8-13. Kelch, R. P., Jenner, M. R., Weinstein, R., Kaplan, S. L. and Grumbach, M. M. (1972). Estradiol and testosterone secretion by human, simian and canine testes, in males with hypogonadism and in male pseudohermaphrodites with the feminizing testes syndrome. J. Clin. Invest. 51, 824-830. Kley, H. K., Wiegelman, W., Nieschlag, E., Solbach, H. G., Zinunerman, H. and Kruskempter, H. L. (1974). LH, FSH and testosterone in plasma following LHRH infusion: a combined test for pituitary and Leydig cell function. Acta Endocrinol. 75, 417427. Lee, V. W. K., Cumming, I. A., de Kretser, D. M., Fmndlay, J. K., Hudson, B. and Keogh, E. J. (1976a). Regulation of gonadotrophin secretion in rams from birth to sexual maturity. I. Plasma LH, FSH and testosterone levels. J. Reprod. Feet. 46: 1-6, 1976. Lee, V. W. K., Cumming, I. A., de Kretser, D. M., Findlay, J. K., Hudson, B. and Keogh, E. J. (1976b). Regulation of gonadotrophin secretion in rams from birth to sexual maturity. II. Response of the pituitary-testicular axis to LHRH infusion. J. Reprod. Fert.46:7-11, 1976. Leonard, J. M., Flocks, A. H. and Korenman, S. G. (1971). Estradiol (E2) secretion by the human testis. Proc. Endoc. Soc., 53rd Meeting, Abstr. 133, San Francisco: The Endocrine Society. Marshall, J. C., Anderson, D. C., Frasier, T. R. and Marsoulis, P. (1973). Human luteinizing hormone in man: studies of metabolism and biological

ET

AL.

action. J. Endocrinol. 56, 431-439. Maurer, W., Volkwein, U. and Tamm, J. (1973). The effect of intravenously administered human chorionic gonadotrophmn on plasma levels of testosterone and 5a-dihydrotestosterone in normal male subjects. Acta Endocrinol. 72, 615-624. Motta, M., Fraschini, F. and Martini, L. (1969). “Short” feedback mechanisms in the control of anterior pituitary function. In Canong, W. F. and Martini, L. (eds.), Frontiers in Neuroendocrinology, Oxford Univ. Press, New York, p. 211-25 3. Rippell, R. H., Johnson, E. S. and White, W. F. (1974). Effect of consecutive injections of synthetic gonadotrophin releasing hormone on LH release in the anestrous and ovariectomized ewe. J. Animal Sci. 39, 907-9 14. Roth, J. C., Grumbach, M. M. and Kaplan, S. L. (1973). Effect of synthetic luteinizing hormone releasing factor on serum testosterone and gonadotropins in prepubertal and adult males. J. Clin. Endocr. Metab. 37, 680-686. Salamonsen, L. A., Jonas, H. A., Burger, H. G., Buckmaster, J. M., Chamley, W. A., Cumming, I. A., Findlay, J. K. and Goding, J. R. (1973). A heterologous radioimmunoassay for FSH: application to measurement of FSH in the ovine estrous cycle and several other species including man. Endocrinology 93, 610-618. Sanford, L. M., Winter, J. S. D., Palmer, W. M. and Howland, B. E. (1974). The profile of LH and testosterone secretion in the ram. Endocrinology 95, 627-63 1. Tixier-Vidal, A. and Farquhar, M. G. (eds.) (1975). The Anterior Pituitary, Academic Press, New York. Wang, C., Youatt, G., O’Connor, S., Dulmanis, A. and Hudson, B. (1974). A simple radioimmunoassay for plasma testosterone plus 5a-dih ydrotestosterone. J. Steroid Biochem. 5, 551-555. Wollesen, F., Swerdloff, R. S. and Odell, W. D. (1974). Dose response to luteinizing releasing hormone (LRH) bolus injection and prolonged infusion in normal human males. Proc. Endoc. Soc., 56th Meeting, Abstr. 53.