Experience with a potent LH-RH agonist, Buserelin, alone and in combination with testosterone for antispermatogenic activity, reversibility and toxicity in langur monkey

CONTRACEPTION EXPERIENCE COMBINATION

WITH WITH

A

POTENT

LH-RH

TESTOSTERONE

REVERSIBILITY

AND

AGONIST, FOR

TOXICITY

BUSERELIN,

ALONE

ANTISPERMATOGENIC IN LANGUR

AND

IN

ACTIVITY,

MONKEY

N.K. Lohiya, K. Sharma, D. Jayaprakash, A.S. Ansari, M. Kumar and S. Sharma Reproductive Physiology Section, Department of Zoology University of Rajasthan, Jaipur-302004, India

ABSTRACT

Chronic intermittent treatment of LH-RH superagonist Buserelln alone or in combination with testosterone enanthate were given to adult male langurs for 90 days to evaluate antispermatogenlc activity of alone and combination therapy, maintenance of normal androgenlclty, possible toxic effects of agonist treatment, related side effects of testosterone supplementation and complete reversibility of the procedure. A gradual decrease in sperm count was recorded in both treatment groups, along motility vitality of spermatozoa. with reduced and the In the oligospermia was achieved in 4 out of 5 animals, combination group, only 2 animals became oligospermic in the agonist alone whereas, group. Significant decrease in serum testosterone levels along with impaired libido and other testosterone withdrawal symptoms were observed in the Buserelln alone group, conversely normal testosterone levels and libido were observed in the combination group. An elevation in haematological variables and serum total protein concomitant with a slight gain in body weight of the animals were recorded in the these changes were not encountered in the agonist combination group; Reversibility of all the altered parameters to control alone group. range was observed in both treatment groups following ‘75 to 90 days of treatment withdrawal. INTRODUCTION

Previous studies have shown that the agonistlc analogues of LHantifertility agent in view of the useful as male RH may be paradoxical inhibitory action of supraphysiological doses [ 11. Neverthe total picture is complicated by the fact that the degree theless, LH-RH agonist, maintenance of of effects of observed and extent peripheral testosterone levels and hence libido, as well as return to are related to the dose and duration of the viable reproductive status, Although there appears to treatment and of the particular agonist [2]. be a commonality of effects in various experimental laboratory animals we are left in a still because of great species variability, [3,41, quandary as to which animal to use in predicting effects in human in men have been preliminary clinical trials clearly. However, with Buserelln alone [5,6] and also in combination with performed Submitted Accepted

for publication November 21, 1989 for publication December 12, 1990

FEBRUARY 1991 VOL. 43 NO. 2

187

testosterone esters [7-lo], but the results were unsatisfactory because uniform azoospermia had not been observed. Thus, the need for more studies in non-human primates prompted us to perform this experiment in adult male langurs; an animal model having close similarity to human in its reproductive exocrine and endocrine profile [ 111. MATERIALS (a)

(Hoechs??? Frankfurt (TE) (Testoiiron Depot,’

AND

METHODS

LH-RH agonist analogue; Buserelin (Hoe 766) F. R. Germany) and (b) testosterone enanthate German Remedied Ltd., Bombay).

Experimental animals : Adult male langurs (Presbytis entellus entellus Dufresne) were caught from the jungle around Jaipur, India. Fully grown animals weighing between 15 and 18 kg were selected. Animals were housed individually in metallic cages (measuring : 0.75 x 0.75 x 1.05 m) and were exposed to the seminatural laboratory conditions of our primate facility. Animals were quarantined for two months, sampling.

during which They were

they

were

trained

for

electroejaculation

and

blood

fed with controlled diet thrice a day; green leaves and roasted or soaked grams in the morning, wheat chapaties during noon and seasonal vegetables and fruits in the evening. Water was provided ad libitum. Veterinary supervision was provided throughout the study period. Experimental

three phases : phase, 90 days; animals

design : The study period consisted of pre-treatment (PT) phase, 30 days; (ii) and (iii) recovery phase, 90 days.

(i)

The animals in each :

were

allocated

into

three

groups

following treatment

containing

five

of this group were treated with vehicle, Group A : animals 0.1% bovine serum albumin + 0.9% NaCl, s.c., during the treatment phase to serve as control. Group B : animals of this group were treated with Buserelin ug/kg b.wt./day, s.c.), during the treatment phase. Group pg/kg days,

C : animals of b.wt./day, s.c.) i.m.),

during

Collection

this in

the

alone

(0.5

group were treated with Buserelin (0.5 with TE (2 mg/kg b.wt./l5 combination

treatment

phase.

evaluation of semen : Semen samples were penile electroejaculation [ 121 and were for semen weight, volume, seminal fluid evaluated macroscopically and microscopically for sperm density, motility volume, pH and colour, and vitality [ 131. obtained

fortnightly

and

by

Biochemical analysis of seminsl plasma : Estimation of seminal (LDH) [ 151, acid phosphaplasma fructose [ 141, lactic dehydrogenase acid [ 171, glycerylphosphorylcholine (GPC) [ 181, and tase [ 161, citric magnesium [ 191 were performed.

188

FEBRUARY 1991 VOL. 43 NO. 2

CONTRACEPTION

every

Libido

month for

:

Animals were introduced to receptive females observation of mounting and copulatory behaviour.

once

Testiarlar histology : Unilateral biopsies were taken before initiating treatment, at the end of treatment and recovery phases, under anaesthesia with sodium thiopentone (25 mg/kg b. wt., i. v. ) (May and Baker (India) Ltd., Bombay, India) in sterile conditions and were processed for light microscopic observations by routine procedure. Hormone assay : Serum testosterone levels were measured monthly at intervals of 08.00, 08.15 and 08.30 hr in triplicates by radioimmunoassay [ 201. The intra-batch coefficient of variation was 8.8%, the inter-batch coefficient of variation was 15.5%, with 10 pg sensitivity. Binding of antisera ranged from 45-65s. Clinical investigations : Blood samples were collected monthly from conscious animals by saphenous vein puncture for haematology [211 and serum biochemistry : alkaline phosphatase [ 161, electrolytes (Na and K+) [22], SGOT and SGPT [23], LDH [ 151, total lipids [24], phospholipids [ 251, total cholesterol [ 261, total protein [27], blood glucose [28], blood urea [29], and bilirubin [30]. monthly

Body weight : Body weight during all phases of study.

analysed

Statistical analysis : Data are expressed as mean f SE and are for statistical difference by using Student’s ‘t’ test.

of

the

animals

were

recorded

RESULTS alone treatment group, one animal selninology : In the Buserelin showed sperm count below 20 million/ml, in another one, sperm count was below 30 million/ml following 90 days of treatment, while the other two animals failed to ejaculate following 75 and 45 days of treatment, The fifth animal maintained sperm count around respectively. 50 to 60 million/ml during 60-90 days of agonist treatment (Fig. 1). motility Vitality and significantly following 60 days in semen weight (control 2.45 p ~0.001)

and

volume

(control

of spermatozoa the of treatment (Fig. 2). f 0.4 2.4 f

in p < 0.001) was also observed treatment. No remarkable alterations pH throughout the experimentation. In pronounced

the

combination

suppression

of

were depleted A marked decline

gm, treatment 1.05 f 0.2 ml, treatment 1.0

this were

(Buserelin spermatogenesis

0.03

gm,

f 0.05 ml, group following 90 days of recorded in semen colour and

+

TE) was

regimen observed

group, as

more compared to

the Buserelin alone group. In this group, severe oligospermia was achieved in 4 out of 5 animals (in 3 animals sperm count was below 5 million/ml and in 1 animal it was below 20 million/ml) and in the fifth animal sperm count did not decrease below 40 million/ml (Fig. 3). parallel

followed a pattern Impairment of sperm vitality and motility to the Buserelin alone group. Only 10% of the sperm were

FEBRUARY 1991 VOL. 43 NO. 2

189

CONTRACEPTION found live with poor to zero motility following 90 days of treatment (Fig. 2). No alterations were recorded in semen weight and volume during different intervals of treatment. All the animals responded to electroejaculation normally. Semen colour and pH did not show any significant variation.

Fig.

+ P<

0.01

A P<

0.001

-

GR-A

(CONTROL

O-Q

GR-8

lBUSERELIN:O~5yg/Kg b-Wdoy,s.c

density 1 Sperm Each recovery. show individual

1 )

treatment and following during pre-treatment, value represents mean k SE. Figures (i) to (v) response.

decrease in the Seminal plasma biochemistry : A significant seminal plasma biochemical constituents, fructose, LDH (Fig. 4)) acid citric acid (Fig. 5)) GPC and magnesium (Fig. 6)) were phosphatase, whereas all these parameters observed in the Buserelin alone group, except GPC remained within control and/or pre-treatment range in the combination group (Figs. 4, 5 and 6). Libido : The animals of the Buserelin alone treated group when exposed to receptive females showed impaired mounting and copulatory behaviour following 60 days of treatment, whereas the animals of the throughout normal sexual drive the exhibited combination group experimentation. Serum testosterane levels : The agonist alone regimen group showed a biphasic pattern with an initial stimulatory and subsequent Serum testosterone levels were elevated from down regulatory phase.

190

FEBRUARY 1991 VOL. 43 NO. 2

CONTRACEPTION baseline during the first 7 days of treatment followed by a gradual decline and the levels remained 50 to 60% of baseline control or pretreatment during 75 to 90 days of agonist therapy. The combination regimen resulted in an initial and then maintained serum increase testosterone levels throughout the study period (Fig. 7). tubules were seen smaller Testicular histology : Seminiferous with mean tubular diameter of 138 * 14 pm in in cross-sectional area, alone and 119 f 16 pm in combination groups, as compared to the vehicle-treated control (206 * 18 urn). Seminiferous tubules showed immature cells spermatogenic sloughing of germ and disruption, thickening of lamina propria. Spermatogonia was the only germ cell type observed in the combination group whereas in the agonist alone group spermatogonia along with some more advanced cell types were also observed. Interstitial tissue also appeared smaller in crossHistological changes observed in sectional area as compared to control. testicular biopsy samples of different animals of treatment groups were semi nological observations. Normal testicular in conformity with the histoarchitecture was observed following 90 days of recovery (Fig. 8).

GROUP A

GROUP B

CONTROL

GROUPC

SUSERELIN: 0.5~afl(p b.*-/ dwjs.c

l

.

P< P<

n,?&

0.05 0.02

+ P
PT

TREAT,

REC.

PT

P<

TREAT.

DURATION

Fig.

2 Sperm

0.001

REC.

IN

PT. TREAT.

REC.

DAYS

vitality and motility of groups, A, B and C during Each value represents treatment and recovery. treatment, f SE.

FEBRUARY 1991 VOL. 43 NO. 2

premean

191

increase in total RBC, Clinical chemistry : A reversible haematocrit serum total protein was haemoglobin, and recorded whereas r~ remarkable variations following the combination regimen, were recorded during the agonist alone treatment. activities of levels of electrolytes, the The serum phosphatase, lactic the transaminases, dehydrogenase and alkaline concentrations of total lipids, phospholipids, total cholesterol, and the blood urea and bilirubin fluctuated within levels of blood glucose, control and/or pre-treatment range in both treatment groups throughout the experimentation. +

P
A

P
-

GR-A

ICONTROL)

-

GR-C

(BUSERELIN:O-S)g/kg

0 PT

30

60

90

TREATMENT

DURATION

Fig.

hwday;

30 so RECOVERY

IN

S.C.

0 PT

90

DAY

during pre-treatment, 3 Sperm density Each value represents mean + SE. individual animal response.

30 60 90 30 M 90 TREAT REC

5

treatment and Figures (i) to

recovery. (v) show

and reversible body EIody weight response : A nonsignificant in some of the animals of the combination weight gain was recorded in the alone group the body weight did not show any group 1 whereas marked difference from control group. Recovery haematological

testosterone,

192

,

:

serum testicular

All

altered parameters, the circulating biochemical and and libido, histoarchitecture

seminological, levels of returned to

FEBRUARY 1991 VOL. 43 NO. 2

CONTRACEPTION normalcy treatment

following groups

75

to

90

days

of

treatment

30

45

withdrawal

60

75

in

both

90

RECOVERY

DURATION

Fig.

4

IN

DA Y s

of fructose and lactic plasma concentration 6 and C during pre-treatment, of groups A, mean + SE. recovery. Each value represents Seminal

dehydrogenase treatment and

DISCUSSION The present study demonstrates that the alone and combination evident by achieving regimen caused inhibition of spermatogsnesis, oligospermia. Some possible mechanisms have been suggested to explain the antigonadal actions of LH-RH agonist [ 311 : (i) chronic administhe anterior pituitary, tration of LH-RH agonist may desensitize diminished circulatory gonadotropins and atrophy of resulting in testicular function, (ii) high dose of LH-RH agonists may cause hyoercausing desensitization of Leydig secretion of LH from the pituitary, ceiis and subsequent spermatogenic impairment, and (iii) LH-RH agonist may exert direct inhibitory actions on the testis. The decline in sperm density was accompanied by a significant The pathogenesis of fall in sperm motility may in sperm motility. inhibition of testosterone biosynthesis and related to profound organs, patterns of accessory sex the impaired secretory hence, Exogenous1 y the LH-RH agonist. specifically of epididymis by fall be

FEBRUARY 1991 VOL. 43 NO, 2

CONTRACEPTION administered TE maintained the peripheral serum testosterone levels at the physiological range in the combination group as evidenced by normal behaviour androgenicity, it is sexual and probably by counteracting the testosterone withdrawal symptoms in a manner similar that to achieved in previous combination studies with our antiandrogenic agents [32, 331 and studies of others [34, 351. Impaired indicative of epididymal function, during the combination GPC levels, regimen, could be explained due to higher threshold requirements of androgens for maintenance of its function than other accessory sex glands [ 361.

*

P
IBUSERELIW:O~~~~~~

m-c

(~u5ER~LI~.d.S)rglk~bw./doy;r.c.

D U RATION

Fig.

iwldoY:s.cl

IN

DAYS

5 Seminal plasma concentrations of acid phosphatase and citric treatment and acid of groups A, B and C during pre-treatment, recovery. Each value represents mean f SE.

The results of this study are in contrast to the effects seen 381 in which subcutaneous in the monkeys [ 3’7, the rhesus administration of 5 pg or even higher doses of Buserelin did not show in pituitary or gonadal function. However, constant any alteration infusion of the agonist was found to be effective [4]. Whereas in men subcutaneous injptions of Buserelin at the same dose level or higher doses of [D-Trp ] LH-RH induced pituitary and gonadal down regulation [I, 39, 40-J. Rabin et al. 1411 have demonstrated that the Doelle et al. [40], administration of testosterone enanthate along with the agonist could compensate the testosterone withdrawal symptoms of the agonist alone

194

FEBRUARY 1991 VOL. 43 NO. 2

CONTRACEPTION

*

PCO.05

P
GR-A

ti

, CONTROL,

GR-8

(EUSERELIN.Q5BgIkg

CR-C

I BUSERELIN:O5~glkgDrld0y.l + TE

Fig.

br,Oov.rc,

2mgll;g

c. Lw,I5d~ys.~

m,

6 Seminal

plasma concentrations of glycerylphosphorylcholine magnesium of groups A, B and C during pre-treatment, and recovery. Each value represents mean f SE. -

GROUP- A CONTROL; VEHICLE TREATED

-----

GRWP-

-.-

GROUP- C BUSERELIN : 0.5 )rg/Kg t&day;

B BUSERELIN : O.Spg/Kg TE

plus

: 2mg/Kg

and treatment

Wdoy;s.c. S.C.

hw45doys; i.m. + PCO.01 A

P

II 0 7

PT

I 30

I 60

7 circulating pre-treatment, mean f SE.

levels

of

testosterone

treatment

FEBRUARY 1991 VOL. 43 NO. 2

and

I 60

I SO

RECOVERY

TREATMENT PERIOD

Fig.

1 30

I SO

PcO.001

IN

DAYS

of groups A, recovery. Each

B and value

C during represent

195

CONTRACEPTION

Fig.

8

Fig.

8a

Fig.

8b

Fig.

8c

Fig.

8d

Fig.

8e

Fig.

8f

196

langur testis during pre-treatment, His&architecture of treatment and recovery phases of groups B and C. HE x 200. Pre-treatment testis of group B (agonist alone) showing all stages of spermatogenesis. Testis after 90 days of agonist alone treatment showing marked disorganisation of reduction of tubular tubular elements, diameter. Premature sloughing of germ cells evident. Testis after 90 days of recovery. Note restored tubular diameter and normal spermatogenesis. Pre-treatment testis of group C (agonist + TE) showing normal process of spermatogenesis. Note Testis after 90 days of agonist plus TE treatment. reduction of seminiferous tubular size and spermatogenic disruption. Testis after 90 days of recovery showing normal tubular size and spermatogenesis.

FEBRUARY 1991 VOL. 43 NO. 2

CONTRACEPTION treatment, without modifying the LH-RH agonist’s influence on sperm density. In the present study the testosterone supplementation could attenute the testosterone withdrawal symptoms of the agonist and not the antispermatogenic potential of the analogue, it could also be seen that there were additional suppressive effects on the spermatogenesis, even though azoospermia could not be achieved. The changes in testicular morphology precede the reduction in sperm output. As yet, not many studies have provided a clear indication of the morphological consequences of the action of LH-RH agonist on the testis. Administration of LH-RH agonist is known to reduce sperm count, LH and FSH receptors in the testis, and to impair the steroidogenic capacity of the Leydig cells. This has been proposed to explain the resulting disruption of the seminiferous epithelium which is normally dependent upon stimulation by testosterone [42-441. Elevated values of RBC, total protein in the combination induced erythropoesis and protein were readily reversible after toxicological parameters revealed untoward effects on electrolytes, metabolism or on the heart, lung,

haemoglobin, haematocrit and serum therapy may be due to testosteroneanabolism [45]. However, the values withdrawal of the treatment. Other that the regimens did not exert any enzymes, lipid and carbohydrate liver and kidney functions.

In supplementation with conclusion, testosterone Buserel in resulted in more pronounced inhibition of spermatogenesis as compared to the agonist alone treatment and maintained normal androgenicity. However, further investigations with varying combination regimens are warranted to achieve uniform azoospermia. ACKNOWLEDGEMENTS

The financial support to this investigation the Indian Council of Medical Research and the Commission, New Delhi. The authors are thankful Saxens for providing facilities.

was provided by University Grants to Professor S.C.

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