Antispermatogenic effects of tolnidamine in langur (Presbytis entellus )

CONTRACEPTION

ANTISPERMATOGENIC IN

LANGUR

EFFECTS (Presbytis

OF TOLNIDAMINE entellus)

NK Lohiya, AS Ansari and P Scorza Barcellotd Reproductive Physiology Section, Department of Zoology University of Rajasthan, Jaipur 302004, India; and *F Angelini Research Institute, Viale Amelia 70 00181, Rome,

Italy

ABSTRACT

(50 mg/kg body weight; twice a week; oral) was Tofnidamine administered for 90 days to adult male langur monkeys (Presbytis entellus entellus Dufresne) to assess its contraceptive potential. Semen weight, volume, seminal fluid volume, colour , pH and libido remained unchanged. Sperm motility, vitality and morphology were impaired with treatment. Sperm severe advancement of density reduced to the oligospermia following 75-90 days of treatment. Increased number of immature germ cells were also noticed. Resumption of changes to following 90 days of cessation of pretreatment range was observed However, density remained low all through the treatment. sperm recovery period of 750 days. Seminal fructose, ACP, LDH and citric A significant depletion in acid concentrations did not change markedly. GPC and magnesium levels was recorded during treatment and early recovery periods. Alterations in germ cells and Sertoli cells were also A progressive but reversible rise in serum creatinine was observed. Other clinical parameters and body weight response revealed evident. drug-related tolnidamine medication no alterations. In conclusion, induced irreversible inhibition of spermatogenesis.

Submitted for publication Accepted for publication

MAY 1991 VOL. 43 NO. 5

December 10, 1990 March 12, 1991

485

CONTRACEPTION INTRODUCTION

Tolnidamine, l-[ (4-chloro-2-methylphenyl)methyl]-1H-indazole-3-carboxylic acid, belongs to the chemical class of lH-indazole-3-carboxylic acids. These agents exert antispermatogenic activity [1] and have two cell targets, Sertoli cells and germ cells, in the seminiferous epithelium [2]. A number of reports indicate that treatment of rodents and rhesus monkey with single dose or short-term studies with multiple doses ranging between 40 and 500 mg/kg produces strong antispermatogenic effects [ 3-61. No long-term investigations, however, have been carried out so far with multiple doses. A 4-week pilot study has also been conducted in rhesus monkey fed tolnidamine orally. Dosages of 200 mg/kg and 400 mg/kg produced aplasia of the germinal epithelium with abnormal renal function [3]. It is not clear from these studies, however, whether testicular inhibition could be achieved with long-term administration of tolnidamine at low doses without impairment of renal function. To determine this, we have carried out seminological and clinical investigations in langur monkey. MATERIALS

Tolnidamine previously

[l].

was

synthesized

AND METHODS

by

the

procedure

as

described

Animals Five adult male langur monkeys (Presbytis entellus entellus Dufresne) weighing between 14 and 18 kg, trapped around Jai-India, were subjected to quarantine for 2 months. During this period, animals were conditioned to handling, blood and semen collection. Animals were housed individually in metallic cages (measuring 0.75 x 0.75 x 1.05 m) Animals were fed with green under seminatural captive conditions. seasonal fruits and wheat chapaties, leaves, roasted/soaked grams, vegetables. Treatment

The study I;;) (iii)

schedule

period

was divided

Pretreatment phase Treatment phase Recovery phase

Animals received tolnidamine with a piece of banana during Parameters

of

in three -

phases:

30 days; 90 days; and 150 days.

(50 mg/kg b. wt. ; twice the treatment phase.

a

week)

orally

study

Semen analysis Semen samples were collected fortnightly by penile electroejaculation for studying seminal characteristics and seminal plasma biochemistry.

485

[7]

MAY 1991 VOL. 43 NO. 5

CONTRACEPTION Seminal characteristics. Semen weight, volume, seminal fluid volume, motility (qualitative and quantitative), density, colour , pH, sperm abnormality and immature germ cell per cent were investigated vitality, [a]. The sperm density was made only in seminal fluid exuded from coagulum .

Seminal plasma biochemistry. Fructose and citric acid 191, acid phosphatase (ACP) [lo], lactic dehydrogenase (LDH) [ 111, magnesium (GPC) [ 131 were estimated in glycerylphosphorylcholine [12] and seminal plasma. Testicular

biopsies

prior to and monthly during biopsies were performed Testicular after sedation with thiopentone treatment recovery periods, and injection (25 mg/kg b. wt. ; i.v.) (May 8 Baker (India) Ltd., Bombay). fixed in Bouin’s fixative, Biopsy samples were immediately dehydrated, cleared and embedded in paraffin wax. Sections, cut at 5 with haematoxylin-eosin and examined under light pm, were stained microscope. Seminiferous tubule cell nuclei diameter

diameter, were also

Leydig cell nuclei diameter measured with an oculometer

and Sertoli [ 141.

Radioimmunoassay Blood samples were collected prior to and every 30 days during Serum was separated and stored at -20°C for treatment and recovery. Serum levels of testosterone were assayed in subsequent analysis. triplicate using specific radioimmunoassay (RIA) method [ 151. Antisera Binding of antisera ranged was raised against T-3 CMO-BSA in sheep. from 45 to 65%. Serum pools gave an intra- and interassay coefficient of variation of 3.45% and 8.67%, respectively. The sensitivyy was 10 to 10,000 counts/minute/tube of (1,2,6,7H)-testosPg. About 8,000 terone were used as tracer. Clinical

investigations

Blood samples were collected monthly for the subsequent determinations and blood and/or serum biochemistry. Haemcglobin, of haematology haematocrit , RBC, WBC and differential leucocyte counts were made. Standard haematological indices were also calculated [ 161. Blood urea [ 171, serum alkaline phosphatase (ALP) [ 181, SGOT, SGP+T [ 191, +LDH and K by (111, bilirubin [20], creatinine [21] and electrolytes (Na flame photometry) were estimated during all phases of the study. Body

In all

weight

the

response

phases

of study,

animals

were

weighed

after

every

2 weeks.

Libido to receptive Animals were introduced was Mounting and copulatory behaviour libido [ 141.

MAY 1991 VOL. 43 NO. 5

females, observed

month. once every in order to test the

487

CONTRACEPTION Statistics

Testosterone values were standard curve linearized as mean f SE and are Student’s paired ‘t’ test

determined from the best fit straight line of on logit-log co-ordinates. Data are expressed analyzed for statistical difference by using [22]. RESULTS

Semen

analysis

Semen weight, volume and seminal fluid volume remained unaltered throughout the study period. Sperm density decreased gradually to severe oligozoospermia within 75-90 days of treatment and remained low

-30 -15 PT

0

15 30 c15 60 75 90 15 30 TREATMENT

*

P
+

P<@OZ

0

P<

A

P
O-01

45 60 75 90 105 120 135 150 RECOVERY

DURATION IN DAYS Fig.

488

1. Sperm density (million/ml and million/ejaculate) shown with pretreatment (PT) , treatment (tolnidamine : 50 respect of periods in oral) and recovery mg/kg b.wt./twice a week; Values are mean * S.E. Vertical bars denote langur monkey. A significant decrease in sperm density up to severe S.E. oligospermia following treatment was noted. Sperm density was low during the recovery period of 150 days.

MAY 1991 VOL. 43 NO. 5

CONTRACEPTION throughout the recovery period of 150 days (Fig. 1). Qualitative as motility reduced gradually during treatment well as per cent sperm A marked reduction in sperm vitality was observed with the schedule. A gradual significant increase in per cent advancement of treatment. germ cells was evident. All the sperm abnormality and immature changes (except sperm density) restored to normalcy following 75-90 semen colour and pH remained days of recovery (Fig. 2). However, unaffected. * Pi005 rP
0

I

-30 -15 0 PT

1530 45 60 75 90 15 30 45 W TREATMENT

7590105120135150

RECOVERY

DURATION IN DAYS

Fig.

2.

abnormality and immature germ Sperm motility, vitality, in langur monkey. tolnidamine administration following parameters during medication changes in all significant early periods of cessation.

cells Note and

LDH and citric acid concentrations remained Seminal fructose, ACP, GPC and magnesium levels were significantly low following unaltered. 45-60 days of treatment and restored to normalcy after 30-45 days of recovery (Fig. 3). Histopathological

Biopsies

of

findings

testes

taken

MAY 1991 VOL. 43 NO. 5

prior

to

drug

administration

showed

normal

CONTRACEPTION features with successive stages of transformation of the seminiferous epithelium into spermatozoa (Figs. 4,5). Seminiferous tubule diameter and Sertoli cell nuclei diameter decreased significantly following 90 days of treatment and 150 days of cessation. cell nuclei Leydig diameters were unaltered (Table I). Histologic examination of the testicular biopsies taken at 90 days of treatment showed various of cellular damage both in germ cells and Sertoli cells. degrees Diminished was evident in most of tubules spermatogenesis the consistently at spermatocyte stage. Depopulation of spermatogonia was evident at certain places. were undergoing Pachytene spermatocytes degeneration. Disorganization and sloughing of immature germ cells were visible. The nuclei became pyknotic. The Sertoli cells nuclei were situated stained. No elongated, narrower, basally and deep1 y morphological changes were noticed in the Leydig cells (Figs. 6,7). At similar microscopic features were seen as 150 days of recovery period, in 90 days of treatment (Figs. 8,9). PRETREATMENT

+

P(O.02

TREATMENT

0

P< 0.01

RECOVERY

1‘15

D I5 45 75 15 45 75 105 135 TREATMENT

Fig.

3.

DURATION

45

TRLATMENT

15 15 45 75 105 135 OURATION(

DAYS)

Decreased levels of GPC and magnesium during treatment and Each bar denotes early periods of recovery were observed. mean * S.E.

Testosterone

levels

The levels of testosterone phases of study (Fig. 10). Clinical

1 DAYS)

were

within

pretreatment

range

in

all

the

investigations

haematological parameters did not Following tolnidamine administration, Serum transaminases, ALP, LDH, bilirubin show appreciable ch$nges. within pretreatment range. and K+) remained and electrolytes (Na

490

MAY 1991 VOL. 43 NO. 5

CONTRACEPTION

Fig.

4.

Fig.

5.

Fig,

6.

Fig.

7.

Fig.

8.

Fig.

9.

of langur testis prior to tolnidamine medication Histology of transforwith successive stages normal features showing mation of seminiferous epithelium to spermatozoa. HE x 400. have Same testis as Fig. 4. Nuclear membrane of Sertoli cells typical infoldings at places. Note faint, fine chromatin granules with a prominent centrally situated nucleolus. HE x 1000. for 90 days. Note testis treated Histology of langur with reduced number of epithelium depopulation of germinal Sertoli cells nuclei were of abnormal shape. spermatogonia. Leydig cells are quite normal. HE x 400. Testis of same animal as in Fig. 6. Sertoli cells nuclei are HE x 1000. elongated, narrower and deeply stained. Testicular histology of langur following 150 days of recovery, comparable to 90 days of treatment. HE x 400. of Sertoli cells is Same testis as Fig. 8. The morphology similar as in Fig. 7. HE x 1000.

MAY 1991 VOL. 43 NO. 5

491

CONTRACEPTION TABLE culometric

measurements administration

I

of testis in langur Leydig nuclei

after tolnidamine monkey

Duration

Seminiferous tubule diameter

Pretreatment

178.65+6.41

5.6320.07

9.4220.10

90

120.56k6.19’

5.3OiO.20

6.96+0.20+

127.79+4.08*

5.5OtO.16

5.81?0.11+

as

n = 5;

0J )

(I.r)

150

days

treatment

days

Values

recovery

are

expressed

cell diameter

mean

+ S.E.;

Sertoli nuclei

cell diameter

(11)

“p

< 0.01,

+ p < 0.001.

PRETREATMENT TREATMENT RECOVERY

1

0 30 60 90 30 60 90 120 150 TREATMENT DURATION (DAYS)

Fig.

492

10.

Circulating during the Testosterone

levels of administration levels did

testosterone in of tolnidamine, not change.

langur monkeys before, and following recovery.

MAY 1991 VOL. 43 NO. 5

CONTRACEPTION Blood urea did not deviate beyond pretreatment values. A gradual but nonsignificant elevation was observed in serum creatinine levels which dropped to pretreatment range following 30 days of recovery (Fig. 11). Body

weight

and libido

appreciable changes introduced to receptive mounting and copulatory throughout the study.

No

PT

Fig.

11.

recorded in body weight. Animals were month, showed normal female, once every to successful copulation behaviour leading

TREATMENT

RECOVERY OUR&TION IN DAY5

creatinine levels following tolnidamine medication. Note a progressive nonsignificant elevation during treatment period.

Serum

OISCUSSION

Results of the present investigation revealed that tolnidamine possesses antispermatogenic activity. Disturbances in the testicular function were evident by severe oligospermia [Z]. Impaired sperm motility, vitality and increased number of immature germ cells in the ejaculates can be demonstrated owing to interference in the post-testicular maturation of spermatozoa [23]. Tolnidamine-induced sperm abnormalities were in accordance with the observations of Heywood and his colleagues [3]. Morphological and physiological maturation of sperm takes place during passage through epididymis [24,25]. Epididymis reacts more sensitively other [26,27] and to androgens than androgen-dependent organs organ is integrity of this dependent both on the physiological testicular fluid and circulating androgens [28]. The impaired functional activities of spermatozoa can also be explained by an interference with the metabolism of epididymis due to the alteration in the testicular fluid affecting its internal milieu, which may have affected the process

MAY 1991 VOL. 43 NO. 5

403

CONTRACEPTION of physiological levels of GPC assumption.

maturation of sperm. A significant reduction in the following tolnidamine treatment further supported this

It is evident that circulating levels of testosterone are required for the maintenance of extra-testicular androgen action, i.e. accessory sex organ function, libido and potency, but the threshold levels of required hormone might be different for different functions. The unimpaired semen weight, volume, seminal fluid volume and different seminal plasma constituents provide a support to the observations of Scorza Barcellona et al. [6], who reported normal functions of ventral prostate and seminal vesicle in rats treated with doses ranging between 35 and 100 mg/kg of tolnidamine. Low levels of GPC and magnesium suggest that the different secretory products of the same organ may not have good correlation [29]. The normal mounting and copulatory behaviour and maintained androgen levels suggested unimpaired libido [4]. In general, when any drug affects the Sertoli cells, an irreversible effect might be achieved [30]. In the present study, morphological alterations in the Sertoli cells as well as in germinal epithelium were found at 90 days of treatment and even after 150 days in recovery of sperm density was evident due of recovery . The failure to permanent alterations in germ cells and Sertoli cells morphology. Blood urea and serum creatinine are two important parameters of renal function. Heywood et al. [3] detected abnormal renal function in rhesus monkeys on day 7 with 400 mg/kg/day and on day 28 with 200 Administration of low doses of tolnidamine in mg/kg/day dose regimen. the present investigation, however, failed to induce any such changes. the azoospermia and the reversibility of sperm density Nevertheless, could not be achieved at the given from severe oligospermia dose long-term animal experiments are suggested with regimen. Further different dose regimens for detailed information on the irreversibility and toxicity and possibility of using this new class of compound as a male contraceptive agent. ACKNOWLEDGEMENTS

Financial support for this investigation was provided Council of Medical Research, New Delhi. We thank Mathur for the use of the facilities.

by the Professor

Indian R.S.

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