MALE ANTIFERTILITY:

AN APPROACH

C. L. Kragt, K. K. Bergstrom, K. T. Kirton and S. E. Porteus Fertility Research, The Upjohn Company Kalamazoo, Michigan 49001

ABSTRACT Experimental data in the rat is presented for an approach to male fertility regulation. The concept involves administration of an antigonadotropic steroid plus a replacement androgen to inhibit spermatogenesis and to maintain normal secondary sex characteristics. A model system utilizing the adult male rat is described. FSH and LH concentrations in the plasma or serum increase 8-10 fold in response to castration. Secondary accessory organs atrophy significantly within 10 days following surgery. The near optimal replacement dosage of testosterone ropionate (TP) in castrated male rats was 40 ug/lOOgbody weight/day. Proverag (300 ug/lOOg BW/day), an antigonadotropic steroid,when administered to castrate males for 10 days decreased plasma LH and FSH concentrations to values observed in intact rats. TP (40 ug/lOOg BW/day) in intact males exerted an effect on accessory organs by the fifth day of treatment. The effect was still increasing after 10 days of treatment. Provera, in intact rats, inhibited accessory organ weights by the fifth day of treatment and no additional inhibition was detected after 10 days. A combination of Provera and TP in the intact male did not significantly affect accessory organ weights after 10 days treatment; however, testicular weights were significantly inhibited. In all animals treated with Provera,significant adrenal atrophy was evident.

Accepted

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1975

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1974

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INTRODUCTION Interest and monetary effortsarebeing directed toward the regulation of population growth by motivating the human male to accept a reversible, voluntary, 100% effective, pharmacological tool (having minimal side effects) which induces temporary sterility (1). Thousands of men have already chosen vasectomy which indicates the existence of a large population willing to voluntarily choose a method which is difficult to reverse (2). Female reproduction has been modified by manipulation of the hypothalamic-hypophyseal-gonadal neuroendocrine feedback system by use of such modified steroids as norethynodrel, mestranol and others. These compounds have been shown to act at a number of biological sites to induce temporary sterility (3). Most of these compounds as well as the natural compounds,estradiol and testosterone,have been shown to suppress gonadotropin (LH, FSH) secretion during midcycle in the intact female, as well as in ovariectomized or orchidectomized animals and in aging humans (3,4,5). One possible approach to a male "pill" would be to modify this neuroendocrine axis with an "antigonadotropic" compound to suppress spermatogenesis and replace any decrease in normal androgen production with exogenous androgen. Numerous investigators have proposed such an approach and much effort is currently concentrated on this problem (6,l). It has been known for decades that steroids and/or non-steroidal compounds with estrogen or estrogenic activity are very potent inhibitors of testicular development in the immature rat (7) and can produce chemical castration in the adult animal. Androgens,on the other hand,can also suppress fertility, however the dosages are much larger than those required for estrogens and the natural androgen may have biphasic effects due to endogenous conversion to estradiol (8). Debate has taken place in the literature on which steroids inhibit FSH and LH release in castrate male rats (9,10,11,12) and intact human subjects (3,4,5). The CIR steroid, estrone, has been shown to be more effective than Cl9 or $0 steroids in preventing post-castration increase in pituitary LH (8). Naftolin -et al. (13) argue that compounds which can be aromatized in the A ring may exert an action via their metabolites. Others argue that all such compounds are equipotent when LH and FSH suppression is measured (12). The model systems chosen to study these actions differ and the demonstration of selectivity of action on either FSH or LH is dependent upon the duration of castration or duration of drug administration or age of animal studied (12,14). Provera has been shown to be antigonadotropic in rats (7), monkeys (15) and man (16). The following studies were designed as initial attempts to determine: a) the optimum replacement dosage of testosterone in castrate male rats, b) the dosage of Provera which would decrease LH and FSH levels in castrate males, C) the time course of the effect of a replacement dosage of testosterone in intact rats on sex organ weight and gonadotropin secretion, d) the time course of the effect of Provera on gonadotropin secretion and accessory organ weight in the intact male, and finally, e) the effect of combining the antigonadotropin (Provera) with the replacement dosage of testosterone on gonadotropin secretion and action in the intact male.

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MATERIALS AND METHODS All rats were 70 days old and weighed about 300g at the start of treatment. In experiments 1, 2 and 4,male rats of the Upjohn Sprague Dawley strain were used, and in experiment 3 male Sprague Dawley rats from Car-worthFarms (Shaver Road, Portage, MI.) were used. The treated rats received daily injections (s.c.) of Provera, testosterone propionate (TP) or Provera + TP until the day of sacrifice. The compounds were dissolved in benzyl alcohol (~5%) in cottonseed oil and the hormone concentrations adjusted so that all rats receivedO.lml/lOOg body weight (BW). Control rats received similar amounts of cottonseed oil. Experiment 1: The rats were divided into 7 groups of 10 rats each. Six of these groups were castrated at 70 days, immediately before the first injection. The intact group and one of the castrate groups received cottonseed oil only, the others were given 50 ug TP; 50 ug TP + 300 ug Provera; 300 pg Provera; 50 pg TP + 125 ug Provera,or 125 pg Provera. All treatments are expressed per 1OOg and all rats were sacrificed after 10 days of treatment. Experiment 2: Intact males were divided into 3 groups of 10 rats each (treated) and 3 groups of 5 rats each (control). All treated rats received 40 ug TP/lOOg BW-and control rats received cottonseed oil. They were given daily injections (s.c.) until the day of sacrifice. One group of treated and one group of controls were sacrificed after 1 day of treatment, another group of each after 5 days of treatment and the last rats were sacrificed after 10 days of treatment. Experiment 3: Intact male rats of the same age were used and the same procedure was followed as in experiment 2, except that the treated rats received 300 ug Provera/lOOg BW instead of TP. Experiment 4: The same basic protocol was also used in the last experiment. Control rats were oil treated, and experimental rats were treated with a combination of Provera (300 ug) and testosterone (40 pg) per 1OOg BW. The time course of efficacy was determined by sacrificing animals at 1, 5 and 10 days after treatment. At the time of sacrifice,all rats were anesthetized with ether and 9 ml of blood was withdrawn from the abdominal aorta. Plasma or serum was frozen until assay for LH and FSH utilizing National Institutes of Arthritis and Metabolic Diseases reagents as described previously (10, 14). During these experiments,it was observed that use of plasma made utilization of the Micromedic Automatic Pipetting Station difficult. Serum was then utilized, and since similar values were obtained for hormones in either hand pipetted plasma samples or machine pipetted serum samples, all values are reported. The seminal vesicles and ventral prostate from each rat were weighed in all experiments; the testes were weighed in all intact animals, and the adrenals were weighed in experiments 3 and 4.

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RESULTS Bilateral castration for 10 days increased plasma LH and FSH concentrations to 8-9 times that observed in intact rats (Table I). The lack of testes also caused atrophy of seminal vesicles and ventral prostates. Fifty ug of testosterone propionate per day per 1OOg body weight maintained sex accessory organ weights and decreased plasma LH and FSH concentrations. This dosage appeared to be supraphysiological since the seminal vesicle and ventral prostate weights were significantly greater than those of intact animals. Provera had some androgen activity as indicated by ventral prostate and seminal vesicle growth at the highest dosage (300 ug). This dosage also decreased plasma LH concentrations in castrate animals to those observed in intact controls and FSH was decreased to about twice normal concentrations. Testosterone propionate treatment of intact males (TableII) for periods of 1, 5 and 10 days duration indicated that a significant androgen effect was detectable after 5 days of treatment and growth of seminal vesicles and ventral prostates continued throughout the lo-day treatment period. The data indicate that these tissues were stimulated to grow beyond normal homeostatic dimensions by the exogenous compound and a plateau of maximal growth was not demonstrated in this experiment. No testicular atrophy was apparent in this study. Plasma LH levels were difficult to interpret since levels were minimally detectable (~30 ng/ml) (14) in intact animals as well as in all treated animals. Since LH was detectable in only 2 of 10 animals after 1 day's treatment, it can only be suggested that the compound was already affecting the hypothalamic-hypophyseal system at that time. Plasma FSH concentrations were unaffected by the treatment. Provera treatment (Table III)ofintact males for 1, 5 and 10 days duration indicated that significant anti-androgen activity, as indicated by seminal vesicle and ventral prostate atrophy, was detectable 5 days after starting the treatment. This effect was probably maximal by that time since the effect of 10 days treatment was not different from that observed after 5 days. Again LH concentrations were minimally detectable and drug effects were difficult to interpret. However, FSH values were easily measured in control rats and a progressive decrease occurred over the lo-day treatment period. A regression of testicular weight may have been present transiently after 5 days of treatment. No decrease in body growth was observed over the treatment period. Control ventral prostate and seminal vesicle weights for these Carworth rats were significantly heavier than those observed in control Upjohn rats in experiments 1, 2 and 4 (Tables I, IIand 19. Adrenal weights were significantly decreased (Pc.01) at both time intervals (5 and 10 days) studied.

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VOL. 11 NO. 1

310.6k7.7 316.Ok6.1

322.7k4.5 323.8k4.0 320.6k4.5 319.2k3.0 315.1+3.3 319.74.4

L.Castrate controls

3.50 ug testosterone propionate

4.50 ug testosterone + 300 pg Provera

5.300 ug Provera

6.50 pg testosterone + 125 pg Provera

7.125 pg Provera

94.5+13.4**

285.6k17.6

126.0_+5.5**

455.6+21

52.5* 4.7

61.5k 4.1

58.8+ 5.5

50.1+ 5.0

469.6-+19

781.7%3

471.6+25

487.4?16

72.52 7.9** 303.5+61.2** 2328.6fl25**

329.5+12.4** 293.Ok21.6

141.1+12.8"*

304.5313.6

FSH ng/ml RP-1

PLASMA

61.0+ 6.0

LH

41.8? 2.6** 500.9+38.1** 3526.6%33*

283.2k20.5

Ventral Prostate Weight (mg)

373.3+17.9** 351.9+26.9*

104.8+ 5.5**

266.7212.9

Seminal Vesicle Weight (m&

+standard errors *Significantly different from intact controls, Pc.05. **Significantly different from intact controls, Pc.01. LH values in treatment groups 3, 4, and 6 are based on averages of values from 7, 9, 8 or 10 animals initially in each group.

308.6k4.8

302.4k6.2

318.6t4.7

317.4k5.7

329.6k6.8

328.9+4.4+

l.Intact Controls

FINAL

INITIAL

TREATMENT GROUP

BODY WEIGHT (g)

EFFECT OF TESTOSTERONE PROPIONATE AND PROVERA IN CASTRATE MALE RATS

TABLE I

5 w

z

control (10 days)

5 in

31O.Ok5.8

312.023.7

304.0+6.2

311.6+10.4

323.4? 5.7

312.5+ 6.1

ND = not detectable ~30 ng/ml. *Significant treatment difference, Pc.05. **Significant treatment difference, Pc.01.

+standard errors

40 ug testosterone for 10 days

control (5 days)

311.2k4.0

40 ug testosterone for 5 days 319.Oi-6.8

316.41 4.8

313.2~6.1

control (1 day)

E cc

3

2

309.2+ 4.9

FINAL

311.6?21+

INITIAL

40 pg testosterone for 1 day

TREATMEfl GROUP

BODY WEIGHT (g)

182.2+16.4X*

483.7k20.0

213.8+13.3**

385.6k15.7

275.9k31.7

316.31t13.2

Seminal Vesicle Weight (mg)

270.6+31.4**

600.5t38.6

282.0+ 6.0*

412.7k33.5

256.8242.0

223.0k16.6

Ventral Prostate Weight (mg)

N.D.

3139.8k145.8

3343.0C132.3

3338.5k109.5

3404.Ok125.2

N.D.

N.D.

N.D.

N.D.

N.D.

3083.0+ 82.8

FSH

449.7*14

458.4222

477.4+10

512.31r18

541.1+30

510.8?21

ng/ml RP-1

LH

PLASMA

3303.6k 62.1

Testes Weight (mg)

EFFECT OF TESTOSTERONE PROPIONATE IN INTACT MALE RATS

TABLE II

CONTRACEPTION

JANUARY

1975 VOL. 11 NO. 1

307.6t4.0

308.4f2.3

306.8k4.5

305.8k2.5

304.4f3.5

control for 1 day

Provera + TP for 5 days

control for 5 days

Provera + TP for 10 days

control for 10 days

3139.0+ 73

Testes Weight (mg)

34.9k.75

FSH

2a.ot3.2 454.7k14.5

38.7C2.0 472.3216.4

34.6f1.4 456.lirli3.9

ng/ml RP-1

LH

PLASMA

32.1k1.3

33.6t1.2 485.1+29.0

3265.0+180** 44.2+2.5** 58.3k5.1 666.7+41.4*

2765.6+ 56

3420.0+102** 43.0+1.8** 42.5-14.6546.7c29.5**

3044.0? 50

42.4k2.6

39.1f1.4

Adrenal Weight (mg)

+standard error *Significantly different, Pc.05. **Significantly different, Pc.01. Body and organ weights are compared to group control; hormone concentrations with pooled controls.

[email protected]+ 7.0 350.2k39.4

342.2i4.4 233.2* 6.9 283.9C16.1

334.Ok5.5 203.9+ 9.3 306.1k38.5

326.0k3.1 207.3? 6.3 246.5k15.5

306.8k4.2 217.6k13.1 300.2+31.3* 3280.0? 16

309.0+3.0+ 307.2k2.6 198.4+ 6.9 224.6t14.6

FINAL

BODY WEIGHT (g)

INITIAL

Ventral Prostate Weight (mg)

Provera + TP for 1 day

TREATMENT GROUP

Seminal Vesicle Weight (mg)

EFFECT OF PROVERA AND TESTOSTERONE PROPIONATE IN INTACT MALE RATS

TABLE IV

P

4

2

CONTRACEPTION

Administration of Provera and testosterone propionate to intact males for periods of 1, 5 and 10 days indicated that the two compounds administered concurrently had an effect on body growth (Table IV) after 10 days of treatment. No change in seminal vesicle weight was observed during the lo-day treatment period. A transitory decrease (Pc.05) in ventral prostate weight on the first day following treatment was noted. Testicular atrophy was significant by the fifth day of treatment (Pc.05) and was greater by the 10th day of treatment (PcO.01). Adrenal weights were suppressed in all groups; however, the differences were significant in only the 5- and lo-day treatmentgroups. Plasma LH levels were at minimally detectable concentrations in all groups while FSH levels were significantly decreased in all treated groups.

DISCUSSION Pharmacological intervention with the fertility of the human male has had little success. Numerous points in the total sequence of events which lead from germ cell formation to insemination can be considered for manipulation. Spermicides in the female tract, mechanical prevention of sperm-deposition by use of condoms or coitus interruptus are all external to the body of the male. Any successful method for fertility regulation in the male must consider many factors such as the proper compound or compounds to achieve optimal pharmacological results with minimal physiological change, time course of administration, routes for delivery, and mutagenic properties of the manipulation. Behavioral parameters outlining acceptance or rejection of the procedure are equally as important. The choice of compound or compounds that could be utilized for pharmacological achievement of antifertility in the male while producing only minimal side effects such as testicular atrophy or adrenal suppression is critical. Gonadotropin inhibiting activity of probable compounds have been characterized in immature male rats. Depression of testicular and accessory sex glands weights can serve as suggestions that reduction in circulating pituitary gonadotropins has occurred or that the target glands have decreased in responsiveness. In such studies rats are usually treated at 25 days of age for a lo-day period and then sacrificed. A number of investigators including one of the authors have shown that at this age the pituitary FSH and LH concentrations of untreated male rats are increasing at very rapid rates. Plasma levels of the two hormones initially decrease and then increase during this age period (17) and the steroid negative feedback system is maximally responsive to testosterone. These changes are reflected in the rapid testicular and sex accessory organ weights and fertility onset (7,14). Administration of drugs in the Upjohn antigonadotropin screen in the immature (25-35 days old) male rat during this period of dynamic pituitary changes has contributed much information which can be utilized to select probable candidates from the many steroids with antifertility properties in males. Figure 1 isaschematic summary of selected data (7) to illustrate this concept. Control testicular weights may range from 1200 to 1500 mg and minimal weights after treatment are about 400 mgs. In general the potent estrogens such as ethinyl estradiol are the most potent inhibitors of testicular growth.

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VOL. 11 NO. 1

99

CONTRACEPTION

(6”‘) lH913M S31S32

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CONTRACEPTION

Provera does inhibit gonad development but at dosages of about 300 pg. The antigonadotropic effect of Provesty a combination of ethinyl estradiol and Provera in a ratio of 1/200,could be attributed to the ethinyl estradiol. Provera apparently has antigonadotropic activity only when administered subcutaneously and not when administered orally; however, it does have some androgen activity when administered by this latter route (7). Nonetheless, Provera was chosen for these initial studies because of its minimal androgen or estrogen activity yet significant antigonadotropic activity at 300 pg/lOOg body weight. The data presented in this report and the fact that it has previously been shown to inhibit formation of basophilic cells in the immature male pituitary suggests that Provera most probably exerts its primary action at that level (18). Differences exist in hypothalamic-hypophyseal negative feedback sensitivity between adult and immature animals (12,14), therefore one should expect some.difficulty when extrapolating from the immature to the adult rat. Differences exist in the enzymatic capacity of the liver and testes which influence the biological activity of exogenous or endogenous hormone at various ages (19). The adult castrated rat was chosen as one model in these experiments because of general agreement in the literature from many laboratories (10,11,14,20) that plasma levels of LH and FSH increase within l-2 days after orchidectomy in the adult rat. Study of the effects of hormones after long term castration has indicated that such model systems are different than those utilizing acutely castrated animals (11). Immediate replacement with exogenous compound after castration may mimic to a greater degree the oral administration of a drug to the intact animal. Pituitary storage of LH begins to increase above control concentration 10 days after castration and is still increasing 2 months or a year after surgery (12). Pituitary FSH decreases transiently for the first 5-7 days and then progressively increases to near normal levels (21,22).Dataobtained by one of the authors in another laboratory (14) indicated that the replacement dosage of TP required after castration would be approximately 50 ug/day/lOOg body weight. This dosage in the current experiments was supraphysiological; 40 yg was a closer yet not optimal therapy. Ramirez and McCann (23) have shown that similar dosages also normalize pituitary LH in adult castrated male rats. In these experiments, the goal was to manipulate those endpoints known to be involved in male fertility with compounds known to possess certain biological activities rather than to develop the treatment duration and optimum dosage needed to induce total infertility. Such experiments would need to be conducted in the future. Provera at dosages which are antigonadotropic in the immature male (7) also completely SUQpressed LH and partially decreased FSH in the castrated male. In intact rats testicular atrophy was evident by 10 days of treatment with Provera in conjunction with TP; however, Provera alone had only transitory effects on the testes. It was clearly an antiandrogen in mature intact animals. It is interesting to speculate that,in the presence of Provera,more testosterone is converted to estradiol which is more effective as a testicular inhibitor.

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CONTRACEPTION Other compounds that have been considered for male antifertility in the literature include estradiol which has been shown to act predominantly by inhibiting synthesis of new FSH in the male pituitary. Administration of as little as 0.5 ug per day for 5 days in adult males will significantly decrease pituitary FSH; however, 10 ug per day is necessary before plasma FSH levels are decreased (22,24). Kalra and Prasad (25) have utilized clomiphene (an antiestrogen with estrogenic activity) in the adult male rat in very high dosages (250 ug/day) as a selective inhibitor of gonadotropin release. This compound has no effect on thyroid or adrenal function in contrast to other steroids including Provera. Pituitary FSH is significantly decreased by this treatment and spermatogenesis is inhibited at the primary spermatocyte stage in the immature rat. Replacement or treatment of animals with androgen (TP) will restore accessory organ weights; however, spermatogenesis was still arrested in step 7 of spermatid formation (26). The literature is highly controversial in the area of the effects of other progestational agents on spermatogenesis. Hydroxyprogesterone caproate and hydroxyprogesterone acetate have been shown to have no effect, while Norethynodrel and ethynylestradiol-3 methyl ether, norethindrone and norethisterone produce reversible inhibition of spermatogenesis in the rat. The efficacy was shown to be related to the ability to suppress pituitary gonadotropins. Progesterone, 17ahydroxypregnenolone and AlI-pregnen-3, 20-dione have also been shown to induce atrophy of the seminiferous epithelium of the rat testes. These studies did not utilize plasma levels of pituitary hormones as an endpoint, only pituitary storage was measured. Most of these compounds also inhibit spermatogenesis in the human (26). With additional data, more insight could be gained into the site of action for antispermatogenic actions by comparison of the selective effects on LH and FSH secretion. In the opinion of the authors, the concept that administration of an antigonadotropic steroid plus replacement androgen to influence pituitary and gonadal function appears to be valid. Optimum compounds and time duration of efficacy for fertility inhibition remain to be determined. REFERENCES 1.

Inventory of Federal Population Research (Fiscal Year 1972) DHEW Publication No. 73-133.

2.

Berelson, B. World Population: lation Council Publication 15.

3.

International Encyclopedia of Pharmacology and Therapeutics: Pharmacology of the Endocrine System and Related Drugs, Progesterone, Progestational Drugs and Antifertility Agents, Sec. 48, Vol. I and II, ed. M. Tausk, Pergammon Press, N.Y. 1971.

4.

Cargille, C. M., Ross, G. I. and Rayford, P. L. Effect of Oral Contraceptives on Plasma Follicle Stimulating Hormone, In: Gonadotropins, Proc. Workshop Vista Hermosa, Morelia, Mexico, 1968.-

Status Report 1974, p. 37.

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5.

Swerdloff, R. S. and Odell, W. D. Gonadotropins Present Concepts in the Human, -In: Medical Progress California Medicine 109:467, 1968.

6.

Segal, S. J. Male Fertility Control Studies: Contraception 8~187, 1973.

7.

Lyster, S.

8.

McDonald, P. C., Rombout, R. P. and Siiteri, P. K. Plasma precursors of estrogen. I. Extent of conversion of plasma A'-androstenedione to estrone in normal males and nonpregnant castrate and adrenalectomized females. J. Clinical End. Metab. 27:1103, 1967.

9.

Ryan, R. J. and Philpott, J. E. Effect of androgens on concentrations of LH and FSH in the male rat pituitary. Proc. Sot. Exp. Biol. Med. 124:240, 1967.

Unpublished observations.

An editorial comment.

The Upjohn Company.

10.

Gay, V. L. and Bogdanove, E. M. Plasma and pituitary LH and FSH in the castrated rat following short term steroid treatment. Endocrinology 84:1132, 1969.

11.

Gay, V. L. and Dever, N. W. Effects of testosterone propionate and estradiol benzoate - alone or in combination - on serum LH and FSH in orchidectomized rats. Endocrinology 89:161, 1971.

12.

Mahesh, V. B., Muldoon, T. G., Eldridge, J. C. and Koroch, K. S. Studies on the regulation of FSH and LH secretion by gonadal steroids, p. 730. In: International Symposium on Gonadotropins, (eds.) B. B. Saxena, C.G. Beling, H. M. Gandy. Wiley-Interscience, N.Y. 1971.

13.

Naftolin, F., Ryan, K. J. and Petro, 2. Aromatization of androstenedione by the anterior hypothalamus of adult male and female rats. Endocrinology 90:925, 1972.

14.

Block, G. J., Masken, J., Kragt, C. L. and Ganong, W. F. Effect of testosterone on plasma LH in male rats of various ages. Endocrinology 94:947, 1974.

15.

Cornette, J. C., Kirton, K. T., and Duncan, G. W. Measurement of medroxyprogesterone acetate (Provera) by radioimmunoassay. J. Clin. Endocr. Metab. 33:459, 1971.

16.

Schwallie, P. C. and Assenzo, J. R. Contraceptive use - efficacy study utilizing medroxyprogesterone acetate administered as an intramuscular injection once every 90 days. Fert. Steril. 24:331, 1973.

17.

Kragt, C. L. and Masken, J. F. Puberty: Physiological Mechanisms of Control. J. of Animal Science 34:Suppl. 1, 1-18, 1972.

18.

Logothetopolous, J., Sharma, B. B. and Kraicer, J. Correlation of anterior pituitary morphology with thyroid function after 6a-methyl-

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17a-hydroxyprogesterone acetate.

Endocrinology 71:660, 1962.

19.

The "Puberty" of the rat liver. II. Denef, C. and DeMoor, P. Permanent changes in steroid metabolizing enzymes after treatment with a single injection of testosterone propionate at birth. Endocrinology 83:791, 1968.

20.

Goldman, B. D., Grazia, Y. R., Kamberi, I. A. and Porter, J. C. Serum gonadotropins concentration in intact and castrated neonatal rats. Endocrinology 88~771, 1971.

21.

Steinberger, E. and Duckett, G. E. Pituitary total gonadotropins, FSH and LR in orchidectomized or cryptorchid rats. Endocrinology 79:912, 1966.

22.

Steinberger, E. and Duckett, G. E. Studies on the mechanisms controlling the release of FSH from the pituitary gland, In: Gonadotropins Proc. Workshop Vista Hermosa, Morelia, Mexico,y968.

23.

Ramirez, V. D. and McCann, S. M. Inhibitory effect of testosterone on luteinizing hormone secretion in immature and adult rats. Endocrinology 76:412, 1965.

24.

Parlow, A. F. Differential action of small doses of estradiol and gonadotropins in the rat. Endocrinology 75:1, 1964.

25.

Kalra, S. P. and Prasad, M.R.W. Effect of FSH and testosterone propionate on spermatogenesis in immature rats treated with clomiphene. Endocrinology 81:965, 1966.

26.

Steinberger, E. Hormonal control of mammalian spermatogenesis. Physiological Reviews 51:1, 1971.

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Male antifertility: an approach.

MALE ANTIFERTILITY: AN APPROACH C. L. Kragt, K. K. Bergstrom, K. T. Kirton and S. E. Porteus Fertility Research, The Upjohn Company Kalamazoo, Michi...
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