Effects of Testosterone and Dihydrotestosterone on Spermatogenesis, Rete Testis Fluid, and Peripheral Androgen Levels in Hypophysectomized Rats*

FERTILITY AND STERILITY Copyright

c

Vol. 28, No. 10, October 1977 Printed in U.s.A.

1977 The American Fertility Society

EFFECTS OF TESTOSTERONE AND DIHYDROTESTOSTERONE ON SPERMATOGENESIS, RETE TESTIS FLUID, AND PERIPHERAL ANDROGEN LEVELS IN HYPOPHYSECTOMIZED RATS*

MARY E. HARRIS, PH.D.t ANDRZEJ BARTKE, PH.D.:J: JUDITH WEISZ, B.CH.§ DAVID WATSON, B.S. Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545

To compare the effects of testosterone (T) and dihydrotestosterone (DHT) on the maintenance and the restoration of spermatogenesis, hypophysectomized (APX) rats were treated daily for 35 days with 0.5 mg of T propionate (TP) or DHT propionate (DHTP) beginning 5 or 33 days after hypophysectomy. In the maintenance experiment, the weights of the testes and the number of early spermatids were significantly lower in DHTP- than in TP-treated animals, while late spermatids were present only in rats treated with TP. In the restoration experiment, TP increased testicular weight and the number of germinal cells, whereas DHTP had very little effect on the testis. In an attempt to explain these findings, we measured androgen levels in the rete testis fluid (RTF) and peripheral plasma of APX rats treated with TP or DHTP. The concentration of T in the RTF of TP-treated rats was nearly 3-fold higher than the level of DHT in the RTF of animals given DHTP. Plasma T levels measured %, 2, 4, and 24 hours after the last of three daily injections of TP were considerably higher than were the corresponding plasma DHT levels in animals given DHTP. In animals treated with free steroids, peripheral androgen levels between % and 4 hours after the last injection were much higher in rats given T than in those given DHT, but thereafter this difference disappeared. We conclude that the difference in the ability of subcutaneously injected TP and DHTP to maintain and to restore spermatogenesis in APX rats was due to a difference in androgen levels in the testes of these animals.

Spermatogenesis can be maintained in hypophysectomized (APX) rats by treatment with testosterone (T), other androgens, or nonandrogenic C-21 steroids. 1-3 However, under physiologic conditions, spermatogenesis appears to depend primarily on the high local concentration of T.4 The action of T on spermatogenesis may involve the reduction ofT to dihydrotestosterone (DHT). This

possibility is suggested by the mechanism of T action in the accessory reproductive glands and by the observations that testicular androgen receptors and testicular androgen-binding protein have high affinity for both T and DHT (reviewed by Steinberger4), that 5a-reductase is present in the seminiferous tubules,5 and that the administration of DHT can initiate spermatogenesis in rats treated with estradiol,3. 6 as well as maintain it in APX rats. 7 It therefore seemed of interest to compare the effects of treatment with T propionate (TP) and DHT propionate (DHTP) on the restoration and the maintenance of spermatogenesis in APX rats. In an attempt to explain the differences between the results obtained with TP and DHTP, we have examined rete testis fluid (RTF) androgen levels in APX rats maintained on· TP

Received April 1, 1977; accepted May 4, 1977. *Supported by Grants HD 06867 and HD 09584 from the National Istitute of Child Health and Human Development and by the Mabel Louise Riley Charitable Trust. tTo whom reprint requests should be addressed. :f:Recipient of Research Career Development Award HD 70369. § Present address: Department of Obstetrics and Gynecology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, Pac 17033.

1113

HARRIS ET AL.

1114

or DHTP and peripheral androgen levels in APX and castrated rats receiving injections of TP, DHTP, T, or DHT.

MATERIALS AND METHODS

Hypophysectomized and intact adult male CD rats were purchased from Charles River Breeding Laboratories, Wilmington, Mass., kept in a lightand temperature-controlled room (14 hours of light, 10 hours of dark; 22 ± 2° C), and fed Agway Charles River formula ad libitum. Testosterone propionate was purchased from Eli Lilly & Co., Indianapolis, Ind. DHTP was prepared from DHT and propionic anhydride in pyridine at room temperature, crystallized twice from ether-heptane and once from methanol-water, and checked for purity by thin-layer chromatography in 2% methanol-methylene chloride. The crystals were dried in an Abderhalden apparatus (boiling toluene) before weighing for use. Experiment 1. Adult APX rats received daily subcutaneous injections of 500 p..g of TP or 500 p..g of DHTP in 0.05 ml of sesame oil for 35 days, beginning 5 days after hypophysectomy (maintenance) or 33 days after hypophysectomy (restoration). The animals were killed 1 day after the last injection. The testes were weighed, fixed in Zenker-formol solution, sectioned at 5 p..m in Paraplast, and stained with hematoxylin and periodic acid-Schiff. Spermatogenesis was assessed quantitatively by counting the nuclei of Sertoli cells, type A spermatogonia (A), pre leptotene spermatocytes (PL), pachytene spermatocytes (P), and step 7 spermatids (ST) in round cross-sections of seminiferous tubules in stage VII of spermatogenesis. The presence or absence of late (step 19) spermatids in the tubules used for counting was also recorded. The results were corrected for the dif-

October 1977 ferences in the nuclear diameter of various cell types and expressed per 10 Sertoli cells. I, 8, 9 Statistical significance of the differences was determined by the X2 test for heterogeneity on pooled crude cell counts. Experiment 2. Adult APX rats received daily subcutaneous injections of 2 mg of TP, 2 mg of DHTP, or sesame oil for 12 days. Twenty hours before the rats were killed their efferent ducts were ligated. About 2 hours before sacrifice each rat received its last daily injection of hormone. Immediately before sacrifice the RTF was collected. lo Blood was collected by cardiac puncture. The concentration of T in RTF and plasma of TPtreated rats and the concentration of DHT in RTF and plasma of DHTP-treated rats were determined by radioimmunoassay Y In this and in subsequent experiments, plasma extracts were not chromatographed before radioimmunoassay. Because the employed antibody to T cross-reacts 100% with DHT and approximately 25% with 5aandrostan-3a,17,8-diol and 5a-androstan-3,8,17,8diol, and the concentration of DHT in the plasma of rats treated with TP is extremely low, the results represent a slight overestimation of T in the plasma of TP-treated rats and a slight overestimation of DHT in the plasma of DHTPtreated rats.

Experiment 3. Adult APX rats received daily subcutaneous injections of2.0 mg ofTP, DHTP, T, or DHT in sesame oil for 3 days. Adult castrated rats were similarly treated with T or DHT but the daily dose was increased to 3.0 mg to compensate for the higher body weights of these animals. Blood samples were collected at lh, 2, 4, and 24 hours after the last injection from the tail vein under light ether anesthesia. In the case of APX rats given T or DHT, additional animals were bled at 8, 12, and 16 hours after the last in-

TABLE 1. Effects of Daily Treatment with 500 pg of TP or DHTP on the Mean Numbers of Type A Spermatogonia (A), Preleptotene Spermatocytes (PL), Pachytene Spermatocytes (P), and Step 7 Spermatids (ST) in Hypophysectomized Rats" Treatment

No. of rats

Wt. of testes

Ratios of cells

% Tubules with

Mean no. of nucleillO Sertoli nuclei

late spermatids

A

PL

P

ST

PL:A

P:PL

ST:P

mg

Intact controls" Maintenance Controls" DHTP TP Restoration Controls DHTP TP

4

3416 ± 80

0.55

22.2

22.7

74.0

100

40

1.02

3.3

4 6 7

556 ± 56 1240 ± 56 1844 ± 80

0.38 0.66 0.63

6.9 20.2 18.0

2.4 11.8 13.9

1.0 37.4 48.1

0 0 85

18 31 29

0.35 0.58 0.77

0.4 3.2 3.5

7 6 7

412 ± 16 556 ± 12 1102 ± 134

0.19 0.20 0.53

4.6 6.5 14.4

0.6 2.4 9.6

0.5 1.8 24.7

0 0 14

24 32 27

0.14 0.37 0.67

0.8 0.8 2.6

"The treatments were started 5 days after hypophysectomy (maintenance) or 33 days after hypophysectomy (restoration). The significance of the differences is found in the text. "Recalculated from the data of Bartke and Lloyd. 9

1115

EFFECTS OF T AND DHT IN HYPOPHYSECTOMIZED RATS

Vol. 28, No. 10

smaller in DHTP- than in TP-treated rats and were similar to those in oil-injected controls. In both maintenance and restoration experiments, the weights of the testes were significantly greater in TP- than in the corresponding DHTPtreated rats (Table 1). Experiment 2. Androgen concentrations in RTF and peripheral plasma of adult APX rats maintained on daily injections of TP were approximately 3-fold higher than the androgen levels in RTF and plasma of adult APX rats maintained on daily injections of DHTP (Table 2). Experiment 3. The concentrations of androgens in the peripheral plasma of APX rats treated with propionates ofT or DHT were fairly stable during the 24 hours following the last of three daily injections (Fig. 1). During this period, androgen concentrations were considerably higher in the plasma of rats givenTP than in those given DHTP. The subcutaneous admi.nistration offree T or DHT to APX or castrated rats resulted in very high peripheral androgen levels within % hour after the injection. These high levels were usually followed by a further increase; maximal levels were observed at 2 or 4 hours after the injection, with a rapid decline thereafter. During the 4-hour period following the last of three daily injections, plasma androgen levels were from 2- to 4-fold higher in rats given T than in those given DHT, but afterward the plasma androgen levels were comparable in the two groups.

TABLE 2. Effect of Daily Treatment with 2 mg ofTP or DHTP on Androgen Concentrations in Rete Testis Fluid (RTF) and Peripheral Plasma of Hypophysectomized Rats" Androgens"

Plasma

RTF

Treatment No. of rats Mean

±

Mean

P

SD

±

P

SD

nglml

nglml

-

Oil DHTP

4 5

0.44 ± 0.88 11.02 ± 7.60

TP

6

30.06 ± 14.76

r

5.94 ± 0.95 <0.01

<0.05 19.64 ± 6.79

aThe treatments were started 2 days after hypophysectomy. bT in TP-treated rats; DHT in DHTP-treated rats. r_, Not determined.

jection. Plasma androgen levels were determined by a radioimmunoassay as described above. RESULTS

Experiment 1. In short-term APX rats given TP, complete spermatogenesis was qualitatively maintained in 85% of the tubules, but the number of spermatocytes and spermatids was less than that in the intact controls (Table 1). In APX rats given DHTP, there were significantly fewer step 7 spermatids and no step 19 spermatids. In long-term APX rats given TP, there was a considerable degree of restoration of spermatogenesis even though the number of germinal cells was much lower than that in the corresponding maintenance group, and late spermatids were present in only 14% of the tubules (Table 1). In contrast, in the long-term APX rats given DHTP, there was essentially no evidence of the restoration of spermatogenesis, except for a slight improvement in the survival rates of spermatogonia (PL:A) and primary spermatocytes (P:PL). The numbers of germinal cells were significantly

DISCUSSION

It is well documented that chronic treatment of adult APX rats with TP can cause nearly quantitative maintenance as well as limited but signifCastrated

Hypophysectomized

Hypophysectomized

-T

-T

-TP 0---<) DHTP

300

<>--0<)

OHT

C>---<)

OHT

:: 250 E

ci.co

200

IJ: 0

150

.

I-

100 50 0

W 0

4

~A-+------ ---

------8

12

16 20 24

o

4

8

12

16 20 24

o

4

8

12

16 20 24

Hours After Injection FIG. 1. Concentrations of androgens in the peripheral plasma of rats treated daily for 3 days with TP, DHTP, T, or free DHT (means ± standard deviations based on three or four determinations per group). Note the additional sampling times (8, 12, and 16 hours) in the central panel.

1116

HARRIS ET AL.

icant restoration of spermatogenesis, 1 and the present results are fully consistent with these findings. No information is available on the effects of DHTP on spermatogenesis, but DHT was reported to maintain spermatogenesis in adult APX rats 3,7 and to initiate spermatogenesis in immature rats treated with estradiol benzoate (EB).3,6 The information on the relative abilities of T and DHT to initiate and to maintain spermatogenesis is incomplete and conflicting. Ahmad et aU' 12 reported that DHT was less active than T when administered by intratesticular Silastic implants but more active than T when injected subcutaneously. Chowdhury and Steinberger3 noted that, in immature EB-treated rats, DHT injected in oil was less active than similarly administered TP but both compounds had similar effects in adult APX rats. Chemes et al. 6 injected androgens as microcrystals suspended in distilled water and found DHT to be more potent than T in initiating spermatogenesis in immature EB-treated rats. In the present study, the degree of maintenance and restoration of spermatogenesis in adult APX rats was significantly greater after treatment with TP than after treatment with DHTP. This difference was associated with significantly higher RTF and peripheral androgen levels in TP-treated animals than in DHTP-treated animals. Hormone levels in the RTF appear to provide a reasonable estimate of the concentration of the same hormones within the seminiferous tubules. lo , 13 Consequently, it is possible to conclude that the germinal cells were dividing and differentiating in an environment much richer in androgens in animals given TP than in those given DHTP. Similarly, peripheral androgen levels were considerably greater in animals receiving injections offree T than in those receiving injections ofDHT. A comparable difference in the peripheral androgen levels was observed in rats given subcutaneous Silastic implants containing T or DHT14 and in animals receiving injections of 2 mg of TP or DHTP daily for 12 days.15 The difference in RTF and peripheral androgen levels after administration of identical amounts of T and DHT is most likely due to the fact that, in the rat, the metabolic clearance rate of DHT is much greater than the metabolic clearance rate ofT.16. 17 No information is available on the possible differences in the rates of absorption of T and DHT from the site of the injection, or on the metabolism of these steroids by subcutaneous tissues. Plasma or RTF levels of 5a-androstan-3a, 17,a-diol (androstanediol) were not measured in

October 1977 the present study, but it is very likely that androstanediol, derived from the metabolism of DHT, contributed to the observed effects ofDHTP on the testis. Androstanediol can restore and maintain spermatogenesis,3, 6 possibly because of its limited but demonstrable conversion to DHT within the seminiferous tubules. 6, 18 It is interesting to speculate that the difference in the responsiveness of the male accessory reproductive glands and the germinal epithelium to injected DHT is partially due tothe difference in the ability of these tissues to convert androstanediol to DHT.18 It is obvious from the present results that the effectiveness of T and DHT at the level of the target tissue cannot be compared by injecting identical doses of these steroids at a distant site. This difficulty is not circumvented by employing longacting esters of these compounds or by controlling the rate of their release with administration in Silastic implants. The comparison of biologic potencies of T and DHT in various experimental systems is further complicated by striking species differences in the rate of DHT metabolism l4 and by the differential ability of these steroids to cross the blood-brain barrier.19 The present results, together with the earlier findings of others, indicate that both T and DHT have the ability to initiate, to maintain, and to' restore spermatogenesis in the rat. The significantly lower effectiveness -of DHTP as compared with that of TP is undoubtedly due to different rates of metabolism of exogenous DHT and T in the rat and to resulting differences in their concentrations in the testis and in the peripheral plasma. In the physiologic situation, Sertoli cells and germinal elements are exposed to both T and DHT, with T being probably far more abundant. 13 ,20 The information available to date does not permit a conclusion as to which of these two androgens is of greater importance at the cellular level. Acknowledgments. We thank Patricia Bell, Alonda Belpulsi, Bruce Croft, and Susan Dalterio for their valuable assistance, and Dr. B. V. Caldwell for the antiserum to testosterone.

REFERENCES 1. Clermont Y, Harvey SC: Effects of hormones on spermatogenesis in the rat. Ciba Found Coloq Endocrinol 16:173, 1967 2. Steinberger E, Chowdhury AK, Tcholakian RK, Roll H: Effects of C21 steroids on sex accessory organs and testes of mature hypophysectomized rats. Endocrinology 96: 1319, 1975

Vol. 28, No. 10

EFFECTS OF T AND DHT IN HYPOPHYSECTOMIZED RATS

3. Chowdhury AK, Steinberger E: Effect of 5a reduced androgens on sex accessory organs, initiation and maintenance of spermatogenesis in the rat. BioI Reprod 12:609, 1975 4. Steinberger E: Hormonal regulation of the seminiferous tubule function. In Hormonal Regulation of Spermatogenesis, Edited by FS French, V Hansson, EM Ritzen, SN Nayfeh. New York, Plenum Press, 1975, p 337 5. Rivarola MA, Podesta EJ, Chemes HE, Cigorraga S: Androgen metabolism in the seminiferous tubule. In Hormonal Regulation of Spermatogenesis, Edited by FS French, V Hansson, EM Ritzen, SN Nayfeh. New York, Plenum Press, 1975, p 25 6. Chemes HE,Podesta E, Rivarola MA: Action of testosterone, dihydrotestosterone and 5a-androstane-3a, 17f3diol on the spermatogenesis of immature rats. BioI Reprod 14:332, 1976 7. Ahmad N, Haltmeyer GC, Eik-Nes KB: Maintenance of spermatogenesis in rats with intra testicular implants containing testosterone or dihydrotestosterone (DHT). BioI Reprod 8:411, 1973 8. Clermont Y, Morgentaler H: Quantitative study of spermatogenesis in the hypophysectomized rat. Endocrinology 57:369, 1975 9. Bartke A, Lloyd CW: Influence of prolactin and pituitary isografts on spermatogenesis in dwarf mice and hypophysectomized rats. J Endocrinol 46:321, 1970 10. Harris ME, Bartke A: Concentration of testosterone in testis fluid of the rat. Endocrinology 95:701, 1974 11. Bartke A, Steele RE, Musto N, Caldwell BV: Fluctuations in plasma testosterone levels in adult male rats and mice. Endocrinology 92:1223, 1973 12. Ahmad N, Haltmeyer GC, Eik-Nes KB: Maintenance of spermatogenesis with testosterone or dihydrotestosterone in hypophysectomized rats. J Reprod Fertil 44:103, 1975

1117

13. Harris ME, Bartke A: Maintenance of rete testis fluid testosterone and dihydrotestosterone levels by pregnenolone and other C21 steroids in hypophysectomized rats. Endocrinology 96:1396, 1975 14. Gay VL: Species variation in the metabolism of dihydrotestosterone: correlation with reported variations in behavioral response (abstr 23). In Program of the Ninth Meeting of the Society for the Study of Reproduction, Philadelphia, August 10-13, 1976, p 27 15. Naess 0, Hang E, Attramadal A, Aakvaag A, Hansson V, French F: Androgen receptors in the anterior pituitary and central nervous system of the androgen "insensitive" (Tfm) rat: correlation between receptor binding and effects of androgens on gonadotropin secretion. Endocrinology 99:1295, 1976 16. Lee DKH, Bird CE, Clark AF: In vivo metabolism of 3H-testosterone in adult male rats: effects of estrogen administration. Steroids 26:137, 1975 17. Van Doorn EJ, Burns B, Wood D, Bird CE, Clark AF: In vivo metabolism of 3H-dihydrotestosterone .and 3H_ androstanediol in adult male rats. J Steroid Biochem 6: 1549, 1975 18. Kao LWL, Perez-Lloret A, Weisz J: Unpublished observations 19. Marynick SP, Havens WW II, Ebery MH, Loriaux DL, Robertson S: Studies on the transfer of steroid hormones across the blood-cerebrospinal fluid barrier in the rhesus monkey. Endocrinology 99:400, 1976 20. Baker HWG, Bailey DJ, Feil PD, Jefferson LS, Santen RJ, Bardin CW: Nuclear accumulation of androgens in perfused rat accessory sex organs and testes. Endocrinology 100:709, 1977