FERTILITY AND STERILITY Copyright ~ 1975 The American Fertility Society Vol. 26, No.7, July 1975 Printed in U.S A. ANDROGEN BIOSYNTHESIS IN EXPERIMENTAL CRYPTORCHIDISM* JUAN CARLOS HOSCHOIAN, PH.D., AND JUAN ANGEL ANDRADA, M.D. lnstituto de Investigaciones Medicas, Universidad de Buenos Aires, Buenos Aires, Argentina Previous reports have been made regarding the histologic characteristics of the process of testicular maturation during normal puberty. The findings have been compared with those in abnormal testes, particularly when the alteration may be due to cryptorchidism.l 2 Morphologic observation of the unilateral ectopic puberal testis shows thickening and sclerosis of the tubular walls, with a limited development of the germinal epithelium and an increased number of immature Leydig cells. Similar changes have been shown in animals with experimental cryptorchidism, especially in the early phase of the experiments, when Leydig cell damage is considerably less important than that to tubular elements. 3 In addition, the androgen production in cryptorchid animals is significantly less than that in normal animals. 4 ' 6 The length of the infertile period following cryptorchidism or thermal treatment appears to be dependent upon the duration of exposure as well as the temperature of exposure. 7 8 The purpose of this study was to analyze the influence of unilateral experimental cryptorchidism on androgen biosynthesis and testicular maturation. Received July 11, 1974. *Supported in part by grants from the Consejo Nacional Investigaciones Cientificas Tecnicas of Argentina and The Population Council of New York. 730 Animals MATERIALS AND METHODS Four groups of 12 albino Hartley strain guinea pigs were used: group 1, postpuberal animals, mean body weight 800 to 900 gm; group 2, small prepuberal animals, mean body weight 200 to 250 gm; group 3, large prepuberal animals, mean body weight 550 to 650 gm; group 4, small prepuberal animals (same as group 2) subjected to a different experimental procedure. Surgical Procedure Group 1. Both eutopic testes from postpuberal control animals were used for histology and steroid biosynthetic studies. Groups 2 and 3. Under ether anesthesia, the skin and peritoneum were opened on the right infra-umbilical hemiabdomen; the right testis was excised and sutured with fine catgut to the anterior abdominal wall near the umbilicus; the left testis was not touched. Thirty days after the operation, the animals were killed; each testis was removed, examined histologically, and analyzed for steroid biosynthesis. Group 4. After 30 days of induced cryptorchidism, the ectopic testis was descended and fixed in its proper place in the scrotum. Seventy days after the second operation, the animals were killed and both the scrotal and the descended ectopic testes were studied.
Vol. 26, No.7 ANDROGEN BIOSYNTHESIS IN CRYPI'ORCHIDISM 731 Histology Each testis was weighed and cut into two pieces. One small piece with tunica albuginea was fixed in Bouin's or Cleland's solution, sectioned, and stained with hematoxylin and eosin for histologic studies. Incubations The other piece of gland was separated from albuginea and homogenized in Krebs-Ringer-phosphate buffer at ph 7.4; 5-ml aliquots of homogenate were incubated in triplicate in the presence of 2.5 JLCi of 3 H-pregnenolone (specific activity, 11.7 Ci/mmole), 1 mg of nicotinamide adenine dinucleotide phosphate, and 5 mg of glucose 6-phosphate; 200 mg of fresh tissue were used for the incubation; it was performed in a Dubnoff shaker, with constant shaking for 1 hour at 36 C in air. At the end of the incubation period, newly formed metabolites were quickly extracted with cold dichloromethane. Separation, identification, and purification of the synthesized steroids were performed following procedures previously described.9 The isotopic dilution method was used to identify radioactive metabolites. The constancy of the specific activities of each isolated compound was considered sufficient proofofpurity (Table 1). RESULTS Group 1: Normal Postpuberal Animals. Incubation of labeled pregnenolone with testes from normal adult guinea pigs led to the formation of several radioactive metabolites; of these, progesterone, 17a-hydroxyprogesterone, 17a-hydroxypregnenolone, 4-androstenedione, 5- androstenediol, and testosterone were isolated and purified. From the results shown in Table 2, it can be seen that pregnenolone was well metabolized by the testicular homogenate to different steroids and that testosterone was the main radioactive compound synthesized. Group 2: Small Prepuberal Animals. In this group, the ectopic testes weighed less than the contralateral testes (90 ± 10 mg versus 150 ± 15 mg; P < 0.001). Immaturity of the germinal line, tubular size, and intertubular spaces were more pronounced in the ectopic testes (Figs. 1 and 2). In addition, pregnenolone was metabolized less efficiently than with the same amount of tissue from adult animals. This difference was even more striking in the ectopic testes than in the scrotal testes. However, the main distinction between mature and immature testes was the high percentage of 4-androstenedione found in both ectopic and eutopic im- Degree of purification TABLE 1. Identification of the Different Radioactive Steroids Isolated after Incubation of Testicular Guinea Pig Tissue with 3 H-Pregnenolone" Solvent and molarity Specific activity Pg p 17-0H Pg 17-0H P.l' 5-Diol Testo mcilmmole After addition of the 5050 907 2060 780 1010 1440 2410 corresponding carried steroid First thin layer Benzene, 94; 5105 910 2100 815 1135 1200 2315 chromatograph methanol, 6 Acetylation: second thin Dichloromethane, 5060 920 2125 760 1070 1310 2350 layer chromatograph 15; methanol, 1; glycerol, 0.04 Hydrolysis: third thin Cyclohexane, 1; 5085 910 2050 1275 layer chromatograph ethyl acetate, 1 "Pg, pregnenolone; P, progesterone; 17-0H Pg, 17a-hydroxypregnenolone; 17-0H P, 17a-hydroxyprogesterone; ~\ 4-androstenedione; 5-Diol, 5-androstenediol; Testo, testosterone.
732 HOSCHOIAN AND ANDRADA July 1975 Group TABLE 2. Percentages of Conversion of 3 H-Pregnenolone into Different Metabolites after Incubation of Testicular Guinea Pig Tissue % Conversion of 3H~pregnenolone into PG p 17-0HPg 17-0HP a 5-Diol Testo 1. Normal postpuberal 11.0 1.3 7.6 0.7 0.7 11.1 29.1 animals 2. Small prepuberal animals Eutopic testes 15.5 3.4 6.9 2.2 21.1 0.8 3.5 Ectopic testes 21.6 4.5 4.6 2.5 24.3 0.6 2.3 3. Large prepuberal animals Eutopic testes 11.9 2.5 5.9 1.2 0.6 10.5 23.1 Ectopic testes 17.4 5.0 4.5 2.5 9-7 4.7 9.6 Values are means. Pg, pregnenolone; P, progesterone; 17-0H Pg, 17a-hydroxypregnenolone; 17-0H P, 17a-hydroxyprogesterone; a, 4-androstenedione; 5-Diol, 5-androstenediol; Testo, testosterone. mature glands. Although mature, normal testes contained negligible amounts of 4-androstenedione, in immature testes there was an accumulation of this metabolic intermediate, with little conversion to testosterone (Table 2). Group 3: Large Prepuberal Animals. The weights of the ectopic and eutopic testes were considerably different (0.25 ± 0.10 gm versus 1.62 ± 0.30 gm; P < 0.001). A mature spermatic line and interstitium were observed in the scrotal testes (Fig. 3A), while the ectopic testes showed only Sertoli cells, some of them vacuolated, and a few spermatogonia in the tubules (Fig. 3B). The spaces contained immature Leydig cells. The steroid studies clearly indicated that, in large prepuberal animals (puberal at the time of operation), testosterone synthesis by scrotal testes is quite similar to that of postpuberal testes. However, FIG. 1. Small prepuberal testis, 30 days after experimental cryptorchidism. Ectopic gland shows immaturity of seminiferous tubules containing undifferentiated germinal syncytium (hematoxylin and eosin; x 100).
Vol. 26, No.7 ANDROGEN BIOSYNTHESIS IN CRYPI'ORCHIDISM 733 FIG. 2. Small prepuberal testis, 30 days after experimental unilateral cryptorchidism. Photomicrographs of characteristic seminiferous tubules of scrotal (A) and ectopic (JJ) testes. More roundshaped tubules, gonadal elements, and pre-sertoli cells with elongated nuclei perpendicular to the basement membrane are seen in B. Leydig cells are more defined and appear more mature in A (hematoxylin and eosin; x 250).
734 HOSCHOIAN AND ANDRADA July 1975 FIG. 3. Large prepuberal testis, 30 days after experimental unilateral cryptorchidism. Photomicrographs of seminiferous tubules of scrotal (A) and ectopic (B) testes. A, tubular size, basement membr.ane, and gonadal elements show different degrees of maturity. The interstitium has normal amounts of steroid-producing cells. B, round-shaped, smaller tubules containing Sertoli cells, some of them vacuolated, and a few spermatogonia without mitosis are seen. Immature Leydig cells can be seen in the intertubular tissue (hematoxylin and eosin; x 250).
Vol. 26, No.7 ANDROGEN BIOSYNTHESIS IN CRYPI'ORCHIDISM 735 FIG. 4. Schematic radiochromatogram of steroid metabolites obtained by incubation of 3M-pregnenolone with testicular homogenates of eutopic (black curves) and ectopic testes (shaded curves) from large prepuberal guinea pigs (group 3). TESTO, testosterone;.i5 DIOL, 5-androstenediol;.i\ 4-androstenedione; P, progesterone; Pg, pregnenolone; 17 OH-Pg, 17a-hydroxypregnenolone. FIG. 5. Postpuberal testis. Small prepuberal guinea pig's testis after 30 days of experimental unilateral cryptorchidism; the ectopic testis was replaced in the scrotum and the animal was killed 70 days later, during puberty. The photomicrograph shows a fully mature testis (hematoxylin.and eosin; x 250).
736 HOSCHOIAN AND ANDRADA July 1975 TABLE 3. Percentages of Conversion of 3H-Pregnenolone into Different Metabolites after Incubation of Testicular Guinea Pig Tissue" Testes % Conversion of 3H-pregnenolone into Pg p 17-0HPg 17-0HP!l' 5-Diol Testo Eutopic 11.2 4.5 3.6 1.7 0.5 6.3 25.2 Formerly ectopic 12.3 5.5 3.0 0.6 1.7 8.2 24.4 a Animals were subjected to experimental unilateral cryptorchidism for 30 days; the organ was then replaced in its normal position until puberty. Pg, pregnenolone; P, progesterone; 17-0H Pg, 17ahydroxypregnenolone; 17-0H P, 17a-hydroxyprogesterone; ~\ 4-androstenedione; 5-Diol, 5-androstenediol; Testo, testosterone. striking differences between ectopic and eutopic testes in the production of 4- androstenedione and testosterone were noted. As Table 2 shows, a significant accumulation of 4-androstenedione and a lower testosterone production were found in the incubated ectopic tissue, similar to results obtained with the immature glands (Fig. 4). Group 4. Small Prepuberal Animals, Different Procedure. No differences were found between eutopic and formerly ectopic glands when the animals became pubescent. The weights of both testes were similar (1.75 ± 0.40 gm for scrotal versus 1.69 ± 0.35 gm for formerly ectopic testes). Histology revealed fully mature glands (Fig. 5). Pregnenolone was efficiently metabolized to different intermediate steroids, and testosterone production was quite similar in both types of glands (Table 3). DISCUSSION Data concerning experimental unilateral cryptorchidism in guinea pigs were obtained in this study. Experiments conducted during different stages of sexual maturation revealed several structural and biosynthetic changes in testicular tissue after 30 days in an ectopic position. In a previous study, 10 adult guinea pig testicular homogenates were incubated for varying lengths of time with pairs of differentially labeled steroids (progesterone and pregnenolone, 17ahydroxyprogesterone and 17a-hydroxypregnenolone, and 4-androstenedione and dehydroepiandrosterone) in order to elucidate the possible pathways involved in testosterone biosynthesis. It was evident that all of these steroids could be converted to testosterone. The results suggested that testosterone is synthesized from pregnenolone and 17a-hydroxypregnenolone by two different pathways, one involving A 4 compounds and the other independent of 4-androstenedione. In the latter case, 5-androstenediol must be considered a possible intermediate in testosterone biosynthesis. Accumulation of 4- androstenedione in the incubation media was not observed in either of these pathways when testes of adult guinea pigs were used. Snipes et al., 11 in incubations of 14 C progesterone with testes from guinea pigs of different ages, observed that more 4-14 C-androstenedione and less 14 C testosterone were synthesized by tissue from younger animals. They concluded that androstenedione is reduced less readily in younger animals. On the other hand, Becker and Snipes 12 showed a shift with age in the steady state concentration of 4-androstenedione and testosterone in incubations of guinea pig testes with 4- androstenedione and testosterone. The predominant steroid was 4-androstenedione in the presence of tissue from immature animals and testosterone in that from adult animals, regardless of the compound added initially. In the present experiments, 4-androstenedione accumulation was evident in incubations with the ectopic and scrotal
Vol. 26, No. 7 ANDROGEN BIOSYNTHESIS IN CRYPI'ORCHIDISM 737 glands of immature animals. A similar accumulation of androstenedione was observed in the ectopic testes of puberal guinea pigs (group 3). Thus, histologic immaturity and hormonal biosynthesis of puberal cryptorchid testes seem to be similar to those observed in scrotal glands of sexually immature guinea pigs. A clear accumulation of 4-androstenedione was present in the media in which tissue from these animals was incubated. The experiments performed in immature animals (testis fixation to the abdominal wall and a return to the scrotal position after 30 days), as expected, did not prevent further development of the testis. When the animals reached puberty, these testes exhibited a cytologic and hormonal maturation similar to that observed in testes left in the scrotal position. Consequently, the age when cryptorchid testes descend is of fundamental importance to the development and maturation of germinal and interstitial structures. SUMMARY This study was conducted to investigate the effects of unilateral cryptorchidism on androgen production and testicular maturation. Experimental cryptorchidism was produced in small and large prepuberal guinea pigs by forcing the testis to remain in the abdomen for a period of 30 days. Small prepuberal animals, which did not reach sexual maturity, showed discrete reductions in the size and weight of the ectopic testes when compared with the scrotal testes. Immaturity of germinal line, tubular size, and intertubular spaces were more pronounced in the ectopic tissue. Both glands produced 4-androstenedione as a primary metabolic product of pregnenolone. Large prepuberal animals, puberal at the time of operation, showed tubular size, spermatic line, and interstitial tissue completely developed in the scrotal gland. Testosterone production was quite similar to that produced in postpuberal testes. The ectopic organ showed only Sertoli cells, some vacuolated, and a few spermatogonia in the tubules. The spaces contained Leydig cells. A significant accumulation of 4-adrenostenedione and a lower testosterone production, compared with that found in the scrotal gland, were observed. The histologic immaturity and the hormonal biosynthesis of the puberal cryptorchid testis parallel findings in scrotal glands from sexually immature guinea pigs. Acknowledgment. The technical assistance of Miss Marcia Coumroglon is acknowledged. REFERENCES 1. De la Baize FA, Mancini RA, Arrillaga F, Andrada JA, Vilar 0, Gurtman AL, Davidson OW: Puberal maturation of the normal human testis. A histologic study. J Clin Endocrinol Metab 20:266, 1960 2. De la Baize FA, Mancini RA, Arrillaga F, Andrada JA, Vilar 0, Gurtman AL, Davidson OW: Histologic study of the undescended human testis during puberty. J Clin Endocrinol Metab 20:286, 1960 3. Nelson WO: Mammalian spermatogenesis: effect of experimental cryptorchidism in the rat and nondescent of the testis in man. Recent Prog Horm Res 6:29, 1951 4. Clegg EJ: Some effects of artificial cryptorchidism on the accessory reproductive organs of the rat. J Endocrinol 20:210, 1960 5. lnano H, Tamaoki Bl: Effect of experimental bilateral cryptorchidism on testicular enzymes related to androgen formation. Endocrinology 83:1074, 1968 6. Llaurado JG, Dominguez OV: Effect of cryptorchidism on testicular enzymes involved in androgen biosynthesis. Endocrinology 72:292, 1963 7. Asdell SA, Salisbury GW: The viability of spermatozoa in the abdominal epididymis and the failure of motile sperms to fertilize ova. Am J Physiol132:791, 1941 8. Casady RB, Legates JE, Myers RM: Correlations between temperature varying from 60"- 950 F and certain physiological responses in young dairy bulls. J Anim Sci 15:141, 1956
738 HOSCHOIAN AND ANDRADA July 1975 9. Hoschoian JC, Brownie AC: Pathways for adrogen biosynthesis in monkey testis. Steroids 49:10, 1967 10. Hoschoian JC: Biosintesis de esteroides suprarrenales y testiculares en el cobayo. II. Sintesis de esteroids testiculares. Rev Asoc Bioq Argentina 184-185:7, 1969 11. Snipes CA, Becker WG, Migeon CJ: The effect of age on the in vitro metabolism of androgen by guinea pig testis. Steroids 6:771, 1965 12. Becker WG, Snipes CA: Shift with age in steadystate concentrations of androstenedione and testosterone in incubations of guinea pig testis. Biochem J 107:35, 1968