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Effects of cryptorchidism and orchidopexy on deoxyribonucleoside-activated nucleotidase (DAN) in the rat testis
hr. J. Biochem. Vol. 19, No. II, pp. 1023-1027,
0020-71 IX/87 $3.00 + 0.00 Journals Ltd
1987
Copyright 0 1987Pergamon
Printed in Great Britain. All rights reserved
EFFECTS OF CRYPTORCHIDISM AND ORCHIDOPEXY ON DEOXYRIBONUCLEOSIDE-ACTIVATED NUCLEOTIDASE (DAN) IN THE RAT TESTIS TRINE B. HAUGEN, FINN OLAV LEVY, VIDAR HANSSON and PER FRITZSON Institute of Medical Biochemistry, University of Oslo, P.O. Box 1112 Blindern, 0317 Oslo 3, Norway [Tel. 02-45-50501 (Received
22 January 1987)
Unilateral cryptorchidism was associated with an increase in specific DAN activity in the abdominal testis of adult rats. 2. In the scrotal, immature testis, there was an age-dependent decrease in specific DAN activity similar to that normally seen in rats. This decrease was less pronounced in the abdominal testis of unilaterally Abstract-l.
cryptorchid rats. 3. Total DAN activity in the abdominal testis showed a decrease in both immature and adult rats, when compared to that of the scrotal testis. 4. Orchidopexy following cryptorchidism in adult rats, resulted in complete restoration of DAN activity in spite of poor recovery of spermatogenesis
INTRODUCTION deoxyribonucleoside-activated nucleotidase The (DAN) which preferentially degrades deoxyribonucleotides, has been studied in various tissues and cells from rats and mice and has been described in detail elsewhere (Fritzson, 1978; Tjernshaugen, 1981; Haugen and Fritzson, 1986a,b; Haugen et al., 1987). This enzyme has both 3’- and S-dephosphorylating activity (Fritzson, 1978) and is distinct from another cytosolic nucleotidase with pHoptimum in the same range (6.&6.5), which acts preferentially on IMP and GMP (Itoh et al., 1967; Fritzson, 1969). By measuring DAN activity in different cell types isolated from the seminiferous tubules of the rat testis we have found that the enzyme activity is mainly located in the somatic cells (Sertoli cells, peritubular cells) (Haugen et al., 1987). The specific DAN activity in cytosol from Sertoli cells was approx. 4 times higher than that found in cytosol from peritubular cells. Little or no activity was found in cytosols from germ cells (primary spermatocytes and round spermatids). As an approach to elucidate whether the presence or absence of germ cells may affect DAN activity in the testis, we studied the enzyme activity in germ cell-depleted testis prepared by means of experimental unilateral cryptorchidism. The condition of cryptorchidism in which the testis is retained within the abdominal cavity, is associated with impairment of spermatogenesis, probably due to the temperature sensitivity of germ cells (Parvinen, 1973; Bergh and Helander, 1978). Orchidopexy is the surgical returning of the testis to the scrotum. Restoration of spermatogenesis has been shown to occur in immature rats when experimental cryptorchidism is followed by orchidopexy (Jegou et al., 1984), whereas the same treatment of adult rats has been shown to result in a poor recovery of spermatogenesis (Jegou et al., 1983). Thus, when adult animals were subjected
to these types of experiments, we could also study how DAN activity in the testis is affected by the temperature, independent of the reappearance of germ cells. The soluble Mn2+-dependent adenylate cyclase (AC) activity, which is specific for haploid germ cells, was used as a marker of germ cell differentiation (Gordeladze et al., 1981; Jahnsen et al., 1981, 1986).
MATERIALS
AND METHODS
Sprague-Dawley rats were made unilaterally cryptorchid at the age of I7 and 60 days, respectively. One of the 60-day groups was subjected to orchidopexy 22 days later. Surgical techniques and treatment of the animals were carried out as described by Karpe et al. (1981). The testes were used for cytosol
preparation
Preparation
immediately
after killing.
of cytosol
Decapsulated testis was homogenized in IO vol of 0.25 M sucrose/50mM Tris-HCI, pH 7.4, with an Ultra-Turrax homogenizer (two IO set bursts). The homogenate was centrifuged at 105,OOOg for 1 hr to obtain the cytosol fraction. DAN assay
DAN activity was determined as described previously (Haugen and Fritzson, 1986). The activation exerted by 5 mM dIno on the dephosphorylation of 2.5 mM 3’-UMP was used for specific enzyme assay. One unit of enzyme is the amount which catalyzes the conversion of I nmol of substrate/min under the specified incubation conditions. Specific enzyme activity is units/mg of protein. Mn2+-dependent
adenylate
Mn*+-dependent
cyclase (AC)
assay
adenylate cyclase (EC 4.6.1. I) activity
was measured essentially as described by Gordeladze and Hansson (1980). The final incubation mixture (50 ~1) contained l&90 pg of cytosol protein, 0.2 mM ATP (including 3 x 106c.p.m. of [aJP]ATP (Amersham), IOmM Mn*+,
TRINE B. HAUGEN et al.
IO24
1 mM CAMP (including 10,000 c.p.m. of [g3H]cAMP (Amersham)), 1.OmM EDTA, and an ATP-regenerating system consisting of creatine phosphate (20 mM), creatine kinase (0.2 mg/ml) and myokinase (0.02 mgjml) in 25mM Tris-HCl buffer, pH 7.3. The incubations were carried out at 32°C for IO min. Cyclic AMP was isolated by Dowex and alumina chromatography (Birnbaumer et al., 1976). Protein and A TP determination
Protein was determined in the cytosol fractions by the method of Lowry et al. (1951) with bovine serum albumin as the standard. The ATP concentration was determined s~ctrophotometri~aily at 260 nm. RESULTS
Fig. 1A shows that there was a marked decrease in specific DAN activity in both the abdominal and scrotal testis of immature rats during the first 20 days following unilateral cryptorchidism. The decrease in specific DAN activity in the scrotal testis was similar to the age-dependent decrease between day 25 and 35 previously observed in non-operated animals (Haugen et al., 1987), whereas the specific activity in the abdominal testis decreased to a lesser extent. By calculating DAN activity per testis (Fig. lC), there
was nearly a 3-fold increase in the scrotal testis activity during the observation period. In the abdominal testis the enzyme activity first showed an increase (up to 4 days after operation) followed by a slight but significant decrease after 18 days when the testis weight had reached a constant level (Fig. IG). The age-dependent increase in the scrotal testis weight (Fig. 1G) was accompanied by a parallel increase in specific Mn 2+-dependent adenylate cyclase (AC) activity reflecting the appearance of haploid germ cells. In the abdominal testis the AC activity was nearly undetectable (Fig. 1E). There were no significant differences between enzyme activities and weights of the scrotal testes and the testes of sham-operated control rats (data not shown). In adult rats (Fig, 1B) there was a transient increase in specific DAN activity in the abdominal testis reaching a maximum at approx. 12 days after operation. The scrotal testis exhibited a constant activity level. The total DAN activity per a~ominal testis showed a considerable decrease to approx. 40% of the activity found at the time of operation (Fig. ID). The dramatic decrease in specific Mn*+-dependent AC activity in the abdominal testis (Fig. 1F) shows that experimental cryptorchidism induces a
6401
D
480
320 160
40
E
Fig. I. Effects of unilateral cryptorchidism on specific DAN activity (A,B), DAN activity per testis (CD), specific Mn?-dependent adenylate cyclase (AC) activity (E,F) and testis weight (G,H) in abdominal (0) and scrotal (0) testis of immature (17 days old) (left) and adult (60 days old) (right) rats. Each point represents the mean rf: SD of the results from 3 individual animals. The enzyme activities were measured in duplicate in each experiment.
1025
Cytosolic nucleotidase
t crypiorchidsm
1
t
orchidopexy
cryplorchidism Time
after
operation
t orchidopexy
(days)
Fig. 2. Effects of unilateral cryptorchidism and subsequent orchidopexy on specific DAN activity (A), DAN activity per testis (B), specific Mn *+-dependent adenylate cyclase (AC) activity (C) and testis weight (D) in cryptorchid/orchidopexic (0) and scrotal (0) testis of adult rats. Each point represents the mean k SD of the results from 4 individual animals, except at 7, 30 and 49 days after orchidopexy, where the numbers of animals examined were 3, 2 and 2, respectively. The enzyme activities were measured in duplicate in each experiment.
rapid depletion of germ cells in the testis. This was roughly paralleled by a 70% decrease in the testis weight (Fig. 1H). Figure 2 shows the effects of cryptorchidism followed by orchidopexy on DAN activity (Fig. 2A and B), Mn2+-dependent AC activity (Fig. 2C) and testis weight (Fig. 2D) in rats made cryptorchid at 60 days of age. The poor recovery of testis weight is in agreement with previous reports (Jegou et al., 1983) and indicates that irreversible changes occur in the testis during cryptorchidism in adult animals. There was only a slight increase in specific AC activity after orchidopexy. In contrast, specific DAN activity in the surgically treated testis showed a marked increase during the observation period reaching a maximum value more than 4-fold the control level (Fig. 2A). The total DAN activity per testis also increased to a level higher than that of the control testis (Fig. 2B). DISCUSSION
DAN activity is primarily or exclusively located in the somatic cells of the rat testis (Haugen et al., 1987). Furthermore, Sertoli cells do not proliferate after day 15 (Clerrnont and Perey, 1957; Steinberger and Steinberger, 1971). Thus, the considerable decrease in specific DAN activity during development (Haugen et al., 1987; Fig. IA) mainly reflect the rapid increase in the proportion of germ cells during this period. Also, the increase in specific activity in adult rats during the first 12 days of cryptorchidism may be ascribed to the depletion of germ cells in the abdominal testis. However, the decrease in specific DAN activity after this period as well as the decrease in specific DAN activity in the abdominal, immature
testis cannot be explained by changes in germ cell population. Based on the assumption that the number of somatic cells is constant during cryptorchidism, expression of total DAN activity per testis may give further insight into the actual content of this enzyme in the somatic cells. The age-dependent increase in this activity in immature rats was prevented during experimental cryptorchidism. Moreover, cryptorchidism resulted in a decrease in DAN activity per testis also in adult rats. These findings are apparently results of a decrease in DAN activity in the somatic cells of the testis, primarily the Sertoli cells. This decrease could be due to a direct effect of the elevated temperature in the cryptorchid state on Sertoli cells or to an indirect effect caused by the disappearance of germ cells. The fact that orchidopexy was associated with a complete restoration and even an overshoot of DAN activity (Fig. 2B), in spite of the poor recovery of spermatogenesis, indicates that the decreased activity is primarily due to the cryptorchid state and not to the disappearance of germ cells. Previous studies have shown that in addition to germ cell damage, several functions of the Sertoli cells are also disrupted during cryptorchidism (Hager& et al., 1978). Furthermore, Jegou et al. (1984) found that when experimental cryptorchidism was followed by orchidopexy in immature rats, the impaired functions were restored. In contrast, orchidopexy following cryptorchidism in adult rats resulted in only partial restoration of spermatogenesis as well as various parameters of Sertoli cell function, such as secretion of androgen-binding protein (ABP) (Jegou ef al., 1983). The effects of orchidopexy on the restoration of spermatogenesis as evaluated by the increase in testis weight were in our studies very
1026
TRINE B. HAUGEN et al.
similar to those obtained by Jegou et af. (1983). The changes in testis weight during cryptorchidism were closely associated with changes in the germ cell specific Mn2+-dependent adenylate cyclase activity. Cellular distribution of DAN and the increase in DAN activity per testis during the initiation of germ cell production suggest that the Sertoli cells may have a function in providing nucleosides for nucleic acid synthesis in germ cells. The decrease in DAN activity during cryptorchidism is then in line with the gradual disappearance of germ cells and the consequent decreased need for nucleosides for nucleotide synthesis in these cells. However, the fact that DAN activity is completely restored during orchidopexy of adult rats, a condition in which spermatogenesis shows poor recovery, indicates that the enzyme activity is not entirely related to germ cell requirements. Another aspect of nucleotidase activity in Sertoli cells which should be considered, is related to the possible role of nucleosides in autocrine and/or paracrine regulation in the testis. For example, Sertoli cells have been shown to possess specific receptors (A 1-receptors) for adenosine, which mediate inhibitory effects on the adenylate cyclase (Monaco et al., 1984; Eikvar et al., 1985). Furthermore, it has been reported that inosine as well as guanosine have stimulatory effects on proinsulin synthesis in isolated pancreatic islets of the rat (Jain and Logothetopoulos, 1977). Inosine has also been shown to have a strong gluconeogenic effect, which is independent of its insulin effect (Haeckel, 1977). These findings raise the question whether nucleosides produced by DAN activity in Sertoli cells may play a regulatory role for Sertoli cell functions and spermatogenesis. Further studies of the mechanisms regulating the enzyme activity may throw light on this possibility. SUMMARY
Unilateral cryptorchidism was associated with striking changes in specific DAN activity in the testis of both immature and adult rats. In immature rats, the specific DAN activity in the abdominal testis decreased to a lesser extent than in the scrotal, whereas in the adult rats an increase in specific DAN activity in the abdominal testis was seen. These observations confirm the previous finding that DAN activity is mainly located in the somatic cells. When expressed per abdominal testis, there was a decrease in total DAN activity in both immature and adult rats. This activity change was reversed by orchidopexy after cryptorchidism in adult animals. The fact that DAN activity was restored in spite of a poor recovery of spermatogenesis, indicates that the effect of cryptorchidism on DAN activity is due to the cryptorchid state and is not germ-cell mediated. Acknowledgements-The skilful technical Marit Haug and Mrs Gerd Rsnneberg ciated.
assistance is greatly
of Mrs appre-
REFERENCES
Bergh A. and Helander H. F. (1978) Testicular development in the unilaterally cryptorchid rat. Ini. J. Androl. 1, 44-58.
Birnbauer L., Yang P.-C., Hunzicker-Dunn M., Bockaert J. and Duran J. M. (1976) Adenylyl cyclase activities in ovarian tissues. I. Homogenization and conditions of assay in Graafian follicles and corpora lutea of rabbits, rats and pigs: regulation by ATP, and some comparative properties. Endocrinology 99, 163-184. Clermont Y. and Perey B. (1957) Quantitative study of the cell population of the seminiferous tubules in immature rats. Am. J. Anat. 100, 241-269. Eikvar L., Levy F. O., Jutte N. H. P. M., Cervenka J., Yoganathan T. and Hansson V. (1985) Effects of adenosine analogs on glucagon-stimulated adenosine 3’,5’-monophosphate formation in Sertoli cell cultures from immature rats. Endocrinology 117, 488491. Fritzson P. (1969) Nucleotidase activities in the soluble fraction of rat liver homogenate. Partial purification and properties of a 5’-nucleotidase with pH optimum 6.3. Biochim. biophys. Acta 178, 534541. Fritzson P. (1978) Regulation of nucleotidase activities in animal tissues. In Advances in Enzyme Regulation (Edited by Weber G.). Vol. 16. pp. 43361. Pergamon Press, Oxford. Gordeladze J. 0. and Hansson V. (1980) Mn’+-dependent adenylyl cyclase (AC) in rat testis: kinetic properties and optimalization of assay conditions. Int. J. Androl. 3, 539-552. Gordeladze J. O., Purvis K., Clausen 0. P. F., Rommerts F. F. G. and Hansson V. (1981) Cellular localization of the Mn’+-dependent adenylyl cyclase (AC) in the rat testis, Int. J. Androl. 4, 172-182. Haeckel R. (1977) Hepatic gluconeogenesis and urate formation from various nucleosides. Adv. exp. Med. Biol. 76A, 488499. Hagenis L., Ritzen E. M., Svensson J., Hansson V. and Purvis K. (1978) Temperature dependence of Sertoh cell function. In Endocrine Approach to Male Contraception (Edited by Hansson V., Ritzen M., Purvis K. and French F. S.), pp. 449458. Scriptor, Copenhagen. Haugen T. B. and Fritzson P. (1986) Measurement and activity of cytosolic deoxyribonucleoside-activated nucleotidase in various cell types from rat liver and spleen. Int. J. Biochem. 18, 167-170. Haugen T. B. and Fritzson P. (1986) Activity of deoxyribonucleoside-activated nucleotidase in cells from various Ivmphoid mouse tissues and in concanavahn Asm&ated lymphocytes. Int. J. Biochem. 18, 965-970. Haueen T. B.. Fritzson P. and Hansson V. (1987) Cellular lolalization and developmental changes ‘of deoxyribonucleoside-activated nucleotidase in the rat testis, Int. J. Biochem. 19, 193-196. Itoh R., Mitsui A. and Tsushima K. (1967) 5’-Nucleotidase of chicken liver. Biochim. biophys. Acta 146, 151-159. Jahnsen T., Gordeladze J. O., Haug E. and Hansson V. (1981) Changes in rat testicular adenylate cyclase activities and gonadotrophin binding during unilateral experimental cryptorchidism. J. Reprod. Fert. 63, 381-390. Jahnsen T., Attramadal H., Karpe B., Ritzen E. M. and Hansson V. (1986) Changes in germ cell adenylate cyclase and protein carboxyl methylase activities in rat testicular tissue during bilateral cryptorchidism and after orchidopexy. J. Reprod. Fert. 77, 317-320. Jain K. and Logothetopoulos J. (1977) Stimulation of proinsulin biosynthesis by purine-ribonucleosides and o-ribose. Endocrinology 100, 923-927. JCgou B., Laws A. 0. and De Kretser D. M. (1983) The effect of cryptorchidism and subsequent orchidopexy on testicular function in adult rats. J. Reprod. Fert. 69, 137-145. Jegou B., Peake R. A., Irby D. C. and De Kretser D. M. (1984) Effects of the induction of experimental cryptorchidism and subsequent orchidopexy on testicular function in immature rats. Biol. Reprod. 30, 179-187.
Cytosolic nucleotidase Karpe B., PIBen L., Hagenls L. and Ritzen E. M. (1981) Recovery of testicular functions after surgical treatment of experimental cryptorchidism in the rat. Znf. J. Androl. 4, 145-160.
Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. L. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Monaco L., Toscano M. V. and Conti M. (1984) Purine modulation of the hormonal response of the rat Sertoli cell in culture. Endocrinology 115, 16161624.
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Parvinen M. (1973) Observation on freshly isolated and accurately identified spermatogenic cells of the rat. Early effects of heat and short-term experimental cryptorchidism. Virchows Arch. Abr. B Zellpath. 13, 38-47. Steinberger A. and Steinberger E. (1971) Replication pattern of Sertoli cells in maturing rat testis in vivo and in organ culture. Biol. Reprod. 4, 84-87. Tjemshaugen H. (1981) Activity of deoxyinosine-activated nucleotidase in lymphoid tissues and circulating cells. Znt. J. Biochem. 13, 417421,
0020-71 IX/87 $3.00 + 0.00 Journals Ltd
1987
Copyright 0 1987Pergamon
Printed in Great Britain. All rights reserved
EFFECTS OF CRYPTORCHIDISM AND ORCHIDOPEXY ON DEOXYRIBONUCLEOSIDE-ACTIVATED NUCLEOTIDASE (DAN) IN THE RAT TESTIS TRINE B. HAUGEN, FINN OLAV LEVY, VIDAR HANSSON and PER FRITZSON Institute of Medical Biochemistry, University of Oslo, P.O. Box 1112 Blindern, 0317 Oslo 3, Norway [Tel. 02-45-50501 (Received
22 January 1987)
Unilateral cryptorchidism was associated with an increase in specific DAN activity in the abdominal testis of adult rats. 2. In the scrotal, immature testis, there was an age-dependent decrease in specific DAN activity similar to that normally seen in rats. This decrease was less pronounced in the abdominal testis of unilaterally Abstract-l.
cryptorchid rats. 3. Total DAN activity in the abdominal testis showed a decrease in both immature and adult rats, when compared to that of the scrotal testis. 4. Orchidopexy following cryptorchidism in adult rats, resulted in complete restoration of DAN activity in spite of poor recovery of spermatogenesis
INTRODUCTION deoxyribonucleoside-activated nucleotidase The (DAN) which preferentially degrades deoxyribonucleotides, has been studied in various tissues and cells from rats and mice and has been described in detail elsewhere (Fritzson, 1978; Tjernshaugen, 1981; Haugen and Fritzson, 1986a,b; Haugen et al., 1987). This enzyme has both 3’- and S-dephosphorylating activity (Fritzson, 1978) and is distinct from another cytosolic nucleotidase with pHoptimum in the same range (6.&6.5), which acts preferentially on IMP and GMP (Itoh et al., 1967; Fritzson, 1969). By measuring DAN activity in different cell types isolated from the seminiferous tubules of the rat testis we have found that the enzyme activity is mainly located in the somatic cells (Sertoli cells, peritubular cells) (Haugen et al., 1987). The specific DAN activity in cytosol from Sertoli cells was approx. 4 times higher than that found in cytosol from peritubular cells. Little or no activity was found in cytosols from germ cells (primary spermatocytes and round spermatids). As an approach to elucidate whether the presence or absence of germ cells may affect DAN activity in the testis, we studied the enzyme activity in germ cell-depleted testis prepared by means of experimental unilateral cryptorchidism. The condition of cryptorchidism in which the testis is retained within the abdominal cavity, is associated with impairment of spermatogenesis, probably due to the temperature sensitivity of germ cells (Parvinen, 1973; Bergh and Helander, 1978). Orchidopexy is the surgical returning of the testis to the scrotum. Restoration of spermatogenesis has been shown to occur in immature rats when experimental cryptorchidism is followed by orchidopexy (Jegou et al., 1984), whereas the same treatment of adult rats has been shown to result in a poor recovery of spermatogenesis (Jegou et al., 1983). Thus, when adult animals were subjected
to these types of experiments, we could also study how DAN activity in the testis is affected by the temperature, independent of the reappearance of germ cells. The soluble Mn2+-dependent adenylate cyclase (AC) activity, which is specific for haploid germ cells, was used as a marker of germ cell differentiation (Gordeladze et al., 1981; Jahnsen et al., 1981, 1986).
MATERIALS
AND METHODS
Sprague-Dawley rats were made unilaterally cryptorchid at the age of I7 and 60 days, respectively. One of the 60-day groups was subjected to orchidopexy 22 days later. Surgical techniques and treatment of the animals were carried out as described by Karpe et al. (1981). The testes were used for cytosol
preparation
Preparation
immediately
after killing.
of cytosol
Decapsulated testis was homogenized in IO vol of 0.25 M sucrose/50mM Tris-HCI, pH 7.4, with an Ultra-Turrax homogenizer (two IO set bursts). The homogenate was centrifuged at 105,OOOg for 1 hr to obtain the cytosol fraction. DAN assay
DAN activity was determined as described previously (Haugen and Fritzson, 1986). The activation exerted by 5 mM dIno on the dephosphorylation of 2.5 mM 3’-UMP was used for specific enzyme assay. One unit of enzyme is the amount which catalyzes the conversion of I nmol of substrate/min under the specified incubation conditions. Specific enzyme activity is units/mg of protein. Mn2+-dependent
adenylate
Mn*+-dependent
cyclase (AC)
assay
adenylate cyclase (EC 4.6.1. I) activity
was measured essentially as described by Gordeladze and Hansson (1980). The final incubation mixture (50 ~1) contained l&90 pg of cytosol protein, 0.2 mM ATP (including 3 x 106c.p.m. of [aJP]ATP (Amersham), IOmM Mn*+,
TRINE B. HAUGEN et al.
IO24
1 mM CAMP (including 10,000 c.p.m. of [g3H]cAMP (Amersham)), 1.OmM EDTA, and an ATP-regenerating system consisting of creatine phosphate (20 mM), creatine kinase (0.2 mg/ml) and myokinase (0.02 mgjml) in 25mM Tris-HCl buffer, pH 7.3. The incubations were carried out at 32°C for IO min. Cyclic AMP was isolated by Dowex and alumina chromatography (Birnbaumer et al., 1976). Protein and A TP determination
Protein was determined in the cytosol fractions by the method of Lowry et al. (1951) with bovine serum albumin as the standard. The ATP concentration was determined s~ctrophotometri~aily at 260 nm. RESULTS
Fig. 1A shows that there was a marked decrease in specific DAN activity in both the abdominal and scrotal testis of immature rats during the first 20 days following unilateral cryptorchidism. The decrease in specific DAN activity in the scrotal testis was similar to the age-dependent decrease between day 25 and 35 previously observed in non-operated animals (Haugen et al., 1987), whereas the specific activity in the abdominal testis decreased to a lesser extent. By calculating DAN activity per testis (Fig. lC), there
was nearly a 3-fold increase in the scrotal testis activity during the observation period. In the abdominal testis the enzyme activity first showed an increase (up to 4 days after operation) followed by a slight but significant decrease after 18 days when the testis weight had reached a constant level (Fig. IG). The age-dependent increase in the scrotal testis weight (Fig. 1G) was accompanied by a parallel increase in specific Mn 2+-dependent adenylate cyclase (AC) activity reflecting the appearance of haploid germ cells. In the abdominal testis the AC activity was nearly undetectable (Fig. 1E). There were no significant differences between enzyme activities and weights of the scrotal testes and the testes of sham-operated control rats (data not shown). In adult rats (Fig, 1B) there was a transient increase in specific DAN activity in the abdominal testis reaching a maximum at approx. 12 days after operation. The scrotal testis exhibited a constant activity level. The total DAN activity per a~ominal testis showed a considerable decrease to approx. 40% of the activity found at the time of operation (Fig. ID). The dramatic decrease in specific Mn*+-dependent AC activity in the abdominal testis (Fig. 1F) shows that experimental cryptorchidism induces a
6401
D
480
320 160
40
E
Fig. I. Effects of unilateral cryptorchidism on specific DAN activity (A,B), DAN activity per testis (CD), specific Mn?-dependent adenylate cyclase (AC) activity (E,F) and testis weight (G,H) in abdominal (0) and scrotal (0) testis of immature (17 days old) (left) and adult (60 days old) (right) rats. Each point represents the mean rf: SD of the results from 3 individual animals. The enzyme activities were measured in duplicate in each experiment.
1025
Cytosolic nucleotidase
t crypiorchidsm
1
t
orchidopexy
cryplorchidism Time
after
operation
t orchidopexy
(days)
Fig. 2. Effects of unilateral cryptorchidism and subsequent orchidopexy on specific DAN activity (A), DAN activity per testis (B), specific Mn *+-dependent adenylate cyclase (AC) activity (C) and testis weight (D) in cryptorchid/orchidopexic (0) and scrotal (0) testis of adult rats. Each point represents the mean k SD of the results from 4 individual animals, except at 7, 30 and 49 days after orchidopexy, where the numbers of animals examined were 3, 2 and 2, respectively. The enzyme activities were measured in duplicate in each experiment.
rapid depletion of germ cells in the testis. This was roughly paralleled by a 70% decrease in the testis weight (Fig. 1H). Figure 2 shows the effects of cryptorchidism followed by orchidopexy on DAN activity (Fig. 2A and B), Mn2+-dependent AC activity (Fig. 2C) and testis weight (Fig. 2D) in rats made cryptorchid at 60 days of age. The poor recovery of testis weight is in agreement with previous reports (Jegou et al., 1983) and indicates that irreversible changes occur in the testis during cryptorchidism in adult animals. There was only a slight increase in specific AC activity after orchidopexy. In contrast, specific DAN activity in the surgically treated testis showed a marked increase during the observation period reaching a maximum value more than 4-fold the control level (Fig. 2A). The total DAN activity per testis also increased to a level higher than that of the control testis (Fig. 2B). DISCUSSION
DAN activity is primarily or exclusively located in the somatic cells of the rat testis (Haugen et al., 1987). Furthermore, Sertoli cells do not proliferate after day 15 (Clerrnont and Perey, 1957; Steinberger and Steinberger, 1971). Thus, the considerable decrease in specific DAN activity during development (Haugen et al., 1987; Fig. IA) mainly reflect the rapid increase in the proportion of germ cells during this period. Also, the increase in specific activity in adult rats during the first 12 days of cryptorchidism may be ascribed to the depletion of germ cells in the abdominal testis. However, the decrease in specific DAN activity after this period as well as the decrease in specific DAN activity in the abdominal, immature
testis cannot be explained by changes in germ cell population. Based on the assumption that the number of somatic cells is constant during cryptorchidism, expression of total DAN activity per testis may give further insight into the actual content of this enzyme in the somatic cells. The age-dependent increase in this activity in immature rats was prevented during experimental cryptorchidism. Moreover, cryptorchidism resulted in a decrease in DAN activity per testis also in adult rats. These findings are apparently results of a decrease in DAN activity in the somatic cells of the testis, primarily the Sertoli cells. This decrease could be due to a direct effect of the elevated temperature in the cryptorchid state on Sertoli cells or to an indirect effect caused by the disappearance of germ cells. The fact that orchidopexy was associated with a complete restoration and even an overshoot of DAN activity (Fig. 2B), in spite of the poor recovery of spermatogenesis, indicates that the decreased activity is primarily due to the cryptorchid state and not to the disappearance of germ cells. Previous studies have shown that in addition to germ cell damage, several functions of the Sertoli cells are also disrupted during cryptorchidism (Hager& et al., 1978). Furthermore, Jegou et al. (1984) found that when experimental cryptorchidism was followed by orchidopexy in immature rats, the impaired functions were restored. In contrast, orchidopexy following cryptorchidism in adult rats resulted in only partial restoration of spermatogenesis as well as various parameters of Sertoli cell function, such as secretion of androgen-binding protein (ABP) (Jegou ef al., 1983). The effects of orchidopexy on the restoration of spermatogenesis as evaluated by the increase in testis weight were in our studies very
1026
TRINE B. HAUGEN et al.
similar to those obtained by Jegou et af. (1983). The changes in testis weight during cryptorchidism were closely associated with changes in the germ cell specific Mn2+-dependent adenylate cyclase activity. Cellular distribution of DAN and the increase in DAN activity per testis during the initiation of germ cell production suggest that the Sertoli cells may have a function in providing nucleosides for nucleic acid synthesis in germ cells. The decrease in DAN activity during cryptorchidism is then in line with the gradual disappearance of germ cells and the consequent decreased need for nucleosides for nucleotide synthesis in these cells. However, the fact that DAN activity is completely restored during orchidopexy of adult rats, a condition in which spermatogenesis shows poor recovery, indicates that the enzyme activity is not entirely related to germ cell requirements. Another aspect of nucleotidase activity in Sertoli cells which should be considered, is related to the possible role of nucleosides in autocrine and/or paracrine regulation in the testis. For example, Sertoli cells have been shown to possess specific receptors (A 1-receptors) for adenosine, which mediate inhibitory effects on the adenylate cyclase (Monaco et al., 1984; Eikvar et al., 1985). Furthermore, it has been reported that inosine as well as guanosine have stimulatory effects on proinsulin synthesis in isolated pancreatic islets of the rat (Jain and Logothetopoulos, 1977). Inosine has also been shown to have a strong gluconeogenic effect, which is independent of its insulin effect (Haeckel, 1977). These findings raise the question whether nucleosides produced by DAN activity in Sertoli cells may play a regulatory role for Sertoli cell functions and spermatogenesis. Further studies of the mechanisms regulating the enzyme activity may throw light on this possibility. SUMMARY
Unilateral cryptorchidism was associated with striking changes in specific DAN activity in the testis of both immature and adult rats. In immature rats, the specific DAN activity in the abdominal testis decreased to a lesser extent than in the scrotal, whereas in the adult rats an increase in specific DAN activity in the abdominal testis was seen. These observations confirm the previous finding that DAN activity is mainly located in the somatic cells. When expressed per abdominal testis, there was a decrease in total DAN activity in both immature and adult rats. This activity change was reversed by orchidopexy after cryptorchidism in adult animals. The fact that DAN activity was restored in spite of a poor recovery of spermatogenesis, indicates that the effect of cryptorchidism on DAN activity is due to the cryptorchid state and is not germ-cell mediated. Acknowledgements-The skilful technical Marit Haug and Mrs Gerd Rsnneberg ciated.
assistance is greatly
of Mrs appre-
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