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Prenatal androgen blockade with flutamide inhibits masculinization of the genitofemoral nerve and testicular descent
Prenatal Masculinization ByDay
Androgen Blockade With Flutamide Inhibits of the Genitofemoral Nerve and Testicular Descent
Way Goh, William
Middlesworth,
Pamela J. Farmer, and John M. Hutson
Melbourne, Australia l Prenatal androgen blockade with the antiandrogen flutamide inhibits the inguinoscrotal phase of testicular descent. The evidence suggests that androgens may act indirectly via the sexually dimorphic genitofemoral nerve (GFN) to control this phase. Rats were exposed to flutamide on gestational days 16 through 19. Seven-day-old rats were subjected to retrograde fluorescent labelling of the GFN combined with immunohistochemistry for calcitonin generelated peptide (CGAP), a neurotransmitter found in the GFN. Fluorescent-labelled and CGRP-immunoreactive neurons in the GFN spinal nucleus were quantified. Sexual dimorphism of the GFN nucleus was absent in the flutamide-treated rats but obviously present in control rats. Furthermore, control male nuclei had 24% more CGRP-immunoreactive neurons and 12% more fluorescent-labelled neurons than did flutamide-treated male nuclei. This study shows that prenatal androgen blockade with flutamide inhibits masculinization of the GFN, with significant reduction of its CGRP content. This supports the proposal that androgens act via the GFN, with CGRP as the second messenger, to control inguinoscrotal testicular descent. Copyright o 1994 by W. B. Saunders Company INDEX WORDS: Cryptorchidism, flutamide, nerve, calcitonin gene-related peptide.
genitofemoral
T
HE INGUINOSCROTAL phase of testicular descent is androgen-dependent, and prenatal androgen blockade with flutamide, an antiandrogen that acts by competitive binding of the androgen receptor, causes failure of this phase in rodents.‘-” It appears that androgens do not have a direct mode of action in controlling this phase but may act via the genitofemoral nerve (GFN).4 An intact GFN has been shown to be essential for completion of this phase. 4,5 The aim of this study was to determine whether prenatal androgen blockade would inhibit normal sexual dimorphism of the GFN spinal nucleus. From the Surgical Research Unit, Royal Children’s Hospital, Melbourne, Australia. Presented at the 40th Annual International Congress of the British Association of Paediatric Surgeons, Manchester, England, July 21-23. 1993. Address reprint requests to John M. Hutson, MD, FRACS, Depatiment of General Surgery, Royal Children S Hospital, Flemington Rd. ParkviNe, Victoria 3052, Australia. Copyright o I994 by W.B. Saunders Company 0022-3468/94/2906-0031$03.0010
836
MATERIALS
AND METHODS
Sprague-Dawley rats were maintained at the Royal Children’s Hospital. Females in estrus were placed with males for 16 hours, then separated and housed individually. Pregnant dams were injected subcutaneously with flutamide (gift from Dr M.C. Railings of Schering Plough Pty Ltd, Sydney, Australia) at a dose of 100 mgikgld, dissolved in 100 ~.LLethanol and mixed with 400 (LL sesame oil, on postcoital days 16 through 19. The control dams were injected with vehicle only. The pregnant dams delivered spontaneously on postcoital day 23. At 7 days of age, the newborn rats were anaesthetised with oxygen and 2% halothane and underwent laparotomy. The GFN on each side was identified retroperitoneally and transected. Crystals of Fast Blue (Sigma Chemical Co, St Louis, MO) were applied to the proximal cut nerve end. The abdominal wound was sutured with 6-O silk. The animals were maintained for 48 hours, then reanaesthetised. The thorax was opened, and transcardiac perfusion was performed with normal saline followed by Zamboni’s fixative. The spinal cord was removed, stored in Zamboni’s tixative for 3 to 7 days at 4“C, then cleared in dimethylsulfoxide and stored in phosphate-buffered saline (PBS) (30% wtivol sucrose) for 1 to 3 days. The tissues were embedded in OCT embedding medium (Miles, Elkhart, IN) and frozen with isopentane over liquid nitrogen; 25-urn frozen serial sections were placed on slides coated with a 1% gelatin mixture and allowed to dry. Sections were viewed under an epifluorescence microscope (Leitz Diaplan), and only neurons with bright fluorescence and clearly defined nuclei were counted as positive.6 The slides were then incubated for 5 minutes with 3% hydrogen peroxide to quench endogenous peroxidase, and were preincubated with 10% normal sheep serum for 30 minutes to block nonspecific staining. Excess serum was removed and the slides were incubated overnight with a primary antibody raised in a rabbit against rat CGRP (Chemicon International Inc, El Segundo, CA: 1:4,000). The next day, following a 15-minute PBS rinse, a biotinylated secondary antibody was added for 2 hours (Amersham RPN 1004, donkey antirabbit; 1:200). The slides were rinsed, and a streptavidinbiotin-peroxidase complex (Amersham RPN 1051; 1:lOO) was added for 30 minutes. After another rinse, DAB (3’,3’-Diaminobenzidine; Sigma Chemical Co) substrate was added for 10 minutes, and the slides were counterstained with haematoxylin and mounted with DPX (DePeX mounting medium; BDH Chemicals, Kilsyth, Australia). Sections were viewed, and neurons with strong CGRPimmunoreactivity and clearly defined nuclei were counted as positive.h Negative controls included either PBS alone or antiserum with excess synthetic rat CGRP instead of the primary antibody. Dorsal root ganglia served as positive controls for CGRP.h Statistical analysis was performed using the unpaired t test and the Mann-Whitney U test.
JournalofPediafric Surgery, Vol29, No 6 (June), 1994: pp 838838
FLUTAMIDE
AND THE GENITOFEMORAL
Flutamlde-treated Rat
NERVE
837
Control Rat
DISCUSSION
300
E g 200 i 6 B s 100 %
0 Fact Blue
CGRP
Fast Blw
CGRP
Labelled
Ponittve
Labelled
Positive
Fig 1. Fast Blue-labelled and CGRP-immunoreactive neurons in genitofemoral nerve spinal nuclei of flutamide-treated (n = 16 of each sex) versus control rats (n = 16 of each sex). Bars represent mean +l SD. Statistical analysis was performed using the unpaired 1 test and Mann-Whitney U test. (m) males; (0) females. lP < ,001; tf < ,001; #P < .02.
RESULTS
Sexual dimorphism of the GFN spinal nucleus was absent in the flutamide-treated rats. There were no significant differences between male and female nuclei in terms of numbers of Fast Blue-labelled or of CGRP-immunoreactive neurons. In contrast, obvious sexual dimorphism was present in the control (vehicletreated) rats. Control male nuclei had almost twice as many Fast Blue-labelled as well as CGRP-immunoreactive neurons than did female nuclei (P < .OOl). Furthermore, control male nuclei had 24% more CGRP-immunoreactive neurons than did flutamidetreated male nuclei (P < .02). Control male nuclei also had 12% more Fast Blue-labelled neurons than did flutamide-treated male nuclei; however, this difference was not statistically significant (Fig 1). The numbers of Fast Blue-labelled and of CGRPimmunoreactive neurons in the flutamide-treated female nuclei are significantly higher in comparison to control female nuclei (P < .OOl) (Fig 1). Examination and dissection of the males from two litters (n = 6), at age 3.5 days, showed a 42% incidence of undescended testes (5 of 12; 1 bilateral and 3 unilateral undescended testes). All undescended testes were situated in a suprainguinal position within a superficial inguinal pouch.‘-’
Prenatal androgen blockade with flutamide causes cryptorchidism in rodents. l-3The incidence of cryptorchidism in this study (42%) is similar to the 40% noted by others. Q More than 80% of such cryptorchid testes have completed the transabdominal phase of descent and are situated at or beyond the inguinal ring.122The inference is that prenatal androgen blockade inhibits the second phase of descent, which occurs postnatally in rodents.’ Sexual dimorphism of the central nervous system is well-documented.’ The evidence suggests that this sexual dimorphism is mediated by the hormonal environment during critical periods of the animal’s development. Specifically, it has been proposed that sex steroids, particularly androgens and oestrogens, act on the developing nervous system to cause structural and functional differences between the sexes.’ In rodents, prenatal flutamide treatment causes demasculinization of the spinal nucleus of the bulbocavernosus muscle and of the dorsolateral nucleus, which innervates the ischiocavernosus muscle.8*9 In contrast, prenatal treatment with testosterone causes masculinization of the spinal nucleus of the bulbocavernosus in female rats.rO Larkins et al6 have shown that the GFN spinal nucleus is sexually dimorphic in normal rodents, with the nucleus being twice as large, and with a fourfold greater number of CGRP-containing neurons in males than in females. This study demonstrates that prenatal androgen blockade with flutamide inhibits the normal sexual dimorphism of the GFN nucleus. It shows that prenatal flutamide treatment demasculinizes the GFN nucleus with a decrease in the numbers of neurons, particularly those containing CGRP. Paradoxically, the results suggest that masculinization of the GFN nucleus occurs in flutamide-treated female rats. The numbers of fluorescent-labelled and CGRP-immunoreactive neurons in the flutamidetreated female nuclei are significantly increased relative to control female nuclei (Fig 1). It is possible that in the female fetus with no endogenous androgens, receptor-bound flutamide may manifest a partial agonist effect similar to the mechanism by which tamoxifen, an antioestrogen that acts by competitive binding to the oestrogen receptor, is capable of a partial oestrogen agonist effect.”
REFERENCES 1. Spencer JR, Torrado T. Sanchez, et al: Effects of flutamide and finasteride on rat testicular descent. Endocrinology 129:741748,199l
2. Husmann DA, McPhaul MJ: Time-specific androgen blockade with flutamide inhibits testicular descent in the rat. Endocrinology 129:1409-1416,199l
a38
3. Goh DW, Momose Y, Middlesworth W. et al: The relationship between CGRP, androgens and gubernacular development in three animal models of cryptorchidism. J Ural 150:571-573, 1993 4. Hutson JM, Baker ML, Griffiths AL, et al: Endocrine and morphological perspectives in testicular descent. Reprod Med Rev 1:165-177,199Z 5. Beasley SW, Hutson JM: Effect of division of genitofemoral nerve on testicular descent in the rat: Aust N 2 J Surg 57:49-51, 1987 6. Larkins SL, Hutson JM, Williams MPL: Localisation of calcitonin gene-related peptide immunoreactivity within the spinal nucleus of the genitofemoral nerve. Pediatr Surg Int 6:176-179, 1991 7. Arnold AP, Gorski RA: Gonadal steroid induction of structural sex differences in the central nervous system. Ann Rev Neurosci 7:413-442, 1984
GOH ET
AL
8. Grisham W, Casto JM, Kashon ML, et al: Prenatal flutamide alters sexually dimorphic nuclei in the spinal cord of male rats. Brain Res 578:69-74,1992 9. Breedlove SM, Arnold AP: Hormonal control of a developing neuromuscular system. I. Complete demasculinization of the male rat spinal nucleus of the bulbocavernosus using the antiandrogen flutamide. J Neurosci 3:417-423,1983 10. Breedlove SM, Arnold AP: Hormonal control of a developing neuromuscular system. II. Sensitive periods for the androgeninduced masculinization of the rat spinal nucleus of the bulbocavernosus. J Neurosci 3:424-432, 1983 11. Neumann F, Elger W: Sexual differentiation. Studies with antiandrogens, in Serio M (ed): Sexual Differentiation: Basic and Clinical Aspects. New York, NY, Raven, 1984, pp 191-208
Androgen Blockade With Flutamide Inhibits of the Genitofemoral Nerve and Testicular Descent
Way Goh, William
Middlesworth,
Pamela J. Farmer, and John M. Hutson
Melbourne, Australia l Prenatal androgen blockade with the antiandrogen flutamide inhibits the inguinoscrotal phase of testicular descent. The evidence suggests that androgens may act indirectly via the sexually dimorphic genitofemoral nerve (GFN) to control this phase. Rats were exposed to flutamide on gestational days 16 through 19. Seven-day-old rats were subjected to retrograde fluorescent labelling of the GFN combined with immunohistochemistry for calcitonin generelated peptide (CGAP), a neurotransmitter found in the GFN. Fluorescent-labelled and CGRP-immunoreactive neurons in the GFN spinal nucleus were quantified. Sexual dimorphism of the GFN nucleus was absent in the flutamide-treated rats but obviously present in control rats. Furthermore, control male nuclei had 24% more CGRP-immunoreactive neurons and 12% more fluorescent-labelled neurons than did flutamide-treated male nuclei. This study shows that prenatal androgen blockade with flutamide inhibits masculinization of the GFN, with significant reduction of its CGRP content. This supports the proposal that androgens act via the GFN, with CGRP as the second messenger, to control inguinoscrotal testicular descent. Copyright o 1994 by W. B. Saunders Company INDEX WORDS: Cryptorchidism, flutamide, nerve, calcitonin gene-related peptide.
genitofemoral
T
HE INGUINOSCROTAL phase of testicular descent is androgen-dependent, and prenatal androgen blockade with flutamide, an antiandrogen that acts by competitive binding of the androgen receptor, causes failure of this phase in rodents.‘-” It appears that androgens do not have a direct mode of action in controlling this phase but may act via the genitofemoral nerve (GFN).4 An intact GFN has been shown to be essential for completion of this phase. 4,5 The aim of this study was to determine whether prenatal androgen blockade would inhibit normal sexual dimorphism of the GFN spinal nucleus. From the Surgical Research Unit, Royal Children’s Hospital, Melbourne, Australia. Presented at the 40th Annual International Congress of the British Association of Paediatric Surgeons, Manchester, England, July 21-23. 1993. Address reprint requests to John M. Hutson, MD, FRACS, Depatiment of General Surgery, Royal Children S Hospital, Flemington Rd. ParkviNe, Victoria 3052, Australia. Copyright o I994 by W.B. Saunders Company 0022-3468/94/2906-0031$03.0010
836
MATERIALS
AND METHODS
Sprague-Dawley rats were maintained at the Royal Children’s Hospital. Females in estrus were placed with males for 16 hours, then separated and housed individually. Pregnant dams were injected subcutaneously with flutamide (gift from Dr M.C. Railings of Schering Plough Pty Ltd, Sydney, Australia) at a dose of 100 mgikgld, dissolved in 100 ~.LLethanol and mixed with 400 (LL sesame oil, on postcoital days 16 through 19. The control dams were injected with vehicle only. The pregnant dams delivered spontaneously on postcoital day 23. At 7 days of age, the newborn rats were anaesthetised with oxygen and 2% halothane and underwent laparotomy. The GFN on each side was identified retroperitoneally and transected. Crystals of Fast Blue (Sigma Chemical Co, St Louis, MO) were applied to the proximal cut nerve end. The abdominal wound was sutured with 6-O silk. The animals were maintained for 48 hours, then reanaesthetised. The thorax was opened, and transcardiac perfusion was performed with normal saline followed by Zamboni’s fixative. The spinal cord was removed, stored in Zamboni’s tixative for 3 to 7 days at 4“C, then cleared in dimethylsulfoxide and stored in phosphate-buffered saline (PBS) (30% wtivol sucrose) for 1 to 3 days. The tissues were embedded in OCT embedding medium (Miles, Elkhart, IN) and frozen with isopentane over liquid nitrogen; 25-urn frozen serial sections were placed on slides coated with a 1% gelatin mixture and allowed to dry. Sections were viewed under an epifluorescence microscope (Leitz Diaplan), and only neurons with bright fluorescence and clearly defined nuclei were counted as positive.6 The slides were then incubated for 5 minutes with 3% hydrogen peroxide to quench endogenous peroxidase, and were preincubated with 10% normal sheep serum for 30 minutes to block nonspecific staining. Excess serum was removed and the slides were incubated overnight with a primary antibody raised in a rabbit against rat CGRP (Chemicon International Inc, El Segundo, CA: 1:4,000). The next day, following a 15-minute PBS rinse, a biotinylated secondary antibody was added for 2 hours (Amersham RPN 1004, donkey antirabbit; 1:200). The slides were rinsed, and a streptavidinbiotin-peroxidase complex (Amersham RPN 1051; 1:lOO) was added for 30 minutes. After another rinse, DAB (3’,3’-Diaminobenzidine; Sigma Chemical Co) substrate was added for 10 minutes, and the slides were counterstained with haematoxylin and mounted with DPX (DePeX mounting medium; BDH Chemicals, Kilsyth, Australia). Sections were viewed, and neurons with strong CGRPimmunoreactivity and clearly defined nuclei were counted as positive.h Negative controls included either PBS alone or antiserum with excess synthetic rat CGRP instead of the primary antibody. Dorsal root ganglia served as positive controls for CGRP.h Statistical analysis was performed using the unpaired t test and the Mann-Whitney U test.
JournalofPediafric Surgery, Vol29, No 6 (June), 1994: pp 838838
FLUTAMIDE
AND THE GENITOFEMORAL
Flutamlde-treated Rat
NERVE
837
Control Rat
DISCUSSION
300
E g 200 i 6 B s 100 %
0 Fact Blue
CGRP
Fast Blw
CGRP
Labelled
Ponittve
Labelled
Positive
Fig 1. Fast Blue-labelled and CGRP-immunoreactive neurons in genitofemoral nerve spinal nuclei of flutamide-treated (n = 16 of each sex) versus control rats (n = 16 of each sex). Bars represent mean +l SD. Statistical analysis was performed using the unpaired 1 test and Mann-Whitney U test. (m) males; (0) females. lP < ,001; tf < ,001; #P < .02.
RESULTS
Sexual dimorphism of the GFN spinal nucleus was absent in the flutamide-treated rats. There were no significant differences between male and female nuclei in terms of numbers of Fast Blue-labelled or of CGRP-immunoreactive neurons. In contrast, obvious sexual dimorphism was present in the control (vehicletreated) rats. Control male nuclei had almost twice as many Fast Blue-labelled as well as CGRP-immunoreactive neurons than did female nuclei (P < .OOl). Furthermore, control male nuclei had 24% more CGRP-immunoreactive neurons than did flutamidetreated male nuclei (P < .02). Control male nuclei also had 12% more Fast Blue-labelled neurons than did flutamide-treated male nuclei; however, this difference was not statistically significant (Fig 1). The numbers of Fast Blue-labelled and of CGRPimmunoreactive neurons in the flutamide-treated female nuclei are significantly higher in comparison to control female nuclei (P < .OOl) (Fig 1). Examination and dissection of the males from two litters (n = 6), at age 3.5 days, showed a 42% incidence of undescended testes (5 of 12; 1 bilateral and 3 unilateral undescended testes). All undescended testes were situated in a suprainguinal position within a superficial inguinal pouch.‘-’
Prenatal androgen blockade with flutamide causes cryptorchidism in rodents. l-3The incidence of cryptorchidism in this study (42%) is similar to the 40% noted by others. Q More than 80% of such cryptorchid testes have completed the transabdominal phase of descent and are situated at or beyond the inguinal ring.122The inference is that prenatal androgen blockade inhibits the second phase of descent, which occurs postnatally in rodents.’ Sexual dimorphism of the central nervous system is well-documented.’ The evidence suggests that this sexual dimorphism is mediated by the hormonal environment during critical periods of the animal’s development. Specifically, it has been proposed that sex steroids, particularly androgens and oestrogens, act on the developing nervous system to cause structural and functional differences between the sexes.’ In rodents, prenatal flutamide treatment causes demasculinization of the spinal nucleus of the bulbocavernosus muscle and of the dorsolateral nucleus, which innervates the ischiocavernosus muscle.8*9 In contrast, prenatal treatment with testosterone causes masculinization of the spinal nucleus of the bulbocavernosus in female rats.rO Larkins et al6 have shown that the GFN spinal nucleus is sexually dimorphic in normal rodents, with the nucleus being twice as large, and with a fourfold greater number of CGRP-containing neurons in males than in females. This study demonstrates that prenatal androgen blockade with flutamide inhibits the normal sexual dimorphism of the GFN nucleus. It shows that prenatal flutamide treatment demasculinizes the GFN nucleus with a decrease in the numbers of neurons, particularly those containing CGRP. Paradoxically, the results suggest that masculinization of the GFN nucleus occurs in flutamide-treated female rats. The numbers of fluorescent-labelled and CGRP-immunoreactive neurons in the flutamidetreated female nuclei are significantly increased relative to control female nuclei (Fig 1). It is possible that in the female fetus with no endogenous androgens, receptor-bound flutamide may manifest a partial agonist effect similar to the mechanism by which tamoxifen, an antioestrogen that acts by competitive binding to the oestrogen receptor, is capable of a partial oestrogen agonist effect.”
REFERENCES 1. Spencer JR, Torrado T. Sanchez, et al: Effects of flutamide and finasteride on rat testicular descent. Endocrinology 129:741748,199l
2. Husmann DA, McPhaul MJ: Time-specific androgen blockade with flutamide inhibits testicular descent in the rat. Endocrinology 129:1409-1416,199l
a38
3. Goh DW, Momose Y, Middlesworth W. et al: The relationship between CGRP, androgens and gubernacular development in three animal models of cryptorchidism. J Ural 150:571-573, 1993 4. Hutson JM, Baker ML, Griffiths AL, et al: Endocrine and morphological perspectives in testicular descent. Reprod Med Rev 1:165-177,199Z 5. Beasley SW, Hutson JM: Effect of division of genitofemoral nerve on testicular descent in the rat: Aust N 2 J Surg 57:49-51, 1987 6. Larkins SL, Hutson JM, Williams MPL: Localisation of calcitonin gene-related peptide immunoreactivity within the spinal nucleus of the genitofemoral nerve. Pediatr Surg Int 6:176-179, 1991 7. Arnold AP, Gorski RA: Gonadal steroid induction of structural sex differences in the central nervous system. Ann Rev Neurosci 7:413-442, 1984
GOH ET
AL
8. Grisham W, Casto JM, Kashon ML, et al: Prenatal flutamide alters sexually dimorphic nuclei in the spinal cord of male rats. Brain Res 578:69-74,1992 9. Breedlove SM, Arnold AP: Hormonal control of a developing neuromuscular system. I. Complete demasculinization of the male rat spinal nucleus of the bulbocavernosus using the antiandrogen flutamide. J Neurosci 3:417-423,1983 10. Breedlove SM, Arnold AP: Hormonal control of a developing neuromuscular system. II. Sensitive periods for the androgeninduced masculinization of the rat spinal nucleus of the bulbocavernosus. J Neurosci 3:424-432, 1983 11. Neumann F, Elger W: Sexual differentiation. Studies with antiandrogens, in Serio M (ed): Sexual Differentiation: Basic and Clinical Aspects. New York, NY, Raven, 1984, pp 191-208