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Mullerian-inhibiting substance deficiency in transgenic mice interferes with postnatal germ cell development: clues for understanding infertility in cryptorchidism
Journal of Pediatric Surgery (2009) 44, 2335–2338
www.elsevier.com/locate/jpedsurg
Mullerian-inhibiting substance deficiency in transgenic mice interferes with postnatal germ cell development: clues for understanding infertility in cryptorchidism☆ Rangga Prasetyo a , Pamela Farmer b , Ian McLennan d , Bridget Southwell a,b , John Hutson a,b,c,⁎ a
Department of Paediatrics, University of Melbourne, Australia Douglas Stephens Surgical Research Laboratory, Murdoch Childrens Research Institute, Australia c Department of Urology, Royal Children's Hospital, Melbourne, Australia d Department of Anatomy and Structural Biology, University of Otago, New Zealand b
Received 25 July 2009; accepted 31 July 2009
Key words: Mullerian-inhibiting substance; Testis; Mouse mutant; Germ cell; Postnatal
Abstract Aim: Mullerian-inhibiting substance (MIS) may have a role in postnatal germ cell development, although this remains unproven. Elucidating the regulatory factors is crucial in finding new treatments for preventing infertility in cryptorchidism. We studied germ cell development in neonatal mice with MIS gene or receptor mutation to determine if germ cell development was affected. Methods: Neonatal (5 MIS mutants, ×1 MIS receptor mutant and 5 wild-type) and 10-day-old mice (×7 MIS mutants, ×1 MIS receptor mutant, 5 wild-type) were killed and prepared for hematoxylin-eosin and Masson trichrome histology of the testis. Testis diameter and tubule diameter were measured by ImageJ, and germ cells were counted in 50 tubules/testis. Results: Total testis and tubular diameters were greater in wild-type vs MIS mutants at days 0 and 10 (P b .01). Gonocytes were decreased in MIS mutants vs wild-type on day 0 (P = .019), and on day 10, the number of type A spermatogonia was slightly decreased (P = .05) and type B spermatogonia were significantly decreased (P b .01). Similar results were seen in the MIS receptor knockout. Conclusion: These results suggest that MIS has a previously unrecognized role in perinatal germ cell development that needs further investigation. Mullerian-inhibiting substance may be a possible future treatment for stimulating germ cell development in cryptorchidism. © 2009 Elsevier Inc. All rights reserved.
Presented at the 42nd Annual Meeting of the Pacific Association of Pediatric Surgeons, Hong Kong, China, May 10-14, 2009. ☆ This study was supported in part by the National Health and medical Research Council (Australia) (grant 436913). ⁎ Corresponding author. Department of Urology, Royal Children's Hospital, Australia. Tel.: +61 3 9345 5805; fax: +61 3 9345 7997. E-mail address: [email protected] (J. Hutson). 0022-3468/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2009.07.058
Infertility remains a significant problem for men with previous cryptorchidism [1] despite significant advances in management. It was once thought that orchidopexy could be delayed to puberty because spermatogenesis did not begin until then. This notion underpinned the recommended age for orchidopexy in much of the late 20th century, until
2336 it was appreciated that there were progressive secondary anomalies occurring in the cryptorchid testis [2]. It gradually became known that the postnatal germ cell undergoes crucial developmental steps in infancy and that these are essential prerequisites for postpubertal spermatogenesis [3]. The crucial steps in postnatal germ cell development are the conversion of neonatal gonocytes into type A spermatogonia and then on to type B spermatogonia and primary spermatocytes [4]. It is now thought that the stem cell for spermatogenesis is the type A spermatogonium, which first appears in the postnatal testis at 3 to 9 months of age [5]. Regulation of this transformation from gonocyte type A spermatogonium has been proposed to be by androgens, during the so-called mini-puberty at 2 to 6 months of age [6]. However, this remains controversial, with androgens having a proven role only in differentiation of primary spermatocytes. By contrast, there is some direct and indirect evidence that mullerian-inhibiting substance (MIS), which is also called anti-mullerian hormone (AMH) may be important [7]. Mullerian-inhibiting substance/anti-mullerian hormone was first predicted to exist by Alfred Jost [8], who postulated its existence to explain regression of the mullerian ducts to prevent uterus and tubes development in a male embryo. Much is now known about MIS/AMH [9], but its role in postnatal germ cell development is illdefined. In some in vitro systems, neonatal germ cell development is augmented by MIS/AMH [7]. Initial examination of MIS/AMH transgenic mutants showed that the males had descended testes and functional sperm [8]. However, whether there are subtle effects on germ cell development was not determined. In this study, we aimed to reexamine the role of MIS/AMH in postnatal germ cell development in MIS/AMH mutant mice.
R. Prasetyo et al.
1. Methods Mice carrying a targeted deletion of the MIS/AMH gene were bred in the laboratory of one of the authors (IM) with ethical approval of the institutional animal care and use committee. Because adult males with homozygous mutations are infertile due to abnormal Wolffian duct connection (and presence of a uterus and upper vagina), homozygous animals for study were bred by mating heterozygote males with homozygote females. Mice with a mutation that functionally inactivated the type II MIS/AMH receptor (MISR) were generated by gene targeting in embryonic stem cells. A 4.4-kb section of the receptor gene encoding the first 6 exons (the extracellular ligand-binding site and transmembrane domain) was deleted and replaced with a neomycin-resistance expression cassette [8]. Homozygous mutants were produced by injection of correctly targeted embryonic stem cell clones into blastocysts to create chimeric mice, followed by a selective breeding program. All animal experimentation was approved by the institutional animal ethics committee. Newborn and 10day-old animals were killed and fixed in formalin. Specimens (5 μm) were prepared for histological examination in the sagittal plane and stained by standard techniques for hematoxylin-eosin and modified Masson trichrome as previously described [10,11]. For trichrome staining, sections were dewaxed, brought to water, and placed in preheated Bouin solution for 20 minutes at 60°C. After washing in running water, nuclear staining was performed using Weighert hematoxylin for 8 to 10 minutes. After another wash, sections were stained in trichrome for 10 to 15 minutes at room temperature, then rinsed in 0.5% acetic acid, dehydrated, cleared, and mounted. One section per testis was selected for measuring and counting. Measurements of testis diameter and tubule diameter were done by Image-J Freeware (National Institutes of Health, Maryland).
Fig. 1 A, Histological sagittal section of testis (T) in wild-type mouse at day 10 (scale bar, 1 mm) (WD, Wolfian duct). B, Similar section of MIS/AMH mutant on day 10, showing smaller testis, and dilated end of mullerian duct (MD) adjacent to the epididymis and vas deferens. C, Histogram showing testicular diameter on days 0 and 10 in wild-type (W) and mutant (M) mice.
Mullerian-inhibiting substance
2337 was used to compare groups, with P b .05 regarded as significant.
2. Results
Fig. 2 Histogram of cross-sectional tubular diameter in wild-type (W) and mutant (M) mice on days 0 and 10.
Sections were viewed under a light microscope (Leica MSG, Wetzler, Germany) attached to a digital camera taken with Leica IM50 program at a range of magnifications (1.6 × 40×). Data were entered into Microsoft Excel and Graphpad prism 3.0 for statistical analysis and graphing. Student t test
Twelve MIS/AMH mutant mice (five newborn, seven 10day-old) were examined and compared with 8 normal wildtype controls (four newborn, four day 10). In addition, 2 MISR mutant mice (one newborn, and one day 10) and 2 corresponding wild-types (one newborn, one day 10) were examined. Macroscopic differences were found in the testis diameter in both newborn and day-10 mice, with mutant animals having significantly smaller testes (P b .001) (Fig. 1). The MIS/AMH mutants and the MISR mutants showed qualitatively similar results. The diameter of the spermatic tubules, like the whole testis diameter, was significantly smaller in MIS/AMH mutant mice compared with wild-types at both day 0 and day 10 (P b .001), with MISR mutants showing a similar qualitative difference (Fig. 2). On germ cell counts, fewer gonocytes were found in the MIS/AMH mutant animals compared with controls at day 0 (P = .02). At 10 days of age, there was a small difference in the number of type A spermatogonia (P = .05) but a more significant decrease in type B spermatogonia (P b .01) (Fig. 3). Only occasional primary spermatocytes were seen in mutant and wild-type animals on day 10. The MISR mutant had similar qualitative
Fig. 3 Histogram of germ cell counts at days 0 and 10 in wild-type (W) and mutant (M) mice. On day 0, all germ cells were gonocytes, whereas on day 10, the populations of germ cells were type A or B spermatogonia.
2338 decreases to the MIS/AMH mutant in the numbers of gonocytes, types A and type B spermatogonia.
3. Discussion This study found that there were subtle but statistically significant differences in testis diameter, tubule diameter, gonocyte numbers, and types A and B spermatocyte numbers in day-0 and day-10 mouse testes between MIS/AMH and wild-type control animals. The MIS/AMH is a 140-hD homodimeric glycoprotein produced by Sertoli cells and responsible for regression of the mullerian ducts in the embryo [11,12]. The known physiological actions of MIS/AMH are currently limited to sexual differentiation in males and mature gonadal function in both sexes [12], although recently, it has been reported to have a role in motor neurone function [9]. A possible role for MIS/AMH in early germ cell development comes from studies of mouse testes in organ culture, where addition of exogenous MIS/AMH caused normal germ cell development [7]. In humans, there is a postnatal rise in serum levels of MIS/ AMH in the second 6 months of life, which coincides with gonocyte transformation [13]. The impaired gonocyte transformation that occurs in cryptorchidism [14] is associated with decreased levels of MIS/AMH, although it is not known whether the latter is cause or effect [15]. An alternative hypothesis is that germ cell development is regulated by gonatrophins and/or androgens during the “mini-puberty” at 3 to 6 months of age [4]. Previous work on transgenic mutants with MIS/AMH deficiency showed that the testes could make some functional sperm, but there was no detailed examination of the postnatal development to determine whether or not this was completely normal [11]. This suggests that MIS/ AMH is not the sole factor regulating germ cell differentiation, but the results described here support the conclusion that MIS/AMH may also be involved in this process. Clearly, much more needs to be understood about postnatal germ cell development, so that new ways to prevent infertility and cancer can be instituted for patients with cryptorchidism [16].
R. Prasetyo et al. In conclusion, careful study of postnatal testicular development in transgenic mice with inactivation of MIS/ AMH or its receptor found inhibition of germ cell development. A better understanding of this effect may help in developing new strategies to augment germ cell numbers in cryptorchid boys to reduce the long-term risk of infertility.
References [1] Hutson JM, Hasthorpe S. Abnormalities of testicular descent. Cell Tissue Res 2005;322(1):155-8. [2] Huff DS, et al. Postnatal testicular maldevelopment in unilateral cryptorchidism. J Urol 1989;142(2 Pt 2):546-8 [discussion 572]. [3] Ritzen EM. Undescended testes: a consensus on management. Eur J Endocrinol 2008;159:87-90. [4] Hadziselimovic F, et al. The significance of postnatal gonadotropin surge for testicular development in normal and cryptorchid testes. J Urol 1986;136(1 Pt 2):274-6. [5] Ong C, Hasthorpe S, Hutson JM. Germ cell development in the descended and cryptorchid testis and the effects of hormonal manipulation. Pediatr Surg Int 2005;21(4):240-54. [6] Virtanen H, et al. Cryptorchidism: classification, prevalence and longterm consequences. Acta Paediatr 2007;96:611-6. [7] Zhou B, Watts LM, Hutson JM. Germ cell development in neonatal mouse testes in vitro requires mullerian inhibiting substance. J Urol 1993;150(2 Pt 2):613-6. [8] Jost A. Problems of fetal endocrinology: the gonadal and hypophysical hormones. Rec Prog Hormone Res 1953;8:379-413. [9] Wang PY, et al. Mullerian inhibiting substance acts as a motor neuron survival factor in vitro. Proc Natl Acad Sci U S A 2005;102(45): 16421-5. [10] Gomori G. A rapid one-step trichrome stain. Am J Clin Pathol 1950;20 (7):661-4. [11] Behringer RR, Finegold MJ, Cate RL. Mullerian-inhibiting substance function during mammalian sexual development. Cell 1994;79(3): 415-25. [12] Teixeira J, Maheswaran S, Donahoe PK. Mullerian inhibiting substance: an instructive developmental hormone with diagnostic and possible therapeutic applications. Endocr Rev 2001;22(5):657-74. [13] Baker ML, Metcalfe SA, Hutson JM. Serum levels of mullerian inhibiting substance in boys from birth to 18 years, as determined by enzyme immunoassay. J Clin Endocrinol Metab 1990;70(1):11-5. [14] Huff DS, et al. Early postnatal testicular maldevelopment in cryptorchidism. J Urol 1991;146(2 ( Pt 2)):624-6. [15] Yamanaka J, et al. Serum levels of mullerian inhibiting substance in boys with cryptorchidism. J Pediatr Surg 1991;26(5):621-3. [16] Rajpert-DeMeyts E. Recent advances and future directions in research on testicular germ cell cancer. Int J Androl 2007;30:192-7.
www.elsevier.com/locate/jpedsurg
Mullerian-inhibiting substance deficiency in transgenic mice interferes with postnatal germ cell development: clues for understanding infertility in cryptorchidism☆ Rangga Prasetyo a , Pamela Farmer b , Ian McLennan d , Bridget Southwell a,b , John Hutson a,b,c,⁎ a
Department of Paediatrics, University of Melbourne, Australia Douglas Stephens Surgical Research Laboratory, Murdoch Childrens Research Institute, Australia c Department of Urology, Royal Children's Hospital, Melbourne, Australia d Department of Anatomy and Structural Biology, University of Otago, New Zealand b
Received 25 July 2009; accepted 31 July 2009
Key words: Mullerian-inhibiting substance; Testis; Mouse mutant; Germ cell; Postnatal
Abstract Aim: Mullerian-inhibiting substance (MIS) may have a role in postnatal germ cell development, although this remains unproven. Elucidating the regulatory factors is crucial in finding new treatments for preventing infertility in cryptorchidism. We studied germ cell development in neonatal mice with MIS gene or receptor mutation to determine if germ cell development was affected. Methods: Neonatal (5 MIS mutants, ×1 MIS receptor mutant and 5 wild-type) and 10-day-old mice (×7 MIS mutants, ×1 MIS receptor mutant, 5 wild-type) were killed and prepared for hematoxylin-eosin and Masson trichrome histology of the testis. Testis diameter and tubule diameter were measured by ImageJ, and germ cells were counted in 50 tubules/testis. Results: Total testis and tubular diameters were greater in wild-type vs MIS mutants at days 0 and 10 (P b .01). Gonocytes were decreased in MIS mutants vs wild-type on day 0 (P = .019), and on day 10, the number of type A spermatogonia was slightly decreased (P = .05) and type B spermatogonia were significantly decreased (P b .01). Similar results were seen in the MIS receptor knockout. Conclusion: These results suggest that MIS has a previously unrecognized role in perinatal germ cell development that needs further investigation. Mullerian-inhibiting substance may be a possible future treatment for stimulating germ cell development in cryptorchidism. © 2009 Elsevier Inc. All rights reserved.
Presented at the 42nd Annual Meeting of the Pacific Association of Pediatric Surgeons, Hong Kong, China, May 10-14, 2009. ☆ This study was supported in part by the National Health and medical Research Council (Australia) (grant 436913). ⁎ Corresponding author. Department of Urology, Royal Children's Hospital, Australia. Tel.: +61 3 9345 5805; fax: +61 3 9345 7997. E-mail address: [email protected] (J. Hutson). 0022-3468/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2009.07.058
Infertility remains a significant problem for men with previous cryptorchidism [1] despite significant advances in management. It was once thought that orchidopexy could be delayed to puberty because spermatogenesis did not begin until then. This notion underpinned the recommended age for orchidopexy in much of the late 20th century, until
2336 it was appreciated that there were progressive secondary anomalies occurring in the cryptorchid testis [2]. It gradually became known that the postnatal germ cell undergoes crucial developmental steps in infancy and that these are essential prerequisites for postpubertal spermatogenesis [3]. The crucial steps in postnatal germ cell development are the conversion of neonatal gonocytes into type A spermatogonia and then on to type B spermatogonia and primary spermatocytes [4]. It is now thought that the stem cell for spermatogenesis is the type A spermatogonium, which first appears in the postnatal testis at 3 to 9 months of age [5]. Regulation of this transformation from gonocyte type A spermatogonium has been proposed to be by androgens, during the so-called mini-puberty at 2 to 6 months of age [6]. However, this remains controversial, with androgens having a proven role only in differentiation of primary spermatocytes. By contrast, there is some direct and indirect evidence that mullerian-inhibiting substance (MIS), which is also called anti-mullerian hormone (AMH) may be important [7]. Mullerian-inhibiting substance/anti-mullerian hormone was first predicted to exist by Alfred Jost [8], who postulated its existence to explain regression of the mullerian ducts to prevent uterus and tubes development in a male embryo. Much is now known about MIS/AMH [9], but its role in postnatal germ cell development is illdefined. In some in vitro systems, neonatal germ cell development is augmented by MIS/AMH [7]. Initial examination of MIS/AMH transgenic mutants showed that the males had descended testes and functional sperm [8]. However, whether there are subtle effects on germ cell development was not determined. In this study, we aimed to reexamine the role of MIS/AMH in postnatal germ cell development in MIS/AMH mutant mice.
R. Prasetyo et al.
1. Methods Mice carrying a targeted deletion of the MIS/AMH gene were bred in the laboratory of one of the authors (IM) with ethical approval of the institutional animal care and use committee. Because adult males with homozygous mutations are infertile due to abnormal Wolffian duct connection (and presence of a uterus and upper vagina), homozygous animals for study were bred by mating heterozygote males with homozygote females. Mice with a mutation that functionally inactivated the type II MIS/AMH receptor (MISR) were generated by gene targeting in embryonic stem cells. A 4.4-kb section of the receptor gene encoding the first 6 exons (the extracellular ligand-binding site and transmembrane domain) was deleted and replaced with a neomycin-resistance expression cassette [8]. Homozygous mutants were produced by injection of correctly targeted embryonic stem cell clones into blastocysts to create chimeric mice, followed by a selective breeding program. All animal experimentation was approved by the institutional animal ethics committee. Newborn and 10day-old animals were killed and fixed in formalin. Specimens (5 μm) were prepared for histological examination in the sagittal plane and stained by standard techniques for hematoxylin-eosin and modified Masson trichrome as previously described [10,11]. For trichrome staining, sections were dewaxed, brought to water, and placed in preheated Bouin solution for 20 minutes at 60°C. After washing in running water, nuclear staining was performed using Weighert hematoxylin for 8 to 10 minutes. After another wash, sections were stained in trichrome for 10 to 15 minutes at room temperature, then rinsed in 0.5% acetic acid, dehydrated, cleared, and mounted. One section per testis was selected for measuring and counting. Measurements of testis diameter and tubule diameter were done by Image-J Freeware (National Institutes of Health, Maryland).
Fig. 1 A, Histological sagittal section of testis (T) in wild-type mouse at day 10 (scale bar, 1 mm) (WD, Wolfian duct). B, Similar section of MIS/AMH mutant on day 10, showing smaller testis, and dilated end of mullerian duct (MD) adjacent to the epididymis and vas deferens. C, Histogram showing testicular diameter on days 0 and 10 in wild-type (W) and mutant (M) mice.
Mullerian-inhibiting substance
2337 was used to compare groups, with P b .05 regarded as significant.
2. Results
Fig. 2 Histogram of cross-sectional tubular diameter in wild-type (W) and mutant (M) mice on days 0 and 10.
Sections were viewed under a light microscope (Leica MSG, Wetzler, Germany) attached to a digital camera taken with Leica IM50 program at a range of magnifications (1.6 × 40×). Data were entered into Microsoft Excel and Graphpad prism 3.0 for statistical analysis and graphing. Student t test
Twelve MIS/AMH mutant mice (five newborn, seven 10day-old) were examined and compared with 8 normal wildtype controls (four newborn, four day 10). In addition, 2 MISR mutant mice (one newborn, and one day 10) and 2 corresponding wild-types (one newborn, one day 10) were examined. Macroscopic differences were found in the testis diameter in both newborn and day-10 mice, with mutant animals having significantly smaller testes (P b .001) (Fig. 1). The MIS/AMH mutants and the MISR mutants showed qualitatively similar results. The diameter of the spermatic tubules, like the whole testis diameter, was significantly smaller in MIS/AMH mutant mice compared with wild-types at both day 0 and day 10 (P b .001), with MISR mutants showing a similar qualitative difference (Fig. 2). On germ cell counts, fewer gonocytes were found in the MIS/AMH mutant animals compared with controls at day 0 (P = .02). At 10 days of age, there was a small difference in the number of type A spermatogonia (P = .05) but a more significant decrease in type B spermatogonia (P b .01) (Fig. 3). Only occasional primary spermatocytes were seen in mutant and wild-type animals on day 10. The MISR mutant had similar qualitative
Fig. 3 Histogram of germ cell counts at days 0 and 10 in wild-type (W) and mutant (M) mice. On day 0, all germ cells were gonocytes, whereas on day 10, the populations of germ cells were type A or B spermatogonia.
2338 decreases to the MIS/AMH mutant in the numbers of gonocytes, types A and type B spermatogonia.
3. Discussion This study found that there were subtle but statistically significant differences in testis diameter, tubule diameter, gonocyte numbers, and types A and B spermatocyte numbers in day-0 and day-10 mouse testes between MIS/AMH and wild-type control animals. The MIS/AMH is a 140-hD homodimeric glycoprotein produced by Sertoli cells and responsible for regression of the mullerian ducts in the embryo [11,12]. The known physiological actions of MIS/AMH are currently limited to sexual differentiation in males and mature gonadal function in both sexes [12], although recently, it has been reported to have a role in motor neurone function [9]. A possible role for MIS/AMH in early germ cell development comes from studies of mouse testes in organ culture, where addition of exogenous MIS/AMH caused normal germ cell development [7]. In humans, there is a postnatal rise in serum levels of MIS/ AMH in the second 6 months of life, which coincides with gonocyte transformation [13]. The impaired gonocyte transformation that occurs in cryptorchidism [14] is associated with decreased levels of MIS/AMH, although it is not known whether the latter is cause or effect [15]. An alternative hypothesis is that germ cell development is regulated by gonatrophins and/or androgens during the “mini-puberty” at 3 to 6 months of age [4]. Previous work on transgenic mutants with MIS/AMH deficiency showed that the testes could make some functional sperm, but there was no detailed examination of the postnatal development to determine whether or not this was completely normal [11]. This suggests that MIS/ AMH is not the sole factor regulating germ cell differentiation, but the results described here support the conclusion that MIS/AMH may also be involved in this process. Clearly, much more needs to be understood about postnatal germ cell development, so that new ways to prevent infertility and cancer can be instituted for patients with cryptorchidism [16].
R. Prasetyo et al. In conclusion, careful study of postnatal testicular development in transgenic mice with inactivation of MIS/ AMH or its receptor found inhibition of germ cell development. A better understanding of this effect may help in developing new strategies to augment germ cell numbers in cryptorchid boys to reduce the long-term risk of infertility.
References [1] Hutson JM, Hasthorpe S. Abnormalities of testicular descent. Cell Tissue Res 2005;322(1):155-8. [2] Huff DS, et al. Postnatal testicular maldevelopment in unilateral cryptorchidism. J Urol 1989;142(2 Pt 2):546-8 [discussion 572]. [3] Ritzen EM. Undescended testes: a consensus on management. Eur J Endocrinol 2008;159:87-90. [4] Hadziselimovic F, et al. The significance of postnatal gonadotropin surge for testicular development in normal and cryptorchid testes. J Urol 1986;136(1 Pt 2):274-6. [5] Ong C, Hasthorpe S, Hutson JM. Germ cell development in the descended and cryptorchid testis and the effects of hormonal manipulation. Pediatr Surg Int 2005;21(4):240-54. [6] Virtanen H, et al. Cryptorchidism: classification, prevalence and longterm consequences. Acta Paediatr 2007;96:611-6. [7] Zhou B, Watts LM, Hutson JM. Germ cell development in neonatal mouse testes in vitro requires mullerian inhibiting substance. J Urol 1993;150(2 Pt 2):613-6. [8] Jost A. Problems of fetal endocrinology: the gonadal and hypophysical hormones. Rec Prog Hormone Res 1953;8:379-413. [9] Wang PY, et al. Mullerian inhibiting substance acts as a motor neuron survival factor in vitro. Proc Natl Acad Sci U S A 2005;102(45): 16421-5. [10] Gomori G. A rapid one-step trichrome stain. Am J Clin Pathol 1950;20 (7):661-4. [11] Behringer RR, Finegold MJ, Cate RL. Mullerian-inhibiting substance function during mammalian sexual development. Cell 1994;79(3): 415-25. [12] Teixeira J, Maheswaran S, Donahoe PK. Mullerian inhibiting substance: an instructive developmental hormone with diagnostic and possible therapeutic applications. Endocr Rev 2001;22(5):657-74. [13] Baker ML, Metcalfe SA, Hutson JM. Serum levels of mullerian inhibiting substance in boys from birth to 18 years, as determined by enzyme immunoassay. J Clin Endocrinol Metab 1990;70(1):11-5. [14] Huff DS, et al. Early postnatal testicular maldevelopment in cryptorchidism. J Urol 1991;146(2 ( Pt 2)):624-6. [15] Yamanaka J, et al. Serum levels of mullerian inhibiting substance in boys with cryptorchidism. J Pediatr Surg 1991;26(5):621-3. [16] Rajpert-DeMeyts E. Recent advances and future directions in research on testicular germ cell cancer. Int J Androl 2007;30:192-7.