INSULIN 3-LIKE HORMONE AND ITS ROLE IN EPIDIDYMO-TESTICULAR DESCENT
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FARUK HADZISELIMOVIC, IBRAHIM ADHAM

Kindertagesklinik (FH), Liestal, Switzerland, and Institute of Human Genetics (IA), University of Gottingen, Gottingen, Germany

ABSTRACT

     Purpose: The role of insulin 3-like (Insl3) hormone signaling in the testicular descent process has been demonstrated. The purpose of the present study was to evaluate epididymal development in Insl3-deficient mice.
     Materials and Methods: Heterozygous and homozygous Insl3 mutants of a mixed CD1 X 129/Sv genetic background were generated by breeding Insl3-/- females with Insl3+/- males, and their genotypes were determined by polymerase chain reaction. On the first postnatal day, newborn males were sacrificed, embedded in paraffin, and cut in 4 µm sections. Sections were stained with hematoxylin/eosin and immunoreacted with anti-α actin antibody.
     Results: An analysis of stained sections indicated an arrest in the development of the epididymis in all homozygous mice. The cauda and corpus of the epididymis were undersized. Compared to the heterozygous epididymis, the homozygous epididymis had fewer peritubular layers and dwarfish musculature. We confirmed this with immunostaining with monoclonal antibodies against α-smooth muscle actin.
     Conclusion: Defective development of the smooth musculature in the epididymis of Insl3 homozygous mutant mice, combined with its high intraabdominal undescended position, supports previous observations regarding the importance of intact epididymis morphology and function for descent of the epididymo-testicular unit.

Key words: cryptorchidism; epididymis; Insl3; mice, mutant strains
Int Braz J Urol. 2007; 33: 407-13

INTRODUCTION

     During early embryonic development of the urogenital tract, mesentery connects the gonads and the Wollfian and Müllerian ducts to the abdominal wall. During male and female development, two parts of the genital mesentery, the cranial suspensory ligament and the caudal genital ligament gubernaculums, are believed to be responsible for sexual dimorphism in the position of testis and ovary (1). It was reported that defects in this developmental process could cause cryptorchidism. Cryptorchidism is the most common disorder of sexual differentiation in humans, with a 3.5% incidence in term newborns (2).
     The mechanism controlling testicular descent in mice was determined by analyzing mouse lines that lack insulin-like 3 (Insl3) hormone and its receptor, Lgr8/Great (3). Bilateral cryptorchidism in Insl3- and Lgr8- deficient mice is due to impaired development of the gubernaculum (4,5). These findings clearly demonstrate the role of Insl3 signaling in the process of testicular descent.
     Restricted expression of Insl3 in pre- and postnatal Leydig cells is mediated by steroidogenic factor 1 (SF1). Insl3 secretion is dependent on the differentiating effect of leutinizing hormone (LH) on Leydig cells and is independent of steroidogenic LH-mediating action (6-8). In Insl3- deficient mice, the development of epididymis that descends with the testis was considered to be normal and had no influence on descent of the epididymo-testicular unit (5). The goal of the present study was to evaluate epididymal development in Insl3- deficient mice and determine whether epididymal development is involved in the process of testicular descent.

MATERIALS AND METHODS

     Heterozygous and homozygous Insl3 mutants of mixed CD1 X 129/Sv genetic background were generated by breeding Insl3-/- females with Insl3+/- males, and their genotypes were determined by polymerase chain reaction (4). On the first postnatal day, newborn males were euthanized with carbon dioxide gas, fixed with Bouin’s fixative, and embedded in paraplast. To evaluate epididymal development in Insl3- deficient mice, 5 Insl3 homozygous and 3 Insl3 heterozygous mice were sectioned at 4 µm. One homozygous mouse was cut in the frontal plane, and the remaining four homozygous and three heterozygous mice were cut in the sagittal plane. Serial sections were stained with hematoxylin and eosin and examined under a light microscope.
     For immunohistologic analysis, selected sections were mounted on slides. After deparaffinization, sections were treated with a 3% hydrogen peroxide/methanol solution to block endogenous peroxidase. Sections were preincubated for 1 h with 5% normal goat serum in 0.05% Triton X-100- phosphate buffered saline (PBS) and incubated overnight at 4°C in 1:200 diluted monoclonal anti-α actin antibody (Dako). Sections were washed with PBS and incubated with peroxidase-conjugated goat anti-mouse antibody at a 1:500 dilution (Dako) for 1 h at room temperature. After washing with PBS, immunoreactivity was detected by incubating the sections in a solution containing 3.3´-diaminobenzidine tetrahydrochloride.

RESULTS

     The testes of all homozygous mice were localized either in a high intraabdominal position in proximity to the kidney (7/10) or in a transversal ectopic position, adjacent to the contralateral partner. In contrast, all 6 testes from heterozygous mice were located at the bladder neck (Figure-1). The gubernacular bulb (scrotal attachment) was less developed in homozygous mice compared to heterozygous mice (Figure-2). In all homozygous mice, epididymis development was arrested. Both the cauda and corpus of the epididymis were severely undersized (Figure-2). Compared to heterozygous mice, the epididymis of the homozygous mice had fewer peritubular layers and displayed a dwarfish musculature. Immunohistological staining for α-smooth muscle actin confirmed this (Figure-2). Immunostaining was absent in the testis and epididymal peritubular muscle layers of Insl3-/- mice, while it was strongly expressed in Insl3+/- mice (Figure-1). Interestingly, blood vessels stained intensely for α-smooth muscle actin in Insl3+/- and Insl3-/- mice, indicating a specific role of Insl3 in myogenesis of peritubular epididymal muscle layers (Figure-2). This novel observation indicates that Insl3 signaling is involved in regulating the development of smooth muscle of the epididymis and testis.



COMMENTS

     The role of the epididymis for descent of the epididymo-testicular unit has been postulated previously in experimental animals and human (9-11). August-Copenhagen-Irish rats have a congenital defect that frequently causes arrested development of a single ipsilateral Wolffian duct (9). If the Wolffian duct fails to form in the early developmental stage, the testis does not descend (9-11). Treatment with LH-releasing hormone induces epididymo-testicular descent in 60% of naturally cryptorchid mice; and in these mice, increased testosterone secretion normalized the underdeveloped cryptorchid epididymis (10).
     In 1984, Frey & Rajfer (12) reported that the distal gubernaculum is an absolute prerequisite for testicular descent, and they attempted to prove this by dissecting the distal part of the gubernaculum, repeating the experiment of Bergh et al. (13). However, shortly before birth, the rodent scrotum is partially inverted in the abdomen, giving an impression of being a part of the distal gubernaculum. It is self-evident that if the scrotum is stunted in its development, as Bergh et al. (13) showed, then the descent cannot take place. In contrast, scrotal development is normal in the vast majority of common cryptorchidism cases.
     Since the gubernaculum in cryptorchid boys regresses after birth, the crucial question is why hormone treatment induces testicular descent, even though there is no gubernaculum in cryptorchid boys. In a placebo-controlled study (14) complete epididymo-testicular descent was achieved in cryptorchid boys who received hormone treatment. Hormone treatment induced a significant increase in serum testosterone (14). Boys with successful descent of the epididymis and testis had a normal-sized epididymis, while the majority of non-responders and surgically treated cryptorchid boys had small, irregular epididymes (15). Appropriate hormone treatment was capable of inducing increased testosterone secretion to stimulate further development of the epididymis and induce its descent into the scrotum (14,15). Our observation of defective development of the smooth musculature in the epididymis in Insl3 homozygous mutant mice, combined with its high intraabdominal undescended position, supports previous observations regarding the importance of intact epididymis morphology and function for descent of the epididymo-testicular unit. Finally, the fact that there are animals with descended epididymes (chinchilla) but undescended testes, but no mammals with descended testes and undescended epididymes, underscores the necessity of epididymal, rather than testicular, descent (16).

CONFLICT OF INTEREST

     None declared.

REFERENCES

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  2. Elder JS: The undescended testis. Hormonal and surgical management. Surg Clin North Am. 1988; 68: 983-1005.
  3. Bogatcheva NV, Truong A, Feng S, Engel W, Adham IM, Agoulnik AI: GREAT/LGR8 is the only receptor for insulin-like 3 peptide. Mol Endocrinol. 2003; 17: 2639-46.
  4. Zimmermann S, Steding G, Emmen JM, Brinkmann AO, Nayernia K, Holstein AF, et al.: Targeted disruption of the Insl3 gene causes bilateral cryptorchidism. Mol Endocrinol. 1999; 13: 681-91.
  5. Nef S, Parada LF: Cryptorchidism in mice mutant for Insl3. Nat Genet. 1999; 22: 295-9.
  6. Gorlov IP, Kamat A, Bogatcheva NV, Jones E, Lamb DJ, Truong A, et al.: Mutations of the GREAT gene cause cryptorchidism. Hum Mol Genet. 2002; 11: 2309-18.
  7. Zimmermann S, Schwarzler A, Buth S, Engel W, Adham IM: Transcription of the Leydig insulin-like gene is mediated by steroidogenic factor-1. Mol Endocrinol. 1998; 12: 706-13.
  8. Bay K, Hartung S, Ivell R, Schumacher M, Jurgensen D, Jorgensen N, et al.: Insulin-like factor 3 serum levels in 135 normal men and 85 men with testicular disorders: relationship to the luteinizing hormone-testosterone axis. J Clin Endocrinol Metab. 2005; 90: 3410-8.
  9. Hadziselimovic F: Mechanism of testicular descent. Urol Res. 1984; 12: 155-7.
  10. Hadziselimovic F. Pathogenesis of cryptorchidism. In Pediatric andrology Eds Kogan SJ, Hafez ESE Martinus Nijhoff, The Hague/Boston/London. 1981; 147-162.
  11. Hadziselimovic F: Letter to the Editor. J Urol. 1984; 131: 688-90.
  12. Frey HL, Rajfer J: Role of the gubernaculum and intraabdominal pressure in the process of testicular descent. J Urol. 1984; 131: 574-9.
  13. Bergh A, Helander H.F, Wahlqvist L: Studies on factors governing testicular descent in the rat-Particularly the role of gubernaculum testis. Inter J Androl. 1978; 1: 342-356.
  14. Bica DT, Hadziselimovic F: The behavior of epididymis, processus vaginalis and testicular descent in cryptorchid boys treated with buserelin. Eur J Pediatr. 1993; 152 (Suppl 2): S38-42.
  15. Bica DT, Hadziselimovic F: Buserelin treatment of cryptorchidism: a randomized, double-blind, placebo-controlled study. J Urol. 1992; 148: 617-21.
  16. Bedford M: Anatomical evidence. for epididymis as a prime mover in the. evolution of the scrotum. Am J Anat. 1978; 152: 483-486.
    17.

____________________
Accepted after revision:
April 2, 2007

_______________________
Correspondence address:
Prof. Dr. Faruk Hadziselimovic
Kindertagesklinik Liestal
Oristalstrasse 87a
4410 Liestal, Switzerland
E-mail: faruk@magnet.ch


EDITORIAL COMMENT

     Although the authors have written to be focused on clarifying the controversy about the role of Insl3 on the development of epididymis, they go further and stress the importance of epididymis for the descent of a testis. However, the epididymis is not mandatory for the descent (1,2). Contrary to the stated in the paper, the ACI rats have descended testes (3,4).
     Their observations revealed a defective myogensis in testis, epididymis and gubernaculum and supported the role of Insl3 for myogenesis in special structures (5,6).
     Their observation of defective myogenesis, together with the controversial role of epididymis, does not support their final conclusion about the importance of insl3 for the descent of epididymo-testicular unit, and how does the defective myogensis affect the descent remain obscure in their conclusion.
     Since Insl3 has a role in the myogenesis, and one of the current explanations of descent involves propulsion by the smooth muscle that depends on myogenesis in the gubernaculum (7), their observation of defective myogenesis in the gubernaculum does not support the epididymal descent as they conclude, but seems to support the place of failed propulsion resulting from defective myogenesis.

REFERENCES

  1. Frey HL, Peng S, Rajfer J: Synergy of abdominal pressure and androgens in testicular descent. Biol Reprod. 1983; 29: 1233-9.
  2. Frey HL, Rajfer J: Epididymis does not play an important role in the process of testicular descent. Surg Forum. 1982; 33: 617-9.
  3. Marshall FF, Ewing LL, Zirkin BR, Cochran RC: Testicular atrophy associated with agenesis of the epididymis in the ACI rat. J Urol. 1982; 127: 155-8.
  4. Hadziselimovic F: Mechanism of testicular descent. Urol Res. 1984; 12: 155-7.
  5. Zimmermann S, Steding G, Emmen JM, Brinkmann AO, Nayernia K, Holstein AF, et al.: Targeted disruption of the Insl3 gene causes bilateral cryptorchidism. Mol Endocrinol. 1999; 13: 681-91.
  6. Koskimies P, Suvanto M, Nokkala E, Huhtaniemi IT, McLuskey A, Themmen AP, et al.: Female mice carrying a ubiquitin promoter-Insl3 transgene have descended ovaries and inguinal hernias but normal fertility. Mol Cell Endocrinol. 2003; 206: 159-66.
  7. Tanyel FC. The descent of testis and reasons for failed descent. Turk J Pediatr. 2004; 46 (Suppl): 7-17.
    8.

Dr. Feridun Cahit Tanyel
Department of Pediatric Surgery
School of Medicine, Hacettepe University
Ankara,Turkey
E-mail: ctanyel@hacettepe.edu.tr

EDITORIAL COMMENT

     According to the current view, normal testicular descent occurs in two phases. The first trans-abdominal phase (until week 15) is mainly depended on insulin-like peptide 3 (INSL3). The second inguinoscrotal phase is completed by week 35 and it is mainly depended on androgen action (1). In addition, androgens are of crucial importance for the development of the Wolffian duct derived organs, p.ex. epididymis. Testicular descent is conducted by gubernaculum, which is attached to the epididymis, which in turn is attached to the testis. Usually cryptorchidism is associated to the defective growth of the gubernaculum. However, epididymis is also important for testicular descent. This becomes evident also in some clinical situations. Sometimes the tip of the epididymis has grown to the scrotum together with the gubernaculum, while the other end of the elongated epididymis is lying in the abdominal cavity with the testis. About one third of the cryptorchid testes are associated with some degree of epididymal abnormalities. In the present study, it is suggested that defective INSL3 action in addition to defective androgen action may cause abnormalities at least to the epididymal smooth musculature. It is unclear if epididymal abnormalities in general or the smooth muscle abnormalities detected in this study have some adverse effect for the fertility. Certainly, the surgeon has to be careful not to divide abnormal epididymis instead of gubernaculum during the operation.

REFERENCE

1. Hutson JM, Hasthorpe S: Testicular descent and cryptorchidism: the state of art in 2004. J Pediatr Surg. 2005; 40: 297-302.

Dr. Seppo Taskinen
Hospital for Children and Adolescents
University of Helsinki
Helsinki, Finland
E-mail: seppo.taskinen@hus.fi

REPLY BY THE AUTHORS

     The two stages of testicular descent are an old story presented by Gier & Marion in 1969 and 1970 (1,2) a long time before John Hutson even began his research. Furthermore, he published that the first phase of testicular descent is under the control of Mullerian Inhibiting Substance (MIS); this assertion is not true. Regarding INSL3 role in epididymo-testicular descent he should not be given credit for INSL3 only because he wrote about it in a review article. It is not his original discovery.

REFERENCES

  1. Gier HT, Marion GB: Development of mammalian testes and genital ducts. Biol Reprod. 1969; 1 (suppl. 1): 1-23.
  2. Gier HT, Marion GB: Development of Mammalian Testis. In: Johnson AD, Gomes W, Vandemark NL (eds.), The Testis I. New York, Academic Press. 1979; pp. 2-43. 
    3.