Suppression of insulin-like3 receptor reveals the role of β-catenin and Notch signaling in gubernaculum development

During male development, the testes move from a high intraabdominal position and descend into the scrotum. The gubernaculum, an inguinoscrotal ligament connecting the testis to the lower abdomen, is believed to play a critical role in this process. The first stage of testicular descent is controlled by insulin like3 hormone (INSL3), produced in testicular Leydig cells. Deletion of Insl3 or its receptor, Rxfp2, in mice causes cryptorchidism. We produced Cre/loxP regulated shRNA transgenic mice targeting RXFP2 expression. We have shown that the transgene was able to reduce Rxfp2 gene expression and thus behaved as a hypomorphic allele of Rxfp2. Variable degrees of uni- and bilateral cryptorchidism was detected in males with the activated shRNA transgene on an Rxfp2+/- background. Conditional suppression of Rxfp2 in the gubernaculum led to cryptorchidism. Gene expression analysis of a mutant cremasteric sac using Illumina microarrays indicated abnormal expression of a significant number of genes in Wnt/β-catenin and Notch pathways. We have demonstrated profound changes in the expression pattern of β-catenin, Notch1, desmin, and androgen receptor (AR), in Rxfp2-/- male embryos, indicating the role of INSL3 in proliferation, differentiation, and survival of specific cellular components of the gubernaculum. We have shown that INSL3/RXFP2 signaling is essential for myogenic differentiation and maintenance of AR-positive cells in the gubernaculum. Males with the deletion of β-catenin or Notch1 in the gubernacular ligament demonstrated abnormal development. Our data indicates that β-catenin and Notch pathways are potential targets of INSL3 signaling during gubernacular development.     

Androgens in relation to prenatal development and postnatal inversion of the gubernacula in rats.

Exposure of male rats to the anti-androgen flutamide during fetal life, from day 10 after conception to the day of birth, allowed quantitatively unaltered development of the gubernacula. Apparently, androgens play no important role or no role at all in their growth. Castration of newborn male rats did not interfere with the inversion during further postnatal life of the gubernacula to create the muscular parts of the scrotum (cremaster muscles). Prenatal exposure to flutamide, followed by castration immediately after birth, also allowed gubernacular inversion and cremaster muscle growth. Neonatal administration of testosterone, after castration at birth, did not enhance gubernacular inversion or promote cremaster muscle growth in infancy or during adulthood. Apparently, postnatal gubernacular inversion and cremaster muscle growth are independent not only of androgens, but also of all testis hormones. Neonatal administration of the potent androgen 5 alpha-dihydrotestosterone propionate suppressed gonadotrophin secretion and, in intact males, inhibited testicular growth. Administration from the day of birth to day 33 delayed testicular descent and enhanced growth of the genital apparatus, but did not affect the size of the cremaster muscles. These experiments indicate that androgens are not involved in the processes that create the cavities into which testes descend to acquire their full reproductive potential.     

The overexpression of the insl3 in female mice causes descent of the ovaries

Testicular descent in mice is dependent upon proper outgrowth of the gubernaculum primordia under the influence of the insulin-like 3 gene product (Insl3). Deletion of this gene prevents gubernaculum growth and causes bilateral cryptorchidism. In vitro experiments have led to the suggestion that Insl3 and androgens together induce outgrowth of the gubernacular primordia. The experiments reported here were designed specifically to determine whether in vivo the Insl3-mediated gubernaculum development is independent of androgens. To that effect transgenic male and female mice were generated that overexpressed Insl3 in the pancreas during fetal and postnatal life. Expression of the transgenic allele in the Insl3-deficient mice rescued the cryptorchidism in male mutant, indicating that the islet beta-cells efficiently processed the Insl3 gene product to the functional hormone. All transgenic females displayed bilateral inguinal hernia. The processus vaginalis developed containing intestinal loops. The Müllerian derivatives gave rise to oviduct, uterus, and upper vagina, and Wolffian duct derivatives were missing, indicating the absence of the androgen- and anti-Müllerian hormone-mediated activities in transgenic females. The ovaries descended into a position over the bladder and attached to the abdominal wall via the well developed cranial suspensory ligament and the gubernaculum. Administration of dihydrotestosterone during prenatal development suppressed formation of the cranial suspensory ligament and thereby allowed the descent of the ovaries into the processus vaginalis. These results suggest that Insl3-mediated activity induces gubernaculum development and precludes a role of androgen in this process. Furthermore, the transgenic females exhibit reduced fertility, which is due to fetal mortality during midgestation.     

Targeted disruption of the Insl3 gene causes bilateral cryptorchidism.

The sexual dimorphic position of the gonads in mammals is dependent on differential development of two ligaments, the cranial suspensory ligament (CSL) and the gubernaculum. During male embryogenesis, outgrowth of the gubernaculum and regression of the CSL result in transabdominal descent of the testes, whereas in the female, development of the CSL in conjunction with failure of the gubernaculum development holds the ovaries in a position lateral to the kidneys. Several lines of evidence suggest that regression of the CSL and induction of gubernaculum development are mediated by testosterone and a yet unidentified testicular factor, respectively. The Insl3 gene (originally designated Ley I-L), a member of the insulin-like superfamily, is specifically expressed in Leydig cells of the fetal and postnatal testis and in theca cells of the postnatal ovary. Here we show that male mice homozygous for a targeted deletion of the Insl3 locus exhibit bilateral cryptorchidism with free moving testes and genital ducts. These malformations are due to failure of gubernaculum development during embryogenesis. In double-mutant male mice for Insl3 and androgen receptor genes, testes are positioned adjacent to the kidneys and steadied in the abdomen by the CSL. These findings demonstrate, that the Insl3 induces gubernaculum development in an androgen-independent way, while androgen-mediated regression of the CSL occurs independently from Insl3.     

GREAT/LGR8 is the only receptor for insulin-like 3 peptide

During male development testes descend from their embryonic intraabdominal position into the scrotum. Two genes, encoding the insulin-like 3 peptide (INSL3) and the GREAT/LGR8 G protein-coupled receptor, control the differentiation of gubernaculum, the caudal genitoinguinal ligament critical for testicular descent. It was established that the INSL3 peptide activates GREAT/LGR8 receptor in vitro. Mutations of Insl3 or Great cause cryptorchidism (undescended testes) in mice. Overexpression of the transgenic Insl3 causes male-like gubernaculum differentiation, ovarian descent into lower abdominal position, and reduced fertility in females. To address the question whether Great deletion complements the mutant female phenotype caused by the Insl3 overexpression, we have produced Insl3 transgenic mice deficient for Great. Such females had a wild-type phenotype, demonstrating that Great was the only cognate receptor for Insl3 in vivo. We have established that pancreatic HIT cells, transfected with the INSL3 cDNA, produce functionally active peptide. Analysis of five INSL3 mutant variants detected in cryptorchid patients showed that P49S substitution renders functionally compromised peptide. Therefore, mutations in INSL3 might contribute to the etiology of cryptorchidism. We have also showed that synthetic insulin-like peptides (INSL4 and INSL6) were unable to activate LGR7 or GREAT/LGR8.     

Abnormalities of testicular descent

Testicular descent occurs in two stages. The transabdominal phase (8–15 weeks) is controlled by enlargement of the caudal genito-inguinal ligament (gubernaculum) and regression of the cranial ligament. Insulin-like 3 from the Leydig cell appears to be the prime stimulator of gubernacular growth, augmented by Müllerian inhibiting substance/anti-Müllerian hormone. Testosterone causes regression of the cranial ligament. The inguinoscrotal phase (25–35 weeks) requires the migration of the gubernaculum from the groin to the scrotum; this migration is guided by the genito-femoral nerve releasing calcitonin gene-related peptide under the influence of androgen. The neonatal gonocyte transforms into a type A spermatogonium at 3–12 months of age, a step that is now known to be crucial for subsequent fertility, as the stem cells for spermatogenesis are created in this structure. This step is blocked in undescended testis and, hence, orchidopexy is currently recommended at 6–12 months of age. Congenital cryptorchidism is caused by the failure of gubernacular migration to the scrotum (1%–2%) but we now recognise that another 1%–2% of boys have acquired cryptorchidism, secondary to the failure of spermatic cord elongation with growth of the boy. These latter cases come to operation at 5–10 years of age. Surgery remains the mainstay of treatment, as hormonal therapy has not been proven to be effective, presumably because testicular descent is a complex anatomical mechanism.     

Disturbed testicular descent in the rat after prenatal exposure to the antiandrogen flutamide.

Exposure of rats in utero to the anti-androgen flutamide resulted in feminization of the external genitalia that was noticeable at birth. This exposure also resulted in a high degree of cryptorchidism during adulthood. In most affected animals, testes were lying in 'ovary position' close to the caudal pole of the ipsilateral kidney. Cryptorchidism occurred despite normal prenatal development of the gubernacular cones and the transformation of these structures, postnatally, into muscular cremaster sacs. Inter- and intralitter variation in the response to prenatal exposure to flutamide was observed as well as intra-individual variation. Cryptorchidism frequently occurred unilaterally with right side cryptorchidism predominating. Cryptorchidism occurred in association with marked suppression of the growth of the ipsilateral epididymis and deferent duct. The possibility is considered that the poor development or absence of these structures contributes to cryptorchidism. Intra-individual variation supports the concept of the local nature of the influence of testis hormones in stabilization and further differentiation of the ipsilateral Wolffian duct derivatives. Cryptorchidism was enhanced when rats were treated postnatally with testosterone or oestradiol. The effect of testosterone was unexpected in view of the generally held hypothesis that androgens enhance testis descent. The effect of oestradiol was as expected: other animal models have been described in which induction of cryptorchidism by oestradiol occurs. Additional treatment with oestradiol caused further suppression of growth of the epididymis and deferent duct. The response to prenatal exposure to flutamide was not altered by further injections of flutamide postnatally. Such injections were without effect in males not exposed to flutamide prenatally except for minor, but statistically significant, testicular enlargement during adulthood. A model is thus presented that describes cryptorchidism as an endogenous developmental disorder.     

Role of aromatase and androgen receptor expression in gonadal sex differentiation of ZW/ZZ-type frogs,Rana rugosa

To elucidate the mechanisms of amphibian gonadal sex differentiation, we examined the expression of aromatase and androgen receptor (AR) mRNAs for days 17-31 after fertilization. The effects of inhibitors and sex steroid hormones were also examined. In ZZ males, expression of AR decreased after day 19, while aromatase expression was low throughout the sampling period. Males treated with 17beta-estradiol (E2) showed increasing aromatase expression after day 21, and formed ovaries. AR antagonist treatment also induced high-level aromatase expression and ovarian differentiation. In males co-treated with an aromatase inhibitor and E2, the undifferentiated gonads developed into testes despite high-level aromatase expression. Males treated with androgen and E2 before and during an estrogen sensitive period, respectively, also formed testes. In ZW females, AR expression persisted at a low-level, while aromatase expression increased after day 18. Short-term treatment with an aromatase inhibitor was ineffective in preventing ovarian differentiation, whereas long-term treatment resulted in testes developing from ovarian structure. Compared with the ZZ males and ZW females, WW females did not exhibit detectable expression of AR, suggesting that the active AR gene(s) itself, or a putative gene regulating AR gene expression, is located on Z chromosomes. From the time lag of aromatase expression between ZW females and ZZ males treated with E2 and the effect of AR antagonist, it was found that in males elevated AR expression suppresses aromatase expression directly or indirectly. Consequently, endogenous androgens, accumulated by blocking estrogen biosynthesis, induced testicular differentiation. The gonadogenesis of males is dependent on sex hormone, whereas that of females has evolved to hormone-independence.     

Environmental effects on hormonal regulation of testicular descent

Regulation of testicular descent is hormonally regulated, but the reasons for maldescent remain unknown in most cases. The main regulatory hormones are Leydig cell-derived testosterone and insulin-like factor 3 (INSL3). Luteinizing hormone (LH) stimulates the secretion of these hormones, but the secretory responses to LH are different: INSL3 secretion increases slowly and may reflect the LH dependent differentiated status of Leydig cells, whereas testosterone response to LH is immediate. Testosterone contributes to the involution of the suspensory ligament and to the inguinoscrotal phase of the descent, while INSL3 acts mainly in transabdominal descent by stimulating the growth of the gubernaculum. INSL3 acts through a G-protein coupled receptor LGR8. In the absence of either INSL3 or LGR8 mice remain cryptorchid. In humans only few INSL3 mutations have been described, whereas LGR8 mutations may cause some cases of undescended testis. Similarly, androgen insensitivity or androgen deficiency can cause cryptorchidism. Estrogens have been shown to down regulate INSL3 and thereby cause maldescent. Thus, a reduced androgen–estrogen ratio may disturb testicular descent. Environmental effects changing the ratio can thereby influence cryptorchidism rate. Estrogens and anti-androgens cause cryptorchidism in experimental animals. In our cohort study we found higher LH/testosterone ratios in 3-month-old cryptorchid boys than in normal control boys, suggesting that cryptorchid testes are not cabable of normal hormone secretion without increased gonadotropin drive. This may be either the cause or consequence of cryptorchidism. Some phthalates act as anti-androgens and cause cryptorchidism in rodents. In our human material we found an association of a high phthalate exposure with a high LH/testosterone ratio. We hypothesize that an exposure to a mixture of chemicals with anti-androgenic or estrogenic properties (either their own activity or their effect on androgen–estrogen ratio) may be involved in cryptorchidism.     

A transgenic insertion causing cryptorchidism in mice

A distinctive feature of gonadal maturation in mammals is the movement to an extraabdominal location. Testicular descent is a complex, multistage process whereby the embryonic gonads migrate from their initial abdominal position to the scrotum. Failure in this process results in cryptorchidism, a frequent congenital birth defect in humans. We report here a new mouse transgenic insertional mutation, cryptorchidism with white spotting (crsp). Males homozygous for crsp exhibit a high intraabdominal position of the testes, associated with complete sterility. Heterozygous males have a wild-type phenotype, and homozygous females are fertile. Surgically descended testes in crsp/crsp males show normal spermatogenesis. Using FISH and genetic analyses, the transgenic insert causing the crsp mutation has been mapped to the distal part of mouse chromosome 5. Transgene integration resulted in a 550-kb deletion located upstream of the Brca2 gene. A candidate gene encoding a novel G protein-coupled receptor (Great) with an expression pattern suggesting involvement in testicular descent has been identified.     

Cryptorchidism: an indicator of testicular dysgenesis?

Cryptorchidism is a common ailment of new-born boys, affecting 1–9% of full term boys at birth. Cryptorchidism has been associated with an increased risk of testicular cancer and reduced fertility. Aetiology of cryptorchidism remains obscure in most cases. Familial occurrence suggests a heritable susceptibility to cryptorchidism; however, seasonal variation in the incidence of cryptorchidism suggests that environmental factors also contribute. Testicular descent is characterised by androgen-dependent regression of cranial suspensory ligament and androgen + insulin-like hormone 3 (Insl3)-dependent gubernacular outgrowth. Even though hormonal defects are rarely detected in patients, both hypo-and hypergonadotropic hormonal patterns have been associated with cryptorchidism. Moreover, cryptorchid boys have significantly reduced serum androgen bioactivity at 3 months of age when normal boys have a strong surge of reproductive hormones. Defects in Insl3 action cause cryptorchidism in male mice, and over-expression in female mice causes ovarian descent. Defects in leucine-rich repeat-containing G-protein-coupled receptor 8/G-protein-coupled receptor affecting testis descent (LGR8/GREAT), the receptor for Insl3, manifest the same phenotype as Insl3 knockout mutants. Even though mutations found in Insl3 and LGR8/GREAT genes are not a common cause of cryptorchidism in patients, it remains to be resolved whether low Insl3 levels during development are associated with cryptorchidism. Cryptorchidism may reflect foetal testicular dysgenesis that may later manifest as subfertility or testicular cancer.This work was supported by the Turku University Central Hospital, the Academy of Finland and the European Commission (contracts BMH4-CT96-0314, QLK4-CT1999-01422, QLK4-CT2001-00269 and QLK4-CT2002-00603).     

A molecular basis for estrogen-induced cryptorchidism

Male sexual differentiation relies upon testicular secretion of the hormones testosterone, Mullerian inhibiting substance, and insulin-3 (Insl3). Insl3 is responsible for testicular descent through virilization and outgrowth of the embryonic gubernaculum. In mouse, prenatal exposure to 17beta-estradiol and the nonsteroidal synthetic estrogen diethylstilbestrol (DES) disturbs the endocrine balance, causing demasculinizing and feminizing effects in the male embryo, including impaired testicular descent (cryptorchidism). In the current study, we show that maternal exposure to estrogens, including 17alpha- and beta-estradiol, as well as DES, specifically down regulates Insl3 expression in embryonic Leydig cells, thereby providing a mechanism for cryptorchidism. These experiments may have implications for the widespread use of estrogenic substances in agriculture and the environment.     

Down-regulation of steroidogenic cytochrome P450XVII in cryptorchid rat testes

The objective of the present study was to investigate the regulation of a key component of testicular androgen biosynthesis, i.e. the cytochrome P450XVII of the steroid-17 alpha-monooxygenase/C17,20-lyase, after surgical induction of bilateral cryptorchidism in vivo. Seven days after induction of cryptorchidism, P450XVII concentrations are diminished (as compared to sham-operated controls) by 64% in isolated purified Leydig cells but only by 44% in the total Leydig cell compartment of the testis, since the Leydig cell yield from cryptorchid testes is by 53% higher than that from control testes. Using microsomal suspensions prepared from testicular homogenates, P450XVII content per testis equivalent is found to be decreased by 36% seven days after incubation of cryptorchidism, whereas the P450XVII concentration per gram testis is not changed due to testicular involution. Fourteen days after induction of cryptorchidism, the induction of the Leydig cell system appears to superimpose on the down-regulation of P450XVII. The study demonstrates both a strong sensitivity of P450XVII to short-term elevation of testicular temperature and a differentiation between effects of cryptorchidism on total testicular content and specific cellular and subcellular concentration of this steroidogenic protein.     

Role of the fetal pituitary in cryptorchidism induced by exogenous maternal oestrogen during pregnancy in mice

A single subcutaneous injection of 5 or 1 mg oestradiol given to pregnant female mice on Day 14 of pregnancy resulted in all male offspring being cryptorchid. Pituitary LH content, testicular weights and structure, seminal vesicle weights and the structure of the reproductive tract as a whole were monitored on the day of birth and at 2, 4, 8 and 14 weeks of age. Apart from an initial significant reduction in pituitary LH at the time of birth, no other marked differences were seen between control and treated animals except that all oestrogen-treated males lacked a gubernaculum and the testes were freely mobile within the abdomen. Hypogonadal (hpg) male mice lacking GnRH are cryptorchid but have a normal gubernaculum and their testes develop and descend normally if treated with gonadotrophins. When the mothers of hpg mice were treated with oestradiol the male offspring lacked a gubernaculum. These results indicate that perturbations of the fetal hypothalamic/pituitary axis play no significant part in oestrogen-induced cryptorchidism in mice.     

Partial characterization of the androgen receptor of the newborn rat gubernaculum

Androgen receptors were measured by sucrose density gradient techniques in low-salt and high-salt extracts of gubernaculum, urogenital sinus, and bladder from 3- to 5-day-old male rats. Specific 5 alpha-[3H] dihydrotestosterone binding was present in both the low-salt and high-salt extracts of gubernacular tissue. The total amount of receptor in gubernaculum was approximately one-fifth the total amount of androgen binding measured in the urogenital sinus (119 fmol/g tissue in gubernaculum vs. 562 fmol/g tissue in urogenital sinus). No androgen receptor was detected in bladder. A 10-fold excess of nonradioactive 5 alpha-dihydrotestosterone competed well for 5 mM 5 alpha-[3H]-dihydrotestosterone binding, whereas 10-fold molar excesses of testosterone, estradiol, and progesterone were ineffective. These results suggest that the gubernaculum may be a target tissue for androgens.