Role of ADAMTS16 metalloproteinase in pathogenesis of cryptorchidism

IntroductionThe etiology of cryptorchidism is still not fully elucidated, but several hypotheses have been proposed. One of the latest concerns is the involvement of metalloproteinase ADAMTS16 in testis descent. The aim of this study was to evaluate both expression and occurrence of selected polymorphisms of metalloproteinase ADAMTS16 in patients diagnosed with cryptorchidism.Material and methodsThe study group consisted of 158 boys (mean age: 4.1 ±2.04 years) who underwent surgery due to undescended testis. Tissue samples from patients with cryptorchidism were obtained from the cremaster muscle, gubernaculum and hernial sac. The reference group consisted of 123 age-related subjects (mean age: 4.1 ±2.41 years) who had no cryptorchidism and underwent surgery for other reasons. Tissue samples from controls were obtained from the cremaster muscle and hernial sac.ResultsThe obtained data indicate that patients with undescended testis have significantly lower expression of Adamts16, especially in the gubernaculum. We also demonstrated a tendency that Adamts16 expression depends on the age of patients; the older the patient was, the higher was the observed expression of Adamts16. These studies also established that polymorphisms rs16875319, rs16875122, and rs58353460 in the Adamts16 gene are not a major determinant to develop cryptorchidism while rs16875054 is associated with increased risk.ConclusionsThese studies highlight ADAMTS16 involvement in cryptorchidism and confirm data obtained in animal models.

KLF6 cooperates with NUR77 and SF1 to activate the human INSL3 promoter in mouse MA-10 leydig cells

Insulin-like 3 (INSL3), a Leydig cell-specific hormone, is essential for testis descent during foetal life and bone metabolism in adults. Despite its essential roles in male reproductive and bone health, very little is known regarding its transcriptional regulation in Leydig cells. To date, few transcription factors have been shown to activate INSL3 promoter activity: the nuclear receptors AR, NUR77, COUP-TFII and SF1. To identify additional regulators, we have isolated and performed a detailed analysis of a 1.1 kb human INSL3 promoter fragment. Through 5′ progressive deletions and site-directed mutagenesis, we have mapped a 10 bp element responsible for about 80% of INSL3 promoter activity in Leydig cells. This element is identical to the CPE element of the placental-specific glycoprotein-5 (PSG5) promoter that is recognized by the developmental regulator Krüppel-like factor 6 (KLF6). Using PCR and western blotting, we found that KLF6 is expressed in several Leydig and Sertoli cell lines. Furthermore, immunohistochemistry on adult mouse testis revealed the presence of KLF6 in the nuclei of both Leydig and Sertoli cells. KLF6 binds to the 10 bp KLF element at −108 bp and activates the −1.1 kb human, but not the mouse, INSL3 promoter. KLF6-mediated activation of the human INSL3 promoter required an intact KLF element as well as Leydig/Sertoli-enriched factors because KLF6 did not stimulate the human INSL3 promoter activity in CV-1 fibroblast cells. Consistent with this, we found that KLF6 transcriptionally cooperates with NUR77 and SF1. Collectively, our results identify KLF6 as a regulator of human INSL3 transcription.

Dynamics of insulin-like factor 3 and its receptor expression in boar testes

Relaxin-like factor (RLF), now mainly known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. As a major step toward understanding the as-yet-unknown function of INSL3 in boars, this study aimed to develop a time-resolved fluoroimmunoassay for boar INSL3, characterize the dynamics of INSL3 expression during development, and demonstrate the expression of the INSL3 hormone–receptor system in the testis. All samples were collected from Duroc boars. The sensitivity of the assay system established was 8.2 pg/well (164 pg/ml), and no cross-reactivity with other hormones, such as porcine relaxin, was observed. Circulating INSL3 was shown to increase progressively during development. INSL3 secreted from the Leydig cells was released not only into the blood circulation but also into the interstitial and seminiferous compartments in sufficient concentrations. A testicular fractionation study revealed that its receptor RXFP2 transcripts were expressed mainly in testicular germ cells. In addition, INSL3 bound to the germ cell membranes in a hormone-specific and saturable manner. These results reveal that INSL3 secreted into the interstitial compartment from the Leydig cells is transported into the seminiferous compartments, where its receptor RXFP2 is expressed mainly in the germ cells to which INSL3 binds, suggesting that INSL3 functions as a paracrine factor on seminiferous germ cells.

The role of RXFP2 in mediating androgen-induced inguinoscrotal testis descent in LH receptor knockout mice

LH receptor knockout (LhrKO) male mice exhibit a bilateral cryptorchidism resulting from a developmental defect in the gubernaculum during the inguinoscrotal phase of testis descent, which is corrected by testosterone replacement therapy (TRT).In vivoandin vitroexperiments were conducted to investigate the roles of the androgen receptor (AR) and RXFP2 signals in regulation of gubernacular development inLhrKO animals. This study demonstrated that AR and RXFP2 proteins were expressed in the gubernaculum during the entire postnatal period. TRT normalized gubernacular RXFP2 protein levels inLhrKO mice. Organ and primary cell cultures of gubernacula showed that 5α-dihydrotestosterone (DHT) upregulated the expression ofRxfp2which was abolished by the addition of an AR antagonist, flutamide. A single s.c. testosterone injection also led to a significant increase inRxfp2mRNA levels in a time-dependent fashion inLhrKO animals. DHT, natural and synthetic insulin-like peptide 3 (INSL3), or relaxin alone did not affect proliferation of gubernacular mesenchymal cells, while co-treatments of DHT with either INSL3 or relaxin resulted in an increase in cell proliferation, and they also enhanced the mesenchymal cell differentiation toward the myogenic pathway, which included a decrease in a mesenchymal cell marker, CD44 and the expression of troponin. These effects were attenuated by the addition of flutamide, siRNA-mediatedRxfp2knockdown, or by an INSL3 antagonist. Co-administration of an INSL3 antagonist curtailed TRT-induced inguinoscrotal testis descent inLhrKO mice. Our findings indicate that the RXFP2 signaling pathway plays an important role in mediating androgen action to stimulate gubernaculum development during inguinoscrotal testis descent.

Factors controlling testis descent

Descent of the testis from an intra-abdominal site in foetal life to an extracorporeal location after birth is a mandatory developmental process to ensure that the mature testis promotes normal spermatogenesis. The two phases of transabdominal and inguinoscrotal descent occur approximately during the first and last thirds of gestation respectively. Key anatomical events to release the testis from its urogenital ridge location and to guide the free gonad into the scrotum are the degeneration of the cranio-suspensory ligament and a thickening of the gubernaculum. Androgens play a role in both these processes, particularly with respect to enabling the testis to traverse the inguinal canal in the final phase of descent. Experiments in animals suggest that androgens mediate this effect via the release of calcitonin gene-related peptide by the genitofemoral nerve, but direct evidence for such a mechanism is lacking in humans. The transabdominal phase of descent is under the control of insulin-like 3 (INSL3), a product of the Leydig cells. Definitive evidence of its role in rodent testis descent is illustrated by the phenotype of bilateral cryptorchidism in Insl3−/− null mice. Circulating levels of INSL3 are higher in boys at puberty, are undetectable in girls and are lower in boys with undescended testes. A minority also have a mutation either in the INSL3 gene or affecting its receptor gene, relaxin/insulin-like family peptide receptor 2 (LGRF8). Other factors that may play a role in testis descent include the anti-Mullerian hormone and members of the HOX gene family. Evidence that the prevalence of undescended testis may be increasing provides a phenotypic readout for the effects of postulated chemicals in the environment interfering in some way with the action of factors that control testis descent. Epidemiological studies point to profound geographical variations in prevalence in countries such as Denmark and Finland. Associations have been found with levels of chemicals labelled as endocrine disruptors being higher in breast milk samples from mothers with cryptorchid boys when compared with controls. The adverse effects of these compounds (e.g. bisphenol A) can be replicated in the offspring of dams exposed during pregnancy. A sensitive marker of an anti-androgen effect of a compound is a reduction in the anogenital distance, an anthropometric measurement that is significantly greater in males compared with females. The observation of an association between the anogenital distance in infant boys and the level of pesticides in the urine of their mothers in late gestation indicates that this has the potential to be a useful surrogate marker of the effects of environmental chemicals on testis descent in human population studies. The rightful place for the testis at birth is in the scrotum in order to provide the temperature differential essential for normal spermatogenesis. Appropriate screening programmes and early surgical intervention are the prerequisites to ensure optimal fertility in adulthood and a considerably lessened risk of testis cancer.

Cryptorchidism in common eutherian mammals

Cryptorchidism is failure of one or both testes to descend into the scrotum. Primary fault lies in the testis. We provide a unifying cross-species interpretation of testis descent and urge the use of precise terminology. After differentiation, a testis is relocated to the scrotum in three sequential phases: abdominal translocation, holding a testis near the internal inguinal ring as the abdominal cavity expands away, along with slight downward migration; transinguinal migration, moving a cauda epididymidis and testis through the abdominal wall; and inguinoscrotal migration, moving a s.c. cauda epididymidis and testis to the bottom of the scrotum. The gubernaculum enlarges under stimulation of insulin-like peptide 3, to anchor the testis in place during gradual abdominal translocation. Concurrently, testosterone masculinizes the genitofemoral nerve. Cylindrical downward growth of the peritoneal lining into the gubernaculum forms the vaginal process, cremaster muscle(s) develop within the gubernaculum, and the cranial suspensory ligament regresses (testosterone not obligatory for latter). Transinguinal migration of a testis is rapid, apparently mediated by intra-abdominal pressure. Testosterone is not obligatory for correct inguinoscrotal migration of testes. However, normally testosterone stimulates growth of the vaginal process, secretion of calcitonin gene-related peptide by the genitofemoral nerve to provide directional guidance to the gubernaculum, and then regression of the gubernaculum and constriction of the inguinal canal. Cryptorchidism is more common in companion animals, pigs, or humans (2–12%) than in cattle or sheep (≤1%). Laboratory animals rarely are cryptorchid. In respect to non-scrotal locations, abdominal testes predominate in cats, dogs, and horses. Inguinal testes predominate in rabbits, are common in horses, and occasionally are found in cats and dogs. S.c. testes are found in cattle, cats and dogs, but are most common in humans.