A transgenic insertion causing cryptorchidism in mice

Overbeek, Paul A.; Gorlov, Ivan P.; Sutherland, Richard W.; Houston, John B.; Harrison, Wilbur R.; Boettger‐Tong, Holly; Bishop, Colin E.; Agoulnik, Alexander I.

doi:10.1002/gene.1029

Cited by 215 publications

(230 citation statements)

References 23 publications

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“…In parallel, the inverse experiment was conducted by increasing the dose of the inhibitor Ades- (1)(2)(3)(4)(5)(6)(7)(8) in the presence of a uniform concentration (2 ng/ml) of hRLF. The two dose-response curves crossed at a concentration of 1.5 ng/ml, indicating that Ades-(1-8) behaves as a competitive antagonist.…”

Section: Resultsmentioning

confidence: 99%

LGR8 Signal Activation by the Relaxin-like Factor

Büllesbach

Schwabe

2005

Journal of Biological Chemistry

102

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The relaxin-like factor (RLF) is thought to be responsible for the intra-abdominal migration of the testis during mammalian development. Our latest studies of RLF and LGR8 have revealed that the N-terminal region of the A chain is not required for receptor binding but is indispensable for cyclic AMP generation. RLF derivatives with six residues deleted from the N terminus of the A chain are active, whereas further truncation, up to the first A chain cysteine (A-10), yields tightly binding ligands devoid of signaling activity. These derivatives are specific competitive inhibitors (RLFi) of RLF. Although receptor binding is dependent upon B chain residues, the N-terminal region of the A chain is a generic trigger of the trans-membrane signaling activity.The relaxin-like factor (RLF), 1 the product of the Insl3 gene (1), appears to be crucial for testicular descent in human neonates. Insl3Ϫ/Ϫ male mice retain the testes in the body cavity and are infertile (2, 3). The same phenotype was observed when the G-protein coupled receptor (GREAT) was deleted (4). These receptors (GREAT, LGR8) that affect the descent of testis in mice (4, 5) and in humans (LGR8) (6) are RLF-specific (7,8). Thus, an experimental system is now available that invites detailed investigation of the ligand/receptor interaction.RLF, isolated from bovine testes, consists of an A chain comprising 26 residues and a B chain of 40 or 45 residues. The chains are linked by three relaxin-like disulfide bonds (9). This native RLF confirms the structure that has been implied for all synthetic molecules. RLF binds its receptor in part through the B chain residue Trp(B-27) (10) and via additional, yet unidentified, residues that would account for the high receptor binding affinity. However, the molecule can be pared down without loss of binding avidity. Full binding intensity, for example, is observed with RLF ending in Trp(B-27)-amide (11).Our studies suggest that the N-terminal region of the A chain initiates the trans-membrane signal. Sequential shortening of the N-terminal end of the A chain reduces cAMP production, which becomes undetectable when 7 or more residues are removed. A chain truncations did not reduce binding avidity so that Ades-(1-7) RLF is a specific competitive inhibitor of RLF function. In this paper we report that the receptor-binding region of RLF is physically separate from the trans-membrane signal initiation site in the N-terminal region of the A chain. EXPERIMENTAL PROCEDURESMaterials-Fmoc amino acid derivatives were purchased from either Advanced ChemTech (Louisville, KY) or Novabiochem. Reagents and solvents for peptide synthesis were obtained from Advanced ChemTech. The LGR8 cloned into the pcDNA3.1.zeo plasmid was a gift from Dr. Hsueh, Department of Obstetrics and Gynecology, Stanford University School of Medicine. The human embryo kidney cell line 293T/17 was obtained from the American Type Culture Collection (ATCC CRL-11286) and used for LGR8 expression.Peptide Synthesis-All RLF chains were synthesized by Fmoc chemistry, usin...

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Section: Resultsmentioning

confidence: 99%

LGR8 Signal Activation by the Relaxin-like Factor

Büllesbach

Schwabe

2005

Journal of Biological Chemistry

102

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“…Disruption of the Insl3 or Lgr8 genes results in bilateral intra-abdominal testes and maldeveloped gubernacular (21,22). Furthermore, over-expression of Insl3 in female mice results in descent of the ovaries to the inguinal region and development of a processus vaginalis (23).…”

Section: Transabdominal Phasementioning

confidence: 99%

Factors controlling testis descent

Hughes¹,

Acerini²

2008

European Journal of Endocrinology

142

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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 insulinlike 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 Insl3K/K 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 con...

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“…9 LGR8 on the other hand, has a more restricted expression pattern, with the strongest level of expression being in the gubernaculae, testis, kidney, and brain. 10,11,12 The wide and divergent distribution of the two RLN receptors implicates their potential roles in a wide range of functions including reproductive, brain, renal, and cardiovascular functions.…”

Section: Introductionmentioning

confidence: 99%