Targeted Disruption of the Insl3 Gene Causes Bilateral Cryptorchidism

Zimmermann, Stephan; Steding, Gerd; Emmen, Judith M. A.; Brinkmann, Albert O.; Nayernia, Karim; Holstein, A. F.; Engel, Wolfgang; Adham, Ibrahim M.

doi:10.1210/mend.13.5.0272

Cited by 516 publications

(243 citation statements)

References 30 publications

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“…This was originally reported using the mouse brain receptor (25,26) but is now also observed for the human relaxin receptors LGR7 and LGR8. 3 For relaxin-LGR7 binding we showed that the large contribution of the arginines to the binding energy is due to their chelation by two carboxyl groups (24). These relaxin binding residues as identified on LGR7 are also present on LGR8 but do not seem to play the important role in the cross-reactivity.…”

Section: Resultsmentioning

confidence: 86%

“…This work signaled a new approach to a long-standing problem of cryptorchidism in humans (3,4) where the problem of testicular retention is exacerbated by a predisposition for testicular cancer (5,6). The availability of synthetic RLF and RLF derivatives (7)(8)(9)(10)(11)(12) has provided the opportunity to investigate several aspects of the role of RLF in gonadal migration.…”

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confidence: 99%

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The Mode of Interaction of the Relaxin-like Factor (RLF) with the Leucine-rich Repeat G Protein-activated Receptor 8

Büllesbach

Schwabe

2006

Journal of Biological Chemistry

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The relaxin-like factor (RLF, also named INSL3) is a critical component in the chain of events that lead to the normal positioning of the gonads in the male fetus. RLF and relaxin share features of the secondary structure to the extent that relaxin cross-reacts with the LGR8, the RLF receptor. Although both hormones interact with their receptors essentially via the B chain, the sharply defined binding cassette of relaxin is not present in RLF. Structure and function analysis of RLF derivatives with single amino acid replacements revealed that the most important binding residues are tryptophan B27, followed by arginine B16 and valine B19. Single alanine replacements for each individual position resulted in a relative receptor affinity of 4.0% (B16), 6.1% (B19), and 0.5% (B27). Tryptophan B27 is located on an extended structure, and arginine B16 and valine B19 are positioned on the exposed surface of the B chain helix. The 3 residues could be brought together to form a contiguous binding area if the C-terminal end of the B chain were free to fold back against the central portion of the B chain helix. Such a movement depends critically on the flexibility of the C-terminal end, which is controlled by positions B23-25. In as much as these major binding residues seem hardly sufficient to explain the strong binding of RLF to LGR8 we searched for and found an extended region where little contributions by individual residues added up to a strong receptor affinity. This mode of interaction could drive the binding energy sufficiently high to account for the picomolar binding constant of RLF and its receptor.Targeted deletion of the relaxin-like factor (RLF) 2 gene (named insl3) in mice by Adham et al. (1, 2) strongly suggests that RLF, a Leydig cell-derived hormone, is a vital factor in the physiological process of gonadal positioning. This work signaled a new approach to a long-standing problem of cryptorchidism in humans (3, 4) where the problem of testicular retention is exacerbated by a predisposition for testicular cancer (5, 6). The availability of synthetic RLF and RLF derivatives (7-12) has provided the opportunity to investigate several aspects of the role of RLF in gonadal migration. This research led to the potentially important discovery of a signal activation site in RLF that does not overlap the RLF binding site. The modified RLF is a potent, highly specific inhibitor of RLF action (12). Although it is clear from our investigations that modifications of the signaling site do not interfere with LGR8 binding, the site or region of RLF that is responsible for a nanomolar binding constant is not known.Native RLF, isolated from bovine testes, is a polypeptide hormone in which a 26-residue A chain is linked by two interchain disulfide bonds to a 41-residue B chain and an intrachain cystine bond in the A chain (13). Previous studies indicated that RLF can be shortened at the C terminus of the B chain up to residue 27 without losing its potency if the C terminus is amidated (11). Receptor binding is retained even af...

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Section: Resultsmentioning

confidence: 86%

mentioning

confidence: 99%

The Mode of Interaction of the Relaxin-like Factor (RLF) with the Leucine-rich Repeat G Protein-activated Receptor 8

Büllesbach

Schwabe

2006

Journal of Biological Chemistry

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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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“…Cryptorchid mice with normal gonadotropin and intratesticular T levels show complete absence of postmeiotic germ cells (23). Why the cryptorchid LuRKO testes showed more advanced spermatogenesis is an intriguing question.…”

Section: Discussionmentioning

confidence: 99%

The low gonadotropin-independent constitutive production of testicular testosterone is sufficient to maintain spermatogenesis

Zhang

Pakarainen

Poutanen

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

123

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Spermatogenesis is thought to critically depend on the high intratesticular testosterone (T) levels induced by gonadotropic hormones. Strategies for hormonal male contraception are based on disruption of this regulatory mechanism through blockage of gonadotropin secretion. Although exogenous T or T plus progestin treatments efficiently block gonadotropin secretion and suppress testicular T production, only Ϸ60% of treated Caucasian men reach contraceptive azoospermia. We now report that in luteinizing hormone receptor knockout mice, qualitatively full spermatogenesis, up to elongated spermatids of late stages 13-16, is achieved at the age of 12 months, despite absent luteinizing hormone action and very low intratesticular T (2% of control level). However, postmeiotic spermiogenesis was blocked by the antiandrogen flutamide, indicating a crucial role of the residual low testicular T level in this process. The persistent follicle-stimulating hormone action in luteinizing hormone receptor knockout mice apparently stimulates spermatogenesis up to postmeiotic round spermatids, as observed in gonadotropin-deficient rodent models on folliclestimulating hormone supplementation. The finding that spermatogenesis is possible without a luteinizing hormone-stimulated high level of intratesticular T contradicts the current dogma. Extrapolated to humans, it may indicate that only total abolition of testicular androgen action will result in consistent azoospermia, which is necessary for effective male contraception. T he circulating gonadotropins and intratesticular androgens and paracrine factors form the regulatory network of development and function of the male reproductive organs and functions, including spermatogenesis (1). Spermatogenesis is a complex cyclic process, where germ cells undergo mitotic divisions (spermatogonia), meiosis (spermatocytes), and morphological differentiation (spermatids, spermiogenesis) in a delicately regulated spatiotemporal fashion in the seminiferous epithelium. The production of an appropriate number of spermatozoa is considered to depend critically on stimulation of the testes by the two pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (1).The mechanisms by which LH and FSH regulate spermatogenesis continue to be of interest in research on male reproductive endocrinology, infertility, and contraception. The need for LH and FSH in the maintenance of normal spermatogenesis remains a matter of debate (1-3), with different interpretations arising from different experimental models. LH receptor (LHR) is expressed in Leydig cells, and, on ligand binding, it stimulates their steroidogenesis (4). It remains a dogma that the high intratesticular level of T is indispensable for the onset, maintenance, and completion of spermatogenesis in the adult testis and for its restoration after experimentally induced azoospermia.Numerous studies have reported that both FSH and T are required for quantitatively and qualitatively normal spermatogenesis in a variety of mamma...

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Targeted Disruption of the Insl3 Gene Causes Bilateral Cryptorchidism

Cited by 516 publications

References 30 publications

The Mode of Interaction of the Relaxin-like Factor (RLF) with the Leucine-rich Repeat G Protein-activated Receptor 8

The Mode of Interaction of the Relaxin-like Factor (RLF) with the Leucine-rich Repeat G Protein-activated Receptor 8

LGR8 Signal Activation by the Relaxin-like Factor

The low gonadotropin-independent constitutive production of testicular testosterone is sufficient to maintain spermatogenesis

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