INSL3 ligand-receptor system in the equine testis.
Abstract: We employed molecular and immunological techniques to investigate the expression of INSL3, a member of the insulin-like superfamily, in prepubertal testis, postpubertal testes exhibiting normal and disturbed spermatogenesis, and cryptorchid testes of male horses. In addition, the partial cDNA coding sequences of the equine homologue of the human relaxin/INSL3-receptor Lgr8 were determined. Nonradioactive in-situ hybridization with a cRNA probe for equine Insl3 and immunohistochemistry with a specific rabbit INSL3 antiserum localized Insl3 transcripts and immunoreactive INSL3 ligand to Leydig cells in all types of testes investigated. Quantitative polymerase chain reaction analysis revealed a down-regulation of Insl3 and an up-regulation of the relaxin/INSL3-receptor expression in unilateral cryptorchid versus descended testes. Western blot analysis of protein extracts from adult normal and cryptorchid testes and prepubertal testes showed a single immunoreactive band at 14.5 kDa, which correlates with the predicted size of equine proINSL3. Densitometric analysis of Western blot data of adult normal testes revealed significantly stronger expression of immunoreactive proINSL3 as compared to extracts derived from cryptorchid or prepubertal testes. Thus, decreased expression of immunoreactive INSL3 in cryptorchid and prepubertal equine testis is transcriptionally regulated. The detection of transcripts for equine Lgr8 in the testis has identified the testis as a potential target of INSL3.
Publication Date: 2003-01-08 PubMed ID: 12606415DOI: 10.1095/biolreprod.102.008466Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
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This research explored the expression and role of INSL3, a hormone like insulin, in the testes of male horses. It found that the hormone is present in all tested states, though its expression varies and it may impact the development and function of the testes.
Research Methodology
- The researchers employed both molecular and immunological techniques to understand the expression of INSL3. This involved examining tissues from prepubertal testis, postpubertal testes with healthy and impaired spermatogenesis (the process of creating sperm), and undescended (cryptorchid) testes from male horses.
- An additional part of their study involved determining the partial cDNA coding sequences of the equine counterpart of the human relaxin/INSL3-receptor, Lgr8.
Localization and Expression of INSL3
- Through in-situ hybridization and immunohistochemistry methods, the researchers pinpointed the position of INSL3-related cells and molecules. They found INSL3 transcripts and the INSL3 ligand localized to Leydig cells in all types of testes investigated.
- Quantitative polymerase chain reaction (qPCR) analysis indicated that INSL3 expression was down-regulated (decreased) in cryptorchid testes and up-regulated (increased) in descended testes.
Western Blot Analysis
- Western blot analysis, another method used to identify and quantify proteins in a sample, was conducted on protein extracts sampled from both adult normal and cryptorchid testes, along with prepubertal testes.
- The result of the Wester Blot revealed a single immunoreactive band at 14.5 kDa, which corresponds to the predicted size of equine proINSL3.
- A densitometric analysis, used for quantifying the results of a Western blot, demonstrated that the expression of proINSL3 was significantly stronger in adult normal testes compared to extracts from cryptorchid or prepubertal testes.
Conclusion & Implications
- The conclusion drawn was that the decreased expression of immunoreactive INSL3 in cryptorchid and prepubertal equine testis is regulated at the transcription level.
- The researchers also identified the presence of transcripts for equine Lgr8 in the testis. This suggests that the testis might be a target for INSL3, hinting at a role for this hormone in the development or function of equine testes.
Cite This Article
APA
Klonisch T, Steger K, Kehlen A, Allen WR, Froehlich C, Kauffold J, Bergmann M, Hombach-Klonisch S.
(2003).
INSL3 ligand-receptor system in the equine testis.
Biol Reprod, 68(6), 1975-1981.
https://doi.org/10.1095/biolreprod.102.008466 Publication
Researcher Affiliations
- Departments of Anatomy and Cell Biology, Martin Luther University Halle-Wittenberg, Faculty of Medicine, Halle, Germany. thomas.klonisch@medizin.uni-halle.de
MeSH Terms
- Animals
- Base Sequence
- Blotting, Western
- Cryptorchidism / metabolism
- Cryptorchidism / veterinary
- DNA Primers
- DNA, Complementary / biosynthesis
- DNA, Complementary / genetics
- Digoxigenin
- Horse Diseases / metabolism
- Horses / physiology
- Immunohistochemistry
- In Situ Hybridization
- Insulin
- Leydig Cells / metabolism
- Male
- Molecular Sequence Data
- Proteins / metabolism
- RNA / biosynthesis
- RNA / isolation & purification
- Reverse Transcriptase Polymerase Chain Reaction
- Sexual Maturation / physiology
- Spermatogenesis
- Testis / metabolism
Citations
This article has been cited 6 times.- Kawate N. Insulin-like peptide 3 in domestic animals with normal and abnormal reproductive functions, in comparison to rodents and humans. Reprod Med Biol 2022 Jan-Dec;21(1):e12485.
- Shakeel M, Yoon M. Functions of somatic cells for spermatogenesis in stallions. J Anim Sci Technol 2022 Jul;64(4):654-670.
- Albrethsen J, Juul A, Andersson AM. Mass Spectrometry Supports That the Structure of Circulating Human Insulin-Like Factor 3 Is a Heterodimer. Front Endocrinol (Lausanne) 2020;11:552.
- Ivell R, Agoulnik AI, Anand-Ivell R. Relaxin-like peptides in male reproduction - a human perspective. Br J Pharmacol 2017 May;174(10):990-1001.
- Minagawa I, Fukuda M, Ishige H, Kohriki H, Shibata M, Park EY, Kawarasaki T, Kohsaka T. Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B-C-A with full biological activity in boars. Biochem J 2012 Jan 1;441(1):265-73.
- Hanna CB, Yao S, Patta MC, Jensen JT, Wu X. Expression of insulin-like 3 (INSL3) and differential splicing of its receptor in the ovary of rhesus macaques. Reprod Biol Endocrinol 2010 Dec 7;8:150.
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