Protein localization of relaxin-like factor in goat testes and its expression pattern during sexual development

Siqin,; Kotani, Mai; Aoshima, Takuya; Nakai, Mari; Fuchigami, Mai; Odanaka, Yuki; Sugawara, Yasuhiko; Yogo, Keiichiro; Nagura, Yoshio; Hamano, Koh‐ichi; Fujita, Masatoshi; Sasada, Hiroshi; Kohsaka, Tetsuya

doi:10.2508/chikusan.81.1

Cited by 8 publications

(18 citation statements)

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“…In contrast, the INSL3 antibody (RLF‐A‐Ab808) was generated in our laboratory in New Zealand White rabbits against a synthetic peptide of 15 amino acid residues of the A‐domain, which shared 100% amino acid homology among porcine, bovine, ovine and caprine INSL3 complementary DNAs (cDNAs). The specificity of this antibody has been described elsewhere (Siqin et al 2010a,b, 2013; Minagawa et al ; Pitia et al ).…”

Section: Methodsmentioning

confidence: 99%

“…Immunohistochemistry was performed using the Vectastain Universal avidin‐biotin complex (ABC) Elite kit (Vector Laboratories, Burlingame, CA, USA) with the biotin blocking kit (Biotin Blocking System; DakoCytomation, Carpinteria, CA, USA) as previously described (Siqin et al 2010a,b, 2013; Pitia et al ). Briefly, deparaffinized 4‐μm testicular sections were incubated with anti‐INSL3 immunoglobulin G (IgG) (0.5 μg/mL), anti‐RXFP2 IgG (5 μg/mL) or antisera preabsorbed with synthetic human INSL3 (hINSL3) (25 μg/mL) (6292 Da; Phoenix Pharmaceuticals, Burlingame, CA, USA) for anti‐INSL3 (Siqin et al ) or recombinant RXFP2 endodomain peptide (400 μg/mL) for anti‐RXFP2 (Pitia et al ; Sagata et al .…”

Section: Methodsmentioning

confidence: 99%

“…), where RXFP2 receptors are expressed on germ cells (Siqin et al . ; Pitia et al . ), suggesting that intra‐testicular INSL3 hormone‐receptor system operates in goat germ cells (Pitia et al .…”

Section: Introductionmentioning

confidence: 99%

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Functional insulin‐like factor 3 (INSL3) hormone‐receptor system in the testes and spermatozoa of domestic ruminants and its potential as a predictor of sire fertility

Pitia

Uchiyama

Sano

et al. 2016

Animal Science Journal

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Insulin-like factor 3 (INSL3) is essential for fetal testis descent, and has been implicated in the testicular and sperm functions in adult males; however, similar functions in domestic ruminants remain largely unknown. This study investigated the functional INSL3 hormone-receptor system in adult ruminant testes and spermatozoa, and explored its potential to diagnose the fertility of sires. Testes and spermatozoa were obtained from fertile bulls, rams and he-goats, whereas subfertile testes and spermatozoa were obtained only from bulls. As expected, INSL3 was visualized in Leydig cells, while we clearly demonstrated that the functional receptor, relaxin family peptide receptor 2 (RXFP2), enabling INSL3 to bind was identified in testicular germ cells and in the sperm equatorial segment of bulls, rams and he-goats. In comparison to fertile bulls, the percentage of INSL3- and RXFP2-expressing cells and their expression levels per cell were significantly reduced in the testes of subfertile bulls. In addition, the population of INSL3-binding spermatozoa was also significantly reduced in the semen of subfertile bulls. These results provide evidence for a functional INSL3 hormone-receptor system operating in ruminant testes and spermatozoa, and its potential to predict subfertility in sires.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Functional insulin‐like factor 3 (INSL3) hormone‐receptor system in the testes and spermatozoa of domestic ruminants and its potential as a predictor of sire fertility

Pitia

Uchiyama

Sano

et al. 2016

Animal Science Journal

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“…The specificity of anti-RLN antiserum was verified by western blotting with immunising antigen (purified porcine RLN) and recombinant RLN-like factor (RLF)/insulin-like factor 3 (INSL3), which was expressed as proform of w16 kDa in E. coli using the expression construct having proRLF/INSL3 sequence inserted into pCold I vector as described by us (Siqin et al 2010). On the other hand, the specificity of anti-RXFP1 antiserum was also verified by western blotting with immunising antigen (recombinant MBP-RXFP1 endodomain) and recombinant MBP-RXFP2 endodomain.…”

Section: Specificity Of Antisera For Rln and Rxfp1mentioning

confidence: 99%

Evidence for expression of relaxin hormone-receptor system in the boar testis

Kato¹,

Siqin²,

Minagawa³

et al. 2010

Journal of Endocrinology

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Although the physiological role of relaxin (RLN) in males remains largely unknown, there is limited evidence that the testis might be a candidate source and target of RLN in boars, as RLN transcripts are detected in the boar testis and it contains RLN-binding sites. To determine whether the boar testis acts as a source and target tissue of RLN, we characterised the expression pattern and cellular localisation of both RLN and its own receptor LGR7 (RXFP1) in boar testes during postnatal development by molecular and immunological approaches. Testes were collected from Duroc boars, and partial cDNA sequences of the boar homologue of human RXFP1 were identified. RLN expression increased through puberty onwards, while RXFP1 expression changed little during development.RLN mRNA and protein expression were restricted to the Leydig cells, whereas both Leydig cells and seminiferous epithelial cells expressed RXFP1 mRNA and protein.Interestingly, RLN was expressed in the testis as an 18 kDa form (the expected size of proRLN), but not as the 6 kDa mature form, during development because of a lack of the enzyme required for proRLN processing. In contrast, RXFP1 was detected at all stages as specific bands of 75 and 91-95 kDa (likely non-glycosylated and glycosylated RXFP1 respectively). Thus, we provide evidence for expression of RLN-RXFP1 ligand-receptor system in the boar testis, suggesting that the testis act as a source and possible target tissue of RLN.

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“…As in other species, INSL3 transcripts are detected only in the testis in the mature male goat [11]. However, no studies have yet been carried out to identify whether INSL3 mRNA is translated into INSL3 protein in the goat testis.…”

Section: Generation Of An Anti-insl3 Peptide Antibodymentioning

confidence: 90%

Recent Advances in Research on the Hormone INSL3 in Male Goats

Kohsaka¹,

Siqin²,

Minagawa³

et al. 2018

Goat Science

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Insulin-like factor 3 (INSL3), previously called relaxin-like factor (RLF), is essential for testis descent during fetal development and has been implicated in the testicular and sperm functions in adult males. However, similar functions in ruminants remain largely unknown. This chapter will cover recent advancement in our understanding of INSL3 in goats. First, testicular Leydig cells were the sole source of INSL3, with INSL3 expression increasing during development. Second, INSL3 was constitutively secreted as a B-C-A single-chain structure with full biological activity. Third, secreted INSL3 was transported into the seminiferous compartments, where its receptor RXFP2 was expressed on germ cells, thus suggesting that the intratesticular INSL3 hormone-receptor system operates in germ cells. Fourth, functional RXFP2 enabling INSL3 to bind was also identified in the spermatozoa and suggested the existence of the extratesticular INSL3 hormone-receptor system in the spermatozoa. Interestingly, percentages of INSL3-binding spermatozoa were significantly reduced in the semen of subfertile bulls compared to that of fertile bulls, suggesting the potential of this system to diagnose fertility in breeding sires. These fascinating findings will give a new perspective in physiological and/or therapeutic actions of INSL3 on male reproductive processes in domestic ruminants, including goats.

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Protein localization of relaxin-like factor in goat testes and its expression pattern during sexual development

Cited by 8 publications

References 10 publications

Functional insulin‐like factor 3 (INSL3) hormone‐receptor system in the testes and spermatozoa of domestic ruminants and its potential as a predictor of sire fertility

Functional insulin‐like factor 3 (INSL3) hormone‐receptor system in the testes and spermatozoa of domestic ruminants and its potential as a predictor of sire fertility

Evidence for expression of relaxin hormone-receptor system in the boar testis

Recent Advances in Research on the Hormone INSL3 in Male Goats

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