**Recent Advances in Research on the Hormone INSL3 in Male Goats Male Goats**

**Recent Advances in Research on the Hormone INSL3 in** 

DOI: 10.5772/intechopen.70079

Tetsuya Kohsaka, Siqin, Itaru Minagawa and Hiroshi Sasada Tetsuya Kohsaka, Siqin, Itaru Minagawa and Hiroshi Sasada

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70079

#### **Abstract**

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, sug‐ gesting 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.

**Keywords:** insulin‐like factor 3 (INSL3), RXFP2, expression, structure, function, testis, spermatozoa, goat

#### **1. Introduction**

Fertilizable sperm production is regulated by the organized actions of germ cell types and stage‐specific gene products and is supported by the complex interplay of many differ‐ ent molecules, including endocrine and paracrine signaling. Of these, insulin‐like factor 3

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© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

(INSL3), previously called relaxin‐like factor (RLF), is a novel member of the insulin/relaxin gene family, and is produced by both fetal and adult testes [1]. INSL3 is essential for fetal tes‐ tis, and has been implicated in the testicular and sperm functions in adult males [2]; however, similar functions in ruminants remain largely unknown. Exploring the function of INSL3 in goats is especially intriguing, because goats are useful pilot animal for studying reproductive physiology in ruminants because of their fecundity and precocious sexual development [3] and other advantageous traits, such as small body size, calm behavior, and ease of handling. Here, we provide an overview of the recent progress in research on INSL3 in male goats.

and immunolocalization. Then, INSL3 from goat testes was purified using a series of chroma‐ tography steps and the native conformation was examined by tandem MS (MS/MS) analysis. Biological activity of purified INSL3 was examined by cell‐based assay based on the cAMP in INSL3 receptor RXFP2‐expressing HEK‐293 cells. Additionally, the secretory pathway of INSL3 was examined by immunoelectron microscopy. Next, we identified cell types express‐ ing its receptor RXFP2 using both laser capture microdissection and immunostaining with RXFP2‐specific antibody and by characterizing its developmental expression pattern and specificity of INSL3 binding. Moreover, we examined the functional RXFP2 protein in the

Recent Advances in Research on the Hormone INSL3 in Male Goats

http://dx.doi.org/10.5772/intechopen.70079

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**2.2. Testicular Leydig cells are the sole source of INSL3 in male goats, with INSL3** 

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. There are so many commercially available antibodies for INSL3, but none of these antibodies cross‐react with goats (http://www.anti‐ bodies‐online.com). Thus, the first step toward elucidating the possible physiological role of INSL3 in goats is to produce its specific antibody. Based on the cDNA sequence [12], we generated an anti‐INSL3 peptide antibody (RLF‐A‐Ab808) in New Zealand White rabbits against the synthetic peptides of 15 amino‐acid residues of the A domain, which shared 100% amino acid homology with boar, bovine, sheep, and goat *INSL3* cDNAs [11]. This peptide antibody was shown by western blotting to recognize the ∼12‐kDa proINSL3 in the testicu‐ lar extracts and the ∼16‐Da recombinant INSL3, which was expressed in *Escherichia coli* by using a construct that expresses the His‐tagged proINSL3 sequence containing the B−C−A domain inserted into the pCold‐I vector (**Figure 1A**). Furthermore, Ouchterlony gel diffusion test found that one precipitin band between the antibody and testicular extracts of goats and boars or recombinant INSL3 was formed, joined at their ends and fused (**Figure 1B**), indi‐ cating that antigens from boars and goats are immunologically identical and are perfectly cross‐reactive with antibody. Additionally, the antibody did not cross‐react with RLN, which

As revealed by *in situ* hybridization, *INSL3* mRNA is expressed in the Leydig cells that are well known as steroid hormone‐producing cells (**Figure 2**), which is consistent with previ‐ ous study [12]. However, it is unclear whether *INSL3* mRNA was translated into the protein therein. Using the INSL3‐specific antibody, INSL3 protein was identified immunohistochemi‐ cally in the Leydig cells (**Figure 2**), with INSL3 expression increasing during sexual develop‐ ment [11]. Therefore, it is reasonable to draw the conclusion that testicular Leydig cells are the sole source of *INSL3* mRNA and protein in male goats, which is consistent with the findings that INSL3 is expressed by Leydig cells of both fetal and adult testes in a number of species [1].

spermatozoa using *in situ* INSL3 ligand binding assay.

**expression increasing during sexual development**

*2.2.1. Generation of an anti‐INSL3 peptide antibody*

is closely related to INSL3.

*2.2.2. Source and expression dynamics of INSL3*
