The relaxin family peptides and their receptors.
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.
- insulin‐like factor 3 (INSL3)
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 different molecules, including endocrine and paracrine signaling. Of these, insulin‐like factor 3 (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 . INSL3 is essential for fetal testis, and has been implicated in the testicular and sperm functions in adult males ; 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  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.
2. Insulin‐like factor 3 (INSL3)
The relaxin family peptides consist of seven distinct gene products, which include relaxin (RLN), H1‐RLN, RLN‐3, INSL3, INSL4, INSL5, and INSL6 (Table 1), of which INSL3, like RLN, has been extensively reviewed elsewhere  and is expressed almost exclusively both in male and female gonads. INSL3 binds with high affinity to and activates its specific receptor, relaxin family peptide receptor 2 (RXFP2), originally called leucine‐rich G protein‐coupled receptor 8 (LGR8) [5, 6]. INSL3 plays an essential role in testis descent during fetal development by acting on the gubernaculum via RXFP2, as revealed by mouse models targeting genes encoding either Insl3 or Rxfp2 . After birth, INSL3 possibly plays a role for maintaining spermatogenesis by functioning as a germ cell survival/anti‐apoptotic factor and as a germ cell proliferation factor in some species [8, 9, 10].
|Relaxin family peptides||Receptors|
|Insulin‐like peptide 3||INSL3||RXFP2|
|Insulin‐like peptide 4||INSL4||?|
|Insulin‐like peptide 5||INSL5||RXFP4|
|Insulin‐like peptide 6||INSL6||?|
2.1. A short summary of research methods
Before moving on to the main subject, we will briefly introduce a short summary of materials and methods that we actually used in this chapter. First, peptide antibody for INSL3 was generated and subsequently used for immunological approaches such as western blotting and immunolocalization. Then, INSL3 from goat testes was purified using a series of chromatography 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 expressing 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 spermatozoa using
2.2. Testicular Leydig cells are the sole source of INSL3 in male goats, with INSL3 expression increasing during sexual development
2.2.1. Generation of an anti‐INSL3 peptide antibody
As in other species,
2.2.2. Source and expression dynamics of INSL3
As revealed by
In common with circulating INSL3,
As in the case of other species, INSL3 expression in goats appears to correspond fundamentally to the developmental status of Leydig cells. With the establishment of a functional hypothalamic‐pituitary‐gonadal (HPG) axis during puberty, goat Leydig cells appear to be most responsive to LH at puberty as characterized by a dramatic increase in Leydig cell number and cell diameters . In addition, in other species, INSL3 expression in Leydig cells has been reported to be induced under the long‐term effects of LH/hCG and is not acutely regulated by LH/hCG and other hormones influencing Leydig cell differentiation, such as insulin‐like growth factor1 (IGF1) [19, 20].
2.3. Goat INSL3 is constitutively secreted from testicular Leydig cells as a B–C–A single‐chain structure with full biological activity
2.3.1. Structure of INSL3
Unlike other peptides from the relaxin/insulin family, very little is known about the native conformation of INSL3. INSL3 is predicted from the cDNA sequence to be biosynthesized as a precursor protein (pro‐INSL3) containing A‐ and B‐domains connected by a C‐domain and is assumed to undergo proteolytic processing to remove the C‐domain peptide. The precursor then matures to an A–B heterodimer linked by two disulfide bonds to form an active hormone, as do the other members of this family of peptides (Figure 4) . This prediction has been supported by the findings that a synthetic INSL3 peptide, which consists of an A–B heterodimer with site‐specific sequential disulfide bonds in some species, such as humans  and rats , stimulates cAMP production by binding to its receptor, RXFP2 . Furthermore, this is corroborated by a report that native INSL3 exists as an A–B heterodimer when isolated from bovine testis .
However, recent study concerning native INSL3 purified from boar testes demonstrated for the first time that the native INSL3 exists as a monomer comprising three domains, B–C–A, with site‐specific disulfide bonds and full biological activity . Therefore, there appears to be not only a bovine form of native INSL3 that exists as an A–B heterodimer , but also a porcine form that exists as a B–C–A single chain . In fact, the molecular mass of INSL3 detected by western blot analysis in tissue extracts from several species, including goats , corresponds to that of pro‐ INSL3 (B–C–A single‐chain form) as deduced from their cDNA sequences. Hence, it is worth proving whether native INSL3 of other species, including goats, exists as a B–C–A single chain that corresponds to a porcine model of native INSL3 but not the bovine model.
When purified the protein using a series of chromatography steps and examined the native conformation by MS/MS (tandem MS) analysis, the native goat INSL3 is isolated as a 12‐kDa protein with a B–C–A single‐chain structure, in which the C‐domain does not undergo proteolytic processing (Figure 5A) . This is consistent with the structural features of porcine native INSL3 comprising a B–C–A single‐chain form  but quite different from those of native bovine INSL3  in that C‐domain does not undergo processing. Clarification of native goat INSL3 and its functional characterization would be a major step toward developing a specific immunoassay system for measuring INSL3 in blood and body fluids, whereby the yet unidentified endocrine, paracrine, and/or autocrine aspects of INSL3 can be explored, thereby giving insight into the potential role of INSL3 in goat testicular function.
2.3.2. Biological activity and secretion of INSL3
INSL3 mediates its effect by stimulating cAMP production through its specific cell‐surface receptor, RXFP2 . A reliable bioassay system based on the cAMP production in HEK‐293 cells transfected with either mouse or human RXFP2 has been employed to examine the biological activity of INSL3. Treatment of RXFP2‐expressing HEK‐293 cells with the goat INSL3 resulted in a dose‐dependent increase in intracellular cAMP production with an EC50 values (approximately 7 nM) comparable with those of the synthetic A–B heterodimeric human INSL3 peptide (Figure 4), indicating the retention of full bioactivity (Figure 5B and C).
Furthermore, western blot analysis of testicular venous and peripheral plasma and also of seminiferous tubular fluid demonstrated that the INSL3 secreted was present as the 12 kDa of molecular nature in blood and body fluid, revealing that most secreted INSL3 is not processed (Figure 5D–F) . These results establish that goat INSL3 is secreted from testicular Leydig cells into the blood and body fluids within the lumen of the seminiferous tubules as a biologically active 12‐kDa B–C–A single‐chain peptide. Therefore, the native goat INSL3 differ from insulin, in which the prohormone undergoes proteolytic processing to form the active hormone, but is quite similar to insulin‐like growth factors (IGFs) , as well as native porcine INSL3 , in that the proforms are not processed into two‐chain peptides and exert full bioactivity.
2.3.3. INSL3 regulation by constitutive secretory pathway
It is important to note what secretory pathways are responsible for regulating INSL3 production and why INSL3 is secreted as a single‐chain peptide. It is now accepted that endocrine cells producing peptide hormone possess regulated or constitutive secretory pathways, and that most secretory proteins that are synthesized as prohormones on the RER are transported to the cis‐Golgi network and then through the trans‐Golgi network (TGN) . In the regulated secretory pathway, prohormones are sorted into secretory granules for processing and storage before their release (Figure 6). In contrast, the constitutive secretory pathway is thought to be the default pathway by which prohormones exit the TGN and are rapidly released without storage [27, 28]. Moreover, the major proteolytic enzymes mediating protein processing are prohormone convertase 1/3 (PC1/3) in the secretory granules in the regulated pathway and furin in the TGN in the constitutive pathway [27, 29]. For example, relaxin, which belongs to the same family as INSL3, is stored in secretory granules [30, 31, 32], and they undergo processing by PC1/3 .
Immunoelectron microscopy demonstrated that INSL3 localizes to the TGN within the goat Leydig cells, where secretory storage granules were undetectable, suggesting that goat INSL3 is not produced by the regulated secretory pathway but by the constitutive secretory pathway in Leydig cells (Figure 6) . This is consistent with the findings that
2.4. INSL3‐receptor RXFP2 network functions in testicular germ cells
2.4.1. An RXFP2‐specific antibody that is suitable for detecting RXFP2
To investigate what effects INSL3 actually exerts in testis, the identification of the possible site(s) of action of INSL3 within the testis is necessary. Identifying the cell type(s) that expresses the receptor RXFP2 and specific INSL3 binding in target cells would be a major step toward understanding the as‐yet‐unknown function of INSL3 in goats. Previous study determined the partial cDNA sequence of goat RXFP2 and demonstrated the high level of expression of this gene in mature testes, suggesting that INSL3 is involved in testicular function . However, the specific cell type(s) that expresses RXFP2 in the testis have remained unidentified because of the absence of reliable anti‐RXFP2 antibodies.
Recently, we successfully identified a highly specific anti‐RXFP2 antibody that was suitable for detecting RXFP2 at protein level by analyzing the characteristics of commercially available RXFP2 antibodies that were directed against different parts of human RXFP2 [9, 35]. Identified RXFP2‐specific antibody (GTX108235; GeneTex), which was directed against the human RXFP2 endodomain (C‐terminal intracellular domain), recognized the cell‐surface antigen by double immunofluorescence in HEK‐293 cells transiently transfected with a FLAG‐tagged mouse
2.4.2. Germ cell types expressing
RXFP2mRNA and protein in the mature testis
Cell types expressing
2.4.3. INSL3 ligand binding to germ cells
As shown in Figure 5D–F, goat INSL3 secreted from testicular Leydig cells was released not only into the blood circulation but also into seminiferous tubules . This finding suggests that INSL3 enters the seminiferous compartment across the blood‐testis barrier (BTB), which is created by inter‐Sertoli tight junctions and adherens junctions, by mechanisms which are still unclear. Similar findings were also found in boar and rat INSL3 [9, 14]. These results taken together reveal that INSL3 secreted from the Leydig cells is transported into the seminiferous compartments and is capable of encountering germ cells, where its receptor RXFP2 is expressed, thus enabling INSL3 to bind (Figure 9). This suggests that INSL3 could act as a paracrine factor in seminiferous germ cells in the goat testis. Unfortunately, it remains unknown what functions INSL3 actually exerts on germ cells in the goat testis. One possibility is that INSL3 plays a role in the maintenance of spermatogenesis by regulating cellular processes such as apoptosis through the receptor RXFP2 on seminiferous germ cells in a paracrine manner. In fact, there is compelling evidence that INSL3 contributes to the maintenance of spermatogenesis by acting as a germ cell survival/antiapoptotic factor in testes of rats  and boars , and as a germ cell proliferation factor in zebrafish .
In contrast, the reason why INSL3 did not bind to Leydig cells is unclear, despite their expression of
2.5. The potential of INSL3 hormone‐receptor system as a novel parameter for predicting fertility in breeding sires
2.5.1. Expression and functionality of RXFP2 in the spermatozoa
INSL3 possibly plays a role in sperm function as suggested by the detection of its receptor
Furthermore, we investigated the functionality of RXFP2 in these spermatozoa using
Although the physiological significance of RXFP2 in the equatorial segment of ruminant spermatozoa, including goats, is still unknown, it might be related to sperm fusion with the oocyte membrane during fertilization. A recent study on knockout mice has demonstrated that gamete fusion is mediated by
2.5.2. Potential to diagnose subfertility in sires
Reproductive efficiency in domestic ruminants is a major factor that affects profitability in meat and dairy industries. In sires, normal sperm production and good semen quality are essential in maximizing reproductive performance. Artificial insemination (AI) with frozen semen has been employed in modern breeding programs of dairy and meat livestock to improve economically important traits. Especially, in cattle, it is prominent. However, even when semen quality parameters such as sperm viability and motility are good, some prized sires are still adversely affected by low fertility (subfertility). Thus, it is important to explore novel molecular markers for an accurate assessment of male fertility from the perspective of sperm production and semen quality.
Although serum INSL3 levels are positively correlated with sperm production , sperm number , and sperm morphology  in humans, we examined the potential to diagnose subfertility from the spermatozoa . Since samples suitable for this analysis were available in bulls, we used bull spermatozoa as an alternative to goat spermatozoa.
While peptide hormone INSL3 is essential for fetal testis, and possibly acts as an important player in testicular and sperm functions in adult males, there has been very little progress in understanding the functions of INSL3 in male ruminants, including goats. In this review, we have undertaken an understanding of both the structure and function of INSL3 in male goats to fill a gap in our knowledge. From a structural point of view, we provided evidence that goat INSL3 is constitutively secreted from Leydig cells as an unprocessed B–C–A form retaining the C‐domain with full biological activity. The reason why the INSL3 exists as a B–C–A form is unclear, but the C‐domain does not appear to interfere with receptor binding and activation. Additionally, clarification of native goat INSL3 will facilitate development of a specific immunoassay system for monitoring INSL3 in blood and body fluids. In contrast, from a functional point of view, we provided the evidence for a functional receptor that binds INSL3 in testicular germ cells and in spermatozoa, implying that the intra‐ and extratesticular INSL3 hormone‐receptor system operate in male goats. We also found the potential of this system as a novel parameter for predicting fertility in breeding sires. However, it remains unknown what functions INSL3 actually exerts on testicular germ cells and on spermatozoa in this species. Finally, the findings outlined here will help in the discovery of new target tissues/organs and receptor‐expressing cells, not only in male goats but also in female goats, thereby giving insight into the potential role of INSL3 in those organs.
This work was supported by a Grant‐in‐Aid for Scientific Research from the Japan Society for the Promotion of Science (grant number 15 K07691 to T. Kohsaka) and by a Special Research Fund from Kitasato University School of Veterinary Medicine (to H. Sasada).