Reproduction is essential for the continuity of the species. Because reproduction is controlled by a complex network of regulatory signals, studying the physiology and pathology of reproduction has long been a primary goal in biomedicine. A major event controlling reproduction is the onset of puberty. Without the successful transition into puberty, an individual remains immature and cannot produce mature gametes for fertilization. After a long search for the factors regulating the onset of puberty, the hypothalamic–pituitary–gonadal axis was reported as a central regulatory system for the initiation of puberty.1
Recently, the kisspeptin/KISS1R system was discovered to be the upstream regulator of the hypothalamic–pituitary–gonadal axis.2 Mechanisms by this system in regulating gonadotropin hormone release have been uncovered and reviewed over the years.3,4 Given that KISS1 and KISS1R genes are not expressed in the brain only,5 scientists have been studying local functions of the kisspeptin/KISS1R system in the ovaries, testes, and other tissues. However, the direct functions of the kisspeptin/KISS1R system, such as ovulation or spermatogenesis in gonads, have not been confirmed because of insufficient and partly discrepant data.6
We succeeded in the production of an antibody against KISS1R in hens, which are good hosts for producing antibodies due to their evolutionary distance from mammals. Then, we used our own anti-KISS1R antibodies and commercial antikisspeptin antibodies to detect the sites of KISS1R and kisspeptin expression in the testes. In addition, we analyzed the patterns of Kiss1 and Kiss1r expression during testicular development and the connection between Kiss1 and other development-related genes to determine the possible role of the kisspeptin/KISS1R system in the testes.
2.1. Synthetic Peptides
KISS1R peptides (H-NASDDPGSAPRPLD-C) were synthesized. For immunogens, KISS1R peptides were conjugated with keyhole limpet hemocyanin.
2.2. Preparation of Antibodies
Modified immunization protocols previously developed were applied in this paper7 (see appendix https://links.lww.com/JCMA/A64).
2.3. Purification of Egg Yolk Antibodies
Immunoglobulin Y (IgY) was purified through polyethylene glycol (PEG) precipitation, as previously described8 (see appendix https://links.lww.com/JCMA/A64).
2.4. Enzyme-linked Immunosorbent Assay
The titers of chicken IgY anti-KISS1R production and its avidity were evaluated using indirect ELISA (see appendix https://links.lww.com/JCMA/A64).
2.5. Animal and Tissue Collection
Institute of Cancer Research mice (ICR mice) were purchased from National Taiwan University, maintained under a 12-hour light cycle, and given a chow diet and water ad libitum. All procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For RNA extraction and immunohistochemistry, testes were obtained from male mice aged 0, 1, 2, 3, 4, 6, 8, and 12 weeks postpartum (wpp). At least 3 mice were enrolled in each group. One side testis from a mouse was used for RNA extraction, and the other side testis was prepared for immunohistochemistry. For protein extraction, the hypothalamus, testes, epididymis, kidney, liver, and heart were obtained from male mice aged 8 wpp. To analyze a specific site, the brain was positioned on a brain blocker with the plane of section of the mouse brain and cut sagitally into 2-mm thick slices containing the hypothalamus area. Furthermore, hypothalamus and ovaries collected from 8-week-old female mice were used as the positive control in immunohistochemical staining. Female mice were pretreated with 10 international units of pregnant mares’ serum gonadotropin for 48 hours to maintain their estrous cycle during the preovulatory stage before sacrifice.
2.6. Primary Mouse Leydig Cell Culture
Mice aged up to 12 weeks were sacrificed through decapitation. The testes were immediately collected and placed in an isolation buffer. The buffer was replaced once to remove red blood cells and tissue debris. Then, testes in the isolation buffer were incubated at room temperature for 5 minutes. The seminiferous tubules were separated by filtration through a sterile stainless-steel net with a nylon mesh; then, the filtrate was centrifuged at 300 g for 5 minutes at room temperature. The dissociated cells were resuspended in 15 mL of Medium 199 (M-199) and incubated at 37°C with 5% of CO2. To identify Leydig cells, 3β-HSD (3 beta-hydroxysteroid dehydrogenase) staining was conducted using a modification of a previous method.9 In total, 2 × 105 cells were seeded onto a 6-well plate and incubated for 24 hours before staining. Cells were allowed to dry on the well for 15 minutes at 37°C. After drying, the cells were covered with a staining solution for 8 hours. Then, the cells were rinsed in PBS and fixed with 4% paraformaldehyde in PBS. These cells were observed at 400× magnification to detect blue-purple formazan granules. Before treatment, the cells were counted and seeded (2 × 105) with M-199 fetal bovine serum on a 6-well plate for 24 hours. Then, the cells were treated with ovine luteinizing hormone (oLH) and a protein kinase A (PKA) inhibitor—RP-cAMPS in a serum-free medium for an additional 24 hours. Later, we extracted RNA from the cells with each treatment for cDNA synthesis and real-time polymerase chain reaction (PCR) analysis.
2.7. Cell Line Culture
MA-10 cells were maintained in Dulbecco’s Modified Eagle Medium/F-12 medium and plated at 2 × 105 cells/well, which allowed to adhere for 24 hours. Then, the cells were treated with or without oLH and RP-cAMPS for the following 24 hours. After treatment, total RNA was extracted from the cells for gene analysis.
The detailed protocol was presented in the appendix, https://links.lww.com/JCMA/A64.
2.9. Western Blot
The detailed protocol was presented in the appendix, https://links.lww.com/JCMA/A64.
2.10. RNA Extraction and cDNA Synthesis
The detailed protocol was presented in the appendix, https://links.lww.com/JCMA/A64.
2.11. Quantitative Real-time PCR
Relative levels of target mRNA were examined using the StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions.
2.12. Statistical Analysis
Each experiment was repeated at least 3 times. Data are expressed as mean ± standard deviation. Data were analyzed by using a Student’s t-test or 1-way ANOVA, followed by Duncan’s method with SigmaPlot. A p value less than 0.05 indicated significance.
3.1. Titer and Specificity of Chicken Anti-KISS1R Antibody and Detection of KISS1R in Various Mouse Tissues
With ELISA assay, we showed that antibodies in serum collected 2 weeks after the third injection reached to the highest titer (Fig. 1A). However, anti-KISS1R antibodies in the yolk showed higher titer than the antibodies in sera did and the highest titer was shown in the fourth week after the third injection.
We used mouse hypothalamus as the positive control to test whether our antibody could specifically recognize its antigen, KISS1R. The density of the band at the size of 43 kDa gradually decreased when the dilution levels of antibodies increased (Fig. 1B). On the other hand, no bands were detected by the antibodies extracted from the hens without immunization. With a dilution ratio of 1:800 000, the antibodies showed the exclusive band for KISS1R at the size of 43 kDa. We also conducted the adsorption test by preincubating our antibody with the antigen. The preincubation with 100 μM immunogenic peptides resulted in a less density of the immunoreactive band than the preincubation with 10 μM or less immunogenic peptides did for the bands (Fig. 1C).
As we confirmed the specificity of the antibody, we applied this antibody on the detection of KISS1R in mouse tissues. The KISS1R immunoreactive band with a molecular weight of 43 kDa was revealed in the hypothalamus and testes and slightly in the epididymis, whereas the specific immunoreactive band was absent from the epididymal fat, kidney, liver, and even heart (Fig. 1D). However, other nonspecific bands were still visualized in the kidney, liver, and heart.
3.2. Immunohistochemical Staining of Kisspeptin and KISS1R in Mouse Testes
Testis sections from mice aged 0–12 wpp were immunostained with commercial rabbit anti-kisspeptin antibodies (Fig. 2A–J) and our own anti-KISS1R antibodies (Fig. 2K–T). From the postnatal third week to 12th week, kisspeptin was particularly expressed in the cytoplasm of Leydig cells located adjacent to the seminiferous tubules (Fig. 2A–H). Immunoreactive cells on the arcuate nucleus of the hypothalamus were used as the positive control (Fig. 2I).
Unlike kisspeptin, KISS1R was observed in the seminiferous tubules from the postnatal third week to the 12th week (Fig. 2K–R). With the magnification of 1000×, KISS1R was clearly observed on the cell membrane of the round spermatids (Fig. 2R). The oviduct was used as the positive control,10 and KISS1R was detected on the ciliated epithelium of the oviduct (Fig. 2S). Twelve-week-old testes incubated without primary antibodies were used as the negative control for immunohistochemical staining (Fig. 2J and T).
3.3. Gene Expression of Kiss1 and Kiss1r During Testicular Development
To address the time frame when the testes express kisspeptin and KISS1R, we analyzed the gene expression of Kiss1 and Kiss1r in the mouse testes using quantitative real-time PCR. Fig. 3A reveals that the testes did not express Kiss1 mRNA until the fourth postnatal week. Throughout the development process, the Kiss1 transcript level gradually increased and reached to the highest level at 8 and 12 wpp. On the other hand, we discovered that Kiss1r mRNA was constitutively expressed in the mouse testes from week 0 to 12 (Fig. 3B).
3.4. Kiss1 Expression Matched the Expression of the Genes Related to Testicular Development
To determine the relevance between Kiss1 expression and testis maturation, we compared the expression of Kiss1 with the expression of the genes related to testicular development. The expression patterns of Lhcgr, Insl3, and Cyp19a1 were similar to that of Kiss1 (Fig. 4A–C). The expressions of Lhcgr, Cyp19a1, and Insl3 drastically increased in levels in the testes at fourth, fourth, and sixth week, respectively. Furthermore, the expression of these genes (Lhcgr, Cyp19a1 and Insl3) were kept at relative high levels till the puberty (8 wpp).
On the contrary, genes (Fshr, Hsd3b, Ar, and Er) relevant to Sertoli cell maturation and hormone signaling had different expression patterns from those of Kiss1. Fshr and Hsd3b genes started reducing their mRNA levels after 1 and 2 wpp, respectively (Fig. 4D and E). The 2 steroid hormone receptor genes, Ar and Er, had minor fluctuations in transcript levels throughout the developmental process (Fig. 4F and G).
3.5. Kiss1 Expression Was Dependent on LH Signaling
We used primary Leydig cells and MA-10 cells as cell models to investigate the relevance of LH to Kiss1 expression. We discovered that the addition of 50 ng/mL oLH increased Kiss1 expression by one and a half fold (Fig. 5A) but did not affect the expression of Kiss1r (Fig. 5B) and Lhcgr (Fig. 5C). Furthermore, Kiss1 expression in MA-10 cells significantly increased when 50 and 100 μM Br-cAMP were added, which was consistent with the results in the primary Leydig cell experiment (Fig. 5D). On the other hand, cotreatment with Br-cAMP and RP-cAMPS suppressed approximately 50% of Br-cAMP-induced Kiss1 expression, while RP-cAMPS alone did not alter Kiss1 expression (Fig. 5E).
Through quantitative reverse transcription-PCR, studies have revealed that KISS1R mRNAs are found in numerous human tissues, and the highest level of KISS1R mRNA expression is observed in the placenta, pancreas, and brain.5,14 In addition, other studies have indicated that KISS1R mRNAs are mainly expressed in the preoptic area of the hypothalamus.15 Our results revealed that KISS1R were expressed in the testes and epididymis. In consistent with the Kiss1r-expressing tissues reported by previous studies,5,14 our results revealed that KISS1R proteins were expressed in the testes and epididymis (Fig. 1). Nevertheless, the expression level of KISS1R proteins may change by species, stage of life, and pathological status.16 Until now, kisspeptin expressed in Leydig cells was observed by our research group17 and Tena-Sempere’s research group.16 However, the location of KISS1R remains controversial. The pattern of KISS1R expression in our study is inconsistent with our previous data,17 which indicated that KISS1R is located on the acrosome rather than the membrane of spermatids. Although more investigations are necessary to clarify the disparities, we confirmed the specificity of our chicken anti-KISS1R antibody through the preabsorption test.
In addition to the altered expression of kisspeptin/KISS1R system during testicular development, we found that the Kiss1 gene had its expression changed along with genes responsible for the reproductive performance in the testis (Figs. 3A and 4). Several functions and landmark events in mouse testicular development have been well-reviewed in the past.18,19 These key functions include hormone secretion, testis descent, cellular differentiation, spermatogenesis, and the formation of the blood–testis barrier. Among these, spermatogenesis is controlled by INSL3 produced in adult Leydig cells20 and an estrogen synthesized by aromatase (CYP19A1) in Sertoli cells.21 Given similar patterns for Kiss1, Insl3, and Cyp19a1 gene expression during testicular development it is likely that spermatogenesis is also dependent with the increasing synthesis of kisspeptin. Moreover, analysis of RXFP2 (the receptor of INSL3) expression indicated that RFXP2 is located on Leydig cells themselves and during both the pre- and post-meiotic stages of germ cells, with the most on postmeiotic spermatids.22,23 These data suggest that INSL3 secreted from Leydig cells can drive its actions for spermatogenesis in spermatids expressing RFXP2. Based on the same gene-induction period, secretory cells, and the receptor-presenting site between Kiss1 and Insl3, we assumed a synergistic effect from kisspeptin and INSL3 on spermatogenesis and expect to confirm this hypothesis in further research.
In addition, Lhcgr has a similar pattern of gene expression to Kiss1. However, the earlier expression pattern of Lhcgr in our study may signify upstream regulation of Kiss1, Insl3, and Cyp19a1. This implication was supported by previous findings on LH receptor knockout mice failing to express INSL3 proteins24 and Cyp19a1.25
As we found that Kiss1 expression was dependent with the treatment of Br-cAMP and the PKA inhibitor (Fig. 5D and E), we suggest that the cAMP/PKA pathway is involved in the regulation of LH on Kiss1 expression. A previous study has reported that mouse Kiss1 promoters contain 2 putative functional cAMP response elements half-sites (TGACT) located −127 and −758 bp upstream of the Kiss1 transcription start site.26 Recently, Song et al. confirmed that the intracellular concentration of cAMP induced by the cotreatment of adenylyl cyclase activator forskolin (fsk) and phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine significantly increased Kiss1 expression in mouse hepatocytes.27 These pieces of evidence strongly support our finding about LH-induced kiss1 expression in the testis and suggest that LH signal with the cAMP/PKA pathway is the primary regulators of Kiss1 expression in tissues including the testis and liver. In fact, the cAMP/PKA pathway is the main mechanism with which LH signal triggers the secretion of sex hormones during puberty.19 Given this same upstream regulation, it will be interesting to further examine how much extent testosterone and kisspeptin interact with each other for testicular development.
Overall, our study verified the local expression of kisspeptin and KISS1R in mice testes. During puberty, the Kiss1 mRNA level increased in both central (hypothalamus) and local (testis) systems11; however, the upstream regulation on Kiss1 expression in the hypothalamus is different from that in the testis. While Kiss1 expression is controlled by sex steroid hormones in the hypothalamus to fulfill sex steroid feedback to GnRH neurons,2,12 it is altered in response to LH signaling in the testis (Fig. 5) and the ovary.13 This signified that Kiss1 mRNA is able to receive and relay information of the environment at least including the hypothalamus and gonads during puberty. Moreover, the difference in signals stimulating Kiss1 mRNA expression indicated that Kiss1 and its encoding kisspeptin may act for various roles in the tissues. Based on the findings in this study, we suggest that the kisspeptin/KISS1R system may play a role in the spermatogenesis of the testes.
There are some limitations in this study. First of all, we used a polyclonal antibody for kisspeptin receptor in our study. The specificity of the polyclonal antibody was not as good as that of monoclonal antibody to the targeted antigen. Another limitation was that we did not have mutant mice with a targeted disruption of the Lhcgr, Kiss1, and Kiss1r genes. Therefore, we are not able to confirm the role of kisspeptin/KISS1R system in the spermatogenesis of the testes directly.
In conclusion, we concluded that LH acted as an upstream initiator to induce Kiss1 expression in mouse Leydig cells through the cAMP/PKA pathway. We determined the synergistic effects of kisspeptin and development-related factors of spermatogenesis because kisspeptin receptors were present on the spermatids, which regulated spermatogenesis with a similar timing and location of gene expression among Kiss1 and several genes (Fig. 6). In addition, the levels of Insl3, Cyp19a1, and Kiss1 gene expression increased simultaneously after increased expression of Lhcgr. However, more studies are necessary to elucidate the definitive role of the kisspeptin/KISS1R system in testicular development.
The authors would like to acknowledge the Laboratory Animal Center at Taipei Medical University for language editing support. This work was supported by grant 106-2320-B-002-040-MY3 (to Dr Chih-Hsien Chiu, National Taiwan University) from the Ministry of Science and Technology, Taiwan. The funding agencies were not involved in designing the study; collecting, analyzing, and interpreting data; or writing the article.
APPENDIX A. SUPPLEMENTARY DATA
Supplementary data related to this article can be found at http://doi.org/10.1097/JCMA.0000000000000264.
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