Downloaded from http://journals.lww.com/jcma by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3jmg6KbmzDt4NVBuxtDw4lCMPV4xbtF0XlL+LvyNrJQ8= on 10/27/2020 Downloadedfrom http://journals.lww.com/jcmaby BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3jmg6KbmzDt4NVBuxtDw4lCMPV4xbtF0XlL+LvyNrJQ8=on 10/27/2020
Journal of the Chinese Medical Association Publish Ahead of Print
DOI: 10.1097/JCMA.0000000000000443
Role of the Kisspeptin/KISS1 Receptor System in the Testicular Development of Mice
Chi-Ming Chianga,f,g, Hsin-Yi Chiua,b,c,d,e, De-Shien Jonga, Leang-Shin Wua, Yue-Jia Leea,*,
Chih-Hsien Chiua,*
aDepartment of Animal Science and Technology, National Taiwan University, Taipei, Taiwan,
ROC
bDivision of Thoracic Surgery, Department of Surgery, Taipei Medical University Hospital,
Taipei, Taiwan, ROC
cDepartment of Medical Education, Taipei Medical University Hospital, Taipei, Taiwan, ROC
dDepartment of Education and Humanities in Medicine, School of Medicine, Taipei Medical
University, Taipei, Taiwan, ROC
eDepartment of Surgery, School of Medicine, Taipei Medical University, Taipei, Taiwan, ROC
fDepartment of Orthopedics Surgery, Cardinal Tien Hospital, New Taipei City, Taiwan, ROC
gProfessional Master Program for Artificial Intelligence in Medicine, College of Medicine,
Taipei Medical University, Taipei, Taiwan, ROC
Author contributions: Dr. Chi-Ming Chiang and Dr. Hsin-Yi Chiu contributed equally to this
work.
Received date: May 10, 2020. Accepted date: August 24, 2020.
Conflicts of interest: The authors declare that they have no conflicts of interest related to the
* Address correspondence. Dr. Chih-Hsien Chiu & Dr.Yue-Jia Lee, Department of Animal
Science and Technology, National Taiwan University, 50, Ln. 155, Section 3, Keelung Road,
Taipei 106, Taiwan, ROC. TEL: +886-2-3366-4161 Fax: +886-336-4160 E-mail:
chiuchihhsien@ntu.edu.tw (C.-H. Chiu) TEL: +1-626-215-0819 Fax: +886-336-4160
E-mail: yuejialee7@gmail.com (Y.-J. Lee)
This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Abstract
Background: Kisspeptin and its receptor KISS1R have been found to be essential regulators of
reproductive function. Previous data have revealed the presence of Kiss1 and Kiss1r mRNAs
in the hypothalamus and the testis of humans and rodents. However, the precise location and
possible physiological role of the kisspeptin/KISS1R system in the testis remain ambiguous.
Methods: We first produced an anti-KISS1R immunoglobulin Y antibody for KISS1R
identification. To detect the exact sites of KISS1R and kisspeptin expression in the testis, we
conducted immunohistochemistry assays on sections of testes. We used real-time polymerase
chain reactions (PCR) to identify Kiss1r in mice and to determine the expression levels of
testicular genes. Finally, to verify the upstream regulation on the Kisspeptin/KISS1 receptor
system, we treated primary mouse Leydig cells and MA-10 cells with luteinizing hormone
(LH) and Br-cAMP, respectively and examined Kiss1 and Kiss1r mRNA expression.
Results: Immunohistochemistry assays revealed that kisspeptin was expressed in Leydig cells
and KISS1R was localized in the seminiferous tubules. With real-time PCR, we found Kiss1r
mRNA was constitutively expressed in the mouse testis from birth until the postnatal fourth
week. Furthermore, mRNA expression of Kiss1 was synchronized with that of Insl3 and
Cyp19a. However, the expression of the LH receptor-encoding gene increased 1 week earlier
than did Kiss1 expression. This indicated that the kisspeptin/KISS1R system in the testis may
be controlled by LH and cAMP signaling pathways. Finally, we confirmed that Kiss1 mRNA
expression was increased in both LH-treated primary Leydig cells and Br-cAMP-treated
MA-10 cells (p < 0.05). On the other hand, cotreatment of both cell lines with Br-cAMP and a
protein kinase A inhibitor RP-cAMP significantly suppressed 50% of Br-cAMP-induced Kiss1
Conclusion: We discovered that Kiss1 expression in mouse Leydig cells was induced by LH
through the cAMP/PKA pathway. Based on the presence of kisspeptin receptors on spermatids,
we inferred that kisspeptin and development-related factors have synergistic effects on
spermatogenesis. Nevertheless, more studies are required to elaborate the role of the
kisspeptin/KISS1R system in testicular development.
Keywords: Kisspeptin, KISS1R, Luteinizing hormone, Spermatogenesis, Testicular
development
1. Introduction
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 (HPG) 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 HPG 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 anti-kisspeptin
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. Methods
2.1 Synthetic peptides: KISS1R peptides (H-NASDDPGSAPRPLD-C) were synthesized. For
immunogens, KISS1R peptides were conjugated with keyhole limpet hemocyanin (KLH).
2.2 Preparation of antibodies: Modified immunization protocols previously developed were
applied in this paper.7 (see appendix)
2.3 Purification of egg yolk antibodies: Immunoglobulin Y (IgY) was purified through
polyethylene glycol (PEG) precipitation, as previously described.8 (see appendix)
2.4 Enzyme-linked immunosorbent assay (ELISA): The titers of chicken IgY anti-KISS1R
production and its avidity were evaluated using indirect ELISA. (see appendix)
2.5 Animal and tissue collection: Institute of Cancer Research mice (ICR mice) were
purchased from National Taiwan University, maintained under a 12-h 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 three 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 h 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 min. 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 min 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 h before staining. Cells were allowed to dry on the well for 15 min at 37°C.
After drying, the cells were covered with a staining solution for 8 h. 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 (FBS) on a 6-well plate for 24 h.
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 h. 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 h. Then, the cells
were treated with or without oLH and RP-cAMPS for the following 24 h. After treatment, total
RNA was extracted from the cells for gene analysis.
2.8 Immunohistochemistry: The detailed protocol was presented in the appendix.
2.9 Western blot: The detailed protocol was presented in the appendix.
2.10 RNA extraction and cDNA synthesis: The detailed protocol was presented in the
appendix.
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, USA) according to
the manufacturer’s instructions.
2.12 Statistical analysis: Each experiment was repeated at least three times. Data are
expressed as mean ± standard deviation. Data were analyzed by using a Student’s t-test or one-way ANOVA, followed by Duncan’s method with SigmaPlot. A p value less than 0.05 indicated significance.
3. Results
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).
ACCEPTED
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 3rd 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 3rd 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 control10 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 2T).
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 Cyp19a1were similar to that of Kiss1 (Fig. 4A–C).
The expressions of Lhcgr, Cyp19a1 and Insl3 drastically increased in levels in the testes at 4th,
4th and 6th 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 4E). The two steroid hormone receptor genes, Ar and Er, had minor fluctuations in transcript levels throughout the developmental process (Fig. 4F and 4G).
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).
4. Discussion
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 studies5,14,
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 (Fig. 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 cells21. 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 5E), 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 two putative functional cAMP response elements (CRE) 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
co-treatment of adenylyl cyclase activator forskolin (fsk) and phosphodiesterase inhibitor
3-isobutyl-1-methylxanthine (IBMX) 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
neurons2,12, it is altered in response to LH signaling in the testis (Fig. 5) and the ovary13.
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. Acknowledgments
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 manuscript.
Reference:
1. Ebling FJ. The neuroendocrine timing of puberty. Reproduction 2005; 129: 675-83. 2. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Crowley WF, et al. A
role for kisspeptins in the regulation of gonadotropin secretion in the mouse. Endocrinology
2004; 145: 4073-7.
3. Colledge WH. Kisspeptins and GnRH neuronal signalling. Trends Endocrinol Metab 2009; 20: 115-21.
4. Han SK, Gottsch ML, Lee KJ, Popa SM, Smith JT, Jakawich SK, et al. Activation of
gonadotropin-releasing hormone neurons by kisspeptin as a neuroendocrine switch for the
onset of puberty. J Neurosci 2005; 25: 11349-56.
5. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, et al. Metastasis
suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature
2001; 411: 613-7.
6. Pineda R, Garcia-Galiano D, Roseweir A, Romero M, Sanchez-Garrido MA, Ruiz-Pino
F, et al. Critical roles of kisspeptins in female puberty and preovulatory gonadotropin surges
as revealed by a novel antagonist. Endocrinology 2010; 151: 722-30.
7. Narat M. Production of antibodies in chickens. Food Technol Biotechnol 2003; 41: 259-67.
8. Atha DH, Ingham KC. Mechanism of precipitation of proteins by polyethylene glycols. Analysis in terms of excluded volume. J Biol Chem 1981; 256: 12108-17.
9. Payne AH, Downing JR, Wong KL. Luteinizing hormone receptors and testosterone synthesis in two distinct populations of Leydig cells. Endocrinology 1980; 106: 1424-9. 10. Cejudo Roman A, Pinto FM, Dorta I, Almeida TA, Hernandez M, Illanes M, et al.
Analysis of the expression of neurokinin B, kisspeptin, and their cognate receptors NK3R and
KISS1R in the human female genital tract. Fertil Steril 2012; 97: 1213-9.
11. Funes S, Hedrick JA, Vassileva G, Markowitz L, Abbondanzo S, Golovko A, et al. The
KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system.
Biochem Biophys Res Commun 2003; 312: 1357-63.
12. Estrada KM, Clay CM, Pompolo S, Smith JT, Clarke IJ. Elevated KiSS-1 expression in the arcuate nucleus prior to the cyclic preovulatory gonadotrophin-releasing hormone/lutenising hormone surge in the ewe suggests a stimulatory role for kisspeptin in oestrogen-positive feedback. J Neuroendocrinol 2006; 18: 806-9.
13. Castellano JM, Gaytan M, Roa J, Vigo E, Navarro VM, Bellido C, et al. Expression of
KiSS-1 in rat ovary: putative local regulator of ovulation? Endocrinology 2006; 147:
4852-62.
14. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul
E, et al. The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of
the orphan G protein-coupled receptor GPR54. J Biol Chem. 2001; 276: 34631-6.
15. Kinoshita M, Tsukamura H, Adachi S, Matsui H, Uenoyama Y, Iwata K, et al.
Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge
and estrous cyclicity in female rats. Endocrinology 2005; 146: 4431-6.
16. Pinilla L, Aguilar E, Dieguez C, Millar RP, Tena-Sempere M. Kisspeptins and reproduction: physiological roles and regulatory mechanisms. Physiol Rev 2012; 92: 1235-316. 17. Hsu MC, Wang JY, Lee YJ, Jong DS, Tsui KH, Chiu CH. Kisspeptin modulates fertilization capacity of mouse spermatozoa. Reproduction 2014; 147: 835-45.
18. O'Shaughnessy PJ, Morris ID, Huhtaniemi I, Baker PJ, Abel MH. Role of androgen and gonadotrophins in the development and function of the Sertoli cells and Leydig cells: data from mutant and genetically modified mice. Mol Cell Endocrinol 2009; 306: 2-8.
19. Nieschlag E, Behre HM, Nieschlag S. Andrology 3rd ed: Springer, Berlin, Heidelberg; 2010.
20. Ivell R, Anand-Ivell R. Biology of insulin-like factor 3 in human reproduction. Hum
Reprod Update 2009; 15: 463-76.
21. Robertson KM, O'Donnell L, Jones ME, Meachem SJ, Boon WC, Fisher CR, et al.
Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene. Proc
Natl Acad Sci U S A 1999; 96: 7986-91.
22. Feng S, Bogatcheva NV, Truong A, Korchin B, Bishop CE, Klonisch T, et al.
Developmental expression and gene regulation of insulin-like 3 receptor RXFP2 in mouse
male reproductive organs. Biol Reprod 2007; 77: 671-80.
23. Filonzi M, Cardoso LC, Pimenta MT, Queiroz DB, Avellar MC, Porto CS, et al.
Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein
distribution in the reproductive tract of the male rat. Reprod Biol Eendocrinol 2007; 5: 29.
24. Anand-Ivell RJ, Relan V, Balvers M, Coiffec-Dorval I, Fritsch M, Bathgate RA, et al.
Expression of the insulin-like peptide 3 (INSL3) hormone-receptor (LGR8) system in the
testis. Biol Reprod 2006; 74: 945-53.
25. Ma X, Dong Y, Matzuk MM, Kumar TR. Targeted disruption of luteinizing hormone beta-subunit leads to hypogonadism, defects in gonadal steroidogenesis, and infertility. Proc
Natl Acad Sci U S A 2004; 101: 17294-9.
26. Zhang X, Odom DT, Koo SH, Conkright MD, Canettieri G, Best J, et al. Genome-wide
analysis of cAMP-response element binding protein occupancy, phosphorylation, and target
gene activation in human tissues. Proc Natl Acad Sci U S A 2005; 102: 4459-64.
27. Song WJ, Mondal P, Wolfe A, Alonso LC, Stamateris R, Ong BW, et al. Glucagon
Figure legends
Fig. 1A. Titer of anti-KISS1R polyclonal antibodies in hen’s serum or egg yolk. The
titer was determined by the reactive binding to the peptide of KISS1R’s extracellular domain
(antigen). The test used 10 μg/mL antigen, 1:100 diluted weekly antiserum or egg yolk, and 1:20,000 diluted anti-immunoglobulin Y secondary antibodies. The unit of titration was
expressed as the optical density at 490 nm. Arrowheads denote immunogen injections. Fig.
1B and 1C. Specificity of anti-KISS1R polyclone antibodies determined through
absorption control. Protein samples were extracted from the hypothalamus of an
8-week-old mouse. 1B) Protein samples were hybridized with sequentially diluted antibodies (1
mg/mL) from the egg yolk of nonimmunized (N) or immunized hens (I). 1C) An antibody
diluted to 1:800,000 (1 mg/mL) from immunized hen was preabsorbed at different
concentrations (0–100 μM) of antigens overnight at 4°C before being hybridized with
protein samples. A dilution of 1:40,000 anti-IgY secondary antibodies were used in the test.
Arrows indicate the presenting bands of KISS1R. Fig. 1D. KISS1R expressed in the
hypothalamus and testis of an 8-week-old mouse. Protein samples were extracted from
the hypothalamus (Hy), testis (T), epididymis (EP), epididymal fat (EF), kidney (K), liver
(L), and heart (H) of an 8-week-old mouse. Arrows indicate the presenting band of KISS1R.
Fig. 2A–J. Kisspeptin is expressed on the Leydig cells of 3 to 12-week-old mouse testis.
A) 0 (birth), B) 1-, C) 2-, D) 3-, E) 4-, F) 6-, G) 8-, and H) 12-week-old male Institute of
Cancer Research mice testis sections were stained for kisspeptin with a specific antibody.
Kisspeptin expressed on Fig. 2I arcuate nucleus in an 8-week-old female mouse was used
as the positive control. Sections incubated without primary antibodies are displayed as the
negative control (Fig. 2J). Fig. 2K–T. KISS1R located on germ cells, particularly
spermatids, in the seminiferous tubules of 2- to 12-week-old mouse testis. K) 0- (birth),
L) 1-, M) 2-, N) 3-, O) 4-, P) 6-, Q) 8-, and R) 12-week-old male ICR mice testis sections
were stained for KISS1R with our own immunoglobulin Y antibodies. The insert panel
presents the original image at 5× magnification. KISS1R expressed on Fig. 2S oviduct
epithelial cells in a 12-week-old female mouse was used as the positive control. Sections
incubated without primary antibodies are shown as the negative control (Fig. 2T). (Arc =
arcuate nucleus; FL = fetal Leydig cell; L = Leydig cell; RS = round spermatid; ST =
seminiferous tubule; SG = spermatogonia; S = spermatocyte; MF = mucosal fold).
Fig. 3 A and B. Quantitative polymerase chain reaction results of Kiss1 and Kiss1r
mRNA expression levels in mouse testis at different ages. Total mRNA was extracted
from the whole testis of mice with ages ranging from 0 to 12 weeks. A) Kiss1 and B) Kiss1r
data are displayed as fold changes compared to the expression levels of Kiss1 and Kiss1r in
12-week-old mice. All mRNA expression levels were normalized with Rpl19 as the internal
control. Bar values are means ± S.D. Bars with different letters are significantly different
and p < 0.05. (n = 3). Fig. 3 C and D. Body and testis weights and their related ratios in
male mice at different ages. C) Body and testis weights of mice and D) the related ratio of
testis and body weights were recorded from birth to 12 weeks of age. Point values are means
± SD. Different letters are significantly different, and p < 0.05 (n = 3).
Fig. 4. Qualitative polymerase chain reaction results of Lhcgr, Insl3, Cyp19a1, Fshr,
Hsd3b, Ar, and Er mRNA expression levels in the testes of mice with different ages.
Total mRNAs were extracted from the whole testis of mice with ages ranging from 0 to 12
weeks. A) Lhcgr, B) Insl3, C) Cyp19a1, D) Fshr, E) Hsd3b, F) Ar, and G) Er data were
shown as fold changes compared to the expression levels of 12-week-old mice. All mRNA
expression levels were normalized with Rpl19 as the internal control. Bar values are means
Fig. 5A–C. Quantitative polymerase chain reaction results of Kiss1, Kiss1r, and Lhcgr
mRNA expression levels in oLH-treated primary Leydig cells. Total mRNAs were
extracted from 15-week-old primary Leydig cells treated without or with 10 and 50 ng/mL
oLH for 24 h. Data are presented as fold changes compared to the expression levels of A)
Kiss1, B) Kiss1r, and C) Lhcgr in the cells of the control group. Fig. 5D and E. Kiss1 mRNA
expression levels were induced by cAMP through protein kinase A (PKA) pathway.
MA-10 Leydig cells were treated with cAMP, RP-cAMPS (PKA inhibitor), or both for 24
h. Data were shown as fold changes compared to the expression levels of Kiss1 in the cells
of the control group. All mRNA expression levels were normalized with RPL19 as the
internal control. Bars with different letters were significantly different, and p < 0.05. Bar
values are means ± SD (n = 3).
Fig. 6. Kisspeptin/KISS1R system local function in the testis. A) Luteinizing hormone
signaling increases testosterone concentration and Kiss1 expression by the cAMP/PKA
pathway in Leydig cells. B) The synergistic effects of kisspeptin and development-related
factors (such as INSL3 and E2) on spermatogenesis may occur in the testis.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
27 Appendix (Methods):
Synthetic peptides
KISS1R peptides (H-NASDDPGSAPRPLD-C) were synthesized by Kelowna International Scientific Inc. (Taipei, Taiwan).
Preparation of antibodies
Two Institut de Sélection Animale brown hens (40-week-old) were immunized through intramuscular injection at multiple sites on the breast. Primary immunization was performed with 400 μg of KISS1R peptide-KLH in 0.5 mL of saline and an equal volume of Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) for each hen. Three boosters with 300 μg of KISS1R peptide-KLH in 0.5 mL of saline and an equal volume of Freund’s incomplete adjuvant were used. The first two boosters were performed at 1-week-intervals, and the third booster was performed 4 weeks after the second booster. The health status of the hens was monitored daily, their blood was tested weekly, and their laid eggs were collected daily. All samples were stored at −20°C or 4°C until further processing.
Purification of egg yolk antibodies
To analyze the average quality of the antibodies in the weeks after immunization, the eggs laid weekly (approximately 5–7 eggs) of each hen were pooled prior to IgY extraction. Because IgY in the serum is selectively transferred to the yolk, we only retained the egg yolk. After recording the total volume of weekly yolks, the yolks were mixed with phosphate-buffered saline (PBS) that was double the yolk volume. Then, 3.5% PEG 6000 (Sigma-Aldrich) of the total volume (yolk + PBS) was added, followed by 10 min of mixing with a rolling mixer. The tubes were centrifuged at 13,000 g at 4°C for 20 min. After centrifugation, the supernatant was passed through a folded filter and was transferred to a new tube. Then, 8.5% of PEG 6000 in grams (calculation based on the new volume) was added to the tube. The tube was rolled with a rolling mixer and centrifuged as aforementioned. The pellet was dissolved in 1 mL of PBS by using a glass stick and a vortex. PBS was added to ensure a final volume of 10 mL. The solution was mixed with 12% PEG 6000 (w/v, 1.2 g), followed by rolling and centrifugation. The pellet was carefully dissolved in 2 mL of PBS buffer, and the solution was dialyzed for 24 h in PBS. Thereafter, the IgY extract was removed from the dialysis bag (Membrane Filtration Products Inc., Seguin, TX, USA) and stored at −20°C until further processing. The protein content (mg/mL) of the samples was measured using the PierceBCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the quality of the antibody was analyzed through simple sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).
28 Enzyme-linked immunosorbent assay (ELISA)
High-affinity microtiter plates (Costar Corning Inc., Corning, NY, USA) were coated with KISS1R peptides (10 mg/mL) in coating buffer (35 mM NaHCO3 and 15 mM Na2CO3, pH 9.6) and incubated overnight at 4°C. Plates were washed twice with washing buffer (6.1 mM Na2HPO4·2H2O, 3.9 mM NaH2PO4·H2O, and 0.1% Tween-20, pH 7.0) and blocked with blocking buffer (0.25% gelatin, 0.15 M NaCl, 0.05 M Tris-base, 6 mM EDTA, and 0.05% Tween-20, pH 8.0) overnight at 4°C. Antibodies (100 mg/mL) in weekly serum or yolk extract were diluted to 1:10,000 in assay buffer, added to the wells in duplicate, and incubated for 1 h at room temperature. After washing, plates were incubated with peroxidase-conjugated goat anti-chicken IgY antibodies (Abcam PLC, Cambridge, UK) and diluted to 1:20,000 in assay buffer for 1 h at room temperature. The color was developed by adding 3.7 mM o-phneylenediamine in 0.03% H2O2 within 5 min, and the color-presenting reaction was stopped by adding 8 N of H2SO4. The optical density was determined at 490 nm on an ELISA reader (DynaTech, Rückersdorf, Germany).
Animal and tissue collection
The Institute of Cancer Research mice (ICR mice) have been created as a fertile mouse line. It is a strain of albino mice originating in SWISS. The mice of this strain were named ICR because they have been sent to various laboratories from the Institute of Cancer Research in the USA.
Pregnant mares’ serum gonadotropin (Sigma-Aldrich) Primary mouse Leydig cell culture
The testes were immediately collected and placed in an isolation buffer [10 mg collagenase and 10 mg bovine serum albumin (BSA) in Hank’s balanced salt solution buffer].
After drying, the cells were covered with a staining solution (1% BSA, 1.5 mM β-nicotinamide adenine dinucleotide, 0.25 mM nitro blue tetrazolium chloride, 0.2 mM dehydroepiandrosterone, and 80% PBS) for 8 h.
RP-cAMPS (Enzo Life Science Inc., Farmingdale, NY, USA): The term-planar chirality used to refer to stereoisomerism resulting from the arrangement of out-of-plane groups with respect to a plane. The configuration of molecular entities possessing planar chirality is specified by the stereo-descriptors Rp and Sp. A center with a clockwise sense of rotation is an R (rectus) center and a center with a counterclockwise sense of rotation is an S (sinister) center. The names are derived from the Latin for 'right' and 'left', respectively.The reason why we used RP-cAMPs in our study is that RP-cAMPs is a potent and specific competitive inhibitor of protein kinase A (PKA) and it can
29
antagonize actions of cAMP. RP-cAMPs has the properties of cell permeability and complete resistance to cyclic nucleotide phosphodiesterase. Therefore, RP-cAMPs becomes a unique tool for researching cAMP-dependent signaling.
Cell line culture
We used MA-10 mouse Leydig tumor cells as the cell model to confirm KISS1R expression on the Leydig cell membrane by using immunohistochemistry, and we investigated the mechanism of luteinizing hormone (LH)-dependent Kiss1 gene expression through real-time PCR.
These cells were maintained in Dulbecco’s Modified Eagle Medium/F-12 medium supplemented with 10% FBS, 2.2 mg/mL NaHCO3, 100U/mL penicillin, and 0.1 mg/mL streptomycin at 37°C and 5% CO2.
Immunohistochemistry
Formalin-fixed mouse tissues were embedded in paraffin, sectioned into 5-mm thick slices, and adhered to poly-L-lysine-coated slides. Tissue sections were deparaffined in xylene, rehydrated in descending concentrations of ethanol, and washed with H2O. Then, they were immersed in 10 mM citrate buffer (pH 6.0) with 0.05% Tween-20 and heated twice in a microwave for 10 min at 750 W at a 5-min interval. The sections were removed and allowed to cool by a brief wash in tap water and then in PBS. After quenching endogenous peroxidase activity with 1% (v/v) H2O2 in methanol for 30 min, the sections were rinsed three times with PBS for 5 min. Nonspecific binding sites were blocked with goat serum in PBS [3% (v/v) normal goat serum and 0.2% (v/v) Triton X-100 in PBS] for 1 h. Commercial rabbit polyclonal antibodies raised against mouse kisspeptin 145 (1:100 dilution; Abcam) were used to visualize kisspeptin. For visualizing KISS1R, we used our chicken anti-mouse KISS1R antibodies diluted at 1:5000. This antibody specificity was validated by presenting gradually obscure bands when the antibodies were preincubated with graded concentrations of antigens in the absorption test. Negative controls for antibodies were established using the blocking buffer alone. After 2 h (KISS1R) or 20 h (kisspeptin) of incubation at 4°C, the antibodies were visualized with a biotinylated secondary antibody directed against rabbit immunoglobulin G (for kisspeptin) or chicken immunoglobulin G and Y (for KISS1R) for 1 h. Slides were washed three times with PBS for 5 min at room temperature and incubated with an avidin–biotin–HRP complex from the Vectastain Universal ELITE ABC Kit (Vector Laboratories, Burlington, ON, Canada) for 30 min according to the manufacturer’s instructions. After the slides were rinsed again, they were incubated for 10–20 min at room temperature with diaminobenzidine to visualize immunostaining. Finally, the slides were rinsed with distilled water twice for 10 min, counterstained with
30
hematoxylin for 30 s, and hydrated with ethanol and xylene before adding a mounting medium (Hecht-Assistant, Sondheim, Germany). Sections were observed under an optical microscope (Axioskop 40, Carl Zeiss, Göttingen, Germany), and images were collected using the AxioCam ERc 5s (Carl Zeiss) digital camera.
Western blot
The tissues or cells were rinsed once with cold PBS and collected. They were ground with a mechanical homogenizer in a cold lysis buffer [150 nM NaCl, 0.1% Triton X-100, 50 mM Tris-HCl (pH 8.0), protease inhibitor, and phosphatase inhibitor]. Protein concentrations were determined using the PierceBCA Protein Assay Kit according to the manufacturer’s instructions. Samples containing 30–60 μg of protein were separated by 15% SDS-PAGE. The separated proteins were transferred to a polyvinylidene fluoride membrane. The membrane was blocked by immersing it in PBS containing 0.01% Tween 20 (PBST) and 2.5% BSA for 8 h at room temperature, followed by incubation with our developed chicken anti-mouse KISS1R antibody (1:200,000 dilution or serial diluted concentrations) in PBST with 0.5% BSA for 18 h at room temperature. Then, the membrane was washed three times with PBST and incubated for 2 h with peroxidase-conjugated goat antirabbit IgG (1:25,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or goat anti-chicken IgY. The membrane was washed with PBST, and bound antibodies were visualized using the enhanced chemiluminescence system (Merck Millipore, Burlington, MA, USA). The images were presented on Kodak X-OMAT film (Eastman Kodak Company, Rochester, NY, USA). RNA extraction and cDNA synthesis
Total RNA was extracted from tissues or cells by using the TRIsure reagent (Bioline Inc., Taunton, MA, USA). We used the PrimeScript RT Reagent Kit (Takara Bio Inc., Shiga, Japan) to synthesize cDNA. Total RNA (500 ng) was mixed with 25 pM oligo(dT) primer, 50 pM random hexamers, an enzyme mix, and a reaction buffer and then incubated at 37°C for 15 min. Reverse transcriptase was inactivated by heating to 85°C for 5 s, and the cDNA products were stored at 4°C for analysis.
ACCEPTED
31 Quantitative real-time PCR
Transcripts were quantified using the Fast SYBR Green Master Mix (Applied Biosystems) in a total volume of 10 μL. Samples were heated for 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C. Then, we conducted melting curve analysis to observe the appearance of nontarget products that could affect the final data.
Statistical analysis
SigmaPlot (Systat Software Inc., San Jose, CA, USA)
