Fig. 2. The 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). 10 μg/mL antigen, 1:100 dilution of weekly anti-serum or egg yolk and 1:20,000 dilution of anti-IgY secondary antibody were used in the test. The unit of titration was expressed as the optical density in 490 nm. Arrowheads denote immunogen injections.
Fig. 2. The titer of anti-KISS1R polyclonal antibodies in hen’s serum or egg yolk.
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Fig. 3. The specificity of anti-KISS1R polyclone antibodies determined by absorption control. Protein samples were extracted from hypothalamus of 8-week-old mouse. A) Protein samples were hybridized with sequentially diluted antibody (1 mg/ml) from egg yolk of non-immunized (N) or immunized hen (I). B) 800,000 dilution of antibody (1 mg/ml) from immunized hen was pre-absorbed by different concentration (0-100 μM) of antigen for overnight at 4°C before hybridizing with protein samples. 1:40,000 dilution of anti-IgY secondary antibody were used in the test. Arrow indicated the presenting bands of KISS1R.
Fig. 3. The specificity of anti-KISS1R polyclone antibodies determined by absorption control.
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Fig. 4. KISS1R is expressed in the hypothalamus and testis of 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 8-week-old mouse. Arrow indicated the presenting band of KISS1R.
Fig. 4. KISS1R is expressed in the hypothalamus and testis of 8-week-old mouse.
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Fig. 5. The Kisspeptin is expressed on 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 ICR mice testis sections were stained for Kisseptin with specific antibody. Kisspeptin expressed on I) arcuate nucleus in 8-week-old female mouse is used as positive control. Section incubated without primary antibody is shown as the negative control (J). (Arc = arcuate nucleus;
FL = fetal Leydig cell; L = Leydig cell; S = spermatid; ST = seminiferous tubule) Fig. 5. The Kisspeptin is expressed on Leydig cells of 3 to 12-week-old mouse testis.
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Fig. 6. KISS1R is located on germ cells, especially spermatids, in seminiferous tubules of 2 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 ICR mice testis sections were stained for KISS1R with our own IgY antibody. The insert panel shows the original image at 5X magnification.KISS1R expressed on I) oviduct epithelial cells in 12-week-old female mouse is used as the positive control. Section incubated without primary antibody is shown as the negative control (J). (Arc = arcuate nucleus; FL = fetal Leydig cell; L = Leydig cell; RS = round spermatid; ST = seminiferous tubule; MF = mucosal fold)
Fig. 6. KISS1R is located on germ cells, especially spermatids, in seminiferous tubules of 2 to 12-week-old mouse testis.
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Fig. 7. qPCR results of Kiss1 and Kiss1r mRNA expression levels in mouse testis at different ages. Total mRNA were extracted from whole testis which range from 0 to 12-week-old age. A) Kiss1 and B) Kiss1r data were shown as fold changes compared to 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. Bar with different letters are significantly different, p < 0.05. (n=3)
Fig. 7. qPCR results of Kiss1 and Kiss1r mRNA expression levels in mouse testis at different ages.
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Fig. 8. Body and testes weights and their related ratios of male mice at different ages.
A) Body and testes weights of mouse and B) the related ratio of testes and body weights were recorded from birth to 12-week-old age. Point values are means ± S.D. Different letters are significantly different, p < 0.05. (n=3)
Fig. 8. Body and testes weights and their related ratios of male mice at different ages.
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Fig. 9. qPCR results of Lhcgr, Insl3 and Cyp19a1 mRNA expression levels in mouse testis at different ages. Total mRNA were extracted from whole testis which range from 0 to 12-week-old age. A) Lhcgr, B) Insl3 and C) Cyp19a1 data were shown as fold change compared to expression levels in 12-week-old mice. All mRNA expression levels were normalized with Rpl19 as the internal control. Bar values are means ± S.D. Bar with different letters are significantly different, p < 0.05. (n=3)
Fig. 9. qPCR results of Lhcgr, Insl3 and Cyp19a1 mRNA expression levels in mouse testis at different ages.
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Fig. 10. qPCR results of Fshr, Hsd3b, Ar and Er mRNA expression levels in mouse testis at different ages. Total mRNA were extracted from whole testis which range from 0 to 12-week-old age. A) Fshr, B) Hsd3b, C) Ar and D) Er data were shown as fold change compared to expression levels in 12-week-old mice. All mRNA expression levels were normalized with Rpl19 as the internal control. Bar values are means ± S.D. Bar with different letters are significantly different, p < 0.05. (n=3)
Fig. 10. qPCR results of Fshr, Hsd3b, Ar and Er mRNA expression levels in mouse testis at different ages.
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Fig. 11. qPCR results of Kiss1, Kiss1r and Lhcgr mRNA expression levels in oLH-treated primary Leydig cells. Total mRNA were extracted from 15-week-old primary Leydig cells treated without or with 10 and 50 ng/ml oLH for 4 hours. Data were shown as fold changes compared to expression levels of A) Kiss1, B) Kiss1r and C) Lhcgr in the cells of control group. All mRNA expression levels were normalized with Rpl19 as the internal control. Bar values are means ± S.D. Bar with different letters are significantly different, p < 0.05. (n=3)
Fig. 11. qPCR results of Kiss1, Kiss1r and Lhcgr mRNA expression levels in oLH-treated primary Leydig cells.
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Fig. 12. Kiss1 mRNA expression levels were induced by cAMP through PKA pathway.
MA-10 Leydig cells were treated with cAMP, RP-cAMPS (PKA inhibitor) or both for 24 hours. Data were shown as fold changes compared to expression levels of Kiss1 in the cells of control group. All mRNA expression levels were normalized with RPL19 as the internal control. Bar with different letters are significantly different, p < 0.05. Bar values are means ± S.D. (n=3)
Fig. 12. Kiss1 mRNA expression levels were induced by cAMP through PKA pathway.
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Fig. 13. The kisspeptin/KISS1R system has local function in the testis. A) LH signaling increases not only testosterone concentration but also kiss1 gene expression by cAMP/PKA pathway in Leydig cells. B) The synergistic action of Kisspeptin and development-related factors (INSL3, E2 and so on) on spermatogenesis might happen in the testis.
Fig. 13. The kisspeptin/KISS1R system has local function in the testis.
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