The Distribution and Expression Patterns of KISS1 and TrkA in Reproductive Axis of Pregnant Oryctolagus cuniculus f. domestica

doi:10.7668/hbnxb.20193038

Abstract

Abstract:

In order to clarify the expression pattern and physiological function of Kiss element (KISS1)and Tyrosine protein kinase A(TrkA) in the reproductive axis of ovulation inducing animals,and analyze the regulatory mechanism of KISS1 and TrkA on the reproduction of ovulation inducing animals.Domesticated rabbits were taken as an ovulation-inducing model.The reproductive axis tissues including hypothalamus,pituitary,ovary and uterus were collected from the pregnant Oryctolagus cuniculus f. domestica(Gestation lasts about 14—18 days).The expression patterns and distribution of KISS1 and TrkA mRNA and proteins was detected using hematoxylin-eosin(H&E)and immunohistochemistry(IHC)staining,semi-quantitative interpretation(RT-PCR),Real-time fluorescent quantitative PCR (qRT-PCR)and Western Blot(WB)in the HPG tissues.The results of IHC showed strongly positive reaction of KISS1 protein was presented in the pituitary basophils and zona pellucida of ovary while the positive expression of TrkA protein presented in the neuroendocrine cells of hypothalamus,basophils of pituitary,granular lutein cell of ovary and lamina propria of uterus.The results of RT-PCR KISS1 was expressed in the reproductive axis of pregnant rabbits,and there was significant difference between the two groups.The expression abundance of TrkA mRNA in hypothalamus,pituitary and ovary was significantly different between the two groups.The qRT-PCR results of KISS1 and TrkA genes were consistent with the RT-PCR results.The results of WB suggested that the relative expression levels of KISS1 protein was only observed in pituitary tissue while the relative expression levels of TrkA protein was presented in the HPG tissues and with a down-regulated trend.The results indicating that KISS1 and TrkA are participated in the regulation of pregnancy in Oryctolagus cuniculus f. domestica,and TrkA has a wider scope of action.

Keywords: Pregnant Oryctolagus cuniculus f. domestica;Reproductive axis;Reproductive hormone;Tyrosine protein kinase A(TrkA);Kiss element(KISS1)

AN Xiaoxiao, ZHANG Bohao, DAI Lijun, BAI Xu, SHI Jun, ZHANG Xia, ZHANG Yong, ZHANG Quanwei, ZHAO Xingxu. The Distribution and Expression Patterns of KISS1 and TrkA in Reproductive Axis of Pregnant Oryctolagus cuniculus f. domestica[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 213-221. doi: 10.7668/hbnxb.20193038.

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Figures/Tables 7

Tab.1 The primer sequences and parameter of RT-PCR

基因 Gene	引物序列(5'—3') Primer sequence(5'—3')	产物长度/bp Fragment lengths	退火温度/℃ Tm	基因登录号 Gene entry number
KISS1	F: TGCCCGAAGGGTCAGCC	154	58.5	XM_008268539.2
	R: GCTGCTGTTCCTCTGTGCCA
TrkA	F: AATGTGGACGGAGGAACAAG	461	58.5	XM_008264290.2
	R: GTGCCGCATGTACTCAAAGA
GAPDH	F: GGTCGGAGTGAACGGATTTG	150	58.5	NM_001082253.1
	R: TTGCCGTGGGTGGAATCATA

Tab.2 The primer sequences and parameter of qRT-PCR

基因 Gene	引物序列(5'—3') Primer sequence(5'—3')	产物长度/bp Fragment lengths	退火温度/℃ Tm	基因登录号 Gene entry number
KISS1	F: CCGGACCCACAGATCAGC	200	60	XM_008268539.2
	R: GTTGTAGGCGGACGAATCC
TrkA	F: GGACAACCCTTTCGAGTTCA	157	60	XM_008264290.2
	R: GAAAAGGCAGGCAAAGACAG
GAPDH	F: GGTCGGAGTGAACGGATTTG	150	60	NM_001082253.1
	R: TTGCCGTGGGTGGAATCATA

Fig.1 Morphological observation of HPG axis tissues in pregnant Oryctolagus cuniculus f. domestica using H&E staining A.Hypothalamus;B.Pituitary;C and D.Ovary;E.Corpus luteum;F.Uterus;The magnification of A—F was 400×.MNEC.Magnocellular neuroendocrine cell;PNEC.Parvocellular neuroendocrine cell;CC.Chromophobe;BC.Basophilic;EC.Eosinophil;PO.Primary oocyte;ZP.Zona pellucida;FC.Follicular cell;PF.Primary follicle;IC.Interstitial cells;FA.Follicular cavity;SG.Stratum granulosum;TFI.Inner layer of follicular membrane;TFE.Outer layer of follicular membrane;SO.Secondary oocyte;GLC.Granular lutein cells;TLC.Theca lutein cells;UG.Uterine glands;LP.Proper layer.The same as Fig.2.

Fig.2 Expression distribution of KISS1 and TrkA proteins in HPG axis tissues of pregnant Oryctolagus cuniculus f. domestica A.KISS1 protein was stained by immunohistochemically;B.TrkA protein was stained by immunohistochemically;C.Negative control (The magnification of A—C was 400×).SCE.Simple columnar epithelium.

Fig.3 Relative expression levels of KISS1 and TrkA mRNA in the HPG axis of pregnant Oryctolagus cuniculus f. domestica with RT-PCR A'.The mRNA expression levels of KISS1 and TrkA in HPG axis tissues of pregnant rabbits were detected by agarose gel electrophoresis.B'.KISS1 mRNA expression level.C'.TrkA mRNA expression level.Different lowercase letters indicated significant difference between groups(P<0.05);different capital letters indicated significant difference between groups (P<0.01).The same as Fig.4—5.

Fig.4 Relative expression levels of KISS1 and TrkA mRNA in the HPG axis of pregnant Oryctolagus cuniculus f. domestica with qRT-PCR

Fig.5 Relative expression levels of KISS1 and TrkA protein in the HPG axis of pregnant Oryctolagus cuniculus f. domestica A.The protein expression levels of KISS1,TrkA and β-actin in HPG axis tissues of pregnant rabbits were detected by polyproacrylamide gel electrophoresis.

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