Dynamic Changes of Plasma Kisspeptin and Expression and Distribution of Kiss1/GPR54 in PHPT Axis of Ganjia Tibetan Sheep During Estrus Cycle

doi:10.7668/hbnxb.20193072

Abstract

Abstract:

The Pineal-hypothalamus-pituitary-thyroid axis(PHPTA)could significantly regulate reproductive activities of animals.In order to investigate the expression of Kiss1/GPR54 system on PHPT axis and its regulation on reproductive activities of Tibetan sheep,24 healthy and non-pregnant Tibetan sheep in estrus cycle were selected as experimental group and 6 non-breeding season Tibetan sheep as control group.The dynamic changes of plasma Kisspeptin were detected by ELISA,and the mRNA and protein expression of Kiss1 and GPR54 in optic nerve,pineal body,hypothalamus,pituitary body and thyroid gland were detected by Real-time quantitative PCR,Western Blot and immunohistochemistry.The results showed that the plasma Kisspeptin secretion in late estrus was significantly higher than that in other periods,and that in each period of breeding season was significantly higher than that in non-breeding season.The mRNA and protein of Kiss1 and GPR54 were expressed in optic nerve and PHPT axis.The relative expression of mRNA and protein of Kiss1 and GPR54 in optic nerve were significantly higher in early estrus than in other periods(P<0.05).The mRNA and its protein of Kiss1 of the pineal body reached its maximum value in estrus,significantly higher than that in reproductive cycle.The mRNA relative expression of Kiss1 and GPR54 of hypothalamus and thyroid gland were significantly increased in late estrus(P<0.05).The mRNA relative expression levels of the two in pituitary body were significantly higher in estrus than in other periods.Immunohistochemical results showed that Kisspeptin and GPR54 were mainly distributed in the glial nucleus and cytoplasm,respectively.Both were strongly positive in pineal cell cytoplasm.Kisspeptin was expressed in neuroendocrine small cells and glial cells in hypothalamus,GPR54 was expressed in cytoplasm of neuroendocrine small cells.Kisspeptin and GPR54 were mainly expressed in the cytoplasm of basophils in pituitary body.In thyroid gland,they were mainly expressed in the cytoplasm of follicular cells.The dynamic changes of plasma Kisspeptin and the differential expressions of Kiss1 and GPR54 in different tissues during estrus cycle of Ganjia Tibetan sheep indicated that Kiss1/GPR54 system was involved in regulating reproductive physiological activities of Ganjia Tibetan sheep.

Key words: Ganjia Tibetan sheep, Seasonal estrus, Kiss1/GPR54, Pineal-hypothalamus-pituitary-thyroid axis

YANG Yawen, GAO Hexuan, LIU Lili, BAO Yingying, HE Yuqin, YANG Zhijie, CHEN Weigang, GE Wenbo. Dynamic Changes of Plasma Kisspeptin and Expression and Distribution of Kiss1/GPR54 in PHPT Axis of Ganjia Tibetan Sheep During Estrus Cycle[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 204-212. doi: 10.7668/hbnxb.20193072.

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Figures/Tables 6

Tab.1 Primer of qRT-PCR

基因 Gene	引物序列(5' —3') Primer sequence(5' —3')	产物大小/bp Product size	退火温度/℃ Annealing temperature	基因登录号 NCBI reference sequence
Kiss1	F:GCCTGATCCCGGCTCCGA	90	58	NM_001306104.1
	R:CGTAGCGCAGGCCGAAGGAG
GPR54	F:TACATCCAGCAGGTCTCGGTG	71	60	NM_001318077.1
	R:ACGTACCAGCGGTCCACACT
β-actin	F:CCATTGAGCACGGCATTGT	184	60	NM_001009784.3
	R:GCAGGGGTGTTGAAGGTCTC

Fig.1 Dynamic changes of plasma Kisspeptin in estrus cycle and anestrus of Ganjia Tibetan sheep A.Estrus(0—36 h)and metestrus(39—72 h);B.Diestrus(4 dM—13 dN),proestrus(14 dM—16 dN)and anestrus(F) ;N.Night ;M.Morning.

Fig.2 Optic nerve and PHPT axis tissues in estrus cycle and anestrus of Ganjia Tibetan sheep relative expression levels of Kiss1 and GPR54 mRNA Significance analysis was conducted in different periods of the same organization, and different lowercase letters indicated significant differences in different periods(P<0.05).The same as Fig.4.

Fig.3 Western Blot results of optic nerve and PHPT axis tissues of Ganjia Tibetan sheep during estrus and anestrus A.Optic nerve;B.Pineal body;C.Hypothalamus;D.Pituitary body;E.Thyroid gland.

Fig.4 Optic nerve and PHPT axis tissues in estrus cycle and anestrus of Ganjia Tibetan sheep relative expression levels of Kisspeptin and GPR54 proteins

Fig.5 Distribution of Kisspeptin and GPR54 immunopositive products(IHC)in optic nerve and PHPT axis tissues of Ganjia Tibetan sheep 1.Glial cell;2.Pineal body cell;3.Neuron;4.Nerve nucleus;5.Eosinophilic cell;6.Basophilic cell ; 7.Chromophobe;8.Follicular epithelial cell;9.Follicular glial cell;10.Parafollicular cell.

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