The Distribution And MRNA Expression Of FSHR And LHR In The Ovary Of Ganjia Tibetan Sheep In The Estrous Cycle

In order to clarify the the reproductive activity role of follicle stimulating hormone receptor and luteinizing hormone receptor in the ovaries during the different stages of the oestrous cycle in Ganjia Tibetan sheep, in this study, the distribution and dynamic changes of mRNA expression of the ovaries at different stages of the estrous cycle of follicle stimulating hormone receptor(FSHR) and luteinizing hormone receptor(LHR) were studied. A total of 32 normal development of healthy and non pregnant female Ganjia Tibetan Sheep were selected, which were 2.5-3.5 years old, and according to the different stages of the estrous cycle was divided into four groups: proestrus, estrus, late estrus, diestrus. Collect ovaries from Ganjia Tibetan sheep at all stages of estrus cycle,(1) extracted total RNA, and the real time fluorescence quantitative PCT(RT-PCR) method with GAPDH as reference gene was established,to study and compare and analyze the FSHR and LHR gene expression differences in mRNA levels in ovaries of Ganjia Tibetan sheep at all stages of estrus cycle;(2) immunohistochemical horseradish peroxidase labeled streptavidin method(SP-HRP) enable to investigate the distribution and localization of FSHR and LHR in ovaries of Ganjia Tibetan Sheep in different Stages in the Estrous Cycle;(3) combined with computer graphic analytical technique to analyze the optical density expression of FSHR and LHR immunoreactive products. The results are demonstrated as fellows:1. The relative expression of FSHR gene and LHR gene in mRNA levels in the ovaries of Ganjia Tibetan sheep were expressed during the whole estrous cycle.But the expression quantity were different and presented a dynamic trend.The relative expression of FSHR mRNA of ovaries in proestrus,estrus,metestrus and diestrus were respectively 46.0±0.21,51.7±0.33,17.7±0.56 and 1.0±0.82.The FSHR mRNA relative expression in estrus was the highest and was very significantly different from that in diestrus(P<0.01).The relative expression of LHR mRNA of ovaries in proestrus,estrus,metestrus and diestrus were respectively 19.8±2.63,30.1±1.81,85.3±0.63 and 1.0±1.37.The LHR mRNA relative expression in metestrus was the highest and was very significantly different from that in diestrus(P<0.01).2. In the ovary of Ganjia Tibetan sheep, the primordial follicles was located in the ovary cortex, and it is smaller.The boundaries of cells were not obvious. The boundaries of primary follicles follicular cells were clear, and were distributed in a ring around the oocyte. Distribution of secondary follicles was deeper in the ovary, increasing the volume of the oocyte nucleus was clear, and the volume and quantity of follicular cells increased, while the boundaries were clear, showing a multi- layered, circular distributed around the oocyte. The tertiary follicles located in the medulla, granule cell boundaries were clear, and cell nucleus were large. Convex surface of mature follicles in the ovary, granulosa cells were thin, and transparent tape was significantly. There was no integral spherical structure in atretic follicles. The cell nucleus was larger, and boundaries were clear. The blood vessels were distributed widespread in the ovary.The vascular endothelial cells were flat and the cell nucleus were narrow, flat.3. The immunolocalization of FSHR and LHR in ovar ies of Ganjia Tibetan Sheep in different stages of estrus cycle(proestrus,estrus,metestrus,diestrus) are basically same.The FSHR and LHR-positive targets were mainly seen in the oocyte, follicle cells of primordial follicles and primary follicles, oocyte, granulosa cells, follicular theca cells of secondary follicle, tertiary follicle and mature follicles, and luteal cells. In addition, it is also expressed in the vascular interstitium and ovarian stromal cel s.4. Applicaed the computer image analysis and found that the FSHR positive cells acerage optical density in the ovary of Ganjia Tibetan sheep in proestrus, estrus, metestrus and diestrus were respectively 0.0616±0.0014, 0.0622±0.0013, 0.0469±0.0099, 0.0473±0.0126.The average optical density of FSHR in estrus was the highest and the lowest in meterus.The FSH expression in proestrus and estrus werw signigicantly higher than that in diestrus(P<0.05).The LHR positive cells average optical density in the ovary of Ganjia Tibetan sheep in proestrus,estrus,metestrus and diestrus were respectively 0.0348±0.0099, 0.0430±0.0062, 0.0575±0.0100, 0.0459±0.0078.The average optical density of LHR was the highest in metestrus and the lowest in proestrus. The LHR average optical density in metestrus was very significantly higher than that in proestrus and estrus(P<0.01).And the LHR expression in diestrus was very significantly higher than that in proestrus(P<0.01).The results provide a test data including molecular and cellular level, for further research of follicle effection and reproductive performance in Ganjia Tibetan sheep at different developmental stages. And provide a scientific basis for studying the regulatory mechanism of the female mammal estrus, ovulation and other reproductive endocrine regulation of the activities, animal breeding and artigicial incressing production performance.