牛卵泡颗粒细胞PRSS35表达模式及功能研究
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摘要
旨在探究丝氨酸蛋白酶35(serine protease 35,PRSS35)在牛卵泡颗粒细胞(granulosa cells,GCs)中的表达及功能。采用B超超声波检测确定牛优势卵泡(dominant follicle,DF)与从属卵泡(subordinate follicles,SF),分离优势卵泡与从属卵泡颗粒细胞;利用qRT-PCR和Western blotting技术检测PRSS35在DF与SF的表达情况;免疫组化技术对PRSS35进行定位研究;设计PRSS35siRNA基因沉默序列,经脂质体转染GCs,测定培养液雌激素(estrogen,E_2)浓度和GCs凋亡情况,研究PRSS35对牛卵泡颗粒细胞生长发育的影响。结果表明:1)PRSS35mRNA在DF中的表达量极显著高于SF(P<0.001);2)PRSS35在牛DF和SF颗粒层与膜层均有表达;3)PRSS35在DF中的表达量极显著高于SF(P<0.01);4)PRSS35基因沉默后,GCs凋亡细胞百分率极显著增高(P<0.001),培养液雌激素浓度极显著降低(P<0.01)。综上表明,PRSS35在牛卵泡颗粒层和膜层均有表达,且在DF表达量极显著高于SF;PRSS35基因沉默后,通过抑制GCs增殖,降低E_2分泌浓度,从而抑制牛卵泡的生长发育。该研究为深入探讨PRSS35与牛卵泡发育的关系奠定基础。
The aim of this study was to explore the expression profile and analyze the function of serine protease 35(PRSS35)in bovine follicular granulosa cells.Cattle dominant follicles(DF)and subordinate follicles(SF)were separated according their sizes measured using B-mode ultrasonography technology.The expression profile of PRSS35 was analyzed using qRT-PCR and Western blotting,and its location in DF and SF was detected by immunohistochemistry.The siRNA sequences of PRSS35 were designed and synthesized and PRSS35-gene-silenced GCs line was obtained.Estrogen(E_2)concentration and cell apoptosis were detected through studying the effects of PRSS35 on growth and development of bovine follicular GCs.The results showed that:1)The expression of PRSS35 mRNA in DF was significantly higher than that in SF(P<0.001).2)The PRSS35 was expressed both in DF and SF granular layer and theca layer.3)The expression of RSS35 protein in DF was significantly higher than that in SF(P<0.01).4)When PRSS35 gene was silenced,the percentage of apoptotic GCs was significantly increased(P<0.001),and the concentration of E_2 in cell culture media was significantly decreased(P<0.01).The results suggested that PRSS35 was expressed both in bovine follicular granular layer and thecalayer,and the PRSS35 expression was significantly higher in DF than that in SF.The proliferation of GCs was inhibited,and the concentration of E_2 was reduced when PRSS35 gene was silenced,indicating the growth and development of bovine follicles were inhibited.This study provides a basis for further studying the relationship between PRSS35 and follicular development in cattle.
The aim of this study was to explore the expression profile and analyze the function of serine protease 35(PRSS35)in bovine follicular granulosa cells.Cattle dominant follicles(DF)and subordinate follicles(SF)were separated according their sizes measured using B-mode ultrasonography technology.The expression profile of PRSS35 was analyzed using qRT-PCR and Western blotting,and its location in DF and SF was detected by immunohistochemistry.The siRNA sequences of PRSS35 were designed and synthesized and PRSS35-gene-silenced GCs line was obtained.Estrogen(E_2)concentration and cell apoptosis were detected through studying the effects of PRSS35 on growth and development of bovine follicular GCs.The results showed that:1)The expression of PRSS35 mRNA in DF was significantly higher than that in SF(P<0.001).2)The PRSS35 was expressed both in DF and SF granular layer and theca layer.3)The expression of RSS35 protein in DF was significantly higher than that in SF(P<0.01).4)When PRSS35 gene was silenced,the percentage of apoptotic GCs was significantly increased(P<0.001),and the concentration of E_2 in cell culture media was significantly decreased(P<0.01).The results suggested that PRSS35 was expressed both in bovine follicular granular layer and thecalayer,and the PRSS35 expression was significantly higher in DF than that in SF.The proliferation of GCs was inhibited,and the concentration of E_2 was reduced when PRSS35 gene was silenced,indicating the growth and development of bovine follicles were inhibited.This study provides a basis for further studying the relationship between PRSS35 and follicular development in cattle.
引文
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[11] LI S H,LIN M H,HWU Y M,et al.Correlation of cumulus gene expression of GJA1,PRSS35,PTX3,and SERPINE2with oocyte maturation,fertilization,and embryo development[J].Reprod Biol Endocrinol,2015,13(1):93.
[12] LETRA A,MENEZES R,FONSECA R F,et al.Novel cleft susceptibility genes in chromosome 6p[J].J Dent Res,2010,89(9):927-932.
[13] LEBLEU V S,TENG Y Q,O'CONNELL J T,et al.Identification of human epididymis protein-4as a fibroblast-derived mediator of fibrosis[J].Nat Med,2013,19(2):227-231.
[14] REYNOLDS A,LEAKE D,BOESE Q,et al.Rational siRNA design for RNA interference[J].Nat Biotechnol,2004,22(3):326-330.
[15] VELAZQUEZ M A,SPICER L J,WATHES D C.The role of endocrine insulin-like growth factor-I(IGF-I)in female bovine reproduction[J].Domest Anim Endocrinol,2008,35(4):325-342.
[16] DROST M.Bubaline versus bovine reproduction[J].Theriogenology,2007,68(3):447-449.
[17] SUNDERLAND S J,CROWE M A,BOLAND M P,et al.Selection,dominance and atresia of follicles during the oestrous cycle of heifers[J].J Reprod Fertil,1994,101(3):547-555.
[18] MANABE N,GOTO Y,MATSUDA-MINEHATA F,et al.Regulation mechanism of selective atresia in porcine follicles:regulation of granulosa cell apoptosis during atresia[J].J Reprod Dev,2004,50(5):493-514.
[19] GINTHER O J.Selection of the dominant follicle in cattle and horses[J].Anim Reprod Sci,2000,60-61:61-79.
[20] KOBAYASHI Y,JIMENEZ-KRASSEL F,IRELAND J J,et al.Evidence of a local negative role for cocaine and amphetamine regulated transcript(CART),inhibins and low molecular weight insulin like growth factor binding proteins in regulation of granulosa cell estradiol production during follicular waves in cattle[J].Reprod Biol Endocrinol,2006,4:22.
[21] GINTHER O J,KOT K,KULICK L J,et al.Sampling follicular fluid without altering follicular status in cattle:oestradiol concentrations early in a follicular wave[J].J Reprod Fertil,1997,109(2):181-186.
[22] SARTORI R,FRICKE P M,FERREIRA J C,et al.Follicular deviation and acquisition of ovulatory capacity in bovine follicles[J].Biol Reprod,2001,65(5):1403-1409.
[23] FORTUNE J E.Ovarian follicular growth and development in mammals[J].Biol Reprod,1994,50(2):225-232.
[24] SHIMIZU T,KOSAKA N,MURAYAMA C,et al.Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary:involvement of apoptosis and hormonal regulation[J].Anim Reprod Sci,2009,116(1-2):28-37.
[25]李鹏飞,孟金柱,郝庆玲,等.胰岛素和FSH对体外培养猪卵泡颗粒细胞雌激素的影响[J].畜牧兽医学报,2017,48(11):2084-2090.LI P F,MENG J Z,HAO Q L,et al.Effects of insulin and FSH on E2 production of pig ovarian follicular granulosa cells in vitro culture[J].Acta Veterinaria et Zootechnica Sinica,2017,48(11):2084-2090.(in Chinese)
[2] FRICKE P M,AL-HASSAN M J,ROBERTS A J,et al.Effect of gonadotropin treatment on size,number,and cell proliferation of antral follicles in cows[J].Domest Anim Endocrinol,1997,14(3):171-180.
[3] SHELDON I M,NOAKES D E,RYCROFT A N,et al.Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle[J].Reproduction,2002,123(6):837-845.
[4] ARMSTRONG D G,GUTIERREZ C G,BAXTER G,et al.Expression of mRNA encoding IGF-I,IGF-II and type 1IGF receptor in bovine ovarian follicles[J].J Endocrinol,2000,165(1):101-113.
[5] SEN A,BETTEGOWDA A,JIMENEZ-KRASSEL F,et al.Cocaine-and amphetamine-regulated transcript regulation of follicle-stimulating hormone signal transduction in bovine granulosa cells[J].Endocrinology,2007,148(9):4400-4410.
[6]李鹏飞,孟金柱,郝庆玲,等.PDF2和ODF1转录组测序筛选牛卵泡发育相关基因[J].畜牧兽医学报,2018,49(2):300-309.LI P F,MENG J Z,HAO Q L,et al.Screening and analysing of genes associated with follicular development in bovine ODF1and PDF2transcriptome[J].Acta Veterinaria et Zootechnica Sinica,2018,49(2):300-309.(in Chinese)
[7] DIAO H L,XIAO S,LI R,et al.Distinct spatiotemporal expression of serine proteases prss23and prss35in periimplantation mouse uterus and dispensable function of prss35 in fertility[J].PLoS One,2013,8(2):e56757.
[8] MIYAKOSHI K,MURPHY M J,YEOMAN R R,et al.The identification of novel ovarian proteases through the use of genomic and bioinformatic methodologies[J].Biol Reprod,2006,75(6):823-835.
[9] GOES F S,HAMSHERE M L,SEIFUDDIN F,et al.Genome-wide association of mood-incongruent psychotic bipolar disorder[J].Transl Psych,2012,2(10):e180.
[10] WAHLBERG P,NYLANDERA,AHLSKOG N,et al.Expression and localization of the serine proteases high-temperature requirement factor A1,Serine Protease 23,and Serine Protease 35in the mouse ovary[J].Endocrinology,2008,149(10):5070-5077.
[11] LI S H,LIN M H,HWU Y M,et al.Correlation of cumulus gene expression of GJA1,PRSS35,PTX3,and SERPINE2with oocyte maturation,fertilization,and embryo development[J].Reprod Biol Endocrinol,2015,13(1):93.
[12] LETRA A,MENEZES R,FONSECA R F,et al.Novel cleft susceptibility genes in chromosome 6p[J].J Dent Res,2010,89(9):927-932.
[13] LEBLEU V S,TENG Y Q,O'CONNELL J T,et al.Identification of human epididymis protein-4as a fibroblast-derived mediator of fibrosis[J].Nat Med,2013,19(2):227-231.
[14] REYNOLDS A,LEAKE D,BOESE Q,et al.Rational siRNA design for RNA interference[J].Nat Biotechnol,2004,22(3):326-330.
[15] VELAZQUEZ M A,SPICER L J,WATHES D C.The role of endocrine insulin-like growth factor-I(IGF-I)in female bovine reproduction[J].Domest Anim Endocrinol,2008,35(4):325-342.
[16] DROST M.Bubaline versus bovine reproduction[J].Theriogenology,2007,68(3):447-449.
[17] SUNDERLAND S J,CROWE M A,BOLAND M P,et al.Selection,dominance and atresia of follicles during the oestrous cycle of heifers[J].J Reprod Fertil,1994,101(3):547-555.
[18] MANABE N,GOTO Y,MATSUDA-MINEHATA F,et al.Regulation mechanism of selective atresia in porcine follicles:regulation of granulosa cell apoptosis during atresia[J].J Reprod Dev,2004,50(5):493-514.
[19] GINTHER O J.Selection of the dominant follicle in cattle and horses[J].Anim Reprod Sci,2000,60-61:61-79.
[20] KOBAYASHI Y,JIMENEZ-KRASSEL F,IRELAND J J,et al.Evidence of a local negative role for cocaine and amphetamine regulated transcript(CART),inhibins and low molecular weight insulin like growth factor binding proteins in regulation of granulosa cell estradiol production during follicular waves in cattle[J].Reprod Biol Endocrinol,2006,4:22.
[21] GINTHER O J,KOT K,KULICK L J,et al.Sampling follicular fluid without altering follicular status in cattle:oestradiol concentrations early in a follicular wave[J].J Reprod Fertil,1997,109(2):181-186.
[22] SARTORI R,FRICKE P M,FERREIRA J C,et al.Follicular deviation and acquisition of ovulatory capacity in bovine follicles[J].Biol Reprod,2001,65(5):1403-1409.
[23] FORTUNE J E.Ovarian follicular growth and development in mammals[J].Biol Reprod,1994,50(2):225-232.
[24] SHIMIZU T,KOSAKA N,MURAYAMA C,et al.Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary:involvement of apoptosis and hormonal regulation[J].Anim Reprod Sci,2009,116(1-2):28-37.
[25]李鹏飞,孟金柱,郝庆玲,等.胰岛素和FSH对体外培养猪卵泡颗粒细胞雌激素的影响[J].畜牧兽医学报,2017,48(11):2084-2090.LI P F,MENG J Z,HAO Q L,et al.Effects of insulin and FSH on E2 production of pig ovarian follicular granulosa cells in vitro culture[J].Acta Veterinaria et Zootechnica Sinica,2017,48(11):2084-2090.(in Chinese)