胰岛素和FSH对体外培养猪卵泡颗粒细胞雌激素的影响
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摘要
旨在研究胰岛素(Insulin)和促卵泡素(Follicular stimulating hormone,FSH)对猪卵泡发育过程的影响。猪屠宰后收集卵巢,选择健康卵泡并分离颗粒细胞(Granulesa cells,GCs),设定对照组和重复组,胰岛素、FSH浓度梯度下Long-term体外培养,7d后显微镜观察细胞增殖情况并计数,竞争法测定雌激素(E_2)浓度。数据分析表明:当FSH为0ng·mL~(-1)时,随着胰岛素浓度的增加,猪卵泡GCs细胞密度增加,细胞计数显示GCs数量呈上升趋势,且胰岛素浓度为100ng·mL~(-1)时,细胞数量最高(P<0.05);当胰岛素为0ng·mL~(-1)时,随着FSH浓度的增加,细胞密度增加,细胞计数显示GCs数量呈上升趋势,且FSH浓度为5和25ng·mL~(-1)时,细胞数量显著高于其它两组(P<0.05);E_2测定结果显示,当FSH为0ng·mL~(-1),胰岛素为100ng·mL~(-1)时,培养液E_2浓度显著高于他不同浓度胰岛素组(P<0.05);当FSH为1ng·mL~(-1),胰岛素为10ng·mL~(-1)时,培养液E2浓度最高,但与不同浓度胰岛素各组差异不显著(P>0.05);FSH浓度为5和25ng·mL~(-1)时,不同浓度胰岛素各组之间E_2浓度差异不显著。猪卵泡发育过程中,胰岛素和FSH均有促颗粒细胞增殖和E_2分泌的能力,FSH超过一定生理浓度会降低卵泡颗粒细胞分泌E_2。
To research the effects of insulin and FSH on porcine ovarian follicular development,ovaries were collected and follicular granulosa cells(GCs)were isolated.Setting control groups and repetitive groups,GCs were cultured for 7 dtreated with different concentrations of insulin and FSH by long-term culture in vitro.Observing the growth situation and counting the numbers of GCs in each well.Competition method was performed to detect the E_2 concentrations in medium.Results showed that,at 0 ng·mL~(-1) concentration of FSH in medium,with the increase ofinsulin concentration,GCs proliferation was more and the number of GCs showed a tendency to increase.The numbers of GCs were the most when the supplement dose of insulin was 100 ng·mL~(-1)(P<0.05).When insulin was 0 ng·mL~(-1),with the increase of FSH concentration,GCs proliferation was more and the number of GCs showed a tendency to increase,and at 5 or 25 ng·mL~(-1) concentration of FSH,the number of GCs were more than other 2 groups(P<0.05).When FSH at 0 ng·mL~(-1),insulin at 100 ng·mL~(-1),E_2 concentration was more than other groups with different concentration of insulin(P<0.05).When insulin was 10 ng·mL~(-1) and FSH was1 ng·mL~(-1),E_2 concentration in the medium was the greatest,but there was no significant differences among groups with different concentrations of insulin(P<0.05).At 5 or 25 ng·mL~(-1) concentration of FSH,there was no significant differences in E_2 concentration among different groups with different concentrations of insulin.Insulin in cooperation with FSH promotes the proliferation and E_2 production of porcine follicular GCs.The E_2 secretion of porcine follicular GCs will decline when FSH exceeded a certain physiological concentration.
To research the effects of insulin and FSH on porcine ovarian follicular development,ovaries were collected and follicular granulosa cells(GCs)were isolated.Setting control groups and repetitive groups,GCs were cultured for 7 dtreated with different concentrations of insulin and FSH by long-term culture in vitro.Observing the growth situation and counting the numbers of GCs in each well.Competition method was performed to detect the E_2 concentrations in medium.Results showed that,at 0 ng·mL~(-1) concentration of FSH in medium,with the increase ofinsulin concentration,GCs proliferation was more and the number of GCs showed a tendency to increase.The numbers of GCs were the most when the supplement dose of insulin was 100 ng·mL~(-1)(P<0.05).When insulin was 0 ng·mL~(-1),with the increase of FSH concentration,GCs proliferation was more and the number of GCs showed a tendency to increase,and at 5 or 25 ng·mL~(-1) concentration of FSH,the number of GCs were more than other 2 groups(P<0.05).When FSH at 0 ng·mL~(-1),insulin at 100 ng·mL~(-1),E_2 concentration was more than other groups with different concentration of insulin(P<0.05).When insulin was 10 ng·mL~(-1) and FSH was1 ng·mL~(-1),E_2 concentration in the medium was the greatest,but there was no significant differences among groups with different concentrations of insulin(P<0.05).At 5 or 25 ng·mL~(-1) concentration of FSH,there was no significant differences in E_2 concentration among different groups with different concentrations of insulin.Insulin in cooperation with FSH promotes the proliferation and E_2 production of porcine follicular GCs.The E_2 secretion of porcine follicular GCs will decline when FSH exceeded a certain physiological concentration.
引文
[1]LV L H,YUE W B,LIU W Z,et al.Effects of sperm capacitation treatments and in vitro culture systems on development of in vitro fertilized embryos derived from prepubertal boer goat oocytes[J].Asian-Austral J Anim Sci,2009,22(7):969-976.
[2]LV L H,YUE W B,LIU W Z,et al.Effect of oocyte selection,estradiol and antioxidant treatment on in vitro maturation of oocytes collected from prepubertal Boer goats[J].Ital J Anim Sci,2010,9(1):e11.
[3]GINTHER O J,BASHIR S T,RAKESH H B,et al.Two-way coupling between FSH and the dominant follicle in heifers[J].Theriogenology,2013,80(5):463-469.
[4]BAERWALD A R,ADAMS G P,PIERSON R A.Characterization of ovarian follicular wave dynamics in women[J].Biol Reprod,2003,69(3):1023-1031.
[5]BOZAN E,ANDRONOWSKA A,BODEK G,et al.The novel effect of hCG administration on luteal function maintenance during the estrous cycle/pregnancy and early embryo development in the pig[J].Pol J Vet Sci,2013,16(2):323-332.
[6]李鹏飞,岳文斌,庞钰莹,等.FSH和胰岛素对绵羊卵巢卵泡颗粒细胞体外培养的影响[J].畜牧兽医学报,2013,44(9):1386-1391.LI P F,YUE W B,PANG Y Y,et al.Effects of FSH and insulin on sheep ovarian follicular granulosa cells in vitro culture[J].Acta Veterinaria et Zootechnica Sinica,2013,44(9):1386-1391.(in Chinese)
[7]TORNER H,BRSSOW K P,ALM H,et al.Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation[J].Theriogenology,2004,61(9):1675-1689.
[8]EPPIG J J.Oocyte control of ovarian follicular development and function in mammals[J].Reproduction,2001,122(6):829-838.
[9]JUENGEL J L,HUDSON N L,HEATH D A,et al.Growth differentiation factor 9and bone morphogenetic protein 15are essential for ovarian follicular development in sheep[J].Biol Reprod,2002,67(6):1777-1789.
[10]SMITH S M,VAUGHAN J M,DONALDSON C J,et al.Cocaine-and amphetamine-regulated transcript activates the hypothalamic-pituitary-adrenal axis through a corticotropin-releasing factor receptor-dependent mechanism[J].Endocrinology,2004,145(11):5202-5209.
[11]李鹏飞,岳文斌,黄洋,等.可卡因-苯丙胺调控转录肽对绵羊卵巢卵泡颗粒细胞雌激素产生的影响[J].畜牧兽医学报,2013,44(6):853-857.LI P F,YUE W B,HUANG Y,et al.Effects of CART on estradiol production in vitro in follicular granulose cells of sheep ovarian[J].Acta Veterinaria et Zootechnica Sinica,2013,44(6):853-857.(in Chinese)
[12]李鹏飞,岳文斌,李富禄,等.可卡因-苯丙胺调控转录肽(CART)对猪卵巢卵泡颗粒细胞雌激素产生的影响[J].畜牧兽医学报,2012,43(12):1879-1886.LI P F,YUE W B,LI F L,et al.Effects of CART on estradiol production of pig ovarian follicular granulosa cells in vitro culture[J].Acta Veterinaria et Zootechnica Sinica,2012,43(12):1879-1886.(in Chinese)
[13]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.
[14]IRELAND J J,ROCHE J F.Development of nonovulatory antral follicles in heifers:changes in steroids in follicular fluid and receptors for gonadotropins[J].Endocrinology,1983,112(1):150-156.
[15]IRELAND J J,ROCHE J F.Development of antral follicles in cattle after prostaglandin-induced luteolysis:changes in serum hormones,steroids in follicular fluid,and gonadotropin receptors[J].Endocrinology,1982,111(6):2077-2086.
[16]IRELAND J J,ROCHE J F.Growth and differentiation of large antral follicles after spontaneous luteolysis in heifers:changes in concentration of hormones in follicular fluid and specific binding of gonadotropins to follicles[J].J Anim Sci,1983,57(1):157-167.
[17]ILHA G F,ROVANI M T,GASPERIN B G,et al.Lack of FSH support enhances LIF-STAT3signaling in granulosa cells of atretic follicles in cattle[J].Reproduction,2015,150(4):395-403.
[18]GARCA GUERRA A,TRIBULO A,YAPURA J,et al.Lengthened superstimulatory treatment in cattle:evidence for rescue of follicles within a wave rather than continuous recruitment of new follicles[J].Theriogenology,2015,84(3):467-476.
[19]SCHAMS D.Luteal peptides and intercellular communication[J].J Reprod Fertil,1987,34:87-99.
[20]MIHM M,AUSTIN E J,GOOD T E M,et al.Identification of potential intrafollicular factors involved in selection of dominant follicles in heifers[J].Biol Reprod,2000,63(3):811-819.
[21]YUAN W,BAO B,GARVERICK H A,et al.Follicular dominance in cattle is associated with divergent patterns of ovarian gene expression for insulinlike growth factor(IGF)-I,IGF-II,and IGF binding protein-2in dominant and subordinate follicles[J].Domest Anim Endocrinol,1998,15(1):55-63.
[22]ATANASOV B,DE KOSTER J,BOMMELL,et al.Pathways of the dominant follicle after exposure to sub-luteal circulating progesterone concentrations are different in lactating dairy cows versus non-lactating heifers[J].Anim Reprod Sci,2015,154:8-15.
[23]SILVA FILHO M L,BEZERRA L R,FERREIRASILVA J C,et al.Influence of biostimulation and temporary weaning on follicular dynamics and pregnancy rates in Nelore cows(Bos taurus indicus)[J].Trop Anim Health Prod,2015,47(7):1285-1291.
[24]RAK A,DRWAL E,KARPETA A,et al.Regulatory role of gonadotropins and local factors produced by ovarian follicles on in vitro resistin expression and action on porcine follicular steroidogenesis[J].Biol Reprod,2015,92(6):142,doi:10.1095/biolreprod.115.128611.
[25]SEN A,LL H,BELLO N,et al.Cocaine-and amphetamine-regulated transcript accelerates termination of follicle-stimulating hormone-induced extracellularly regulated kinase 1/2and Akt activation by regulating the expression and degradation of specific mitogen-activated protein kinase phosphatases in bovine granulosa cells[J].Mol Endocrinol,2008,22(12):2655-2676.
[26]LV L H,JIMENEZ-KEASSEL F,SEN A,et al.Evidence supporting a role for cocaine-and amphetamine-regulated transcript(CARTPT)in control of granulosa cell estradiol production associated with dominant follicle selection in cattle[J].Biol Reprod,2009,81(3):580-586.
[27]HUGHES F M Jr,GOROSPE W C.Biochemical identification of apoptosis(programmed cell death)in granulosa cells:evidence for a potential mechanism underlying follicular atresia[J].Endocrinology,1991,129(5):2415-2422.
[28]SILVA J M,PRICE C A.Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450aromatase and cholesterol side-chain cleavage in bovine granulosa cells in vitro[J].Biol Reprod,2000,62(1):186-191.
[29]RAMAN R S,CHAN P J,CORSELLI J U,et al.Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection[J].Hum Reprod,2001,16(5):831-835.
[30]SHORES E M,PICTON H M,HUNTER M G.Differential regulation of pig theca cell steroidogenesis by LH,insulin-like growth factor I and granulosa cells in serum-free culture[J].J Reprod Fertil,2000,118(2):211-219.
[2]LV L H,YUE W B,LIU W Z,et al.Effect of oocyte selection,estradiol and antioxidant treatment on in vitro maturation of oocytes collected from prepubertal Boer goats[J].Ital J Anim Sci,2010,9(1):e11.
[3]GINTHER O J,BASHIR S T,RAKESH H B,et al.Two-way coupling between FSH and the dominant follicle in heifers[J].Theriogenology,2013,80(5):463-469.
[4]BAERWALD A R,ADAMS G P,PIERSON R A.Characterization of ovarian follicular wave dynamics in women[J].Biol Reprod,2003,69(3):1023-1031.
[5]BOZAN E,ANDRONOWSKA A,BODEK G,et al.The novel effect of hCG administration on luteal function maintenance during the estrous cycle/pregnancy and early embryo development in the pig[J].Pol J Vet Sci,2013,16(2):323-332.
[6]李鹏飞,岳文斌,庞钰莹,等.FSH和胰岛素对绵羊卵巢卵泡颗粒细胞体外培养的影响[J].畜牧兽医学报,2013,44(9):1386-1391.LI P F,YUE W B,PANG Y Y,et al.Effects of FSH and insulin on sheep ovarian follicular granulosa cells in vitro culture[J].Acta Veterinaria et Zootechnica Sinica,2013,44(9):1386-1391.(in Chinese)
[7]TORNER H,BRSSOW K P,ALM H,et al.Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation[J].Theriogenology,2004,61(9):1675-1689.
[8]EPPIG J J.Oocyte control of ovarian follicular development and function in mammals[J].Reproduction,2001,122(6):829-838.
[9]JUENGEL J L,HUDSON N L,HEATH D A,et al.Growth differentiation factor 9and bone morphogenetic protein 15are essential for ovarian follicular development in sheep[J].Biol Reprod,2002,67(6):1777-1789.
[10]SMITH S M,VAUGHAN J M,DONALDSON C J,et al.Cocaine-and amphetamine-regulated transcript activates the hypothalamic-pituitary-adrenal axis through a corticotropin-releasing factor receptor-dependent mechanism[J].Endocrinology,2004,145(11):5202-5209.
[11]李鹏飞,岳文斌,黄洋,等.可卡因-苯丙胺调控转录肽对绵羊卵巢卵泡颗粒细胞雌激素产生的影响[J].畜牧兽医学报,2013,44(6):853-857.LI P F,YUE W B,HUANG Y,et al.Effects of CART on estradiol production in vitro in follicular granulose cells of sheep ovarian[J].Acta Veterinaria et Zootechnica Sinica,2013,44(6):853-857.(in Chinese)
[12]李鹏飞,岳文斌,李富禄,等.可卡因-苯丙胺调控转录肽(CART)对猪卵巢卵泡颗粒细胞雌激素产生的影响[J].畜牧兽医学报,2012,43(12):1879-1886.LI P F,YUE W B,LI F L,et al.Effects of CART on estradiol production of pig ovarian follicular granulosa cells in vitro culture[J].Acta Veterinaria et Zootechnica Sinica,2012,43(12):1879-1886.(in Chinese)
[13]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.
[14]IRELAND J J,ROCHE J F.Development of nonovulatory antral follicles in heifers:changes in steroids in follicular fluid and receptors for gonadotropins[J].Endocrinology,1983,112(1):150-156.
[15]IRELAND J J,ROCHE J F.Development of antral follicles in cattle after prostaglandin-induced luteolysis:changes in serum hormones,steroids in follicular fluid,and gonadotropin receptors[J].Endocrinology,1982,111(6):2077-2086.
[16]IRELAND J J,ROCHE J F.Growth and differentiation of large antral follicles after spontaneous luteolysis in heifers:changes in concentration of hormones in follicular fluid and specific binding of gonadotropins to follicles[J].J Anim Sci,1983,57(1):157-167.
[17]ILHA G F,ROVANI M T,GASPERIN B G,et al.Lack of FSH support enhances LIF-STAT3signaling in granulosa cells of atretic follicles in cattle[J].Reproduction,2015,150(4):395-403.
[18]GARCA GUERRA A,TRIBULO A,YAPURA J,et al.Lengthened superstimulatory treatment in cattle:evidence for rescue of follicles within a wave rather than continuous recruitment of new follicles[J].Theriogenology,2015,84(3):467-476.
[19]SCHAMS D.Luteal peptides and intercellular communication[J].J Reprod Fertil,1987,34:87-99.
[20]MIHM M,AUSTIN E J,GOOD T E M,et al.Identification of potential intrafollicular factors involved in selection of dominant follicles in heifers[J].Biol Reprod,2000,63(3):811-819.
[21]YUAN W,BAO B,GARVERICK H A,et al.Follicular dominance in cattle is associated with divergent patterns of ovarian gene expression for insulinlike growth factor(IGF)-I,IGF-II,and IGF binding protein-2in dominant and subordinate follicles[J].Domest Anim Endocrinol,1998,15(1):55-63.
[22]ATANASOV B,DE KOSTER J,BOMMELL,et al.Pathways of the dominant follicle after exposure to sub-luteal circulating progesterone concentrations are different in lactating dairy cows versus non-lactating heifers[J].Anim Reprod Sci,2015,154:8-15.
[23]SILVA FILHO M L,BEZERRA L R,FERREIRASILVA J C,et al.Influence of biostimulation and temporary weaning on follicular dynamics and pregnancy rates in Nelore cows(Bos taurus indicus)[J].Trop Anim Health Prod,2015,47(7):1285-1291.
[24]RAK A,DRWAL E,KARPETA A,et al.Regulatory role of gonadotropins and local factors produced by ovarian follicles on in vitro resistin expression and action on porcine follicular steroidogenesis[J].Biol Reprod,2015,92(6):142,doi:10.1095/biolreprod.115.128611.
[25]SEN A,LL H,BELLO N,et al.Cocaine-and amphetamine-regulated transcript accelerates termination of follicle-stimulating hormone-induced extracellularly regulated kinase 1/2and Akt activation by regulating the expression and degradation of specific mitogen-activated protein kinase phosphatases in bovine granulosa cells[J].Mol Endocrinol,2008,22(12):2655-2676.
[26]LV L H,JIMENEZ-KEASSEL F,SEN A,et al.Evidence supporting a role for cocaine-and amphetamine-regulated transcript(CARTPT)in control of granulosa cell estradiol production associated with dominant follicle selection in cattle[J].Biol Reprod,2009,81(3):580-586.
[27]HUGHES F M Jr,GOROSPE W C.Biochemical identification of apoptosis(programmed cell death)in granulosa cells:evidence for a potential mechanism underlying follicular atresia[J].Endocrinology,1991,129(5):2415-2422.
[28]SILVA J M,PRICE C A.Effect of follicle-stimulating hormone on steroid secretion and messenger ribonucleic acids encoding cytochromes P450aromatase and cholesterol side-chain cleavage in bovine granulosa cells in vitro[J].Biol Reprod,2000,62(1):186-191.
[29]RAMAN R S,CHAN P J,CORSELLI J U,et al.Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection[J].Hum Reprod,2001,16(5):831-835.
[30]SHORES E M,PICTON H M,HUNTER M G.Differential regulation of pig theca cell steroidogenesis by LH,insulin-like growth factor I and granulosa cells in serum-free culture[J].J Reprod Fertil,2000,118(2):211-219.