In order to investigate the mechanism of progestin and antiprogestin in the regulation of ovarian steroidogenesis, a dual chamber culture system was prepared with the amnion membrane of human placenta. Isolated porci...In order to investigate the mechanism of progestin and antiprogestin in the regulation of ovarian steroidogenesis, a dual chamber culture system was prepared with the amnion membrane of human placenta. Isolated porcine granulosa and thecal cells from 4~6 mm diameter follicles were grown on both sides of the amnion, respectively, and co cultured with or without LNG and RU486. After 48 h incubation, the mRNAs of FSH receptor (FSH R) and LH receptor (LH R) of both cells were observed by in situ hybridization. The results showed that granulosa cells expressed both FSH R mRNA and LH R mRNA, while thecal cells expressed LH R mRNA only. Under the stimulation of FSH, both LNG and RU486 increased FSH R mRNA expression of granulosa cells. Under the stimulation of LH, LNG enhanced LH R mRNA expression of thecal cells; while RU486 decreased its expression. When granulosa and thecal cells were exposed to FSH and LH both, the actions of LNG and RU486 in thecal cells showed the same result as that stimulated by LH alone. In granulosa cells LNG decreased LH R mRNA expression, while RU486 increased its expression. These data suggest that: (1) granulosa cells expressed FSH R mRNA significantly; (2) both the progestin and antiprogestin directly acted on the mRNA expression of gonadotropin receptors of ovarian cells, but effects were different; (3) the response of granulosa or thecal cells to the action of LNG and RU486 was not the same. The mechanism needs to be further investigated.展开更多
The functional significance of the endometrial hCG/ LH receptors has been related to a rapid release of prostaglandins. However, as compared to gonads and myometrium, in-endometrium mechanisms of transmembrane signall...The functional significance of the endometrial hCG/ LH receptors has been related to a rapid release of prostaglandins. However, as compared to gonads and myometrium, in-endometrium mechanisms of transmembrane signalling of the hCG/LH receptors are probably not conventional and remain unclear. Here we investigated, in vivo, the potential of hCG to interact with, and stimulate the membrane effector enzyme, adenylyl cyclase (AC), in human endometrium. Hormonal and nonhormonal activation of AC was tested in membrane fractions prepared from endometrial biopsies obtained from patients undergoing evaluation cycles for hormone replacement therapy (HRT) and controlled ovarian hyperstimulation (COH). AC activity was determined by the direct conversion of the substrate ATP into cAMP under unstimulated conditions and in the presence of the non-hormonal activators guanyl nucleotide and forskolin. Also AC activity was tested in the presence of hCG under conditions allowing maximal enzyme stimulation. Isoproterenol and prostaglandin E2 (PGE2) were included for comparison. Immunoblot analyses demonstrated the presence of hCG/LH receptors and Gsα protein and other members of the G protein family in the membrane fractions. Endometrial membranes also exhibited high levels of AC activity compared to luteal membranes used as control. Stimulation by GMP-P(NH)P alone was 196 ± 63 (n = 8) (pmol/mg/ min ± SD). Neither hCG nor isoproterenol showed stimulation of endometrial AC (210 ± 65, and 197 ± 53, respectively;n = 66 assays). But PGE2 stimulated the enzyme system significantly (264 ± 63, p < 0.05;n = 66 assays). These data show that membrane fractions from human endometrium express all the AC system components, namely, hCG/LH receptors, Gsα protein and AC;however, hCG does not stimulate the endometrial AC system. Our data indicate that, in great contrast to gonadal receptors, endometrial hCG/ LH receptors are not coupled to the transmembrane AC effector. The well known release of eicosanoids in response to hCG suggests that these receptors are functional in human endometrium but throughout a signalling system different from AC. This enzyme is certainly coupled to and directly activated by eicosanoids and other embryonic signals.展开更多
By using DNA 3'-end labeling, immunocytochemistry and mRNA in situ hybridization detection techniques , the expression of inhibin subunits and LH receptor in the granulosa cells and tissue-type plasminogen activat...By using DNA 3'-end labeling, immunocytochemistry and mRNA in situ hybridization detection techniques , the expression of inhibin subunits and LH receptor in the granulosa cells and tissue-type plasminogen activator (tPA) in the oocytes has been studied in relation to follicular development and atresia. The results demonstrated that: (i) tPA activity in the oocytes of normal developing follicles is undetectable, and increases significantly in the follicle undergoing atresia; (ii) the production of inhibin subunits in granulosa cells is negatively correlated with the expression of oocyte tPA activity, indicating that they may be an important regulator of oocyte tPA production and follicular development; (iii) in atretic follicles, granulosa cells do not express LH receptor and inhibin subunits. It is therefore suggested that tPA may play a role in oocyte self-destruction and clearance in some of atretic follicles, and inhibin of granulosa-origin might be an inhibitory factor for the translation of tPA in the oocyte.展开更多
Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare ...Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in preovulatory follicles, IGF-I stimulated LHR mRNA expression. These results show that the interaction between ECF and IGF-I may be involved in the regulation of atresia of follicles at different stages of development.展开更多
Relationship between insulin-like growth factor-l receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL) . In the CL of estrous cy...Relationship between insulin-like growth factor-l receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL) . In the CL of estrous cycle rat, LHR mRNA positive CL expressed high level of mRNA of IGF-IR. While the expression of LHR mRNA decreased on estrus, the CL still expressed relatively high level of IGF-IR mRNA. In pseudopregnant rat CL, the expression level of LHR mRNA was low on day 1, the most intense signals were detected on day 8, the signals of LHR mRNA became undetectable on day 14. In contrast to LHR expression, the high level of IGF-IR mRNA was observed in pseudopregnant CL of day 1, and thereafter its signals were detected from day 2 to day 14. Pregnant rat CL expressed both LHR and IGF-IR mRNAs. IGF-I stimulated LHR expression in CL. PGF2ainhibited expression of IGF-IR and LHR. PGE2 negated the inhibiting effects of PGF2α. These data suggest that IGF-I may be involved in regulating CL function, and maintaining CL structure through changes in expression of its receptors. Inhibited expression of IGF-IR by PGF2α may be part of mechanisms for regression of CL.展开更多
Bone morphogenetic proteins（BMPs） play critical roles in follicle growth and development.BMPs initiate signaling by assembling BMP receptors and activating Smads,which in turn alter expression of target genes.The me...Bone morphogenetic proteins（BMPs） play critical roles in follicle growth and development.BMPs initiate signaling by assembling BMP receptors and activating Smads,which in turn alter expression of target genes.The mechanism underlying the regulation of the expression of BMP receptors and Smads during follicle development in pigs is still unknown.By quantitative real-time PCR,the mRNA expression of BMP receptors and Smads in granulosa cells（GC） was investigated.Cells were obtained from small porcine follicles（SF;3 mm diameter） and dominant follicles（DF;6 mm diameter）;ActR1A and BMPR2 mRNA levels in DF were significantly higher（P0.05） than that in SF,whereas BMPR1B,Smad4 and Smad7 expression tended to decrease（P0.05）.The levels of BMPR1A,ActR2,Smad1,Smad5,and Smad8 mRNA did not differ between DFs and SFs.To investigate the effect of LH on BMP receptors in GC,cells obtained from porcine DFs were cultured in medium supplemented with different doses of luteinizing hormone（LH）.High doses of LH（4 IU mL-1） significantly decreased the concentration of estradiol（E2） and progesterone（P4） in medium and the expression of Cyp19a1（P450 aromatase,P450arom） and Cyp11a1（cholesterol side-chain cleavage enzyme P450,P450scc）,while significantly increased viable cell numbers and up-regulated expression of cyclin dependent kinase-4（CDK4） and cyclin D2.However,LH had no effect on the expression of BMP receptor genes.Thus,the present study indicates that the expression of members of the BMP signaling pathway in porcine GC is regulated during follicle development and the expression of BMP receptors are not regulated by LH in porcine GCs.展开更多
文摘In order to investigate the mechanism of progestin and antiprogestin in the regulation of ovarian steroidogenesis, a dual chamber culture system was prepared with the amnion membrane of human placenta. Isolated porcine granulosa and thecal cells from 4~6 mm diameter follicles were grown on both sides of the amnion, respectively, and co cultured with or without LNG and RU486. After 48 h incubation, the mRNAs of FSH receptor (FSH R) and LH receptor (LH R) of both cells were observed by in situ hybridization. The results showed that granulosa cells expressed both FSH R mRNA and LH R mRNA, while thecal cells expressed LH R mRNA only. Under the stimulation of FSH, both LNG and RU486 increased FSH R mRNA expression of granulosa cells. Under the stimulation of LH, LNG enhanced LH R mRNA expression of thecal cells; while RU486 decreased its expression. When granulosa and thecal cells were exposed to FSH and LH both, the actions of LNG and RU486 in thecal cells showed the same result as that stimulated by LH alone. In granulosa cells LNG decreased LH R mRNA expression, while RU486 increased its expression. These data suggest that: (1) granulosa cells expressed FSH R mRNA significantly; (2) both the progestin and antiprogestin directly acted on the mRNA expression of gonadotropin receptors of ovarian cells, but effects were different; (3) the response of granulosa or thecal cells to the action of LNG and RU486 was not the same. The mechanism needs to be further investigated.
文摘The functional significance of the endometrial hCG/ LH receptors has been related to a rapid release of prostaglandins. However, as compared to gonads and myometrium, in-endometrium mechanisms of transmembrane signalling of the hCG/LH receptors are probably not conventional and remain unclear. Here we investigated, in vivo, the potential of hCG to interact with, and stimulate the membrane effector enzyme, adenylyl cyclase (AC), in human endometrium. Hormonal and nonhormonal activation of AC was tested in membrane fractions prepared from endometrial biopsies obtained from patients undergoing evaluation cycles for hormone replacement therapy (HRT) and controlled ovarian hyperstimulation (COH). AC activity was determined by the direct conversion of the substrate ATP into cAMP under unstimulated conditions and in the presence of the non-hormonal activators guanyl nucleotide and forskolin. Also AC activity was tested in the presence of hCG under conditions allowing maximal enzyme stimulation. Isoproterenol and prostaglandin E2 (PGE2) were included for comparison. Immunoblot analyses demonstrated the presence of hCG/LH receptors and Gsα protein and other members of the G protein family in the membrane fractions. Endometrial membranes also exhibited high levels of AC activity compared to luteal membranes used as control. Stimulation by GMP-P(NH)P alone was 196 ± 63 (n = 8) (pmol/mg/ min ± SD). Neither hCG nor isoproterenol showed stimulation of endometrial AC (210 ± 65, and 197 ± 53, respectively;n = 66 assays). But PGE2 stimulated the enzyme system significantly (264 ± 63, p < 0.05;n = 66 assays). These data show that membrane fractions from human endometrium express all the AC system components, namely, hCG/LH receptors, Gsα protein and AC;however, hCG does not stimulate the endometrial AC system. Our data indicate that, in great contrast to gonadal receptors, endometrial hCG/ LH receptors are not coupled to the transmembrane AC effector. The well known release of eicosanoids in response to hCG suggests that these receptors are functional in human endometrium but throughout a signalling system different from AC. This enzyme is certainly coupled to and directly activated by eicosanoids and other embryonic signals.
基金Project supported by the National Natural Science Foundation of China (Grant Nos. 39770099 and 39770290)Chinese Academy of Sciences and Rockefeller Foundation/WHO HPR research project.
文摘By using DNA 3'-end labeling, immunocytochemistry and mRNA in situ hybridization detection techniques , the expression of inhibin subunits and LH receptor in the granulosa cells and tissue-type plasminogen activator (tPA) in the oocytes has been studied in relation to follicular development and atresia. The results demonstrated that: (i) tPA activity in the oocytes of normal developing follicles is undetectable, and increases significantly in the follicle undergoing atresia; (ii) the production of inhibin subunits in granulosa cells is negatively correlated with the expression of oocyte tPA activity, indicating that they may be an important regulator of oocyte tPA production and follicular development; (iii) in atretic follicles, granulosa cells do not express LH receptor and inhibin subunits. It is therefore suggested that tPA may play a role in oocyte self-destruction and clearance in some of atretic follicles, and inhibin of granulosa-origin might be an inhibitory factor for the translation of tPA in the oocyte.
文摘Expression of mRNAs for IGF-I, IGF-binding protein-2 (IGFBP-2), and LH receptor (LHR) as well as their regulations during induced follicular atresia was determined. 26-day-old female rats received 15 IU pregnant mare serum gonadotropins (PMSG). Through detection, it was demonstrated that apoptosis occurred in some small antral follicles after 48 h of PMSG treatment. At 96 h, apoptosis occurred in preovulatory follicles. At 120 h, numerous apoptotic cells appeared in preovulatory follicles. IGF-I was mainly expressed in preantral and small antral follicles from 48 to 120 h. At 48 and 96 h, the theca cells of preantral and antral follicles expressed high level of IGFBP-2 mRNA. At 48 h, there were strong signals of LHR mRNA in granulosa cells, but the LHR signals in granulosa cells significantly decreased at 96 and 120 h (p<0.001). Both epidermal growth factor (EGF) and IGF-I inhibited apoptosis in preantral and antral follicles. Meanwhile, it was observed that EGF promoted IGF-I mRNA expression, and in preovulatory follicles, IGF-I stimulated LHR mRNA expression. These results show that the interaction between ECF and IGF-I may be involved in the regulation of atresia of follicles at different stages of development.
文摘Relationship between insulin-like growth factor-l receptor (IGF-IR) and luteinizing hormone receptor (LHR) mRNA expression as well as their regulation was determined in rat corpora lutea (CL) . In the CL of estrous cycle rat, LHR mRNA positive CL expressed high level of mRNA of IGF-IR. While the expression of LHR mRNA decreased on estrus, the CL still expressed relatively high level of IGF-IR mRNA. In pseudopregnant rat CL, the expression level of LHR mRNA was low on day 1, the most intense signals were detected on day 8, the signals of LHR mRNA became undetectable on day 14. In contrast to LHR expression, the high level of IGF-IR mRNA was observed in pseudopregnant CL of day 1, and thereafter its signals were detected from day 2 to day 14. Pregnant rat CL expressed both LHR and IGF-IR mRNAs. IGF-I stimulated LHR expression in CL. PGF2ainhibited expression of IGF-IR and LHR. PGE2 negated the inhibiting effects of PGF2α. These data suggest that IGF-I may be involved in regulating CL function, and maintaining CL structure through changes in expression of its receptors. Inhibited expression of IGF-IR by PGF2α may be part of mechanisms for regression of CL.
基金supported by the National High Tech-nology Research & Development Program of China(2006AA10Z136)
文摘Bone morphogenetic proteins（BMPs） play critical roles in follicle growth and development.BMPs initiate signaling by assembling BMP receptors and activating Smads,which in turn alter expression of target genes.The mechanism underlying the regulation of the expression of BMP receptors and Smads during follicle development in pigs is still unknown.By quantitative real-time PCR,the mRNA expression of BMP receptors and Smads in granulosa cells（GC） was investigated.Cells were obtained from small porcine follicles（SF;3 mm diameter） and dominant follicles（DF;6 mm diameter）;ActR1A and BMPR2 mRNA levels in DF were significantly higher（P0.05） than that in SF,whereas BMPR1B,Smad4 and Smad7 expression tended to decrease（P0.05）.The levels of BMPR1A,ActR2,Smad1,Smad5,and Smad8 mRNA did not differ between DFs and SFs.To investigate the effect of LH on BMP receptors in GC,cells obtained from porcine DFs were cultured in medium supplemented with different doses of luteinizing hormone（LH）.High doses of LH（4 IU mL-1） significantly decreased the concentration of estradiol（E2） and progesterone（P4） in medium and the expression of Cyp19a1（P450 aromatase,P450arom） and Cyp11a1（cholesterol side-chain cleavage enzyme P450,P450scc）,while significantly increased viable cell numbers and up-regulated expression of cyclin dependent kinase-4（CDK4） and cyclin D2.However,LH had no effect on the expression of BMP receptor genes.Thus,the present study indicates that the expression of members of the BMP signaling pathway in porcine GC is regulated during follicle development and the expression of BMP receptors are not regulated by LH in porcine GCs.
