Serotonin-induced stimulation of progesterone production by cow luteal cells in vitro

The addition of acetylcholine or histamine (10(-7) to 10(-4) M), gamma-aminobutyric acid, a dopamine agonist, and melatonin (10(-7) to 10(-5) M) did not alter basal or LH-stimulated progesterone production (P greater than 0.05). The addition of the specific beta 2-adrenergic agonist terbutaline and salbutamol did not significantly elevate progesterone production. Treatment of luteal cells with serotonin (5-HT), 10(-6) to 10(-4) M, increased the production of progesterone (P less than 0.05). This stimulated production was inhibited by the addition of mianserin (10(-5) M, a 5-HT antagonist; P less than 0.05). Isoproterenol (10(-7) to 10(-4) M) also resulted in significant increases in progesterone production (P less than 0.05). The combined treatments of 5-HT + LH, isoproterenol + LH, or isoproterenol + 5-HT did not result in a further increase in progesterone above that observed in response to LH or isoproterenol alone (P greater than 0.05). The isoproterenol-induced progesterone production could not be blocked by butoxamine (10(-5) M, a beta 2-antagonist), or practolol (10(-5) M, a beta 1-antagonist), but was inhibited by propranolol (10(-5) M, a general beta-antagonist; P less than 0.05). The response to isoproterenol was unaffected by mianserin (10(-5) M). These results demonstrate a possible role for 5-HT in the regulation of steroidogenesis by the corpus luteum of the cow. Furthermore, these results suggest that serotonin-induced progesterone production is a receptor-mediated event.     

Lipoprotein-stimulated and estradiol-maintained progesterone secretion by dissociated rabbit luteal cells in vitro

Elevated activity of 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMG-CoA reductase) was observed in the rabbit ovary and corpus luteum during pregnancy. Based on this study, it was proposed that de novo cholesterol synthesis rather than the uptake of exogenous plasma cholesterol (lipoproteins) was of primary importance in providing steroid substrate for progesterone synthesis by the rabbit luteal cell. Using a perifusion system, the present study challenges this hypothesis by demonstrating that both low- and high-density lipoproteins (at protein concentrations of 100 micrograms/ml and 50 micrograms/ml, respectively) were able to acutely stimulate progesterone production by dissociated rabbit luteal cells. The increase in progesterone synthesis was due to increased cholesterol substrate and not to protein-enhanced progesterone release. The ability of luteal cells to respond to lipoproteins was dependent on both dose- and sequence of treatment, with high-density lipoprotein (HDL) being unable to stimulate progesterone production if preceded by perifusion with low-density lipoprotein (LDL) or HDL. In addition, 17 beta-estradiol appeared to regulate lipoprotein utilization by attenuating the LDL response after 1 h of perifusion. We conclude that lipoproteins may provide cholesterol substrate for progesterone biosynthesis in vitro and that 17 beta-estradiol, in addition to maintaining progesterone production by luteal cells, may also regulate lipoprotein utilization. Thus, maintenance of steady progesterone secretion in response to estradiol supercedes that of LDL-stimulated progesterone secretion by rabbit luteal cells in vitro. This study suggests an interaction between estrogen and lipoproteins that may prove physiologically important in regulating progesterone production by rabbit luteal cells in vivo.     

Decrease of estrogen receptors induced by 17 beta-estradiol and progesterone in cultured rabbit endometrial cells

A whole cell technique to measure estradiol receptors in cultured rabbit endometrial cells is described. The estradiol receptors measured appear to be composed of at least two components: one component has high affinity but low capacity, while the other component has low affinity but high capacity. Using the assay, the effects of estradiol and progesterone pretreatment were examined on the estradiol receptor levels. It was found that both of the hormones decreased the number of estrogen receptors in the cultured cells. The finding that estradiol decreased its own receptors was unexpected and its possible relevance is discussed.     

Steroidogenic response of rat and pig luteal cells to estradiol-17 beta and catecholestrogens in vitro.

The corpora lutea of several species contain estrogen receptors, but the role of estrogens in luteal function is unclear in most species. In this study, we investigated the direct effect of estradiol-17 beta (E2) and catecholestrogens (2-OHE2 or 4-OHE2) on rat and pig luteal steroidogenesis using in vitro cultures of small (SLC) and large (LLC) luteal cells prepared by elutriation. SLC and LLC were cultured at 37 degrees C for 36 h in serum-free media and treated with E2, 2-OHE2, or 4-OHE2; LH; forskolin (FORS); dibutyryl cAMP (dbcAMP); or combinations thereof. In the rat, E2 (2.5-10 micrograms/ml) inhibited progesterone (P4) production by both cell types dose-dependently. P4 production by rat SLC increased with increasing dose of 4-OHE2 up to the 2.5-microgram dose, then decreased to near control level at the 10-microgram dose. In LLC, P4 production in the presence of 4-OHE2 decreased initially (up to 2.5 micrograms/ml 4-OHE2), then increased at the 10-microgram dose. LH, FORS, and dbcAMP stimulated P4 production by SLC and LLC. For SLC, the stimulatory effects of LH and 4-OHE2 (2.5 micrograms) were comparable but lower than those of FORS and dbcAMP. For LLC, the effects of 4-OHE2 (10 micrograms), LH, and FORS were comparable but lower than those of dbcAMP. In time-course experiments, E2 inhibition of P4 production was observed at 36 and 72 h but not 6 h of culture for SLC and at all time points for LLC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxytocin and vasopressin have no effect on progesterone production and cyclic AMP accumulation by rat luteal cells in vitro

Enzymically dispersed luteal cells obtained from PMSG-hCG-treated immature pseudopregnant rats were incubated with oxytocin and vasopressin. In response to increasing doses of hCG the rat luteal cells produced progesterone and accumulated intracellular cAMP in a dose-dependent manner. A neuropeptide GnRH agonist (4 X 10(-6) M) produced a significant inhibition of hCG-stimulated progesterone production and of accumulation of intracellular cAMP. However, neither the basal nor the hCG-stimulated rate of progesterone production and level of intracellular cAMP was affected by the neurohypophysial peptides tested. Therefore, it is concluded that oxytocin and vasopressin do not have a direct action on steroidogenesis by rat luteal cells.     

Effect of cyclodextrin-encapsulated beta-carotene on progesterone production by bovine luteal cells

Experiments were conducted to examine the effect of cyclodextrin-encapsulated beta-carotene on basal or cholesterol (cyclodextrin-encapsulated), LH and dibutyryl cyclic AMP (dbcAMP)-stimulated progesterone production by bovine corpus luteum cells isolated from mid-luteal heifer ovaries by collagenase digestion. Cells were cultured with serum-free DMEM/Ham's F12 medium in serum pre-treated plastic culture dishes for periods of up to 11 days. Medium was replaced after 24h and thereafter every 48 h. Beta-carotene was added to cultures in a carrier molecule, dimethyl-beta-cyclodextrin, to facilitate dissolution. All treatments were started on day 3 of culture. Treatment of cells with 1 or 2 micromol/l beta-carotene resulted in sharp inhibition of progesterone production. On the contrary, treatment of cells with 0.1 micromol/l beta-carotene resulted in significant stimulation (P<0.05) of both basal and cholesterol-stimulated progesterone secretion. The effect of beta-carotene on LH or dbcAMP-stimulated progesterone production was also examined. Treatment of cells with LH or dbcAMP always resulted in stimulation of progesterone secretion (P<0.001). However, cells treated with LH plus beta-carotene or dbcAMP plus beta-carotene both produced significantly (P<0.01) less progesterone relative to those cells treated with LH or dbcAMP alone on days 7, 9 and 11 of culture. These results indicate that beta-carotene can enhance luteal steroidogenesis when present at low concentrations but is inhibitory at higher concentrations and that encapsulation of beta-carotene in cyclodextrin is an effective method of supplying it to cells in culture.     

GABA影响大鼠卵巢黄体细胞孕酮的生成

实验用离体培养方法观察ＧＡＢＡ对大鼠黄体细胞孕酮及羟自由基（．ＯＨ）生成的影响。结果表明：ＧＡＢＡ抑制黄体细胞孕酮的生成,同时也促进黄体细胞．ＯＨ的生成。ＧＡＢＡ对孕酮的抑制作用可能与腺苷酸环化酶系统及ＧＡＢＡＡ型受体有关,而与蛋白质合成无关。     

Effect of ovine conceptus and endometrial secretory products on luteal progesterone production in vitro

The objectives of this study were the following: (i) to determine if ovine conceptus secretory products are directly luteotrophic to luteal tissue in vitro and (ii) to determine if ovine conceptus secretory products stimulate endometrial tissue to secrete a luteotropin in vitro. Conceptus-conditioned medium (CCM) was prepared by incubating day 14 ovine conceptuses in minimal essential medium (MEM) for 24 h and harvesting the supernatant. Endometrium-conditioned CCM (E-CCM) and endometrium-conditioned medium (ECM) were prepared by incubating dispersed ovine endometrial cells from day 9-10 cycling ewes in CCM or MEM, respectively, for 16 h and harvesting the supernatants. Media, conditioned as described, were incubated at various dilutions with dispersed luteal cells from day 9-10 cycling ewes for 90 min or 6 h in the absence or presence of 50 ng/mL ovine luteinizing hormone (oLH). CCM did not alter progesterone (P4) production in the 90-min incubation but did increase (p less than 0.05) P4 production in the 6-h incubation (1:4, 1:8, 1:16 dilutions). When coincubated with oLH, CCM did not increase P4 production above that stimulated by oLH alone. The effect of E-CCM was similar to CCM or ECM and did not differ significantly from basal. It is concluded that the day 14 ovine conceptus does secrete a factor that is able to directly stimulate P4 secretion by luteal cells in a 6-h, but not a 90-min, incubation. Conceptus secretory products did not stimulate endometrial cells to secrete a luteotropin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of cultured rat vaginal epithelial cells by 17 beta-estradiol and progesterone

We have previously described a technique to obtain short-term cultures of epithelial cells from Wistar rat vaginae. In order to improve the efficiency and life span of these cultures, in the present study we have cultured the vaginal cells with lethally irradiated 3T3 cell feeder layers. Under this condition, cells can grow for several weeks while retaining epithelial characteristics and can eventually be subcultured. The proliferative effect of the ovarian hormones in these cultures was studied using two different approaches, [Methyl-3H]Thymidine (3HTdr) incorporation and increase in cell number. Both assays indicated a proliferative effect of 17 beta-estradiol and progesterone at physiological concentrations. This proliferative effect was also shown in feeder layer-free cultures, ruling out an indirect effect through the mesodermal cells. The capacity of the hormones to modify terminal differentiation in the culture was also studied, using colony stratification as an indicator of differentiation. Progesterone and fetal calf serum had an inhibitory effect on terminal differentiation, whereas 17 beta-estradiol induced a stimulatory action. This culture model allowed us to show a direct effect of the ovarian hormones on vaginal cells in vitro and seems to be a useful model to study hormone-cell interactions in vitro.     

Differential effects of calcium on progesterone production in small and large bovine luteal cells

We studied the effects of calcium (Ca2+) ions in progesterone (P) production by separated small and large luteal cells. Corpora lutea were collected from 31 heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. P accumulation in cells plus media was determined after incubating 1 x 10(5) small and 5 x 10(3) large cells for 2 and 4 h respectively. Removal of Ca2+ from the medium did not influence basal P production in the small cells (P greater than 0.05). However, stimulation of P by luteinizing hormone (LH), prostaglandin E2 (PGE2), 8-bromo-cyclic 3',5' adenosine monophosphate (8-Br-cAMP) and prostaglandin F2 alpha (PGF2 alpha) was impaired (P less than 0.05) by low Ca2+ concentrations. LH and PGE2-stimulated cAMP production was not altered by low extracellular Ca2+ concentrations, and PGF2 alpha had no effect on cAMP. In contrast, basal as well as LH and forskolin-stimulated P production were attenuated (P less than 0.05) in Ca2(+)-deficient medium in the large cells. However, P production stimulated by 8-Br-cAMP was not altered in Ca2(+)-deficient medium. Steroidogenesis in large cells was also dependent on intracellular Ca2+, since 8-N, N-diethylamineocytyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release and/or action, suppressed (P less than 0.05) basal, LH and 8-Br-cAMP stimulated P. In contrast, basal P in small cells was not altered by TMB-8; whereas LH-stimulated P was reduced 2-fold (P less than 0.05). The calcium ionophore, A23187, inhibited LH-stimulated P in small cells and both basal and agonist-stimulated P in large cells. These studies show that basal P production in small cells does not require Ca2+ ions, while hormone-stimulated P production in small cells and both basal and hormone-stimulated P in large cells do require Ca2+. The inhibitory effect of Ca2+ ion removal was exerted prior to the generation of cAMP in the large cells, but distal to cAMP generation in hormone-stimulated small cells. The calmodulin/protein kinase C antagonist, W-7, also inhibited both basal and hormone-stimulated P production in both small and large luteal cells, indicating that P production in luteal cells also involves Ca2(+)-calmodulin/protein kinase C-dependent mechanisms.     

Effect of A-nor steroids and oestradiol on progesterone production by human luteal cells

Cell suspensions were prepared from human corpora lutea obtained during the mid-luteal phase. Progesterone production was assessed after short-term incubation of luteal cell suspensions. Luteal cells were very sensitive to hCG, the concentration required for 50% maximum response being 0.01 i.u./ml, and the response was 5 times higher than the basal production. Oestradiol (1-100 microM) induced a significant dose-related decrease in both basal and hCG-stimulated progesterone production. The A-nor steroidal compounds anordrin and AF-45 reduced hCG-stimulated progesterone production only at the high concentration of 100 microM. The ED50 values were approximately 3 microM, 75 microM and 100 microM for oestradiol, AF-45 and anordrin respectively. Anordrin showed no significant effects on basal progesterone production. In addition, oestradiol markedly inhibited the activity of 3 beta-hydroxysteroid dehydrogenase in luteal cells, expressed by the conversion of pregnenolone to progesterone, but the inhibitory effects of anordrin and AF-45 were negligible or relatively low. The effects of anordrin and AF-45 were different from those of oestradiol on progesterone production by human luteal cells in vitro, indicating that neither substance is likely to be a useful luteolytic agent in women.     

Effect of histone H2A on progesterone production by bovine luteal cells

Histone H2A competitively inhibits binding of GnRH to high affinity rat ovarian receptor sites and blocks gonadotropin-stimulated steroid and cAMP accumulation during culture of rat granulosal or luteal cells. The objective of our study was to examine the progesterone suppressive effects of histone H2A on bovine luteal cells. In the first study, luteal cells were treated at Time = 0 h with a partially purified preparation of bovine ovarian histone H2A (3 ng GnRH equivalents, 800 micrograms protein), equivalent amounts of GnRH (3 ng), or BSA (800 micrograms) and incubated for a total of 4 h. At Time = 2 h, cells were treated with 5 ng bovine LH (bLH) or with medium. Histone H2A completely blocked both basal and LH-induced accumulation of progesterone compared with untreated cultures or cultures treated with bLH. Neither BSA nor GnRH suppressed LH-induced progesterone accumulation. In the second study, histone H2A was added to cultures at Time = 0 h and bovine luteal cells were cultured for 8 h. After 2 h of treatment, histone H2A (3 ng GnRH equivalents) was removed from selected cultures and replaced with fresh medium. Four hours later cultures were treated with 5 ng bLH or medium. LH treatment of cultures from which histone H2A had been removed resulted in an increase in accumulation of progesterone compared with control cultures treated throughout the treatment period with histone H2A. The third study examined the effect of 9-181 pg GnRH equivalents (1.7-34 micrograms protein) of a highly purified preparation of bovine ovarian histone H2A on basal and LH-induced progesterone production during 2 or 3 h of culture.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of cyclooxygenase 2 in the protective effect of 17beta-estradiol on hypercholesterolemic rabbit aorta

The involvement of cyclooxygenase (COX) in the effects of 17beta-estradiol was investigated on hypercholesterolemic rabbits aorta. Acetylsalycilic acid, nimesulide, or SQ22536 was used as respective antagonist of COX-1, COX-2, or adenylate cyclase using aortic rings precontracted with phenylephrine and exposed to cumulative concentrations of acetylcholine (ACh). The relaxation effect of ACh was impaired by hypercholesterolemia and restored by an 8-week 17beta-estradiol treatment. In the control group treated with estrogen, nimesulide, acetylsalycilic acid, or SQ22536 slightly reduced the response to ACh. In hypercholesterolemic rabbits treated with estrogen, nimesulide significantly reduced the maximal relaxation and shifted to the right the relaxation curve of ACh, whereas acetylsalycilic acid did not modify the maximal response to ACh but displaced slightly the concentration-response curve. SQ22536 reduced the relaxant effect of ACh down to the level obtained in the presence of nimesulide. These results suggest that the protective effect of 17beta-estradiol against hypercholesterolemia involved COX-2/adenylate cyclase pathway.     

Steroidogenic capacity and ultrastructural morphology of cultured ovine luteal cells

Corpora lutea were surgically collected from superovulated ewes 36 h post-injection of human chorionic gonadotropin (hCG) (Day 2), dissociated (0.2% collagenase), plated, and maintained in culture Days 2-10 in Medium 199 supplemented with 5% calf serum. Accumulation of progesterone in the cultures did not decrease (p greater than 0.05) from Day 3 (17.5 +/- 5.1 nmol/10(6) cells) to Day 10 (4.8 +/- 1.7 nmol/10(6) cells). Calf serum (5%) in the medium supported greater (p less than 0.05) progesterone production than fetal calf serum (5%) or medium without added serum. Steroidogenic cells did not increase (Days 2-10) in numbers, but increased (p less than 0.01) in mean cell diameter (Day 2, 11.7 +/- 0.4 micron; Day 10, 24.5 +/- 1.6 micron). Steroidogenic capacity on Day 10 of cells cultured Days 2-10 (in vitro) was not different (p greater than 0.05) from that of cells collected from the ovary on Day 10 (in vivo); however, steroidogenic cells recovered from plates had greater (p less than 0.01) mean cell diameters (24.5 +/- 1.6 micron, in vitro, compared to 15.2 +/- 1.0 micron, in vivo). Transmission electron microscopy revealed that cultured cells (Days 5, 10) possessed less smooth endoplasmic reticulum but more lipid droplet inclusions, ribosomes, and rough endoplasmic reticulum than cells obtained in situ (Day 10). Electron-dense secretory granules were rarely seen. Although subcellular morphology of ovine luteal cells in culture was altered, these changes did not appear to significantly affect the ability of these cells to produce progesterone.     

Measurement of glucuronometabolites of 17 beta-estradiol and progesterone in diluted overnight urine. An approach to the study of luteal insufficiency

The determination of the concentrations of estrone-3-glucuronide, pregnanediol-3-glucuronide and luteinizing hormone has been performed in early morning urine samples of 14 normal menstruating women using a timed and measured volume urine collection procedure. In order to investigate the variability of the urinary hormonal concentrations due to day-to-day differences in diuresis, the absolute hormonal concentrations have been corrected either for the urinary creatinine excretion or for the volume of urine voided during the night. The results demonstrate that both correction factors are able to reduce substantially the coefficient of variation values in comparison to the absolute hormonal concentrations. The urinary test of ovarian function has been performed in 11 infertile women affected by luteal insufficiency using the same procedure, and the hormonal profiles showed some alterations in both estrone-3-glucuronide and pregnanediol-3-glucuronide concentrations in comparison to the hormonal profiles of the normal subjects. Such alterations were significant in the single subject when integrated values of the hormonal data in defined time intervals were investigated.     

Effects of prostaglandin F2 alpha on agonist-induced progesterone production in cultured bovine luteal cells

The present study examines the effects of prostaglandin F2 alpha (PGF2 alpha) on basal and agonist-stimulated progesterone (P4) production utilizing long-term, serum-free cultures of bovine luteal cells. During the first 24 h of culture, PGF2 alpha had no significant effect on P4 production, and was unable to inhibit either luteinizing hormone (LH)- or dibutyryl cAMP (dbcAMP)-stimulated increases in P4. Treatment with PGF2 alpha on Day 1 produced a moderate, nonsignificant (P greater than 0.05) inhibition of cholera toxin (CT)- and forskolin (FKN)-stimulated P4 synthesis. Beyond Day 1 of culture (Days 3-11), PGF2 alpha continued to have no significant effect on basal P4 production, but suppressed all stimulatory effects of LH, dbcAMP, CT and FKN. Treatment with indomethacin inhibited prostaglandin synthesis by the cultured cells and also elevated levels of P4 from Days 3 to 11 of culture. Concurrent treatment with PGF2 alpha suppressed the steroidogenic effect of indomethacin. From these studies it was concluded that in cultured bovine luteal cells, PGF2 alpha does not affect basal P4 production, but is able to inhibit agonist-stimulated P4 production at a site beyond the accumulation of cAMP. This inhibitory effect is not apparent during the first 24 h of culture, but appears after Day 1 and persists throughout the remaining 10 days of the culture period.     

Dose-and-time dependent effect of 2,3,7,8-tetrachlorodibenzo-P-dioxin (TCDD) on progesterone secretion by porcine luteal cells cultured in vitro.

In the current study, to characterize TCDD action during luteal phase of the ovarian cycle, the direct effect of TCDD was investigated in vitro using a system of monolayer cell culture. Luteal cells isolated from mid-developing corpora lutea were cultured with four different doses of TCDD (0.1, 1.0, 10.0 and 100 nM). The dose of 0.1nM TCDD had no effect on progesterone (P4) secretion by luteal cells while the doses of 10nM and 100nM in the same, statistically significant manner decreased P4 secretion (p <0.05). The inhibitory effect of TCDD was dependent not only on doses by also on experimental conditions. In cells treated every day for 72 hrs of culture with 0.1nM TCDD, P4 secretion was 71% of basal secretion. 100nM TCDD added only at the beginning of the culture and nor repeated when medium was changed every 24 hrs decreased P4 secretion to 81.8% of basal secretion. The most inhibitory effect was observed in experiments in which 100nM TCDD was added at the beginning of the culture and medium was not changed for 72 hrs. Secretion of P4 was only 33.9% of that by control cultures. In order to show the time-dependent response to TCDD in terms of P4 secretion, luteal cells were cultured for 24,48, 72 hrs with 0.1 and 100nM TCDD. 85%, 75% and 72% of basal progesterone secretion was noted after 24, 48 and 72h respectively in 0.1nM TCDD-treated cells. In 100nM TCDD treated cells the decrease of progesterone secretion was 57%, 67% and 82% of basal secretion after 24, 48 and 72 hrs of culture. These experiments suggest that TCDD by suppressing progesterone secretion by corpora lutea can cause adverse reproductive effects such as early pregnancy failure. Endocrine disrupters that interfere with progesterone production can act as abortifacients.     

c-erbB_2对大鼠黄体细胞hCG诱导的孕酮分泌的影响

采用离体细胞体外孵育法 ,研究反义c erbB2 寡脱氧核苷酸 (antisensec erbB2 ODN)对大鼠黄体细胞hCG诱导的孕酮分泌的影响 ,及其与外源性cAMP和Ca2 以及蛋白抑制剂放线菌酮 (CYX)之间的关系。结果表明 ,反义c erbB2 以剂量相关方式抑制黄体细胞hCG诱导的孕酮的产生 ,同时使c erbB2 蛋白染色阳性的黄体细胞百分数下降 ,无义tatODN没有相应的作用。10 -4 mol/L的二丁酰cAMP能明显反转反义c erbB2 ODN对孕酮产生和c erbB2 表达的抑制作用 ,钙离子通道阻断剂维拉帕米和蛋白抑制剂CYX对此抑制作用有协同效应。该实验说明c erbB2 参于hCG诱导黄体细胞生孕酮作用     

c—erbB2对大鼠黄体细胞hCG诱导的孕酮分泌的影响

采用离体细胞体外孵育法,研究反义ｃ－ｅｒｂＢ２寡脱氧核苷酸（ａｎｔｉｓｅｎｓｅ ｃ－ｅｒｂＢ２ ＯＤＮ）对大鼠黄体细胞ｈＣＧ诱导的孕酮分泌的影响,及其与外源性ｃＡＭＰ和Ｃａ＾２＋以及蛋白抑制剂放线菌酮（ＣＹＸ）之间的关系。结果表明,反义ｃ－ｅｒｂＢ２以剂量相关方式抑制黄体细胞ｈＣＧ诱导的孕酮的产生,同时使ｃ－ｅｒｂＢ２蛋白染色阳性的黄体细胞百分数下降,无义ｔａｔ ＯＤＮ没有相应的作用。１０＾－４