Assessing estrogenic activity of pyrethroid insecticides using in vitro combination assays

Pyrethroid insecticides are among the most commonly used classes of insecticides worldwide, but their endocrine disrupting activities remain unclear. Therefore, in the present study, we examined the estrogenic activities of pyrethroid insecticides in E-screen and competition binding assays. In addition, we measured estrogen receptor (ER) protein and pS2 mRNA levels in human breast cancer cells (MCF-7 BUS) to clarify the mechanism of their estrogenicity. Seven pyrethroid insecticides (bioallethrine, cypermethrin, deltamethrin, fenvalerate, permethrin, sumithrin, and tetramethrin) were tested because of their worldwide usage. In addition, 17beta-estradiol was tested as a positive control. As expected, 17beta-estradiol significantly increased MCF-7 BUS cell proliferation at concentrations of 10(-11) M and above. Of the pyrethroid insecticides tested, only sumithrin increased MCF-7 BUS cell proliferation in a dose-dependent manner; the maximum induction of cell proliferation was observed at a dose of 10(-5) M. In the anti-estrogenic activity test, bioallethrin, fenvalerate, and permethrin significantly inhibited 17beta-estradiol-induced MCF-7 BUS cell proliferation at 10(-6) M, a concentration comparable to the effective dose (10(-9) M) of ICI 182,780, a pure ER antagonist. However, none of the pyrethroid insecticides competitively inhibited the binding of [(3)H]estradiol to rat uterus ERs in competition binding assays. Both 17beta-estradiol (10(-10) M) and sumithrin (10(-5) M) decreased the levels of cytosolic ERalpha and ERbeta protein expression significantly as compared with the vehicle control. In addition, 17beta-estradiol (10(-10) M) increased pS2 mRNA expression markedly, and sumithrin significantly increased pS2 mRNA levels in a dose-dependent manner. The other six compounds tested in the present study did not affect ER protein levels or pS2 mRNA levels. These results suggest that certain pyrethroid insecticides may be considered to be estrogen-like chemicals that act through pathways other than direct ER binding, and may function as endocrine modulators in both wildlife and humans.     

Conflict of estrogenic activity by various phthalates between in vitro and in vivo models related to the expression of Calbindin-D9k

Phthalates are suspected to disrupt the endocrine system, especially through estrogenic effects. In the present study, we investigated the effects of various phthalates and compared them with those of estrogenic compounds that disrupt the female reproductive system. To assess the effects of these phthalates, alteration of the Calbindin-D9k (CaBP-9k) gene was measured as a biomarker because rat CaBP-9k gene carries an estrogen response element (ERE) which is involved in estrogen responsiveness of the gene during the estrous cycle. In this study, phthalates were tested for estrogenic properties in in vitro and in vivo models. First, the E-Screen assay was used to measure the proliferation of MCF-7 cells, a human breast cancer cell line. Treatments with 17beta-estradiol (E2; 9-fold) and 17alpha-estradiol (EE; 9-fold) induced MCF-7 cell proliferation at concentrations of 10(-9) M. Phthalates induced an increase in MCF-7 proliferation at concentration of 10(-6) M up to 10(-4) M. Nbutyl benzyl phthalate (BBP; 6-fold vs. vehicle), dicyclohexyl phthalate (DCHP; 8-fold), 2-ethylhexyl phthalate (DEHP; 6-fold) and di-n-butyl phthalate (DBP; 7-fold) at the concentration of 10(-4) M induced in an increase in MCF-7 proliferation after 6 d of treatment compared to vehicle. However, significant increase in MCF-7 proliferation was induced by diethyl phthalate (DEP). Second, we investigated the expression of CaBP-9k in the uterus of immature rats after oral treatment with BBP, DCHP, DEHP, DBP or DBP (600 mg/kg per day) in this in vivo model, because the immature rat model is highly sensitive to exposure to estrogenic chemicals. None of the phthalates induced the expression of CaBP-9k mRNA and its protein in the neonatal uterus as analysed by Northern and Western blot analyses, respectively. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce CaBP-9k expression in the in vivo system, suggesting that the assays of estrogenic effects of various phthalates conducted in vitro and in vivo expression of CaBP-9k may produce conflicting results.     

Determination of the estrogenic activity of wild phytoestrogen-rich Pueraria mirifica by MCF-7 proliferation assay

The aim of this study was to evaluate the estrogenic activity of tuberous samples of wild, phytoestrogen-rich Pueraria mirifica collected from 28 out of 76 provinces of Thailand by MCF-7 proliferation assay. The plant extracts were administered to MCF-7, ER alpha positive human mammary adenocarcinoma cell cultures, for 3 days at dosages of 0.1, 1, 10, 100 and 1,000 microg/ml and were compared with 17 beta-estradiol at concentrations of 10(-12)-10(-6) M. The mean P. mirifica population at 1 mug/ml exhibited significant proliferation. Two plant samples exhibited levels of proliferation in MCF-7 that were similar to 17beta-estradiol. The mean P. mirifica populations at 100 and 1,000 microg/ml exhibited significant cytotoxicity in MCF-7. Analysis of the estrogenic activity of puerarin, representative of major isoflavonoids in P. mirifica tubers, revealed proliferation in MCF-7 only at the highest dose (10(-6) M) that was 10(2)-10(5) times less active than 17 beta-estradiol. Puerarin and 17 beta-estradiol at concentration of 10(-12)-10(-6) M exhibited no cytotoxicity in MCF-7.     

A sensitive bioassay for detection of dietary estrogens in animal feeds

Estrogen-responsive proliferation in the MCF-7 cell line was used as a bioassay for detection of dietary estrogens. The bioassay procedure was adapted to screen for estrogenic activity in feedstuffs that have been associated with hyperestrogenism in livestock. Methanolic feed extracts were added to the cell culture medium at microliter/ml concentrations for 4 days, after which the cell proliferation response was measured as DNA content. The half-maximal response for estradiol occurred at 2 pM, or 0.54 pg/ml. For zearalenone, a weaker estrogen, the half-maximal response occurred at approximately 200 pM, or 64 pg/ml. The bioassay was calibrated against a number of known estrogens (estradiol, diethylstilbestrol, zearalenone, zearalanol [cattle implant], beta-zearalenol, zearalane), including the naturally occurring phytoestrogens (formononetin, genistein, daidzein, biochanin A, and coumestrol). The estrogenic activity of feed samples was expressed as equivalents of zearalenone (ppm zearalenone) that would have to be present to equally stimulate proliferation of the MCF-7 cells. The sensitivity of the bioassay was 0.05-0.1 ppm equivalents of zearalenone in feed, well below the threshold level associated with reproductive problems. The feed additive melengestrol acetate (MGA) showed no estrogenic activity in this assay. Estrogenic activity of feed extracts was confirmed by competitive inhibition with the antiestrogens tamoxifen or LY156758 (keoxifene) to show that stimulation of growth by feed extracts was through an estrogenic mechanism. Confirmation of known estrogens was by tandem mass spectroscopy. The assay is a sensitive and reliable screening procedure for detecting estrogenic activity in feedstuffs.     

Melengestrol acetate alters muscle cell proliferation in heifers and steers

In vitro experiments were performed to investigate the effects of melengestrol acetate (MGA) or progesterone (P4) on bovine muscle satellite cells and C2C12 myoblasts. Addition of MGA at physiological and supraphysiological concentrations resulted in a dose-dependent decrease (P < 0.05) in DNA synthesis as measured by [3H]-thymidine incorporation (TI). Similarly, P4 addition (0.01 nM) reduced (P < 0.05) TI. Addition of MGA (10 nM) increased (P < 0.05) IGF-I mRNA abundance but did not affect myogenin mRNA. Progesterone addition (10 nM) increased myogenin mRNA abundance (P < 0.05). In C2C12 cultures, P4 addition resulted in a dose-dependent decrease in TI. The antiprogestin RU486, in combination with MGA or P4, also resulted in reduced (P < 0.05) TI. Treatment with RU486 alone had a negative effect (P < 0.05) on TI that was similar to the progestins. Treatment of C2C12 myoblasts with MGA (100 nM) resulted in an increase (P < 0.05) in myogenin mRNA. These studies suggest that progestins may reduce satellite cell proliferation, ultimately affecting carcass composition.     

Potential role of G-protein-coupled receptor 30 (GPR30) in estradiol-17beta-stimulated IGF-I mRNA expression in bovine satellite cell cultures

Androgenic and estrogenic steroids enhance muscle growth in animals and humans. Estradiol-17beta (E2) and trenbolone acetate (TBA) (a synthetic testosterone analog) increased IGF-I mRNA expression in bovine muscle satellite cell (BSC) cultures. The goal of this study was to evaluate the mechanisms responsible for this increase by evaluating the effects of ICI 182 780 (an E2 receptor antagonist), flutamide (an androgen receptor inhibitor), G1 (a GPR30 agonist), and BSA-conjugated E2 on E2 and/or TBA-stimulated IGF-I mRNA expression in BSC cultures. Flutamide completely suppressed TBA-stimulated IGF-I mRNA expression in BSC cultures. ICI 182 780 did not suppress E2-stimulated IGF-I mRNA expression and 100nM ICI 182 780 enhanced (93%, p<0.05) IGF-I mRNA levels in BSC cultures. G1 (100nM) stimulated IGF-I mRNA expression (100%, p<0.05) but had no effect on proliferation in BSC cultures. E2-BSA, which cannot cross the cell membrane, stimulated IGF-I mRNA expression (approximately 100%, p<0.05) in BSC but even at extremely high concentrations had no effect on proliferation. In summary, our data indicate the E2-stimulation of proliferation and E2-stimulation of IGF-I mRNA expression in BSC cultures occur via different mechanisms. Our previous results showing that ICI 182 780 inhibited BSC proliferation and results of the current study showing lack of response to E2-BSA or G1 suggest that E2-stimulated proliferation in BSC cultures is mediated through classical estrogen receptors. Stimulation by ICI 182 780, G1 and E2-BSA suggests the E2-stimulated IGF-I mRNA expression in BSC cultures is mediated through the GPR30 receptor.     

Mechanism of action of noradrenaline on secretion of progesterone and oxytocin by the bovine corpus luteum in vitro

The present studies were conducted: (1) to determine which beta-adrenoceptor subtypes are involved in progesterone and oxytocin (OT) secretion, (2) to examine whether noradrenaline (NA) acts directly on the cytochrome P-450scc and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and (3) to study the effect of prostaglandin F2 alpha (PGF2 alpha) on NA-stimulated steroidogenesis in luteal cells. The effect of NA on progesterone secretion from luteal slices of heifers on days 8-12 of the oestrous cycle was blocked by both atenolol (beta 1-antagonist) and ICI 118.551 hydrochloride (beta 2-antagonist). OT secretion was blocked only after treatment with ICI 118.551 hydrochloride (P < 0.05). Dobutamine (10(-4)-10(-6) M), a selective beta 1 agonist and salbutamol (10(-4)-10(-6) M), a selective beta 2 agonist, both increased progesterone production (P < 0.01) with an efficiency comparable to that produced by NA (P < 0.01). The increase of OT content in luteal slices was observed only after treatment with salbutamol at the dose of 10(-5) M (P < 0.01). Dobutamine had no effect on OT production at any dose. A stimulatory effect of NA on cytochrome P-450scc activity (P < 0.05) was demonstrated using 25-hydroxycholesterol as substrate. 3 beta-HSD activity also increased following NA (P < 0.01) or pregnenolone (P < 0.05) and in tissue treated with pregnenolone together with NA (P < 0.01). PGF decreased progesterone synthesis (P < 0.05) and 3 beta-HSD activity (P < 0.01) in tissue treated with NA. We conclude that NA stimulates progesterone secretion by luteal beta 1- and beta 2-adrenoceptors, while OT secretion is probably mediated only via the beta 2-receptor. NA also increases cytochrome P-450scc and 3 beta-HSD activity. PGF inhibits the luteotropic effect of NA on the luteal tissue.     

Ability of intravaginal progesterone inserts and melengestrol acetate to induce estrous cycles in postpartum beef cows

Postpartum anestrous interval in beef cows is a major factor contributing to reproductive failure during a defined breeding season. Our objectives were to determine the ability of a controlled internal drug-releasing device (CIDR, 1.9 g of progesterone), a normal dose of melengestrol acetate (MGA, 0.5 mg x cow(-1) x d(-1)), or a high dose of MGA (4.0 mg x cow(-1) x d(-1)) to induce ovulation and to eliminate short estrous cycles. Multiparous beef cows (n = 100) were equally assigned to one of four treatments: CIDR, normal MGA, high MGA, or control by age, days postpartum, body condition, and body weight. All cows were fed carrier (0.9072 kg x cow(-1) x d(-1)) with (normal MGA, 0.55 mg/kg; high MGA, 4.41 mg/kg) or without MGA for 7 d (d -6 to 0). On d -6, CIDR were inserted and then removed on d 0. Estrous behavior was monitored continuously from d -6 until 29 using HeatWatch electronic mount detectors. Blood was collected on d -13, and three times weekly from d -6 to 29. Treatment influenced (P = 0.03) the percentage of cows that were detected in standing estrus. Beginning on d 2, more CIDR-treated cows had exhibited standing estrus compared with high MGA-treated or control cows, but CIDR- and normal MGA-treated cows did not differ. The percentage of CIDR-treated cows that had ovulated was greater (P < 0.05) than the percentage of normal MGA-treated, high MGA-treated, or control cows beginning on d 4. The percentage of cows that exhibited standing estrus before the first postpartum ovulation (CIDR = 65%, normal MGA = 57%, high MGA = 35%, control = 30%) did not differ (P = 0.09) among treatments. Luteal life span following the first ovulation postpartum and the percentage of cows with a normal luteal life span (i.e., progesterone > 1 ng/mL for > or = 10 d) was greater (P < 0.01) in CIDR-treated cows (14.0 +/- 0.8 d; 20/20, 100%) compared with normal MGA-treated (6.2 +/- 1.0 d; 3/13, 23%), high MGA-treated (9.6 +/- 1.0 d; 8/14, 57%), or control cows (6.1 +/- 0.9 d; 4/17, 24%), and greater (P < 0.03) in high MGA-treated cows than in normal MGA-treated or control cows. In the present study, treatment of early postpartum suckled beef cows with CIDR induced ovulation and initiated estrous cycles with a normal luteal life span in more cows than did treatment with MGA. Treatment with MGA (normal or high dose) did not induce ovulation earlier than in control cows, but a high dose of MGA increased the percentage of cows with normal luteal life spans following the first ovulation postpartum.     

miR-495-3p对山羊卵巢颗粒细胞功能的影响

旨在探究miR-495-3p对山羊卵巢颗粒细胞功能的影响及作用机制.本研究选取健康的3～4月龄大足黑山羊母羊,收集卵巢颗粒细胞,利用miR-495-3p模拟物(mimics)和抑制物(inhibitor)构建过表达和抑制模型,通过流式细胞术检测细胞凋亡和周期,ELISA分析颗粒细胞的雌二醇(E2)和孕酮(P4)分泌,采...     

A comparison of progestin-based protocols to synchronize estrus in postpartum beef cows

Two progestin-based protocols for estrus synchronization in postpartum beef cows were compared following treatment administration on the basis of estrous response, interval to and synchrony of estrus, and pregnancy. Cows were assigned to one of the two treatment protocols by age, body condition score (BCS), and days postpartum (DPP). The MGA Select-treated cows (MGA Select; n = 109) were fed melengestrol acetate (MGA; 0.5mg x cow-1 x d(-1)) for 14 d, fed carrier for 8 d, GnRH (100 microg of Cystorelin) was injected i.m. 12 d after MGA withdrawal, and PG (25 mg of Lutalyse) was administered i.m. 7 d after GnRH. Cows assigned to the 7-11 Synch protocol (7-11 Synch; n = 111) were fed carrier for 15 d, fed MGA for 7 d, injected with PG on d 22 (d 7 of MGA), injected with GnRH on d 26, and injected with PG on d 33. Mean BCS (4.8 +/- 0.1, MGA Select; 4.7 +/- 0.1, 7-11 Synch) and DPP (40 +/- 1, MGA Select; 40 +/- 1, 7-11 Synch) did not differ between treatments. Blood samples were collected 8 d and 1 d before feeding of MGA or carrier to determine the pretreatment estrous cyclicity (progesterone > or = 1 ng/mL; 10/109 [9%], MGA Select; 12/111 [11%], 7-11 Synch), and again at PG on d 33 to evaluate treatment response (81/109 [74%], MGA Select; 84/111 (76%), 7-11 Synch). Serum concentrations of progesterone at PG on d 33 differed (P < 0.01) between treatments (3.3 +/- 0.3 ng/mL [MGA Select] vs. 1.7 +/- 0.1 ng/mL [7-11 Synch]). HeatWatch was used for 6 d after PG on d 33 to detect estrus, and AI was performed 12 h after the onset of estrus. Estrous response did not differ between treatments (100/109 [92%], MGA Select; 101/111 [91%], 7-11 Synch). Mean interval to estrus (65 +/- 2.7 h, MGA Select; 52 +/- 1.8 h, 7-11 Synch) and synchrony of estrus differed (P < 0.01) between treatments. Synchronized conception and pregnancy rates (61/100 [61%], 61/109 [56%], MGA Select; 71/101 [70%], 71/111 [64%], 7-11 Synch), and final pregnancy rates (94/109 [86%], MGA Select; 99/110 [90%], 7-11 Synch) did not differ between treatments. In summary, estrous response and fertility did not differ among cows assigned to the MGA Select or 7-11 Synch protocols. Synchrony of estrus, defined as the variance in the interval to estrus from PG, however, was improved following treatment with the 7-11 Synch protocol.     

Effects of prostaglandin F(2alpha) and nitric oxide on the secretory function of bovine luteal cells

The objective of the present study was to investigate the influence of prostaglandin F(2alpha) (PGF (2alpha)) and nitric oxide (NO) on production of steroids and PGs by culturing bovine luteal cells obtained from ovaries on days 8-12 of the estrous cycle with a nitric oxide (NO) donor (Spermine NONOate), and a NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester dihydrochloride: L-NAME). When the cells were exposed for 24 h to PGF(2alpha) (10(-7)-10(-5) M), production of progesterone (P(4)) increased significantly at all doses used (P<0.05). Moreover, PGF(2alpha) stimulated PGF(2alpha) production (P<0.01), depressed testosterone (T) production (P<0.05), but did not affect synthesis of prostaglandin E(2) (PGE(2)). Spermine NONOate decreased P(4) production to 66%, 47% and 34% of the control concentration after treatment with 10(-5) M, 10(-4) M and 10(-3) M, respectively, but did not affect T production, and increased PGF(2alpha) synthesis (P<0.05) and PGE(2) (P<0.01) at all doses used. L-NAME increased production of P(4) (P<0.01) but did not affect (P>0.05) secretion of T, PGF(2alpha) and PGE(2). Estradiol-17beta (E(2)) was detectable on the level of sensitivity of assay and was not significantly altered by any treatments. The overall results suggest that PGF(2alpha) and NO produced locally in bovine CL play roles in the regulation of the secretory function of the bovine CL as auto/paracrine factors.     

A comparison of progestin-based protocols to synchronize ovulation and facilitate fixed-time artificial insemination in postpartum beef cows

The experimental objective was to compare pregnancy rates after fixed-time AI in postpartum suckled beef cows following administration of two progestin-based protocols to synchronize ovulation. Cows (n = 424) at three locations (n = 208, 122, and 92 per location) were stratified by age, BCS, and days postpartum (DPP) and assigned randomly to one of the two treatment protocols. The MGA Select-treated cows (MGA Select; n = 213) were fed melengestrol acetate (MGA, 0.5 mg x cow(-1) x d(-1)) for 14 d and carrier for 8 d, and then GnRH (100 microg i.m. Cystorelin; d 26) was injected 12 d after MGA withdrawal, and PG (25 mg i.m. Lutalyse) was administered 7 d after GnRH. Cows assigned to the 7-11 Synch protocol (7-11 Synch; n = 209) were fed carrier for 15 d and MGA for 7 d, and then injected with PG on d 22 (d 7 of MGA), GnRH on d 26, and PG again on d 33. Artificial insemination was performed at fixed times for cows in both treatments at 60 or 72 h after d 33 PG for 7-11 Synch and MGA Select groups, respectively. All cows were injected with GnRH (100 microg of i.m. Cystorelin) at AI. There was no treatment x location interaction for age (P = 0.90), BCS (P = 0.64), or DPP (P = 0.93), and the results were therefore pooled for the respective treatments (age [7-11 Synch, 5.5 +/- 0.2; MGA Select, 5.5 +/- 0.2], BCS [7-11 Synch, 5.7 +/- 0.1; MGA Select, 5.6 +/- 0.1], and DPP [7-11 Synch, 41.1 +/- 1.1; MGA Select, 42.1 +/- 1.1]). Blood samples were collected 8 and 1 d before MGA or carrier to determine pretreatment estrous cyclicity (progesterone >or=1 ng/mL; 7-11 Synch, 59/209 [28%]; MGA Select, 54/213 [25%]; P = 0.50) and again on d 33 PG to evaluate treatment response as a percentage of cows with progesterone concentrations in serum >or=1ng/mL (7-11 Synch, 184/209 [88%]; MGA Select, 177/213 [83%]; P = 0.15). Pregnancy rates resulting from fixed-time AI did not differ (P = 0.25) between treatments (7-11 Synch, 128/209 [61%]; MGA Select, 142/213 [67%]), nor did pregnancy rates (P = 0.77) at the end of the breeding season (7-11 Synch, 198/208 [95%]; MGA Select, 204/213 [96%]). These data indicate that pregnancy rates were comparable after fixed-time AI, following administration of the 7-11 Synch and MGA Select protocols. Both protocols provide opportunities for beef producers to use AI and eliminate the need to detect estrus.     

Synchronization of estrus in beef cattle with norgestomet and estradiol valerate

Fifty-six cows received a norgestomet implant and an injection of norgestomet and estradiol valerate; half (n = 28) received 500 IU equine chorionic gonadotrophin (eCG) at implant removal, 9 d later. A third group (n = 25) received 2 doses of cloprostenol (500 micrograms) 11 d apart. Estrous rate was higher (P < 0.05) for cows given norgestomet and estradiol plus 500 IU eCG (75.0%) than for those receiving cloprostenol (44.0%); for those receiving norgestomet and estradiol alone, it was intermediate (67.8%). Pregnancy rates to artificial insemination (after estrus or timed) were higher (P < 0.05) for cows given norgestomet and estradiol than for those given cloprostenol (23 of 28, 82.1% vs 13 of 25, 52.0%), and intermediate (67.8%) for those given norgestomet and estradiol plus eCG. In a second experiment, for heifers treated with norgestomet and estradiol plus eCG (n = 15) or with 2 doses of cloprostenol (n = 16), estrous rates were 66.7% vs 56.2% (P > 0.5), ovulation rates were 100.0% vs 81.2% (P = 0.08), intervals from implant removal or cloprostenol treatment to estrus were 48.0 +/- 4.4 hours vs 61.3 +/- 7.0 hours (P = 0.12) and to ovulation were 70.4 +/- 4.4 hours vs 93.2 +/- 7.5 hours (P < 0.01), respectively; pregnancy rates were 41.7 and 35.7%, respectively (P > 0.5). Norgestomet and estradiol were as good as (heifers) or superior to (cows) a 2-dose cloprostenol regimen. In cows given norgestomet and estradiol, injecting eCG at implant removal did not significantly improve estrous or pregnancy rates.     

RNA干扰水牛MTPN基因对颗粒细胞增殖、凋亡及激素分泌的影响

本研究旨在探讨敲低MTPN基因对体外培养水牛颗粒细胞增殖、凋亡及雌激素、孕酮分泌的影响。应用RNAi技术敲低颗粒细胞中MTPN基因的表达水平,通过实时荧光定量PCR方法检测体外培养水牛颗粒细胞中MTPN基因及增殖和周期相关基因的表达情况,CCK-8法检测细胞增殖,借助流式细胞仪检测细胞周期的分布,采用ELISA试剂盒检测细胞培养液中雌激素与孕酮含量。结果显示,经siRNA(si-MTPN)转染颗粒细胞后,MTPN基因相对表达量下降60%(P<0.01);细胞增殖受到显著抑制(P<0.05),G1期细胞数量下降,S期细胞数量上升,G2期细胞数量极显著上升(P<0.01),细胞被阻滞在G2期;增殖与周期相关基因Cyclin D2、Cytochrom C表达量显著上升(P<0.05),Caspase9、Fas基因表达量极显著上升(P<0.01);ELISA检测雌激素和孕酮分泌水平均显著下降(P<0.05)。综上表明,敲低MTPN基因能通过调控相关基因的表达抑制体外培养水牛颗粒细胞的增殖及雌激素、孕酮的分泌水平,为阐明MTPN基因参与家畜卵泡发生的分子机制提供参考。     

The effect of interferon-tau and ovarian steroids on the proliferation of bovine endometrial cells in vitro

The goal of this study was to evaluate the effect of various concentrations of interferon-tau (IFN-tau) with or without steroid hormones, 171 estradiol or progesterone, on the proliferation of bovine endometrial cells in vitro. Endometrial epithelial and stromal cells were isolated from the uterus of cows during the early estrus cycle (2-3 days) and incubated with different doses of IFN-tau with or without steroid hormones. The proliferation was determined by the MTT test in 48, 96, and 144 h of incubation. An antiproliferative activity of IFN-tau was observed both in epithelial and stromal cells cultured in RPMI 1640 medium supplemented with 10% FBS or. serum replacement. However, epithelial cells were more sensitive to antiproliferative action of interferon-tau. It;s activity was dose-and time-dependent. The inhibition of epithelial cell proliferation by 50% (ED50) was achieved at concentrations of 500 U/ml, 340 U/ml, and 8.8 U/ml of IFN-tau after 48, 96, and 144 h of incubation, respectively. None of the doses of IFN-tau (10-10.000 U/ml) used inhibited stromal cell proliferation in 50%. The most effective dose of IFN-tau inhibiting stromal cell proliferation was 10.000 U/ml, which decreased cell growth by 17.08%, 22.87%, and 2.6% after 48, 96, and 144 h of incubation, respectively. Steroid hormones, 17beta estradiol and progesterone, added to the culture of stromal cells with or without IFN-tau did not significantly modulate stromal cell growth. In contrast, a high concentration of progesterone (10(-5) M) alone significantly enhanced stromal cell growth. Progesterone at low, physiological concentrations (10(-7)- 10(-9) M) ameliorated the antiproliferative activity of IFN-tau, especially at the 10(-9 )M concentration. At this concentration, the stimulatory effect on stromal cell growth was observed. The mechanisms of such response are not entirely clear but may arise from the influence of IFN-tau on progesterone down regulation of its own receptor. Depicted activity of IFN-tau may find usefulness in therapy of neoplastic disorders.     

Impact of norgestomet supplementation during early luteal phase on subsequent luteal profiles and conception rate in buffalo: a preliminary study

The current study was aimed to establish the impact of progesterone supplementation (norgestomet progestagen) between days 4 to 10 post-ovulation on subsequent luteal profile and conception rate in buffaloes. The 28 Murrah buffaloes of second to fourth parity, having normal reproductive organs, were estrus synchronized by double PGF_2α protocol at 11 days apart. The buffaloes were inseminated during mid- to late estrus and thereafter repeated at 24 h interval. The buffaloes were randomly assigned into two groups: (1) control (no treatment, n?=?14) and (2) treatment group (CRESTAR ear implant, n?=?14). The CRESTAR ear implant (3 mg, norgestomet progestagen) was inserted subcutaneous between days 4 to 10 post-ovulation. The ovaries were scanned at estrus and thereafter on days 4, 10, 16, 21, and 40 post-ovulation to examine the preovulatory follicle (POF) and corpus luteum (CL) diameter. Each ultasonography was followed by blood sample collection for analysis of plasma progesterone concentrations following ovulation. The conception rate was similar (p?>?0.05) between treated and control buffaloes. The pregnant buffalo of the control group had larger (p?<?0.05) POF diameter than nonpregnant counterparts. The CL diameter was similar (p?>?0.05) in both treated and untreated control as well as in their pregnant and nonpregnant buffaloes of the respective groups. The plasma progesterone concentrations were higher (p?<?0.05) in the treatment group on the day 10 post-ovulation as compared to the control buffaloes. It is concluded that norgestomet supplementation had no impact on conception rate and CL diameter but enhances the plasma progesterone concentrations following treatment in buffaloes.     

Relationship of pre- and post-ovulatory gonadotropin concentrations to subnormal luteal function in postpartum beef cattle

Early weaning of calves from anestrous cows results in formation of short-lived corpora lutea (CL) unless the animals are pretreated with a progestagen (norgestomet). This study was conducted to investigate the relationship between pre- and post-ovulatory gonadotropin secretion and luteal lifespan. Postpartum beef cows were assigned randomly into two groups, control (n = 5) and norgestomet (implant given at weaning for 9 d; n = 7). Calves from all cows were weaned 30 to 33 d postpartum. Coccygeal artery cannulas were placed into cows in the control group 1 d prior to weaning and 2 d before implant removal in cows in the norgestomet group. Plasma for determination of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol-17 beta (E) and progesterone (P) was collected daily at 10-min intervals for 6 h from weaning (control) or the day prior to implant removal (norgestomet) to estrus (d 0) and on d 2, 4 and 6 following estrus. Average interval (X +/- SE; P less than .05) from weaning to estrus or implant removal was 4.2 +/- .8 and 2.3 +/- .2 d for the control and norgestomet groups, respectively. Estrous cycle length for the control group was 12.4 +/- 1.8 d compared with 20.4 +/- .3 d for the norgestomet group (P less than .05). Four of five control cows had an estrous cycle length of 7 to 14 d; all cows in the norgestomet group and the remaining control cow had an estrous cycle of normal length (16 to 21 d).2+ estrus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Estrogen and Progesterone on Canine Endometrial Stromal Cells Proliferation and Progesterone Receptor Expression

In this study different digestion and isolation methods were applied to obtain canine endometrial stromal cells (ESCs), different concentration levels were set for estrogen (E₂) and progesterone (P₄), then MTT method was used to measure their effect on cell proliferation in vitro, also cell immunohistochemistry was used for cell identification and measurement of effect on progesterone receptors (PR) expression.The results indicated that E₂ (15, 30 and 100 pg/mL) showed no significant regulations on both cell proliferation and PR expression, P₄ (15 and 30 ng/mL) had significant promoting effect on proliferation of ESCs (P<0.05), P₄ (3, 15 and 30 ng/mL) showed significant inhibitory effect on PR expression (P<0.05), the regulation level was related to concentration and acting time.     

Resynchronization of estrus in beef cattle: ovarian function, estrus and fertility following progestin treatment and treatments to synchronize ovarian follicular development and estrus

The objective was to optimize rebreeding of nonpregnant, previously inseminated beef cattle. In Experiment 1, 43 cows received a used intravaginal progesterone-releasing insert (IVPRI; Days 0-7) 12.3 d after ovulation and received concurrently no treatment, 100 microg gonadotropin releasing hormone (GnRH), 1 mg estradiol cypionate (ECP), or 150 mg progesterone. Emergence of a new ovarian follicular wave was most synchronous (P < 0.0001) in the GnRH group. In Experiment 2, 675 heifers were given GnRH or no treatment on Day 0, fed melengestrol acetate (MGA; 0.5 mg/head/d) from Days 0-5 (Day 0 = 13-14 d after timed insemination; TAI), given 0.5 mg ECP or nothing on Day 7, and reinseminated 6-12 h after onset of estrus. Estrus was more synchronous (P < 0.05) in heifers given GnRH versus no treatment on Day 0. In Experiment 3, 317 TAI heifers were resynchronized with either MGA or a used IVPRI with or without ECP on Day 7; estrus was more synchronous (P < 0.05) and pregnancy rates were higher (54.1% versus 39.2%, P < 0.05) in heifers given a used IVPRI than those fed MGA. For resynchronization of heifers, pregnancy rates were not significantly improved with GnRH treatment, but were higher with a used IVPRI than with MGA.     

Effects of 21-day treatment with melengestrol acetate (MGA) with or without subsequent prostaglandin F2 alpha on synchronization of estrus and fertility in beef cattle

Beef cattle were treated to synchronize estrus using one of three procedures, and effects on subsequent endocrine responses and fertility were studied. Procedures were 1) feeding .5 mg.head-1.d-1 of melengestrol acetate (MGA) for 21 d (M), 2) feeding .5 mg.head-1.d-1 of melengestrol acetate for 21 d followed 14 d later by a single injection of prostaglandin F2 alpha (M + P) and 3) two injections of prostaglandin (PGF) 14 d apart (P). In Exp. 1, 94 beef cows were assigned to be artificially inseminated 12 h after detection of estrus. Procedures for synchronizing estrus did not affect the proportion of cows observed in estrus within 7 d (mean = 70.2%). However, conception rate of cows treated with MGA alone was lower (P less than .01) than that of cows treated with PGF alone (31.8 vs 78.3%). The conception rate of cows in the M + P group was intermediate (57.1%) but greater than that of cows treated with MGA alone (P less than .10). In Exp. 2, 18 heifers were observed for estrus four times daily and bled daily from 1 wk before predicted estrus until second estrus or 35 d post-treatment. Heifers treated with MGA alone maintained lower concentrations of progesterone and higher concentrations of estradiol-17 beta before first estrus than heifers treated with MGA and PGF or PGF alone (P less than .01). Conception rate following insemination was lower after long-term feeding of MGA than after two injections of PGF. Delaying insemination until after a PGF-shortened cycle 14 d after MGA resulted in an intermediate conception rate.(ABSTRACT TRUNCATED AT 250 WORDS)