DNA末端定量及其应用的研究

为定量DNA断裂末端评价DNA的降解程度,以饱和标记(TdT)法定量DNA末端最大标记量(L_max);并用流式细胞分析(FCA)、原位DNA末端标记(TUNEL)和琼脂糖电泳检测或标记DNA降解片段.结果发现: a.TdT法最低检测限5 ng DNA,线性范围为5～5 000 ng,较检测DNA降解片段的琼脂糖电泳敏感200倍以上;b.地塞米松(DEX)诱导淋巴瘤(Raji)细胞凋亡产生的L_max呈DEX剂量与诱导时间依赖性增加;c.自发性高血压大鼠(SHR)心肌的L_max呈年龄依赖性增加并与正常对照鼠(WKY)相比差异非常显著(P＜0.01);d.TdT法定量L_max与FCA、TUNEL或电泳分析的相关性良好(r＞0.98).TdT法定量L_max敏感、特异、准确,可应用于分子生物学、细胞生物学,尤其是细胞凋亡动力学DNA降解评价的定量研究.     

瑶药红牛膝质量控制方法研究

瑶药红牛膝为苋科植物柳叶牛膝(Achyranthes longifolia)的根及根茎,为瑶族地区习用药材,目前仅有个别省区收载了其质量控制方法。该文对广西壮族自治区区内采集的14批红牛膝药材进行了性状鉴别、显微鉴别和薄层色谱鉴别,并采用飞诺美Gemini-NX C₁₈色谱柱(250 mm × 4.6 mm,5 μm),以乙腈-水溶液(15:85)进行等度洗脱,柱温25 ℃,流速1.0 mL·min^-1,检测波长243 nm,建立红牛膝中β-蜕皮甾酮的HPLC含量测定方法。结果表明:(1)红牛膝药材性状、显微鉴别特征明显,显微鉴别可见栓内层、维管束、异常维管束、导管、射线细胞、草酸钙砂晶或方晶等。(2)薄层色谱分离好,β-蜕皮甾酮和人参皂苷R_O对应的斑点清晰。(3)含量测定中,β-蜕皮甾酮在5.606～224.224 μg范围内线性关系良好(r=0.999 9),平均加样回收率为97.68%,RSD为1.37%(n=6)。(4)14批样品中β-蜕皮甾酮含量为0.005%～0.158%,平均含量为0.095%。该研究建立了完整的显微鉴别、薄层鉴别及含量测定方法,为合理评价红牛膝质量和临床合理用药提供科学依据。     

秦岭天然林凋落物去除对土壤团聚体稳定性及细根分布的影响

研究凋落物去除对秦岭天然林土壤团聚体稳定性及细根分布特征的影响机制,为森林生态治理提供理论依据。结果表明:(1)凋落物去除导致云杉林10-40 cm土层土壤中粒径大于0.25 mm水稳性团聚体含量(WR_0.25)较对照显著降低(P<0.05);混交林10-40 cm土层的大于0.25 mm机械稳定性团聚体含量(DR_0.25)显著降低(P<0.05);红桦林表层0-10 cm土壤团聚体的平均质量直径(MWD)显著降低(P<0.05);(2)凋落物去除导致三种天然林0-20 cm土层的细根生物量密度(FRBD)较对照显著降低44.18%-57.24%,细根体积(FRV)显著降低24.64%-60.41%;三种天然林中红桦林0-40 cm各土层的FRV最高;(3)凋落物去除导致云杉林0-40 cm土层的土壤容重较对照显著增加5.24%-13.04%,三种天然林0-40 cm土层的土壤有机碳含量显著降低7.92%-25.21%,全氮含量显著降低10.17%-18.10%;(4)相关分析表明,云杉林土壤的团聚体破坏率(PAD)和土壤不稳定团粒指数(E_LT)与FRBD、细根根长密度(FRLD)和FRV均呈极显著负相关,混交林土壤的PAD与FRBD呈极显著负相关关系(r=-0.814),红桦林土壤团聚体MWD与FRBD、FRLD和细根比根长(FSRL)均呈显著负相关关系(r=-0.777、-0.771和-0.786)。综上,凋落物去除在总体上降低了天然林土壤团聚体的稳定性及有机碳和全氮含量,并且不利于林木细根的生长。     

高分辨电泳分离短串联重复序列DNA片段

采用多相缓冲系统,在成层胶T＝5%,C＝2.6%, 分离胶T＝8%,C＝5%的条件下用聚丙烯酰胺凝胶电泳对人类短串联重复序列(STR)DNA片段进行分离.其中,成层胶内主要缓冲成分为2-二羟乙基亚胺-三羟甲基甲烷(Bistris)、H₂SO₄及N、N-2(羟乙基)甘氨酸(Bicine) ; 而分离胶以Tris、H₂SO₄及2-二羟乙基亚胺-三羟甲基甲烷(Bistris)为主,构成多相缓冲系统.DNA片段在成层胶中被有效地压缩, 在分离胶内又可完全解压缩,使其按片段大小分离;从而达到提高分辨率的目的.     

Cyclic AMP and theophylline enhance DNA synthesis in L cells stimulated with anti-actin IgG and [(IgG)2protein A]2 complex by recruiting cells into S-phase

Surface binding of anti-actin IgG alone or in a M_r = 716 000 [(IgG)₂Protein A]₂ complex results in a stimulation of DNA synthesis and cell growth in L cells. Cyclic-AMP (0.01–1.0 mM) added to such cell cultures augmented DNA synthesis as measured by incorporation of [³H]thymidine into DNA. Theophylline (0.1–1.0 mM), a phosphodiesterase inhibitor which prevents enzymatic breakdown of cAMP, had similar effects, but cGMP (0.01–1.0 μM) reversed the effects of cAMP and theophylline upon DNA synthesis. Analysis of the cell cycle by flow cytometry revealed that antibody produced a shift (7%) of cells from the G₁-phase to the S-phase (DNA-synthetic) of the cell cycle at 72 hr of incubation. Addition of cAMP (0.5 mM) to cell cultures, however, produced significant shifts of antibody stimulated cells from G₁-phase to S-phase at all time points measured, i.e., 24 (12%),48 (22%),72 hr (23%). Thus, antibody recruited cells into S-phase of the cell cycle and cAMP greatly augmented the effect. These observations suggest that the mechanism of activation of L cell growth by antibody to surface antigens involves a recruitment of cells into the DNA-synthetic phase and that the effect may be mediated by cAMP.     

外源H2O2对盐碱胁迫下苹果矮化砧木幼苗生理特性的影响

以2年生苹果矮化砧木M9 T337为试材,采用盆栽试验法,设置浇灌清水(CK)和盐碱胁迫(0.1 mol/L NaCl+NaHCO₃溶液)+ 喷施5种浓度的H₂O₂ ［0(T₁)、0.2 mmol/L(T₂)、0.4 mmol/L(T₃)、0.6 mmol/L(T₄)、0.8 mmol/L(T₅)］ 处理,测定各处理叶片叶绿素含量、光合气体交换参数、渗透调节物质含量、抗氧化酶活性和细胞膜透性,并利用相关性与主成分分析进行综合评价,以探讨外源过氧化氢(H₂O₂)增强其盐碱耐性的生理机制。结果表明：(1)随着盐碱胁迫(T₁)的时间延长,M9 T337幼苗叶片叶绿素a(Chl a)含量、叶绿素b (Chl b)含量、叶绿素总量(Chl t)、净光合速率(P_n)、气孔导度(G_s)、蒸腾速率(T_r)、可溶性蛋白(SP)含量均呈逐渐下降趋势;胞间CO₂浓度(C_i)、可溶性总糖(TSS)含量、脯氨酸(Pro)含量、过氧化氢酶(CAT)活性、抗坏血酸过氧化物酶(APX)活性、相对电导率(REC)、丙二醛(MDA)含量均呈上升趋势;超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性均呈先升后降趋势。(2)与CK相比,盐碱胁迫+外源H₂O₂(T₂- T₅)处理后M9 T337幼苗叶片各指标均呈现不同幅度变化,且存在明显浓度效应,并以T₃(0.4 mmol/L H₂O₂)处理叶片的Chl a、Chl b、Chl t、SP和G_s降幅最小,C_i、REC、MDA升幅最小,TSS、Pro、APX升幅最大。(3)M9 T337幼苗叶片P_n与T_r、G_s、Chl a、Chl b、Chl t、SP、SOD、POD呈显著正相关,与C_i、MDA、CAT、APX、REC呈显著负相关。(4)综合评价表明,各处理对M9 T337幼苗叶片生理特性的效应依次为：CK>T₃>T₄>T₂>T₅>T₁。研究发现,叶面喷施适宜浓度H₂O₂可有效改善盐碱胁迫下M9 T337幼苗光合能力,显著提高抗氧化酶活性和渗透调节物质的含量,降低细胞膜透性,从而达到缓解盐碱胁迫的作用,并以0.4 mmol/L H₂O₂处理效果最佳。     

热激诱导对高温胁迫下百合相对电导率及电阻抗参数的影响

为了研究电阻抗图谱法对百合耐热性测定的有效性，采用45 ℃温水热激百合鳞茎0、15、30、45、60 min后，测定植株叶片的相对电导率和电阻抗参数对38 ℃ 高温胁迫的反应。结果表明，随胁迫时间延长各处理的相对电导率均呈先降低后升高的趋势；4和5 d时各处理的相对电导率均低于对照，其中热激30 min的值最低。表明热激诱导可提高百合的耐热性。随高温胁迫时间延长，高频电阻率(r)、低频电阻率(r₁)、胞外电阻率(r_e)、胞内电阻率(r_i)和弛豫时间（τ）的变化趋势相似，整体均呈先上升后下降的趋势。相关分析表明，植株受不同时间热胁迫时，r_e 与相对电导率呈负相关，r_i 与相对电导率呈正相关，其中r_e 是测定百合耐热性最适宜的参数。     

蚕豆幼苗光合特性对土荆芥挥发性物质胁迫的响应

以蚕豆(Vicia faba)为受体,采用盆栽试验评价了入侵植物土荆芥(Chenopodium ambrosioides)挥发油及其两个主要成分α-萜品烯和对伞花素对受体光合特性的影响。结果表明:土荆芥挥发油及其两个主要成分不同程度地影响了蚕豆叶片的特性。挥发油处理显著降低了净光合速率(P_n)、气孔导度(G_s)、蒸腾速率(T_r)、最大光化学效率(F_v/F_m)、实际光化学效率(ΦPSⅡ)和叶绿素含量,但增加了胞间CO₂浓度(C_i),这种效应表现为剂量和时间双重效应,高剂量挥发油处理的这种效应是不可逆的; 与对照相比,α-萜品烯处理组的P_n、F_v/F_m和ΦPSⅡ降低,C_i、G_s和T_r上升,停止处理后,各参数均趋于对照水平; 整体来看,对伞花素对蚕豆幼苗的光合特性影响不大。上述研究结果说明,土荆芥化感胁迫对受体光合特性的影响是诸多化感物质协同作用的结果,并非由单一组分决定。     

基于叶绿体基因组数据的芸香科属间系统发育初步分析（专题约稿）

该研究在人工控制水分条件下,设置3个干旱胁迫处理,选用3个主栽油菜品种‘陇油10号’、‘陇油2号’、‘青杂5号’幼苗进行盆栽试验,测定干旱胁迫条件下叶片相对含水量、叶绿素含量、光合气体交换参数及叶绿素荧光参数等指标,考察各指标在干旱胁迫过程中的变化特征,并通过主成分分析(PCA)和隶属函数法评价品种的抗旱性及其主要响应因子,以揭示西北地区油菜幼苗响应干旱胁迫的光合调控机制。结果表明：(1)各品种油菜幼苗的叶片相对含水量(RWC)均随干旱胁迫程度的递增而逐渐降低,最大水分亏缺(WSD)却逐渐上升。(2)各品种油菜幼苗叶片的叶绿素a含量、叶绿素总含量随着干旱胁迫程度的递增而先增加后递减,且同一种幼苗在不同处理间差异显著。(3)各品种油菜幼苗叶片的净光合速率(P_n)、水分利用效率(WUE)、单株生物量、气孔导度(G_s)、胞间CO₂浓度(C_i)均在受到干旱胁迫时迅速降低,且同一品种幼苗在不同处理间差异显著,而其叶片蒸腾速率(T_r)在干旱胁迫下无显著变化。(4)各品种油菜幼苗叶片光化学猝灭系数(qP)和非光化学猝灭系数(NPQ)随着干旱胁迫程度的递增先增加后递减,最大光化学效率(F_v/F_m)和电子传递速率(ETR)在受到干旱胁迫时迅速降低,且同一品种幼苗在不同处理间差异显著。(5)主成分分析结果表明,在油菜幼苗受到干旱胁迫时RWC、C_i、G_s、P_n、WUE、叶绿素总含量、叶绿素a含量和NPQ起主要调控作用;隶属函数法综合评价表明,3个品种油菜幼苗耐旱能力由高到低依次为‘陇油10号’＞‘陇油2号’＞‘青杂5号’。     

新型MGB探针在沙眼衣原体实时PCR检测中的应用

为建立基于TaqMan-MGB探针的沙眼衣原体DNA荧光定量PCR检测方法,探讨其临床应用价值,用 PCR法扩增沙眼衣原体隐蔽质粒pLVG440 2 464～2 980 nt段,并克隆入pMD18-T载体用作参比模板,设计一对引物和一个TaqMan-MGB探针,优化反应条件,建立沙眼衣原体DNA荧光定量PCR检测系统,并运用该系统同时应用连接酶链式反应(LCR)法对临床标本进行检测.结果显示所建立的沙眼衣原体DNA荧光定量PCR检测系统,最低检测限度为1 DNA拷贝每反应;在10⁰～10⁹ DNA拷贝每反应范围内,C_t值(每个反应管内的荧光信号达到设定的域值时所经历的循环数）和DNA拷贝数呈线性关系（r＞0.990）;对临床标本检测结果同LCR分析结果吻合率为100%.以上结果表明,所建立的基于TaqMan-MGB探针的沙眼衣原体DNA荧光定量PCR检测系统具有敏感性高、特异性强和线性检测范围广等特点,适用于对沙眼衣原体进行大规模筛选.     

Relationship between concentrations of cortisol in ovarian follicular fluid and various biochemical markers of follicular differentiation in cyclic and anovulatory cattle

Concentrations of cortisol were determined in pooled fluid of small (less than 10 mm) and large (greater than or equal to 10 mm) follicles of cyclic cattle (Exp. 1), and in fluid of the largest follicle of 17 post-partum anovulatory cows (Exp. 2). In Exp. 1, concentrations of cortisol in small follicles were greater (P less than 0.05) than in large follicles (14.7 versus 13.2 ng/ml), and varied significantly with stages of the cycle; small and large follicles had the highest cortisol concentration during the early luteal phase of the cycle. Large follicles had 2-fold greater concentrations of oestradiol than did small follicles, whereas small follicles had 2-fold greater concentrations of androstenedione than did large follicles. Across pools of follicular fluid, cortisol concentrations were correlated only to androstenedione concentrations (r = 0.65, P = 0.07). In Exp. 2, concentrations of cortisol did not significantly differ between oestrogen-active (oestradiol greater than progesterone in follicular fluid) and oestrogen-inactive (progesterone greater than oestradiol) follicles, although oestrogen-active follicles had a 24-fold greater concentration of oestradiol than did oestrogen-inactive follicles. Cortisol concentrations were correlated to hCG binding capacity of thecal cells (r = -0.35, P = 0.08) and to follicular diameter (r = 0.45, P less than 0.05). These results suggest that normally fluctuating concentrations of cortisol in follicular fluid of cattle play little or no active role in follicular differentiation in vivo.     

Characterization of in vitro growth of bovine preantral ovarian follicles: A preliminary study

Preantral follicles were mechanically extracted from bovine ovaries collected at slaughter. On average 90 early growing follicles were collected per ovary. The follicles were cultured for 7 days in Dulbecco's modified Eagle medium F-12 nutrient mixture + 10% fetal calf serum + 10% newborn calf serum. The individual follicular diameter and the follicular DNA content were recorded at the start and the end of culture. The follicular DNA content was estimated by a microfluorometric method using the fluorochrome, 4'-6 diamidino-2-phenylindol-2HCl (DAPI). After culture, 86% of the follicles looked morphologically normal. Of the surviving follicles, 73% showed an increase in diameter varying between 5 and 30 mum, and the follicular DNA content increased from 1 to 72% under the same culture conditions. These results indicate that bovine preantral follicles can survive in vitro and even grow in a single medium.     

Magnetic resonance image attributes of the bovine ovarian follicle antrum during development and regression

The magnetic resonance images and maps of bovine ovaries acquired at defined phases of follicular development and regression were studied to determine whether magnetic resonance image attributes of the follicular antrum reflect the physiological status of dominant and subordinate ovarian follicles. Ovariectomies were performed at day 3 of wave one, day 6 of wave one, day 1 of wave two and at >/= day 17 after ovulation. The timings of ovariectomies were selected to acquire growing, early static, late static and regressing follicles of the first wave and preovulatory follicles of the ovulatory wave. Pre-selection and subordinate follicles were also available for analysis. Serum samples were taken on the day of ovariectomy and follicular fluid samples were taken after imaging. Numerical pixel value and pixel heterogeneity in a spot representing approximately 95% of the follicular antrum were quantified in T(1)- and T(2)-weighted images. T(1) and T(2) relaxation rates (T(1) and T(2)), proton density, apparent diffusion coefficients and their heterogeneities were determined from the computed magnetic resonance maps. The antra of early atretic dominant follicles showed higher T(2)-weighted mean pixel value (P < 0.008) and heterogeneity (P < 0. 01) and lower T(2) heterogeneity (P < 0.008) than growing follicles. Subordinate follicles in the presence of a preovulatory dominant follicle had higher T(1), T(1) heterogeneity, proton density, proton density heterogeneity, and lower mean pixel value in T(1)-weighted images than subordinate follicles of the anovulatory wave (P < 0.04). T(1) relaxation rate heterogeneity and proton density heterogeneity were positively correlated with follicular fluid oestradiol concentration (r = 0.4 and 0.3; P < 0.04). T(2) relaxation rate heterogeneity was positively correlated with follicular fluid progesterone concentration (r = 0.4; P < 0.008). Quantitative differences in magnetic resonance image attributes of the antrum observed among phases of follicular development and regression coincided with changes in the ability of the dominant follicle to produce steroid hormones and ovulate, and thus were indicative of physiological status and follicular health.     

Follicular dynamics prior to and during superovulation in heifers

The present ultrasonographic study examined the relationship between certain follicular parameters and the superovulatory response in gonadotropin-stimulated heifers. Thirty heifers received a total of 35 mg FSH twice daily for 4 d and 0.75 mg cloprostenol were given to induce luteolysis and estrus at 72 h after the initial FSH injection. Transrectal ultrasonography was performed once daily from 1 or 2 d before the initial FSH injection and until the day of estrus. The number of small (2 to 4 mm), medium (5 to 9 mm), and large (>/=10 mm) size follicles as well as the diameter of the large follicles were recorded. Embryos were recovered non-surgically 6 or 7 d after estrus, and the number of corpora lutea was determined by palpation per rectum. Heifers with >2 or 0.05). The number of large follicles and the sum of medium and large follicles were positively correlated (r=0.43 and r=0.54, respectively; P<0.05) with the number of corpora lutea palpated on the day of embryo recovery (6 to 7 d after estrus). In conclusion, there was an effect of the day relative to initiation of FSH treatment on all follicular categories in heifers responding positively to superovulation, and there was no effect of side (left or right ovary) or of corpus luteum diameter (ipsilateral or contralateral).     

Modulation of intrafollicular oestradiol in explanted hamster follicles does not affect oocyte meiotic status

Mature antral follicles were collected from PMSG-primed hamsters before the LH surge on Day 4 of the oestrous cycle and were incubated individually for 6 or 10 h under 95% O2 and 5% CO2. The relationship between follicle size and follicular fluid volume was established, and both the oestradiol output during incubation and the LH sensitivity of the follicle with respect to induction of meiotic resumption were determined. The data obtained from these initial studies were then applied to experiments in which the oestrogenic status of follicles was altered by pressure injection of solutions into the antral cavity to result in known concentrations in follicular fluid of oestradiol (0-500 microM), the anti-oestrogens, tamoxifen (0-200 microM) or enclomiphene citrate (0-500 microM), or an oestradiol-specific antiserum (1:200 dilution of serum). The proportion of immature oocytes (% GV) was determined cytogenetically after follicular incubation. Significant correlations were established between follicle size and both follicular fluid volume (r = 0.90) and follicular oestradiol accumulation (r = 0.96). The minimum concentration of LH tested which induced maximal meiotic resumption (0% GV) was 0.5 microgram/ml. The % GV was not significantly reduced by intrafollicular injection of either of the anti-oestrogens over the concentration ranges tested, or of the oestradiol-specific antiserum, which was injected in an amount adequate to bind all oestradiol accumulated during the incubation. Intrafollicular injection of oestradiol failed to increase the % GV after follicular incubation in the presence of 0.5 microgram LH/ml. From these results we conclude that it is unlikely that oestradiol plays a primary role in the maintenance of meiotic arrest in follicle-enclosed hamster oocytes in vitro.     

The effect of short-term nutritional supplementation of ewes with lupin grain (Lupinus luteus), during the luteal phase of the estrous cycle on the number of ovarian follicles and the concentrations of hormones and glucose in plasma and follicular fluid

An experiment was conducted using 16 cyclic, Welsh Mountain ewes during the luteal phase of the estrous cycle to determine the effect of a 5-day period of feeding a high-energy high-protein diet (lupin grain; 500 g/day) on folliculogenesis and on the plasma concentrations of glucose, insulin, follicle stimulating hormone (FSH) and estradiol-17beta, and on the follicular fluid concentrations of glucose, inhibin A, estradiol-17beta, androstenedione and progesterone. Average weight did not differ between lupin-fed and control groups during the experiment. There was a trend for the number of small and large follicles to increase in the lupin-fed group. The plasma concentrations of glucose (P=0.012) and insulin (P=0.007) were higher during the feeding period in lupin-fed ewes. The plasma concentrations of FSH and estradiol-17beta were not significantly different. The mean follicular fluid concentration of glucose (small follicles; <3.5 mm) from lupin-fed ewes was elevated (P=0.010) and progesterone lowered (P=0.034) compared to controls. The follicular fluid concentrations of estradiol-17beta, androstenedione and inhibin A were not significantly different. The follicular fluid concentration of estradiol-17beta was positively correlated with androstenedione (r=-0.241; P=0.001) and inhibin A (r=0.734; P< or =0.001) and glucose was negatively correlated with inhibin (r=-0.241; P=0.01), but not estradiol (r=0.075; P=0.410) or androstenedione (r=0.050; P=0.564). The lupin grain supplement increased the number of follicles as expected, but this increase was not significant. These changes were reflected in follicular fluid where lupin feeding increased the concentration of glucose and decreased the concentration of progesterone in follicles less than 3.5mm in diameter. These data suggest that the local ovarian actions of nutrients have a role in the mediation of nutritional influences on folliculogenesis.     

A comparison of hormone levels in follicle-lutein-cysts and in normal bovine ovarian follicles

Insulin-like growth factors 1 and 2 (IGF-1 and 2), oxytocin, progesterone, estradiol and ubiquitin were measured in bovine follicle-lutein-cysts and in follicular fluid after the classification of ovarian follicles by size (Class I = <4 mm; Class II = 5-8 mm; Class III = 9-12 mm; Class IV = preovulatory; Class V = cystic). It was found that IGF-1 concentrations increased during growth from 280 ng/ml in small follicles to 489 ng/ml in preovulatory follicles; IGF-2 appeared to remain constant in follicular fluid and in cysts (275 ng/ml). Oxytocin values were low in Class I, II and III follicles (30 pg/ml) but increased in preovulatory and cystic follicles (75 pg/ml). Estradiol increased significantly only in preovulatory follicles. Ubiquitin, a protein reflecting cellular replicative activity, could be found in bovine follicular fluid in high concentrations: 1.6 mug/ml in Class I,II and III follicles with the highest amounts in preovulatory follicles (2.3 mug/ml). In contrast with normal follicles, cysts were found to have a minimal concentration of ubiquitin (0.3 mug/ml). Progesterone levels were 5 times higher in cysts (325 ng/ml) and IGF-1 concentrations were markedly higher in cystic follicles (881 ng/ml) than in the other follicles. Simultaneously, maximum gene expression for IGF-1 was found in granulosa/lutein cells of cystic follicles (Class V), suggesting de novo synthesis of IGF-1. Between the different follicle classes progesterone, oxytocin and IGF-1 concentrations correlated positively (r=0.82). Hormonal levels in follicle-lutein-cysts indicated an arrested stage of insufficient luteinization as a possible result from the premature release of LH or from the release of amounts of LH inadequate to cause ovulation.     

Superovulatory response in a bovine model of reproductive aging

Two experiments were done to test the hypotheses that aging in cattle is associated with a reduced number of follicles recruited into an ovarian follicular wave, and a reduction in the ovarian response to gonadotropin treatment. Older cows (13-16 years of age) and their daughters (3-6 years of age) were treated with FSH for ovarian superstimulation four times over two consecutive years (31 and 33 superstimulations in old and young cows, respectively, experiments and years combined). In Experiment 1, ovulation was induced using LH. In Experiment 2, cumulus-oocyte complexes were collected by ultrasonographic-guided follicle aspirations before expected ovulations. The ovarian follicular and ovulatory responses were monitored daily by ultrasonography. Fewer 2-5mm follicles (P<0.01) were detected at the expected time of follicular wave emergence in older cows than in their daughters. After superstimulation, older cows had fewer follicles >or=6mm (P<0.01), and tended (P=0.1) to have fewer ovulations than their daughters (32+/-4 versus 40+/-3, respectively). There was a positive correlation in the response of individual cows to successive superstimulatory treatments (r>0.8; P<0.0001) and the number of detected ovulations from one year to the next (r=0.6; P=0.04). In conclusion, aging was associated with fewer 2-5mm follicles at follicular wave emergence and a lesser follicular and ovulatory response after superstimulatory treatment. The follicular and ovulatory response after superstimulation was repeatable within individuals, regardless of age.     

Changes in the cumulus-oocyte complex of subordinate follicles relative to follicular wave status in cattle

We investigated factors that affect cumulus-oocyte complex (COC) morphology and oocyte developmental competence in subordinate follicles on different days after follicular wave emergence in beef heifers. In Experiment 1, heifers (n = 13) were assigned at random to COC aspiration during the growing/static (Days 1 to 3) or regressing (Day 5) phase of subordinate follicle development (follicular wave emergence = Day 0). Follicular wave emergence was induced by transvaginal ultrasound-guided follicular ablation, ovaries were collected at slaughter, all follicles > or = 2 mm except the dominant follicle were aspirated, and COC were microscopically evaluated for morphology. There was a greater percentage of COC with expanded cumulus layers on Day 5 (42.4%) than on Days 1 to 3 (2.2%). In Experiment 2, heifers (n = 64) at random stages of the estrous cycle had all follicles > or = 5 mm ablated and 4 d later, 2 doses of PGF were injected 12 h apart; heifers were monitored daily by ultrasonography for ovulation (Day 0 = follicular wave emergence). Heifers were assigned to the following time periods for oocyte collection from subordinate follicles: Days 0 and 1 (growing phase), Days 2, 3 and 4 (static phase), and Days 5 and 6 (regressing phase). Ovaries were individually collected at slaughter, and all follicles > or 2 mm except for the dominant follicle were aspirated. The COC were morphologically evaluated and then matured, fertilized and cultured in vitro. Expanded COC were more frequent during the regressing phase (53.4%) than the growing or static phase (14.4 and 17.8%, respectively; P < 0.05). While the proportions of COC with > or = 4 layers of cumulus cells and denuded oocytes were higher (P < 0.05) in the growing and static phases, the production of morulae was highest (P < 0.05) with COC collected from subordinate follicles during the regressing phase. In Experiment 3, heifers (n = 18) were assigned at random to oocyte collection from subordinate follicles 3 and 4 d (static phase) or 5 and 6 d (regressing phase) after follicular wave emergence. The heifers were monitored ultrasonically for ovulation (Day 0 = follicular wave emergence); COC were collected from all follicles (> or = 5 mm) except for the dominant follicle by transvaginal ultrasound-guided follicle aspiration 3 to 6 d later. Recovered oocytes were stained and examined microscopically to evaluate nuclear maturation. A higher proportion of oocytes collected on Days 5 and 6 showed evidence of nuclear maturation (50%) than on Days 3 and 4 (8.3%; P < 0.05). Results support the hypothesis that COC morphology and oocyte developmental competence change during the growing, static and regressing phases of subordinate follicle development.     

Morphological and functional characterization of large antral follicles in three breeds of sheep with different ovulation rates

Morphological and functional features of large ovarian follicles from three breeds of sheep, with different ovulation rates (Finnish Landrace N = 12, Finnish Landrace X Scottish Blackface N = 16, Merino X Scottish Blackface N = 16) were compared by integrating three techniques; ink labelling, in-vitro oestradiol production and morphological classification. The follicles were removed at two stages of the follicular phase, 1 (PG + 1) or 2 (PG + 2) days after PGF-2 alpha treatment and compared after monitoring their rates of growth with the use of ink labelling. After ovariectomy all follicles greater than or equal to 1 mm in diameter were dissected, and the 8 largest were incubated individually for 2 h to assess their ability to secrete oestradiol and testosterone. After incubation the follicles were processed for histological examination and checked for atresia. An analysis of the follicle population was based on in-vitro oestradiol secretion rates in all three breeds; an oestrogen-active population producing 500-8100 pg oestradiol/ml/h and an oestrogen-inactive population producing 0-499 pg oestradiol/ml/h. A comparison of the 3 approaches demonstrated agreement on 94.3 +/- 1.2% of occasions. Ink-labelling demonstrated that all follicles identified as oestrogen-active were increasing in size. Within oestrogen-active follicles significant correlations were detected between oestradiol production and testosterone production (r = 0.42), oestradiol production and granulosa cell number (r = 0.45) and between oestradiol production and mitotic index (r = -0.38). A regression model fitting breed, stage of atresia, granulosa cell number, in-vitro testosterone production and mitotic index demonstrated that granulosa cell number is a characteristic which contributes significantly to the variation of in-vitro oestradiol production in oestrogen-active and oestrogen-inactive follicles. There was no significant difference between breeds in the mean number of ink-labelled follicles growing from Day PG - 1 to Day PG + 1. There was a significant difference between the breeds in the number of ink-labelled follicles growing between Days PG + 1 and PG + 2 (Days 1 and 2 of the follicular phase), the number being similar to the ovulation rate for the breed. The majority of the oestrogen-active follicles had been recruited by Day PG - 1, although in the Finnish Landrace genotypes more than 30% were recruited on or after Day PG + 1 compared to less than 10% in Merino x Scottish Blackface ewes.(ABSTRACT TRUNCATED AT 400 WORDS)