Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200? implanted heifers

Background

Heifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments: non-implanted (n = 5), Revalor 200 for 28 d (n = 5), or Revalor 200 for 84 d (n = 6). Small follicle (1 to 5 mm) granulosa cells were isolated from each pair and incubated with phosphate buffered saline (n = 16) or 100 ng/mL follicle stimulating hormone (n = 16) for 24 h.

Results

Granulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar (P = 0.22) to non-implanted heifers, while medium estradiol concentrations were increased (P < 0.10) at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein (P = 0.05) mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 (P < 0.10) or 84 d (P < 0.05) compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase (P = 0.57) and aromatase (P = 0.23) were demonstrated in implanted or non-implanted heifers.

Conclusions

These results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.     

三个山羊品种FSHR基因部分序列的克隆与分析

以波尔山羊、莱芜黑山羊、崂山奶山羊为研穷对象,对其FSHR基因5’端调控区及第一外星子部分序列进行克隆测序。结果表明,波尔山羊与莱芜黑山羊、崂山奶山羊三者序列的同源性为98．13%,相应的突变率为1．87%。三个品种间共有27处发生碱基缺失/插入,且碱基突变区段位于基因的5’端区转录启动调控区,主要集中在-210～—290bp之间,莱芜黑山羊与崂山奶山羊之间仅出现3个碱基的突变。     

PMSG对母兔卵巢机能的影响

以三种不同剂量的国产孕马血清促性腺激素(PMSG)对28只成年哈白母兔进行处理，剂量分别为30IU，50IU和80IU，间隔48小时后耳静脉一次性注射人类绒毛膜促性腺激素(HCG)，剂量均为80IU，再隔48小时后手术冲卵，观察母兔卵巢动态变化。结果表明三种剂量30IU、50IU和80IU处理后的母兔平均排卵点数分别为9．5个、10．8个和15．2个，其中以80IU的PMSG剂量组的排卵点个数为最高和其他两组比较差异显著(P<0．05)；48小时后各组的卵巢上可见卵泡数分别平均为43.0个、33.4个和35.9个，其中以30IU的PMSG组的未成熟卵泡数为量多，和其他两组比较差异显著(P<0.05)，各组的充血卵泡数分别为2.5个、2.0个和9.4个。80IU的PMSG剂量组的充血卵泡高于30IU和50IU剂量组，差异极显著(P<0.     

Ovarian activity in F1 prepubertal ewe lambs under tropical conditions

Seventy crossbred ewe lambs, born from Pelibuey (PB) dams mated with five sire breeds: White Dorper × PB (WD × PB), Black Head Dorper × PB (BHD × PB), Ile de France × PB (Ile × PB) Katahdin × PB (KT × PB) and Pelibuey (PB × PB), were used to determine age and weight to puberty, follicular populations during prepubertal development, ovulation rate at first ovulation and function of the first corpus luteum (FCL). Ewe lambs were maintained under grazing conditions and were supplemented with 300 g head^− 1 day^− 1 of a concentrate with 14% CP and 3.0 Mcal ME kg^− 1 of DM. From 145 days of age, monthly observations were performed by endoscopy to monitor follicular populations and luteal structures. Ovarian follicles were classified according to size into small (≥ 1 and < 3 mm), medium (≥ 3 and ≤ 4 mm) and large (> 4 mm). Additionally, weekly observations by ultrasonography were performed in four ewes per genotype to assess follicular populations. Luteal function from first ovulation was determined by circulating concentrations of progesterone. Ovulation rate was not affected by genotype (P > 0.05). All breed groups had the same pattern of follicular populations, where most of follicles were small, followed by medium and a small proportion of large follicles. Maximum diameter of follicles was similar for all genotypes during prepubertal development (P > 0.05), with an average range of 3.9 ± 0.20 to 4.4 ± 0.20 mm. The BHD × PB reached puberty at the youngest age of 240.9 ± 13.0 days, compared to all other genotypes (P < 0.05), 259.1 ± 11.7 for WD × PB, 279.3 ± 9.6 for KT × PB and 289.0 ± 15.3 days for PB × PB and 308.6 ± 11.9 days for Ile × PB ewe lambs. Ile × PB had greater weight to first ovulation (31.5 ± 1.19 kg) as compared to other breed groups (P < 0.01). The largest percentage of FCL was for KT × PB ewes (100%) (P < 0.05), followed by BHD × PB (88.9%), PB × PB (83.3), WD × PB (78.6%) and Ile × PB (77.8%). There were no differences in ovulation rate attributable to type of lambing from which ewe lambs came from (P > 0.05). BHD × PB ewe lambs reached puberty at an earlier age than other in this study. KT × PB ewe lambs had the highest percentage of FCL in their first ovulation. Breed of sire Ile de France increased weight to puberty and decreased FCL at first ovulation. In conclusion, the BHD × PB ewe showed puberty at an earlier age than other genotypes, while the KT × PB had the highest percentage of functional corpus luteum in their first ovulation. Ewe lambs coming from single lambings reach puberty at a heavier weight than those coming from multiple lambings. The ovulation rate in the first ovulation of ewe lambs is not affected by genotype and type of lambing.     

IGF-1 receptor signaling pathways and effects of AMPK activation on IGF-1-induced progesterone secretion in hen granulosa cells

IGF-1 plays a key role in the proliferation and differentiation of granulosa cells. However, the molecular mechanism of IGF-1 action in avian granulosa cells during follicle maturation is unclear. Here, we first studied IGF-1 receptor (IGF-1R) expression, IGF-1-induced progesterone production and some IGF-1R signaling pathways in granulosa cells from different follicles. IGF-1R (mRNA and protein) was higher in fresh or cultured granulosa cells from the largest follicles (F1 or F2) than in those from smaller follicles (F3 or F4). In vitro, IGF-1 treatment (10(-8)M, 36h) increased progesterone secretion by four-fold in mixed F3 and F4 (F3/4) granulosa cells and by 1.5-fold in F1 granulosa cells. IGF-1 (10(-8)M, 30min)-induced increases in tyrosine phosphorylation of IGF-1R beta subunit and phosphorylation of ERK were higher in F1 than in F3/4 granulosa cells. Interestingly, IGF-1 stimulation (10(-8)M, 10min) decreased the level of AMPK Thr172 phosphorylation in F1 and F3/4 granulosa cells. We have recently showed that AMPK (AMP-activated protein kinase) is a protein kinase involved in the steroidogenesis in chicken granulosa cells. We then studied the effects of AMPK activation by AICAR (5-aminoimidazole-4-carboxamide ribonucleoside), an activator of AMPK, on IGF-1-induced progesterone secretion by F3/4 and F1 granulosa cells. AICAR treatment (1mM, 36h) increased IGF-1-induced progesterone secretion, StAR protein levels and decreased ERK phosphorylation in F1 granulosa cells. Opposite data were observed in F3/4 granulosa cells. Adenovirus-mediated expression of dominant negative AMPK totally reversed the effects of AICAR on IGF-1-induced progesterone secretion, StAR protein production and ERK phosphorylation in both F3/4 and F1 granulosa cells. Thus, a variation of energy metabolism through AMPK activation could modulate differently IGF-1-induced progesterone production in F1 and F3/4 granulosa cells.     

不同因素对高产荷斯坦奶牛体内性控胚胎生产效率和质量的影响

[目的]试验旨在探讨不同种类的促卵泡素(FSH)超数排卵处理、不同精液类型人工输精处理以及不同季节等前处理方式对高产荷斯坦奶牛体内性控胚胎生产效率的影响,为高产胚胎牛后代的繁育做出贡献。[方法]试验选取208头优质供体牛使用加拿大(Folltropin-v)和比利时进口的促卵泡素(FSH)进行超排,性控冻精和性控鲜精进行人工输精,春(3—5月)、夏(6—8月)、秋(9—11月)、冬(12—2月)四季进行胚胎生产。[结果]结果表明,使用产地为加拿大(Folltropin-v)促卵泡素进行超数排卵头均可用胚胎数和I级胚胎数(包括桑葚胚I级、囊胚I级)均显著高于比利时(P<0.05);通过使用性控冻精和性控鲜精对供体牛进行人工输精头均可用胚胎数分别为和I级胚胎数差异均不显著(P>0.05);春、夏、秋、冬四季进行胚胎生产头均可用胚胎数和I级胚胎数夏季均显著低于其他三季(P<0.05)。[结论]因此,使用加拿大促卵泡素(Folltropin-v)超数排卵、性控冻精进行人工输精避免在夏季进行胚胎生产可获得质量较高的高产荷斯坦奶牛体内性控胚胎。     

云南半细毛羊不同发育阶段皮肤毛囊性状的发育规律研究

本研究选择了 58只母羔 ( 38只单羔、2 0只双羔 ) ,分别于初生、断乳、6月龄、周岁时采取皮肤样品 ,采用横切和纵切连续切片法 ,在皮脂腺水平上观测皮肤毛囊群结构及毛囊性状发育规律。结果表明 :云南半细毛羊皮肤毛囊群结构为三元型 ,毛囊群呈长椭园形 ,皮脂腺较发达 ,毛囊较少弯曲 ,生长方向一致。毛囊性状初生时与断乳、6月龄、周岁时差异极显著 (P <0 0 1 ) ,其余各年龄段间除皮肤厚度和真皮层厚差异显著 (P <0 0 5)外 ,其余差异都不显著 ,说明毛囊性状在断乳 ( 4月龄 )时已基本稳定 ,断乳时的毛囊性状即可代表周岁时的毛囊性状。初生、断乳、6月龄、周岁时的皮肤总毛囊数 (个 /mm2 )分别为4 7 4 4± 7 73、2 9 0 0± 6 4 9、2 9 2 9± 6 4 2和 2 9 79± 6 0 7,S/P值分别为 3 52± 0 91、6 0 0± 1 1 3、6 6 3± 0 95和 7 39± 1 2 6。单双羔在毛囊发育上有不同 ,这种差别差异不显著 ,但数量上的差异一直持续到断乳     

驴,马卵泡发育及黄体形成的超声显像初步研究

本实验采用实时Ｂ型超声显像法，初步观察了１６头母驴和１７匹马发情期卵泡发育及黄体形成。卵泡为一轮廓完整的液性器官，卵泡液呈无回声（暗区）。卵泡壁在排卵前变薄，回声增强，轮廓更加清晰。７５％的马卵泡在排卵前２天由椭圆形变为梨形或锥形，大部分驴的卵泡始终保持椭圆形。９０％以上的驴和／或马的卵巢上有多个卵泡发育，多卵泡发育比单卵泡发育排卵迟缓。驴的最大卵泡从排卵前５天开始迅速增大，于排卵前１天达到最大（