为研究大鼠副交感神经对妊娠早期子宫内组织胺(HA)和血清白血病抑制因子(LIF)的影响,选取180～200日龄健康性成熟Wistar雌性大鼠20只,随机分为对照组和处理组,处理组采用腹腔注射阿托品阻断副交感神经,对照组腹腔注射0.9％生理盐水.结果表明:阿托品处理后妊娠5d子宫囊胚数量明显减少、囊胚发育延迟;妊娠6d虽然囊胚数量接近正常对照组数量,但胚胎凸起仍小于正常妊娠6d状态.子宫肥大细胞(MC)释放HA结果显示,阻断副交感神经激发MC释放的HA量显著高于正常状态,且与MC自发释放的HA变化趋势不同;正常对照组血清LIF含量的变化趋势是随着妊娠时间的增加而减少,而处理组出现相反的趋势.试验结果提示,在着床窗口期副交感神经通过对MC释放HA等介质调控子宫的生理功能,HA和LIF分泌规律紊乱对胚胎着床的影响更为显著,导致囊胚发育延迟,甚至丢失.

为研究6-羟多巴胺(6-OHDA)损毁大鼠子宫交感神经的剂量及其对子宫肥大细胞分布的影响,选取180～200日龄健康的性成熟Wistar雌性大鼠24只,随机分为对照组和处理组,处理组尾静脉注射25,50,100 mg/kg的6-OHDA.结果显示:按照一次性注射100 mg/kg 6-OHDA的方法可以损毁支配子宫的交感神经,而50 mg/kg未能损毁子宫内膜固有层的神经.肥大细胞观察结果显示,其主要分布在内环、外纵肌之间的血管周围,随着6-OHDA剂量的不断加大,肥大细胞数量呈减少趋势,3个处理组对比差别显著,对照组略高于25 mg/kg组.血清中组织胺含量呈相反的变化趋势.结果表明,可采取一次性注射100mg/kg的6-OHDA来建立交感神经损毁模型,交感神经影响子宫内肥大细胞的分布,对于机体组织胺释放的调控不同于子宫.

采用SABC法检测济宁青山羊生后发育过程中生长激素受体(growth hormone receptor,GHR)与胰岛素样生长因子Ⅰ受体(insulin like growth factorl receptor,IGF-ⅠR)在乳腺组织中的分布;GAPDH作为内参基因,qRT PCR法检测GHR mRNA与IGF-ⅠR mRNA相对表达量.结果显示:GHR和IGF-ⅠR阳性物质主要在乳腺组织内的导管上皮细胞、基质细胞和血管肉皮细胞的胞质中表达,细胞核偶有表达;GHR和IGF-ⅠR阳性细胞光密度值与其mRNA表达趋势基本一致,但IGF-ⅠR的表达量低于GHR;GHR和IGF-ⅠR在出生当天表达水平较低,60日龄(初情期)和120日龄(性成熟)出现峰值(P＜0.05),120日龄后逐渐下降.结果表明:济宁青山羊生后发育时期,GHR与IGF-ⅠR对乳腺的生长发育具有重要的调节作用,初情期和性成熟期可能是乳腺生长发育最快的阶段.

In this work, we examined the ovarian development and its relationship with steroid hormone receptors levels and the precocious puberty in JiNing Gray goats by using optical microscopy, immunohistochemistry, quantitative real-time RT-PCR (qPCR) and Western blotting. We found that in the ovaries of neonatal kids, high level of receptors for estrogen (ERα and ERβ) and progesterone (PR) and their mRNA were observed along with growing follicles. From 0 to 30 days of age, the weight and volume of ovaries increased significantly and the boundary between the inner and outer cortex disappeared, while the expression of ERα, ERβ and PR and their mRNA decreased sharply. When 60 days old, the animals began to ovulate; the expression of ERα, ERβ and PR and their mRNA significantly increased, and the animals reached puberty. On day 90, the animals manifested sexual maturity with biggest mature follicles 6.18mm in diameter, the expression of ERβ and PR protein and their mRNA was maintained at a high level, with decreased expression of ERα and its mRNA. Before puberty, the expression of ovarian ERα (prepubertal dominant receptor) and it's mRNA was significantly higher than that of ERβ (dominant receptor after sexual maturity). The results showed that JiNing Grey goats' ovaries had fast development and early maturation, and ERα, ERβ and PR protein and mRNA expression in the ovary had distinct specificity for time and space, which may be closely related to the strain's progenitive characteristics.

To explore how uterine innervations affect expression of Dickkopf-3 (DKK-3) during peri-implantation, we first examined the consequence of uterine neurectomy on embryo implantation events. We observed that amputation of autonomic nerves innervating the uterus led to the failure of on-time implantation in rats. We then analyzed the effect of neurectomy on expression of DKK-3 further using immunohistochemistry and quantitative real-time reverse transcription polymerase chain reaction. We observed that disconnection of autonomic nerve innervation significantly increased DKK-3 expression in the endometrium before and during invasion of the blastocyst. We also observed high levels of DKK-3 immunoreactivity in the vasculature of the uterus during peri-implantation. Thus, we speculate that DKK-3 may relate to implantation. Besides, our findings provide a new line of evidence that DKK-3 may be regulated by the autonomic nervous system.

分析了高校图书采购招标存在的几个问题,并提出了改进图书招标工作的几点建议.

To clarify the molecular evolution and characteristic of beta estrogen receptor (ER beta) gene in lining Gray goat in China, the entire ER beta gene from Jining Gray goat ovary was amplified, identified and sequenced, and the gene sequences were compared with those of other animals. Functional structural domains and variations in DNA binding domains (DBD) and ligand binding domains (LBD) between Jining Gray goat and Boer goat were analyzed. The results indicate that the ER beta gene in Jining Gray goat includes a 1584 bp sequence with a complete open-reading-frame (ORF), encoding a 527 amino acid (aa) receptor protein. Compared to other species, the nucleotide homology is 73.9-98.9% and the amino acid homology is 79.5-98.5%. The main antigenic structural domains lie from the 97th aa to the 286th aa and from the 403rd aa to the 527th aa. The hydrophilicity and the surface probability of the structural domains are distributed throughout a range of amino acids. There are two different amino acids in the DBD and three different amino acids in the LBD between Jining Gray and Boer goats, resulting in dramatically different Spatial structures for ER beta protein. These differences may explain the different biological activities of ER beta between the two goat species. This study firstly acquired the whole ER beta gene sequence of Jining Gray goat with a complete open reading frame, and analyzed its gene evolutionary relationship and predicted its mainly functional structural domains, which may very help for further understanding the genome evolution and gene diversity of goat ER beta. (C) 2014 Elsevier Ltd. All rights reserved.

The concentrations of melatonin( MT) in blood plasma were measured by radioimmunoassay during the annual cycle of two local goat breeds in Shandong with low( Yimeng Black goat) and high( Jining Gray goat) fecundity. The results showed that the secretion of the MT reached the maximum at midnight,Melatonin contents associated with seasonal changes. At night,the average concentration of melatonin in the summer solstice was the highest than other seasons( P 0. 05),Jining Gray goat reached a highly significant level( P 0. 01); no difference between the vernal equinox and the autumn equinox( P 0. 05) was observed. Different with the winter solstice,Jining Grey goats MT concentrations were higher than Yimeng black goat in the other three seasons at each sampling point,furthermore,the same results appear in theaverage levels at night.

Immunohistochemical Strept Avidin Biotin-Peroxidase Complex (SABC) three-step method was used to investigate the distribution patterns and developmental changes of GnRH and GnRHR immunopositive (GnRH-ip and GnRHR-ip) cells in the pituitary of Ji Ning Gray goats during 0-180 days of age. The results showed that GnRH-ip and GnRHR-ip cells were detected only in the pars distalis of adenohypophysis. There were no positive cells in the pars intermedia and neurohypophysis. GnRH-ip and GnRHR-ip cells were not observed in the anterior pituitary at birth day and 30 days of age. At 60 days, a number of GnRH-ip and GnRHR-ip cells were found in the anterior pituitary. GnRH-ip cells were pale brown which scattered or clustered around the sinusoid capillary; GnRHR-ip cells were brown and the cytomembrane was darker. The size and percentage of GnRHip and GnRHR-ip cells increased with the age growth. The numbers of GnRH-ip and GnRHR-ip cells after sexual maturity were significantly bigger than that before sexual maturity. The results above suggested that GnRH and GnRHR in the pituitary of Ji Ning Gray goats play a pivotal role in the sexual development and sexual maturity. (C) 2013 PVJ. All rights reserved

The research was conducted to prepare the polyclonal antibody against goat estrogen receptor β(ERβ) and build the specific immunohistochemitry assay to detect ERβ in the goat's ovary and uterus.The ERβ gene was amplified from Jining grey goat ovary by RT-PCR.The cloning sequence was identified by enzyme digestion and sequencing,then the gene was cloned into a prokaryotic expression vector pET32a(+) to construct recombinant plasmid named as pET32a(+)-ERβ.The expression of fusion protein was induced by IPTG in E.coli BL21(DE3) system and then identified by SDS-PAGE and Western blotting.After purification with Ni-NTA under denaturing condition,the fusion protein was applied as antigen to immunize New Zealand White rabbits for preparing specific polyclonal antibody.The titer of the antibody was detected by ELISA,and the specificity of the antibody was detected by Western blotting;the tissue sections of goat's ovary and uterus were assayed with immunohistochemistry method mediated by the prepared antibody.The results showed the protein of ERβ was expressed as fusion protein,53 kDa.Western blotting result indicated that ERβ fusion protein could specially reacted with polyclonal antibody against human ERβ.The titer of the antibody detected with ELISA was 1:213,and the prepared antibody combined to ERβ was specific.the results of immunohistochemistry assay indicate ERβ mainly exists in the granule cells of ovary follicles and the epithelial cells and smooth muscle cells of the uterus endometrial layer.In conclusion,the polyclonal antibody against goat ERβ are successfully obtained in the present study and the ERβ distribution in the ovary and uterus of Jining Grey goat was firstly reported with immunohistochemistry assay mediated by the antibody against goat estrogen receptor β.

To detect the location of ERα,ERβand PR and the expression of ERα,ERβand PR mRNA in the uterus of Jining Grey goats during postnatal development.In the uterus of different group,the SABC methods were used to study the distribution of ERα,ERβand PR,and the real time fluorescent quantitative PCR were used to research the relative expression of ERα,ERβand PR mRNA with GAPDHas an internal gene.ERα,ERβand PR were malinly localized in the nuclei of the luminal epithelium,glandular epithelium,stromal cells,endothelium and smooth muscle cells of the uterus,and occasionally in the cytoplasm.The trends of the optical density in the immunopositive cells of ERα,ERβand PR were similar with that of the expression of ERα,ERβand PR mRNA.But the level of the expression of ERβwas less than that of ERα.From the birth day to the 30th day,the expression of ERαand ERβremained at a lower level then increased gradually on the 60th day,ERαand ERβreached its peak from the 90th day to the 120th day,and afterwards maintained at a high level.While the expression of PR was higher on the birth day,than reached its lowest level on the 30th day and significantly increased after puberty,and two peaks were found during the sexual maturity and maturation.ERα,ERβand PR have an important role in regulating the proliferation and differentiation of the endometrial and the myometrial.

The single nucleotide polymorphisms of GDF9(growth differentiation factor 9)gene on goats was tested,which provided theoretical basis for studying the correlations among GDF9gene with lambing number.According to the sequence of GDF9gene(GenBank:EF 446168),three primer pairs were designed to detect single nucleotide polymorphism of exon 2ofGDF9gene in both high fecundity goat breed(Jining Grey goat)and low fecundity goat breeds(Laoshan dairy goat,Laiwu native goat)by PCR-SSCP. The products amplified by primer 1and primer 3displayed polymorphism.For primer 1,three genotypes(AA,AB and BB)were detected in all three goat breeds.The sequencing results indicated that there was one single nucleotide mutation(G26→A26)at exon 2of GDF9gene in goats,but this mutation did not cause amino acid change.Frequency of allele A was 0.8917,frequency of allele B was 0.1083in Jining Grey goats.The does with genotype AA had 0.539(P0.01)or 0.629(P0.01)kids more than those with genotype AB or BB in Jining Grey goats,respectively.For primer r3,three genotypes(CC,CD and DD)were detected in all fivegoat breeds.The sequencing results showed that there was one single nucleotide mutation(G795→A795)at exon 2of GDF9gene in goats,and this mutation resulted in an amino acid change:valine→isoleucine.Frequency of allele C was 0.9167,frequency of allele D was 0.0833in Jining Grey goats.The does with genotype CC had 0.568(P0.01)or 0.62(P0.01)kids more than those with genotype CD or DD in Jining Grey goats,respectively.These results preliminarily showed that the GDF9gene is either a major gene that influences the prolificacy in Jining Grey goats or a molecular marker in close linkage with such a gene.

To investigate the variation law of mRNA expression levels of FSHR gene and LHR gene in uterus of low productive Goats,total RNA was extracted from the uterus of YiMeng Black goat and Real Time Fluorescence Quantitative PCR method(real-time qRT-PCR) was established so as to analyse mRNA levels of FSHR gene and LHR gene in uterus of four estrous cycle phases in YiMeng Black Goat.The results showed that mRNA of FSHR gene and LHR gene were expressed in the uterus during the four estrous cycle phrases.The expression of FSHmRNA was higher than that of LHRmRNA.In cornua uteri,the expression of FSHmRNA was highest in metestrus and lowest in proestrus;in uteri caruncle the expression of FSHmRNA was the highest in diestrus and lowest in proestrus and there was no obvious difference among the four phases(P0.05).In cervical,the expression of FSHmRNA was highest in estrus and lowest in diestrus,which is significantly different with other phases(P0.05).In cornua uteri,uteri caruncle and cervical,the expression of LHRmRNA was highest in diestrus,which is significantly different with the rest phases(P0.05).In uterine body,the expression of FSHmRNA and LHRmRNA were low and no obvious law was observed.

In the present study,the distribution,morphological characteristics and developmental changes of Ghrelin-immunopositive(Ghrelin-ip) cells in the gastrointestinal tract of goat were investigated.Using the method of immunohistochemistry,Ghrelin-ip cells were showed in the gastrointestinal tract of Jining Gray goat at the ages of day 0,30,60,90,120,150 and 180.Ghrelin-ip cells classified into open-and closed-type cells were found to be localized in the mucosa epithelium,glands and lamina propria of abomasums,duodenum,jejunum,ileum,cecum and colon,but not in rumen,reticulum,omasum and rectum.The greatest number of Ghrelin-ip cells was found in the abomasums and the Ghrelin-ip cell density gradually decreased from the abomasums to the colon.The percentage of closed-type cells to all immunopositive cells also decreased from the abomasums to the colon.With the goat growth,the densities of Ghrelin-ip cells in the abomasums and jejunum increased from ages of day 0 to 120,but slightly decreased at ages of day 150 and 180.There was also a steady increase in the number of Ghrelin-ip cells from postnatal day 0 to 90 in duodenum,which reached a peak and remained steady.Very few Ghrelin-ip cells were found in the ileum,cecum and colon,and the densities increased with the age growth.The change of Ghrelin-immunopositive cells in the gastrointestinal tract of growing goats suggests that Ghrelin may be involved in gastrointestinal tract development.

<正>为揭示不同发育阶段雌性济宁青山羊肠道肥大细胞分布状况,本试验采用组织化学方法对1、2和5月龄山羊小肠、大肠的肥大细胞分布进行了比较研究。结果显示:1、2月龄小肠前段在固有层内,粘膜下层、肌层少见;后段在粘膜下层内较多,而固有层、肌层和浆膜层少。大肠段主要分布在肌层的肌纤维之间,粘膜下层内偶见,固有层内无肥大细胞存在。5月龄阶段在固

The objective of the experiment was to study the localition and the average integrated optical density of ERα and PR immunopositive cells and the abundance of ERα mRNA and PR mRNA in mammary gland during postnatal development in Jining Gray goat.The SP immunohistochemical staining and Image analysis methods were used to localize ERα and PR immunoprotective(-ip) cells in mammary gland of Jining Gray goat.The relative expression of ERα mRNA and PR mRNA was analyzed by using the real time fouorescence quantitative PCR with GAPDH as an internal gene.ERα and PR had similar variations,immunoprotective staining was mainly localized in the nucleus of blood vessels endothelial cells,stromal cells and ductal epithelial cells of mammary gland,occasionally in cytoplasmic,PR-ip substance also found in alveolar epithelial cells in 180 days.The average optical density was gradually increased from birth day to sexual maturity,and it was significantly higher in sexual maturity than that in birth day and puberty(P0.01),it was not different with that in 180 days(P0.05).The results of qRT-PCR showed that the abundance of ERα mRNA was gradually increased from birth day to sexual maturity,and it was significantly higher in sexual maturity than that in birth day and puberty(P0.01),there was a slightly decrease from sexual maturity to 180 days.While the abundance of PR mRNA from birth day to sexual maturity was lower and there was no significant difference during these groups.In 180 days,it was significantly higher than the other three groups(P0.01).These results indicated that the estrogen receptors of alph and the progesterone receptors could promote the growth of glandular,the hyperplasia of epithelial cells and the development of lobulo-alveolar during postnatal development in Jining Gray goat.

In order to clarify the mechanisms of melatonin affected goat reproduction traits,and compared the different expressed amount of melatonin receptors(MTR1A)in ovary tissues between Jining Grey Goat with high superfoetation and Yimeng Black Goat with single birth.Real-time fluorescent quantitate differential display PCR technique were used to detected the expressed amount of melatonin receptors(MTR1A)in ovary tissure of Jining Gray Goat and Yimeng Black Goat in oestrus stage,while GAPDH internal standards were used as control.The results showed that MTR1A mRNA were expressed in ovary tissue of both goats,and the expressed amount of MTR1A mRNA was lower at Jining Gray Goat ovary than that of it at Yimeng Black Goat ovarys.The results suggested that MTR1A mRNA were expressed in both goat ovary obviously,the differential expression of MTR1A may affect the development,maturity and ovulating of goat ovary.This provided the molecular genetic data to clarity the mechanism of MTR1A action at goat reproduction.

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In order to clarify the genetic mechanisms of melatonin affected goat reproduction traits,differential display PCR,cloning and sequencing techniques were used to detected the expressions of melatonin receptors(MelR1a) in ovary tissure of Jining Gray Goat and Yimeng Black Goat in oestrus stage,while MelR1a genes from the two kinds of goats were cloning and sequencing.The results showed that MelR1a genes were expressed in ovary tissues of the both goats,and the bases sequences of MelR1a genes were the same.The results suggest that MelR1a genes expressed in both goat ovaries play an important role in controlling the reproduction traits obviously,and make clear that Mel1a is conservative.