梅山与长大母猪下丘脑-垂体-卵巢轴Kiss1和GPR54基因的表达差异

研究早熟与晚熟品种母猪下丘脑-垂体-卵巢轴Kiss1和GPR54基因的表达差异。选用8头梅山母猪与12头长大(LY)母猪为研究对象,梅山母猪于70 d和100 d屠宰,LY母猪于70、100 d和199 d屠宰,收集血清及下丘脑、垂体、卵巢组织样品。ELISA检测血清瘦素(Leptin)和雌二醇(E2)水平,PCR克隆梅山与LY母猪Kiss1基因编码序列,实时荧光定量PCR检测母猪下丘脑、垂体、卵巢组织Kiss1和GPR54基因表达水平。结果表明:梅山与LY母猪Kiss1基因编码序列相似性为100%;梅山与LY母猪初情期下丘脑Kiss1基因表达量极显著高于垂体与卵巢(P<0.01),卵巢GPR54基因表达水平显著高于下丘脑与垂体(P<0.05)。梅山母猪下丘脑-垂体-卵巢轴Kiss1基因表达水平都显著高于相同日龄和初情期LY母猪(P<0.05),梅山母猪血清Leptin水平极显著高于相同日龄LY母猪(P<0.01)。而E2水平显著高于100 d与初情期LY母猪(P<0.01)。Leptin与下丘脑Kiss1和GPR54基因表达呈显著正相关(P<0.05),而E2仅与下丘脑Kiss1基因表达有极显著正相关关系(P<0.01);梅山与LY母猪Kiss1基因编码区序列相似性为100%,梅山母猪下丘脑-垂体-卵巢轴上kiss1基因表达量较LY母猪高,主要原因可能是初情日龄早,下丘脑Kiss1基因表达水平的高低与血清Leptin和E2浓度密切相关。     

L-精氨酸对热处理MLTC-1细胞氧化、凋亡及睾酮合成相关基因表达的影响

试验旨在研究热处理对睾丸间质细胞的影响及L-精氨酸对热处理睾丸间质细胞抗氧化、抗凋亡及睾酮合成相关基因表达的影响。试验选用小鼠睾丸间质瘤细胞系(MLTC-1)细胞,将细胞分为4组,分别为对照组(C)、热处理组(HT)、5μg/mL L-精氨酸+热处理组(5H)和10μg/mL L-精氨酸+热处理组(10H),处理结束后收集培养上清液或细胞备用。结果显示:与C组相比,热处理显著降低MLTC-1细胞GSH-Px活力(P<0.01),当添加10μg/mL L-精氨酸后,GSH-Px活力得到恢复,并显著增加(P<0.01);热处理诱发细胞凋亡,添加L-精氨酸后,细胞凋亡数量明显降低;与HT组相比,添加5μg/mL L-精氨酸显著增加抗凋亡基因PI3K和Bcl-2的基因表达量(P<0.05);与HT组相比,5H组睾酮合成相关基因,StAR和Cyp17a1基因表达量极显著增加(P<0.01),SF-1的基因表达量也显著增加(P<0.05)。以上结果说明:L-精氨酸可缓解热处理诱导的MLTC-1细胞凋亡,增强MLTC-1细胞的抗氧化和抗凋亡能力,并可调控热处理MLTC-1细胞睾酮合成相关基因的表达。     

Term placenta shows methylation independent down regulation of Cyp19 gene in animals with retained fetal membranes

Retention of fetal membranes (RFM) is the major post-partum disorder in dairy cattle. Cyp19 gene encodes the aromatase enzyme responsible for catalyzing the rate limiting step in estrogen biosynthesis, an important hormone for placental maturation and expulsion. The present study was aimed for comparative analysis of Cyp19 gene expression and its epigenetic regulation in placental cotyledons of animals with and without RFM. Significantly lower expression of Cyp19 gene was found in placental samples of RFM affected animals in comparison to normal animals. Methylation analysis of 5 CpG dinucleotides of placenta specific Cyp19 gene promoter I.1 and proximal promoter, PII showed hypo-methylation of both PI.1 and PII in term placenta of normal and diseased animals. In conclusion, a mechanism other than promoter methylation is responsible for decreased aromatase expression in placental cotyledons of animals suffering from RFM.     

Kisspeptin,c‐Fos and CRFR Type 2 Co‐expression in the Hypothalamus after Insulin‐Induced Hypoglycaemia

Normal reproductive function is dependent upon availability of glucose and insulin‐induced hypoglycaemia is a metabolic stressor known to disrupt the ovine oestrous cycle. We have recently shown that IIH has the ability to delay the LH surge of intact ewes. In the present study, we examined brain tissue to determine: (i) which hypothalamic regions are activated with respect to IIH and (ii) the effect of IIH on kisspeptin cell activation and CRFR type 2 immunoreactivity, all of which may be involved in disruptive mechanisms. Follicular phases were synchronized with progesterone vaginal pessaries and at 28 h after progesterone withdrawal (PW), animals received saline (n = 6) or insulin (4 IU/kg; n = 5) and were subsequently killed at 31 h after PW (i.e., 3 h after insulin administration). Peripheral hormone concentrations were evaluated, and hypothalamic sections were immunostained for either kisspeptin and c‐Fos (a marker of neuronal activation) or CRFR type 2. Within 3 h of treatment, cortisol concentrations had increased whereas plasma oestradiol concentrations decreased in peripheral plasma (p < 0.05 for both). In the arcuate nucleus (ARC), insulin‐treated ewes had an increased expression of c‐Fos. Furthermore, the percentage of kisspeptin cells co‐expressing c‐Fos increased in the ARC (from 11 to 51%; p < 0.05), but there was no change in the medial pre‐optic area (mPOA; 14 vs 19%). CRFR type 2 expression in the lower part of the ARC and the median eminence was not altered by insulin treatment. Thus, disruption of the LH surge after IIH in the follicular phase is not associated with decreased kisspeptin cell activation or an increase in CRFR type 2 in the ARC but may involve other cell types located in the ARC nucleus which are activated in response to IIH.     

Kisspeptin as a master player in the central control of reproduction in mammals: An overview of kisspeptin research in domestic animals

The hypothalamo‐pituitary‐gonadal (HPG) axis is the regulatory system for reproduction in mammals. Because secretion of gonadotropin‐releasing hormone (GnRH) into the portal vessels is the final step at which the brain controls gonadal activities, the GnRH neuronal system had been thought to be central to the HPG axis. A newly discovered neural peptide, kisspeptin, has opened a new era in reproductive neuroendocrinology. As shown in a variety of mammals, kisspeptin is a potent endogenous secretagogue of GnRH, and the kisspeptin neuronal system governs both the pulsatile GnRH secretion that drives folliculogenesis, spermatogenesis and steroidogenesis, and the GnRH surge that triggers ovulation in females. The kisspeptin neuronal system is therefore considered a master player in the central control of mammalian reproduction, and kisspeptin and related substances could therefore be valuable for the development of novel strategies for the management of fertility in farm animals. To this end, the present review aimed to summarize the current research on kisspeptin signaling with a focus on domestic animals such as sheep, goats, cattle, pigs and horses.     

山羊卵巢褪黑素信号特异性载体pcDNA3.1-Pro/GDF9-ASMT与pcDNA3.1-Pro/Cyp17-MTNR1A/B的构建

本研究旨在构建褪黑素（melatonin，MLT）合成酶乙酰血清素甲基转移酶（acetylserotonin methytransferase，ASMT）的卵母细胞特异表达载体GDF9-ASMT和MLT受体（MTNR1A与MTNR1B）的颗粒（黄体）细胞特异表达载体Cyp17-MTNR1A、MTNR1B，以期为进一步生产高繁殖力转基因山羊提供载体材料。首先，分别扩增出山羊生长分化因子9（GDF9）与细胞色素P450家族17亚家族A成员1（Cyp17a1）启动子序列备用；然后，扩增ASMT、MTNR1A及MTNR1B基因CDS全长序列，并通过酶切将ASMT连接至GDF9启动子下游，将MTNR1A和MTNR1B连接至Cyp17a1启动子下游，制备出Pro/GDF9-ASMT、Pro/Cyp17-MTNR1A与Pro/Cyp17-MTNR1B表达元件；最后通过酶切连接将上述表达元件整合至pcDNA3.1（+）载体中，从而构建出pcDNA3.1-Pro/GDF9-ASMT、pcDNA3.1-Pro/Cyp17-MTNR1A和pcDNA3.1-Pro/Cyp17-MTNR1B真核表达载体。经测序验证，本研究扩增出的GDF9和Cyp17a1启动子长度分别为2 496和2 497 bp，与GenBank数据库中山羊GDF9和Cyp17a1基因起始密码子上游2 500 bp区域的同源性均为98%；扩增出的ASMT、MTNR1A与MTNR1B的CDS序列长度分别为1 037、1 100和1 130 bp，与GenBank所递交的标准序列同源性分别为98%、98%和99%，氨基酸序列同源性分别为97%、98%和99%。上述载体的成功构建可为进一步生产褪黑素高繁殖力转基因山羊提供参考。     

The Construction of the Goat Ovary Melatonin Signal Specific Vectors pcDNA3.1-Pro/GDF9-ASMT and pcDNA3.1-Pro/Cyp17-MTNR1A and MTNR1B

This study intended to construct an oocyte-specific expression vector (GDF9-ASMT) for melatonin (MLT) synthetase acetylserotonin methytransferase (ASMT),and granule (luteal) cell-specific expression vector (Cyp17-MTNR1A,MTNR1B) for MLT receptors MTNR1A and MTNR1B,the construction of the above vectors provided carrier materials for further production of transgenic sheep with high fertility.First,the goat GDF9 and Cyp17a1 promoter sequences were amplified separately.Then,the full-length CDS sequences of ASMT,MTNR1A and MTNR1B genes were amplified,and ASMT gene was connected to the downstream of GDF9 promoter by enzyme-cutting,with MTNR1A and MTNR1B respectively connected to the downstream of Cyp17a1 promoter to prepare Pro/GDF9-ASMT,Pro/Cyp17-MTNR1A and Pro/Cyp17-MTNR1B expression elements.Finally,the above-mentioned expression elements were integrated into the pcDNA3.1(+) vector by enzyme digestion,thereby constructing pcDNA3.1-Pro/GDF9-ASMT,pcDNA3.1-Pro/Cyp17-MTNR1A and pcDNA3.1-Pro/Cyp17-MTNR1B eukaryotic expression vectors.After sequencing verification,the GDF9 and Cyp17a1 promoters amplified in this study were 2 496 and 2 497 bp in length respectively,and they were 98% homologous to the 2 500 bp region upstream of the start codons of the goat GDF9 and Cyp17a1 genes in GenBank database.The amplified ASMT,MTNR1A and MTNR1B CDS sequence lengths were 1 037,1 100 and 1 130 bp,and the homology with the standard sequences submitted by GenBank were 98%,98% and 99%.The amino acid sequence homologies was 97%,98%,99% respectively.The construction of the above vectors provided references for further production of melatonin transgenic sheep with high fertility.     

Steroidogenesis and the influence of MAPK activity during in vitro maturation of porcine cumulus oocyte complexes

The aim of this study was to investigate steroidogenesis within porcine cumulus oocyte complexes during in vitro maturation and to examine the possible influence of the mitogen-activated protein kinase (MAPK). Porcine cumulus oocyte complexes were matured in vitro with and without the MAPK kinase inhibitor U0126 for 0, 5, 26 and 46 h. The 17β-estradiol and progesterone concentration in the culture medium were then determined. In addition, the mRNA levels of StAR, Cyp11A1, 3β-HSD and Cyp19A1 in cumulus cells were analysed by RT-PCR. Using an immunoblot, the MAPK phosphorylation in cumulus cells and oocytes was examined. During the first 26 h of in vitro maturation, 17β-estradiol secretion was predominant, whereas, after a culture period of 46 h, the progesterone secretion decreased conspicuously. Under the influence of U0126, the secretion of 17β-estradiol increased progressively during the complete maturation period, while progesterone secretion was completely inhibited. The mRNA levels of StAR and Cyp11A1 were not altered by U0126; however, corresponding to the hormone secretion, the gene expression of Cyp19A1 was up-regulated and the expression of 3β-HSD down-regulated. The results suggested an influence of the MAPK on steroidogenesis in cumulus cells comparable to a luteinization factor. Hormone synthesis in cumulus cells during oocyte maturation seems to be regulated by altering expression of Cyp19A1 and 3β-HSD.     

Chilled and post‐thaw storage of sperm in different goldfish types

The effective storage time of sperm after stripping (for 48 hr in 6‐hr intervals) and after thawing (for 6 hr in 2‐hr intervals) in Black moor, Oranda and Calico goldfish types was investigated. Variations in sperm density were also measured in all lines. The efficiency of a sperm cryopreservation method formerly developed for common carp was recorded in all three goldfish lines. Motility parameters ((pMOT, %), curvilinear velocity (VCL, μm/s) and straightness (STR, %)) of Black moor sperm did not decrease significantly during 48 hr of storage. A significant reduction in the Oranda type compared to the fresh control was observed in pMOT after 42 (23 ± 2%) and VCL after 36 (94 ± 12 μm/s) hours (pMOT 84 ± 5%, VCL 150 ± 11 μm/s). In the Calico type, pMOT decreased significantly already after 18 (42 ± 26%) and VCL after 6 (105 ± 8 μm/s) hours (fresh: pMOT 92 ± 5%, VCL 151 ± 6 μm/s). A high pMOT immediately following thawing was measured in Oranda (46 ± 12%) and Calico (55 ± 15%) types, whereas a reduced pMOT was recorded in Black moor (24 ± 19%). In Calico, pMOT showed a significant reduction after 6 hr (19 ± 11%) in comparison with the initial value, with no changes observed in VCL and STR. None of the parameters changed in the Black moor and Oranda types. Evidence was found that different goldfish lines have different sperm quality and characteristics. Further studies can investigate the possible effects of chilled and post‐thaw storage on the fertilizing capacity of sperm in the Black moor, Oranda and Calico goldfish types.     

Serum kisspeptin: New possible biomarker for sexual behaviour and sperm concentration in buffalo bulls

The study was designed to decipher the inter-relationship between peripheral hormones (kisspeptin and testosterone), sexual behaviour and seminal variables of Murrah buffalo bulls (n = 134). In this study, we recorded that 13%, 37%, 40%, 6% and 4% Murrah buffalo bulls had reaction time of <30, 31–60, 61–180, 181–300 and >300 s, respectively. Further, it was observed that 4%, 85% and 10% buffalo bulls were sexually aggressive, active and dull, respectively, during semen collection. The courtship behaviour was not found to be desirable for the bulls used for the semen collection. Mean of ejaculate volume, sperm concentration and mass motility (0–5 scale) were 3.57 ml, 977.11 million/ml, 2.7, respectively. Correlation studies revealed that the reaction time was positively correlated with courtship behaviour and body weight, and negatively correlated with sexual aggressiveness and sperm concentration. Serum kisspeptin in buffalo bulls, measured for the first time, was found to 3.8 ± 0.7 ng/ml. Serum kisspeptin and testosterone level are negatively correlated to each other and kisspeptin level influenced the sexual behaviour (reaction time, sexual aggressiveness and penile erection) of study bulls. Serum kisspeptin was higher in the buffalo bulls with higher sperm concentration indicating its role in spermatogenesis. In conclusion, for the first time basic information related to sexual behaviour of Murrah buffalo bulls in large population along with its inter-relationship with peripheral hormones (kisspeptin and testosterone) has been documented.     

Dynorphin-Kappa Opioid Receptor Signaling Partly Mediates Estrogen Negative Feedback Effect on LH Pulses in Female Rats

Accumulating evidence suggests that the arcuate nucleus (ARC) kisspeptin/neurokinin B (NKB)/dynorphin (KNDy) neurons play a role in estrogen negative feedback action on pulsatile gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release. The present study aimed to determine if dynorphin (Dyn) is involved in estrogen negative feedback on pulsatile GnRH/LH release. The effect of the injection of nor-binaltorphimine (nor-BNI), a kappa-opioid receptor (KOR) antagonist, into the third cerebroventricle (3V) on LH pulses was determined in ovariectomized (OVX) adult female rats with/without replacement of negative feedback levels of estradiol (low E₂). The mean LH concentrations and baseline levels of LH secretion in nor-BNI-injected, low E₂-treated rats were significantly higher compared with vehicle-treated controls. On the other hand, the nor-BNI treatment failed to affect any LH pulse parameters in OVX rats without low E₂ treatment. These results suggest that Dyn is involved in the estrogen negative feedback regulation of pulsatile GnRH/LH release. The low E₂ treatment had no significant effect on the numbers of ARC Pdyn (Dyn gene)-,Kiss1- and Tac2 (NKB gene)-expressing cells. The treatment also did not affect mRNA levels of Pdyn and Oprk1 (KOR gene) in the ARC-median eminence region, but significantly increased the ARC kisspeptin immunoreactivity. These findings suggest that the negative feedback level of estrogen suppresses kisspeptin release from the ARC KNDy neurons through an unknown mechanism without affecting the Dyn and KOR expressions in the ARC. Taken together, the present result suggests that Dyn-KOR signaling is a part of estrogen negative feedback action on GnRH/LH pulses by reducing the kisspeptin release in female rats.     

Investigation of the usability of kisspeptin and oxidative stress parameters in the early diagnosis of asymptomatic cystic endometrial hyperplasia in dogs

This study aimed to investigate the differences in oxidative stress index (OSI) and kisspeptin levels in clinically asymptomatic dogs with cystic endometrial hyperplasia (CEH) compared to healthy and pregnant dogs, and to determine the usability of the obtained results in the diagnosis of asymptomatic CEH. The study comprised three groups; a healthy (n = 8), a pregnant (n = 10) and a CEH (n = 10). All dogs in the three groups were included in the study at the 30 ± 3th day after estrus, and blood samples were collected for analysis of kisspeptin, total antioxidant status (TAS), total oxidant status (TOS), progesterone (P4), estradiol (E2) and some biochemical parameters (TSH; thyroid stimulating hormone, ALT; alanine aminotransferase, AST; aspartate aminotransferase, ALP; alkaline phosphatase, LDH; lactate dehydrogenase, CRE; creatine and BUN; blood urea nitrogen). In addition, OSI value was calculated. P4 and ALT and BUN levels were significantly lower and higher in CEH group than the pregnant group, respectively (p < .05). While kisspeptin and TAS levels were significantly lower in CEH group compared to the healthy and pregnant groups (p < .01), OSI level increased dramatically. In conclusion, it was confirmed that CEH clearly affected kisspeptin and OSI levels, and it is thought that these parameters may be an alternative diagnostic tool for the detection of CEH after further studies.     

Resveratrol Downregulates Cyp2e1 and Attenuates Chemically Induced Hepatocarcinogenesis in SD Rats

Cyp2e1 plays an important role in chemically induced hepatocarcinogenesis. Resveratrol (REV) is known to prevent diethylnitrosamine (DEN)-induced hepatocarcinogenesis, but its effects on this process induced by DEN and 2-acetylaminofluorene (2-AAF) and the role of Cyp2e1 remain unclear. In this study, glutathione S-transferase placental form (GST-P)-positive foci were used as a marker of hepatocarcinogenesis. REV or diallyl disulfide (DADS, an inhibitor of Cyp2e1) significantly reduced both the area and number of GST-P-positive foci induced by DEN and 2-AAF. Treatment with REV or DADS also markedly decreased the expression of Cyp2e1 in the rat liver. By immunohistochemical staining of serial liver sections, we found that the expression of Cyp2e1 in GST-P-positive foci showed three distinct patterns: decreased in GST-P foci, increased in GST-P foci when compared with surrounding liver tissue and mixed type. The number of GST-P foci with increased Cyp2e1 expression was greater than the number of GST-P foci with decreased Cyp2e1. Protein levels of GST-P and Cyp2e1 were also higher in foci compared with surrounding liver tissue. REV or DADS significantly reduced the expression of GST-P and Cyp2e1 in both foci and surrounding liver tissue. Taken together, these results suggested that REV has a significant inhibitory effect on chemically induced hepatocarcinogenesis, which may be attributed to downregulation of Cyp2e1.     

DNA methylation and expression of imprinted genes are associated with the viability of different sexual cloned buffaloes

The DNA methylation of imprinted genes is an important way to regulate epigenetic reprogramming of donor cells in somatic cell nuclear transfer (SCNT). However, the effects of sexual distinction on the DNA methylation of imprinted genes in cloned animals have seldom been reported. In this study, we analysed the DNA methylation status of three imprinted genes (Xist, IGF2 and H19) from liveborn cloned buffaloes (L group, three female and three male), stillborn cloned buffaloes (S group, three female and three male) and natural reproduction buffaloes (N group, three female and three male), using bisulphite sequencing polymerase chain reaction (BS‐PCR). The expression levels of these imprinted genes were also investigated by quantitative real‐time PCR (QRT‐PCR). The DNA methylation levels of H19 were not significantly different among the groups. However, the Xist in female and IGF2 in male of the S group were found to be significantly hypomethylated in comparison with the same sexual buffaloes in L group and N group (p < .05). Furthermore, the expression levels of Xist, IGF2 and H19 in the stillborn female cloned buffaloes of S group were significantly higher than that of the female buffaloes in the L group and N group (p < .05). The expression levels of IGF2 and H19 in the stillborn male cloned buffaloes in the S group were significantly higher than that of the male buffaloes in the L group and N group (p < .05). These results indicate that Xist may be associated with the viability of female cloned buffaloes, and IGF2 may also be related to the viability of male cloned buffaloes.     

Gene expression and mutation assessment provide clues of genetic and epigenetic mechanisms in liver tumors of oxazepam-exposed mice

Liver tumors from a previous National Toxicology Program study were examined using global gene expression and mutation analysis to define the mechanisms of carcinogenesis in mice exposed to oxazepam. Five hepatocellular adenomas and 5 hepatocellular carcinomas from male B6C3F1 mice exposed to 5000 ppm oxazepam and 6 histologically normal liver samples from control animals were examined. One of the major findings in the study was upregulation of the Wnt/β-catenin signaling pathway. Genes that activate β-catenin, such as Sox4, were upregulated, whereas genes that inhibit Wnt signaling, such as APC and Crebbp, were downregulated. In addition, liver tumors from oxazepam-exposed mice displayed β-catenin mutations and increased protein expression of glutamine synthetase, a downstream target in the Wnt signaling pathway. Another important finding in this study was the altered expression of oxidative stress-related genes, specifically increased expression of cytochrome p450 genes, including Cyp1a2 and Cyp2b10, and decreased expression of genes that protect against oxidative stress, such as Sod2 and Cat. Increased oxidative stress was confirmed by measuring isoprostane expression using mass spectrometry. Furthermore, global gene expression identified altered expression of genes that are associated with epigenetic mechanisms of cancer. There was decreased expression of genes that are hypermethylated in human liver cancer, including tumor suppressors APC and Pten. Oxazepam-induced tumors also exhibited decreased expression of genes involved in DNA methylation (Crebbp, Dnmt3b) and histone modification (Sirt1). These data suggest that formation of hepatocellular adenomas and carcinomas in oxazepam-exposed mice involves alteration of the Wnt signaling pathway, oxidative stress, and potential epigenetic alterations.     

Neuroendocrine,Metabolic and Genomic Cues Signalling the Onset of Puberty in Females

Puberty is the result of a dynamic interaction between genetic factors and environmental cues, all of which lead to the attainment of reproductive capacity. Thus, significant changes in hormone secretion occur from the pre‐pubertal to the pubertal stage. The objective of this review is to provide an update of some endocrine, physiological, metabolic and genetic concepts involved in the establishment of the hypothalamic‐hypophyseal‐gonadal axis function promoting the onset of the reproductive function during puberty. To achieve this purpose, basic aspects of the function of the hypothalamic‐hypophyseal‐gonadal axis, the control of the axis by neurotransmitters and the interaction between reproductive function and metabolic status will be considered. Finally, the role of the novel kisspeptin system and the GPR54 receptor as modulators of puberty will be considered, in addition to the hierarchical expression of the main genes acting as regulators of the onset of puberty.