Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary: Involvement of apoptosis and hormonal regulation

In the mammalian ovary, the microvasculature in the thecal layer of follicles is associated with follicular development. Apelin and its receptor, APJ, are expressed in the tissues and organs which include the vasculature. The aims of the present study were to examine the mRNA expression of apelin and the APJ receptor in granulosa cells and theca tissue of bovine follicles and the effects of steroid hormone and gonadotrophins on the expression of these genes in cultured granulosa cells and theca cells. The expression of apelin mRNA was not found in the granulosa cells of bovine follicles. The expression of the APJ gene was increased in granulosa cells of estrogen-inactive dominant follicles. The expression of apelin mRNA increased in theca tissues of estrogen-inactive dominant follicles. APJ expression in theca tissues increased with follicle growth. Progesterone stimulated the expression of APJ mRNA in the cultured granulosa cells. FSH stimulated the expression of APJ mRNA in the cultured granulosa cells. LH induced the expression of apelin and APJ receptor mRNAs in cultured theca cells. Taken together, our data indicate that the APJ receptor in granulosa cells and both apelin and the APJ receptor in theca tissues are expressed in bovine ovary, that APJ in granulosa cells may be involved in the appearance of the cell apoptosis, and that LH stimulates the expression of apelin and APJ genes in theca cells.     

Cell-specific localisation of apoptosis in the bovine ovary at different stages of the oestrous cycle

Apoptosis was localized in all ovarian cell types of 23 cows in various stages of the oestrous cycle, using the detection of active caspase-3, in situ end labelling (TUNEL) and DNA fluorescent staining (DAPI). Very few apoptotic cells were found in primordial, primary, secondary and vital tertiary follicles. In contrast, apoptosis in atretic tertiary follicles was much more frequent, and high apoptotic scores were recorded when using the TUNEL technique and lower scores with the caspase-3 assay. Cystic atretic follicles showed in general a higher apoptotic score than obliterative atretic follicles, with intermediate to high scores in granulosa cells and lower scores in theca cells. In corpora lutea, large and small lutein cells had intermediate to high scores using the caspase-3 assay, and intermediate to low scores using the TUNEL assay. Irrespective of the detection method, the scores were higher in lutein cells than in the capsular stroma cells. In all ovarian structures examined, variations in apoptotic scores were seen in the different cycle stages, suggesting a cycle-dependent influence on apoptosis, although correlations with plasma progesterone concentrations were low.     

Cell-specific localization of progesterone receptors in the bovine ovary at different stages of the oestrous cycle

This immunohistochemical study describes the localization of progesterone receptors (PR) in the bovine ovary of 23 cows at different stages of the oestrous cycle. In primordial, primary and secondary follicles the score for PR in the follicle cells increased progressively with the maturation of the follicle. In vital tertiary follicles and cystic atretic follicles a moderate score for PR was found, while in obliterative atretic follicles the score was much lower. Scores were high in corpora hemorrhagica, low in corpora lutea and still lower in corpora albicantia. Low PR scores were also found in the tunica albuginea and surface epithelium. Cyclic variations of PR immunoreactivity were manifest in most ovarian tissues. Follicular scores for PR were high in oestrus and decreased during the following stages, whereas scores in corpora lutea cells varied according to a characteristic pattern with high levels during oestrus and metoestrus. The variations in the scores for PR in the different ovarian cell types suggest a cell-specific and cycle-dependent influence of progesterone. A negative correlation was found between the PR scores and the plasma progesterone concentration.     

A missense mutation in the bovine leptin receptor gene is associated with leptin concentrations during late pregnancy

The leptin receptor (LEPR) gene consists of 20 exons divided over 1.75 Mb. Parts of bovine LEPR exon 4 (79 bp), exon 11 (95 bp) and exon 20 (513 bp) of 20 cows (Holstein-Friesian) were sequenced (AJ580799; AJ580800; AJ580801) in an attempt to find polymorphisms. In exons 4 and 11 no SNPs were found. In exon 20, a T to C missense mutation was found at nucleotide 115, which causes an amino acid substitution at residue 945 (T945M). Frequencies for alleles C and T were 0.93 and 0.07 respectively, in a population of 323 Holstein-Friesian cows and TT animals were not detected. Using genotypes of these cows an association study was performed for leptin concentrations during late pregnancy and lactation. Leptin concentrations were determined by radioimmunoassay (RIA). The T945M mutation showed an association with circulating leptin concentrations only during late pregnancy (P < 0.05) but not during lactation (P > 0.05). The CC genotype had higher concentrations than the CT genotype during this period. A combined effect with previously described leptin polymorphisms on prepartum leptin concentrations was observed, with one genotype combination having significantly lower levels of leptin up to 50 days, but interaction effects were not significant. The T945M polymorphism may have induced a structural change in the intracellular domain of the LEPR, which may have influenced the signal transduction pathway. However, the effect was found only for the heterozygous genotype because the TT genotype was not detected in this population of 323 Holstein-Friesian cows.     

Relationship between leptin receptor and polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders, which is involved in the multi-system disease, and its etiology is still not clearly understood. It is currently considered that not only the genetic factors but also the environment factors play a crucial role in the pathogenesis of PCOS. Obesity plays an important role through the insulin, leptin and endocannabinoid system in the pathological process of PCOS, leading to more severe clinical manifestations. The aim of our present study is to investigate whether there is association between single nucleotide polymorphisms (SNPs) of Gln223Arg and Pro1019Pro in the leptin receptor gene (LEPR) and PCOS in a Korean population. Interestingly, a significant association was found between the Pro1019Pro in LEPR gene and PCOS, and a highly significant association was found between the Gln223Arg in LEPR gene and PCOS (P = 0.033, OR = 1.523, 95% confidence interval and P < 0.0001, OR = 0.446, 95% confidence interval). Moreover, genotype combination and haplotype analyses indicate that Gln223Arg and Pro1019Pro polymorphisms of LEPR are significantly associated with the risk of PCOS.     

Mutation in the Sp1 motif of the bovine leptin gene affects its expression

Leptin is expressed mainly by adipocytes and plays a crucial role in the regulation of energy expenditure, food intake, and adiposity. Using PCR-heteroduplex analysis and sequencing, we investigated a C/G substitution in the promoter region of the bovine leptin gene. Application of the electrophoretic mobility shift assay showed that the C→G transversion decreased the leptin gene promoter binding capacity for nuclear proteins. With real-time PCR and Western blotting, we showed that the leptin expression level was higher in cattle with the CC than with the GG genotype.     

Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation

Leptin is a product of the ob gene that is produced primarily by adipose tissue. Leptin and its receptors are found within the ovary, but it is unclear what function this hormone has in the ovary. Using immunohistochemistry, we determined that leptin is found in most cell types in the murine ovary, with the highest staining levels observed in the oocyte. Leptin receptor was also expressed in all of the main ovarian cell types, with the thecal cell layer exhibiting the highest staining levels. Leptin administration did not affect spontaneous or induced maturation of either isolated denuded oocytes or cumulus-oocyte complexes, but it did significantly increase the rate of meiotic resumption in preovulatory follicle-enclosed oocytes (P < 0.01). Measurements of cAMP within oocytes cultured with leptin showed that this enhanced ability to resume meiosis does not occur via activation of phosphodiesterase 3B and subsequent cAMP reduction. These results provide evidence that leptin affects oocyte maturation when the oocyte is cultured within its normal follicular environment. It is suggested that leptin may induce the production of another factor, possibly from thecal cells, that directly or indirectly acts on the oocyte to initiate germinal vesicle breakdown in this species.     

Molecular cloning and expression of bovine (Bos taurus) leptin receptor isoform mRNAs

We characterized Bos taurus leptin receptor (Ob-R) isoform mRNAs as well as their expression in different tissues, including some adipose depots (perirenal, subcutaneous and intermuscular adipose tissues). Based on the GenBank database sequences of the bovine partial Ob-R, primers were designed to amplify cDNAs of bovine Ob-R isoforms. The full-length cDNAs of bovine the Ob-R isoforms were cloned by combination with 3'-and 5'-RACE. Three bovine Ob-R isoform cDNAs were cloned and the sequence analyses revealed that these cDNAs were bovine Ob-R isoforms, i.e., the long form (Ob-Rb), the middle form (Ob-Ra) and the short form (Ob-Rc). The open reading frames of Ob-Ra, Ob-Rb and Ob-Rc gene were 2688, 3498 and 2673 bp, respectively. The deduced amino acid sequences suggested that the isoforms were single transmembrane proteins, and differed in the C-terminal amino acid sequences. The amino acid sequence of these bovine Ob-R isoforms showed 73-75% identity compared with the corresponding mouse isoforms. The tissue-specific expression of the bovine Ob-R isoforms were measured by semi-quantitative RT-PCR. Expression of Ob-Rb was highest in liver, heart, spleen and kidney, with lower expression in lung and testis, and slight expression in muscle. Ob-Ra was highly expressed in liver and spleen, whereas moderate expression was observed in heart, testis, and muscle, and its expression was the lowest in lung and kidney. Ob-Rc mRNA was expressed in the liver, heart, testis, kidney and muscle, but not in the lung and spleen. In adipose tissues, higher expression of Ob-Ra and Ob-Rb mRNA was observed in intermuscular adipose tissue than in subcutaneous or perirenal adipose tissues. Ob-Ra mRNA level was positively correlated with Ob-Rb mRNA level in the adipose tissues (r=0.81, P<0.05). The results demonstrated that each Ob-R isoform mRNA was differentially expressed in various tissues of cattle, which may be involved in the difference of peripheral actions for leptin.     

不同生理阶段女性阴道微生态状况分析

目的 通过对女性阴道微生态的检查,全面评估不同生理阶段的女性阴道微生态状况,为临床对女性阴道感染性疾病的诊断和治疗提供较全面客观的实验室依据。方法 选取2017年1月1日至2017年6月30日在山东省立医院妇产科门诊就诊的女性患者2311例,分别采集患者的阴道分泌物,通过相差显微镜、革兰染色和干化学检测等方法,分别对患者阴道分泌物的清洁度、菌群密集度、多样性,pH值、AV评分、Nugent评分和功能评价等进行分析。结果 （1）2311例患者中育龄期患者组1806例,围绝经期患者组380例,老年绝经期患者组125例,其中育龄期患者组阴道优势菌为G+大杆菌（26.57%,480/1806）,菌群密集度（+++/++++,76.97%,1390/1806）、菌群多样性（++/+++,27.96%,505/1806）与围绝经期患者组和老年绝经期患者组比较,差异均有统计学意义（Ps<0.01）。围绝经期患者组阴道清洁度（III/IV度,50.26%,191/380）,pH值（pH≥4.6,72.89%,277/380）,菌群密集度（未见,3.42%,13/380）,过氧化氢（+,84.74%,322/380）,唾液酸苷酶（+‒,14.21%,54/380;+,27.89%,106/380）,与育龄期患者组比较,差异均有统计学意义（Ps<0.01）;围绝经期患者组阴道优势菌为G‒小杆菌（20.26%,77/380）,革兰染色不定球杆菌（27.10%,103/380）、菌群多样性（+,78.16%,297/380）与育龄期患者组和老年绝经期患者组比较,差异均有统计学意义（Ps<0.01）。老年绝经期患者组阴道pH值（pH≥4.6,85.60%,107/125）,无优势菌（25.60%,32/125）,优势菌为G+球菌（39.20%,49/125）,菌群密集度（未见,20.80%,26/125;+/++,48.80%,61/125）、菌群多样性（未见,20.80%,26/125）、白细胞酯酶（+,77.60%,97/125）,与育龄期患者组和围绝经期患者组比较,差异均有统计学意义（Ps<0.01）。（2）老年绝经期患者组AV（中度,17.6%,22/125;重度,5.6%,7/125）、BV（中介,56%,70/125）均显著高于育龄期患者组和围绝经期患者组;育龄期患者组VVC（23.20%,419/1806）和CV（8.47%,153/1806）的发病率均显著高于围绝经期患者组和老年绝经期患者组;围绝经期患者组TV（7.10%,27/380）的发病率显著高于育龄期患者组和老年绝经期患者组。结论 通过分析2311例女性患者阴道微生态状况,发现女性不同生理阶段阴道的微生态情况和各种阴道炎的发病率有差异,因而阴道微生态评价能够为临床的诊断和及时治疗提供依据,更快速地解决患者的病痛,提高患者的生活质量。     

Induction of leptin receptor expression in the liver by leptin and food deprivation

Leptin resistance is a common feature of obesity and the metabolic syndrome. However, the regulated expression of the leptin receptor (Ob-R) has not been studied in detail. Expression profiling of liver mRNA in leptin-treated wild-type mice revealed a marked increase in leptin receptor mRNA levels, which had not previously been described. This was confirmed by isoform-specific real-time PCR, which showed a >25-fold increase in the mRNAs encoding the short forms (Ob-Ra, Ob-Rc) and a >10-fold increase in the mRNA encoding the long (Ob-Rb) form of the leptin receptor in liver. In parallel, we also observed induction of plasma-soluble leptin receptor (SLR) protein by leptin administration, pair feeding, and short term food restriction. However, induction of SLR by leptin is abolished in mice with selective deletion of Ob-R from liver using Cre-LoxP technology. These data suggest that the liver is a major source of Ob-R mRNA expression under conditions of negative energy balance. Membrane-bound Ob-R is then shed into the circulation as SLR. Our study thus reveals an unexpected role of the liver in modulating total circulating leptin levels and possibly its biological activity.     

The cardiac expression of Mas receptor is responsive to different physiological and pathological stimuli

The Mas protooncogene encodes a G protein-coupled receptor that has been described as a functional receptor for the cardioprotective fragment of the renin-angiotensin system (RAS), Angiotensin (Ang)-(1-7). The aim of this current study was to evaluate the responsiveness of Mas expression in hearts during different physiological and pathological conditions in rats. Physical training was considered a physiological condition, while isoproterenol-induced hypertrophy, myocardial infarction and DOCA-salt model of hypertension were used as pathological models of heart injury. The expression of Mas was analyzed by western blotting. Although swim-trained rats presented significant cardiac hypertrophy, our physical training protocol was unable to induce changes in the expression of Mas. On the other hand, cardiac hypertrophy and damage elicited by isoproterenol treatment led to a reduction in Mas expression. Myocardial infarction also significantly decreased the expression of Mas after 21 days of myocardial ischemia. Additionally, Mas expression levels were increased in hearts of DOCA-salt rats. Our present data indicate that Mas expression is responsive to different pathological stimuli, thereby suggesting that Mas receptor is involved in the homeostasis of the heart, as well as in the establishment and progression of cardiac diseases.     

Gene expression profiling of dendritic cells in different physiological stages under Cordyceps sinensis treatment

Cordyceps sinensis (CS) has been commonly used as herbal medicine and a health supplement in China for over two thousand years. Although previous studies have demonstrated that CS has benefits in immunoregulation and anti-inflammation, the precise mechanism by which CS affects immunomodulation is still unclear. In this study, we exploited duplicate sets of loop-design microarray experiments to examine two different batches of CS and analyze the effects of CS on dendritic cells (DCs), in different physiology stages: naïve stage and inflammatory stage. Immature DCs were treated with CS, lipopolysaccharide (LPS), or LPS plus CS (LPS/CS) for two days, and the gene expression profiles were examined using cDNA microarrays. The results of two loop-design microarray experiments showed good intersection rates. The expression level of common genes found in both loop-design microarray experiments was consistent, and the correlation coefficients (Rs), were higher than 0.96. Through intersection analysis of microarray results, we identified 295 intersecting significantly differentially expressed (SDE) genes of the three different treatments (CS, LPS, and LPS/CS), which participated mainly in the adjustment of immune response and the regulation of cell proliferation and death. Genes regulated uniquely by CS treatment were significantly involved in the regulation of focal adhesion pathway, ECM-receptor interaction pathway, and hematopoietic cell lineage pathway. Unique LPS regulated genes were significantly involved in the regulation of Toll-like receptor signaling pathway, systemic lupus erythematosus pathway, and complement and coagulation cascades pathway. Unique LPS/CS regulated genes were significantly involved in the regulation of oxidative phosphorylation pathway. These results could provide useful information in further study of the pharmacological mechanisms of CS. This study also demonstrates that with a rigorous experimental design, the biological effects of a complex compound can be reliably studied by a complex system like cDNA microarray.     

Plasma leptin-binding activity and hypothalamic leptin receptor expression during pregnancy and lactation in the rat

Leptin, the 16-kDa peptide hormone product of the ob gene, regulates body weight via the hypothalamus but also influences several aspects of reproductive function. Results of previous studies have suggested that pregnancy is a state of leptin resistance, because food consumption remains stable or increases despite a progressive rise in plasma leptin across most of gestation. In the present study, we assessed whether this apparent leptin resistance during rat pregnancy was due to either increased plasma leptin-binding activity and/or reduced expression of hypothalamic leptin receptor. Plasma leptin increased from 2.2 +/- 0.4 ng/ml before pregnancy to a maximum at midgestation (4.2 +/- 0.8 ng/ml on Day 12) and then fell by Day 22 and remained low throughout lactation. Despite the higher plasma leptin levels in pregnancy, food consumption increased from a minimum of 13.6 +/- 0.5 g/day before pregnancy to a peak of 21.9 +/- 0.6 g/day on Day 19, then fell before parturition (11.9 +/- 0.4 g/day on Day 22). At least part of the increase in plasma leptin during pregnancy was attributable to a marked increase (P < 0.001) in plasma leptin-binding activity between diestrus and late pregnancy, which then fell after birth but remained at midpregnancy levels to at least Day 12 of lactation. Hypothalamic expression of mRNA encoding the long form of the leptin receptor (Ob-Rb) was elevated in early pregnancy (Day 7) but returned to prepregnancy levels by midgestation and remained stable thereafter. The results of this study confirm that pregnancy in the rat is a state of relative leptin resistance, which is due primarily to increased plasma leptin-binding activity rather than to changes in hypothalamic Ob-Rb expression.     

The expression and putative role of brain-derived neurotrophic factor and its receptor in bovine sperm

The neurotrophin family of proteins promote the survival and differentiation of nerve cells and are thought to play an important role in development of reproductive tissues. The objective of the present study was to detect the presence of Brain-derived neurotrophic factor (BDNF) and its receptor TrkB in bovine sperm, and explore the potential role of BDNF in sperm function. We demonstrated that both the neorotrophin BDNF and the tyrosine kinase receptor protein TrkB were expressed in ejaculated bovine sperm. Furthermore, BDNF per se was secreted by sperm. Insulin and leptin secretion by bovine sperm were increased (P < 0.01) when cells were exposed to exogenous BDNF, whereas insulin was decreased by K252a. Therefore, we inferred that BDNF could be a regulator of sperm secretion of insulin and leptin through the TrkB receptor. Sperm viability and mitochondrial activity were both decreased (P < 0.05) when the BDNF/TrkB signaling pathway was blocked with K252a. Furthermore, BDNF promoted apoptosis of bovine sperm through TrkB binding (P < 0.05). In conclusion, these observations provided evidence that BDNF secreted by bovine sperm was important in regulation of insulin and leptin secretion in ejaculated bovine sperm. Furthermore, BDNF may affect sperm mitochondrial activity and apoptosis, as well as their viability.     

褐飞虱flightin的原核表达及其差异表达检测

flightin最早发现于果蝇Drosophila melanogaster的间接飞行肌中,并且定位于粗肌丝。这种蛋白对维持肌节的结构和功能起到了重要的作用,但其在具有长短翅型分化的褐飞虱Nilaparvata lugens Stl的不同翅型间差异并不清楚。本研究以长翅型雌虫褐飞虱cDNA为模板,通过PCR扩增得到褐飞虱flightin基因ORF全长,将其连接到表达载体pGEX-6P-1中以与谷胱甘肽S-转移酶(GST)融合表达。将表达载体转入大肠杆菌表达株Rosseta,在不同温度、不同浓度IPTG的条件下诱导表达flightin,得到了最优表达条件,获得了高水平可溶性表达。在用GST抗体进行Western blotting验证GST-flightin融合重组蛋白表达的正确性后,我们通过GST柱纯化了的GST-flightin,进而用纯化后的蛋白免疫新西兰兔制备了高特异性的多克隆抗体。最后,我们用制备的多克隆抗体检测了长、短翅型雌成虫和不同发育阶段的褐飞虱体内flightin的表达差异。结果显示,flightin仅在长翅型成虫中表达,在短翅型雌成虫中未检测到其明显表达,而且flightin只在成虫期表达。本研究为进一步研究褐飞虱的flightin与其它蛋白互作、翅肌发育和翅型分化打下了基础。