石斑鱼垂体腺苷酸环化酶激活多肽和生长激素释放激素基因的cDNA克隆及表达分析

垂体腺苷酸环化酶激活多肽 (PACAP)和生长激素释放激素 (GHRH)均属于血管活性肠肽家族成员 ,且两者前体基因在脊椎动物的鸟类、两栖类、鱼类中由同一基因编码 ,而哺乳动物是由两个不同基因编码。已有几例关于鱼类编码PACAP和GHRH基因克隆的报道 ,而关于重要海水养殖鱼类石斑鱼的PACAP和GHRH基因未见报道。克隆了PACAP GHRH前体cDNA序列 ,该前体有两种剪接方式 ,包括一个长序列和一个短序列 ,其中长序列编码PACAP和GHRH ,短序列缺失 10 5个碱基 ,仅编码PACAP而缺失编码GHRH的外显子区 ,同样情况在虹鳟和沟鲶中也有报道。通过半定量RT PCR方法对石斑鱼PACAP GHRH前体mRNA在胚胎发育和发育早期以及各部位的表达情况进行了分析。胚胎发育分析结果表明 ,从神经胚期开始 ,PACAP GHRH前体mRNA大量表达 ,提示该蛋白质在神经发育或神经营养方面具有重要作用。PACAP GHRH前体基因在中枢系统的表达量远高于外周组织。在鱼类的眼和鳃发现PACAP GHRH前体分布。     

Evidence for pituitary adenylate cyclase-activating peptide as a direct immunoregulator in teleost head kidney

In mammals, pituitary adenylate cyclase activating polypeptide (PACAP) is a potent anti-inflammatory factor, showing that it inhibits the expression and release of proinflammatory cytokines and enhances the production of anti-inflammatory factors. However, whether fish PACAP plays similar regulatory roles as seen in mammals remains unclear. In the present study, expression of PACAP-specific receptor PAC1-R was shown in grass carp head kidney and spleen, supporting that PACAP may have a direct effect on fish immune cells. To test this hypothesis, the immunoregulatory role of grass carp PACAP (gcPACAP) was examined in head kidney leucocytes (HKLs). Results showed that gcPACAP inhibited basal and further attenuated lipopolysaccharide (LPS)-stimulated cell viability of HKLs, indicating that gcPACAP may possess similar inhibitory property at cellular level as seen in mammals. Curiously, in vitro and in vivo studies revealed that gcPACAP stimulated proinflammatory factors (IL-1β and TNF-α) but not IL-10 mRNA expression in HKLs and head kidney. Moreover, bacterial infection and LPS enhanced IL-1β, TNF-α and IL-10 mRNA expression in grass carp head kidney and HKLs, respectively, and these stimulatory effects were not influenced by gcPACAP. These findings suggest that PACAP plays distinct roles, at least does not function as an anti-inflammatory factor, in fish compared with that in mammals.     

Genomic structure, polymorphism and expression analysis of growth hormone-releasing hormone and pituitary adenylate cyclase activating polypeptide genes in the half-smooth tongue sole (Cynoglossus semilaevis)

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase activating polypeptide (PACAP) regulate development and somatic growth in teleosts; they may be associated with sexual growth dimorphism in the half-smooth tongue sole (Cynoglossus semilaevis). We found that the full-length GHRH and PACAP gene sequences obtained from females and males consist of 4160, 4159, 2425, and 2446 bp, respectively, each of which includes four exons and three introns. When we analyzed normal females and males and extra-large male adults, GHRH and PACAP mRNA were found to be predominantly expressed in the brain; the expression levels were highest in normal males. The extra-large males exhibited the lowest mRNA levels of both GHRH and PACAP. Sex differences in GHRH and PACAP mRNA expression during development were also examined in a full-sib family; GHRH and PACAP mRNA were detected at all 27 times sampled from 10 to 410 days old. The GHRH expression levels in females were significantly higher than in males at most of the stages between 20 and 100 days old, while lower than those of males after 120 days old. Five microsatellite loci were identified in GHRH and PACAP genes. We used these five polymorphic markers to genotype 224 individuals, and no significant differences were found between females and males from the Bohai Sea, the Yellow Sea and hatchery samples.     

Endothelial deletion of ADAM17 in mice results in defective remodeling of the semilunar valves and cardiac dysfunction in adults

Global inactivation of the metalloproteinase ADAM17 during mouse development results in perinatal lethality and abnormalities of the heart, including late embryonic cardiomegaly and thickened semilunar and atrioventricular valves. These defects have been attributed in part to a lack of ADAM17-mediated processing of HB-EGF, as absence of soluble HB-EGF results in similar phenotypes. Because valvular mesenchymal cells are largely derived from cardiac endothelial cells, we generated mice with a floxed Adam17 allele and crossed these animals with Tie2-Cre transgenics to focus on the role of endothelial ADAM17 in valvulogenesis. We find that although hearts from late-stage embryos with ablation of endothelial ADAM17 appear normal, an increase in valve size and cell number is evident, but only in the semilunar cusps. Unlike Hbegf^?/? valves, ADAM17-null semilunar valves do not differ from controls in acute cell proliferation at embryonic day 14.5 (E14.5), suggesting compensatory processing of HB-EGF. However, levels of the proteoglycan versican are significantly reduced in mutant hearts early in valve remodeling (E12.5). After birth, aortic valve cusps from mutants are not only hyperplastic but also show expansion of the glycosaminoglycan-rich component, with the majority of adults exhibiting aberrant compartmentalization of versican and increased deposition of collagen. The inability of mutant outflow valve precursors to transition into fully mature cusps is associated with decreased postnatal viability, progressive cardiomegaly, and systolic dysfunction. Together, our data indicate that ADAM17 is required in valvular endothelial cells for regulating cell content as well as extracellular matrix composition and organization in semilunar valve remodeling and homeostasis.     

PACAP and NGF cooperatively enhance choline acetyltransferase activity in postnatal basal forebrain neurons by complementary induction of its different mRNA species

Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.     

Cellular localization of an embryonic interferon,ovine trophoblastin and its mRNA in sheep embryos during early pregnancy

The ovine embryo produces an interferon named ovine Trophoblastin (oTP) which is involved in the maternal recognition of pregnancy and ensures the maintenance of progesterone secretion by the corpus luteum. We have used indirect immunohistofluorescence and in situ hybridization on histological sections to investigate the fate of this protein and its mRNA in ovine embryos from days 3 to 25 of pregnancy. The level of expression was measured by image analysis of the autoradiographs after in situ hybridization. Both techniques clearly demonstrated that oTP and its mRNA were specifically localized in the extra-embryonic trophoblast. Neither the embryonic cells, nor the yolk sac or the amniotic tissues produced the protein or its mRNA. The protein could be detected by d 11 of pregnancy in the elongated blastocyst. Maximum of expression is observed at d 14 and the level decreased by d 16 of pregnancy. The arrest of expression occurred in the regions of trophoblast which have established cellular contacts with the uterine epithelium during the implantation process.     

In vivo expression of interferon tau mRNA by the embryonic trophoblast and uterine concentrations of interferon tau protein during early pregnancy in the cow

In this study, we have measured uterine concentrations of interferon tau and intensity of embryonic interferon tau mRNA expression between day 14 and 18 in cows. While interferon tau concentrations rose dramatically (P < 0.001) from day 14 to 18, there was no significant increase in the intensity of expression of interferon tau mRNA by the trophoblast. When results were analyzed on the basis of embryo size, well elongated embryos (>10 cm) produced significantly (P < 0.001) more interferon tau than smaller embryos but showed similar levels of interferon tau mRNA expression. These results demonstrate that the increase in interferon tau concentrations responsible for the maternal recognition of pregnancy results from the increase in embryo size during elongation and not from any upregulation of mRNA expression.     

Carbon disulfide exposure at peri-implantation disrupts embryo implantation by decreasing integrin β3 expression in the uterine tissue of pregnant mice

Carbon disulfide (CS₂) exposure might disrupt embryo implantation in females during pregnancy but the relevant mechanism remains unclear. Since integrin β₃ has shown to be one of the cell adhesion molecules involved in embryo implantation, the current study examined the effect of CS₂ exposure during the peri-implantation period on the protein and mRNA expression of integrin β₃ and its DNA methylation degree in the uterine tissue of gestational mice. Exposure was on the 3rd day of gestation (GD3), GD4, GD5 and GD6, respectively, one time administration (631.4 mg/kg). The number of implanted embryos on GD9 was observed and compared with the control, it was decreased by 32.92%, 56.11%, 56.11% and 51.21%, respectively, which revealed that GD4 and GD5 were the most sensitive time for embryotoxicity. The protein and mRNA expression of integrin β₃ were detected each day after exposure till the GD7 endpoint. It down-regulated the protein and mRNA expression of integrin β₃ in uterine tissue and there was a positive correlation between the number of embryos and the expression of protein on the first and second day after exposure. However, the degree of integrin β₃ DNA methylation was not affected, which was detected by bisulfite sequencing polymerase chain reaction (BSP) method.     

Is caprine arthritis encephalitis virus (CAEV) transmitted vertically to early embryo development stages (morulae or blastocyst) via in vitro infected frozen semen?

The aim of this study was to determine, in vivo, whether in vitro infected cryopreserved caprine sperm is capable of transmitting caprine arthritis-encephalitis virus (CAEV) vertically to early embryo development stages via artificial insemination with in vitro infected semen. Sperm was collected from CAEV-free bucks by electroejaculation. Half of each ejaculate was inoculated with CAEV-pBSCA at a viral concentration of 10⁴ TCID₅₀/mL. The second half of each ejaculate was used as a negative control. The semen was then frozen. On Day 13 of superovulation treatment, 14 CAEV-free does were inseminated directly into the uterus under endoscopic control with thawed infected semen. Six CAEV-free does, used as a negative control, were inseminated intrauterine with thawed CAEV-free sperm, and eight CAEV-free does were mated with naturally infected bucks. Polymerase chain reaction (PCR) was used to detect CAEV proviral-DNA in the embryos at the D7 stage, in the embryo washing media, and in the uterine secretions of recipient does. At Day 7, all the harvested embryos were PCR-negative for CAEV proviral-DNA; however, CAEV proviral-DNA was detected in 8/14 uterine smears, and 9/14 flushing media taken from does inseminated with infected sperm, and in 1/8 uterine swabs taken from the does mated with infected bucks. The results of this study confirm that (i) artificial insemination with infected semen or mating with infected bucks may result in the transmission of CAEV to the does genital tack seven days after insemination, and (ii) irrespective of the medical status of the semen or the recipient doe, it is possible to obtain CAEV-free early embryos usable for embryo transfer.     

Hormonal asynchrony and embryonic development

Luteinizing hormone (LH), progesterone and estradiol profiles in peripheral blood serum were compared among parous and nonparous females with normal, abnormal or no embryonic development. Hormonal profiles between parous and nonparous females of the same embryonic status did not differ and the data were combined. Estrous cycle length was longer (P<.05) in parous (22.3±.4 days) than nonparous females (21.0±.4 days). Females with normal developing embryos had a higher serum progesterone concentration at Days 3 and 6 and a lower ratio of estradiol to progesterone than did females with abnormal embryonic development. Females with a normal embryo had higher (P<.05) preovulatory LH peaks than females with abnormal development or no recovery of an oocyte or embryo (34.3±4.7, 11.8±6.8 and 13.3±2.5 ng/ml, respectively). The interval from onset of estrus to LH peak was 8.9±2.1, 13.7±3.7 and 13.5±6.2 hr for females with normal, abnormal or no recovery of an embryo. The lower LH peak, the longer interval from onset of estrus to LH peak, and lower progesterone concentration in peripheral blood serum in females with abnormal embryos or no recovery indicated that these females had a hormonal asynchrony. The hormonal asynchrony may produce an undesirable uterine environment for male and female gametes or embryos which resulted in fertilization failure or embryonic death. In the second experiment, more transferable embryos were obtained when superovulated females received prostaglandin F_2α (PGF_2α) intravenously rather than intramuscularly. Administering PGF₂α intravenously rather than intramuscularly may have caused the demise of the corpus luteum sooner and thereby produced a more normal uterine environment which allowed more embryos to develop normally.     

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

As an activator of adenylate cyclase, the neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) impacts levels of cyclic AMP, a key second messenger available in brain cells. PACAP is involved in certain adult behaviors. To elucidate PACAP interactions, a compendium of microarrays representing mRNA expression in the adult mouse whole brain was pooled from the Phenogen database for analysis. A regulatory network was computed based on mutual information between gene pairs using gene expression data across the compendium. Clusters among genes directly linked to PACAP, and probable interactions between corresponding proteins were computed. Database “experts” affirmed some of the inferred relationships. The findings suggest ADCY7 is probably the adenylate cyclase isoform most relevant to PACAP's action. They also support intervening roles for kinases including GSK3B, PI 3-kinase, SGK3 and AMPK. Other high-confidence interactions are hypothesized for future testing. This new information has implications for certain behavioral and other disorders.     

Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation

Notochord is an embryonic midline structure that serves as mechanical support for axis elongation and the signaling center for the surrounding tissues. Precursors of notochord are initially induced in the dorsal most mesoderm region in gastrulating embryo and separate from the surrounding mesoderm/endoderm tissue to form an elongated rod-like structure, suggesting that cell adhesion molecules may play an important role in this step. In Xenopus embryo, axial protocadherin (AXPC), an orthologue of mammalian Protocadherin-1 (PCDH1), is indispensable for the assembly and separation from the surrounding tissue of the notochord cells. However, the role of PCDH1 in mammalian notochord remains unknown. We herein report that PCDH1 is expressed in the notochord of mouse embryo and that PCDH1-deficient mice form notochord normally. First, we examined the temporal expression pattern of pcdh1 and found that pcdh1 mRNA was expressed from embryonic day (E) 7.5, prior to the stage when notochord cells detach from the surrounding endoderm tissue. Second, we found that PCDH1 protein is expressed in the notochord of mouse embryos in addition to the previously reported expression in endothelial cells. To further investigate the role of PCDH1 in embryonic development, we generated PCDH1-deficient mice using the CRISPR-Cas9 system. In PCDH1-deficient embryos, notochord formation and separation from the surrounding tissue were normal. Structure and marker gene expression of notochord were also unaffected by loss of PCDH1. Major vascular patterns in PCDH1-deficient embryo were essentially normal. These results suggest that PCDH1 is dispensable for notochord formation, including the tissue separation process, in mammalian embryos. We successfully identified the evolutionary conserved expression of PCDH1 in notochord, but its function may differ among species.     

Expression of pituitary adenylate cyclase activating peptide in the uterine cervix, lumbosacral dorsal root ganglia and spinal cord of rats during pregnancy

The uterine cervix is highly innervated by the sensory nerves containing neuropeptides which change during pregnancy and are regulated, in part, by estrogen. These neuropeptides act as transmitters both in the spinal cord and cervix. The present study was undertaken to determine the expression pattern of the neuropeptide pituitary adenylate cyclase activating peptide (PACAP) in the cervix and its nerves during pregnancy and the influence of estrogen on this expression using immunohistochemistry, radioimmunoassay and RT-PCR. PACAP immunoreactivity was detected in nerves in the cervix, lumbosacral (L6-S1) dorsal root ganglia (DRG) and spinal cord. PACAP immunoreactivity was highest at day 15 of pregnancy in the cervix and dorsal spinal cord, but then decreased over the last trimester of pregnancy. However, levels of PACAP mRNA increased in the L6-S1 DRG at late pregnancy relative to early pregnancy. DRG of ovariectomized rats treated with estrogen showed increased PACAP mRNA synthesis in a dose-related manner, an effect partially blocked by the estrogen receptor (ER) antagonist ICI 182,780. We postulate that synthesis of PACAP in L6-S1 DRG and utilization in the cervix and spinal cord increase over pregnancy and this synthesis is the under influence of the estrogen-ER system. Since PACAP is expressed by sensory nerves and may have roles in nociception and vascular function, collectively, these data are consistent with the hypothesis that sensory nerve-derived neuronal factors innervate the cervix and play a role in cervical ripening.     

Reproductive cycle and maternal–embryonic nutritional relationship of shovelnose guitarfish Pseudobatos productus in the Gulf of California

Samples of the shovelnose guitarfish Pseudobatos productus were collected on board a vessel and at landings of artisanal commercial fisheries in the Gulf of California from May 2004 to June 2007. Samples of 650 females, 2047 embryos and 484 uterine eggs were examined. The reproductive cycle is annual, ovulation and parturition occur in July, the uterine eggs are in diapause for 9 months (July–March) before an accelerated growth of embryos of 3 months. Histological analyses of the uterine wall of pregnant females suggested that no secretions were used for embryo nourishment. The standard percentage of water content was 48·6% in fertilized eggs and 80·75% in full‐term embryos. Dry mass loss during embryonic development was 16·3% and the chemical balance of development was 0·84. This indicates that P. productus is a strictly lecithotrophic, viviparous species, that makes no maternal contribution of nutrients during embryonic development.