[Optimization of urban ecological spatial structure based on network analysis: A case study of Changzhou City, China]. / 基于网络分析的城市生态空间结构优化­­ä»¥å¸¸å·žå¸‚为例.

The rapid development of economy and society leads to the rapid expansion of cities, resulting in the atrophy of urban ecological space and the decline of ecological function, as well as a serious threat to urban ecological security. It is of great significance for the sustainable development of a city to systematically analyze the structure of urban ecological space and put forward targeted protection and optimization measures. Taking Changzhou City as the research area and considering the natural ecological function and social service function of urban ecological space, we constructed two ecological networks, the "source-corridor" ecological network based on natural ecology and the "supply-demand" ecological network based on human ecology. For the "source-corridor" ecological network, quantitative analysis was mainly carried out from the importance of nodes, network connectivity and stability. For the "supply-demand" ecological network, quantitative analysis was mainly carried out from the importance of nodes, supply-demand equilibrium and stability. The results showed that the levels of connectivity and stability of the "source-corridor" ecological network in the main urban area of Changzhou were not high, the stability level of the "supply-demand" ecological network was general, and there was spatial mismatch between service supply and demands. From the perspective of connectivity and stability improvement, an optimization scheme of "source-corridor" ecological network with 12 additional source nodes and 57 corridors was proposed. From the perspective of supply-demand balance and stability improvement, an optimization scheme of "supply-demand" ecological network with 22 new supply nodes was proposed. Compared with the original "source-corridor" ecological network, the connectivity level of the optimized network was improved by 10%, and the network stability was improved by 0.05. Compared with the initial "supply-demand" ecological network, the service level of the optimized network was improved by 4%, and the network stability was improved by 0.10. Finally, we integrated the two ecological networks, and formulated the implementation plan of protection and management for both the current protected patches and the new ecological nodes.

Induction of right open reading frame kinase 3 (RIOK3) during ovulation and luteinisation in rat ovary.

Atypical protein serine kinase RIOK3 is involved in cellular invasion and survival. The spatiotemporal expression pattern and regulatory mechanisms controlling expression of Riok3 were investigated in the rat ovary during the periovulatory period. Immature female rats (22-23 days old) were treated with pregnant mare's serum gonadotropin (PMSG) to stimulate follicular development, followed 48h later by injection with human chorionic gonadotrophin (hCG). Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after hCG administration. Both real-time polymerase chain reaction (PCR) and in situ hybridisation analysis revealed that Riok3 was highly induced in both granulosa cells and theca-interstitial cells by hCG. Riok3 expression was induced in theca-interstitial cells at 4h after hCG. However, the expression of Riok3 mRNA was stimulated in granulosa cells at 8h. Both protein kinase C inhibitor (GF109203) and the protein kinase A inhibitor (H89) could block the stimulation of Riok3 mRNA by hCG. Furthermore, Riok3 induction is dependent on new protein synthesis. Inhibition of prostaglandin synthesis or progesterone action did not alter Riok3 mRNA expression, whereas inhibition of the epidermal growth factor (EGF) pathway downregulated Riok3 expression. In conclusion, our findings suggest that the induction of the RIOK3 may be important for ovulation and luteinisation.

Induction of tumor suppressor KCTD11 during periovulatory period in rat ovary.

The tumor suppressor gene KCTD11 plays a critical role in cell proliferation, differentiation and invasion. The current study investigated the regulation and the spatiotemporal expression pattern of Kctd11 in the rat ovary during the periovulatory period. Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after hCG administration using an established gonadotropin-primed immature rat model that induces follicular development and ovulation. Real-time quantitative PCR analysis revealed that mRNA for Kctd11 was significantly induced both in theca-intersititial and granulosa cells after hCG treatment although their temporal expression patterns differed. In situ hybridization analysis demonstrated that Kctd11 mRNA expression was induced in theca-intersititial cells at 6 h after hCG, and the expression remained elevated until 12 h after hCG. Kctd11 mRNA was stimulated in granulosa cells at 6 h and reached the highest expression at 12 h. There was negligible Kctd11 mRNA signal observed in newly forming corpora lutea. In addition, the data indicate that both the protein kinase A and the protein kinase C pathway regulate the expression of Kctd11 mRNA in granulosa cells. Either forskolin or phorbol 12 myristate 13-acetate can mimic hCG induction of Kctd11 expression. Furthermore, the stimulation of Kctd11 by hCG requires new protein synthesis. Inhibition of progesterone action and the EGF pathway blocked Kctd11 mRNA expression, whereas inhibition of prostaglandin synthesis had no effect. Our finding suggest that the induction of the Kctd11 may be important for theca and granulosa cell differentiation into luteal cells.

Expression and regulation of high mobility group AT-hook 1 (HMGA1) during ovulation and luteinisation in rat ovary.

High mobility group AT-hook 1 (HMGA1) is able to regulate gene expression and function as a tumour suppressor. The spatiotemporal expression pattern of HMGA1 was investigated in this study. Immature female rats (22-23 days old) were treated with 10IU, s.c., pregnant mare's serum gonadotrophin to stimulate follicular development, followed 48h later by injection with 5IU, s.c., human chorionic gonadotrophin (hCG). Whole ovaries or granulosa cells were collected at various times after hCG administration (n=3 per time point). Real-time polymerase chain reaction and western blot analysis revealed that HMGA1 was highly stimulated in the ovary by 4-12h after hCG treatment. In situ hybridisation analysis demonstrated that Hmga1 mRNA expression was induced in granulosa cells between 8 and 12h after hCG treatment. There was negligible Hmga1 mRNA signal observed in newly forming corpora lutea. In addition, the data indicated that both the protein kinase (PK) A and PKC pathways regulated Hmga1 expression in rat granulosa cells. In rat granulosa cell cultures, upregulation of Hmga1 was dependent on new protein synthesis because Hmga1 was inhibited by cycloheximide. Furthermore, Hmga1 mRNA expression in rat granulosa cell cultures was inhibited by AG1478, whereas NS398 and RU486 had no effect, suggesting that Hmga1 expression was regulated, in part, by the epidermal growth factor pathway. In summary, the findings of this study suggest that induction of Hmga1 may be important for theca and granulosa cell differentiation into luteal cells.

Induction of Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 During the Periovulatory Period in the Rat Ovary.

Ectonucleotide pyrophosphatase/phosphodiesterase 3 ( Enpp3) is involved in multiple physiological processes, such as morphological changes and inflammatory processes. The present study investigated the spatiotemporal expression pattern and regulatory mechanisms controlling expression of Enpp3 in the rat ovary during the periovulatory period. Immature female rats were injected with pregnant mare serum gonadotropin to stimulate follicular development. Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after human chorionic gonadotropin (hCG) administration. Real-time polymerase chain reaction analysis revealed that messenger RNA (mRNA) for Enpp3 was highly induced in both granulosa cells and theca-interstitial cells by hCG. In situ hybridization analysis demonstrated that Enpp3 mRNA expression was induced in theca cells at 4 hours after hCG, and the expression remained elevated until 12 hours after hCG. The expression of Enpp3 mRNA was stimulated in granulosa cells at 8 hours and reached the highest expression at 12 hours. Localization of Enpp3 mRNA was observed in newly forming corpora lutea by in situ hybridization. The hCG-stimulated expression of Enpp3 mRNA was blocked by a protein kinase C inhibitor (GF109203) instead of the protein kinase A inhibitor (H89). Furthermore, Enpp3 induction is dependent on new protein synthesis. Inhibition of progesterone action did not alter Enpp3 mRNA expression, whereas inhibition of prostaglandin synthesis or the epidermal growth factor pathway diminished Enpp3 mRNA levels. In conclusion, our findings suggest that the induction of the Enpp3 mRNA may be important for the morphological changes and inflammatory response during ovulation and luteinization.

Expression and regulation of the differentiation regulators ERBB Receptor Feedback Inhibitor 1 (ERRFI1) and Interferon-related Developmental Regulator 1 (IFRD1) during the periovulatory period in the rat ovary.

The current study investigated the regulation and the spatiotemporal expression pattern of Errfi1 and Ifrd1, genex encoding factors that regulate differentiation and cessation of cell division, in the rat ovary during the periovulatory period. Immature female rats (22-23 days old) were injected with pregnant-mare serum gonadotropin to stimulate folliculogenesis, followed by human chorionic gonadotropin (hCG) to induce ovulation. Ovaries, granulosa cells, theca-interstitial cells, or cumulus oocyte complexes (COCs) were collected at various times after hCG administration (n = 3 per time point). Expression analysis revealed that Errfi1 and Ifrd1 were highly induced in the ovary, although their spatiotemporal expression differed: In situ hybridization analysis demonstrated that Errfi1 mRNA expression was initially induced in theca-interstitial cells at 4 and 8 hr after hCG, then transitioned to granulosa cells at 12 hr, and decreased in newly forming corpora lutea at 24 hr. Ifrd1 mRNA, on the other hand, was primarily induced in granulosa cells, and expression remained elevated in newly forming corpora lutea. Interestingly, Errfi1 and Ifrd1 were also expressed in the COC, suggesting a potential role in cumulus cell expansion or oocyte maturation. Inhibition of progesterone or prostaglandin synthesis reduced Errfi1 and Ifrd1 transcription, whereas inhibition of epidermal growth factor signaling inhibited only Errfi1 mRNA abundance. Down-regulation of both genes led to further suppression of progesterone. Our findings thus suggest that the stimulation of Errfi1 and Ifrd1 may be important for theca and granulosa cell differentiation and COC expansion. Mol. Reprod. Dev. 83: 714-723, 2016 © 2016 Wiley Periodicals, Inc.

Gene expression pattern and hormonal regulation of small proline-rich protein 2 family members in the female mouse reproductive system during the estrous cycle and pregnancy.

Small proline-rich proteins (SPRR) are known to construct the cornified cell envelope (CE) in the stratified squamous epithelial cell. Their functions in the simple epithelium such as the uterine epithelium are not clear hitherto. In the present study, the mRNA expression patterns of sprr2 family members in the mouse uterus and vagina during the estrous cycle and pregnancy as well as their regulation by steroids were investigated. Using semi-quantitative RT-PCR, it was revealed that the transcripts of sprr2b, 2e and 2g genes were up-regulated in the proestrous and estrous uteri, and sprr2d was up-regulated only in the estrous uterus. In the vagina, transcription of sprr2a, 2b, 2d, 2e and 2k genes were up-regulated at the metestrous stage. Northern blot analysis demonstrated that the overall expression of sprr2 was highly up-regulated in the estrous uterus and the metestrous vagina. During pregnancy, the sprr2 mRNA in the uterus was sharply repressed from day 3 postcoitus on, and began to be induced around labor time. In situ hybridization showed that the sprr2 transcripts were localized in uterine luminal and glandular epithelial cells as well as vaginal stratified epithelial cells. In ovariectomized mice, the expression of sprr2a, 2d, 2e and 2f genes in the uterus were induced by estrogen, and the effect of estrogen on sprr2d and 2e expression could be partly abolished by progesterone. The data indicate that the sprr2 genes have unique regulation patterns in different reproductive tissues under different physiological conditions, and the encoded proteins might play diverse functions in the female reproductive system.

Expression of SWAP-70 in the uterus and feto-maternal interface during embryonic implantation and pregnancy in the rhesus monkey (Macaca mulatta).

SWAP-70 is a unique signaling protein involved in multiple processes including lymphatic cell activation, migration, adhesion, and cytoskeleton organization. Its role in reproductive system remains to be unclear. In the present study, the spatial and temporal expression of SWAP-70 in the uterus during normal menstrual cycle as well as on the feto-maternal interface during pregnancy was investigated in the rhesus monkey by in situ hybridization and immunohistochemistry. It was shown that SWAP-70 was mainly expressed in glandular epithelial cells of uterine endometrium, and the level peaked at the mid-secretory stage. At the beginning of embryonic implantation, SWAP-70 was intensely expressed at the implantation site, mainly localized in glandular and luminal epithelial cells, as well as in primary trophoblasts and epithelial plaque. High level of SWAP-70 was observed in villous cytotrophoblast (VCT), syncytiotrophoblast (ST), column cytotrophoblast, trophoblast shell, interstitial trophoblast, and endovascular trophoblast during gestational days 15-25. From gestational day 50 to term, expression of SWAP-70 decreased evidently and was restricted in VCT cells. What's more, SWAP-70 co-localized with F-actin on the feto-maternal interface, especially in highly motive extravillous trophoblasts. The data indicate that SWAP-70 may be involved in regulating motility of trophoblast cells during embryonic implantation and placentation.

Analysis of substrate specificity and endopeptidyl activities of the cathepsin B-like proteinase from Helicoverpa armigera.

The cathepsin B-like proteinase from Helicoverpa armigera (HCB) is involved in the degradation of yolk proteins during embryonic development. In order to gain insight into the substrate specificity of this proteinase, various proteins from animals and plants were tested as substrates. The specific cleavage sites of this enzyme on endopeptide bonds were assayed using bovine serum albumin (BSA) as a substrate. Results showed that BSA was degraded into several fragments, which suggests that HCB cleaves BSA at specific endopeptidyl sites. The amino acid sequences of the BSA derived peptides were determined, revealing cleavage of the bonds between residues Arg81-Glu82, Val423-Glu424 and Gly430-Lys431. This suggests that the minimum requirement for a scissile bond to be recognized by HCB is the presence of an ionic amino acid at the P1 ' position and the P1 position can vary. These observations suggest that HCB cleaves bonds at the N-terminal side of ionic amino acid residues giving HCB a wide range of substrates, though other factors dictating the substrate specificity of this enzyme remains to be clarified. Our results provide new evidence that HCB functions as an endopeptidase on some proteins.

Determination of genes involved in the early process of embryonic implantation in rhesus monkey (Macaca mulatta) by suppression subtractive hybridization.

Embryonic implantation is a temporally and spatially restricted process that involves a precise cross talk between the embryo and the receptive maternal endometrium. Underlying the complex changes in the uterus during implantation is the alteration in gene expression pattern, which is not fully understood for the primates. In the present study, suppression subtractive hybridization (SSH) was performed to screen genes that were differentially expressed in the implantation site of the pregnant rhesus monkey, and a subtractive cDNA library was constructed. Furthermore, with dot blot analysis, reverse Northern blot analysis, and semiquantitative reverse transcription-polymerase chain reaction, 76 of 376 clones randomly selected from the library were proven to be differentially expressed in the implantation site. With DNA sequencing and BLAST analysis against the GenBank/EMBL database, it was demonstrated that the cDNA fragments carried by 73 clones shared high homology with 31 human genes. Among them, 15 positive clones represented the S100A10 gene and 10 positive ones corresponded with the secreted frizzled-related protein 4 gene. The other two clones shared homology with one human EST. There was one clone homologous to a human DNA sequence, which indicated that it might be a novel gene. To our knowledge, this is the first report to determine genes involved in the early implantation stage in the rhesus monkey with high throughput technology.