Cell Biology
2013;1(1) : 9-17
Published online May 20, 2013 (https://www.doczj.com/doc/1610631441.html,/j/cb)
doi: 10.11648/j.cb.20130101.12
Involvement of pro-apoptotic and pro-autophagic pro-teins in Granulosa cell death
EscobarML, EcheverríaOM, CasasaAS, García G, AguilarSJ, Vázquez-NinGH*
Laboratorio de Microscopía Electrónica. Departamento de Biología Celular. Facultad de Ciencias. Universidad Nacional Autónoma de
México (UNAM).
Email address:
vazqueznin@ciencias.unam.mx(V-NinGH)
To cite this article:
Escobar M L, Echeverría O M;Casasa A S, García G , Aguilar S J, Vázquez-NinGH. Involvement of Pro-Apoptotic and Pro-Autophagic Proteins in Granulosa Cell Death Cell Biology Vol. 1, No. 1, 2013, pp. 9-17. doi: 10.11648/j.cb.20130101.12
Abstract: Follicular atresia is a process present in all mammals studied. It involves the oocyte and granulosa cells. The apoptotic cell death has been implicated in follicular atresia. Now it is known that the autophagy is a programmed cell death. In this work, atretic follicles of Wistar rats’ ovaries were analyzed, to evaluate the routes of granulosa cell death during the follicular atresia. The apoptosis and autophagy presence was studied by means of ultrastructural and immunohis-tochemical techniques, and by molecular procedures. During atresia, follicular cells undergo the standard processes of cell death, apoptosis and autophagy, as well as a process in which features of both occur in the same cell. Other processes of cell death affect only granulosa cells and involve such features as contraction of cell volume, an increase of the lumen of the nuclear envelope and the endoplasmic reticulum, and loss of contacts with the oocyte, which is also altered. Keywords: Atresia, Apoptosis, Autophagy, Granulosa Cell
1. Introduction
The follicle is a morphological and physiological unit that can remain unchanged, may grow into an ovulating follicle, or may suffer regressive changes called atresia. Follicular atresia is the process through which ovarian follicles not selected for ovulation are eliminated. Ovarian follicles in the process of atresia occur frequently in the ovaries of newborn, prepubertal and adult female mammals. The eventual fate of the follicle depends on a balance in the expression and actions of the factors that promote the proli-feration, growth and differentiation of follicular cells, and those that induce programmed cell death. FSH binds to its granulosa cell receptors to promote the survival and growth of ovarian follicles by stimulating proliferation and estra-diol secretion and inhibiting cell death by up-regulating the expression of anti-apoptotic proteins [1].
During follicular atresia, the oocyte and granulosa cells undergo processes of cell death. In primordial follicles, the onset of the process of atresia seems to depend on altera-tions of the oocyte[2]. Immunohistochemical and ultra-structural analyses of the processes of cell death in oocytes in newborn and prepubertal female rats have demonstrated the presence of abundant autophagosomes, apoptotic bodies, a positive reaction to the TUNEL method, active caspase-3 and Lamp-1, and the absence of large clumps of compact chromatin associated with the nuclear membrane [3]. In adult rats, markers of both apoptosis and autophagy were found in the same oocyte [4]. The structural features of the process of oocyte death are characterized by the presence of clear vacuoles and autophagosomes, and the absence of large clumps of compact chromatin and apoptotic bodies [5, 6]. The main cytochemical feature of the oocytes was their positive reaction to the TUNEL method, which changed according to the age of the rats studied [3].
The apoptotic process of cell death (programmed cell death type I) has been implicated in follicular atresia, as several studies have shown the different morphological characteristics of that process with, and the expression of several proteins related to the apoptotic cell death process has been identified in granulosa cells during follicular progress [7]. Apoptotic cell death is carried out by proteas-es called caspases; enzymes that are classified into two groups: initiator caspases (such as 8 and 9), and effector caspases (3, 6, 7). Once activated, these proteases cleave to different cellular proteins [8].
Programmed cell death process type II –autophagy– is characterized morphologically by the presence of a large
10 EscobarML et al.: Involvement of Pro-Apoptotic and Pro-Autophagic Proteins in Granulosa Cell Death
number of autophagosomes with cytoplasmic content in different degrees of degradation [9].
Molecular mechanisms involved in autophagy suggest the participation of a tg genes [10]. In mammals, the micro-tubule-associated protein 1 light chain 3 (LC3), the Atg8 homologue, has been used as an indicator of autophagy. The phosphatidylethanolamine (PE)-conjugated Atg8 pro-tein plays a structural role in autophagy and is a useful tool for evaluating that process [11]. Beclin-1 (Atg6) is another autophagy-related protein with a fundamental role in auto-phagy [12], it is involved in the initiation of the autophagy [13, 14], in the formation [15] and maturation [16] of auto-phagosomes.
The aim of the present study is to analyze the structural, ultrastructural, cytochemical, and molecular aspects of granulosa cell alterations together with modifications of the interrelations among rat granulosa cells and between them and the oocyte.
2. Materials and Methods
2.1. Experimental animals
60 female Wistar rats were used in the study. They were kept under a 12 h light-darkness cycle with water and food ad libitum. All animals were handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
2.2. Ovary tissue processing for optical microscopy Ovaries were processed as described elsewhere [4]. They were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) at pH 7.2 for 2 hours at room temperature, embedded in paraffin, and serially sectioned. Five μm-thick sections from each ovary were aligned on glass microscope slides covered with poli-l-lysine (Sigma, St Louis, MO), and observed under a Nikon Eclipse E600 Microscope. Images were recorded with a Nikon Digital Camera DXM1200F.
2.3. Electron microscopy process
Ovaries were fixed in 2.5% glutaraldehyde-4% formal-dehyde in phosphate-buffered saline (PBS) at pH 7.2 for 2 hours. After rinsing in the same buffer, tissues were post-fixed in 1% osmium tetroxide (OsO4) in a PBS buffer at pH 7.2 for 1 hour. Tissues were then dehydrated using a graded ethanol series and embedded in Epon (Embed-812, Electron Microscopy Science; Hatfield, PA). Semi-thin sections were stained with toluidine blue. Selected areas were thin-sectioned and mounted on grids, which were counterstained with uranyl acetate and lead citrate. Sections were examined under a JEOL 1010 electron microscope operated at 100 KV. Digital images were taken with a Ha-mamatsu camera. 2.4. Immunodetection assays
Ovaries were fixed in 4% formaldehyde in PBS at pH 7.2 for 2 hours. The tissues were dehydrated using a graded ethanol series and embedded in paraffin. The paraffin-embedded samples were then sectioned for use in carrying out immunolocalizations of the active caspase-3, Beclin-1, and LC3 proteins, and to perform the TUNEL technique. Antigen unmasking was carried out by microwaving the tissue sections in citrate-buffer at 0.1 M and pH 6 (BioGe-nex, Fremont, CA) in a Panasonic microwave oven for 3 min at 1,300 W, and then for 6 min at 780 W. After cooling, the sections were washed in PBS and incubated with prima-ry antibodies.
2.5. Simultaneous Beclin-1 and active caspase-3 immuno-
localization
Deparaffinized tissue sections were washed in PBS and incubated simultaneously with anti-active caspase-3 (Sigma, St Louis, MO) and anti-Beclin-1 (Abcam, Cambridge, UK) (1:100) in PBS dilution for 18 h at 4°C. Negative controls were elaborated by omitting the primary antibodies. After washing, the slides were incubated for 1 h under darkness at room temperature with an anti-rabbit immunoglobulin coupled to Alexafluor488(Invitrogen Carlsbad, CA) and anti-sheep immunoglobulin that in turn was coupled to Alexafluor 594 (Invitrogen; Carlsbad, CA). Next, the prep-arations were washed and counterstained with DAPI (Sig-ma, St Louis, MO) to evaluate DNA distribution. Slides were covered with mounting media for fluorescence micro-scopy (Vectashield Mounting Medium, Vector Labs., Bur-lingame, CA).
2.6. Simultaneous TUNEL procedure and LC3 immuno-
detection
Deparaffinized tissue sections were washed in PBS and simultaneously incubated with primary antibody anti-LC3 (Af?nity BioReagents; Rockford, IL) (1:200) in PBS dilu-tion for 18 h at 4°C. After washing, slides were incubated with an anti-rabbit immunoglobulin coupled to Alexafluor 488 (Invitrogen; Carlsbad, CA) for 1 h under darkness at room temperature. Then the preparations were washed and the evaluation of DNA fragmentation was performed using the Apoptag Red In Situ Apoptosis Detection Kit (Milli-pore; Co, USA). Sections were labeled with biotin-dUTP by incubation with a reaction buffer containing terminal deoxinucleotidyltransferase for 1 h at 37°C. Biotinylated nucleotides were detected with a streptavidin-Rhodamine conjugate. Tissues were counterstained with DAPI (Sigma, St Louis, MO) to evaluate DNA distribution. Slides were covered with Vectashield Mounting Medium for fluores-cence microscopy. The negative controls were carried out omitting the primary antibody or the terminal deoxinucleo-tidyltransferase.
Cell Biology 2013, 1(1):9-17 11
2.7. Annexin-V assay
The apoptotic granulosa cells were identified by fluores-cence microscopy using the Annexin-V/FITC Apoptosis Detection Kit (Sigma, St Louis, MO) according to the manufacturer’s instructions. 5 μl of Annexin-V/FITC and 1 μl of propidium iodide to the cellular fraction were added. Then the preparation was incubated at 37°C, protected from light, for exactly 10 min. The cells in early apoptotic process were stained by the Annexin-V/FITC alone.
2.8. Western Blot analysis
The protein expression level was evaluated using the Western Blot assay. The granulosa cells were isolated from the ovaries of the Wistar rats, which were disaggregated in 0.1% trypsin (GIBCO; Grand Island, NY) in a calcium- and magnesium-free solution for 15 min at 37°C. The ovarian cells were then incubated at 37°C in an atmosphere con-taining 5% CO2 for 24 h on 35 mm culture plates (Nunc; Denmark), with DMEM GlutaMAX(GIBCO; Grand Island, NY) supplemented with 0.1% albumin (Sigma, St Louis, MO) and 4% fetal bovine serum (GIBCO; Grand Island, NY). The cells were incubated for 24 h to allow the granu-losa cells to adhere to the bottom of the dish. Next, the dishes were washed and the granulosa cells were incubated for 15 min in lysis buffer (50 mMTris–Cl, pH 7.5; 150 mMNaCl, 0.1% SDS, 1 mM PMSF, 0.5% sodium deoxy-cholate, and 1% Nonidet P-40), supplemented with the complete protease inhibitor cocktail (Roche, Mannheim, Germany). Total proteins were measured by Bradford As-say: 50 μg of total proteins were loaded onto a 12% SDS-PAGE gel before being transferred to polyvinylidene ?uoride (PVDF) membranes, which were incubated for 1 h at room temperature in blocking buffer. Then the mem-branes were incubated with anti-active caspase-3 and anti-LC3 antibodies at a dilution of 1:5000. Afterwards, the proteins were tagged by incubation with peroxidase-conjugated secondary antibody (Jackson, Newmarket, UK) at 1:10,000 in blocking buffer for 1 h at room temperature. Using Horse Radish Peroxidase (HRP) as the substrate (Immobilon Western, Millipore Co, USA), speci?c labeling was detected by chemiluminescence. Amersham Bios-ciences Hyper?lm(Amersham Biosciences, UK) was ex-posed to the membranes to detect chemiluminescence.
2.9. DNA fragmentation by electrophoresis
The presence of a ladder pattern of DNA degradation during the apoptotic process of cell death was evaluated by electrophoresis of ovarian DNA, which was obtained from the rats’ ovaries using DNAzol (Invitrogen; Carlsbad, CA), following the manufacturer’s instructions. DNA electro-phoresis was carried out in a 2% agarose gel and visualized by ethidium bromide. 2.10. mRNA by reverse transcriptase-polymerase chain
reaction (RT–PCR)
Total RNA from the rats’ ovaries was prepared using Trizol (Invitrogen; Carlsbad, CA), following the manufac-turer’s instructions. RT–PCR analysis was performed in a Mastercycler gradient (Eppendorf, Germany). For cDNA synthesis, OligodT (Applied Biosystem) was used as a primer following the condition for Transcriptor Reverse Transcriptase (Roche; Mannheim, Germany). The PCR contained 1X Taq polymerase buffer, 0.2 μM each of dNTPs, 0.5 ml of Taq Polymerase (Roche Applied Science; New Jersey), and 20 mM of the following primers: Cas-pase-3 Fwd GCCTGTCCTGGATAAGACCA, Rev TTGACTCAGAAGCCGAAGGT; LC3 Fwd TGGCCCTGAAATACGAAGTC, Rev GGCAG-TAGTCGCCTCTGAAG; β-Actin Fwd GTATGCCTCTGGTCGTACCA, and Rev CTTCTGCATCCTGTCAGCAA, as reported previously (6). The conditions for PCR were as follows: 2 min of initial denaturation at 94°C for 32 cycles (94°C for 45 s, 61°C for 45 s, 72°C for 45 s). In the case of β-actin, an annealing temperature of 60°C was used. Amplified PCR products were resolved in 1% agarose gel electrophoresis. Negative controls were provided by omitting reverse tran-scriptase from the cDNA synthesis reaction.
2.11. Quantitative analyses
The estimations were conducted by taking into account all the atretic follicles in 384 sections examined. The total of granulosas cells were counted per each follicle. The intensity of label to Beclin-1 and LC3 immunodetections was measured using the Image Processing and Analysis Java (ImageJ) program[17],from 1200 granulosa cells of different atretic follicles. The background of the slide was evaluated with ImageJ program tools. For comparison of intensity of fluoresce between the normal and altered auto-phagy process, one way ANOVA was conducted with a P<0.05.
3. Results
To define the morphological characteristics of the atretic follicles in rat ovaries, we made an examination by a light and electron microscope of ovary sections.
The initial features of the process of cell death in granu-losa cells include the contraction of cell volume and the separation among them with the loss of most cell-to-cell contacts. As contraction progresses, the remaining cell contacts take place between prolongations of neighboring follicular cells that cross a large intercellular space. A greatly reduced number of prolongations of the follicular cells penetrate the pellucida to make contact with the cell membrane of the oocyte. In the figure1b, an arrow points to one of these remaining prolongations. Numerous granulosa cells contain clear cytoplasmic vacuoles, indicating the
12 EscobarML et al.: Involvement of Pro-Apoptotic and Pro-Autophagic Proteins in Granulosa Cell Death
onset of an autophagic process of cell death (Fig.1b). In the normal follicles the granulosa cells are bonded together as is shown in the Fig. 1a.
Figure 1. Growing follicles. (a) Is showing a normal follicle. (b) Follicle in process of atresia showing ample abnormal spaces between deformed granulosa cells. Clear vacuoles are frequent in the cytoplasm of these cells. The relationships between granulosa cells and the oocyte are almost completely lost, except for a few prolongations of the former, one of which is indicated by the arrow.Oo-oocyte; gc-granulosa cell; zp-zonapellucida. Scale bar 2 microns.
Figure 2. Primordial follicles.a) Non atretic follicle. The granulosa cells are in contact one another and maintain the relationship with the oocyte (empty arrow). The cytoplasm of the oocyte has few autophagosomes, considered as physiological autophagy. (b) The cytoplasm of the oocyte is extensively vacuolated and it is contracted, indicating that it is undergoing a process of autophagic death(empty arrow). However, the granulosa cells are almost normal except for a few clear vacuoles. The arrows point to autophagic vacuoles containing residues of organelles.Oo-oocyte; gc-granulosa cell. Scale bar 2 microns.
Figure 3. Light microscopic images of antral follicles. (a) and (b) are showing healthy follicles. (c) Antral follicle in an advanced stage of atresia. The cavity of the follicle contains granulosacells in an advanced process of cell death. The rectangle indicates the area enlarged in figure d. In (d) the arrows point to cells with dark, compacted nuclei and clear cytoplasmic vacuoles. Arrowheads point to severely altered cells in the last stages of the apoptotic process of cell death. gc-granulosa cells. Scale bars 50 microns.
The atresia of primordial follicles is characterized by morphologically normal granulosa cells and important autophagic alterations of the oocyte in the form of clear vesicles, some of which contain cytoplasmic debris (Fig.2b). These features suggest that the process of atresia is triggered in these follicles by alterations of the oocyte, which are not present in a normal follicle (Fig.2a).
In healthy follicles, the granulosa cells are attached be-tween them, the oocyte is rounded (Figs. 3a and 3b), and there are no altered cells in the antral cavity. In antral fol-licles undergoing atresia the granulosa cells suffer processes of cell death that are typical of either apoptosis or autophagy (Fig. 3). Their nuclei have abundant compact chromatin and their cytoplasm is frequently vesiculated (Fig. 3d). Cells in the final stages of the death process lose contacts with other granulosa cells and undergo fragmenta-tion of the cytoplasm through budding; however there is no apparent destruction of the cell membrane. Both nuclei without cytoplasm and portions of cytoplasm without nuc-lei can be found in the antral cavity (Fig.3d).
The ultrastructural evaluation of granulosa cells in atretic follicles evidenced several alterations, including the clas-sical apoptotic morphology (Fig.4a); elevated amount of autophagic vacuoles in the cytoplasm. However, their nuc-lei are notapparentlyhighly altered (Fig.4b). High magnifi-cation of the autophagic cell cytoplasm allowsappreciating vesicles with cytoplasmic content in different degree of degradation (Fig.4c). A third type of ultrastructuralaltera-tions was evidenced in cells with simultaneous characteris-tics of apoptosis and autophagy, since they have highly compact chromatin corresponding to apoptosis cell death and anumerous autophagic vesicles (Fig.4d). Our ultra-structural observations indicate that the granulosa cells of atreticfollicles are dying with morphological characteristics corresponding to apoptosis, autophagy and with simultane-ous characteristics of both cell death processes.
Figure 4. Electron microscopy views of altered granulosa cells. Classical apoptotic characteristics as compact chromatin are present (a). Numerous vesicles are present in several granulosa cells (b). High magnification of the cytoplasm shows the autophagic vesicles (c). Some granulosa cells with ultrastructural characteristics of apoptosis and autophagy (d). cc-compact chromatin; C-cytoplasm; N-nucleus; av-autophagic vesicle.
Scale bars 2 microns.
Cell Biology 2013, 1(1):9-17 13
Figure 5.Granulosa cells of follicles during the process of atresia. (a) Normal granulosacellswith normal endoplasmic reticulumas can be seen in high magnification in a’. In some granulosa cells the lumen of the nuclear envelope and the cisterns of the endoplasmic reticulum are ex-tremely swollen (b, b’, c and c’). Other neighboring follicular cells appear to be almost normal. Arrows point the endoplasmic reticulum. N-nucleus. Scale bars 2 microns.
Some altered cells showed swelling of the lumen of the nuclear envelope and of the cisterns of the endoplasmic reticulum. These cells co-exist with normal granulosa cells and other follicular cells that are undergoing distinct processes of cell death (Figs.5b and 5c). These alterations are not present in the normal cells (Fig.5a).
To corroborate our structural and ultrastructural observa-tions we immunodetected specific proteins of apoptosis and autophagy cell death in the same cell. To identify autopha-gy we have immunodetectedBeclin-1 and LC3 proteins. We used the active-caspase-3 immunolocalization, and TUNEL technique to evaluate the apoptosis.
Taken in account that the autophagy is a physiological process, the basal autophagic process was discriminated from autophagic cell death, measuring the fluorescence intensity corresponding to the Beclin-1 and LC3 immuno-detections (graphs 1a and 1b). Statistical analyses showed that the fluorescence signals between the two groups were significantly different.
Graph 1Quantitation of fluorescence. (a) Beclin-1immunodetection. (b) LC3 immunodetection. The intensity of labeling in the altered granulosa cells was significantly different from that in the normal ones. Standard error is represented in each bar. a and b are significantly different (P<0.05, ANOVA; n=1200). Figure 6. Light microscopic images of atretic follicles. (a) Antralfollicle. The phase contrast image shows thegeneral morphology of the follicle. The granulosa cells are detached from their neighbors. The dotted square region is the zone shown at a higher magnification. DAPI is evidencing the chromatin disposition. Some cells are positive to active caspase-3 (in green). Some of these cells are also positive to Beclin-1 (in red, empty arrow). Active caspase-3 and Beclin-1 are simultaneously present in few granulosa cells (arrow heads), as the merging of the images reveals. (b) Atretic follicle with antrum as the negative control to active caspase-3 and Beclin-1 immunodetections. Scale bars 20 microns.
Figure7.Phase contrast image showing the morphology of granulosa cells in a large antral follicle in an advanced process of atresia (a), the dotted square is the zone shown at high magnification. Some cellular shapes evidenced by the phase contrast are delineated by dotted lines (b). The arrow heads point to granulosa cells that are positive only for the auto-phagic marker LC3 (c). Some contracted cells that are positive only for the apoptotic marker TUNEL (arrows in d). The empty arrow points to one cell positive for autophagic and apoptotic markers; LC3 (green) and TUNEL (red) respectively (e). Scale bars (a) 100 microns; all the others 10 microns.
Two simultaneous light microscope immunolocalizations of active caspase-3 and Beclin-1, as LC-3 and TUNEL technique were carried out to localizeapoptosis and auto-phagy in the same cells. The results reveal that some-granulosa cells of follicles in the process of atresia are simultaneously positive for both markers. Figure6 shows the presence of active caspase-3 (apoptosis) and Beclin-1 (autophagy) in a section of anantral follicle. Some cells are only positive to active caspase-3 or to Beclin-1, and simul-taneous positive cells were found to a lesser extent
(high
14 EscobarML et al.: Involvement of Pro-Apoptotic and Pro-Autophagic Proteins in Granulosa Cell Death magnifications Fig.6). Simultaneous LC3 and TUNEL
detection showed that some cells are positive to the auto-
phagic marker LC3 (Fig. 7c); other cells are positive to the
TUNEL technique (Fig.7d), and few of them are positive to
both markers (Fig.7e). These findings indicate that proteins
of both processes of cell death are already present in fol-
licles when the process of cell deathis carried out.
Markers of apoptosis and autophagy are not always
present to the same extent in all granulosa cells. Most of
those cells are positive to markers of apoptosisor autophagy,
and few are double positive to both apoptosis and autopha-
gy (table 1).
Table 1. Percentage of granulosa cells positive to the different proteins to
identify apoptosis and autophagy processes; n=11,188.
label
% posi-
tive
granulosa
cells
label
% posi-
tive
granulosa
cells
Active Caspase-3 67.29 TUNEL 8.54 Beclin-1 0.61 LC3 35.99 Act Casp-3+Beclin-1 32.08 TUNEL+LC3 4.30
Figure 8.Annexin-V assay in granulosa cells.The phase contrast image shows the general morphology. The granulosa cells positive to the Annex-in-V incorporation (arrows) and negative to propidium iodide were apop-totic cells; the cells positive to both markers were considered as necrotic (arrow head). There were cells negative to both marks, considered as healthy. Scale bar 20 microns.
The tendency and variation of the percentage of the markers to apoptosis and autophagy cell death were similar in each individual analyzed.
The apoptosiswas evaluated in isolated cells by means of the Annexin-V labeled with FITC incorporation, which binds to the phosphatidylserine located on the external side of the cell membrane of the isolated cells. Supra-vital pro-pidium iodide labeling was used to identify dying cells with damaged cell membrane. Our results showed that the gra-nulosa cells are positive to the Annexin-V incorporation (Fig. 8), evidencing the apoptosis process.
After analyzing these ultrastructural and immunohisto-chemical characteristics, we evaluated the molecular and biochemical properties of granulosa cell populations. The mRNA corresponding to the pro-apoptotic caspase-3 and pro-autophagic LC3 genes was evaluated via the RT-PCR technique. Results indicate the expression of both genes in the same granulosa cell population (Fig. 9a).
The mature, functional stage of the proteins correspond-ing to the mRNA of LC3 and caspase-3 was evaluated with Western Blot. The level of autophagy was assessed by detecting the conversion of LC3-I to LC3-II, which corre-lates with the amount of autophagosomes in follicular cells. To investigate the participation of apoptosis, we assessed the presence of active caspase-3 in the granulosa cells. Results suggest that the active form of caspase-3 is present in follicular cells (Fig. 9a).
Figure 9.(a) RT-PCR to identify mRNA from caspase-3 (1), LC3 (2) and β-actin (3). The negative (-) control was developed by omitting the reverse transcriptase from the cDNA synthesis reaction. (b) Western Blot for LC3, active caspase-3 and GADPH proteins. Lysates from granulosa cell populations indicate the protein bands that correspond to the two LC3 forms. Bands are detected at 18 and 16 kDa and correspond to mem-brane-bound LC3-I and LC3-II, respectively. Active caspase-3 is detected at 17 kDa. GADPH protein is detected at 16 kDa.
(c) Agarose gel electro-phoresis of DNA extracted from granulosa cells, showing apoptotic DNA fragmentation in this cellular population.
Finally, we evaluated the fragmented DNA using aga-rose gel electrophoresis, which showed the typical DNA ladder pattern of apoptosis in the granulosa cell population (Fig. 9c).
4. Discussion
Apoptosis and autophagy are two programmed cell death events involved in tissue homeostasis and development. In follicular atresia, cell death plays an important role in maintaining normal follicular maturation. Studies of folli-cular atresia in several species have shown that granulosa cells display typical traits of apoptosis, such as nuclei with chromatin margination, pyknotic nuclei, fragmented DNA, and apoptotic bodies[reviewed in 18]. Apoptotic cell death was found to occur simultaneously with mitosis within the same follicle; a result consistent with the notion that atresia is determined by a dynamic equilibrium between cell divi-sion and cell death [19].Conventionally, the death of granu-losa cells in mammalian ovaries during the process of folli-cular atresia was thought to be apoptotic in nature; however, morphological characteristics of other forms of cell death have been seen in birds [20].Observations from the present study demonstrate that granulosa cells from mammalian ovaries die through a variety of processes; i.e., both pro-grammed cell death processes apoptosis and autophagy.
Cell Biology 2013, 1(1):9-17 15
The apoptotic morphology includes cellular contraction, chromatin condensation [21]. The main biochemical cha-racteristics of apoptosis are the activation of cysteine-aspartic acid-specific proteases (caspases) [22; 23; 24] and DNA fragmentation [21], which are processes possible to identify by several techniques as TdTdUTP nick end labe-ling (TUNEL) assay [25], DNA agarose gel electrophoresis [26, 27], and by the loss of the cytoplasmic membrane asymmetry with the phosphatidylserine externalization [28]. In this work we use morphological and biochemical criteria to characterize the apoptotic cells.
Autophagy is involved in physiological processes as re-cycling intracellular compounds, in a cytoprotective man-ner when occurs at basal levels [reviewed in 29]. When autophagic process increases, the cell may be engaged in an irreversible process of death with autophagic features [30]. There are several strategies to discriminate the basal levels of autophagy from the increased autophagy conducting to the cell death [31]. In the present study we include the evaluation of the amount of autophagosomes by means of transmission electron microscopy, the presence of Atg proteins (as Beclin-1 and LC3) by means of immunofluo-rescence, and the LC3 lipidation revealed by the Western blot technique [reviewed in 32]. It is important to mention that to define the autophagy cell death, we have taken into account morphological and biochemical characteristics of the altered cells. Furthermore, we could distinguish auto-phagy from apoptosis, by means of their different biochem-ical and morphological characteristics.
To assert that both processes of cell death, apoptosis and autophagy are present in a granulosa cell it was necessary to identify morphological and/or biochemical characteris-tics of both processes in the same cell. It means that the altered cells were simultaneously positive to Beclin-1 and active caspase-3, LC3 and TUNEL or that the ultrastructur-al features of these cells showed an exacerbated amount of autophagic vesicles and a highly compacted nucleus. Normal granulosa cells are connected by gap junctions that facilitate the transfer of nutrients, growth factors and oxygen between them [reviewed in 33]. Our structural and ultrastructural observations show that in some follicles the first signs of the alteration of granulosa cells are the con-traction of cytoplasm, changes in shape, and the partial or total loss of contact among them. All these characteristics correspond to the apoptosis process, and the localization of pro-apoptotic factors such as active caspase-3, DNA frag-mentation as identified by the TUNEL technique, andAn-nexin-V incorporation, supporting the idea that a signifi-cant number of cells death by apoptosis. However, the ultrastructural alterations observed in the present work, show that not only apoptotic features exists in dying granu-lose cells, there are also a high quantity of autophagic ve-sicles in the granulosa cells. These findings are coincident with the increased localization of the pro-autophagic Bec-lin-1 and LC3 proteins in the granulosacells undergoing cell death. We have speculated that theextreme restriction of the flow of nutrients as consequence of the loss of con-tact between the granulosa cells in the process of cell death,couldinduce the over expression of pro-autophagic proteins, such as Beclin-1 and LC3 in granulosa cells dur-ing the atresiaprocess. This continuous limitation of nu-trients provokes a high expression of these pro-autophagic proteins directingthe cells toward an autophagic cell death, which is evidenced by numerous autophagosomes in the cytoplasm of the granulosa cells observed with the electron microscopy.
Additionally, we found ultrastructural characteristics of both types of cell death in the same cell as highly compact chromatin corresponding to a hallmark of apoptosis with combination with numerous autophagic vesicles. The im-munohistochemical analyses showed double positive cells to active caspase-3 and to Beclin-1 proteins, as TUNEL and LC3 markers, demonstrating that although to a lesser extent this alternative rout of cellular elimination is present in granulosa cells of atreticfollicles.All these patterns of cell death process were observed along the different folli-cular developmental stages.
Our morphological and immunohistochemicalobserva-tions were further strengthened by biochemical analyses and molecular assays, which showed that the mRNAs of the caspase-3 and LC3 areexpressed in isolated granulosa cell populations. Moreover, the expression of active cas-pase-3 and LC3 proteins during follicular atresia, evaluated by Western Blot, further confirms our morphological find-ings. In addition, the conversion of LC3 to the lapidated form (LC3-II) proved the presence of autophagy in the granulosa cells. Finally, DNA fragmentation observed in theagarose gel electrophoresis of DNA extracted fromgra-nulosa cells of rat ovaries corroborates the TUNEL obser-vations, as the apoptotic bodies observed at ultrastructural level. Choi et al. have observed in a modified model, that the induction of autophagy in granulosa cells triggers the initiation of apoptosis; suggesting that both processes are involved in regulating granulosa cell death [34]. It is inter-esting to note that the present work was realized in intact animals under physiological environmental, without artifi-cial stimulation, thus our results indicate the prevalence of several routes of cell death during the follicular atresia in the rat ovary.
Our results show that the morphological and biochemical characteristics of apoptosis and autophagy are present dur-ing granulosa cell death, and they exclude the necrosis process, since we did not find ultrastructural evidences of breakdown of cytoplasmic membrane or degraded DNA in the agarose gel electrophoresis. The Annexin-V assay demonstrated the membrane integrity in cells negative to propidium iodide and positive to the phosphatidylserine externalization. All these results are indicative that necrosis does not participate in follicular atresia.
We observed images of a process of granulosa cell death under electron microscopy that involve the dilatation of the cisterns of the endoplasmic reticulum and the nuclear
16 EscobarML et al.: Involvement of Pro-Apoptotic and Pro-Autophagic Proteins in Granulosa Cell Death
envelope. No other alterations, such as those seen in apop-tosis or autophagy, were found in these cells, suggesting that granulosa cells may dye by a process not found in other tissues.
Autophagic cell death is a multi-step process regulated by various signaling pathways that can be activated when the ER is under stress conditions [35]. However, it is also well known that apoptosis can be activated via ER stress, which ends with activation of the apoptotic machinery [36]. Theseultrastructural alterations of granulosa cells observed in this study may be explained by the presence of pro-autophagy and pro-apoptotic proteins in the same cell, since both types of cell death –apoptosis and autophagy-could be initiated by a step that entails ER alterations. Thus, our results suggest that both mechanisms participate in cellular elimination, as the double immunolocalizations also show.
The present work demonstrates the participation of pro-teins belonging to apoptosis and to autophagy in the same cell or in different cells during the follicular atresia. How-ever, it was not possible to define which of both processes of cell death is activated in first place, because our observa-tions were carried out in an in situ environment which image is an instant of the cell life. On the other hand, these procedures allowed us to obtain ultrastructural evidences of processes of cell death not found in other cell types, as a large increase of the lumen of nuclear envelope, and of the cisterns of the endoplasmic reticulum. However, it is ne-cessary a study carried out with different methods to eva-luate a possible sequence of different stages of the processes of cell death.
5. Conclusions
Our observations demonstrate that granulosa cells decay not only by means of the typical processes of apoptosis or autophagy, but also through alterations not found in other organs. All our observations lead us to propose that during follicular atresia in rat ovaries cell death occurs through a combination of processes that involves apoptosis and auto-phagic cell death, a mixed event that includes both routes in the same cell and also by processes not found in other tis-sues.
Acknowledgments
This work was supported by the grants CONACYT 180526 and PAPIIT IN212912. The authors would like to thank Ernestina Ubaldo Pérez for technical assistance. They also thank Paul C. Kersey Johnson for reviewing the English word usage and grammar. References
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2012谷歌pr值计算方法大揭秘（将启用新颖度算法） 一，2012年谷歌友情链接计算新方法 + 十大谷歌参数值（为了方便理解姑且定义为参数） A B N代表不同的站点 很少有SEO人员懂的这种终极算法，这种谷歌参数超级重要，就算没有友情链接，只要将参数作到极致，完全可以提高pr值.这就是量变而引起的质变，也就是说不设置一个友情链接，pr照样可以提高到5,这篇文章我会好好解答其中的奥妙，以及谷歌在2012年pr值计算方面的新改变。 二，对pr计算公式的解析 谷歌对于网站友情链接或者是页面pr的计算方式是： PR(A)=(1-d)+d(PR(t1)/C(t1)+PR(t2)/C(t2)..........PR(tn)/C(tn))+（参数） 大家不知道这个d0.85是啥意思，d为固定的常数，也就是0.85 而PR(A)则是你网站可以获得的pr值，PR(t1)是交换网站的pr值，C(t1)则表示该网站所含有的导出的友情链接数，以此类推。专业名词叫阻尼因数(damping factor)，实际上是计算PR分值所设置的系数，Google 的阻尼因数一般是0.85PR(A)表示的是从一个外部链接站点t1上，依据Pagerank(tm)系统给你的网站所增加的PR分值PR(t1)表示该外部链接网站本身的PR分值;C(t1)则表示该外部链接站点所拥有的外部链接数量，很多SEO们的行家们迷信这个基础算法，而忽视了后边的参数，如果简单的利用这个基本的公式计算，发现会有一个漏洞，就是老网站获得的PR值会高，新网站即使很受欢迎仍然不能获得PR！
实则不然！大家都会发现三个怪现象， 1，有的网站刚上线没几天pr会更新到4 ， 2，有的网站友情链接做的越来越多，而pr却不断的在下降 3，有的网站根本没有友情链接，pr却也很高 这就是中国式的常规猜想，而忽略了后边的后浪参数，所以搜索引擎会有很复杂的算法来解决。不要深究算法，因为你不是搜索引擎工程师，研究提高PR没有更大的意义，所谓万变不离其宗，网站要获得高的PR值，友情链接不管是质量和数量上都有优势的时候才能走向PR的巅峰。同样，光靠参数，pr也可以走向一个巅峰。 三，友情链接因参数而不同 交换友链要考虑三方面的因素： （1）pr越高越好（最好是让高PR值的网站首页链接上你的网站，这是大部分人在做的） （2）pr不太高，但是导出连接比较少（很多做友情链接都不能彻底明白这一点，但通过计算公式很多人就明白了，pr不太高，但导出链接少的，甚至比pr高的网站，但为啥pr高是首要因素呢？因为导出链接少的pr还凑合的网站比较稀少） （3）权威网站的主要页面，或者叫后浪参数比较高的页面（很多做友情链接的都忽视这一点，网站是否权威，网站权威性与网页权威性这两个概念是有所区此外。网站权威性是由一张张高质量的网页、网站声望、用户口碑等等身分形成。搜索引擎判定一张网页的主要性，可能会优先判定网站的权威性。基于网站的权威性，再判定某一网页
视频切换特效 1.( )使画面像纸一样被重复折叠从而显示另一画面。 2.( )使画面以屏幕的一边为中心从后方转出覆盖另一画面。 3.( )使画面以屏幕的一边为中心从前方转出覆盖另一画面。 4.( )使画面从屏幕的中心逐渐展开覆盖另一画面。 5.( )使画面从屏幕的中心旋转出现而覆盖另一画面。 6.( )使画面像窗帘一样被拉起出现另一画面。 7.( )使画面以立方体的两个面进行过渡转换。 8.( )使两个画面作为页面，通过翻转实现转换。 9.( )使画面从屏幕的中心旋转缩小消失，出现另一画面。 10.( )使画面以关门的方式覆盖另一画面。 11.( )使画面从中心以矩形不断放大覆盖另一画面。 12.( )使画面以十字形向外移动显示另一画面。 13.( )使画面以圆形逐渐放大覆盖另一画面。 14.( )使画面以一个或多个形状打开覆盖另一画面。 15.( )使画面以星形不断放大覆盖另一画面。 16.( )使画面以交叉点擦除显示另一画面。 17.( )使画面以菱形逐渐放大覆盖另一画面。 18.( )画面从屏幕中心逐渐向四角撕开并卷起显示另一画面。 19.( )画面以翻页的形式从一角卷起显示另一画面(背面为灰色)。 20.( )画面以卷纸的形式从一边卷起显示另一画面(卷轴)。 21.( )画面以透明翻页的形式从一角卷起显示另一画面。 22.( )画面在中心分成四块分别卷起显示另一画面。 23.( )淡入淡出效果。 24.( )画面以点的方式叠加后逐渐显示另一画面。 25.( )画面逐渐变白后，由白色逐渐显示另一画面。 26.( )两画面以加亮模式交叉叠加后逐渐显示另一画面。 27.( )画面以随机反色显示的形式消失，逐渐显示另一画面。 28.( )两画面以亮度叠加消融的方式切换。 29.( )画面逐渐变为黑色后，由黑色逐渐显示另一画面。 30.( )画面从不同方向挤压显示另一画面。 31.( )一个画面淡出，另一个画面放大流入。 32.( )一个画面在另一个画面中心横向伸展开来。 33.( )一个画面从一边以伸缩状展开覆盖另一个画面。 34.( )画面沿着“Z”字形交错扫过另一画面。 35.( )画面从中央以开关门的方式转到另一画面。 36.( )画面以棋盘状消失过渡到另一个画面。 37.( )画面从水平、垂直或者对角线的方向以条状覆盖另一画面。 - 1 -
1．Brightness & Contrast（亮度与对比度） 本视频滤镜效果将改变画面的亮度和对比度。类似于电视中的亮度和对比度的调节，但在这里调整则是对滑块的移动， 2．Channel Mixer（通道合成器） 使用本视频滤镜效果，能用几个颜色通道的合成值来修改一个颜色通道。使用该效果可创建使用其他颜色调整工具很难产生的颜色调整效果，通过从每个颜色通道中选择其中一部分就能合成为高质量的灰度级图像，创建高质量的棕褐色或其他色调的图像，已经交换或复制通道， 3．Color Balance（色彩平衡） 本视频滤镜效果利用滑块来调整RGB颜色的分配比例，使得某个颜色偏重以调整其明暗程度。本过滤属于随时间变化的特技， 4．Convolution Kernel（回旋核心） 本视频滤镜效果使用一道内定的数学表达式，通过矩阵文本给内定表达式输入数据，来计算每个像素的周围像素的涡旋值，进而得到丰富的视频效果。可以从提供的模式菜单中选择数据模式进行修改，也可以重新输入新的值（只要效果认为在Convolution Matrix（回旋矩阵）文本框中，中心的数字为该像素的亮度计算值（所输入的数字会乘以像素的亮度值），周围的数字是该像素周围的像素所需要的计算值，在此框内所输入的数值会乘以周围像素的亮度值。 在Misc（杂项）框架中的Scale文本框中输入的数将作为除数，像素点包括周围像素点的像素点与输入给“环境中心像素点的数据矩阵”对应点的乘积之和为被除数。Offset文本框是一个计算结果的偏移量（与所得的商相加）。 所有数值允许的输入范围很大，可从-99999～999999，但实际使用的没有这么大，要根据演示效果而定。 假如发现定义的一套数据很实用，并且以后还要使用，可单击Save按钮将其存储起来，并记住文件夹和文件名（或存储到软盘上）。下次通过单击Load按钮将其装载到当前的数据定义格式中，即可使用。 假如想使用内定的图像转换数据格式，应该单击图7-20中间上方的按钮，从弹出的菜单中选择相应的选项。内定的图像转换数据格式是十分有用的工具，它可让图像变得模糊或清楚。为能充分说明问题，图7-21列出了几种典型的效果供参考。这是从图7-20中选择的典型代表。这些视频效果在制作静态图片的特别节目时非常有用。它类似于Adobe Photoshop 中的过滤效果，只是在这里用于电影制作方面。假如不了解Premiere这个绝活，可能还会想到使用Photoshop进行处理。而假如将一幅照片通过Photoshop加工后再用于电影制作，势必会造成磁盘空间上的浪费和时间浪费。在此可看出Premiere的高效能力。5．Extract（提取） 当想利用一张彩色图片作为蒙板时，应该将它转换成灰度级图片。而利用此视频滤镜效果，可以对灰度级别进行选择，达到更加实用的效果。图7-22是提取视频片断的蒙板成分的对话框。 有2个滑块，带有图线的2个黑3角的滑块用来选定原始画面中的被转换成白色的灰度，Softness滑块用来调节画面的柔和程度。通过Invert复选框可以将已定的灰度图片进行反相。左下角的黑色梯形图（反相时为白色）随着Softness的滑动而变化，当变为三角形时，表明已达到原始画面的效果；当为梯形时，表明对原始画面的明暗分界进行了改动。6．Levels（色彩级别） 本视频滤镜效果将画面的亮度、对比度及色彩平衡（包括颜色反相）等参数的调整功能组合在一起，更方便地用来改善输出画面地画质和效果。图7-23是其调整对话框。
视频转场特效 在Adobe Premiere Pro中，根据功能可分为10大类多达73种的转场特效。每一种转场特效都有其独到的特殊效果，但其使用方法基本相同。 如果根据转场影响边数，转场方式可以两大类：单边转场和双边转场。单边转场方式只影响相邻编辑点的前一个或后一片断，其空白区域会透出低层轨道画面，但低层画面只是被动透出而已；而双边转场则需要两个片断的参与。 单边转场的添加需要先用鼠标选中一种转场方式然后再按下CTRL键，将其拖至某一片断的开头或结尾。双边转场只需左键拖至片断相邻处。其转场的标志有差异，注意区分。双连转场有三种对齐方式，左、中、右，但左、右对齐与单边转场有差异。 本节就转场特效的具体内容以分组的形式予以详尽的分析与论述。 1.1 3D Motion转场特效 1．Cube Spin（立方体旋转）特效 这种特效用来产生类似于立方体转动的过渡效果，但是该效果中的立方体转动使得图像会产生透视变形，立体感非常强烈，如图1-1所示。 2．Curtain（舞台拉幕）特效 这种特效用来产生一段素材像被拉起的幕布一样消失，同时另一段素材显露出来的效果，如图1-2所示。 图1-1 Cube Spin特效图1-2 Curtain特效 3．Doors（开关门）特效 这种特效用来产生一段素材位于门后，随着位于门上的另一段素材的开关而显示的效果，如图1-3所示。 4．Flip Over（翻转）特效 这种特效用来产生一段素材像一块板一样翻转，并显示出另一段素材的效果，如图1-4所示。
图1-3 Doors特效图1-4 Flip Over特效 5．Fold Up（折叠）特效 这种特效用来产生一段素材像一张纸一样被折叠起来，逐渐显露另一段素材的效果，如图1-5所示。 6．Spin（旋转）特效 这种特效用来产生一段素材旋转出现在另一段素材上的效果，如图1-6所示。 图1-5 Fold Up特效图1-6 Spin特效 7．Spin Away（变形旋转）特效 这种特效与上述的旋转特效类似，不同之处在于另一段素材旋转出现时画面有透明变形，如图1-7所示。 8．Swing In（摆入）特效 这种特效用来产生一段素材如同摆锤一样摆入，逐渐遮住另一段素材的效果，如图1-8所示。 图1-7 Spin Away特效图1-8 Swing In特效
premiere pro2.0视频转场特效英汉对照 1、3D过渡（3D motion） 英文名中文名备注 Cube spin 立方体旋转立方体旋转切换 Curtain 窗帘图像A呈拉起的链子状消失，图像B出现 Doors 关门图像A、B呈开门状切换 Flip over 翻页图像A翻转到图像B Fold up 折叠图像A像纸一样折叠到图像B Spin 翻转中心向左右扩展图像B旋转出现在图像A上Spin away 旋转中心旋转出图像A旋转离开 Swing in 内关门图像B像摆钟一样摆入 Swing out 外关门与上面一样，方向相反 Tumble away 筋斗翻出图像A像筋斗云一样翻出 2、溶解（Dissolve） 英文名中文名备注 Additive Dissolve 附加溶解图像A溶为图像B，有亮度叠加 Cross Dissolve 交叉溶解图像A与图像B同时淡化溶合 Dip to Black 加入暗场溶解 Dither Dissolve 淡入淡出图像A以点的形式逐渐淡化到图像B Non-Additive Dissolve 无附加溶解亮度映像的图像溶合 Random Invert 随机颠倒附加上了颗粒溶解图像A以随机板块反转消 失，图像B以随机板块反转出现 3、圈入（Iris） 英文名中文名备注 Iris Box 盒装圈出中心点在画面中央 Iris Cross 十字形圈出图像B呈十字形在图像A上展开 Iris Diamond 菱形圈出图像B呈钻石形在图像A上展开 Iris Points 四点圈出从四个角圈出整个画面 Iris Round 圆形圈出图像B呈圆形在图像A上展开 Iris Shapes 形状圈出可以设置菱形、椭圆形或者矩形 Iris Star 五角星圈出图像B呈星形在图像A上展开 4、映射（Map） 英文名中文名备注 Channel Map 通道映射可以设置三基色、alpha、灰度等通道 Luminance Map 亮度映射画面1亮度降低 5、卷页(Page Peel) 英文名中文名备注 Center Peel 中心卷页图像A从中心分裂成四块卷开，显露出图像B Page Peel 翻页图像A带背景色卷走，显露出图像B Page Turn 页面翻转类似上一个，不过图像A卷走时，背景仍然是图像A Peel Back 由中心依次向四个角翻页图像A由中心呈四块分别卷走，显露出图像B Roll Away 滚轴卷出图像A像一张纸一样卷走，显露出图像B 6、滑行（Slide） 英文名中文名备注
1、3D过渡（3D motion） Cube spin 立方体旋转 Curtain 窗帘 Doors 关门 Flip over 翻页 Fold up 折叠 Spin 翻转 Spin away 旋转 Swing in 内关门 Swing out 外关门 Tumble away 筋斗翻出 2、溶解（Dissolve） Additive Dissolve 附加溶解 Cross Dissolve 交叉溶解 Dip to Black 加入暗场溶解Dither Dissolve 淡入淡出 Non-Additive Dissolve 无附加溶解Random Invert 随机颠倒 3、圈入（Iris） Iris Box 盒装圈出 Iris Cross 十字形圈出 Iris Diamond 菱形圈出 Iris Points 四点圈出 Iris Round 圆形圈出 Iris Shapes 形状圈出 Iris Star 五角星圈出 4、映射（Map） Channel Map 通道映射 Luminance Map 亮度映射 5、卷页(Page Peel) Center Peel 中心卷页 Page Peel 左上角卷页 Page Turn 页面翻转 Peel Back 由中心依次向四个角翻页Roll Away 滚轴卷出 6、滑行（Slide） Band Slide 带状滑行 Center merge 向中心收缩至消失Center split 十字形撕开画面 Multi-Spin 多图旋转 Push 推出 Slash slide 斜线滑行 Slide 滑行 Sliding Bands 带状滑行 Sliding Boxes 移动带状滑行Split 撕开画面 swap 滑行交换 Swirl 盘旋 7、特技 Direct 硬切 Displace 替换 Image Mask 图片遮罩 Take 硬切 Texture 贴图 Three—D 三维 8、伸展（Stretch） Cross Stretch 交叉伸展 Funnel 漏斗收缩 Stretch 伸展 Stretch In 伸展进入 Stretch Over 伸展覆盖 9、擦除 Band Wipe 带状擦除 Barn Doors 左右推开 Checker Wipe 方格擦除 Checkerboard 积木碎块 Clock Wipe 时钟擦除 Gradient Wipe 渐进擦除 Inset 插入 Paint Splatter 泼溅油漆 Pinwheel 转轮风车 Radial Wipe 射线擦除 Random Wipe 随机擦除 Random Blocks 随机碎片擦除 Spiral Boxes 旋转擦除消失 Venetian Blinds 百叶窗 Wedge Wipe 楔形擦除 Wipe 擦除 Zig-Zag Blocks Z字形擦除 10、变焦（Zoom） Cross Zoom 交叉变焦 Zoom 变焦放大 Zoom Boxes 方格放大 Zoom Trails 拖尾缩小 1
Seo中pr值的意思 Seo中pr值的意思？这是seo学习过程中必须了解的一个常识，pr值越高说明该网页越受欢迎。pr值是什么。影响网页PR值的因素有很多，但主要的有： 一、网站被三大知名网络目录DMOZ，Yahoo和Looksmart收录 众所周知，Google的PageRank系统对那些门户网络目录如DMOZ 、Yahoo和Looksmart尤为器重。特别是对DMOZ。一个网站上的DMOZ链接对Google的PageRank来说，就好象一块金子一样有价值。如果你的网站为ODP收录，则可有效提升你的页面等级。 如果你的网站为Yahoo和Looksmart所收录，那么你的PR值会得到显著提升。如果你的网站是非商业性质的或几乎完全是非商业性质的内容，那么你可以通过https://www.doczj.com/doc/1610631441.html, 使你的网站为著名的网络目录Looksmart所收录。 如果你是一个网站管理员，而你的网站又已经收录在三大知名的开放目录DMOZ、Yahoo和Looksmart中，我想你的网站的PR值一定比较高，而且搜索排名也不会差。 二、Google抓取您网站的页面数量 让搜索引擎尽量多的抓取你网站的网页，这样搜索引擎才会认为你网站的内容非常丰富，因为搜索引擎喜欢内容丰富的网站，才会认为你的网站很重要。而且这些内容最好是原创的文章，因为搜索引擎同样喜欢原创的东西。 三、网站外部链接的数量和质量 Google在计算PR值时，会将网站的外部链接数量考虑进去，但并不是说一个网站的外部链接数越多其PR值就越高，因为网页的PR值并不是简单地由计算网站的外部链接数来决定的，还要考虑外部链接的质量，与相关网站做交换链接的分值要比与一般网站做敛接的分值高。让我们来看一下PR值的计算公式： PR(A)=(1-d)+d(PR(t1)/C(t1)+…+PR(tn)/C(tn)) 其中PR(A)表示的是从一个外部链接站点t1上，依据PageRank系统给你的网站所增
Distort扭曲特效 --Bezier warp贝赛尔曲线弯曲 --Bulge凹凸镜 --CC Bend It 区域卷曲效果 --CC Bender 层卷曲效果 --CC Blobbylize 融化效果 --CC Flo Motion 两点收缩变形 --CC Griddler 网格状变形 --CC Lens 鱼眼镜头效果，不如Pan Lens Flare Pro --CC Page Turn 卷页效果 --CC Power Pin 带有透视效果的四角扯动工具，类似Distort/CornerPin --CC Ripple Pulse 扩散波纹变形，必需打关键帧才有效果 --CC Slant 倾斜变形 --CC Smear 涂抹变形 --CC Split 简单的胀裂效果 --CC Split 2 不对称的胀裂效果 --CC Tiler 简便的电视墙效果 --Corner pin边角定位 --Displacement map置换这招 --Liquify像素溶解变换 --Magnify像素无损放大 --Mesh warp液态变形 --Mirror镜像 --Offset位移 --Optics compensation镜头变形 --Polar coordinates极坐标转换 --Puppet木偶工具 --Reshape形容 --Ripple波纹 --Smear涂抹 --Spherize球面化 --Transform变换 --Turbulent displace变形置换 --Twirl扭转 --Warp歪曲边框 --Wave warp波浪变形 Expression Controls表达式控制特效 --Angel control角度控制 --Aheckbox control检验盒控制 --Color control色彩控制 --Layer control层控制 --Point control点控制 --Slider control游标控制
黑龙江省外贸发展现状、技术评析与优化研究 目录 摘要1 1 近十年黑龙江省外贸发展现状2 1.1 黑龙江省外贸现状简介3 1.2 黑龙江省外贸商品的优劣势分析3 1.3 黑龙江省的对外贸易条件分析6 1.4 黑龙江省外贸比较优势和贸易条件的关系10 2 黑龙江省外贸比较优势与GDP贡献率的相关性评析11 2.1 黑龙江GDP的贡献率11 2.2 比较优势和GDP 贡献率的相关分析12 3 黑龙江对外贸易发展存在的优势分析14 3.1黑龙江对外贸易优势原因分析16 3.2 黑龙江省对外贸易发展方式转变的结果进行评价16 3.3 黑龙江省对外贸易发展方式转变的原因分析17 3.4 黑龙江省对外贸易发展方式转变的主要指标评价18 4 黑龙江对外贸易发展中存在的问题22 4.1 对外开放程度不足，外贸对经济增长的贡献率低22 4.2 没有良好的环境进行外资吸引，过低的外资流入22 4.3 区位优势发挥不健全，自身的特色贸易并未彻底形成22 4.4 外贸结构与产业结构为了促进良性机制的发展并未形成基本的模式23 5黑龙江对外贸易发展优化措施23 5.1 开拓市场23 5.2 调整结构25 5.3商业贸易多元化发展26 参考文献28
黑龙江省外贸发展现状、技术评析与优化研究 摘要：通过对黑龙江省2004-2013年间外贸进出口数据的分析，对黑龙江省的外贸发展进行简要的分析，建设和发展黑龙江省的经贸区，进行多元建设来进行对外贸易，将会对黑龙江省外贸经济的发展产生极大地促进作用。 关键字：外贸发展黑龙江分析优化 大卫·李嘉图这位英国古典经济学派集大成者在1817年的代表作《政治经济学及赋税原理》中提取出比较优势理论，并在未来百年内将此优势理论称之“国际贸易理论的基石”。在这样的理论指导下，英国顺利完成产业革命，并在19世纪成功成为全球的第一个“世界工厂”。在整个贸易实践中，发展中国家若只是遵循比较优势理论来推动社会的发展，很容易陷入到比较优势陷阱当中。初级产品和劳动密集产品的发展主要集中在发展中国家，从比较优势理论的角度上分析，相比于发达国家的资本和技术密集型产品为主要出口产品的贸易相比处于相当不利位置，甚至有可能会出现贸易利益流失的情况。 现阶段国内对于比较优势理论的研究还不是很多，但是实证研究确实不少，对其研究方法也是角度方法各一。国内主要代表研究作品主要有：张鸿（2006）采用RCA指数和NEPR（纯出口比较优势指数），
Video Effects 【视频效果】 一. Adjust【调整】 1. Lighting effects【照明效果】 2. Levels【色阶】 二. Blur&Sharpen【模糊&锐化】 1. Fast blur【快速模糊】 2. Directional blur【方向模糊】 3. Gaussian blur【高斯模糊】 三. Channel【通道】 1. Set matte【设置蒙版】 四. Color Correction【色彩校正】 1. RGB Curves【RGB曲线】
2. Change color【更改颜色】 3.Color balance HLS【色彩平衡HLS】 4. Change to color【转换颜色】 5. Luma curve【亮度曲线】 五.Distort【扭曲】 1. Offset【偏移】 2. Wave warp【波形弯曲】 3. Magnify【放大】
4. Twirl【旋转扭曲】 5. Spherize【球面化】 6. Corner pin【边角固定】 7. Lens distortion【镜头扭曲】 六.Generate【生成】 1. Circle【圆】 2. Ramp【渐变】 3. Grid【网格】 4. Lens flare【镜头光晕】 5. Lighting【闪电】
七. Generate【键控】 1. Four-Point Garbage Matte【四点蒙版键】 2. Luma【亮度键】 3. Image Matte Key【图像遮罩键】 4. Chroma Key【色度键】 5. Color Key【颜色键】 6. Blue Screen Key【蓝屏键】 7. Track Matte Key【轨道蒙版键】 8. Non Red Key【非红色键】 八. Noise&Grain【噪波与颗粒】 1. Noise【噪波】 2. Noise Alpha【通道噪波】 九. Perspective【透视】 1. Basic 3D【基本3D】 2. Drop Shadow【投影】 3. Bevel Edges【斜角边】
光伏电站发电效率的计算与监测 1、影响光伏电站发电量的主要因素 光伏发电系统的总效率主要由光伏阵列的效率、逆变器的效率、交流并网效率三部分组成。 1.1光伏阵列效率： 光伏阵列的直流输出功率与标称功率之比。光伏阵列在能量转换与传输过程中影响光伏阵列效率的损失主要包括：组件匹配损失、表面尘埃遮挡损失、不可利用的太阳辐射损失、温度的影响以及直流线路损失等。 1.2逆变器的转换效率: 逆变器输出的交流电功率与直流输入功率之比。影响逆变器转换效率的损失主要包括：逆变器交直流转换造成的能量损失、最大功率点跟踪（MPPT）精度损失等。 1.3交流配电设备效率： 即从逆变器输出至高压电网的传输效率，其中影响交流配电设备效率的损失最主要是：升压变压器的损耗和交流电气连接的线路损耗。 1.4系统发电量的衰减： 晶硅光伏组件在光照及常规大气环境中使用造成的输出功率衰减。 在光伏电站各系统设备正常运行的情况下，影响光伏电站发电量的主要因素为光伏组件表面尘埃遮挡所造成太阳辐射损失。 2、光伏电站发电效率测试原理 2.1光伏电站整体发电效率测试原理 整体发电效率E PR公式为： E PDR PR PT = —PDR为测试时间间隔(t?)内的实际发电量；—PT为测试时间间隔(t?)内的理论发电量；
理论发电量PT 公式中： i o I T I =，为光伏电站测试时间间隔(t ?)内对应STC 条件下的实际有效发电时间； －P 为光伏电站STC 条件下组件容量标称值； －I 0为STC 条件下太阳辐射总量值，Io =1000 w/m 2； －Ii 为测试时间内的总太阳辐射值。 2.2光伏电站整体效率测试（小时、日、月、年） 气象仪能够记录每小时的辐射总量，将数据传至监控中心。 2.2.1光伏电站小时效率测试 根据2.1公式，光伏电站1小时的发电效率PR H i H i PDR PR PT = 0I I i i T = —PDRi ，光伏电站1小时实际发电量，关口计量表通讯至监控系统获得； —P ，光伏电站STC 条件下光伏电站总容量标称值； —Ti ，光伏电站1小时内发电有效时间； —Ii ，1小时内最佳角度总辐射总量，气象设备采集通讯至监控系统获得； —I 0=1000w/m 2 。 2.2.2光伏电站日效率测试 根据气象设备计算的每日的辐射总量，计算每日的电站整体发电效率PR D D PDR PR PT = 0I I T = —PDR ，每日N 小时的实际发电量，关口计量表通讯至监控系统获得； —P ，光伏电站STC 条件下光伏电站总容量标称值； —T ，光伏电站每日发电有效小时数
PR 内置特效一览表 1.预置 A.卷积内核：查找边缘、模糊、模糊更多、浅浮雕、浮雕、 锐化、锐化更多、锐化边缘、高斯模糊、高斯锐化 B.斜角边：厚斜边、薄斜边 C.旋转扭曲：旋转扭曲入、旋转扭曲出 D.曝光过度：曝光入、曝光出 E.模糊：快速模糊入、快速模糊出 F.画中画：25%画中画 G.马赛克：马赛克入、马赛克出 2.音频特效 A.5.1：选频、多功能延迟、Chorus、DeClicker、DeCracker、 DeEsser、DeNoiser、Dynamics、EQ、Flanger、 MultibandCompressor、低通、低音、Phaser、PitchShifter、Reverb、Spectral NoiseReduction、去除指定频率、参数 平衡、反相、声道音量、延迟、音量、高通、高音 B.立体声：选项、多功能延迟、Chorus、DeClicker、 DeCracker、DeEsser、DeHummer、DeNoiser、Dynamics、EQ、Flanger、MultibandCompressor、低通、低音、Phaser、PitchShifter、Reverb、平衡、Spectral NoiseReduction、 使用右声道、使用左声道、互换声道、去除指定频率、参
数平衡、反相、声道音量、延迟、音量、高通、高音 C.单声道：选项、多功能延迟、Chorus、DeClicker、 DeCracker、DeEsser、DeuHummer、DeNoiser、 Dynamics、EQ、Flanger、MultibandCompressor、低通、低音、Phaser、PitchShifter、Reverb、Spectral NoiseReduction、去除指定频率、参数平衡、反相、声道 音量、延迟、音量、高通、高音 3.音频过度：交叉渐隐：恒定功率、恒定增益、指数型淡入淡 出 4.视频特效 A.CPU特效：卷叶折射、波纹（圆形） B.变换：垂直保持、垂直翻转、摄像机视图、水平保持、水 平翻转滚动、羽化边缘、裁剪 C.噪波与颗粒：中间值、噪波、噪波Alpha、噪波HLS、自 动噪波HLS、蒙版与刮痕 D.图像控制：灰度系数（Gamma）校正、色彩传递、色彩匹 配、颜色平衡、（RGB）、颜色替换、黑白 E.实用：Cineon转换 F.扭曲：偏移、变换、弯曲、放大、旋转、波形弯曲、球面 化、紊乱置换、边角固定、镜像、镜头扭曲 G.时间：抽帧、时间偏差、重影 H.模糊与锐化：复合模糊、定向模糊、快速模糊、摄像机模
知识点4－5 对流传热系数关联式 【学习指导】 1．学习目的 通过本知识点的学习，了解影响对流传热系数的因素，掌握因次分析法，并能根据情况选择相应的对流传热系数关联式。理解流体有无相变化的对流传热系数相差较大的原因。 2．本知识点的重点 对流传热系数的影响因素及因次分析法。 3．本知识点的难点 因次分析法。 4．应完成的习题 4-11 在一逆流套管换热器中，冷、热流体进行热交换。两流体进、出口温度分别为t1=20℃、t2=85℃；T1=100℃、T2=70℃。当冷流体流量增加一倍时，试求两流体的出口温度和传热量的变化情况。假设两种情况下总传热系数不变，换热器热损失可忽略。 4-12 试用因次分析法推导壁面和流体间自然对流传热系数α的准数方程式。已知α为下 列变量的函数： 4-13 一定流量的空气在蒸汽加热器中从20℃加热到80℃。空气在换热器的管内湍流流动。压强为180kPa的饱和蒸汽在管外冷凝。现因生产要求空气流量增加20％，而空气的进出口温度不变，试问应采取什么措施才能完成任务，并作出定量计算。假设管壁和污垢热阻可忽略。 4-14 常压下温度为120℃的甲烷以10m/s的平均速度在列管换热器的管间沿轴向流动，离开换热器时甲烷温度为30℃，换热器外壳内径为190mm，管束由37根ф19×2的钢管组成，试求甲烷对管壁的对流传热系数。
4-15 温度为90℃的甲苯以1500kg/h的流量流过直径为ф57×3.5mm、弯曲半径为0.6m的蛇管换热器而被冷却至30℃，试求甲苯对蛇管的对流传热系数。 4-16 流量为720kg/h的常压饱和蒸汽在直立的列管换热器的列管外冷凝。换热器的列管直径为ф25×2.5mm，长为2m。列管外壁面温度为94℃。试按冷凝要求估算列管的根数（假设列管内侧可满足要求）。换热器的热损失可以忽略。 4-17 实验测定列管换热器的总传热系数时，水在换热器的列管内作湍流流动，管外为饱和蒸汽冷凝。列管由直径为ф25×2.5mm的钢管组成。当水的流速为1m/s时，测得基于管外表面积的总传热系数为2115W/(m2.℃)；若其它条件不变，而水的速度变为1.5m/s时，测得系数为2660 W/(m2.℃)。试求蒸汽冷凝的传热系数。假设污垢热阻可忽略。 对流传热速率方程虽然形式简单，实际是将对流传热的复杂性和计算上的困难转移到对流传热系数之中，因此对流传热系数的计算成为解决对流传热的关键。 求算对流传热系数的方法有两种：即理论方法和实验方法。前者是通过对各类对流传热现象进行理论分析，建立描述对流传热现象的方程组，然后用数学分析的方法求解。由于过程的复杂性，目前对一些较为简单的对流传热现象可以用数学方法求解。后者是结合实验建立关联式，对于工程上遇到的对流传热问题仍依赖于实验方法。 一、影响对流传热系数的因素 由对流传热的机理分析可知，对流传热系数决定于热边界层内的温度梯度。而温度梯度或热边界层的厚度与流体的物性、温度、流动状况以及壁面几何状况等诸多因素有关。 1．流体的种类和相变化的情况 液体、气体和蒸汽的对流传热系数都不相同，牛顿型流体和非牛顿型流体也有区别。本书只限于讨论牛顿型流体的对流传热系数。 流体有无相变化，对传热有不同的影响，后面将分别予以讨论。 2．流体的特性
关于premiere中音频特效的翻译和使用方法介绍 1.Balance（平衡）：改变左右声道的音量大小。 Bypass （旁路） 2.Bandpass（带通滤波器） center（中心频率） Q值:Q= center / bandwidth,主要用来限定某些频率音频的输出 3.Bass（低音）：调节音频中的低音部分，消弱高频部分的影响 boost（推子）：增大音频中低音（低频）部分的影响（音量） 4.Channel Volume（通道音量）：用来调节左右声道的音量的大小，参数只有Left和Right 5.Chorus（合唱）：用来模拟产生大环境合唱的效果。模拟很多声音和乐器同时工作，带有一定的延迟和回声 6.Declicker（消音器）：去除喀嚓声 7.Decrackler（消音器）：去除爆破音 8.Deesser：去除唇齿音，去咝咝音 9.DeHummer：去除嗡嗡声 10.Denoiser：去或降低噪音 11.Delay（延迟） delay：设置延迟时间 feedback：设置回响 Mix：设置延迟声音的混合度 12.Dynamics（动态调整）：以不同的模式调整音频 13.EQ（均衡器）：通过控制音频中的频率成分在调整音频输出效果。 主要将音频的频率分成五个段落来调节。 14.Fill right/Fill left：向左或者向右填充音频声道。 立体声中，利用左（右）声道去填充或覆盖右（左）声道里的音频。 15.flanger:声音的延迟和叠加（就是产生一个与原音频一样的音频带有一定的延迟与原音频混合）。 16.Hightpass(高通滤波器)：控制在一个数值之上的所有频率能够输出。 Cutoff设置一个频率值。 17.Invert（倒置）：将音频所有通道的相位（Phase）倒转。 18.Lowpass（低通滤波器）：与HP相对 19.Multibandcompressor（多频带压缩）：把音频中的频率分成多段，通过改变某一段或者多段，从而来影响音频的输出效果。 ~ 1 / 2 ~ 可以使用该效果模拟电话中的人声等。 20.Multitap Delay（多重延迟）：对音频添加多个级别Delay的效果。 21.Notch（V形滤波器）：类似于Bandpass 22.Parameticra EQ（参数均衡器）：类似于EQ，但是功能和参数都比EQ少，只控制某一频段的音频。 23.Phaser(相位器):将音频某部分频率的相位发生反转，并与原音频混合。（sin & rect& Tri等低频震荡方式） 24.Pitch Shifter（声音变调）：改变声音的音调。可以模拟卡通鼠等声音 25.Reverb（混响）：模拟声音在房间里的效果和氛围。 26. Spectral Noise Reduction (频谱降噪)：使用特殊的算法来消除素材片段中的噪声。
自己编的代码 Matlab中自带的函数 clc;clear all; %用自己编的函数 pic1=imread('188_2.jpg');%修改数字可以看到另一幅图片的效果pic1=rgb2gray(pic1); subplot(221);imshow(pic1);title('均衡化前的图像'); subplot(222);imhist(pic1);title('均衡化前的直方图'); L=256;%设置灰度级为256 [width,height]=size(pic1); %求nk nk=zeros(1,L); for i=0:L-1 num=find(pic1==(i+1)); nk(i+1)=length(num);
end %求pr（rk）=nk/MN pr=zeros(1,L); for i=1:L pr(i)=nk(i)/(width*height); end %pc存储的就是累计的归一化直方图 pc=zeros(1,L); for i=1:L for j=1:i pc(i)=pc(i)+pr(j); end end sk=zeros(1,L); for i=1:L sk(i)=round((L-1)*pc(i)); end %求pr(sk)，即计算现有每个灰度级出现的概率并显示在屏幕上for i=0:L-1 pr(i+1)=sum(pc(find(sk==i))); end pr %显示pr值 %替换原有图片 pic2=pic1; for i=1:L pic2(find(pic2==(i-1)))=sk(i); end subplot(223);imshow(pic2);title('均衡化后的图像'); subplot(224);imhist(pic2);title('均衡化后的直方图'); %用matlab自带的函数 pic1=imread('188_2.jpg');%先把要处理的图像读入 pic1=rgb2gray(pic1);%转化成灰度图像 %显示灰度图像与直方图 figure;
风格化类视频特效 风格化视频效果主要是对素材进行艺术化处理的效果。【风格化】文件夹中包含13个视频效果，分别是： 【Alpha发光】：使素材Alpha通道产生发光效果。 参数详解： 发光：设置素材发光的大小。 亮度：设置素材发光明亮的程度。 起始颜色：设置素材发光开始的颜色。 结束颜色：设置素材发光结束的颜色。 淡出：勾选选项，产生发光颜色逐渐衰减的平滑过渡效果。 【复制】：可以在画面中创建多个图像副本。 【彩色浮雕】：使素材在不去除颜色的基础上产生立体浮雕效果。 参数详解： 方向：设置浮雕效果的方向。 起伏：设置浮雕效果的尺寸大小。 对比度：设置浮雕效果的对比度。 与原始图像混合：设置与原始素材的混合程度。 【抽帧】：通过改变素材的颜色层次，从而调整素材的颜色效果。 【曝光过度】：模拟相机曝光过度的效果。 【查找边缘】：利用线条效果将素材对比高的区域勾勒出来。 【浮雕】：使素材产生立体浮雕效果。
【画笔描边】：使素材模拟出笔触绘画的效果。 参数详解： 描边角度：设置画笔描边的角度。 画笔大小：设置画笔尺寸的大小。 描边长度：设置每个描边笔触的长度大小。 描边密度：设置描边的密度。 描边浓度：设置描边笔触的随机性。 绘画表面：设置笔触与画面的位置和绘画的进行方式，包括【在原始图像上绘画】、【在透明背景上绘画】、【在白色上绘画】和【在黑色上绘画】4种方式。 与原始图像混合：设置与原始素材的混合程度。 【粗糙边缘】：使素材边缘变得粗糙。 【纹理化】：在当前图层中创建指定图层的浮雕纹理的效果。 【闪光灯】：在素材中创建有规律时间间隔的闪光灯效果。 【阈值】：可以调整素材为黑白效果。 【马赛克】：可以调整素材为马赛克效果。
BCC插件中英文对照表 一、BCC 3D Objects三维物体 BCC Extruded EPS 内置图形挤压成3D图形BCC Extruded Spline挤压样条曲线 BCC Extruded Spline Curves挤出的样条曲线BCC Extruded Text挤压文本 BCC Extrusion Bevel Curves挤压锥曲线BCC Extrusion Side Curves挤压边曲线BCC Extrusion T ext Paths挤压文本路径BCC Layer Deformer层变形 BCC Title Studio 文字标题效果插件BCC Type On Text类型文本 二、BCC Art Looks视觉艺术 BCC Artist's Poster招贴画 BCC Bump Map凹凸贴图 BCC Cartoon Look漫画 BCC Cartooner艺术画效果 BCC Charcoal Sketch木炭画 BCC Halftone中间色 BCC Median中间的
BCC Pencil Sketch素描 BCC Posterize色调分离 BCC Spray Paint Noise喷漆噪声BCC Tile Mosaic马赛克瓷砖 BCC Water Color水彩画 三、BCC Blur滤镜 BCC Directional Blur方向模糊滤镜BCC Fast Lens Blur快镜头模糊BCC Gaussian Blur高斯模糊BCC Lens Shape透镜 BCC Motion Blur动态模糊 BCC Pyramid Blur金字塔模糊BCC Radial Blur径向模糊滤镜BCC Spiral Blur旋转模糊 BCC Unsharp Mask反锐化模糊BCC Z-Blur 四、BCC Color & Tone色彩与色调BCC 3 Way Color Grade色彩分级BCC Brightness-Contrast亮度对比BCC Color Balance色彩平衡BCC Color Correction色彩校正BCC Color Match色彩搭配 BCC Colorize着色
Premiere 置视频特效解析 一、变换类特效 1.垂直保持：【垂直保持】特效可以使图像在垂直方向上滚动，但滚动速度速度将要快。 2.垂直翻转：运用该特效可以将画面沿中心翻转180度，产生垂直翻转效果。 3.摄像机视图：该特效可以模拟摄像机在不同的角度对图像进行拍摄所产生的视图效果。 单击该特效右侧【设置】按钮，将弹出摄像机视图设置对话框。该对话框中的参数与特效控制中的参数相同。
【经度】设置摄像机拍摄时的垂直角度 【纬度】设置摄像机拍摄时的水平角度 【垂直滚动】让摄像机绕自身中心轴转动，使图像产生摆动的效果【焦距】设置摄像机的焦距，焦距越短视野越宽 【距离】设置摄像机与图像之间的距离 【缩放】设置对图像的放大或缩小程度 【填充】设置图像周围空白区域填充的色彩 【填充Alpha通道】：勾选该复选框，使图像产生一个Alpha通道【复位】单击该按钮，将所有设置恢复到默认 4.水平保持：该特效可以使图像在水平方向上产生倾斜 【偏移】用来设置图像的水平偏移程度
5.水平翻转：该特效可以将画面沿垂直中心翻转，产生水平翻转效果 6.羽化边缘：该特效可以使图像边缘产生羽化效果 【数量】用来设置边缘羽化的程度 7.裁剪：该特效根据指定的数值对图像进行修剪，但裁剪可以使剪切后的图像进行放大处理 【左侧】、【顶部】、【右侧】、【底部】：分别指图像左、上、右、下4个边界，用来设置4个边界的裁剪程度 【缩放】勾选该复选框，在裁剪时将同时对图像进行缩放处理 二、图像控制类特效 1.灰度系数（Gramma）校正：该特效可以通过改变图像中间色调的亮度，实现在不改变图像高亮区域和低暗区域的情况下，让图像变得更明亮或更暗 【灰度系数（Gramma）】用来修正颜色的Gramma值，值越大，图像越暗；越小图像越亮 2.色彩传递：该特效可以将图像中指定颜色保留，而其他颜色转化成灰度效果