Sperm motility in the horseshoe crab. IV. Extracellular ions and intracellular pH are not mediators of motility initiation

Upon dilution into sea water, Limulus spermatozoa undergo a brief flurry of motility (duration < 60 sec), after which they are nonmotile until encountering a sperm motility initiating peptide (SMI) that emanates from eggs. Utilizing highly purified SMI extracts and simplified seawater formulations (from which individual ions have been deleted), we found that no specific extracellular ion is required for either dilution-initiated or SMI-initiated motility. Indeed, deletion of one ion (Na⁺) produced dilution-initiated motility of very long duration (several hours). When motility is initiated by SMI (in normal seawater) there is an increase in intracellular pH (pH_i), as indicated by the fluorescent probe, 9-amino acridine; however, this pH, change is not a trigger for motility. As a more general method examining ion movements, the fluorescent probe diS-C₃-(5) was used to qualitatively measure changes in the membrane potential of spermatozoa. Although crude SMI extracts caused membrane depolarization, further purification resulted in an almost complete separation of this activity from SMI, thus showing that SMI activation is apparently an electroneutral event. (The membrane-depolarizing factor has a molecular weight > 30,000 and does not initiate acrosome reactions.) Experiments utilizing the ionophore A23187 and Ca⁺²-blocking agents (verapamil and TMB-8) provided tentative evidence that mobilization of intracellular Ca⁺² may be required for motility initiation. These results show that neither changes in pH_i nor the influx of specific extracellular ions are direct mediators of SMI-initiated motility; however, experiments with pharmacologic agents indicate a possible role for intracellular Ca⁺².     

Sperm motility in the horseshoe crab,Limulus polyphemus L: II. Partial characterization of a motility initiating factor from eggs and the effects of inorganic cations on motility initiation

Egg extracts (obtained by washing intact Limulus eggs with either distilled water or artificial seawater, ASW) contain a sperm motility initiating factor (SMI). The SMI is heat stable (withstands boiling to dryness), passes through a dialysis membrane, and is retained by G-10 Sephadex (indicating a molecular weight of less than 700). Qualitative analysis (by X-ray fluorescence spectroscopy) and quantitative analysis (by atomic absorption spectroscopy) of SMI extracts revealed the presence of four divalent cations (Ca, Mg, Ni, and Cu) and one monovalent cation (K) that affect sperm motility. When assayed individually at high concentrations, all of the divalent cations initiate sperm motility and K⁺ inhibits motility initiation by the divalent cations. However, none of the divalent cations are present at concentrations high enough to produce the observed SMI activity, and since K⁺ is present when motility is initiated by SMI, K⁺ must not normally be an inhibitor. Therefore, if inorganic cations are involved in normal sperm motility initiation in Limulus, they are acting in conjunction with some other low molecular weight factor.     

Sperm motility in the horseshoe crab,Limulus polyphemus L: I. Sperm behavior near eggs and motility initiation by egg extracts

Limulus spermatozoa are nonmotile when spawned and become motile only after encountering a sperm motility initiating factor (SMI) exuded by the egg. SMI extracts (produced by washing intact eggs with distilled water, lyophilizing the supernatant to dryness, and redissolving the dried extract in artificial seawater, ASW) initiate sperm motility in the absence of eggs. Utilizing such SMI extracts, sperm motility initiation was found to be unaffected by changes in temperature from 16 to 30°C, pH from 6.3 to 8.6, and salinity from 85 to 125% ASW. Within these ranges, sperm motility initiation was an “all-or-nothing” response, with greater than 99% of the spermatozoa becoming motile. Also, each sperm swam with apparently the same speed (at a given temperature) until spontaneously stopping within 10 min after the addition of SMI extracts. Evidence was found that SMI may bind irreversibly to a receptor, which is inactivated within a few seconds or minutes, leading to the observed cessation of motility. Observations of sperm behavior near intact eggs showed no evidence of chemotaxis. Spermatozoa observed to swim toward intact eggs progressed with a uniform speed and were motile less than 5 sec from initiation of motility until attaching to the egg. The presence of an all-or-nothing response to SMI, the independence of sperm motility to experimental parameters, and several other characteristics of the animal and its spermatozoa make Limulus a potentially excellent model animal for examination of sperm motility control mechanisms.     

Agreement between measures of total motility and membrane integrity in stallion sperm

Increasing seminal plasma concentrations in extended stallion semen were utilized to model decreasing sperm motility over time. Level of agreement was determined between flow cytometric measurement of sperm membrane integrity, using a combination of SYBR-14 and propidium iodide, and computer-assisted analysis of sperm motility. Values for total sperm motility (TMOT;%) and membrane integrity (SMI;%) were similar (∼80%) at Time 0 within all sperm treatments. However, TMOT was lower than SMI after 24 and 48 h of storage in treatments with >20% seminal plasma. At Time 0, agreement (bias and absolute difference) between TMOT and SMI was high (-0.7 and 5.6%, respectively), but decreased after 24 (10.8 and 15.1%, respectively) and 48 h (23.0 and 23.8%, respectively) of cooled storage as motility declined more rapidly than SMI. We concluded that TMOT and SMI measured separate aspects of sperm quality.     

Secretory origin and temporal appearance of the porcine beta-microseminoprotein (sperm motility inhibitor) in the boar reproductive system

A specific antiserum against the porcine sperm motility inhibitor (SMI) was used in Western blotting analysis of tissue homogenates to reveal the possible origin of SMI in the boar reproductive system at different ages. The ages of the boar used were day 0, day 15, day 30, day 60, day 100, day 120, day 135, day 150, and day 210. The tissue homogenates of the day 60 and older showed immunoreaction. The results were further checked by indirect immunohistochemical staining and observed under light microscope. The SMI antigen appeared in the epithelial cells and in the lumen of the secretory ducts of the prostate gland. These results indicate that porcine SMI is synthesized only by the postnatal prostate gland. The homogenate of the prostate gland of day 100 was also used for the purification of SMI. The prostatic SMI was co-eluted with the seminal SMI in the reversed phase HPLC. Mass spectrometric analysis of the prostatic SMI revealed a molecular weight of 10,066. These results indicate that the prostatic SMI is identical to that purified from seminal plasma (Jeng et al., 1993; Biochem Biophys Res Communi 191:435-440).     

The Limulus sperm motility-initiating peptide initiates acrosome reactions in sea water lacking potassium

During fertilization in Limulus, the spermatozoa first attach to the egg and then undergo an acrosomal reaction. In this reaction, the acrosomal vesicle exocytoses, and a long, preformed acrosomal filament is extruded (and subsequently penetrates the egg chorion). The egg surface component that triggers the acrosome reaction has not yet been solubilized; therefore, previous studies have examined either spontaneous acrosome reactions or acrosome reactions that were triggered by eggs (or insoluble egg fragments), elevated extracellular Ca2+, or Ca2+ ionophores. In this study, we report a new method for initiating acrosome reactions in Limulus sperm. When the Limulus sperm motility-initiating peptide (SMI) is added to sperm in K+-free sea water, greater than 90% acrosome reactions are initiated within 5 min. However, less than 5% acrosome reactions occur either in K+-free sea water lacking SMI or when SMI is added to sperm in either normal sea water or K+- and Ca2+-free sea water. Experiments with K+ ionophores (nigericin and valinomycin), a K+ channel blocking agent (tetraethyl ammonium), an Na+ ionophore (monensin), and reagents that increase the intracellular pH (monensin, nigericin, and NH4Cl) indicate that changes in intracellular K+, Na+, or H+ do not mediate SMI-initiated acrosome reactions. The K+/Ca2+ ratio determines whether or not SMI will initiate acrosome reactions, with greater than 50% acrosome reactions being initiated when this ratio is below 0.3. In that K+ movement does not appear to be the critical event, possibly the K+/Ca2+ ratio either determines the rate of Ca2+ entry or controls the conformation of sperm surface molecules to allow SMI to initiate acrosome reactions in low K+.     

Measurement of the sperm motility index via the sperm quality analyzer and its relationship to other qualitative sperm parameters

Sperm parameters such as the concentration and percentage of motile spermatozoa are commonly used to assess semen quality. The sperm quality analyzer (SQA) is a device that detects variations in the optical density of motile spermatozoa, providing a sperm motility index (SMI) that is based on various sperm parameters including the concentration, morphology and acrosomal status of motile spermatozoa. The relationship between SMI values of frozen-thawed bovine spermatozoa undergoing swelling in a hypoosmotic medium (100 mOsm/L) and other sperm parameters were evaluated. Frozen semen specimens from 3 bulls were thawed and washed with Ham's F-10 supplemented with 3% BSA and split into 3 (0.2 mL) aliquots. The aliquots were diluted with 1.0 mL of Ham's F-10 (Aliquot 1), isotonic sodium citrate (Aliquot 2), and hypotonic sodium citrate (Aliquot 3). The osmotic pressure of the media used for dilution of Aliquots 1 and 2 was 300 mOsm/L, while that for Aliquot 3 was 100 mOsm/L. Following dilution, the aliquots were incubated for 30 min and manually assessed at 5-min intervals for the percentage and grade of motility (Grades 0 to 4) as well as for the percentage of swollen spermatozoa. Sperm samples were simultaneously evaluated by SQA to obtain the SMI values at the same 5-min intervals during the 30-min incubation. Significant correlations were observed between SMI values and other sperm parameters in Aliquot 3 (P < 0.05). The results indicated that the SMI values obtained from frozen-thawed bovine spermatozoa exposed to a 100 mOsm/L diluent, which causes optimal swelling of spermatozoa, are highly correlated to other sperm parameters. The SQA unit, as applied in this study, can be used for rapid and reliable screening of sperm samples.     

Storage of red deer epididymides for four days at 5 degrees C: effects on sperm motility,viability, and morphological integrity

The purpose of this study was to assess the sperm motility, the plasma membrane integrity and the morphology of red deer spermatozoa when maintained within epididymides stored for 4 days at 5 degrees C, and to evaluate whether such stored spermatozoa are able to withstand a refrigeration process. Thirty pairs of testes, with attached epididymides, were collected from 30 hunter-killed mature stags (Cervus elaphus hispanicus), and spermatozoa from each one of the pairs were immediately collected in Triladyl medium, evaluated and refrigerated (Control Group). The remaining testes and epididymides were gradually cooled to 5 degrees C and stored for 1, 2, 3, and 4 days (Experimental Groups), after which spermatozoa were processed as described previously for the control group. The effects on spermatozoa that had been stored within epididymides for various times were determined by assaying sperm motility index (SMI), plasma membrane integrity and sperm morphology (SM). In the same way, SMI and SM were assessed after spermatozoa refrigeration at 5 degrees C for 3 hours in different groups (SMI-R, SM-R). There was no significant decrease in plasma membrane integrity of spermatozoa recovered from epididymides stored at 5 degrees C for 4 days. Similarly, the percentage of morphologically normal spermatozoa remained unaffected during the first 3 days of storage. In contrast, during storage sperm motility evaluation revealed significantly (P<0.05) lower SMI values for samples from epididymides stored 2, 3, and 4 days (47.7+/-3.6, 45.5+/-4.4, 44.1+/-5.2) than that of the control group (57.6+/-1.6). Similar results were obtained after refrigeration of spermatozoa in Triladyl at 5 degrees C. These data suggest that it might be possible to recover functional spermatozoa from red deer epididymides stored at 5 degrees C during several days when epididymal spermatozoa cannot be collected and cryopreserved immediately.     

Validation of the sperm quality analyzer (SQA) for dog sperm analysis

In the present study, a simple and inexpensive unit (the Sperm Quality Analyzer-SQA), was evaluated for dog sperm analysis. Our objective was to propose a cheap, accurate and convenient device to be used in veterinary practices involved with dog fertility assessment and artificial insemination. The device was tested by analyzing repeatability and accuracy at different sperm concentrations and motility characteristics. The Sperm Motility Index (SMI), a numeric index provided by the SQA, was compared with the results obtained using a computer-aided sperm analyzer (Hamilton Thorn IVOS 10). The correlation between SMI and some sperm parameters as well as predictive values of the SMI were established. The dog sperm data provided by the SQA were consistent and repeatable (coefficient of variability below 10% for all concentrations tested). The SMI was significantly dependant on motile sperm concentration and a positive significant correlation was established for the different motile sperm concentrations from a concentration of 25 x 10(6) up to over 200 x 10(6) cells/mL. Zero motility did not affect SMI because non-motile cells, regardless of their concentration, do not cause any fluctuations in the optical density (OD). Over the tested 200 x 10(6) cells/mL value, a correlation still could be observed but it was not statistically significant, possibly because of a saturation of the system. In dog semen, the correlation is better between SMI values and the number of motile spermatozoa than with the overall motile concentration. Based on this observation, a predictive value was given to the SMI allowing for a sorting of dog ejaculates in 3 sperm categories (SMI <100, 100250) each characterized by a range of sperm number and motility. If a positive correlation between the SMI categories and fertility has been demonstrated in humans, such a correlation needs to be established in dogs.     

Glucose regulation of guinea-pig sperm motility.

Washed guinea-pig spermatozoa from the vas deferens re-acquired progressive motility within 1-2 min of incubation in minimal culture medium containing pyruvate and lactate. When glucose was added, either at the beginning or after 15 min of incubation, the cells showed stimulated motility (increased straight-line velocity, linearity and beat-cross frequency, P less than 0.01). Re-acquisition of progressive motility was preceded by a significant (P less than 0.005) transient increase in sperm concentration of cyclic adenosine 5'-phosphate (cAMP) with or without glucose in the medium. Papaverine caused another large significant (P less than 0.001) increase in cAMP concentration; and 5.56mM glucose with papaverine caused a further stimulation in cAMP beyond that with papaverine alone (P less than 0.005). Although 0.05 or 5.56mM glucose plus alpha-chlorohydrin stimulated sperm motility, they did not further stimulate the increase in cAMP after 30 s of incubation. Thus, there was no apparent correlation between the glucose-stimulating effect on sperm motility and the enhancement of cAMP at 30 s. However, there was a close correlation between glucose-stimulated motility and enhancement of ATP (P less than 0.05) by glucose even under incubation conditions in which glucose caused the Crabtree effect (decrease in respiration rate).     

The motility of human spermatozoa as influenced by prostasomes at various pH levels.

Human semen contains several components among which spermatozoa, membranous vesicles called 'prostasomes', secreted by the prostate gland and unorganized material. Prostasomes possess an unusual lipid composition, contain a number of proteins and small molecules and have been claimed to take a part in the immune response, in seminal fluid liquefaction and in sperm motility. Since sperm may come in contact with an acidic environment in the vagina, it may be of some interest to know whether prostasomes may affect spermatozoon motility or may protect spermatozoa upon the exposure to an acidic milieu. Human semen was supplied by donors. From whole semen we collected spermatozoa by centrifugation and used the supernatant to prepare prostasomes (centrifugation at 105,000 g for 120 min, followed by purification step on Sephadex G 200); spermatozoa were then collected by a swim-up procedure and exposed to an acidic pH medium (from 5 to 7) in the presence or absence of prostasomes. Spermatozoa motility was subsequently assessed with a superimposed image analysis system (SIAS). Results indicate that the motility of spermatozoa was affected by the pH value of the medium. Acidic media reduced the percentage of motile cells and decreased the straight line velocity of spermatozoa (VLS). Prostasomes had a protective effect and increased the percentage of motile cells. However, they did not change the characteristics of motility (curvilinear and straight). Prostasomes may be considered as a system for counteracting the negative effects of acidic pH values that may be present in the vagina after coitus.     

Identification of proctolin in the central nervous system of the horseshoe crab, Limulus polyphemus

A proctolin-like peptide was isolated from the prosomal CNS of the chelicerate arthropod, Limulus, and purified using size exclusion, ion exchange and high performance liquid chromatography. Coincident bioassay (cockroach hindgut) and radioimmunoassay were employed to identify fractions which contained proctolin-like material. Proctolin-like activity coeluted with synthetic proctolin with all three chromatographic techniques employed. When applied to either the Limulus heart or hindgut preparations, purified Limulus proctolin produced excitatory responses which were indistinguishable from those produced by the synthetic peptide. Purified samples of the Limulus proctolin-like peptide were subjected to Edman degradation and tandem mass spectrometry and the amino acid sequence of the Limulus peptide was determined to be identical to that of cockroach proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH). The presence of proctolin in the Limulus CNS and its biological action on the isolated heart and hindgut suggest a physiological role for this peptide in the regulation of cardiac output and hindgut motility.     

Identification and characterization of a sperm motility promoting glycoprotein from buffalo blood serum

Early investigators reported the occurrence of unidentified protein factors in biological fluids that may regulate sperm motility essential for fertility potential. This study reports for the first time purification of a forward motility stimulating protein (FMSF-I), to apparent homogeneity, from a biological fluid (buffalo blood serum) and its characterization. FMSF-I is the major motility protein of buffalo serum: a rich source of the factor. FMSF showed high protein specificity and affinity for activating forward motility of goat cauda epididymal spermatozoa. The motility promoter at 0.5 microM level showed maximal activity when nearly 60%-70% of spermatozoa expressed forward motility. It is a 66 kDa monomeric acidic protein rich in aspartate, glutamate, and leucine with isoelectric point of 3.7. FMSF: a Mg2+ -dependent protein binds to concanavalin A-agarose and the glycoprotein nature of FMSF has been confirmed by PAS staining. The factor lost activity completely when treated with alpha-mannosidase showing that the sugar part of the protein is essential for its biological activity. FMSF has no species specificity for its motility-activating potential. Sperm surface has specific receptors of FMSF, which is strongly immunogenic. The factor is present in testis and epididymis although liver is its richest source. Motility promoting efficacy of FMSF is markedly higher than the well-known non-protein motility activators: theophylline and bicarbonate or their combination. FMSF is a physiological activator of sperm motility and as a slaughterhouse byproduct it has potentiality for solving some of the problems of animal breeding, conservation of endangered species, and human infertility: a global social problem.     

Effects of cell motility and chemotaxis on microbial population growth.

A mathematical model is developed to elucidate the effects of biophysical transport processes (nutrient diffusion, cell motility, and chemotaxis) along with biochemical reaction processes (cell growth and death, nutrient uptake) upon steady-state bacterial population growth in a finite one-dimensional region. The particular situation considered is that of growth limitation by a nutrient diffusing from an adjacent phase not accessible to the bacteria. It is demonstrated that the cell motility and chemotaxis properties can have great influence on steady-state population size. In fact, motility effects can be as significant as growth kinetic effects, in a manner analogous to diffusion- and reaction-limited regimes in chemically reacting systems. In particular, the following conclusions can be drawn from our analysis for bacterial populations growing at steady-state in a confined, unmixed region: (a) Random motility may lead to decreased population density; (b) chemotaxis can allow increased population density if the chemotactic response is large enough; (c) a species with superior motility properties can outgrow a species with superior growth kinetic properties; (d) motility effects become greater as the size of the confined growth region increases; and (e) motility effects are diminished by significant mass-transfer limitation of the nutrient from the adjacent source phase. The relationships of these results for populations to previous conclusions for individual cells is discussed, and implications for microbial competition are suggested.     

Characterization of epididymal spermatozoa motility rate, morphology and longevity of springbok (Antidorcas marsupialis), impala (Aepyceros melampus) and blesbok (Damaliscus dorcus phillipsi): pre- and post-cryopreservation in South Africa

Cryopreservation of antelope epididymal spermatozoa could play a vital role in future breeding by developing a successful protocol for cryo-conserving them. The aim of this study was to characterize morphology, motility rates and longevity of epididymal spermatozoa from springbok, impala and blesbok. Cauda epididymal spermatozoa were collected post-mortem from both testicles of free-ranging springbok (n=18), impala (n=21) and blesbok (n=21), and divided into two groups (pre- and post-cryopreservation). Spermatozoa were cryopreserved in Biladyl supplemented with 20% egg yolk and 7% glycerol under field conditions. Pre-freeze and post-thaw sperm quality was evaluated. The longevity of thawed spermatozoa was evaluated under culture conditions that support domestic cattle in vitro fertilization. There was a significant difference between pre-freeze and post-thaw sperm motility index (SMI) (p<0.05), plasma membrane integrity (p<0.05) and acrosome integrity (p<0.05) for all species. Post-thaw SMI and plasma membrane integrity were comparable between species (p>0.05). The effects of cryopreservation on sperm cell morphology differed between species and between specific abnormal morphology. Blesbok had the least abnormalities in post-thaw spermatozoa. Cryopreservation substantially reduced the survivability and motility rates of antelope species. Blesbok spermatozoa tolerated cryopreservation and thawing process better than impala and springbok. The antelope cauda epididymal sperm maintained viability and acrosome integrity for at least 4h following incubation under conditions that support domestic cattle in vitro fertilization (IVF) with a decline in longevity over time across species however; species responded differently over time in terms of plasma membrane integrity and acrosome integrity. The antelope species may have different in vitro culture requirements, indicating differences in sperm physiology between the species. This research could contribute species-specific protocol development for IVF thus promoting ex-situ conservation strategies of African antelope species in South Africa.     

Caffeine-stimulated ATP-reactivated motility in a detergent-treated bovine sperm model.

Mammalian sperm cells contain most of the components of a cyclic AMP-mediation system. To determine if the cyclic AMP-dependent protein kinase has a role in the control of bovine sperm motility, a sperm model was developed that was permeable to exogenously added ATP. Treatment of bovine epididymal spermatozoa with dithiothreitol and Brij-35 (polyoxyethylene alcohol), a nonionic surfactant, resulted in a sperm model with caffeine-stimulated, ATP-reactivatable motility. The results of the data obtained using this sperm model can be summarized as follows. (1) Brij partially solubilized the cyclic AMP-dependent protein kinase activity and released nearly half of the total acid-extractable nucleotides of the cells. (2) Brij treatment severely damaged the sperm mitochondria as judged by their lack of respiration. (3) Brij-treated spermatozoa lose their motility but were reactivated with ATP; the reactivated motility was stimulated by caffeine. (4) Despite the caffeine stimulation of motility in Brij-treated spermatozoa, increased protein phosphorylation did not accompany reactivation of motility, nor could a cyclic AMP effect be demonstrated on reactivated motility or on kinase activity in the sperm model.     

Tumor autocrine motility factor is an angiogenic factor hat stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine that primarily stimulates tumor cell motility via receptor-mediated signaling pathways and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for anti-angiogenic treatment.     

Protonmotive force and motility of Bacillus subtilis.

Motility of Bacillus subtilis was inhibited within a few minutes by a combination of valinomycin and a high concentration of potassium ions in the medium at neutral pH. Motility was restored by lowering the concentration of valinomycin or potassium ions. The valinomycin concentration necessary for motility inhibition was determined at various concentrations of potassium ions and various pH's. At pH 7.5, valinomycin of any concentration did not inhibit the motility, when the potassium ion concentration was lower than 9 mM. In the presence of 230 mM potassium ion, the motility inhibition by valinomycin was not detected at pH lower than 6.1. These results are easily explained by the idea that the motility of B. subtilis is supported by the electrochemical potential difference of the proton across the membrane, or the protonmotive force. The electrochemical potential difference necessary for motility was estimated to be about -90 mV.     

Tumor autocrine motility factor is an angiogenic factor that stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine which primarily stimulates tumor cell motility via receptor-mediated signaling pathways, and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for antiangiogenic treatment.