Semi-automated analysis of manually reconstructed tracks of progressively motile human spermatozoa

Several alternative algorithms for computer-assisted derivationof measurements of movement characteristics from manually reconstructedtracks of progressively motile human spermatozoa were compared.Fifty tracks were reconstructed at 30 Hz from video recordingsand analysed using traditional manual methods and by four combinationsof computer algo rithms. The best algorithm set was identified(‘Video-mot.mdpt’) and the values for the curvilinear,average path and linear velocities (V_CVL, V_AVE and V_LIN respectively),the amplitude of lateral displacement of the sperm head aboutthe axis of progression (A_H) and the number of times the spermhead crossed the average path (the ‘beat/cross frequency’,BXF) obtained using it were compared to those obtained by manualanalysis. There was a considerable time saving when the computer-assistedmethod was used and the values it gave for the various movementcharacteristics showed good correspondence with those obtainedmanually. In addition, repeated data entry and analysis wasfound to be highly reproducible. When the tracks were re-plottedat 6 Hz (as used by the multiple-exposure photomicrography methodfor sperm motility analysis) insufficient information remainedin the tracks for reliable determination of anything other thanV_LIN We conclude that the Videomot.mdpt program provides reliablevalues for the movement characteristics of progressively motilehuman spermatozoa, and believe it will be of great value inthe validation of commercial systems providing automated spermmovement analysis and in laboratories which do not have accessto such costly equipment.     

Intestinal motility responses to neuropeptide γ in vitro and in vivo in the rat: comparison with neurokinin 1 and neurokinin 2 receptor agonists

We have studied the effect of a novel tachykinin, neuropeptide γ(NPγ) on small intestinal motility in the rat. Experiments were done in vitro on longitudinal muscle strips of duodenum, and in vivo on the migrating myoelectric complex (MMC) of the small intestine. In vitro, contractile effects of NPγ were compared with those of a selective neurokinin 1 (NK1) receptor agonist, substance P methyl ester (SPME), and a selective neurokinin 2 (NK2) receptor agonist, Nle¹⁰-NKA(4–10)(NleNKA). NPγ, SPME and NleNKA caused concentration-dependent contractions (P < 0.001). NPγ was eight-fold more potent than NleNKA, and 118-fold more potent than SPME. Contractile responses to NPγ were reduced by hexamethonium (P < 0.01) and atropine (P < 0.05). The non-selective NK receptor antagonist spantide I only slightly reduced the contractile response to NPγ, as did the selective NK1 antagonist GR 82334, and the selective NK2 antagonist L-659877 and MEN 10376. In vivo, effects of NPγ on the MMC were compared with those of the natural tachykinins substance P (SP) and neurokinin A (NKA). NPy disrupted the MMC and induced irregular spiking in a dose-dependent manner from 25 to 100 pmol kg^-1 min^-1 i.v. (P < 0.05). The effect of NPγ was more prominent than that of NKA at equal doses, while SP had no effect. Our findings show that NPγ exerts potent stimulatory effects on small intestinal motility, most likely mediated directly via distinct NK receptors on smooth muscle cells, but also indirectly via a cholinergic link.     

Effect of hepatocyte growth factor on sperm motility

PROBLEM: Hepatocyte growth factor (HGF) exists abundantly in seminal plasma and its receptor, c-met, is expressed on spermatozoa. Considering its motogenic activity, we speculated that HGF might affect the movement ability of spermatozoa. METHODS: Recombinant HGF was added to washed spermatozoa and their movements were analyzed using a computer-assisted sperm analyzer. The concentration of HGF in the seminal plasma of infertile patients (n = 83) was measured by ELISA, and the data were compared with their hormonal profile and semen parameters. RESULTS: The HGF physiological concentration (1 ng/mL) maintained the motility of sperm after a long incubation, though the difference was not statistically significant. Recombinant HGF did not affect the linearity or frequency of movement, which suggested that it does not evoke the hyperactivation of spermatozoa. The concentration of HGF in seminal plasma did not correlate with any clinical parameter of the patients. CONCLUSIONS: These findings contradict the theory that HGF controls the movement of sperm. The main role of this axis in the male reproductive system might be maturation in the epididymis.     

Induction of capacitation in human spermatozoa in vitro by an endometrial sialic acid-binding protein

A Ca²⁺-dependent sialic acid-binding protein (SABP) of humanendometrium, which specifically bound to human sperm head plasmamembrane in vitro, was found to increase the percentage motilityand acrosome-reacted pattern of uncapacitated spermatozoa. Theprotein was synthesized in the endometrium and secreted intothe uterine fluid. This intra-uterine factor, which is apparentlyadvantageous in vitro in inducing human sperm capacitation,may play a significant role in promoting the postrelease maturationof ejaculated spermatozoa by enhancing ⁴⁵Ca uptake into spermatozoaby a pathway which is insensitive to calcium-channel blockers.However, the ⁴⁵Ca uptake could be enhanced on exposure to thedivalent cation ionophore A23187 and inhibited in the presenceof the calmodulin inhibitor trifluoperazine. The SABP also inducesan increase in intracellular Ca²⁺ in spermatozoa, as seen byFURA-2 AM studies. Furthermore, overlay studies show human SABPto be a Ca²⁺-binding protein. The data presented here suggestthat SABP induces invitro sperm capacitation and the subsequentacrosome reaction by increasing intracellular Ca²⁺ concentration.     

Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events

Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1–5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCα to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal. This revised version was published online in July 2006 with corrections to the Cover Date.     

TPA-induced cohort migration of well-differentiated human rectal adenocarcinoma cells: cells move in a RGD-dependent manner on fibronectin produced by cells, and phosphorylation of E-cadherin/catenin complex is induced independently of cell-extracellular matrix interactions

 We have already presented a two-dimensional cell motility assay using a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1 as a motility model of tumour cells of epithelial origin. In this model, L-10 cells showed locomotion as a coherent sheet when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA), and we called this type of movement ”cohort migration”. Electron and immunoelectron microscopic study of the migrating cell sheets demonstrated localized release from cell–cell adhesion only at the lower portion of the cells with loss of E-cadherin immunoreactivity, and this change was associated with increased tyrosine phosphorylation of the E-cadherin–catenin complex, including β-catenin. Cell–extracellular matrix (ECM) interactions involved in this TPA-induced cohort migration and their effect on tyrosine phosphorylation of the E-cadherin-catenin complex have now been investigated. L-10 cell cohort migration was almost completely inhibited by addition of Arg-Gly-Asp (RGD) peptide into the medium, and thus RGD dependent. Cohort migration was stimulated on type I and IV collagens, fibronectin (FN)- and laminin-coated substratum, but was inhibited by RGD only on FN-coated surface. By using immunofluorescent techniques, FN was demonstrated preferentially around migrating cells, and a protein synthesis inhibitor, cycloheximide, inhibited the migration by about 75%. FN produced by L-10 cells were found to be mostly EDA+ FN when analysed by RT-PCR. Moreover, anti-FN antibody, but not anti-vitronectin antibody, inhibited the TPA-induced cohort migration almost completely. Thus, it was likely that L-10 cells produced FN themselves and moved on the FN substrate in an RGD-dependent manner. However, stimulation of migration by type I collagen coating and inhibition by RGD treatment did not affect the tyrosine phosphorylation of the E-cadherin–catenin complex induced by TPA, indicating that cell–cell interactions were adjusted to suit cell migration, irrespective of the condition of cell–ECM adhesion, during TPA-induced cohort migration. Received: 31 December 1997 / Accepted: 2 April 1998     

Disturbed pyloric motility in Ws/Ws mutant rats due to deficiency of c-kit expressing interstitial cells of Cajal

Interstitial cells of Cajal (ICC) are believed to lnitlate the basic contractile activity of the gastrointestlnal tract. Interstitial cells of Cajal express c-kit receptor tyroslne kinase and are deficient in Ws/Ws mutant rats with a small deletion of the c-kit gene . As Ws/Ws rats show remarkable bile reflux to the stomach, the contraction pressure of the pylorus was compared between Ws/Ws and control +/+ rats. The contraction pressure of the pylorus was measured using a mlcrotransducer, which was Inserted through a pln-hole in the anterlor wall of the stomach under anesthesla. The magnitude of bile reflux was estimated by measurlng the content of bile acids In the stomach. The c-kit messenger RNA-expressing cells were detected by in sltu hybrldlzatlon. Frequency and the maxlmum pressure of the contractlon were comparable between Ws/Ws and +/+ rats, but the duration of the contractlon was significantly shorter In Ws/Ws rats than In +/+ rats. The number of c-kit messenger RNA-expresslng ICC in the pylorus of Ws/Ws rats was 1.7% that of +/+ rats. The bile reflux observed in Ws/Ws rats was attributed to the decrease in the duration of the pyloric contraction, which appeared to result from the deficlency of c-kit messenger RNA-expressing ICC.     

Malignant effusions are sources of fibronectin and other promigratory and proinvasive components

Metastatic dissemination is the primary cause of death in ovarian cancer (OvCa) patients, and dissemination to pleural and peritoneal effusions is a common clinical event. Effusion samples were collected from 15 OvCa patients. Twenty-six samples were collected prospectively, two were archival, and eight were taken from patients with other malignancies. Twenty-nine samples were from malignant ascites, and seven specimens were pleural fluids. In addition, six ascites and two pleural fluids from noncancer patients were studied as effusion controls. Effusion supernatants were tested for migration-stimulation activity, using A2058 human melanoma cells as the index responder cell. Malignant samples induced a 400-1200% increase in migration. Sixty percent of the migration was inhibited by incubation of the malignant fluid with antifibronectin (FN) antibody, in contrast to 75% inhibition of control fluid-stimulated migration (P = 0.017). Gelatin zymography and Western blot analyses showed that latent and activated MMP-2 and MMP-9 collagenases, and tissue inhibitor of metalloproteinase-2 (TIMP-2) were present in all malignant fluids. Serial samples were taken from several patients, and a trend for correlation between MMPs and clinical behavior of the tumors was shown. Free TIMP-2 correlated with CA-125 levels in two patients for whom serial samples were available. The demonstration of promigratory and proinvasive activity in malignant effusions is consistent with their association with other metastatic disease in OvCa patients and their function as a haven for metastatic cells.     

Poor development of outer dense fibers as a major cause of tail abnormalities in the spermatozoa of asthenoteratozoospermic men.

For a reasonable analysis of sperm motility, consideration should be given to the morphology of the flagellum. Among various tail disturbances, the present paper describes a structural shaft defect that is characterized by variations in diameter, poor outline and/or lacking endpiece. Electron microscopical examinations, especially the negative staining technique, have shown that in addition to membrane alterations there is one essential cause for the limited extension of the outer dense fibres. This disturbance may be of genetic origin or due to toxic or inflammatory influences. Further molecular biological studies are required to detect the actual cause of this defect, for which there is so far no adequate therapy.