Διερεύνηση του ρόλου αμινοξέων πεπτιδίων και γλυκοζαμινογλυκανών στο ανθρώπινο σπέρμα
Creators: name: Παπαχαρίτου, Σταμάτης Χ.
Publisher: name: Aristotle University of Thessaloniki
Year: 2006
Subjects: Αμινοξέα
Πεπτίδιο
Γλυκοζαμινογλυκάνες
Aminoacids
Peptide
Glycozaminoglucan

description: Στόχος είναι η διευκρίνηση μηχανισμών που εμπλέκονται στην αύξηση της κινητικότητας των σπερματοζωαρίων όπως ο ρόλος των καναλιών Cu++ και η διερεύνηση του ρόλογυ μορίων του εσωκυττάριου χώρου(GAGs, MMP και TIMP) και η συσχέτισή τους με τις παραμέτρους του σπέρματος. Χρησιμοποιήθηκαν δείγματα ανθρώπινου σπέρματος και μετά την ανάλυσή τους σύμφωνα με τα κριτήρια της WHO επωάθηκαν με διάφορες συγκεντρώσεις KCL, NMDA και AMPA. Στα δείγματα τακτοποιήθηκε η ύπαρξη MMP-2, 9 και pro MMP-2 k 9 καθώς και μορφών GAGs. Διαπιστώθηκε πως η προσθήκη KCL διεγείρει την κινητικότητα σε συγκεντρώσεις &gt;0,1 mM. Η προσθήκη NMDA σε συγκεντρώσεις από 0,01 έως 1 μΜ προκαλεί δοσοεξαρτώμενη μείωση της. Το ΑΜΡΑ δεν προκαλεί επίδραση. Παρατηρήθηκε συσχέτιση μεταξύ αριθμού σπερματοζωαρίων και της δραστηριότητας pro MMP-9 και θετική μεταξύ βαθμού κινητικότητας και της δραστηριότητας pro MMP-9, pro MMP-2 και ΜΜΡ-2. Στο σπερματικό υγρό τακτοποιήθηκαν η δερματάνη και η θειϊκική χονδροϊτίνη. Αλλά δεν υπάρχει συσχέτιση της με τις παραμέτρους του σπέρματος
description: Although epidemiological data regarding subfertility in the greek population are limited, it is estimated that more than 300.000 couples are facing fertility problems. According to the international literature, 33% of the subefertility cases attributed to problems in the male. Today, there is a trend to promote the application of assisted reproduction techniques (it is estimated that 12-15.000 attempts/annum are performed throughout the country) and especially intracytoplasmic sperm injection (ICSI) as panacea for every man with either primary or secondary subfertility, future approaches should be less expensive and should target to the treatment of the individual male problem itself. Due to the lack of sufficient data regarding the underlying molecular mechanisms of morphometric and molecular parameters of fertility in males, such as spermatozoan motility, the aim of the thesis was the elucidation of the role of Ca+2 channels and the exploration of extracellular matrix molecules (GAGs, MMP and TIMP) in relation to sperm parameters. Our results showed that KCl stimulates the motility in concentrations &gt;0.1 mM and specifically from 1 to 100 mM. The peak value in motility is attained during the first 10 min. Curvilinear and straight line velocities were increased dose-dependently. In contrast, NMDA proved to affect both curvilinear and straight line velocities. At concentrations &gt;0.01 μΜ (specially between 0.01-1 μM) it induced a dose-dependent increase in motility, while at concentrations &gt;1μΜ, it resulted to a dose-dependent derease in motility. The constant velocity's change takes place within approximately in 60 min. AMPA did not have any pharmacologically significant effect. Regarding GAGs, there were not traced in any homogenized spermatozoan sample. Though, total GAGs polyacrylamide gel electrophoresis in 8-20% linear gradient gels (30μg uronic acids) from seminal fluid showed that they migrated as broad zones with molecular weight between 180-10 kDa, no statistical significant differences were found in their distribution between samples showing different sperm concentration or %motility. Total GAGs (4 μg uronic acids) were further studied with their cellulose acetate electrophoresis in a pyridine and HCOOH buffer as well as in barium acetate buffer producing similar results. In the samples were identified dermatan sulfate and chondroitin sulfate C. Total GAGs electrophoresis (30 μg uronic acids) in cellulose acetate in the presence of pyridine and HCOOH buffer as well as in barium acetate buffer produced similar results, that is there are no statistical significant differences regarding GAGs distribution between samples with different sperm concentration and % motility. No correlation was found between GAGs level in seminal fluid and any of the sperm parameters (p &gt; 0,05). No association was found between GAGs and varicocele's presence, either. Regarding MMP, we found negative correlation between the sperm count and pro-MMP-9 and MMP-9 activity; positive correlation between the grade of sperm movement and proMMP-9, proMMP-2 and MMP2 activities; no correlation was found between the number of spermatozoa or their %motility and any MMP. We also show a positive correlation between ΜΜΡ levels and % sperm concentration abnormalities, but no correlation was found with any other parameter (p &gt; 0,05). No statistical correlation was found between MMP and diagnosed varicocele, either. Finally, TIMP-1 and 2 were found in seminal fluid but no statistical significant correlation was found between their concentrations and any sperm parameter or varicocele's presence (p &gt; 0,05).