Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: In recent years there has been an increase in obesity and diabetes mellitus (DM).
These conditions have for a long time been associated with infertility. Obesity is
characterized by high levels of circulating leptin and cytokines as well as insulin
resistance. Type I DM is associated with low or no insulin whereas, Type II DM is
characterised by insulin resistance. As the prevalence of obesity and DM continues
to rise, it is likely that the incidence of infertility associated with these pathological
conditions will likewise increase. The effects of insulin and leptin on male
reproductive function have been reported on the endocrine and spermatogenesis
level, but their effects on cellular level of human ejaculated spermatozoa are yet to
be elucidated.
This study presents data on the role of insulin and leptin on human ejaculated
spermatozoa and their interaction with cytokines and nitric oxide. In the first part of
the study, we established the suitable concentrations of glucose, insulin and leptin
that could be administered to human spermatozoa in vitro. Glucose concentration of
5.6 mM was chosen as the suitable concentration to be administered to human
spermatozoa because it has previously been reported in the literature; furthermore, it
is within the range of the physiological glucose levels found in the blood of fasting
humans. Insulin and leptin concentrations of 10 μIU and 10 nmol were chosen
respectively because they gave much improved sperm function and this was within
the range of insulin and leptin levels previously measured in human ejaculated
spermatozoa. This was followed by investigating the signalling pathway of insulin and
its beneficial effects on human spermatozoa function. Endogenous insulin secretion
from human ejaculated spermatozoa was blocked by nifedipine and its receptor
tyrosine phosphorylation effects were inhibited by erbstatin while phosphatidylinositol
3-kinase (PI3K) phosphorylation activity was inhibited by wortmannin. Exogenous
insulin administration significantly increased human sperm motility parameters as
well as the sperm ability to acrosome react. The inhibition of endogenous insulin
release from spermatozoa as well as the inhibition of the insulin receptor substrate
(IRS) tyrosine phosphorylation significantly decreased motility parameters and the
ability of spermatozoa to acrosome react.
The study also investigated the effects of insulin and leptin on human sperm motility,
viability, acrosome reaction and nitric oxide (NO) production. Both insulin and leptin
significantly increased sperm motility parameters, acrosome reaction and NO
production. The NO production induced by insulin and leptin was via PI3K signalling
as evidenced by a reduction in NO levels when PI3K activity was inhibited by
wortmannin. To investigate whether insulin and leptin could improve motility
parameters of asthernozoospermic and teratozoospermic spermatozoa, the
spermatozoa were separated into two fractions by means of a double density
gradient technique. The gradient system was able to separate spermatozoa into high
morphologically abnormal and less motile spermatozoa similar to that of
asthernozoospermic and teratozoospermic patients as well as a more motile fraction.
Insulin and leptin significantly increased the motility parameters of spermatozoa from
the immature and less motile fraction.
The fourth part of the study was aimed at investigating the effects of the cytokines,
tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), on human sperm
motility, viability, acrosome reaction and NO production. The study shows that TNF-α
and IL-6 significantly reduced motility parameters and acrosome reaction in a dose4
and time-dependent manner. These cytokines were also shown to significantly
increase NO production from human spermatozoa. The decreased motility
parameters induced by these cytokines could be attributed to their ability to induce
excessive NO production. It is not yet clear how they inhibit spermatozoa to undergo
the acrosome reaction.
The fifth part of the study was to investigate the expression and localization of
glucose transporter 8 (GLUT8) in human spermatozoa. This study shows that GLUT8
is constitutively expressed and located in the midpiece region of the human
spermatozoa. The study also showed that stimulating spermatozoa with insulin led to
an increase in GLUT8 expression as well as translocation to the acrosomal region.
In the last part of the study we wanted to investigate why the increase in NO
generation by spermatozoa due to insulin and leptin stimulation is accompanied with
increased sperm function whereas NO increased due to TNF-α and IL-6 stimulation
is accompanied with decreased sperm function. We observed that TNF-α and IL-6
not only increased NO production but also ROS production. This study speculates
that the decrease in sperm motility and acrosome reaction when TNF-α and IL-6
were administered was due to the concomitant high increase in NO and ROS they
induced.
In conclusion, this study has established in vitro beneficial effects of insulin and leptin
in normozoospermic and asthernozoospermic human sperm function. These
hormones influence sperm function via the PI3K signalling pathway in two ways.
Firstly, by increasing GLUT8 expression and translocation thereby possibly
increasing glucose uptake and metabolism and secondly, by increasing NO
production. The study has also established that TNF-α and IL-6 have detrimental
effects on human spermatozoa in a dose and time dependent manner. These effects
are mediated via their ability to stimulate both NO and ROS production in human
spermatozoa. This study reports that GLUT8 is expressed in the midpiece region of
human spermatozoa and that insulin stimulation upgrades its expression and leads to
its translocation to the acrosomal region. / AFRIKAANSE OPSOMMING: Oor die afgelope jare was daar `n toename in obesiteit en diabetes mellitus (DM).
Hierdie toestande word reeds vir ’n geruime tyd geassosieer met onvrugbaarheid.
Obesiteit word gekenmerk deur verhoogde sirkulerende vlakke van leptiene en
sitokiene sowel as insulien weerstandigheid. Tipe I DM word geassosieer met lae of
geen insulien terwyl Tipe II DM gekenmerk word deur insulien weerstandigheid. Soos
wat die voorkoms van obesiteit en DM toeneem, is dit waarskynlik dat die insidensie
van onvrugbaarheid wat met hierdie patologiese toestande geassosieer word,
gevolglik ook sal toeneem. Die effek van insulien en leptien op die manlike
voortplantingsfunksie is alreeds aangetoon op endokriene en spermatogenese vlak,
maar hul effekte op sellulêre vlak van menslike geëjakuleerde spermatosoë is nog
onduidelik.
Die studie vertoon data oor die rol van insulien en leptien op die menslike
geëjakuleerde spermatosoë en hul interaksie met sitokiene en stikstofoksied (NO). In
die eerste gedeelte van die studie, het ons ’n toepaslike konsentrasie van insulien en
leptien bepaal wat aan menslike spermatosoë in vitro toegedien kan word. Glukose
konsentrasies van 5,6 mM is bepaal as die gepaste konsentrasie om aan menslike
spermatosoë toe te dien, omdat dit beter resultate tot gevolg het; verder is dit
vergelykbaar met fisiologiese glukose vlakke in die bloed van `n vastende persoon.
Insulien en leptien konsentrasies is op 10 μIU en 10 nm onderskeidelik vasgestel,
aangesien dit tot beter resultate gelei het, en omdat dit vergelykbaar was met
insulien en leptien vlakke wat reeds voorheen in menslike geëjakuleerde
spermatosoë gemeet is. Dit was gevolg deur `n ondersoek na die insulien
seintransduksie pad en sy voordelige effekte op menslike spermatosoë funksie.
Endogene insulien afskeiding deur menslike geëjakuleerde spermatosoë was deur
nifedipien geïnhibeer en sy reseptor tirosien fosforilasie effekte was deur erbstatin
geïnhibeer terwyl fosfatidielinositol 3-kinase (PI3K) fosforilasie deur wortmannin
geïnhibeer is. Eksogene insulien toediening het menslike sperm-motiliteit parameters
betekenisvol laat toeneem asook die vermoë van sperme om die akrosoomreaksie te
ondergaan. Die inhibisie van endogene insulien afskeiding deur spermatosoë sowel
as die inhibisie van die insulien reseptor substraat (IRS) tirosien fosforilasie het die
motiliteit parameters en die akrosoomreaksievermoë van spermatosoë verlaag.
Die studie het ook die effekte van insulien en leptien op menslike sperm-motiliteit,
-lewensvatbaarheid, -akrosoomreaksie en -NO produksie nagevors. Beide insulien
en leptien het sperm-motiliteit parameters, -akrosoomreaksie en -NO produksie
betekenisvol verhoog. NO produksie is deur insulien en leptien via PI3K
seintransduksie geïnduseer, soos bewys deur die verlaging waargeneem in NO
vlakke toe PI3K aktiwiteit deur wortmannin geïnhibeer was. Om vas te stel of insulien
en leptien die motiliteit parameters van asthenozoospermiese en teratozoospermiese
spermatosoë kon verbeter, het ons spermatosoë in twee fraksies met ’n dubbel
digtheid gradiënt geskei. Die gradiënt sisteem was daartoe instaat om die
spermatosoë in ’n onvolwasse, (morfologies abnormaal en minder motiel - soortgelyk
aan dié van asthenozoospermiese en teratozoospermiese pasiënte), sowel as ’n
volwasse meer motiele fraksie te skei. Insulien en leptien het die motiliteit parameters
van spermatosoë van die onvolwasse en minder motiele fraksie verhoog.
Die vierde gedeelte van die studie was daarop gemik om die effekte van die sitokiene
tumor nekrose faktor alfa (TNF-α) en interleukin-6 (IL-6) op menslike sperm-motiliteit,
-lewensvatbaarheid, -akrosoomreaksie en -NO produksie, te ondersoek. Die studie
het getoon dat TNF-α en IL-6 motiliteit parameters en akrosoomreaksie in ’n tyd- en
dosis-afhanklike wyse betekenisvol verlaag het. Hierdie sitokiene was ook in staat
om NO produksie in menslike spermatosoë te verhoog. Die verlaging in motiliteit
parameters wat deur hierdie sitokiene geïnduseer is, kan toegeskryf word aan hul
vermoë om die produksie van oormatige hoeveelhede NO te stimuleer. Dit is nog nie
duidelik hoe hulle die akrosoomreaksie in spermatosoë kan inhibeer nie.
Die vyfde gedeelte van die studie het dit ten doel gehad om die uitdrukking en
lokalisering van die glukose transporter 8 (GLUT8) in menslike spermatosoë te
ondersoek. Hierdie studie kon aantoon dat GLUT8 konstitutief uitgedruk is en in die
middelstuk van die menslike spermatosoë voorkom. Die studie bewys ook dat
stimulering van die spermatosoë met insulien tot `n toename in GLUT8 uitdrukking
sowel as translokasie na die akrosomale area, lei.
In die finale gedeelte van die studie wou ons ondersoek waarom die toename in NO
produksie in spermatosoë (as gevolg van insulien en leptien stimulasie) deur `n
toename in spermfunksie gekenmerk word, terwyl die toename in NO produksie (as
gevolg van TNF-α en IL-6 stimulasie) deur ’n afname in spermfunksie gekenmerk
word. Ons het waargeneem dat TNF-α en IL-6 nie alleen NO produksie nie, maar ook
reaktiewe suurstof spesies (ROS) produksie verhoog het. Ons vermoed dat die
afname in sperm motiliteit en akrosoomreaksie met TNF-α en IL-6 toediening, die
gevolg van die gelyktydige verhoging in NO en ROS was.
In gevolgtrekking kan ons sê dat hierdie studie die voordelige in vitro effekte van
insulien en leptien op asthenozoospermiese en teratozoospermiese menslike
spermfunksie aangetoon het. Hierdie hormone beïnvloed spermfunksie via die PI3K
seintransduksie pad op twee maniere. Eerstens, deur `n toename in GLUT8
uitdrukking en translokasie, met die gevolg dat glukose opname en metabolisme
moontlik verhoog is, en tweedens, deur die toename in NO produksie. Die studie het
ook vasgestel dat TNF-α en IL-6 nadelige effekte op menslike spermatosoë in `n
dosis- en tyd-afhanklike wyse het. Hierdie effekte vind plaas a.g.v. hul vermoë om
beide NO en ROS produksie in menslike spermatosoë te induseer. Die studie toon
aan dat GLUT8 uitdrukking in die middelstuk van die menslike spermatosoon
voorkom en dat insulien stimulasie GLUT8 uitdrukking opreguleer en tot translokasie
na die akrosomale area lei.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/1083 |
| Date | 12 1900 |
| Creators | Lampiao, Fanuel |
| Contributors | Du Plessis, S.S., Strijdom, H., University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Medical Physiology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Stellenbosch |
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