Identifikation und funktionale Charakterisierung - Logo Dragon-Ivf ...

Zusammenfassung
dieses Teilprojektes standen die bislang unbeschriebenen elektrophysiologischen
Eigenschaften kultivierter Spermatogonien. In einem Spermatogonien-Sertolizell-
Kokultursystem konnten sowohl spannungsaktivierte K + - und Ca 2+ -Kanäle unterschiedlichen
Typs, als auch P2X-Rezeptoren identifiziert und charakterisiert werden. Darüber hinaus
konnten zahlreiche Hinweise auf die Expression von TRP-Kanälen in Spermatogonien
erbracht werden.
Durch die Kombination zellbiologischer und molekularbiologischer Methoden, sowie
hochauflösender bildgebender Verfahren und elektrophysiologischer Ableittechniken konnten
in der vorliegenden Arbeit sowohl neue Erkenntnisse über die Rolle von olfaktorischen
Rezeptorproteinen im Kontext der Keimzellentwicklung, Spermienphysiolgie und
Fertilisation gewonnen, als auch die Physiologie muriner Spermatogonien erstmals in Kultur
elektrophysiologisch beschrieben werden.
6.1 Summary
Odorant receptors (ORs) play a central role in the detection and differentiation of odor
molecules. In addition to OR expression in sensory neurons of the olfactory epithelium, some
OR proteins are also found in ectopic tissues, e.g. in testes. In 2003, a human testicular
odorant receptor (OR1D2) was for the first time identified, recombinantly expressed, and
functionally characterized (Spehr et al., 2003).
During my graduate studies, I have examined different aspects of chemosensory
communication mechanisms in male germ cells at different stages of development. On the
molecular level, I characterized the receptive field of OR1D2 to provide novel structural
insight into the OR ligand binding pocket. Using recombinant expression in a heterologous
cell system (HEK293 cells) and live-cell Ca 2+ -imaging, the OR1D2 activation profile was
investigated by testing structurally related and modified ligand candidates. These experiments
resulted in the identification of five novel OR1D2 ligand molecules as well as the first
experimental in-vitro data showing that OR-ligand interaction is based on a ,lock and key’
concept , rather than on molecular vibrations as proposed by the ‘vibration theory’.
In a follow-up study, I screened constituents of various human bodily fluids (e.g. follicular
fluid, vaginal secretions) to identify endogenous ligands for both OR1D2 and several other
testicular ORs that had been identified by members of the department for cell physiology
(OR4D1, OR7A5 und PSGR). In a collaborative approach, various molecular constituents of
follicular fluid and vaginal secretions were identified by gas chromatography. Using
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