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| Home > Publications database > Molekularbiologische und immunologische Charakterisierung von Chemorezeptoren in Säugetier-Spermien |
| Dissertation / PhD Thesis/Book | PreJuSER-26445 |
2002
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/2563
Report No.: Juel-4002
Abstract: Chemotaxis of sperm is well established in marine invertebrates, such as sea urchin (Morisawa, 1994). The chemotactic egg-derived peptide resact of the sea urchin $\textit{Arbacia punctulata}$ binds to a membrane-bound receptor-guanylyl cyclase. Mammalian sperm show chemotactic behaviour in response to factors released by the egg itself and/or the female reproductive tract. Neither the chemotactic factors nor the respective membrane receptors of sperm have been identified. Current hypotheses on the chemotactic signaling pathways rely an models for odorant sensation in olfactory sensory neurons (OSN). Thus, G-protein coupled odorant receptors (OR) or the receptor guanylyl cyclase-D might play a role in chemotaxis of mammalian sperm. In the first part of this PhD Thesis, 12 different putative OR have been identified by RT-PCR in germ cells of male mice. Four complete OR genes were cloned from genomic DNA. The twelve amplified ORs are members of ten subfamilies revealing that testicular ORs do not constitute a specific subfamily. Except for TOR8 that belongs to class I OR (fish-like), the other receptors are members of class II OR (mammalian-like). All ORs are also expressed in OSN. Furthermore 6 ORs are expressed in additional tissues (i.e. heart, brain). Most ORs are already expressed in the prepubertal testis. This finding points out that - in addition to a functional role in mature sperm - ORs may be involved in the development and organization of the germ cell epithelium. Northernblot experiments revealed that two ORs (TOR13 and TORSI) are more abundantly expressed than the other ORs. By $\textit{in situ}$ hybridization-experiments transcripts of TOR13 were localized to spermatogonia and spermatocytes. Cloning of the TOR13 and TORSI genes showed that the TOR 13 gene codes for a C-terminal truncated receptor protein. Because it is unknown whether an OR lackeng the seventh transmembrane domain and the C-terminus is functional, only the TORS 1 gene was further characterized. Southemblot analysis revealed that TORSI is the only gene of a new cluster. The gene could be assigned to chromosome 11. TORS1 could not be functionally expressed in a heterologous system although the receptor protein was synthesized and posttranslationally modified in the host cells. In conclusion, TORSI is a candidate for a conditional gene $\textit{knock-out}$. This approach might provide a deeper insight into the function of ORs in sperm cells. In the second part, G$_{\alpha olf}$, the interaction partner of OR in OSN, has been cloned from testicular cDNA. By Westernblot experiments, G$_{\alpha olf}$ could be detected in sperm. G$_{\alpha olf}$ was immunhistochemically localized to the acrosomal part of the head. Adenylyl cyclase III (ACIII), the effector of G$_{olf}$ in OSN, could not be detected in sperm by Westernblot experiments. Thus unlike in OSN, OR in sperm do not activate ACIII by means of G$_{\alpha olf}$. Consequently, in sperm ORs must participate in a different signaling cascade. In the third part, I investigated whether a receptor GC and a cGMP-regulated ion channel (CNG-channel) are expressed in sperm. A signaling cascade comprising a receptor GC could be involved in chemotaxis such as in sea urchin sperm. Transcripts of the GC-D could be identified in cDNA of sperm precursor cells. The ion channel $\alpha$-subunit mCNGA3 und the modulatory $\beta$-subunit mCNGB3 have been also amplified from germ cell cDNA. In immunhistochemical experiments, the GC-D was localized to the $\textit{principal piece}$ of the sperm tail. Thus, it is tempting to speculate that the GC-D and the CNG ion channel are components of a signaling pathway that regulates the swimming behaviour of mammalian sperm cells.
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