IZW-Berlin : Research - RG 4 (Fertility and Seasonality)

Research Group 4: Reproduction Biology

Projects 2: Andrology

2.1. Teratozoospermia in felids (DFG Je 163/9-1).

Teratozoospermia is relatively common among various felids and is causal for reduced fertility especially in some endangered species. The domestic cat teratozoospermic model shows hyper-active spermatogenesis in conjunction with high proportions of sperm with head, midpiece and flagellar defects. Teratozoospermia in cats does not only compromise sperm quality, but is also characterized by disruption of testicular function. Teratozoospermic cats produce more sperm by virtue of more sperm producing tissue, more germ cells per Sertoli cell, and reduced germ cell loss during spermatogenesis. Since apoptosis plays a crucial role in eliminating defect spermatozoa during spermatogenesis, and estrogens are involved in apoptosis regulation, the aim of this present project is to better understand the role of estrogens during spermatogenesis and sperm maturation in the epididymis of teratozoospermic domestic cats. We test the hypothesis, that either the expression patterns of ERalpha, beta and/or P450aromatase or the molecular structure of these proteins are different in normo- versus teratozoospermic individuals. We use a well characterized domestic cat model which perfectly reflects the situation in wild type species. We will gain new insights into the general impact of estrogens on spermatogenesis and sperm maturation and into the possible influence of the estrogen signaling system on teratozoospermia in felids.

Results/Publications

Jewgenow K, Neubauer K, Blottner S, Schön J, Wildt DE, Pukazhenthi BS (2009): Reduced Germ Cell Apoptosis during Spermatogenesis in the Teratospermic Domestic Cat. J Androl. 30(4):460-468. doi: 10.2164/jandrol.108.006726
Schön J, Neumann S, Jewgenow K. (2009): Influence of estrogens during sexual maturation: differential expression of the alpha receptor in the cat epididymis. Reprod Dom Anim. Reprod Dom Anim 44 (Suppl.2), 294-301; doi: 10.1111/j.1439-0531.2009.01391.x
Baudi DLK, Jewgenow K, Pukazhenti BS, Spercoski KM, Santos AS, Reghelin ALS, Candido MV, Javorouski ML, Müller G, Morais RN (2008). Influence of cooling rate on the ability of frozen-thaw spermatozoa to bind to heterologous zona pellucida as assessed by competitive in vitro binding assays in the Ocelot (Leopardus pardalis) and Tigrina (Leopardus tigrinus). Theriogenology 69: 204-211. doi:10.1016/j.theriogenology.2007.09.013
Blottner S, Jewgenow K (2007): Moderate Seasonality in Testis Function of Domestic Cat. Reprod Dom Animals, 42/5, 536-540. DOI: 10.1111/j.1439-0531.2006.00817.x
Pukazhenthi BS, Neubauer K, Jewgenow K , Howard J, Wildt DE (2006): Teratospermia in Felids - Etiology, Impacts and Biological Basis. Theriogenology 66, 112-121.

Cooperation

Institute of Veterinary Biochemistry, Freie Universität Berlin, Germany
Center for Species Survival, Department of Reproductive Sciences, Smithsonian’s National Zoological Park, Conservation & Research Center, Front Royal, Virginia, USA

Current research topic:

Quantity in expense of quality – role of estrogens for spermatogenesis and sperm maturation in teratozoospermic cat (DFG Je 163/

2.2 Sperm lipids – basics and adaptations

The principles of fertilization are similar for spermatozoa of many species. They have to interact with the male and female genital tract, to overcome immunological barriers, to penetrate the egg shell, to fuse with the oocyte and to catalyse the early embryonal development. The lipid composition and architecture of sperm membranes influence the interactions of spermatozoa with proteins and lipids up to the characteristic sperm fusion processes (acrosome reaction, oocyte fusion). The knowledge of general principles and species-dependent strategies of sperm lipid function at fertilization will help to establish customized methods in the field of assisted reproduction, particularly for endangered or threatened species (evaluation of sperm quality, preservation of gametes, artificial insemination, in vitro fertilization).

Figure: Ram spermatozoa after cryopreservation - thirty minutes after labeling with C6-NBD-phosphatidylserine (green fluorescence) and propidiumiodide (red fluorescence) in the presence of 20 mM dithionite. Fluorescence of C6-NBD-phosphatidylserine remained preserved in thepresence of dithionite only in viable cells (not stained by propidiumiodide, note the motile green tails). The fluorescent analogue of phosphatidylserine is actively enriched on the cytoplasmic leaflet of the cell membrane (from Müller et al. (1999), J Cell Sci. 112, 11- 20).

Schiller J, Müller K, Süß R, Arnhold J, Gey C, Herrmann A, Leßig J, Arnold K, Müller P (2003): Analysis of the Lipid Composition of Bull Spermatozoa by MALDI-TOF Mass Spectrometry and thin-layer chromatography - A cautionary note. Chemistry and Physics of Lipids 126, 85-94
Kurz A, Viertel D, Herrmann A, Müller K (2005): Localisation of phosphatidylserine in boar sperm cell membranes during capacitation and acrosome reaction. Reproduction 130, 615-626
Fuchs B, Müller K, Göritz F, Blottner S, Schiller J (2007): Characteristic oxidation products of choline plasmalogens are detectable in cattle and roe deer spermatozoa by MALDI-TOF mass spectrometry. Lipids 42(11), 991-998. DOI 10.1007/s11745-007-3108-7
Fuchs B, Jakop U, Göritz F, Hermes R, Hildebrandt T, Schiller J, Müller K (2008): MALDI-TOF "fingerprint" phospholipid mass spectra allow the differentiation between ruminantia and feloideae spermatozoa. Theriogenology 71, 568-575. http://dx.doi.org/10.1016/j.theriogenology.2008.08.023

Current research topics

Identification and reconstitution of detergent-resistent membrane domains in mammalian sperm cells and their function for fertilization (DFG MU 1520/2)
Analysis of sperm lipids and lysolipids – individual and species-dependent differences in relation to sperm quality, metabolism and reproductive system
Interaction of synthetic antimicrobial peptides with the sperm cell membrane (AIF cooperation project with FG 3, IFN Schönow e.V. and FMP Berlin)
Analysis of individual sperm lipid patterns within the project “Testicular function and lipid profile of epididymis sperm of normospermic and teratospermic domestic cats” (CAPES, Brazil)

Cooperation

Institut für Biologie/Biophysik der Humboldt-Universität zu Berlin
Institut für medizinische Physik und Biophysik der Universität Leipzig
Institut für Fortpflanzung landwirtschaftlicher Nutztiere Schönow e.V.
Forschungsinstitut für molekulare Pharmakologie im Forschungsverbund Berlin e.V.
Federal University of Paraná, Centro Politécnico, Sector of Biological Science, Department of Physiology, Curitiba, Brazil

2.3 Seminal fluid and the implementation of sperm function

Seminal fluid has crucial effects on the function of spermatozoa. In the female genital tract, it exerts immunsuppressive effects and acts stimulatory within the fertilization cascade. In dependence of the mammalian species, different lipid and protein components were found in the seminal fluid. We are interested to explore similar and specific functions of seminal fluid components and their species-specific contribution to the general principles of fertilization.

Figure: The removal of sperm lipids by the seminal fluid protein PDC-109, measured by MALDI-TOF mass spectrometry after exposition of epididymal bull sperm to protein

Results / Publications

Greube A, Müller K, Töpfer-Petersen E, Herrmann A, Müller P (2004): Interaction of Fn type II proteins with membranes: the stallion seminal plasma protein SP-1/2. Biochemistry 43, 464-472
Tannert A, Kurz A, Erlemann KR, Müller K, Herrmann A, Schiller J, Töpfer-Petersen E, Manjunath P, Müller P (2007): The bovine seminal plasma protein PDC-109 extracts phosphorylcholine containing lipids from the outer membrane leaflet. Europ. Biophys. J. 36, 461-476.
Tannert A, Töpfer-Petersen E, Herrmann A, Müller K, Müller P (2007): The lipid composition modulates the influence of the bovine seminal plasma protein PDC-109 on membrane stability. Biochemistry 46 (41), 11621 -11629. DOI: 10.1021/bi7011299

Cooperation

Institut für Fortpflanzung landwirtschaftlicher Nutztiere Schönow e.V.
Institut für Biologie/Biophysik der Humboldt-Universität zu Berlin

2.4 Improvement of (functional) assays to evaluate spermatogenesis and sperm quality with respect to individual fertilizing ability and development of customized methods for (low temperature) preservation of semen from different species

To assess the fertilizing ability of sperm samples, all aspects of the fertilization cascade have to be considered. Our aim is the refinement of a comprehensive set of functional assays (applicable to semen of many species) to provide objective parameters for sperm capabilities. In continuation to previous investigations, the quantitative and qualitative characterization of spermatogenesis will complete the semen analysis if testis tissue is available.
The maintenance of sperm function during sperm preservation is the prerequisite for a successful artificial insemination, for in vitro fertilization and, at least partly, for intracytoplasmatic sperm injection. Therefore, we search for those species-dependent sperm properties that cause the different sensibility towards cooling and freezing, and aim to develop customized methods for the semen preservation in different species.


Figure: Motility analysis of epididymal spermatozoa from domestic cat by the SpermVision system (Minitüb GmbH). Colored lines characterize the different categories of sperm movement pattern.		Figure: Active mitochodria of spermatozoa from domestic cat are stained with Rhodamine 123 and appear green, dead spermatozoa are stained by propidiumiodide and appear red (Imaging system, Olympus

Results/Publications

Reid CE, Hermes R, Blottner S, Goeritz F, Wibbelt G, Walzer C, Bryant BR, Portas TJ, Streich WJ, Hildebrandt TB (2008): Split-sample comparison of directional and liquid nitrogen vapour freezing method on post-thaw semen quality in white rhinoceroses (Ceratotherium simum simum and Ceratotherium simum cottoni). Theriogenology 71, 275-291.
Müller K, Müller P, Pincemy G, Kurz A, Labbe C (2008): Characterization of sperm plasma membrane properties after cholesterol modification. Consequences for cryopreservation of rainbow trout spermatozoa. Biol Reprod 78, 390-399

Current research topics

Establishment of sperm motility analysis (SpermVision, Minitüb GmbH, Tiefenbach) for different species and its application in investigating the effects of culture media as well as extender components on sperm motility
Improvement of low-temperature preservation of cold-sensitive semen - biochemical characterization, cell-physiological and biophysical effects of extender lipid components (Cooperation project with IFN Schönow e.V. and Minitüb GmbH, Tiefenbach)
Development of functional tests to evaluate the quality of honey bee semen and for its long-term storage (Cooperation with LIB Hohen Neuendorf e.V.)
Long term observation of sperm quality and spermatogenesis in a captive population of roe deer (Cooperation with FG 1 and FG 5, field station Niederfinow)

Cooperation

Institut für Fortpflanzung landwirtschaftlicher Nutztiere Schönow e.V.
Länderinstitut für Bienenkunde Hohen Neuendorf e.V.
Minitüb GmbH, Tiefenbach
INRA, Joint Research Unit for Fish Physiology, Biodiversity and the Environment, France