Contribution of pathogen and non pathogen bacteria in reactive oxygen species production by...

Poster presentations

Effect of genistein upon capacitationof cryopreserved bovine spermatozoa

V. A. Aires, E. Hinsch and K.-D. Hinsch

Centre of Dermatology and Andrology, Justus Liebig Univer-sity, Giessen, Germany

Introduction. Calcium regulation and proteintyrosine phosphorylation play an important roleduring sperm capacitation and acrosome reaction.Genistein, a protein tyrosine kinase (PTK) inhi-bitor, reduces tyrosine phosphorylation of spermproteins and inhibits progesterone- and zonapellucida-induced acrosome reaction in mammals.Previously, we demonstrated that pre-incubationof cryopreserved bovine spermatozoa with geni-stein inhibits progesterone- and ZP3-6 peptide-induced acrosome reaction. Our preliminary datashowed no major changes in protein tyrosinephosphorylation during capacitation. However,protein tyrosine phosphorylation was more intensein cryopreserved spermatozoa than in fresh sper-matozoa. This could be an indication for �pre-capacitation� due to cryopreservation. Because theassessment of changes of protein tyrosine phoph-orylation might not be the adequate indicator forcapacitation in cryopreserved spermatozoa, weapplied the chlortetracycline (CTC) fluorescenceassay.Materials and methods. Cryopreserved bovinespermatozoa were incubated for 4 h with dimethylsulphoxide (DMSO) (0.2 l1 ml)1) or genistein(2 lg ml)1). Two independent experiments yieldedan increased proportion of capacitated spermatozoa(B pattern) after 4-h incubation.Results. The percentage of capacitated sperma-tozoa at t0 was 8 ± 0.82% and 5.25 ± 0.85% forcontrol spermatozoa and genistein treated semen,respectively. After a 4-h capacitation period wefound 35.5 ± 6.44% capacitated spermatozoa inthe control sample and 33 ± 4.08% in the probepre-incubated with genistein.Conclusion. Our preliminary results suggest thatgenistein has no effect upon capacitation in cryo-preserved bovine spermatozoa.

Effect of anti-porin type 2 antibodies uponbovine sperm motility and acrosomal status

Asmarinah, K.-D. Hinsch, V. A. Airesand E. Hinsch


Introduction. The pore-forming protein porin(voltage dependent anion channel, VDAC) wasfound in bacterial and outer mitochondrial mem-branes as well as in the plasma membrane ofsomatic cells. Numerous porin functions werereported in somatic cells, e.g. regulation of ATP-fluxes in outer mitochondrial membranes or mito-chondrial apoptosis. Previously, we reported theoccurrence of porin type 2 in late stages of bovinespermatogenesis; transmission electron microscopyrevealed the presence of porin in the sperm tailand in the plasma membrane of sperm heads.In this preliminary study we determined the effectof anti-porin 2 antibodies upon bovine spermfunction.Material and methods. After incubating sper-matozoa with anti-porin antibodies we assessedsperm motility using the CASA system. Theacrosomal status of live spermatozoa was deter-mined applying the Hoechst 33285/PSA-FITCstain. The mean percentage of motile sperm andthe acrosomal status were determined 4 h aftertreatment with anti-porin 2 antibodies; rabbit IgGwas used as negative control.Results. Sperm motion analysis revealed that themean percentages of motile sperm of samplestreated with anti-porin type 2 antibodies (71.7%)did not show any statistically significant differencescompared with control spermatozoa (69.9%). Sam-ples of bovine semen that had been pre-incubatedwith anti-porin type 2 antibodies showed anelevated number of spermatozoa undergoing acro-somal exocytosis (17.1%) compared with spermato-zoa that had been treated with rabbit IgG (11%), orbuffer alone (9.9%). However, as a result of the highSD obtained in three experiments, the results werenot statistically significant.

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Conclusion. Our data indicate that porin mightplay a role in signal transduction events that occurduring acrosome reaction.

Principal cells in the cauda epididymidisresorb zinc eliminated from spermatozoa

C. Baldauf1, W. Miska1, W. Weidner2, W.-B. Schill1

and R. Henkel

1Centre of Dermatology and Andrology; 2Urological Clinic,Justus Liebig University, Giessen, Germany

Introduction. In mammalian spermatozoa, zincis mainly located in the outer dense fibres (ODF),which are important for the generation of motility.This element is bound to the SH-groups of cysteinewhich are present in ODF proteins to protect themfrom oxidation. It is subsequently eliminated formthe flagella during epididymal sperm transit. Theobjective of this study was to investigate theelimination of zinc during epididymal transit.Material and methods. To investigate zinc-binding substances present in the epididymis welabelled bovine epididymal fluid from caput, corpusand cauda with 65Zn and measured radioactivityafter gel filtration. In addition, chelate columnchromatography was performed. Moreover, histo-logical sections from caput, corpus and caudaepididymidis as well as from the vas deferens wereanalysed by means of an autometallographicmethod.Results. Chromatography experiments showed azinc binding protein with a molecular mass of 150–160 kDa, which was co-located with radioactivity.Enrichment of these proteins was found afterchelate column chromatography. By calculatingthe radioactivity according to the protein content,the zinc-binding ability of epididymal fluid from thecaput epididymidis revealed an approximately fivetimes higher activity than in the cauda. In thehistological sections, zinc was visualized in theadluminal compartment of the principal cells. Inthe cauda, this labelling appeared more basally.While no staining was observed in the caput, thestaining intensity increased dramatically from thecorpus to the cauda and vas deferens.Conclusion. In order to obtain functional ODF,elimination of zinc from the flagellum with subse-quent formation of disulphide bridges in the ODFproteins is mandatory. This stiffens the ODF andleads to a better use of the available energy. Weidentified a zinc-binding protein fraction in epi-didymal fluid. The fact that the zinc-bindingcapacity of the epididymal fluid is approximately

five times higher in the caput than in the caudasuggests that zinc is immediately mobilized from thespermatozoa when reaching the caput epididymidisfrom the testes. However, resorption from the fluidonly commences in the corpus, increases in thecauda and continues in the proximal part of the vasdeferens.Acknowledgement. This project was supportedby the German Research Foundation (DFG) (He2167/7-1).

Expression of CREM isoforms in humanand equine testis with normal and impairedspermatogenesis

S. Blocher1, R. Behr2, G. F. Weinbauer3,M. Bergmann1 and K. Steger1

1Institute of Veterinary Anatomy, Histology and Embryology,Justus Liebig University, Giessen; 2Institute of Anatomy andCell Biology, University of Essen; 3Covance LaboratoriesGmbH, Munster, Germany

Introduction. During spermatogenesis, histonesare replaced by transition proteins (TP) whichsubsequently are exchaged by protamines (Prm).The cAMP response element modulator (CREM)protein is a transcription factor that is involved inthe regulation of the expression of TP 1 as well asPrm 1 and 2. The CREM gene consists of 15 exons.Alternative splicing results in both activator andrepressor transcripts. CREM repressors were foundin spermatogonia, spermatocytes and elongatedspermatids. CREM activators were solely presentin round spermatids. The aim of this study was toinvestigate whether differences exist in the CREMgene expression between normal and impairedspermatogenesis.Material and methods. RT–PCR was appliedusing human and equine testis homogenates exhib-iting normal and impaired spermatogenesis.Results. Both in human and horse exhibitingnormal spermatogenesis one activator and up tofour repressors of CREM transcripts were identi-fied. By contrast, in testes with spermatogenic arrestat the level of round spermatids (man and horse)CREM activators were not expressed. In man theCREM repressor pattern was either unchanged(two testes homogenates) or only two of therepressor isoforms were amplified (five testes homo-genates). Only one repressor isoform could be seenin horse.Conclusion. (i) CREM activator and repressorexpression does not differ in man and horse.(ii) Spermatogenic arrest is associated with a

Poster presentations 3

ANDROLOGIA 35, 2–12 (2003)

down-regulation of CREM activators. This con-firms earlier studies dealing with TP 1, Prm 1 and2 gene expression and suggests that down-regula-tion of CREM activators is causally related tospermatogenic arrest at the level of roundspermatids.

Development-dependent expression oftissue-kallikrein and the bradykinin B2

receptor in rat testis

S. Blocher, W.-B. Schill and T. K. Monsees

Center of Dermatology and Andrology, Justus LiebigUniversity, Giessen, Germany

Introduction. In the rat testis, all components ofthe tissue kallikrein kinin system (tKKS) have beendetected. The inactive precursor, kininogen, andseveral kinin-degrading proteases, the kininases,were found some years ago. Recently, we detectedtissue kallikrein (tK) and the bradykinin B2

receptor (B2R) in the rat testis. Here we describethe stage- and development-dependent expressionof tK and the B2R. This investigation was per-formed to better understand the role of tKKS inspermatogenesis.Material and methods. Stage- and age-depend-ent expression to tK and B2R was determined byimmune histochemical staining of testis slices(polyclonal antibodies). Age-dependent expressionof B2R protein and mRNA was shown by immu-noblots (monoclonal antibody) and RT–PCR of rattestis homogenates.Results. Immunostaining for tK was found onround, elongating and elongated spermatids asso-ciated with the acrosomal cap. It started to occuron step 8 and lasted until step 18 of spermatids.tK first occurred in 28-day-old rat testis, when thefirst round spermatids appear. Specific immuno-staining for B2R was detected on pachytenespermatocytes from stages VIII to XIII and onelongating spermatids from stage IX to XII. Itsspecific staining on germ cells began in 18-day-oldrats, when the first pachytene spermatids occurred.Peritubular cells of 4- and 18-day-old rats werealso stained. Immunoblots showed three bandsthat are specific for the B2R protein. Two of thesebands, at approximately 42 and 45 kDa, weredetected in all homogenates. These bands repre-sent the unglycosylated/unphosphorylated and thepartially glycosylated B2R protein, respectively.Testis from late pubertal (38 days), post-pubertal(53 days) and adult rats (158 days) displayed athird band of approximately 70 kDa. This band

represents the activated dimer form of the 45 kDaB2R protein. The mRNA of B2R was detectedin testis homogenates from rats of all ages(4–158 days).Conclusion. All components of a local tKKS weredetected in the rats testis, thus a physiological roleseems to be likely. The development- and stage-dependent expression of tissue kallikrein and thebradykinin B2R in the testicular seminiferousepithelium suggests a local function of the tKKSin the regulation of rat spermatogenesis.Acknowledgements. Supported by GermanResearch Foundation (DFG Mo 693/3-1). Theauthors thank the group of Wemer Muller-Esterl(Frankfurt/Main) for a probe of the polyclonal B2Rantibody.

Morphofunctional damage of mammaliansperm incubated with organophosphoricagropesticides

E. Bustos-Obregon*, J. Caballero and C. Ortiz

Laboratory Biology of Reproduction (ICBM), Faculty ofMedicine, University of Chile, Santiago, Chile

Introduction. Organophosphoric (OP) agropesti-cides affect many organs in mammals but little isknown on their effects upon spermatozoa ofdomestic animals. In vitro observations are reportedafter 1-h incubation of bull frozen (Bu) or boar fresh(Bo) sperm in parathion (PT) or its metabolite,paraoxon (PO), upon vitality (Vit), acrosome reac-tion (AR), membrane permeability (HOS) andchromatin decondensation (CD), as summarizedin the following table (Table 1) of results (*P < 0.5;**P < 0.001).Conclusions. OP agropesticides altered spermparameters, PT being more active than PO. ARwas not noticeably affected. However, the spermplasma membrane was modified by higher doses ofPT, as shown by the HOS test in bull and boar.This may be explained by the lipophilic nature ofOP. CD was not modified in bull sperm, probablydue to the presence in this species of protamine Iand II in their nuclei, rich in cysteine and agrinine,which may account for greater resistance ofchromatin after treatment with decondensingagents. In summary, OP pesticides affect the spermfertilizing ability. The type of damage seems to bespecies-specific concerning chromatin packing andstability.

*Supported by the A. von Humboldt Foundation, Bonn, Germany.

4 Poster presentations

ANDROLOGIA 35, 2–12 (2003)

Frequent DAZ1/DAZ2 deletions in menwith severe oligozoospermia

S. Fernandes1,2, J. Goncalves3, K. Hullen1,J. Zeisler1, E. Rajpert De Meyts4, N. E. Skakkebaek4,

B. Habermann5,W. Krause5, A. Barros2 andP. H. Vogt1

1Institute of Human Genetics, University of Heidelberg,Germany; 2S Genetica, F.M. Porto, Portugal; 3INS Dr RicardoJorge, Lisboa, Portugal; 4Department of Growth and Repro-duction, Rigshospitalet, University of Copenhagen, Denmark;5Department of Andrology, University of Marburg, Germany

Introduction. One of the strongest candidates forthe azoospermia factor (AZF) is the (Deleted inAZoospermia, DAZ) gene family, exclusivelyexpressed in male germ cells and mapped to theAZFc region located in distal Yq11 (Vogt et al.,1996). AZFc deletions are the most common knowngenetic cause of male infertility as they were foundin men with idiopathic azoospermia or severeoligozoospermia with a frequency between 4 and20%. Analysis of the DAZ gene cluster by restric-tion mapping. Fibre-FISH and sequence analysis indifferent men identified a variable number of DAZgene copies in AZFc: three DAZ genes (Yen, 1998),four DAZ genes (Saxena et al., 2000), seven DAZgenes (Glaser et al., 1998), suggesting the presence ofDAZ pseudogenes. We therefore set out to identifythose DAZ gene copies which are essential forhuman male fertility.Material and methods. BAC and PAC cloneswere isolated from the DAZ locus and placed into acontig of the AZFc region. The structure of the DAZgene copy in each BAC/PAC clone was typed by aset of copy-specific single nucleotide variants (DAZ-SNVs), single tagged sites (STS) and copy-specificrestriction maps of the DYS1 Locus defining therepetitive exon structure of each DAZ gene.

Results and Conclusions. Thirteen gene copy-specific DAZ haplogroups were established andused for analysis fo deletions of specific DAZ genecopies in the AZFc locus of 63 patients withidiopathic severe oligozoospermia. In five of themwe found a deletion of the DAZ1 and DAZ2 genes,one of which was identified as a de novo deletionbecause it was absent in the patient’s father. EachDAZ gene deletion was confirmed by the deletionof the corresponding DYS1 genomic restrictionfragments (EcoRV and TaqI) specific for DAZ1 andDAZ2. The same DAZ deletions were not found inany of the 107 fertile control samples. It is thereforeconcluded that deletion of the DAZ1/DAZ2 genesin five of the 63 oligozoospermic patients isresponsible for the patients� reduced sperm num-bers (Fernandes et al., 2002).

References

Fernandes S, Huellen K, Goncalves J, Dukal H, Zeisler J,Rajpert De Meyts E, Skakkebaek NE, Habermann B, Kra-use W, Sousa M, Barros A, Vogt PH (2002) High frequencyof DAZ1/DAZ2 gene deletions in patients with severeoligozoospermia. Mol Hum Reprod 8:286–298.

Glaser B, Yen PH, Schempp W (1998) Fibre-fluorescence in situhybridization unravewls apparently seven DAZ genes or pseu-dogenes clustered within a Y-chromosome region frequentlydeletedinazoospermicmales.ChromosomeRes6:481–186.

Saxena R, de Vries JW, Repping S, Alagappan RK, SkaletskyH, Brown LG, Ma P, Chen E, Hoovers JM, Page DC (2000)Four DAZ genes in two clusters found in the AZFc region ofthe human Y chromosome. Genomics 67:256–267.

Vogt PH, Edelmann A, Kirsch S, Henegariu O, Hirchmann P,Kiesewetter F, Kohn FM, Schill WB, Farah S, Ramos C,Hartmann M, Hartschuh W, Meschede D, Behre HM,Castel A, Nieschlag E, Weidner W, Grone HJ, Jung A, EngelW, Haidl G (1996) Human Y chromosome azoospermiafactors (AZF) mapped to different subregions in Yq11. HumMol Genet 5:933–943.

Yen PH, Chai NN, Salido EC (1997) The human DAZ genes,a putative male infertility factor on the Y chromosome, are

Table 1. Seminal parameters under organophosphoric treatment

% Vit. Bo Vit. Bu AR Bo AR Bu HOS Bo HOS Bu CD Bo CD Bu

Control 68.75 38.04 10.98 11.40 41.56 39.05 14.01 60.78

0.05 PT 68.65 33.37 12.39 10.40 38.32 34.24 18.48 59.42

0.1 PT 68.54 35.39 10.68 11.91 35.64 33.11 22.44 52.28

0.2 PT 66.36 31.39 12.48 12.60 33.07 28.82* 24.46** 61.71

0.4 PT 63.92 25.16* 15.12 13.94 27.42** 25.34** 30.04** 65.64

0.8 PT 56.56* 17.81** 20.85** 12.19 21.82** 23.39** 34.27** 58.92

Control 69.58 41.75 10.26 10.48 43.83 35.19 15.2 65.35

0.05 PO 70.51 39.06 10.81 8.35 38.36 33.38 18.52 57.81

0.1 PO 70.51 38.16 10.02 8.78 38.87 33.14 20 64.28

0.2 PO 68.3 37.3 10.94 8.37 35.49 31.38* 24.13 64.1

0.4 PO 68.15 38.36 12.42 9.46 32.74** 30.36* 21.96* 63.42

0.8 PO 58.2 36.24 22.48* 8.75 26.92** 27.2** 32.88** 73.82


ANDROLOGIA 35, 2–12 (2003)

highly polymorphic in the DAZ repeat regions. MammGenome 8:756–759.

PHGPx is the mitochondrial capsuleselenoprotein of mammalian sperm

L. Flohe1, C. Foresta2, A. Garolla2, A. Roveri3,F. Ursini3, J. Wissing1 and M. Maiorino3

1Department of Biochemistry, Technical University of Bra-unschweig, Germany; 2Department of Medical and SurgicalSciences; 3Department of Biological Chemistry, University ofPadova, Italy

Introduction. Selenium is highly concentrated inthe mitochondrial capsule of spermatozoa. Thetrace element is thought to be contained in astructural protein �MCS�, thereby assuring struc-tural and functional sperm integrity. Reinvestiga-tion of the chemical nature of MCS revealed thatthe mitochondrial capsule consisted of at least 50%of enzymatically inactive, oxidatively cross-linkedphospholipid hydroperoxide glutathione peroxidase(PHGPx) (Ursini et al., 1999), a selenoprotein that isabundantly expressed as an active peroxidase inround spermatids (Maiorino et al., 1998). PHGPxactivity can be rescued from the capsule material byreductive treatment.Methods. The clinical relevance of sperm PHGPxwas investigated in a pilot trial correlating rescuedPHGPx activity (Roveri et al., 2002) with fertility-related parameters in infertile subjects (n ¼ 75) andhealthy volunteers (n ¼ 37). Genomic PHGPxDNA was amplified by PCR from white bloodcells of selected subjects and sequenced.Results. Also in human sperm, the inactive formaccounted for more than 95% of total PHGPx. TheSperm PHGPx content was inversely correlatedwith sperm count, morphological alterations andmotility, but less markedly with vitality. PHGPxgene polymorphism was frequent in both controlsand infertile subjects.Conclusions. Low PHGPx content of sperm isassociated with impaired male fertility. The reasonsfor decreased PHGPx content in infertile subjectsremain elusive.

References

Maiorino M, Wissing JB, Brigelius-Flohe R, Calabrese F,Roveri A, Steinert P, Ursini F, Flohe L (1998) Testosteronemediates expression of the selenoprotein PHGPx by induc-tion of spermatogenesis and not by direct transcriptionalgene activation. FASEB J 12:1359–1370.

Roveri A, Flohe L, Maiorino M, Ursini F (2002) Phospholipid-hydroperoxide gluthathione peroxidase in sperm. MethodsEnzymol 347:208–212.

Ursini F, Heim S, Kiess M, Maiorino M, Roveri A, Wissing J,Flohe L (1999) Dual function of the selenoprotein PHGPxduring sperm maturation. Science 285:1393–1396.

Alteration of immune cell parametersduring the development of experimentalautoimmune orchitis in rats

S. Graenz1, A. Lewen1, M. P. Hedger2, J. Seitz3,G. Aumuller3 and A. Meinhardt1

1Department of Anatomy and Cell Biology, University ofGiessen, Germany; 2Monash Institute of Reproduction andDevelopment, Clayton, Australia; 3Department of Anatomyand Cell Biology, University of Marburg, Germany

Introduction. Experimental autoimmune orchitis(EAO) has been studied as an animal model forhuman immunological male infertility. In ourmodels, EAO was induced by injection of syngeneictesticular homogenate in adjuvant in two differentrat strains (Wistar/Kyoto and Lewis) to investigatethe role of the genetic pre deposition on theoutcome of the disease.Material and methods. Groups of animals werekilled 25, 35, 50 and 80 days after immunizationand serum and testes were collected. H–E stainingswere made to determine the degree of testiculardamage and cell infiltration. Frozen sections werestained immunohistochemically with the mouseanti-rat monoclonal antibodies ED1 (lysosomalantigen specific to monocytes, dendritic cells andsome macrophages), ED2 (resident macrophages),OX8 (CD8+ cells), R73 (abT cell receptor), CD25(IL-2 receptor) and OX62 (dendritic cells). Immu-nostained cells containing a nuclear profile werequantified using stereological methods. In addition,antibodies directed against Ki-67 (proliferationmarker), Ox-6 (MHCII+ cells) as well as Ox-33and IgG/IgM (B-cells) were applied.Results and discussion. A strong increase ofED1- and ED2-positive cells was found in theinflamed testes. In addition, for the first time adendritic cell (Ox62) population could be detectedin the rat testis, which increased substantially(430%) in EAO animals. Furthermore, we observedan accumulation of CD4, CD8 and TCR positivecells over the experimental time. Most likely, theincrease of cells over time and during EAO is basedon migration rather than proliferation as generallyimmune cells did not show any staining with theproliferation marker Ki-67.


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Serum antibodies against testicular antigens weredetermined using Western blot analysis. Both ratstrains displayed a different reaction pattern. Wistarrats generally showed a substantially higherresponder rate than Lewis rats (87% versus 13%80 days after immunization), although the latter area common model for other autoimmune diseases.Reactions of autoantibodies were particularlystrong during the early experimental phase inWistar rats, whereas the reaction was less pro-nounced in the later phase when testicular damagewas most prominent. In contrast, the rate ofhistologically proven EAO responders was constantin Lewis rats although the development of auto-antibodies increased steadily with experimentaltime. Auto-antigens were determined using West-ern blot analysis and 2D-SDS-PAGE. Proteins at amolecular weight of 84, 55 and 40 kDa were themajor detected testicular auto-antigens.

The differential expression of IL-7, IL-1 recep-tor1 and IL-8 receptor b as determined by cDNAarray analysis suggests a role for these factors duringthe pathogenesis of EAO.

Relationship between in vitro chromatindecondensation and fertilization ratein an IVF programme

M. E. Hammadeh, K. Golzer, P. Rosenbaumand W. Schmidt

Department of Obstetrics and Gynecology, University ofSaarland, Homburg/Saar, Germany

Introduction. Chromatin condensation anddecondensation are prerequisite for fertilization.The process fo chromatin decondensation occurswhen sperm has entered the oocyte cytoplasm.Failure of sperm decondensation in the oocytes mayresult from a subtle sperm abnormality that isunrecognizable by conventional analysis, such asstructural or biochemical defect associated withchromatin packaging or organization during sper-matogenesis. The aims of this study were (i) to findout the relationship between in vitro nuclear chro-matin decondensation (NCD) after semen incuba-tion with detergent and polyanion (SDS/heparin)and the fertilization rate after in vitro fertilization(IVF) and (ii) to determine whether this test couldbe used to predict the fertilization potential ofspermatozoa in an IVF programme.Material and methods. Thirty-five couplesundergoing IVF therapy were included in this pro-spectively designed study. Each semen sample wasdivided into two aliquots after semen liquefication

and assessment according to guidelines of WHO(1999). The first aliquot was processed by PurSpermgradient centrifugation and the spermatozoaobtained were used for oocyte insemination. Thesecond aliquot was washed with Borat solution(50 mm) by centrifugation at 2500 g for 10 min,followed by incubation with SDS (1%) and heparin(0.15 mm) solution for 2 h. Smears were made at thefollowing intervals: Immediately, 30, 60 and120 min. Two hundred spermatozoa were eval-uated for NCD at each time period (immediately,30, 60 and 120 min) after staining the slide withAcridine orange.Results. The mean percentage of in vitro nucleardecondensation of spermatozoa increased signifi-cantly after incubation for 10, 60 and 120 min withSDS/heparin from 7.6 ± 3.5% in the native semensamples to 32.9 ± 17.0, 37.8 ± 20.2 and 50.8 ±24.1%, respectively. On the other hand, 287 oocyteswere retrieved from the female partners and 158oocyte were fertilized (55.6 ± 32.5%) fertilizationrate. However, no correlation was found betweenNCD and fertilization rate in IVF at any timeinvestigated.Conclusion. In vitro nuclear chromatin deconden-sation assay using SDS/heparin could not predictthe fertilization potential of spermatozoa in an IVFprogramme. Further research using other substancethat mimic the cytoplasmic factor in the oocyte iscurrently performed in our laboratory.

Reference

World Health Organization (1999) WHO Laboratory Manualfor the Examination fo Human Semen and Sperm-CervicalMucus Interaction, 4th edn. Cambridge University Press,Cambridge.

Role of follicle-stimulating hormonein the control of foetal Sertoli celltransferrin expression

S. Migrenne1, C. Racine1, A. Dierich2

and R. Habert1

1EA 3513 and INSERM-CEA, Universite Paris, France;2IGBMC-INSERM U.184, Illkirch, France

Introduction. The onset of the Sertoli cellpopulation is well characterized in rodents, but itsregulation is poorly understood. Follicle-stimulatinghormone (FSH) is an important mitogenic factor(Orth, 1984) and its role in foetal Sertoli celldifferentiation remains to be investigated. In thepresent study, we evidenced that transferrin can be


ANDROLOGIA 35, 2–12 (2003)

used as a foetal Sertoli cell differentiation marker.This marker was then used to analyse Sertoli celldevelopment in both decapitated rat foetuses andFSH receptor-deficient neonatal mice.Material and methods. Transferrin mRNA waschecked by reverse transcription–polymerase chainreaction in testes and different tissues. Whensemiquantification was desired, transferrin wasco-amplified with b- actin and analysed by NIHImage 1.60. To evaluate the acute effect of FSH ontransferrin expression, foetal rat testes were incu-bated for 3 h in DMEM/F12 (1/1) with or withoutrecombinant FSH (200 mU ml)1) (Migrenne et al.,2001). Rat foetuses were decapitated at 16.5 daypost conception (dpc) (Migrenne et al., 2001), i.e.before the onset of gonadotrophin secretion, andtheir testes were removed on 21.5 dpc, the last dayof gestation. Testes from FSHRKO mice (Dierichet al., 1998) were removed at birth.Results. Transferrin mRNA was detected in ratfoetal testes as early as 14.5 dpc and in all tissuesstudied, except in the tongue. Acute in vitrostimulation by FSH increased transferrin mRNAlevels 1.6- and 1.9-fold, respectively, in 16.5 and21.5 dpc testes.

Foetal decapitation in the rat and FSH receptordeficiency in the mouse decreased the number ofSertoli cells by 22 and 30%, respectively, comparedwith control litter mates. Surprisingly, the level oftransferrin mRNA was significantly increased in ratdecapitated foetuses. It did not significantly differbetween deficient mice and their normal littermates. Considering the decreased Sertoli cellnumber, these results suggest that the lack of FSHchronic effect in both rat and mouse increases thelevel of transferrin mRNA in each Sertoli cell.Conclusions. These data provide first evidencethat transferrin mRNA is detected in rat foetal testisas early as 14.5 dpc and that transferrin can be usedas a marker of foetal Sertoli cells differentiationsince its mRNA level is acutely stimulated by FSH.

Interestingly, we also report here that FSHphysiologically decreases the transferrin mRNAlevel. This effect is opposite to that observed inpre-pubertal testes (Suire et al., 1995).

References

Diedrich A, Sairam MR, Monaco L, Fimia GM, GansmullerA, LeMeur M, Sassone-Corsi P (1998) Imparing follicle-sti-mulating hormone (SFH) signaling in vivo: targeted disrup-tion of the FSH receptor leads to aberrant gametogenesisand hormonal imbalance. Proc Nat Acad Sci USA95:13612–13617.

Migrenne S, Pairault C, Racine C, Livera G, Geloso A, HabertR (2001) Luteinizing hormone-dependent activity and

luteinizing hormone-indepdendent differentiation of rat fetalLeydig cells. Mol Cell Endocrinol 172:193–202.

Orth JM (1984) The role of follicle-stimulating hormone incontrolling Sertoli cell proliferation in testes of fetal rats.Endocrinology 115:1248–1255.

Suire S, Fontaine I, Guillou F (1995) Follicle stimulating hor-mone (FSH) stimulates transferrin gene transcription in ratSertoli cells: cis and trans-acting elements involved in FSHaction via cyclic adenosine 3¢5¢-monophosphate on thetransferrin gene. Mol Endocrinol 9:765–766.

Subcellular characterization of ejaculatedspermatozoa with disturbed asymmetryof the plasma membrane

U. Paasch, S. Grunewald, T. Jopeand H.-J. Glander

EAA Center, University Clinics, Leipzig, Germany

Introduction. Disturbed asymmetry of the plasmamembrane can be detected by binding of annexin Vto externalized phosphatidyl serine (PS). Spermato-zoa showing this phenomenon are expected to havereduced cell vitality (apoptosis?) and fertilizationcapacity. Therefore, it was our objective to investi-gate the presence and the extent of related phe-nomena of the apoptotic machinery.Material and methods. Seventy-four freshsperm samples from healthy donors were investi-gated in two sperm fractions depending on theannexin-microbead binding (ANMB) during pas-sage through a magnetic field (MiniMACS�) independence of Fas (CD95) domains, detection ofultrastructural changes by electron microscopy,FACS analyses of active caspases 8, 9, 1 and 3(aCP, CaspaTagTM; Western blotting, WB), and bymonitoring the integrity of the mitochondrialtransmembrane potential (MTP) by lipophilic cat-ions (5,5¢,6,6¢-tetrachloro-1,1¢,3,3¢-tetraethylbenzi-mid-azolyl carbocyanine chloride).Results. The subpopulations of sperm emerged independence of their membrane intergrity byMACS are characterized as follows [ANMB+

versus ANMB), X ± SEM, (%)]: Fas 48.9 ± 19.3versus 0.1 ± 0.3 (P < 0.01); aCP8 65.3 ± 6.9 ver-sus 10.3 ± 2.2 (P < 0.01); aCP9 68.3 ± 5.6 versus7.4 ± 3.3 (P < 0.01); aCP1 48.1 ± 5.8 versus 9.3 ±6.1 (P < 0.01); aCP3 50.8 ± 9.9 versus 13.2 ± 6.7(P < 0.01); MTP 90.6 ± 8.2 versus 6.8 ± 4.5(P < 0.01). Protein analysis by WB confirmed acti-vation of CP within the ANMB+ fraction. Electronmicroscopy revealed binding of the microbeadswithin the fraction of ANMB+ sperm only. ANMBwere bound at the acrosomal and post-equatorialregion, respectively.


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Conclusions Ultrastructural analysis revealedbinding of ANMB at both the acrosomal and thepost-equatorial regions of spermatozoa. Ligation ofthese microbeads results in retention within themagnetic field (ANMB+) and characterizes thosespermatozoa to have significantly increasedamounts of detectable death receptor domainsFas, activation of CP 8, 9, 1 and 3 and dissipationof MTP compared with sperm free of ANMB. Inconclusion, cells having their membranes disturbedare distinguished by activation of known elementsof receptor-mediated (Fas and CP8) as well asmitochondrial (CP9 and CP1) triggered pathways,the so called programmed cell death. Finally,terminal events of this signalling cascade as activa-tion of CP3 and dissipation of MTP were observed.

Protein kinase control of regulatoryvolume response in boar spermatozoa

A. M. Petrunkina and E. Topfer-Petersen

Institute for Reproductive Medicine, Veterinary School ofHannover, Hannover, Germany

Introduction. Activation of mechanisms regula-ting ionic concentrations and cell volume mayrequire changes in protein kinase activity. Intactsperm cells swell in response to hypo-osmoticconditions. Following that, a cell volume reductionoccurs (regulatory volume decrease, RVD). Inboar, bull and human sperm this step is affectedprimarily by quinine-sensitive ion channel. Themolecular mechanisms behind this phenomenonare yet poorly understood. That RVD is animportant physiological function was shown bystudies of sperm from c-ros tyrosine kinasereceptor knockout mice. Latter infertile spermshowed characteristics commensurate with a vol-ume regulatory lesion, observed even within thecauda epididymides. The aim of this study was totrace signalling pathways involved in boar spermvolume response.Materials and methods. Sperm samples weremeasured under iso-osmotic (300 mosmol kg)1)and hypo-osmotic (180 mosmol kg)1) conditions inHEPES buffered saline solutions at 39 �C using CellCounter CASY1 (Scharfe System GmbH, Reutlin-gen, Germany). To check the involvement of PTKin the regulative processes, general and specificprotein kinase inhibitors were added to testingsolutions: staurosporine (20 lm), H-89 (20 lm, spe-cific PKA inhibitor), lavendustin (20 lm, tyrosinekinase inhibitor), GF-109203X (50 lm, specificPKC inhibitor) and genistein (100 lm). Further,

forskolin (50 lm) was used, which is known to havean inhibiting effect on Cl)-channels.Results. Under control conditions cell volume wasmaintained, and/or the down regulation of the cellvolume took place after 20-min exposure to hypo-osmotic conditions. Sperm treated with forskolinfailed to show RVD, pointing out that Cl)-channelsare involved in volume regulation. Addition ofstaurosporine, lavendustin A and GF-109203X tothe incubated sperm cell suspensions resulted in anup to 4.3-fold increase of the relative volumeresponse (after 20 min under hypo-osmotic condi-tions). Genistein had no effect; the addition of H-89resulted in increased iso-osmotic volume, butaccelerated rather than delayed RVD.Discussion and conclusions. From the resultsobserved it can be concluded that RVD in boarsperm is triggered by at least one PTK-dependentpathway. A tyrosine phosphorylation step seems tooccur when volume regulation is required. Itappears that PKC rather than PKA is involved inthe signalling cascade leading to regulation underhypo-osmotic conditions. PKA activity seems to beimportant for iso-osmotic regulation; its inactiva-tion, contrarily, may improve the regulatoryresponse under hypo-osmotic conditions. Missingeffect of the more general TKI genistein appears tobe conform with the fact that phosphorylation ofboar sperm proteins was not blocked by thisinhibitor. Genistein could not thereby affect trig-gering RVD. Identification and functional charac-terization of the still unknown phosphorylatedproteins involved in the signal transduction in boarsperm (at different maturational stages) are underfurther investigation.

Contribution of pathogen and nonpathogen bacteria in reactive oxygenspecies production by polymorphonucleargranulocytes: effect on sperm motility

R. Sanchez1, L. Soto1, J. Villegas1, C. Boehme1,T. Iglesias1 and W. Miska2

1Department of Preclinical Sciences, Department of InternalMedicine, Center of Reproductive Biotechnology, Faculty ofMedicine, Universidad de la Frontera, Temuco, Chile; 2Centreof Dermatology and Andrology, Justus Liebig University,Giessen, Germany

Introduction. Leucocytospermia has been associ-ated with reduced sperm motility and decreasedsperm–oocyte fusion capacity. This effect is mainlymediated by reactive oxygen species (ROS), whichinduce cellular injury, including DNA damage,


ANDROLOGIA 35, 2–12 (2003)

lipid peroxidation and cellular death at highconcentrations. The present study evaluated theeffects on ROS production and sperm motilityduring incubation of polymorphonuclear (PMN)granulocytes with pathogen or nonpathogen bac-teria.Materials and methods. Human spermatozoaselected by Percoll gradient (10 · 106 ml)1) wereincubated with 2.5 · 106 PMN isolated from blood.Either 100 lmol phorbol-12-myristate-13-acetate(PMA), 30 · 106 Escherichia coli, 30 · 106 S. aureus,or 30 · 106 S. epidermidis was added to the incuba-tion mixture. ROS level and sperm motility weredetermined at time 0 and after 60-min incubation.Results. In PMN granulocytes the basal ROSproduction showed no variation after 60 min. (41.6versus 30.6) The rate of ROS production wasslightly increased in samples with PMN granulo-cytes and sperm (86.7 versus 114.6). In both groupssperm motility remained unchanged. The rate ofROS after stimulation with PMA was significantlyhigher (4380) and inversely correlated with spermprogressive motility (decrease 15%). Similar resultswere obtained in samples with E. coli (0 min: 77.7;60 min: 3102); S. aureus (0 min: 86.7; 60 min: 1207)and S. epidermidis (0 min: 86.7; 60 min: 1185).Progressive motility decreased by 6, 10 and 15%respectively.Conclusions. Escherichia coli, S. aureus andS. epidermidis activate the oxidative metabolism ofleucocytes, as evidenced by the production of highROS concentrations. Similarly to PMA, bothpathogen and nonpathogen bacteria induce highproduction of ROS in PMN and, consequently, adecrease in sperm motility. Exact definition of theeffect of ROS on spermatozoa during bacterialinfection will contribute to the knowledge of factorsinvolved in impaired male fertility.Acknowledgement. Supported by Fondecyt:Project No 1010 729–2001 and No 7010 729.

Detection of lymphocyte endothelial–epithelial cell adhesion molecule(LEEP-CAM) in human testis

H.-C. Schuppe1, G. Wienrich2, K. Pauls3,M. Schon2, M. B. Brenner4 and M. P. Schon2,3

1Centre of Dermatology and Andrology, Justus Liebig Uni-versity, Giessen, Germany; 2Department of Dermatology,Heinrich Heine University, Dusseldorf, Germany; 3Depart-ment of Dermatology, Otto von Guericke University, Magde-burg, Germany; 4Division of Rheumatology, Immunology andAllergy, Brigham and Women’s Hospital and Harvard MedicalSchool, Boston, MA, USA

Introduction. Studies in experimental animalsindicate that the testis is an immunologicallyprivileged site where germ cell antigens are protec-ted from autoimmune attack and foreign tissuegrafts may survive for extended periods. Thetesticular environment, however, does not precludeinflammatory reactions caused by infections, toxins,trauma, or tumours. With regard to the regulationof testicular immune functions in health anddisease, trafficking and specific localization of Tlymphocytes have to be considered as crucialcomponents. Here, we demonstrate LEEP-CAM,a recently identified receptor mediating lymphocyteadhesion to both endothelial cells and selectedepithelia, for the first time in human testis.Materials and methods. Testicular biopsiesfrom infertile men included normal tissue, variousdegrees of hypospermatogenesis or spermatogenicarrest, inflammatory reactions, Sertoli cell-only(SCO) syndrome, carcinoma in situ (CIS) andimmature testis. The material was fixed in Bouin’ssolution and embedded in paraffin. Immunohisto-chemical detection of LEEP-CAM was performedwith monoclonal antibody (mAb) 6F10 (mouseIgMj) by means of the avidin–biotin–peroxidasecomplex method. A nonbinding mouse IgMj andhuman skin were used for control purposes. In amodified Stamper–Woodruff assay, binding ofactivated lymphocytes (PHA-blasts) to deparaffi-nized sections of normal testicular tissue and itsinhibition by mAb 6F10 were assessed.Results. In all specimens examined, expression ofLEEP-CAM was observed on endothelial cells oftesticular blood vessels, including those within thelamina propria of tubuli seminiferi. In contrast,Leydig cells and other interstitial components didnot reveal any immunoreactivity. Sections ofnormal testis showed strong expression of LEEP-CAM within seminiferous tubules. Staining wasmainly confined to the adluminal compartmentwith some extensions to the basement membrane,whereas single immature germ cells and spermato-zoa in the tubular lumen were negative. Moreover,LEEP-CAM was not detectable in tubules withcomplete loss or absence of the adluminal com-partment, as well as SCO syndrome, immatureseminiferous tubules, or focal hypoplastic cords inadult testis. In some SCO specimens, very fewSertoli cells showed residual LEEP-CAM expres-sion. Disorganized CIS tubules revealed variablestaining. Binding of PHA-blasts to testicular tissue,i.e. normal seminiferous epithelium, was reduced by61% after pre-incubation with mAb 6F10 ascompared with control sections in the Stamper–Woodruff assay (P < 0.02).


ANDROLOGIA 35, 2–12 (2003)

Discussion. Our observations indicate thatLEEP-CAM is constitutively expressed on vascularendothelium in human testis. The immunohisto-chemical staining pattern in seminiferous tubulessuggests that LEEP-CAM is confined to Sertoli cellswithin normal adult germinal epithelium. Aslymphocytes do not migrate into this compartmentunder healthy conditions, LEEP-CAM may be partof the complex array of adhesive interactionsbetween Sertoli cells and germ cells. On the otherhand, LEEP-CAM could play an important role forperitubular and intratubular localization of activa-ted T cells during induction of testicular inflamma-tion.

Localization of a new polypeptidein mammalian outer dense fibres

B. Seefeldt-Schmidt, R. Henkel, W.-B. Schilland W. Miska


Outer dense fibres (ODF), one of the mostprominent cytoskeletal structures in the flagellumof mammalian spermatozoa, are composed of amedulla surrounded by a cortical layer. Damage tothis organelle leads to male infertility. In previousexperiments we demonstrated that bovine ODF arepredominantly composed of four proteins rangingfrom 11 to 85 kDa, whereas a 27 kDa-proteincontributes to the composition of the medulla. Inthe present study, we investigated ODF polypep-tides with molecular masses lower than 27 kDa.Materials and methods. ODF from bovinespermatozoa were isolated by SDS/DTT treatmentand analysed by SDS-polyacrylamide gel electro-phoresis. In the low-molecular mass range, twoprotein bands at 14 and 11 kDa were found. Bothpolypeptides were analysed for amino acid contentand were partially sequenced. Polyclonal antibodieswere raised against the first 20 N-terminal aminoacids of the 11 kDa ODF-protein, purified, char-acterized and used for localization of the corres-ponding polypeptide at the ultrastructural level.Results. Immunocytochemistry at electron micro-scopial level revealed specific labelling of surfacestructures of the ODF by the peptide-antibody,indicating that the 11 kDa polypeptide contributesto the formation of a cortex. The striated columnsin the connecting piece of the sperm tail were alsospecifically labelled.

Our results demonstrates for the first time that a11 kDa polypeptide contributes to the corticalstructure of bovine ODF.

Mitochondrial glycerol-3-phosphatedehydrogenase: its testis expressionand regulation

N. Shiryaeva, J. M. Weitzel and H. J. Seitz

Department of Biochemical Endocrinology, IMBM, Universityof Hamburg, Germany

Introduction. The FAD-dependent mitocondrialglycerol-3-phosphate dehydrogenase (mGPDH) isessential to the transport of the reducing equivalentsderived from glycolysis (or fructolysis, in the case ofsperm) to the mitochondrial compartment for thesynthesis of ATP. It has been shown that multiple(three) promoters of the mGPDH exist for theexpression to be regulated in a tissue-restrictedmanner. Promoter A is used in the brain, brownadipose tissue and pancreas, promotor B is ubiquit-ous promoter C proved to be testis-specific.

To evaluate if potential defects in the testis-specific mGPDH promotor C could contribute toreduce metabolic activity in the sperm followed byinfertility, we investigated a pattern of gene expres-sion and regulation in the rat testis.Materials and methods. In the present study,nonradioactive in situ mRNA hybridization andimmunohistochemistry were performed to deter-mine the cellular location of the mGPDH mRNAand protein in the testis. The tissue specificity of thepromoter activity was studied using transienttransfections. Band shift and supershift assays wereused to determine the transcription factors that areinvolved in the testis-specific mGPDH expression.Results. The mGPDH mRNA and proteinexpression within the testis is distinctly germ cell-specific and stage-specific. A testis-restricted tran-scriptional activator CREM-tau (cAMP-responseelement modulator) was found to bind specificallyto the mGPDH promoter. The tissue-restrictedusage of the promoter was confirmed by series oftransient trasfections in HepG2 (hepatoma cells),primary hepatocytes and TM3 (Leydig) cell lines.Conclusions. The mGPDH gene proved to beexpressed within the male germ cells in a delayedtranslation manner that is typical for a number oftestis-restricted genes. The transcription start ishighly probably initiated by the unique CREM-taufactor.


ANDROLOGIA 35, 2–12 (2003)

Seasonal expression of transforminggrowth factor-b3 (TGFb3) in roe deer(Capreolus capreolus) testis

A. Wagener, S. Blottner and J. Fickel

Institute for Zoo and Wildlife Research, Berlin, Germany

Introduction. Roe deer is a seasonal breedercharacterized by involution and recrudescene oftesticular tissue during the annual cycle. Themechanisms of these histomorphological changesas well as the seasonal onset and offset of sperma-togenesis are assumed to be triggered by growthfactors which are key molecules for the regulation ofcell proliferation and differentiation. It has beensuggested that TGFb3 might be involved in theonset of spermatogenesis and/or in the interactionsbetween pre-meiotic and post-meiotic germ cells.Therefore, the testicular expression of TGFb3 wasinvestigated.Materials and methods. Fresh samples of roedeer tests taken bimonthly (n ¼ 2–4 per month) by

castration were immediately snap frozen in liquidnitrogen. After isolation of total RNA and itsreverse transcription, TGFb3 gene expression levelswere analysed using quantitative PCR. Expressionwas quantified with a plasmid-cloned TGFb3fragment. Nonparametric anova was used tocalculate the variation among monthly means.Furthermore, TGFb3 mRNA was localized byin situ hybridization.Results. TGFb3 shows a specific seasonal expres-sion pattern with an almost five times higher levelbefore (June) and during the rut in Augustcompared with levels during the nonbreedingseason (December and February). The geneexpression differences among the months weresignificant (P < 0.05). The high expression level ofTGFb3 in summer is most likely because of theincreasing numbers of spermatocytes at this time, inwhich TGFb3 mRNA was mainly localized.Conclusions. These results indicate that in roedeer testis TGFb3 involved in the development ofspecific spermatogenetic cell types rather than inthe onset of spermatogenesis.


ANDROLOGIA 35, 2–12 (2003)

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