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MALE REPROD.UCTIVE TRACT/SPERM FUNCTIONMALE REPRODUCTIVE TRACT/SPERM FUNCTIONFINAL CONSTRUCTION OF SPERM ACROSOME MAY BE ANACTIN-MEDIATED PROCESSMiniie Lin, Chris Scarlett and R. John AitkenCooperative Research Centre for Conservation and Management ofMarsupialsSchool ofBiological and Chemical Sciences, The University ofNewcastle, NSW 2308, AustraliaIntroductionIt is now known that a number of most dynamicprotrusions at the somatic cell periphery are actinmicrofilament-containing structures, and theconstantly assembling and disassembling of themicrofilament (F-actin) within the cell protrusionsdetermine the structural reorganisation and functionsof the cell (1). Posttesticular change in the form ofthe acrosome as a concomitant of sperm passagethrough the .epididymis has been found in someeutherian mammals. The changes of the acrosomerange from a major reorganization expressed in theguinea pig and chinchilla to more modest changeseen in the rabbit, some primates (not human) andsome rodents (2). However, recently we confirmedthat some marsupial spermatozoa display the mostradical reorganization seen in any species so far (3).Therefore, the marsupial acrosome is an idealstructure to study the role of actin filaments on thefinal construction ofthe mammalian acrosome duringsperm maturation in the epididymis.Materials and MethodsSpermatozoa were freed from the testis and caput,corpus and cauda epididymis from 5 tammarwallabies for examinations. For EM, tissues werefixed in 2.5 % (v/v) glutaraldehyde and 2%paraformaldehYde in O.lm cacodylate buffer forovernight at 4°C, then treated with 1% osmiumtetroxide for 4 hr. After processing through thedehydration and critical point drying, the tissues werecoated with gold and examined in a JSM 840 SEM.For F- actin immunofluorescences, spermatozoa werefixed for 60 min in 4% formaldehyde in PBS at 4°C,and air dried on Poly-L-Iysine coated sliders;permeabilised with cold methanol and acetone (-20°C) for 10min each; stained with 50J.lg/ml Phalloidin­FITC conjugate solution in PBS for 60 min at RT;after 3 washes with PBS, F-actin fluorescence wasdetected under a Ziss Axiovert 10 fluorescentmicroscope.Results and DiscussionsIn the tammar wallaby, the acrosome is a sheet oftissue with elongated 'scoop' shaped protrusionswhen spermatozoa leave the testis. The acrosomalprotrusions condensed into a compact button-likeorganelle as the spermatozoa transit through theepididymis (Fig. 1, top row). The occurrence of actinfilaments within the acrosome was temporally andspatially associated to the process of the acrosomeshaping in the epididymis (Fig. 1, bottom row). Actinfilaments were assembled when acrosomalprotrusions started to fuse together in the caputepididymis, and disassembled after the acrosomecondensation was completed in the distal corpus andcauda epididymis. Future work will be directed atidentification and characterising sperm-specificproteins involved in the actin assembly anddisassembly during sperm epididymal maturation. Itis ~ticipated that the identification ofthose proteins,which may be exploited for the manipulation of malefertility, particularly in those mammalian species,such as in rabbits, mice and some marsupials, whoseacrosome requires a morphological maturation in theepididymis.Fig. 1. Fonnation ofthe wallaby acrosome (thetop row) and occurrences ofF-actin filamentsin the acrosome (the bottom row) as spermtransit from the testis to caput, corpus, andcauda epididymis (arranged from left to right inboth rows). A, acrosome., Reference:(1) Carraway et aI., (1998) In "Signalingand the Cytoskeleton", p 8-30, Springer.(2) Fawcett & Bedford (1979) In "TheSpermatozoon", P 11-19, Urban &Schwarzenberg.(3) Lin & Rodger (1999) 1. Anat. 194:223­232.DEVELOPMENT OF PSA-IO MONOCLONAL ANTIBODY REACTIVITY WITHBRUSHTAIL POSSUM (TRICHOSURUS VULPECULA) SPERMATOZOA DURINGEPIDIDYMAL MATURATIONM.S. Harris a and J.C. Rodger bCooperative Research Centre for the Conservation and Management ofMarsupialsaLandcare Research, Lincoln, NZ and bMacquarie University, NSW, AustraliaIntroductionThe PSA-10 monoclonal ant:J.body (mAb) binds to antigen(s) on the fibrous sheath and midpiece fibrenetwork (MFN) of mature tammar wallaby (Macropus eugenii) 'spermatozoa and the fibrous sheath.,midpiece fibre network and acrosome ofmature bmshtail possum (Trichosurus vulpecula) spermatozoa(1). This study examined the development ofPSA-l0 mAb immunoreactivity with the acrosome andmidpiece ofpossum spermatozoa during epididymal maturation.MethodsSpermatozoa were collected from the testes and proximal and distal regions ofthe head, body and tailof the epididymides of three possums after mincing the tissue in phosphate buffered saline (PBS)containing protease inlnbitors. Sperm were pooled by region, fixed in 4% paraformaldehyde andlabelled with PSA-I0 ascites fluid or myeloma ascites as described previously (1). The fluorescencepatterns on 100 randomly selected sperm were counted for each ofthree subsamples of sperm. pooledfrom different regions of the tract. Data was analyzed using a two-tailed Fisher exact test. Freshlydissected tissue from the possum and wallaby male reproductive tract was fixed in 4%paraformaldehyde for 5 h on ice before being processed for post-embedding immunogold labellingwith PSA-l0 ascites and myeloma ascites control (1).ResultsPSA-IO acrosomal immunor~activity was evident on most possum spennatozoa from the proximalhead ofthe epididymis but not those from the testis (Table 1). PSA-I0 immunoreactivity with restrictedregions of the midpiece was detected first on some possum sperm from the proximal·head of theepididymis (Table 1). The proportion of sperm showing midpiece immunoreactivity increased withepididymal transit, with the entire posterior midpiece ofall spermatozoa from the proximal body oftheepididymis being labelled. Immunogold labelling of the possum midpiece was associated first withgranular material surrounding the mitochondrial sheath, before the formation of the midpiece fibrenetwork. Labelling of the :MFN was evident after formation in the distal head of the epididymis.Immunogold labelling ofthe possum acrosome was associated with the outer acrosomaI membrane.Table 1. The percentage of fixed possum spermatozoa (n=3) from different regions of the malereproductive tract demonstrating acrosomal, midpiece and principal piece immunoreactivity with PSA­10 ascites fluid.Percentage ofpossum sperm demonstrating PSA-I0 mAbOrigin ofsperm inmale reproc Immunoreactivity with given structure (Mean % ±SE)Acrosome Midpiece Principal PieceTestis O±O O±O 100±0Proximal Head Epididymis 92.9±1.6* 11.9±2.0* 100±0Distal Head Epididymis 98.8±1.2* 96.0±2.1* 100±0ProximalBody Epididymis 100±0 100±0* 100±0* WIthin column, values are sIgmficantly different from preVIOUS (P