N OCIETY' - the Society for Reproductive Biology

In vitro culture of brushtailed possum epididymal epithelium and induction of epididymal spermmaturation in co-cultureMinjie Lin, Xiyi Zhang, Ray Murdoch and R. John AitkenMarsupial CRC, Dept of Biological Sciences, The University of Newcastle, NSW 2308, AustraliaIntroductionIn mammals, testicular sperm undergo a maturation processin the epididymis to acquire motility and the ability forfertilisation. However, sperm maturation in marsupials is amuch longer and more complex process than that of theireutherian counterparts (1). This process is poorlyunderstood, mainly because it cannot as yet be investigatedin vivo. Techniques of in vitro co-culture of sperm andepididymal cells have been developed in eutherians duringthe past 2 decades, but not in marsupials species (2). Thispaper describes for the fITst time an in vitro cell culturesystem for a marsupial species, which supports epididymalepithelial cells from the brushtailed possum, and evidence ofsperm maturation induced by the cultured epididymal cells.Materials & MethodsThe epididymis from 5 possums were used to establish thecell culture system with a medium modified from eutheriancultures (3,4), and maintained at 36.5-37°C in 5% C02 in airwith humidity at 100%. Immunofluorescence ofcytokeratins was used as a marker for identification ofepithelial cells. Morphology of cultured cells was examinedby light and electron microscopy. Immature sperm obtainedfrom the proximal caput epididymis were co-cultured with7-day old cultured epithelial cells for the in vitro maturationstudy. Development of sperm motility and changes of spermhead orientation were examined as indicators of spermmaturation in the co-culture preparations.Results and DiscussionAll cell cultures of caput, corpus and cauda epididymisformed confluent epithelial monolayers with densely packedpolygonal cells. The cells remained viable for more than 2months in culture, and displayed typical epithelial characterspeculiar to each segment of the epididymis. EMobservations confIrmed that after 12 days in culture, thecells retained many structural features characteristic ofepithelial cells in the intact epididymis. These featuresincluded very slender and branched stereocilia andmicrovilli located on the apical surface of the cells, welldevelopedrough endocytoplasmic reticulum network,bundles of 10-nm filaments, Golgi apparatus, mitochondria,numerous secretory and pinocytosis vesicles coalescing withlysosomes, desmosome and tight junctions holdingneighbours cells to form the monolayers. This structuralintegrity suggested that the cultured epithelial cellsmaintained the secretory and resorptive functions of theepididymal principal.cells in intact organs.The maintenance of functional integrity of the cultured cellswas also evidenced by the in vitro maturation study. After 2days in coculture with caput, corpus and cauda epididymalcell monolayers, a proportion of sperm (significantly higherthan that in control incubations) exhibited changes of headorientation from immature T shape to the maturestreamlined shape accompanied by the development of a fastflagellum beat with slow or medial progressive motility (Fig1 & Table 1).ConclusionsIt is envisaged that the present sperm co-culture system formarsupial species will greatly increase our understanding ofsperm-epididymal cell interactions in these species. It isanticipated that this system will also facilitate theidentification of specific epithelial factors, which may beexploited for the manipulation of male fertility.References(1) Lin M, RodgerJC (1999) J Anat 194: 223-232(2) Moore HPM, Pacey AA (1997) J Reprod Fert Sup 19: s3(3) Moore et aI. (1992) Fert Steril58: 776-783(4) Carballada R, Saling PM (1997) J Reprod Fert 110: 171Fig. 1. The population of streamlined-shape spermatozoa was higher(proximately 5-8 times) in Day 4 coculture of epididymal cells than thatofsmall intestine cells. * p~ 0.05; **, p~ 0.001.~ 252.s 20III~!. 15•" CP.5 10eI 5U)30..---------------~**_ OaY2/*EliEI Oay-41*IIISmallIntealMIIcaput Corpus caudaEpldlclymla Epldldymla EplcllclymlsCell Types In Day 2 and Day 4 Co-culturesTable 1: Motility development in coculture preparations.Cultured c:ell types Coc:ulturetime Sperm motility c:haracter(days)1 Twitching and slow tail beatingCaput epididymis 3 Fast tail beating with progression