Galeolaria caespitosa is a native tube dwelling marine invertebrate which broadly distributes along the intertidal zone of south-eastern Australian coast. It is a broadcast-spawning species whose abdomen is divided by peritonea into over 80 segments which are filled up with developing gametes throughout the year. The spermatogenesis of Galeolaria has been described in our lab at the ultrastructural level. Spermatogonia are proliferated in cell clusters attaching to the ciliated peritonea, together with primary spermatocytes forming a distinct germinal center located at the dorsal-central region of each segment. Spermatids detached from germ cell clusters in the germinal centre and completed the remainder of spermatogenesis while floating freely in coelomic fluid, without any physical support from somatic nursing cells. This independence enabled us to repeat this process in vitro, so as to further uncover the key molecules involved in self-programmed spermiogenesis.
Within 36-hour cultivation in male coelomic fluid, ten steps of spermiogenesis were observed. To determine if the male coelomic fluid contained necessary signaling molecules for spermiogenesis, a series of attempts at in vitro culture of spermatids have been undertaken. Spermatids were cultured in female and boiled male coelomic fluid respectively. These cultures had resulted in failure of differentiation or the arrest of spermatids in less developed stages, which indicated the key factors of spermiogenesis were gender-specific and heat-sensible. In order to narrow down the protein candidates in the male coelomic fluid, the protein extracted from male and female coelomic fluid will be separated and compared through SDS-PAG gel. The protein bands only exist in the male fluid will be analysed under mass spectrometry. Moreover, male coelomic fluid will be fractionated using size-exclusive chromatography and each fraction of the fluid will be used to incubate the spermatids. The fractions which can support the differentiation of spermatids will be analysed under mass spectrometry.