The Function Of Arl3 During Spermiogenesis

Spermiogenesis is a complex and tightly regulated biological process, during which, haploid germ cells undergo dramatic morphological changes, and eventually form functional spermatozoa. Elongation and condensation of the nucleus, formation of the acrosome and flagella, disposal of unnecessary cytoplasm and the transient appearance of manchette are included in these changes. The most punctual change among these processes is the development of manchette, which starts during the eighth Step of spermiogenesis and vanishes soon after the completion of the elongation and condensation of the spermatid nuclear. The manchette provides a transient storage for structural and signaling proteins which contribute to the developing of sperm tails and participate in the elongation and condensation of the nucleus.In the early stage of the study, we constructed a proteome profile for the mouse first wave of spermatogenesis by means of proteomics, and screend a number of important proteins associated with spermiogenesis. Arl3 protein which belongs to the Arf family was chosen for further research. Arf family is composed of at least 22 members which include 6 Arfs, 2 Sars and 14 Arl (Arf-like) proteins. At least six Arf family members were found in very early eukaryotes, including orthologues of Arf, Sar, Arl2, Arl3, Arl6 and Arl8. The main function of Arfs is related to membrane traffic. A number of newly published papers suggest that several Arl proteins may be involved in different aspects of microtubule-dependent functions. In somatic cells, Arl3 located in the centrosome and microtubule-rich structure in the process of cell division. Strikingly, when a constitutively'active'form of LdARL-3A (blocked under the GTP-bound form) of Leishmania donovani was overexpressed, the promastigotes were immobile with a very short ?agellum.In the proteome profile of spermiogenesis, Arl3 started to express in neonatal mouse testes, but the expression level was low and lasted to 3 weeks old. When the mouse was 4 weeks old, the expression level of Arl3 increased significantly and remained to adulthood period. As we all know, spermiogenesis begins from 3 weeks after born, so Arl3 was likely to participate in spermiogenesis. Immunohistochemical results suggested that, Arl3 was mainly located in pachytene spermatocytes, elongated spermatids and germ cells between these two stages. Immunofluorescence results suggested that in spermatogonia, diffusing distribution in cytoplasm; in the spermatocyte , spotty distribution on one side of the cell, similar to the centresome; in round spermatid phase, distributed in the cytoplasm; during elongating spermatid stage, Arl3 specifically located in posterior region of the nucleus which was similar to the manchette; when the nuclear elongation and condensation was completed, the manchette disappearing, and Arl3 relocated at the axoneme of the sperm tail. Laser Scanning Confocal Microscope showed that the fluorescent signal surrounded the spermatid head, which was similar to the manchette. Moreover, Arl3 colocated withα-tubulin. As a result, Arl3 was present on the manchette. Further, we studied functions of Arl3 by means of microinjection combined with RNAi. We found lots of deformed spermatozoa including abnormal head, neck and coiled tail. So Arl3 played an important role during spermiogenesis, and may be as a new protein of manchette involved in formation of sperm head and tail.In summary, Arl3 is important for spermiogenesis. Teratospermia is one of the common causes of infertility, so exploring the founction of Arl3 provide us a powerful clue to study male sterility.