Morphological Characterization Of Organelle Remodeling And Autophagy During Spermatogenesis Of Chinese Soft-Shelled Turtle,Pelodiscus Sinensis

Spermatogenesis is a physiological process which takes place in the epithelium of the seminiferous tubule.During the spermatogenesis,diploid cell(spermatogonia)transforms into haploid cell(spermatozoa)via three phases;mitosis,meiosis,and spermiogenesis.The final phase of spermatogenesis is spermiogenesis;a highly sophisticated process that occurs without cell division comprised of numerous morphological events/changes occurs along with the conversion of round spermatids into elongated spermatozoa.Organelle remodeling or rearrangement,acrosome biogenesis,nuclear shaping and cytoplasmic trimming are the major morphological events/changes which occurs during spermiogenesis.Autophagy is an evolutionarily conserved cell reprograming machinery and has shown its active involvement in spermatogenesis.However,the complex morphological events and involvement of autophagy during spermiogenesis remains unknown in reptilian species.Chinese soft-shelled turtle is an ancient type of reptile widely raised in China and to the less extent in other parts of the world.In China,the Chinese soft-shelled turtle is considered as one of the important reptilian species due to its economic and medicinal value.It is of utmost importance to know the morphological events/changes that occurs during the spermiogenesis in this particular species,due to the fact that it will be very helpful for the proper evaluation of the testes in terms of health or disease which will contribute to more productive animals in the future.Our current study is first one to address the cellular remodeling of mitochondria and the role of autophagy in the acrosome formation,mitochondrial cristae formation during the turtle spermiogenesis.Ultrastructural analysis revealed that sperm mitochondria follows a series of fascinating morphological changes,leading to a mature onion-like shape during spermiogenesis.During this sophisticated remodeling process,a non-membranous structure(MAG)was observed which was closely associated with the mitochondria.We also explored the involvement of autophagy in normal formation of acrosome and mitochondrial cristae in spermiogenesis.Inhibition of autophagy caused severe ultrastructural damages in the acrosome,nucleus and mitochondria.This study also reported the existence of CD63 enriched exosomes in seminiferous tubules and their relationship with autophagy during turtle spermatogenesis.Moreover,a cellular comparison of in vivo autophagy was performed on germ cells of fowl and turtle,sticking to the fact that birds are closest relatives of turtles.Experiment-1 Remodeling of mitochondria during spermiogenesis of Chinese soft-shelled turtle(Pelodiscus sinensis)Mitochondria are vital cellular organelles that have the ability to change their shape under different conditions,such as in response to stress,disease,changes in metabolic rate,energy requirements and apoptosis.In the present study,we observed remodeling of mitochondria during spermiogenesis and its relationship with mitochondria associated granules(MAG).At the beginning of spermiogenesis,mitochondria are characterized by their round shape.As spermiogenesis progresses,the round-shaped mitochondria change into elongated and then swollen mitochondria,subsequently forming a crescent-like shape and finally developing into onion-like shaped mitochondria.We also noted changes in mitochondrial size,location and patterns of cristae at different stages of spermiogenesis.Significant differences(P<0.0001)were found in the size of the different-shaped mitochondria.In early spermatids transitioning to the granular nucleus stage,the size of the mitochondria decreased,but increased subsequently during spermiogenesis.Changes in size and morphological variations were achieved through marked mitochondrial fusion.We also observed a non-membranous structure(MAG)closely associated with mitochondria that may stimulate or control fusion during mitochondrial remodeling.The end product of this sophisticated remodeling process in turtle spermatozoa is an onion-like mitochondrion.The acquisition of this kind of mitochondrial configuration is the robust strategy for long-term sperm storage in turtles.Experiment-2 Autophagy is required for formation of acrosome and mitochondrial cristae during spermiogenesis in turtleAutophagy is a subcellular process that is extensively involved in spermiogenesis.In this study,our aim was to study the role of autophagy in the formation of acrosome and mitochondrial cristae during the spermiogenesis of Chinese soft-shelled turtle.Animals were divided into two groups(control and 3-MA treated).Autophagy was blocked with 3-MA,which is one of classical autophagy inhibitor.The morphological abnormalities of acrosome and mitochondria due to inhibition of autophagy were observed under transmission electron microscopy(TEM).In the early spermiogenesis(Golgi and cap phases),damaged macrovesicle was observed,and its proper expansion over the nucleus failed to form a normal acrosomal cap.As spermiogenesis proceeded,the malformation of the acrosome in spermatids became more severe.In the late spermiogenesis(acrosomal and maturation phases),defective acrosome with damaged acrosomal membrane was observed which was detached from the nucleus.Along with malformed acrosome,abnormal nucleus which failed the elongation having oval or round shaped morphology was also observed.Moreover,morphological damages to the mitochondrial cristae were observed.Lacuna formation,half and complete loss of cristae were observed in the mitochondria of developing spermatids of 3-MA treated group.We proposed that autophagy is required for formation of the acrosome and mitochondrial cristae during turtle spermiogenesis.Experiment-3 Cellular evidence of CD63 enriched exosome in seminiferous tubules during spermatogenesis of Chinese soft-shelled turtle.Seminiferous tubule is the location of spermatogenesis to produce mature spermatozoa as a result of mitotic proliferation,meiosis and cellular differentiation.The transformation of specific male-haploid-germ-cell require a deliberate communication for coordination.As a communicator,the role of vesicles is still not fully understood.In this study,we reported CD63 enriched exosome by immunohistochemistry,immunofluorescence,western blotting and cellular evidence of MVBs(multivesicular body)by TEM during spermatogenesis that is associated with developing spermatids in turtle.The expression of CD63 enriched exosome in ST were higher during late than early spermatogenesis.CD63 localized in the basal membrane,spermatogonia,Sertoli cells during early spermatogenesis and luminal region with developing spermatids during late spermatogenesis.Ultrastructure revealed MVBs existed in cytoplasm of Sertoli cell at basal membrane,and within developing germ cells.In spermatid,Chrysanthemum flower center(CFC)generated isolated membrane involved in autophagosome formation,while the fusion of MVBs and autophagosome was also observed.The 3-MA treatment,CD63 signals severely expressed in seminiferous tubules by IF and WB.In conclusion,our study suggests that CD63 enriched exosomes were synthesized and finely released into seminiferous tubule's basal and lumen region when spermatogenesis was active.MVBs and autophagosomes were found in male haploid germ cell,whereas exosome and autophagy pathway were coordinately functional.Collectively,our data provides cellular evidence of exosomes in seminiferous tubules and inhibition of autophagy enhanced exosomes production during spermatogenesis.Experiment-4 Cellular comparison of in vivo autophagy between fowl and turtle during spermatogenesisSpermatogenesis is a complex cellular process which includes many subcellular events that are essential for the production of healthy spermatozoa.Autophagy is a physiological process which plays significant role in the process of spermatogenesis.In the present study,we have characterized and compared in vivo autophagy during the spermatogenesis of domestic fowl and Chinese soft-shelled turtle.In order to cellular comparison of autophagy,immunofluorescence and ultrastructural analysis were performed.In both species,immunofluorescence staining of LC3 indicated strong localization within spermiogenic cells(intermediate and late spermatids)and primary spermatocytes.However,poorly expressed in cells(spermatogonia)that are located near the basement membrane.While p62 showed opposite tendency to LC3 and more strongly localized with round spermatids.Furthermore,ultrastructural analysis revealed,well-developed autophagosomes along with multivesicular bodies(MVBs)were more prominent in primary spermatocytes and spermiogenic cells of both species.Whereas autophagosomes were absent in the spermatogonial cells.Moreover,other vesicular structures like early endosome,amphisome were also observed during spermatogenesis.The above findings collectively suggest that,in both species,autophagy was active in a similar way,and the expressions of autophagy specific markers were increased from basal to luminal region of seminiferous tubules.We proposed that autophagy may be involved in multiple functions to sustain the avian and reptilian spermatogenesis.