3. Protein Deubiquitination in Spermatogenesis
Deubiquitinases (DUBs) are cysteine proteases, which remove and recycle ubiquitin. Till now, more than 100 DUB enzymes have been identified in human 
. DUBs participate in various biological processes such as apoptosis, cell cycle progression, and protection of proteins from degradation. DUBs are classified into six catalogues: USP (ubiquitin-specific processing proteases), UCH (ubiquitin C-terminal hydrolases), JAMM (Jab1/Pab1/MPN domain-containing metalloenzymes), OUT (Otu-domain ubiquitin aldehyde-binding proteins), MCPIPs (Monocyte chemotactic protein-induced proteases) and Ataxin-3/Josephin 
. Some DUBs have been demonstrated to play a requisite role during spermatogenesis, which largely belong to the USP and UCH family 
USP7 localizes preferentially to the XY body in early pachytene spermatocytes but gradually decreases its level as meiosis progresses, which is highly concurrent with the expression manner of SCML2, a testis-specific polycomb protein 
. Biochemically, USP7 forms a complex with SCML2 to counteract histone H2A ubiquitination in the XY chromatin during meiosis. In SCML2-deficient mice, USP7 is absent from H2A ubiquitination sites, leading to augmented H2A monoubiquitination, which in turn causes spermatogenic impairments characterized by massive pachytene spermatocyte apoptosis 
USP8 is expressed in both brain and testis and interacts with MSJ1, STAM2, EEA1 and VSP54 
. In mouse testis, USP8 shows remarkable increase during post-meiotic differentiation and forms small plaques. USP8 localizes specifically to the nuclear envelope of round spermatids, as well as the centrosome and acrosome vesicle of elongating spermatids 
. In mouse spermatids, USP8 associates with MSJ1 and the 20S CP to move towards the developing acrosome and centrosome 
. STAM2 gives rise to the endosomal-sorting complex ESCRT-0 and EEA1 is an early endosome antigen. Colocalization of USP8 with STAM2 and EEA1 is found at acrosomal vacuole and acrosome surface, respectively 
. VSP54 is responsible for retrograde transport from early endosomes, and during post-meiotic differentiation, it follows the same migration trajectory as USP8 until complete acrosome formation is achieved 
. Taken together, these results suggest that USP8 is a key participant of the endosome pathway during acrosome generation.
USP2 is restrictedly expressed in elongating spermatids, possessing speculative function during post-meiotic differentiation. USP2 deficiency in mice leads to defective sperm whose motility is highly vulnerable to environmental changes 
. Besides, these sperm exhibit poor fertilization capacity due to failure in binding or penetrating to the zona pellucida. USP14 is important for post-meiotic differentiation. In Drosophila
, USP14 deficiency impairs spermatid individualization, during which syncytial spermatids are separated into individual cells 
. USP14 deficiency causes loss of synchronization and abnormal distribution of the actin cones. Besides, USP14-deficient mice exhibit significantly reduced sperm counts as well as severe sperm malformation such as multiple nucleus, missing of sperm head or dual sperm tails 
Mammalian USP9X is the functional orthologue of drosophila
deubiquitinating enzyme fat facets (Faf), which interacts with and impedes the degradation of a DEAD-box RNA helicase Vasa, a highly-conserved marker for germ cells 
. USP9X is predominantly expressed in spermatogonia and weakly expressed in early spermatocytes before pachytene stage. USP9X-deficiency in mouse germ cells leads to male infertility with various abnormalities along the progression of spermatogenesis: reduced number of spermatocytes, degenerated spermatids with residual body-like structures, as well as aberrantly retained mature spermatozoa in seminiferous tubules 
. However, the maintenance and proliferation of spermatogonia were less affected in USP9X-deficient testes, indicating a critical role of USP9X since mitosis-to-meiosis transition in male germ cells.
has been considered as a potential infertility gene due to its restricted expression in mammalian testis and X-chromosome-localization (single copy in males) 
. Several studies have reported the polymorphisms in USP26
associated with non-obstructive azoospermia or asthenozoospermia, suggesting a causal relationship with human male infertility. However, most of the identified USP26
polymorphisms can’t disrupt its enzymatic function 
. In addition, USP26
is indispensable for mouse fertility in both sexes 
. It has recently been found the effects of USP26
mutation on male fertility dependent on the genetic background of mice 
mutants in DBA/2, rather than C57BL/6 background exhibit impaired spermatogenesis with obvious deficiency and malformation of spermatozoa, resulting in infertile or sub-fertile males. These results implicate that strain-specific genetic components interact with USP26
mutations to interfere spermatogenesis; such components may also exist in human but further investigations are needed.
UCHL3 expression exhibits differentiation-dependent pattern during spermatogenesis with minimal amount in spermatogonia but to a sequentially increasing extent in meiotic pachytene spermatocytes and post-meiotic spermatids 
. Besides, a later study has detected intensive UCHL3 expression in sperm acrosomes and flagella, suggesting the role of UCHL3 in regulating both meiosis and post-meiotic differentiation 
. According to the results, OA patients show significantly lower UCHL3 content and activity as compared to normozoospermia controls; UCHL3 condition is slightly better in A patients but still far from normal level 
. Meanwhile, it is proposed that the amount and activity of UCHL3 are positively related to a series of fertility indicators including sperm counts, sperm concentration and sperm motility, highlighting the importance of this DUB during spermatogenesis.
UCHL1 shares high sequence similarity with UCHL3, however, its distribution pattern in male germ cells is totally different, indicating distinct function during spermatogenesis 
. Predominant UCHL1 expression is found in spermatogonia as well as in Sertoli cells. The level of UCHL1 precisely determines spermatogonia fate of either self-renewal or meiotic differentiation as indicated by two markers Plzf and c-Kit, respectively 
. In mouse testis, UCHL1 overexpression leads to drastic loss of post-meiotic germ cells and a large amount of arrested pachytene spermatocytes retards in seminiferous tubules which further undergo apoptosis 
. On the contrary, UCHL1 deficiency significantly increases the number of spermatogonia and preleptotene spermatocytes while immensely inhibits germ cell apoptosis during the first round of spermatogenesis. Additionally, these mice have reduced sperm motility and more abnormal spermatozoa even though complete sterility is not observed 
. Taken together, UCHL1 and UCHL3 express strongly but reciprocally during spermatogenesis, where the former mediates fate determination of spermatogonia as well as elimination of defective spermatozoa to maintain testicular homeostasis, while the latter facilitates post meiotic maturation to produce fertilization-competent spermatozoa.
Besides the DUBs in USP and UCH families, CYLD (cylindromatosis) is also found to be required for spermatogenesis by regulating early wave of germ cell apoptosis mainly in spermatogonia, a requisite process to eliminate excessive germ cells and thereby maintain the balance between germ cells and Sertoli cells 
. CYLD directly deubiquitinates the receptor-interacting protein 1 (RIP1), to prevent the activation of IKK and NF-𝜅B signaling and the expression of downstream anti-apoptotic genes, ultimately promote cell apoptosis 
. Loss of CYLD leads to reduced spermatozoa, failure in radial organization of round spermatids, as well as malformation of acrosomes in elongating spermatids. On the contrary, spermatogonia and early spermatocytes are aberrantly accumulated in CYLD-deficient seminiferous tubules.