Sperm proteostasis: Can-nabinoids be chaperone's partners?

The male gamete is a highly differentiated cell that aims to fuse with the oocyte in fertilization. Sperm have silenced the transcription and translational processes, maintaining proteostasis to guarantee male reproductive health. Despite the information about the implication of molecular chaperones as orchestrators of protein folding or aggregation, and the handling of body homeostasis by the endocannabinoid system, there is still a lack of basic investigation and random controlled clinical trials that deliver more evidence on the involvement of cannabinoids in reproductive function. Besides, we noticed that the information regarding whether recreational marijuana affects male fertility is controversial and requires further investigation. In other cell models, it has recently been evidenced that chaperones and cannabinoids are intimately intertwined. Through a literature review, we aim to explore the interaction between chaperones and cannabinoid signaling in sperm development and function. To untangle how or whether this dialogue happens within the sperm proteostasis. We discuss the action of chaperones, the endocannabinoid system and phytocannabinoids in sperm proteostasis. Reports of some heat shock and lipid proteins interacting with cannabinoid receptors prove that chaperones and the endocannabinoid system are in an intimate dialogue. Meanwhile, advancing the evidence to decipher these mechanisms for introducing innovative interventions into routine clinical settings becomes crucial. We highlight the potential interaction between chaperones and cannabinoid signaling in regulating proteostasis in male reproductive health.

The molecular chaperone cysteine string protein is required for monomeric SNARE proteins to assemble in trans-complexes during human sperm acrosomal exocytosis†.

Membrane fusion in sperm cells is crucial for acrosomal exocytosis and must be preserved to ensure fertilizing capacity. Evolutionarily conserved protein machinery regulates acrosomal exocytosis. Molecular chaperones play a vital role in spermatogenesis and post-testicular maturation. Cysteine string protein (CSP) is a member of the Hsp40 co-chaperones, and the participation of molecular chaperones in acrosomal exocytosis is poorly understood. In particular, the role of CSP in acrosomal exocytosis has not been reported so far. Using western blot and indirect immunofluorescence, we show that CSP is present in human sperm, is palmitoylated, and predominantly bound to membranes. Moreover, using functional assays and transmission electron microscopy, we report that blocking the function of CSP avoided the assembly of trans-complexes and inhibited exocytosis. In summary, here, we describe the presence of CSP in human sperm and show that this protein has an essential role in membrane fusion during acrosomal exocytosis mediating the trans-SNARE complex assembly between the outer acrosomal and plasma membranes. In general, understanding CSP's role is critical in identifying new biomarkers and generating new rational-based approaches to treat male infertility.

A connection between reversible tyrosine phosphorylation and SNARE complex disassembly activity of N-ethylmaleimide-sensitive factor unveiled by the phosphomimetic mutant N-ethylmaleimide-sensitive factor-Y83E.

N-ethylmaleimide-sensitive factor (NSF) disassembles fusion-incompetent cis soluble-NSF attachment protein receptor (SNARE) complexes making monomeric SNAREs available for subsequent trans pairing and fusion. In most cells the activity of NSF is constitutive, but in Jurkat cells and sperm it is repressed by tyrosine phosphorylation; the phosphomimetic mutant NSF-Y83E inhibits secretion in the former. The questions addressed here are if and how the NSF mutant influences the configuration of the SNARE complex. Our model is human sperm, where the initiation of exocytosis (acrosome reaction (AR)) de-represses the activity of NSF through protein tyrosine phosphatase 1B (PTP1B)-mediated dephosphorylation. We developed a fluorescence microscopy-based method to show that capacitation increased, and challenging with an AR inducer decreased, the number of cells with tyrosine-phosphorylated PTP1B substrates in the acrosomal domain. Results from bioinformatic and biochemical approaches using purified recombinant proteins revealed that NSF-Y83E bound PTP1B and thereupon inhibited its catalytic activity. Mutant NSF introduced into streptolysin O-permeabilized sperm impaired cis SNARE complex disassembly, blocking the AR; subsequent addition of PTP1B rescued exocytosis. We propose that NSF-Y83E prevents endogenous PTP1B from dephosphorylating sperm NSF, thus maintaining NSF's activity in a repressed mode and the SNARE complex unable to dissociate. The contribution of this paper to the sperm biology field is the detection of PTP1B substrates, one of them likely being NSF, whose tyrosine phosphorylation status varies during capacitation and the AR. The contribution of this paper to the membrane traffic field is to have generated direct evidence that explains the dominant-negative role of the phosphomimetic mutant NSF-Y83E.

Altered renal expression of Na(+) transporters and ROMK in protein-deprived rats.

Potassium depletion has been associated with altered sodium reabsorption in tubule segments. We studied if the altered abundance of Na(+) transporters and ROMK are associated with distal potassium secretion that contributes to the development of hypokalemia in protein-deprived rats. After weaning, Wistar rats were fed with a low-protein diet (8%, LP) for 14 days and then recovered with a normal-protein (NP) diet (24%, RP). An age-matched control group was fed with an NP diet (24%, NP). We showed hypokalemia, lower glomerular filtration rate and higher FEK(+) in the LP group. Immunoblotting revealed that the type 3 Na(+)/H(+) exchanger in the cortex was decreased in the LP group. However, the type 2 Na(+)-K(+)-2Cl(-) cotransporter was increased in the outer stripe of the outer medulla in the LP group. The abundance of the aldosterone-regulated Na(+)-Cl(-) cotransporter (NCC) and epithelial Na(+) channel (ENaC) was higher in the LP group and was associated with higher plasma aldosterone level. ROMK protein levels were increased. Na(+)/K(+)-ATPase protein levels were the same in both groups. After the recovery period, the expression of Na(+) transporters and ROMK returned to control values. We conclude that increased expression of NCC, ENaC subunits, and ROMK contributed to distal potassium secretion leading to enhanced potassium excretion, which may explain the hypokalemia resulting from LP feeding. A role of aldosterone may be suggested.

Na+/K+ -ATPase stabilization by Hsp70 in the outer stripe of the outer medulla in rats during recovery from a low-protein diet.

A low-protein (LP) diet induces injury from energy depletion in renal epithelial cells. Overexpression of heat-shock proteins has been implicated in the restoration of the cytoskeletal anchorage of Na(+)/K(+)-ATPase. We tested if Hsp70 stabilizes renal Na(+)/K(+)-ATPase attachment to the cytoskeleton from the cortex and the outer stripe of the outer medulla (OSOM) in rats during recovery from a LP diet. Rats were fed with a LP diet (8% protein) for 14 days, and then the rats were recovered with a 24% protein (RP) diet. The control group received a 24% protein (NP) diet. Increased Na(+)/K(+)-ATPase dissociation was demonstrated in soluble fraction from OSOM with lower ATP content as a result of LP diet vs NP. Meanwhile, decreased Hsp70 levels in the same fraction were shown. Translocation of Hsp70 to the cytoskeletal injured fraction associated with stabilization of Na(+)/K(+)-ATPase was shown in OSOM from LP after in vitro co-incubation of the cytoskeletal fraction of LP and non-cytoskeletal fraction of RP. These effects were abolished by the addition of the anti-Hsp70 antibody. Absence of Na(+)/K(+)-ATPase detachment from its cytoskeletal anchorage was demonstrated in proximal duct segments from cortex in LP. Co-immunoprecipitation showed that the amount of Na(+)/K(+)-ATPase co-precipitating with Hsp70 increased in the OSOM as a result of the LP diet. In the cortex tissues from rats fed the LP and the RP diet, the interaction of both proteins were similar to the control groups. Our results indicate that Hsp70 has a critical role in protecting the integrity of the cytoskeletal anchorage of Na(+)/K(+)-ATPase during recovery from ATP-depleted injury resulting from LP in OSOM.