AMPK identified for the first time in Human Sperm: The “Shock and Live” approach links the creation of all human life with Progeria & HIV-1

AMPK identified for the first time in Human Sperm: The “Shock and Live” approach links the creation of all human life with Progeria & HIV-1

https://www.linkedin.com/pulse/ampk-identified-first-time-human-sperm-shock-live-approach-finley?articleId=6199709017560928257#comments-6199709017560928257&trk=prof-post 

Sperm cell showing AMPK, highlighted in green, localized across the sperm tail and at the entire acrosome in the sperm head. Adapted from: See reference [1].

In line with recent findings demonstrating that cellular stress-induced activation of the master metabolic regulator AMPK is critical for oocyte meiotic resumption and maturation (processes that are essential for subsequent oocyte activation), T cell activation, and extension of lifespan, a recent study published in September of 2016 identified for the first time that AMPK is present in the whole human spermatozoa (i.e. sperm). AMPK localized along the tail, the midpiece, and at the entire acrosome, with inhibition of AMPK significantly decreasing sperm motility in all motility parameters analyzed [1].  Given that an increase in intracellular calcium levels, which activate AMPK, is critical for oocyte activation, sperm acrosome reaction, and T cell activation, and that several compounds that improve accelerated cellular aging defects in Hutchinson-Gilford progeria syndrome also promote oocyte meiotic induction, induce the acrosome reaction in human sperm, and activate AMPK, the results from this ground-breaking study provide compelling evidence that AMPK likely occupies a central node facilitating the creation of all human life, the potential eradication of HIV-1, and the amelioration of symptoms associated with accelerated aging.          

Similar to oocyte activation, which is indispensable for the creation of all human life, the sperm acrosome reaction is also indispensable for the creation of all human life outside of a clinical setting (human oocyte fertilization and embryonic development can be achieved with sperm that have not undergone the acrosome reaction via in vitro fertilization techniques such as ICSI) [2]. The sperm acrosome reaction, an exocytotic process that is indispensable for oocyte penetration and fertilization, is characterized by the release of contents (e.g. proteolytic enzymes) of the acrosome (a cap-like structure residing in the anterior portion of the sperm head) that facilitates penetration of the outer protective coat of the egg, thus allowing fusion of sperm and egg cell membranes [3].  Interestingly, as an increase in intracellular calcium, similar to oocyte activation, is critical for induction of the acrosome reaction, physiological induction of the acrosome reaction by compounds found in the vicinity of the oocyte (e.g. progesterone) or pharmacological inducers (e.g. the calcium ionophore A23187) of the acrosome reaction each share a common mechanism of promoting intracellular calcium increases in sperm [3,4].

Because an intracellular calcium increase plays a pivotal role in both oocyte activation and induction of the acrosome reaction and because AMPK activation has also been shown to promote oocyte meiotic resumption, maturation, and likely oocyte activation, the acrosome reaction in sperm would also be predicted to be critically dependent on AMPK activation [4].  Indeed, Calle-Guisado et al. identified AMPK for the first time in human spermatozoa (i.e. mature motile sperm), with localization of AMPK at the entire acrosome, midpiece, and tail [5].  The authors used semen obtained from healthy human donors by masturbation and spermatozoa motility was analyzed using motility parameters including curvilinear velocity (VCL), average path velocity (VAP), straight-line velocity (VSL), percent of motile and progressive spermatozoa, amplitude of lateral head movement (ALH), beat cross of flagellum frequency (BCF), and linearity (LIN) [5].  Using both immunoflourescence and Western blotting techniques, the authors initially demonstrated that AMPK protein levels were highly expressed in freshly ejaculated human sperm, with localization of AMPK at the entire acrosome, the midpiece, and along the tail sperm.  Strikingly, using the same techniques along with antibodies to detect the phosphorylated/activated form of AMPK (pThr172-AMPK), the authors also showed that activated AMPK is also located along human spermatozoa, with prominent staining at the most apical portion of the acrosome and at the sperm tail, providing compelling evidence that activation of AMPK likely plays a critical role in the induction of the acrosome reaction and in facilitation of sperm motility [5].  Indeed, separation of sperm fraction into low and high motility sperm for each donor revealed that activated AMPK is predominant and present with higher intensity in highly motile sperm compared to low motility sperm. Additionally, after 20 hours of treatment with compound C, a pharmacological inhibitor of AMPK, the authors noted a significant decrease (65%) in both the percent of motile sperm present and the percentage of sperm with progressive motility (67% decrease).  20 hours of treatment with compound C also led to a significant decrease in the coefficients of BCF (56% reduction), ALH (45% reduction), and LIN (60% reduction) as well as a statistically significant reduction (approximately 50%) in all sperm velocity parameters tested (VCL, VAP, and VSL) [5].

Interestingly, published at around the same time as the study by Calle-Guisado et al., a study published by Aparicio et al. also identified AMPK in human spermatozoa as well as several functionally active autophagy-related proteins, indicating that autophagy may play a crucial role in cell survival and motility [6].  Interestingly, AMPK has been shown to be a critical regulator of autophagy via phosphorylating and activating ULK1, a key initiator of autophagic induction [7].  Autophagy is an evolutionarily conserved process that maintains cellular homeostasis through targeted degradation of damaged or long-lived intracellular proteins and organelles. Aparicio et al. showed that the autophagic proteins LC3, Atg5, Atg16, Beclin 1, and p62 were present in human spermatozoa as well as AMPKα 1/2 and mTOR, two critical regulators of cellular metabolism [6].  Activation of autophagy significantly increased spermatozoa viability and motility whereas autophagy inhibition decreased viability, motility, and the intracellular concentrations of calcium and ATP.  A majority (72%) of freshly incubated sperm cells were stained for LC3 (protein widely used to study autophagic induction) in the acrosome, further indicating that activated AMPK, which also demonstrates prominent staining in the acrosome, is likely critical for the induction of the acrosome reaction [5,6].  Curiously, the ratio of LC3-II/LC3-I (indicator of autophagic induction) was increased by both chloroquine, an autophagy inhibitor that activated AMPK in sperm, and rapamycin, an autophagy activator and mTOR inhibitor that has also been shown to increase the lifespan of several model organisms [6,8]. As the authors also demonstrated that rapamycin increased progressive motility and the percentage of rapid spermatozoa, whereas Calle-Guisado et al. showed that activated AMPK showed prominent staining at the sperm acrosome and tail, it would be expected that rapamycin would also activate AMPK [5,6]. Indeed, two recent studies published in 2016 revealed for the first time that rapamycin potently induced AMPK activation in normal elderly mice in vivo [9,10].  Because rapamycin and AMPK activation have been shown to promote autophagy by activating ULK1, the results from these two studies detecting AMPK for the first time in human spermatozoa provides compelling evidence that AMPK activation is likely a critical determinant for enhancing motility and promoting the acrosome reaction in sperm [7,9].   

As noted above, the induction of the acrosome reaction and oocyte activation are both critically dependent on an increase in intracellular calcium levels.  Interestingly, the acrosome reaction and oocyte activation also share many of the same intracellular signaling mediators, including phospholipase C (PLC), diacylglycerol (DAG), and  protein kinase C (PKC) [3,4].  Because AMPK activation is essential for oocyte meiotic induction, maturation, and likely oocyte activation, and because activated AMPK displays prominent staining at the sperm acrosome and is critical for sperm motility, it would be expected that compounds that induce the acrosome reaction in sperm will also induce oocyte activation and activate AMPK.  Indeed, calcium ionophores including A23187 have been shown to induce the acrosome reaction in human spermatozoa, activate AMPK, and induce human oocyte activation, leading to the birth of normal healthy children [11-13]. Interestingly, reactive oxygen species (ROS)/oxidative stress, which also activates AMPK, has been shown to induce the acrosome reaction in human sperm and the free radical-generating agent menadione has been shown to induce AMPK-dependent meiotic resumption in cumulus-enclosed and denuded mouse oocytes via promotion of oxidative stress, indicating that induction of cellular stress (i.e. “Shock”) via increases in the levels of ROS or intracellular calcium likely induces AMPK-mediated oocyte meiotic induction/maturation and oocyte activation as well as induction of the acrosome reaction in sperm (i.e. “Live”) [11,14,15].  

Furthermore, as the induction of cellular stress (e.g. intracellular calcium increases, ROS, etc.) leads to oocyte activation and the acrosome reaction in sperm, the exposure of HIV-1 latently infected monocyte or lymphocyte cell lines to hydrogen peroxide (H2O2)/ROS has also been shown to reactivate HIV-1 and the calcium ionophore A23187 has been shown reactivate latent HIV-1 as well [16,17]. Additionally, because AMPK plays a critical role in T cell activation (and thus reactivation of latent HIV-1 that resides in T cells) and because knockdown of AMPK or the upstream AMPK kinase CaMKK2 significantly inhibits HIV-1 replication, cellular stress-induced AMPK activation appears to also link oocyte activation, sperm acrosome reaction, and reactivation of latent HIV-1 (facilitating immune system detection and destruction of the virus) [18,19].

Lastly, cellular stress-induced AMPK activation likely also links oocyte meiotic induction, oocyte activation, sperm acrosome reaction, and amelioration of accelerated cellular aging defects associated with the genetic disorder Hutchinson-Gilford progeria syndrome (HGPS).  Strikingly, 1α,25-dihydroxyvitamin D3, the most potent metabolite of vitamin D, has been shown to delay premature senescence and significantly improve accelerated aging in patient-derived HGPS cells, activate AMPK, and induce the acrosome reaction in human sperm [20-22].  MG132, a proteasome inhibitor, has also been shown to improve symptoms of accelerated aging in HGPS fibroblasts, activate AMPK, reactivate latent HIV-1, and induce oocyte meiotic resumption [23-26].  Additionally, methylene blue and retinoic acid, both of which activate AMPK, have been shown to ameliorate accelerated cellular aging defects in HGPS fibroblasts, with methylene blue also inducing oocyte meiotic induction and retinoic acid enhancing maturation and activation of both mouse and human oocytes [23,27-34].

The results from the aforementioned studies engenders a provocative and novel proposition that oocyte activation, a prerequisite for the creation of all human life, and the acrosome reaction in sperm share a common mechanism of AMPK activation that is promoted by the induction of cellular stress (e.g. ROS, intracellular calcium increase, AMP/ADP:ATP ratio increase, etc).  As first proposed in my most recent publication, this cellular stress-induced activation of AMPK also links oocyte activation and reactivation of latently infected HIV-1 reservoirs, facilitating detection and destruction of the virus by the immune system [4].  Additionally, AMPK-activating compounds (e.g. vitamin D, MG132, methylene blue) that have been shown to induce oocyte meiotic resumption and the acrosome reaction in sperm also ameliorate accelerated cellular aging defects in HGPS, a genetic disorder characterized by excessive accumulation of a toxic protein (called progerin) that is also produced in much smaller quantities by normal humans [35].  Also, as noted in my most previous LinkedIn Post, H2O2/ROS induced by the bacterium Wolbachia in a mosquito strain that serves as a vector for dengue and Zika viruses has also been shown to enhance antiviral immune responses in those mosquitoes, decreasing dengue virus replication and transmission [36].  Because AMPK activation in that mosquito strain by compounds that increase cellular stress leads to an increase in lifespan and enhancement of immune responses and because H2O2, ROS, or intracellular calcium increases have been shown to induce oocyte activation, the acrosome reaction in sperm, and reactivate latent HIV-1, the beneficial effects of cellular stress-induced AMPK activation may not only link the creation of all human life with HGPS and latent HIV-1 reactivation, but may indeed represent a unifying theme that crosses species boundaries.        

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36. https://www.linkedin.com/pulse/probiotic-activates-ampk-mosquitoes-inhibiting-zika-dengue-finley?trk=pulse_spock-articles