New strategies for germ cell cryopreservation: Cryoinjury modulation

1. Apoptosis

Apoptosis, or programmed cell death, is a crucial process in maintaining tissue homeostasis. It contributes to cell turnover, embryonic development, and the functioning of the reproductive system [17,18]. During apoptosis, cells undergo a series of morphological changes, such as cell shrinkage and the fragmentation of cells, organelles, and DNA into apoptotic bodies [19,20]. This process can be initiated by two primary pathways. The extrinsic pathway is activated by cell-surface death receptors, such as tumor necrosis factor (TNF) receptor 1, Fas, and death receptors, which interact with their respective ligands, including TNF-α, Fas ligand, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In contrast, intrinsic apoptosis is initiated when pro-apoptotic proteins from the B-cell lymphoma 2 (BCL-2) family permeabilize the outer mitochondrial membrane within cells [21]. Both apoptotic pathways lead to the activation of a family of proteases and caspases. In the extrinsic pathway, the activation of cell-surface death receptors results in the recruitment of the adaptor protein Fas-associated protein with death domain (FADD). The activated FADD then interacts with procaspases 8 and 10 to form cleaved caspase, marking the onset of apoptosis. In the intrinsic pathway, the activation of BCL-2 proteins leads to the permeabilization of the outer mitochondrial membrane, a step often referred to as the point of no return. This allows for the release of cytochrome C into the cytoplasm. Once in the cytoplasm, cytochrome C forms apoptosomes with apoptotic protease activating factor 1. These apoptosomes enable the cleavage of procaspase-9. The cleaved caspases 8, 10, and/or 9 then proceed along a common apoptosis execution path, which involves the activation of caspase-3 and/or caspase-7 [21].

2. Necroptosis

Necroptosis is a regulated, caspase-independent form of cell death that serves as an alternative pathway to bypass resistance to apoptosis. Cells undergoing necroptosis exhibit necrotic characteristics, such as swelling and rupture of the cellular membrane. This pathway can be initiated by death receptors, toll-like receptors, TNF, Z-DNA binding protein 1, or certain viral infections, particularly when the activation of caspase-8 is compromised [22-24]. While necroptotic cell death can be triggered by pro-inflammation or as an alternative to apoptosis, our focus is on the former, given that necroptosis induced by cryoinjury is seldom associated with viral infection or inflammation. Furthermore, apoptosis and necroptosis are closely regulated by each other, with caspase-8 playing a pivotal role as a mediator of both apoptotic and necroptotic pathways. This is because caspase-8 not only regulates apoptosis but also serves as a key component of the ripoptosome [25]. In the absence of caspase-8 activity, receptor-interacting protein kinase (RIPK) 1 autophosphorylates RIPK3, leading to the formation of the ripoptosome. The RIPK1/RIPK3 complex then phosphorylates mixed lineage kinase domain-like protein (MLKL), resulting in the creation of the necrosome. This structure triggers cell death by destabilizing mitochondria, perturbing the membrane, and causing cell lysis [26-28].

3. Autophagy: a double-edged sword

Autophagy is a process in which cellular components such as mitochondria, organelles, and endoplasmic reticula are degraded by lysosomes, allowing them to be recycled and used in cellular function during periods of stress or starvation. This makes autophagy a crucial player in maintaining cellular homeostasis. The autophagy process can be broken down into five stages: (1) initiation; (2) nucleation and phagophore formation; (3) phagophore expansion; (4) fusion with the lysosome; and (5) degradation [29]. The primary regulators of autophagy are the mammalian target of rapamycin, which functions as an inhibitor, and adenosine monophosphate-activated kinase, which acts as an activator. The initiation of autophagy triggers the regulation of nucleation by various protein complexes, including autophagy-related proteins. Subsequently, phagophores marked with microtubule-associated protein 1A/1B-light chain 3 (LC3) mature into autophagosomes. These autophagosomes then fuse with lysosomes to create autolysosomes, where the cargo is degraded [30].

While autophagy is typically viewed as a survival mechanism, its deregulation has also been linked to a non-apoptotic form of cell death known as either ADCD or autophagic cell death [7,31-33]. Morphologically, ADCD is characterized by the extensive presence of autophagic components in the cytoplasm, devoid of other forms of programmed cell death. This process can be halted either pharmacologically or genetically [31,33]. ADCD can occur via an autophagic flux-dependent or flux-independent pathway. The former, known as autosis, is a non-apoptotic form of cell death that is autophagy-dependent and results from excessive or uncontrolled autophagy levels [34]. The latter is triggered by glycosylceramidase beta 1 and leads to autophagic cell death [35].

1. Targeting CPAs

Apoptosis can be initiated by two distinct pathways: the extrinsic and intrinsic pathways. These pathways can be activated during low-temperature freezing, as evidenced by the protective role of specific cysteinase inhibitors (for example, Z-Val-Ala-Asp-(OMe)-fluoromethylketone [Z-VAD-FMK], a pan-caspase inhibitor; Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone [Z-IETD-FMK], a caspase-8 inhibitor; and Z-Leu-Glu(O-Me)-His-Asp(O-Me)-fluoromethylketone [Z-LEHD-FMK], a caspase-9 inhibitor) when used as CPAs in germ cell freezing. Ha et al. [57] revealed that the proliferation capacity of SSCs was increased in frozen SSCs treated with a mixture of 200 mM trehalose and 15 µM Z-VAD-FMK. This treatment also reduced early apoptosis after thawing through pan-caspase inhibition. In a similar vein, Niu et al. [58] reported a decrease in caspase activity and early apoptosis in vitrified porcine oocytes treated with Z-IETD-FMK and Z-LEHD-FMK. In 2020, Colombo et al. [59] also discovered that Z-VAD-FMK reduced DNA damage and caspase activity in vitrified oocytes. These findings suggest that inhibiting apoptotic proteolytic activation allows frozen germ cells to minimize cell death induced by cryoinjury. This conclusion is consistent with the findings of Jung et al. [60], who showed that extrinsic apoptosis, which involves the proteolytic activation of caspase-8, is increased in frozen SSCs. Therefore, Z-IETD-FMK can significantly enhance the cryoprotective efficacy of SSCs by suppressing caspase-8 activity during freezing. Furthermore, bongkrekic acid, an inhibitor of mitochondrial permeability transition pore opening, has been shown to improve the freezing efficacy of stallion sperm. This is achieved by reducing caspase activity and increasing the mitochondrial membrane potential [61].

Antioxidants are often used as CPAs to reduce apoptosis, as excessive ROS generation during freezing can cause cell death or damage upon thawing. For instance, vitrification of bovine oocytes with 50 µM of coenzyme Q10, an antioxidant, significantly improved oocyte survival and mitigated premature cortical granule (CG) exocytosis, thereby preserving the CG migration pattern [62]. Similarly, freezing SSCs with 100 µM of melatonin, a mitochondria-targeted antioxidant, greatly enhanced survival, reduced ROS production, and decreased intrinsic apoptosis [63]. Vitamin C, known for its potent antioxidant properties due to its strong reducing power, has been found useful in preserving DNA integrity, sperm motility, acrosome membranes, and plasma after freezing bovine sperm with vitamin C [64]. Vitamin E, a lipophilic chain-breaking antioxidant, has also been effectively used as a CPA for the freezing of ram sperm [65-67]. Moreover, resveratrol, a powerful non-flavonoid antioxidant, has shown beneficial effects on post-thaw sperm survival, fertility, and quality by inhibiting ROS production [68-70]. In addition, previous research has suggested that an increase in intracellular TNF-α after freezing is implicated in TNF-α–mediated necroptotic cell death [53]. The activation of the RIP complex also induces ROS production in the mitochondria and mediates plasma membrane rupture [71]. As such, the necroptosis inhibitor necrostatin-1 has shown beneficial effects on the proliferation rate while reducing necroptosis and apoptosis after thawing in SSC cryopreservation; it exerts these impacts by reducing ROS generation, apoptosis, and RIP1 activity [10]. The addition of pifithrin µ (a p53 inhibitor) or 3-methyladenine, which blocks autophagosome formation by inhibiting type III phosphatidylinositol 3-kinase, has been found to enhance cryoprotective effects and help maintain normal stem cell characteristics after thawing during SSC freezing [9]. Mitophagy, a selective autophagy that removes aged and damaged mitochondria, is also activated in frozen oocytes to improve survival by eliminating damaged mitochondria [55]. In 2020, Feng et al. [72] demonstrated that melatonin inhibits autophagosome formation and regulates the levels of autophagy-related proteins, thereby reducing cryoinjury-induced excessive autophagy in goat SSC freezing.

Interestingly, autophagy does not consistently safeguard frozen germ cells; in fact, excessive autophagy can lead to cryoinjury-induced cell death [73]. Furthermore, while each pathway regulator may have cryoprotective effects resembling those of CPAs, the intricate nature of these pathways prevents complete rescue from cryoinjury-induced cell death. This is due to our limited understanding of the complex interplay among these pathways. This underscores the need for further molecular mechanistic studies to enhance our comprehensive understanding of cryoinjury-induced cell death.

2. Pre-freeze or post-thaw care

Efforts have been made to develop effective CPAs to prevent cryoinjury during the freezing process. However, these CPAs only partially mitigate cryoinjury, as damage continues to occur even after thawing. This highlights the necessity for careful handling and recovery of frozen cells and tissues post-thaw [12]. In this context, Hwang and Hochi [14] discovered that a brief post-thaw culture of oocytes with Y-27643, a Rho-associated protein kinase inhibitor, prevented the formation of multiple asters. This formation is a key factor limiting the embryonic potential of vitrified-warmed bovine oocytes [14]. Additionally, Girka et al. [13] found that treatment with 5 and 10 µM Y-27632 effectively improved spindle rescue following vitrification-warming. In 2018, Pero et al. [74] proposed that the use of 20 mM Z-VAD-FMK during in vitro culture post-thawing could partially reduce cryoinjury-induced apoptosis by suppressing active caspase-3. Pagano et al. [16] demonstrated that treating with 100 µM Z-VAD-FMK enhanced the mass motility of bovine sperm prior to freezing and maintained sperm membrane integrity post-thawing. Finally, Jung et al. [15] reported that supplementing post-thaw media with 200 µM Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethylketone (Z-DEVD-FMK) enhanced the proliferation potential of frozen SSCs, safeguarding them against ROS generation and apoptosis following cryo-thawing.

Numerous studies have demonstrated that incorporating antioxidants into media during in vitro maturation prior to freezing can enhance embryo development in various species, including mice [75,76], sheep [77], and cattle [62,78]. Furthermore, a range of antioxidants, such as selenium, manganese (III) tetrakis (4–69 benzoic acid) porphyrin (MnTBAP), pentoxifylline, N-acetyl-L-cysteine (NAC), and leptin, have been found to mitigate oxidative stress related to cryoinjury-induced damage when added to the in vitro culture media used in sperm freezing. Gavella and Lipovac [79] found that pre-incubating human sperm with 3.7 or 10 mM pentoxifylline, a phosphodiesterase inhibitor that also protects against lipid peroxidation by hydrogen peroxide, for 30 minutes at 37 °C reduced oxidative stress and enhanced fertility potential post-thawing. Oeda et al. [80] reported that incubating human semen with 1 mg/mL NAC for 20 minutes at room temperature significantly lowered ROS levels and improved sperm motility. Boroujeni et al. [81] found that rinsing with 5 μg/mL selenium after thawing can enhance human sperm parameters and survival. Fontoura et al. [82] found that adding leptin, a hormone that regulates food intake behavior, to human sperm prior to freezing supports antioxidant enzyme activity and provides additional antioxidant protection. Additionally, Treulen et al. [83] demonstrated that pre-incubating with MnTBAP, a cell-permeable superoxide dismutase mimetic, for 4 hours at 38 °C improved stallion sperm motility and reduced oxidative stress, while maintaining normal fertilization potential post-thawing.

While the addition of antioxidants or apoptosis inhibitors to in vitro culture media may help reduce oxidative stress, the use of these compounds in freezing is not always effective. Prolonged exposure or high concentrations can disrupt the appropriate physiological balance, as apoptosis is instrumental in removing damaged cells in adults and regulating embryonic development [84]. Similarly, excessive antioxidant supplementation may disrupt the oxidation-reduction balance, resulting in reductive stress [85]. However, the apoptosis or oxidative stress induced by cryoinjury often results in considerably more additional apoptotic/oxidative stress, which affects cell survival [86]. Therefore, the inclusion of antioxidants or apoptosis inhibitors could be beneficial in alleviating cell death caused by cryoinjury, provided optimization and functional tests for fertility are conducted.