Participation of apoptotic markers in the process of maturation and elimination of spermatozoa in the epididymis of the Corynorhinus mexicanus bat

The Corynorhinus mexicanus bat is characterized by a specific form of reproductive asynchrony between males and females. After mating, some sperm remain in the male's epididymis, the organ where the sperm had matured. It has not yet been determined if apoptotic markers participate in the process of the maturation and/or elimination of these cells, so studying this topic is essential for our understanding of this species. Male bats were collected during three stages: Before mating; during the Mating phase; After mating and the final phase, which we call, Storage. Their epididymides were removed, weighed and measured. Sperm were extracted and the following sperm parameters were evaluated: active caspases, phosphatidylserine externalization, and mitochondrial membrane potential. Sperm from the testes enter the epididymis during Before mating, causing the organ to grow. During Mating phase, spermatozoa present a large amount of active caspases with externalization of phosphatidyl serine, even while still alive. This suggests that these two markers could participate in maturation and elimination, respectively.

The Corynorhinus mexicanus bat is a species characterized by seasonal asynchronous reproductive pattern with temporary asynchrony between the reproductive functions of males and females, as spermatogenesis takes place in summer (May-August) but females are receptive well into autumn (November), several months after the male gonads have regressed completely (León-Galván et al., 2005, 1999).To ensure reproductive success, males store sperm in the epididymal cauda for 3 months (September−November), until the mating phase, when maturation is completed (Arenas-Ríos et al., 2005;León-Galván et al., 2005;Rodríguez-Tobón et al., 2016).After the mating phase, the unejaculated sperm remains in the epididymis for a few more months, then they must be eliminated (Gasparini et al., 2017).However, the mechanisms by which sperm are eliminated and whether they remain alive and/or active during the final period of storage in the epididymis are unknown.
Apoptosis is a type of cell death that takes place in an orderly manner, preventing inflammatory processes, and that can be activated in response to various types of cellular stress such as DNA damage, deprivation of growth factors, environmental signals, such as radiation, toxins, hypoxia, hyperthermia and reactive oxigen species (ROS) (Elmore, 2007 andGreen &Llambi, 2015).In somatic cells, this type of death causes: nuclear fragmentation, loss of plasma membrane integrity, loss of mitochondrial membrane potential (Δψm), loss of phosphatidylserine (PS) residues, exposure of caspases 3 and 7 (cysteinyl-aspartate-specific proteases), also known as "effector caspases," the detection of the cell in discrete fragments (referred to as "apoptotic bodies") and their subsequent engulfment by phagocytes (Elmore, 2007;Galluzzi et al., 2015;Green & Llambi, 2015;López-Trinidad, 2017).Some of these processes that trigger apoptosis could be removing dysfunctional cells in the epididymis.However, spermatozoa are not capable of dying by apoptosis, since, after they leave the testis, they lose the ability to synthesize proteins, and unlike what happens with somatic cells, the gamete does not fragment into apoptotic bodies, therefore what the concept of apoptosis does not include the spermatozoon.However, it has been suggested to use the presence of apoptotic signals or markers in spermatozoa from the epididymis, since apoptotic signals have been reported in these cells such as caspase 3 and 7 activity, externalization of PS (EPS), DNA fragmentation and loss of membrane potential mitochondrial (Elmore, 2007;Galluzzi et al., 2015;Kroemer et al., 2009 andTalarczyk-Desole et al., 2016).
Apoptotic markers in epididymal sperm were analyzed in a previous study evaluating DNA damage, active caspases 3 and 7, EPS and loss of mitochondrial membrane potential with the aim of relating them to aging in Wistar rats.The results of this work showed that apoptotic markers in sperm do not increase with the age of rats (López-Trinidad et al., 2017).However, although these markers are associated with apoptosis, the authors suggested that the presence of caspases 3 and 7 may have other functions besides programmed cell death, such as the acquisition of fertile capacity by the sperm in the epididymis, a process known as epididymal sperm maturation (Chabory et al., 2010).This hypothesis is based on studies that have shown that caspases 3 and 7 do not only participate as indicative of cell death but are also involved in the process of terminal differentiation in some cell types (Aram et al., 2017, 2003and Nhan et al., 2006).
In other studies, associated with markers of apoptotic cell death have found a gradual increase in the EPS in the sperm membrane of viable cells during transit through the epididymis in proportional relation to the fertilization rate, probably because the oocyte has recognition sites for EPS (Rival et al., 2019).Once apoptotic signaling begins, caspases 3 and 7 will participate in the loss of asymmetry of the plasma membrane phospholipid distribution causing the EPS, which corresponds to the mark known as "eat-me" to be phagocytosed.In spermatozoa, it has been shown that the EPS in the head and flagellum region of the spermatozoon correspond to damaged cells and those that present the mark in the acrosomal region are viable cells (Gadella & Harrison, 2002).However, EPS does not only participate as a cell elimination signal, but also as a modification of the sperm plasma membrane required for successful fertilization (Rival et al., 2019).
It is important to note that other events that occur during an apoptotic event is the activity of a DNase, ICAD (Caspase Activated Deoxyribonuclease Inhibitor), which causes nuclear DNA fragmentation, so it could also be considered as an indicator of apoptosis, without neglecting that this event can be triggered by various factors (Portella-Ruiz & Gonzales, 2016).As well as imbalances in the antioxidant system, they are the cause of mitochondrial dysfunction, being also considered as an early marker of programmed cell death in somatic cells, and it has also been identified in spermatozoa (Barroso et al., 2006).
In continuously reproducing species such as the rat, it is very difficult to determine whether apoptotic markers are involved in sperm death or possibly in epididymal sperm maturation.However, the reproductive pattern of the bat C. mexicanus has been characterized with great detail through histological, physiological and biochemical studies that have made it possible to distinguish the specific times and the different stages through which sperm passage during its transit and storage in the epididymis, before, during and after the copulation period (Cervantes et al., 2008;Rodríguez-Tobón et al., 2016, 2020).This knowledge helps to elucidate the role of apoptotic markers in sperm maturation and elimination processes.
Therefore, in this study we set out to determine whether apoptotic markers are present in sperm from C. mexicanus bat and whether they participate in the process of sperm maturation and/or elimination during the prolonged period of epididymal storage.1.19 mM KH 2 PO 4 .Sigma).Eosin-Nigrosine's solution (E-N) (0.67 g of eosin, 0.9 g of NaCl and 10 g of nigrosine Sigma, dissolved in 100 mL of water).

| Collection of specimens
The bats were collected in the "El Túnel" refuge located 10 km E of Tlaxco, Tlaxcala, Mexico (19°37′14′'N, 98°02′02"W, 3220 m).A total of four samplings were carried out to cover the period of prolonged sperm storage (October−February) (León-Galván et al., 2005;Rodríguez-Tobón et al., 2016).All handling was carried out in accordance with NOM-059-SEMARNAT-2010 and the scientific standards established in 2016 (Sikes, 2016).The processing of the biological material was carried out in accordance with the guidelines of the Ethics Committee of the Division of Biological and Health Sciences of the Autonomous Metropolitan University-Iztapalapa (Mexico).Collection permit SGPA/DGVS/07397/19 was obtained from the General Directorate of Wildlife, a Secretaria de Medio Ambiente y Recursos Naturales (SEMARNAT) agency.

| Epididymis and sperm obtention
The specimens were transported to the laboratory while still alive but subsequently decapitated.The bats were not anesthetized because the effect of the anesthetic could influence the physiology of their sperm.An incision was made in the pelvic-ventral region to extract the epididymides.Both organs from each individual were measured with a vernier weighed on an analytical balance, then dissected into three anatomical regions (caput, corpus, and cauda) according to the procedure described previously (Cervantes et al., 2008;Mendoza-Sánchez et al., 2023and Rodríguez-Tobón et al., 2016, 2020).In the cephalic region can find the union with the efferent ducts; the corpus, is very narrow and the cauda, is the most robust region of the three, it is clearly identified, since the curvature of the vas deferens marks the point where this region begins.The spermatozoa of each individual were analyzed separately and both epididymis were used.

Each region was subsequently placed in individual Eppendorf
tubes with 1 mL of Ringer's solution to be minced later.The spermatozoa were released from the epididymal tissue for 5 min and centrifuged (500g, 5 min).The supernatant was removed, and 1 mL was calibrated with Ringer's solution.Ringer's solution has been used in various works of the research group (Rodríguez-Tobón et al., 2016, 2020) and the reason for its use is not to modify the physiological state of the cell.

| Evaluation of sperm parameters
Standard methods were used to evaluate the sperm parameters, largely following the laboratory manual for the examination and processing of human semen (WHO, 2010), but with adaptations for the type of sample analyzed in the study (León-Galván et al., 2005and Rodríguez-Tobón et al., 2016, 2020) The parameters of sperm motility, viability and concentration were recorded.
Motility was determined by taking a 10 μL sample from each region of the epididymis and counting 100 cells in a bright field microscope at x40 magnification to categorize them as motile or nonmotile.To measure sperm viability, a smear was performed using 10 μL of sample and 10 μL of E-N solution.A count of 100 cells was performed under bright field microscopy (Olympus BX-41) (x40) to differentiate dead (pink color) from live sperm (without color).For the parameter of sperm concentration, a 1:50 dilution was used, and counts were performed in a Neubauer chamber under bright field microscopy at x40 magnification (Rodríguez-Tobón et al., 2016, 2020).

| Caspase detection
To detect active caspases 3 and 7, an aliquot of 5 × 10 6 sperm was taken, incubated with FLICA, and processed according to the manufacturer's instructions.FLICA is a compound permeable that passes through a plasma membrane and binds to active caspase.
Any unbound FLICA diffuses out of the cell and is washed away; binds only active caspase, there is no interference from procaspases or inactive forms of the enzyme (Amstad et al., 2001;Slee et al., 1999 andThornberry et al., 1997).A total of 10 000 cells were used to identify the presence of these caspases using the FACScalibur flow cytometer (Becton Dickinson).FLICA optimally excites at 488−492 nm and has a peak emission at 515−535 nm.

| Phosphatidyl serine externalization
Aliquots of 5 × 10 6 spermatozoa were taken and incubated with Annexina Vfor 5 min at 37°C, then analyzed following the manufacturer's instructions.Annexin V is a Ca 2+ dependent phospholipid-binding protein with high affinity for PS, it is a noncell-permeable protein, so it binds to PS that has been redistributed and is found in the outer layer, event that occurs in cell death events Koopman et al., 1994;Martin et al., 1995 andMoss et al., 1991).Subsequently, the samples were analyzed by flow cytometry to detect labeling.Optimally excites at 488 nm and has a peak emission at 518 nm.

| Disruption of mitochondrial function
Samples of 5 × 10 6 spermatozoa/mL were stained for 18 min at 37°C with Mitotracker Green.This compound can diffuse passively across the plasma membrane, once they enter cells they contain a slightly thiol-reactive chloromethyl moiety to label active mitochondria, accumulating in them.Following the manufacturer's instructions and subsequently analyzed by flow cytometry.Optimally excites at 490 nm and has a peak emission at 516 nm.

| Statistical analysis
Results for the sperm samples (10 6 /mL) of the three epididymal

| RESULTS
The length and weight of the epididymis of male C. mexicanus bats change during the reproductive phase.Figure 1  presented as percentages that were transformed to arco-sin ([x] 1/2 ).
Figure 2 shows our results for EPS.Before mating, exteriorization was low in all regions of the epididymis (F (2,6) = 3.52, p = 0.06), being the least in the cauda (0.001 arco-sin), but it occurred most frequently during Mating phase (F (2,6) = 6.33, p = 0.03) and After mating (t = 0.72, p = 0.38) reaching values of 0.68 arco-sin and 0.54 arco-sin at the corpus level, respectively.In the last phase, Storage, the cauda presents a value of 0.42 arco-sin.When comparing the EPS between the different reproductive phases in the same epididymal region, a decrease is shown at the caput level as epididymal storage progresses (t = 4.25, p = 0.01), on the contrary, at the corpus level an increase is observed towards the Mating phase and a decrease towards the After mating (F (2,6) = 7.2, p = 0.02).Finally, at the cauda level, although a variation is observed as time progresses, there are no statistical differences (F (3,8) = 3.5, p = 0.06).
It has been postulated that caspases may play a role in sperm maturation.To find out if these enzymes are present in the sperm of the C. mexicanus, we analyzed epididymal sperm using flow cytometry (Supporting Information: Figure 2) to find out if there is an increase or decrease in caspases in relation to the different study stages and between epididymal regions.The number of resulting events is presented as percentages that were transformed to arco-sin ([x] 1/2 ).
Mitochondrial functionality may reflect sperm activity.To find out if its functionality is associated with the integrity the sperm of the C. mexicanus, we analyzed its integrity in epididymal sperm using flow cytometry (Supporting Information: Figure 3) to find out how it is in relation to the different study stages and between epididymal regions.
The number of resulting events is presented as percentages that were transformed into arc-sin ([x] 1/2 ).In the Mating phase, a high mitochondrial membrane potential was observed that varied for the three regions of the epididymis (F (2,6) = 8.59, p = 0.01) and increased from caput (1.11 arco-sin) to cauda (1.48 arc-sin).During the After mating stage, there are no differences between regions (t = 1.89, p = 0.13) showing less heat in the caput (1.24 arco-sin) than in the cauda (1.41 arc-sin).In the final stage (Storaged), the cauda shows a value of 1.12 arc-sin.When comparing the integrity of the membrane obtained between the different storage phases in the same region of the epididymis, no variation is observed at the corpus level (t = 2.11, p = 0.10), with a value of 1.44 arco-sin for the Mating phase and a value of 1.24 arco-sin for After mating.At the cauda level, a statistical variation between stages is observed (F (2,6) = 31.32,p = 0.0006), which decreases from caput to cauda, with 1.48 arco-sin in Mating phase, 1.41 arco-sin in After mating and 1.12 arco-sin in Storage.

| DISCUSSION
C. mexicanus presents a monoestrous seasonal reproductive patter, with a temporary asynchrony between the reproductive functions.Spermatogenesis occurs in summer, while maximum development of the epididymides and accessory sex glands, courtship, and mating all occur in autumn (León-Galván et al., 2005), the period when females are sexually receptive (León-Galván et al., 2005, 1999).As a consequence of this asynchrony, sperm is stored in the epididymis for approximately 6 months (September−February). (Arenas-Ríos et al., 2005;León-Galván et al., 2005;Rodríguez-Tobón et al., 2016)so that the male's cycle coincides with that of the female, allowing successful mating and ensuring fertilization (León-Galván et al., 2005, 1999).This means that the male's gametes must be available and viable at the time of mating.However, it has been reported that sperm maturation in the epididymis of C.
Our results show that epididymal weight was greatest during Before mating phase, but gradually decreased until After mating and Storage phases, while peak length occurs during Mating phase.Based on earlier reports, we know that spermatogenesis begins in May and ends in August, when the epididymis receives the sperm to begin their maduration, leading to an increase in size at the beginning of the Before mating phase (Rodríguez-Tobón et al., 2016).Our findings for sperm concentrations in the epididymis matched predictions; that is, a greater number of sperm during Before mating and Mating phase, and fewer during After mating and Storaged (Table 1).One of the most evident physiological changes in sperm during its passage through the epididymis is the acquisition of motility (Lewis & John Aitken, 2001;Rodríguez-Tobón et al., 2016;Sullivan & Mieusset, 2016).Table 1 shows that the sperm analyzed herein were motile during all stages, with higher motility in the cauda, similar to the findings reported by Rodríguez-Tobón et al. (2016), where the motility of the spermatozoa obtained from the caudal region of the epididymis remained above 80%, 80%, as occurs in the Before mating phase.Similarly, sperm viability was maintained at around 80% in all epididymal regions and in all reproductive phases.In this regard, it is important to note that the gametes remained alive even after 6 months of storage in the epididymis (Cervantes et al., 2008;León-Galván et al., 2005), as occurs in the Storage phase.
Although the exact mechanisms that allow sperm to remain viable for so long are unknown, it has been hypothesized for strictly hibernating bats that the high osmolarity of the epididymal fluid could favor a state of quiescence or inactivity in the sperm that allows them to remain alive for long periods (Crichton et al., 1993(Crichton et al., , 1994)).
However, for the case of the C. mexicanus bat, which is not a strict hibernator, the participation of antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase has been proposed as auxiliaries in regulating the redox state of the epididymal luminal medium (Arenas-Ríos et al., 2005).That being so, additional studies are required to elucidate the mechanisms of prolonged epididymal sperm storage in this bat species.
EPS in spermatozoa has recently been associated with the maturation of spermatozoa in the epididymis.Rival et al. (2019) show that EPS in the membrane of mouse sperm increases as its transits through the epididymis, since if this were not the case then gamete fertility would decrease.However, that study was conducted with a species characterized by continuous reproduction in which epididymal sperm maturation ends before the spermatozoa reach the caudal region.Our results did not show this behavior, as externalization was lower in the cauda region, perhaps reflecting the observation that the spermatozoa of C. mexicanus are not fully mature when they reach the cauda (Cervantes et al., 2008;Rodríguez-Tobón et al., 2020, 2016), as occurs in other species (Lewis &John Aitken, 2001 andSullivan &Mieusset, 2016), however, EPS are present in the Mating phase.The spermatozoa have to remain in the cauda to complete their maturational process (James et al., 2020).However, further research is needed to verify that EPS increases directly proportional to sperm maturation.
In other cell models, EPS is a signal for phagocytes (by the immune system) to phagocytose sperm cells without triggering an inflammatory response (Elmore, 2007;Fadok et al., 2001;Green & Llambi, 2015).In the epididymis, the principal cells are responsible for removing damaged sperm from the epididymal lumen (Robaire et al., 2006).Although the recognition mechanism between these two cell types is unknown, we propose that EPS could participate in the process of removal of epididymal sperm that were not ejaculated during Mating phase in this species of bat.However, more studies are needed to verify this assumption.
Another important factor that remains to be elucidated concerns large number of sperm that showed active caspases, especially during Before mating and Mating phases (Figure 3).These data could be interpreted as indicating the death of the sperm; however, Table 1 shows that the sperm remain alive in both the corpus and cauda.López-Trinidad et al. (2017) reported this same behavior in rat epididymal sperm.Various cell types (such as keratinocytes or lens fibers) undergo post-meiotic terminal differentiation that involves the elimination of most of their organelles (Nhan et al., 2006).This process of requires the action of active effector caspases to execute rapid, irreversible changes during cell differentiation.This hypothesis has been called "catastrophic proteolysis" (Nhan et al., 2006), a phenomenon similar to what could be occurring during sperm formation and maturation.However, more research is required to corroborate this event.
The mitochondrial membrane potential in the sperm of C. mexicanus was high (Figure 4).Several authors have reported that oxidative phosphorylation in the mitochondria is not the main source of the ATP used for the physiological functions of sperm (Miki, 2007) but it contributes to the production of ROS which promote the protein phosphorylation (Park & Pang, 2021), an essential process for sperm maturation (Vernet et al., 2001), especially in this animal model, since during Mating phase the sperm must have a certain degree of maturation and the ROS could favor this process.

| CONCLUSION
Sperm from the testes begin to enter the epididymis during Before mating, causing this organ to grow, but they do not yet have the ability to fertilize.Later, during Mating phase in this specie the bat, the spermatozoa present large amounts of active caspases and EPS, even while still alive.This suggests that these two markers could help in the processes of maturation before and during the copulation period and later elimination during the storage respectively, but additional research is needed to elucidate these claims.
FLICA (Vybrant ® FAM Caspase-3 and −7 Assay Kit (V35118) Molecular Probes).Annexina V-FLUOS Staining kit (Roche).Mitotracker Green (MitoTracker ® Mitochondrion-Selective Probes, Invitrogen-Molecular Probes).Ringer's solution (94.6 mM NaCl, 4.78 mM KCl, 1.71 mM CaCl 2 •2H 2 O, 1.19 mM MgSO 4 •7H 2 O, regions (caput, corpus, and cauda) were compared, as well as the collection dates from the four reproductive phases.A Shapiro −Wilk statistical test was used to verify the normality of the data.When the data showed normality, a Tukey−Kramer Multiple-Comparison Test was performed.When this was not the case, a Kruskal−Wallis Z test was used with p < 0.05.The percentages data were transformed to arco-sin ([x] 1/2 ).All tests were run in the GraphPad Software.Due to pandemic issues (COVID-19), it was not possible to use the Before mating data for mitochondrial membrane tests.
21 a,b 86.33 ± 5.50 a,b 80.33 ± 5.50 b Note: Mean value are represented ±SD.Letters represent significant differences between phases of storage (Kruskal−Wallis Z test, p < 0.05).Asterisks represent a significant difference with respect to the anterior epididymal anatomical region (Kruskal−Wallis Z test, p < 0.05).F I G U R E 2 Externalization of phosphatidylserine (EPS) in the plasmatic membrane of sperm obtained from the caput, corpus, and cauda of the epididymis of the bat C. mexicanus in the four reproductive phases (Before mating, n = 3; Mating phase, n = 3; After mating, n = 3; Storage, n = 3).The percentages data were transformed to arco-sin ([x] 1/2 ).Violin plot are shown to visualize the distribution of the data, the horizontal line in each bar represents the median.Letters represent significant differences between epididymal regions (Kruskal−Wallis Z test, p < 0.05).Asterisks represent a significant difference with respect to the anterior storage phase in the same epididymal region (Kruskal−Wallis Z test, p < 0.05).

Figure 3
Figure3shows our results for active caspases 3 and 7. Before mating, although the presence of caspases varies between epididymal regions, being the highest in the corpus (0.61 arco-sin) and lowest in the cauda (0.41 arco-sin), there are no statistical differences (F (2,6) = 0.18, p = 0.83).During the Mating phase, it was observed more frequently and with an increasing variation (F (2,6) = 6.33, p = 0.03) from caput (0.99 arco-sin) to cauda, reaching a value of 1.43 arco-sin.In the After mating stage, values of 0.27 arco-sin and 0.58 arco-sin are shown for corpus and cauda respectively, but there is no difference between them (t = 1.33, p = 0.25).In the last phase, Storage, the cauda presents a value of 0.27 arc-sin.When comparing the percentages of caspases obtained between the different storage phases in the same epididymal region, a variation is shown at the caput level as epididymal storage progresses;

F
I G U R E 3 Active caspases 3 and 7 in sperm obtained from the caput, corpus, and cauda of the epididymis of the bat Corynorhinus mexicanus bat in the four reproductive phases (Before mating, n = 3; Mating phase, n = 3; After mating, n = 3; Storage, n = 3).The percentages data were transformed to arco-sin ((x) 1/2 ).Violin plot are shown to visualize the distribution of the data, the horizontal line in each bar represents the median.Letters represent significant differences between epididymal regions (Kruskal−Wallis Z test, p < 0.05).Asterisks represent a significant difference with respect to the anterior storage phase in the same epididymal region (Kruskal−Wallis Z test, p < 0.05).

F
Mitochondrial membrane potential in sperm obtained from the caput, corpus, and cauda of the epididymis of the bat Corynorhinus mexicanus bat in the three reproductive phases (Before mating, n = 3; Mating phase, n = 3; After mating, n = 3; Storage, n = 3).The percentages data were transformed to arco-sin ([x] 1/2 ).Violin plot are shown to visualize the distribution of the data, the horizontal line in each bar represents the median.Letters represent significant differences between epididymal regions (Kruskal−Wallis Z test, p < 0.05).