Cell signaling in sperm midpiece ensures quiescence and survival in cauda epididymis

in Reproduction
Authors:
Archana DeviDivision of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, India
Academy of Scientific and Innovative Research, CSIR-HRDC Campus, Ghaziabad, India

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Bhavana KushwahaDivision of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, India
Academy of Scientific and Innovative Research, CSIR-HRDC Campus, Ghaziabad, India

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Jagdamba P MaikhuriDivision of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, India

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Rajender SinghDivision of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, India
Academy of Scientific and Innovative Research, CSIR-HRDC Campus, Ghaziabad, India

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Gopal GuptaDivision of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, India
Academy of Scientific and Innovative Research, CSIR-HRDC Campus, Ghaziabad, India

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Correspondence should be addressed to G Gupta; Email: g_gupta@cdri.res.in
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Sperm in most mammalian species including rat, mice and human are kept completely quiescent (motionless) and viable for up to a few weeks in the cauda epididymis before ejaculation. Vigorous motility is initiated almost instantly upon sperm release from cauda during ejaculation. The molecular mechanisms that suppress sperm motility but increase cell survival during storage in cauda epididymis are not known. Intracellular signaling via phosphorylation cascades is quick events that may regulate motility and survival of transcriptionally inactive sperm. Pathscan intracellular signaling array provided the preliminary picture of cell signaling in quiescent and motile rat sperm, indicating upregulation of cell-survival pathways in quiescent sperm, which were downregulated during motility activation. Interactome of signaling proteins involved in motility activation was constructed by Search Tool for the Retrieval of Interacting Genes (STRING) software, which identified mitogen activated protein kinase-p38 (MAPK-p38), AKT, mTOR and their downstream target p70S6K as the key kinases regulating sperm function. Further validation was achieved by western blotting and pathway activators/inhibitors. Immunofluorescence localized the kinase proteins in the sperm mid-piece region (mitochondria), a known extra-nuclear target for these signaling pathways. Activators of these kinases inhibited sperm motility but increased viability, and vice versa was true for inhibitors, in most of the cases. Activators and inhibitors also affected sperm mitochondrial membrane potential, ATP content and reactive oxygen species (ROS) levels. Data suggest that sperm motility and survival are inversely complementary and critically regulated by intracellular cell signaling. Aberrant cell signaling in caudal sperm may affect cell survival (sperm concentration) and motility of ejaculated sperm.

Abstract

Sperm in most mammalian species including rat, mice and human are kept completely quiescent (motionless) and viable for up to a few weeks in the cauda epididymis before ejaculation. Vigorous motility is initiated almost instantly upon sperm release from cauda during ejaculation. The molecular mechanisms that suppress sperm motility but increase cell survival during storage in cauda epididymis are not known. Intracellular signaling via phosphorylation cascades is quick events that may regulate motility and survival of transcriptionally inactive sperm. Pathscan intracellular signaling array provided the preliminary picture of cell signaling in quiescent and motile rat sperm, indicating upregulation of cell-survival pathways in quiescent sperm, which were downregulated during motility activation. Interactome of signaling proteins involved in motility activation was constructed by Search Tool for the Retrieval of Interacting Genes (STRING) software, which identified mitogen activated protein kinase-p38 (MAPK-p38), AKT, mTOR and their downstream target p70S6K as the key kinases regulating sperm function. Further validation was achieved by western blotting and pathway activators/inhibitors. Immunofluorescence localized the kinase proteins in the sperm mid-piece region (mitochondria), a known extra-nuclear target for these signaling pathways. Activators of these kinases inhibited sperm motility but increased viability, and vice versa was true for inhibitors, in most of the cases. Activators and inhibitors also affected sperm mitochondrial membrane potential, ATP content and reactive oxygen species (ROS) levels. Data suggest that sperm motility and survival are inversely complementary and critically regulated by intracellular cell signaling. Aberrant cell signaling in caudal sperm may affect cell survival (sperm concentration) and motility of ejaculated sperm.

Introduction

In most mammals (including mouse, rat and human), sperm motility is activated at the time of ejaculation and motile sperm are deposited directly into the female genital tract. Potentially motile and fully mature sperm are stored quiescent (motionless) in the cauda epididymis in a viable state for up to 6 weeks, with highly suppressed metabolism (James et al. 2020) that delivers enough ATP to maintain resting membrane potential (viability). The internal fertilization process in mammals is an energy extensive exercise for the sperm that need to propel through a considerable distance in the female genital tract to reach the site of fertilization (Zhang et al. 2016), and therefore they spend nearly 70% of their energy in motility. Hence activation of a burst of vigorous sperm motility at ejaculation is crucial for male fertility. Undoubtedly, asthenozoospermia is a major cause of male infertility in majority of infertile men with abnormal semen parameters (Shahrokhi et al. 2020). Once activated during ejaculation, it is practically impossible to reversibly inhibit sperm metabolism and motility (Jones & Murdoch 1996) under normal conditions; yet potentially motile sperm are stored completely quiescent in cauda epididymis for several weeks (Turner 2008). The molecular mechanism that suppresses sperm motility/metabolism in the cauda epididymis and initiates it during ejaculation is still an enigma.

The second important aspect of sperm storage in cauda epididymis is its longevity. It has been well established that sperm may spend up to a few weeks in the cauda epididymis without losing viability and motility potential, before being ejaculated (Jones 2004), while the superfluous/defective sperm are eliminated by altered signaling. The nature’s robust cell-survival processes ensure prolonged sperm viability and functionality under extreme stressful conditions of cell crowding (Kempinas & Lamano-Carvalho 1988), limited nutrition (Turner 2002), suppressed respiration (Murdoch et al. 1999), hyperosmotic environment (Si et al. 2009) and continuous oxidative stress (Aitken et al. 2012) in the cauda epididymis. However, the molecular processes that maintain sperm viability under such stressful conditions have not been elucidated.

Signaling pathways operated by the phosphorylation of tyrosine, serine and threonine residues of proteins are the major regulators of scaffold anchoring proteins (Mugabo & Lim 2018), e.g. AKAP4, that play a deciding role in mammalian sperm flagellar motility (Miki et al. 2002). On the other hand, it has been clearly shown in other biological systems that intracellular signaling through reversible phosphorylation modulates mitochondrial function for ATP production (Lucero et al. 2019), which is a crucial deciding factor for vigorous sperm motility (Tourmente et al. 2015). Furthermore, an optimal concentration of mitochondrial reactive oxygen species (ROS) keeps a balance between sperm quiescence and apoptosis in cauda epididymis (Aitken et al. 2012), which may help in cell survival by gearing up the cell’s antioxidant defense system as well as activating intracellular cell-survival pathways by maintaining optimum levels of ROS (Gutiérrez-Uzquiza et al. 2012). Generally, mitogen-activated protein kinase (MAPK) pathways are mostly associated with cell-survival signaling in cancer cells. With this background we used an intracellular cell-signaling array to identify the major signaling pathways that are modified during motility initiation of caudal sperm at ejaculation in the rat model. Some of the key targets were picked up for further validation in rat sperm.

Materials and methods

Reagents and antibodies

Sorbitol (S-1876), retinoic acid (R-2625), MHY1485 (500554) and rapamycin (R-8781) were purchased from Sigma-Aldrich. SB203580 (ab12016), LYS6K2 (ab146199), SC79 (ab146428) and Akti-1/2 (ab142088) were purchased from Abcam. Phospho-p38MAPK (4511S), p38MAPK (9212S), phospho-AktSer473 (4060S), Akt (9272S), p-mTOR (5536S) and mTOR (2983S) antibodies were purchased from Cell Sgnaling Technology (CST) phospho-p70S6K (701064), p70S6K (PA5-28597) antibodies were from Invitrogen. Hank’s balanced salt solution (HBSS) and all other chemicals/reagents were purchased from Sigma-Aldrich, unless stated otherwise.

Isolation of quiescent and motile sperm from rat cauda epididymis

Sprague–Dawley male rats (aged 16–20 weeks) were obtained from the Laboratory Animal Division of CSIR-Central Drug Research Institute, Lucknow, India. All animal experiments and protocols were approved by the Institutional Animal Ethics Committee for the Use of Laboratory Animals, CSIR-CDRI, Lucknow, India. Immediately at autopsy, one epididymis was excised and rinsed briefly in PBS, small incisions were made in the cauda segment (Supplementary Fig. 1, see section on supplementary materials given at the end of this article) and placed in HBSS for 10–15 min with light shaking at 37°C for sperm to swim out. The sperm gained vigorous motility (motile sperm sample). Simultaneously, the other epididymis was wrapped in aluminum foil and placed on ice for 15 min to cool the sperm in situ. Sperm cooled in the epididymis maintain membrane and motility parameters which are comparable to ejaculated sperm (Martínez-Fresneda et al. 2019). Thereafter a few small nicks were made and the cauda segment was shaken vigorously in ice-cold HBSS. Here, the sperm could not attain motility and remained quiescent (Kumar et al. 2016). Both the samples were subsequently washed with sterile, ice-cold PBS and centrifuged at 700 g for 5 min under refrigeration, resuspended in cold PBS and were used for further experiments. For motility measurements, caudal sperm were directly diluted in HBSS and assessed for motility parameters in the presence or absence of pathway activators/inhibitors (Supplementary Fig. 2).

Intracellular signaling array analysis of quiescent and motile rat sperm

An antibody-based Pathscan intracellular signaling membrane array (#14471, CST), was used for simultaneous detection of 18 key intracellular signaling molecules in rat sperm. Quiescent and motile rat sperm were washed thrice in PBS at 4°C and lysed according to the manufacturer's instructions. The protein concentrations were measured by Bradford assay and diluted to 250 µg/mL. The multiplexed array membranes were incubated in these samples separately at 4°C overnight with orbital shaking. The blots were developed with Enhanced Chemiluminescence (ECL) kit (EMD Millipore) and imaged in Image Quant LAS4000 (GE healthcare). Spot intensities of the targets are based on either their phosphorylation or cleavage. ‘Quantity one’ software (Bio-Rad) was used to quantitate spot intensity, which was then normalized to the internal positive controls provided on the membrane.

Protein–protein interaction network analysis

The differentially activated intracellular signaling proteins in quiescent and motile sperm were further analyzed by constructing their protein–protein interaction (PPI) map to predict their interactions, using the STRING online software (STRING 11.0 database). The constructed interactome predicts the type and strength of interactions among identified targets. For each protein, the related gene name was inserted into the input and Rattusnorvegicus was selected as the reference organism. The generated output network of proteins show connections with each other and highlights the probable mode of regulation of these proteins (Kushwaha et al. 2021).

Validation of quiescent sperm preparation by immunohistochemistry of caudal sperm in situ

At autopsy rat cauda was excised, rinsed and fixed in 4% formalin. Later tissues were dehydrated, embedded in paraffin and sectioned with a microtome. The sections (thickness 5 µm) were stained with hematoxylin and eosin (H&E) for routine morphological examination. For immunohistochemistry, tissue sections were deparaffinized and hydrated followed by antigen retrieval with sodium citrate buffer (pH 6) for 30 min at 95°C. Thereafter, sections were treated with 3% hydrogen peroxide for 10 min to inhibit endogenous peroxidase activity and further incubated with the blocking solution (2% BSA in PBS containing 0.1% tween (PBST)) for 1 h to block nonspecific antigen-binding sites. Subsequently, sections were incubated with phospho-p70S6K antibody at 1:100 dilution in a humidified chamber at 4°C overnight. The sections were washed three times with PBST and incubated with 1:500 dilution of horseradish peroxidase (HRP) conjugated secondary antibody for 1 h at room temperature. After washing with PBST, antibody detection was performed with diaminobenzidine + substrate chromogen system (DAB150 kit, Merck Millipore), counterstained with hematoxylin (for nuclear staining), dehydrated and mounted. Images were captured under a phase contrast microscope (Nikon eclipse 80i). For comparison, motile sperm released from cauda epididymis were taken up for 3,3′-diaminobenzidine (DAB) staining. Cells were isolated in HBSS at 37°C and fixed with 4% PFA, permeabilized with 0.5% Triton-X-100 followed by 3% hydrogen peroxide treatment and further processed as mentioned above for tissue sections. For immunohistochemical detection p-p70S6K was selected as it is the downstream target for several other signaling pathways and also has a strong signal in quiescent sperm, which could easily be detected in situ.

Western blotting

Sperm samples were lysed in radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitor cocktail, incubated at 4°C for 2 h followed by sonication. After sonication, samples were centrifuged at 20,000 g at 4°C for 30 min and protein concentration of supernatant was estimated by Bradford assay, mixed with loading buffer and boiled at 100°C for 10 min. An equal amount of protein was loaded in each well and resolved on 8–12% SDS polyacrylamide gels by electrophoresis. Further, the protein bands were transferred onto a PVDF membrane (Immobilon-P PVDF membrane, Millipore) and were incubated for 1 h with 2% BSA in Tris buffer saline containing 0.1% tween (TBST) to block non-specific sites. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies at appropriate dilutions: phospho-p38MAPK (1:1000), p38MAPK (1:1000), phospho-p70S6K (1:1000), p70S6K (1:1000), Akt (1:1000) and pAktSer473 (1:1000), mTOR (1:1000), phospho-mTOR (1:1000) in TBST with 2% BSA. Thereafter, membranes were washed three times with TBST for 10 min each and incubated in HRP-linked to an appropriate secondary antibody at a dilution of 1:3000 (for all) for 1 h at room temperature. After incubation, membranes were washed three times with TBST and developed on Image Quant LAS4000 using ECL kit.

Immunofluorescence

Rat sperm (quiescent, motile and treated) were centrifuged at 1000 g for 5 min and pellets were re-suspended in PBS. Then, the cells were placed on 0.1% poly-l-lysine coated slides and fixed with 4% paraformaldehyde for 1 h at room temperature. Thereafter, cells were washed three times for 5 min each with PBS and permeabilized with 0.5% Triton X-100 for 15 min, washed again for three times and blocked with 2% BSA for 1 h at room temperature to prevent non-specific binding. After that, samples were incubated overnight in a humidified chamber with primary antibodies: p-p38 (1:200), p-p70S6K (1:500), p-AktSer473 (1:400) and p-mTOR (1:50), at 4°C. Subsequently, cells were washed three times with PBST and incubated with fluorescence-tagged (Alexa546) secondary antibodies for 1 h in darkness. After incubation, slides were washed with PBST and a small drop of antifade reagent with DAPI was placed on the slide for nuclear staining. The stained sections were examined under a fluorescence microscope (Nikon 80i), and images were captured with the NIS Elements F3.0 camera. The fluorescence intensities were also measured in the captured images by ‘Image-J’ software for quantitative analysis.

Motility analysis of sperm treated with p38MAPK, AKT, mTOR and p70S6K activators and inhibitors

Caudal rat sperm were diluted directly with HBSS containing p38MAPK activator sorbitol (0.5 M), p38MAPK specific inhibitor SB203580 (25 µM), p70S6K activator retinoic acid (1 mM), p70S6K inhibitor LYS6K2 (100 µM), Akt activator SC79 (50 µM), Akt inhibitor Akti-1/2 (50 µM), mTOR activator MHY1485 (50 µM) and mTOR inhibitor rapamycin (25 µM). Caudal sperm diluted in HBSS served as control (motile). Stock solutions of activators and inhibitors were diluted with HBSS to working concentrations. Motility parameters [total motility (%), progressive motility (%), average path velocity (VAP, µ/s), straight line velocity (VSL, µ/s), curvilinear velocity (VCL, µ/s), amplitude of lateral head displacement (ALH, µ) and beat cross frequency (BCF, Hz)] were studied using the Hamilton Thorne Computer Assisted Sperm Analyzer (CASA; IVOS, version 12.3, Hamilton Thorne Inc. USA). Ten microliters sample were placed on pre-warmed (37°C) slides to record motility.

Mitochondrial membrane potential analysis

The sperm mitochondrial membrane potential (MMP) was measured using JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimi-dazolylcarbocyanine iodide) fluorescent dye and flow cytometry. The cyanide-based cationic fluorescent JC-1 dye measures ΔΨm by translocating into the cells and forming aggregates of JC-1 with orange-red fluorescence in mitochondria with polarized membranes. In depolarized mitochondria, JC-1 exists in monomers which emit green fluorescence. Briefly, sperm were washed and re-suspended in PBS equilibrated with 10 µM JC-1 at 37°C for 30 min. After washing with PBS re-suspended in the same buffer and MMP was recorded by a flow cytometer (FACS Calibur, BD Biosciences) at Ex/Em = 488/530 nm; 10,000 gated events were analyzed per sample.

Sperm ATP content

Sperm ATP content was measured using an ATP Assay Kit (Abcam, ab83355). Briefly, equal number of sperm were taken and washed with cold PBS. Cell lysates were prepared according to the manufacturer’s instructions, added to wells with reaction mixture and incubated for 30 min in darkness at room temperature. Values of ATP levels were determined by using ATP standard curve plotted by fluorometric analysis of standard ATP concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 nmol. Fluorescence readings were recorded at Ex/Em = 535/587 nm on a microplate reader. Analyses were performed in triplicate (n = 3).

Estimation of ROS

To detect intracellular ROS level in control and treated sperm, nonfluorescent, cell-permeable dye 2′,7′-dichlorofluorescein diacetate (DCFDA) was used, which on oxidation by ROS, is irreversibly converted to highly fluorescent 2',7'-dichlorofluorescein (DCF). The fluorescence intensity directly indicates the amount of ROS. An equal number of sperm cells were washed with PBS and re-suspended in 10 µM DCFDA for 30 min at 37°C. At the end of the incubation period cells were washed with PBS, and fluorescence was measured by Flow cytometer (FACS Calibur, BD Biosciences) at 525 nm in the FL-1 green channel; 20,000 gated events were analyzed per sample.

Rat sperm viability test by trypan-blue staining

Dye exclusion method was used to determine rat sperm viability in control and treated cells. The sperm cells were isolated from cauda epididymis in vehicle (HBSS, control) or solutions of inhibitors/activators of different pathways (in HBSS) at concentrations mentioned above, and incubated for 60 min at 37°C. Supravital staining with 2% Trypan blue was used, which is a diazo dye derived from toluidine, at the end of the experiment. This negatively charged vital stain shows great binding affinity for membranes and specifically colors dead cells, whereas live cells with intact cell membrane exclude the dye. Sperm suspensions were mixed with the dye for 3 min at room temperature and were visually examined under a light microscope (Nikon eclipse 80i). Dead (stained) and live (unstained) sperm cells were counted in ten different fields (total ~200 cells).

Statistical analysis

Results were analyzed by nonparametric, two-tailed Student’s t-test using PRISM GraphPad 8.1.2 software and have been expressed as mean ± s.e.P-values <0.05 were considered as statistically significant.

Results

Quiescent sperm maintain active intracellular signaling in rat cauda epididymis

The intracellular signaling array (18 targets) revealed marked upregulation of phosphorylation/cleavage of several targets in quiescent sperm as compared with motile sperm. The major targets that were kept phosphorylated in caudal sperm included p38 MAPK (pT180/pY182), HSP27 (pSer78), p70S6K (Thr389), AKT (Ser473), mTOR (pSer2448) Bad (pSer112), GSK-3β (pSer9), JNK/SAPK (pT183/pY185), PRAS40 and p53 (pSer15). The cleavage levels of PARP (at D214) and Caspase-3 (at D175) were also higher in quiescent sperm cells. All these targets were dephosphorylated during motility activation. The cleavage of PARP and Casp-3 were also declined in motile cells (Fig. 1A, B and C).

Figure 1
Figure 1

Alterations in rat sperm cell intracellular signaling during motility initiation. (A) Intracellular signaling array analysis of 18 key phosphorylated/cleaved targets (proteins) in quiescent and motile rat sperm cell lysates; (B) Array layout chart, (C) Target proteins, their modification sites and types; (D) Proteins–protein interaction (PPI) network of array proteins by STRING software, the color of lines denote mode of action; (E) Mutual interactions among the four key targets viz. p38MAPK, AKT, mTOR and p70S6K that apparently play the central role in the intracellular interactome.

Citation: Reproduction 162, 5; 10.1530/REP-21-0202

PPI-network analysis of sperm proteins identified in signaling array

To predict interactions among sperm cell-signaling proteins identified by the antibody array, a PPI network was constructed using the STRING software, which assembles the PPI interactome on the basis of molecular actions and confidence levels. The different colors of lines denote mode of action and thickness represents the strength of interaction (Fig. 1D). All the array targets were found to be grossly interacting with each other to form the sperm interactome. We identified four key targets viz. p38MAPK, AKT, mTOR and p70S6K, which apparently played a central role in this interactome (Fig. 1E). These signaling proteins exhibited major change in activity during motility initiation of quiescent spermatozoa. The signaling pathways governed by the selected targets are interconnected by several modes of action with other signaling proteins, chiefly by phosphorylation and were thus taken up for further validation.

Validation of a key intracellular signaling target in rat caudal sperm in situ

To further validate our finding in the isolated quiescent sperm preparation, we also checked the expression level of one important downstream target of intracellular cell signaling in quiescent caudal-sperm, in situ. The activation (phosphorylation) level of p70S6K in quiescent sperm was evaluated by immunohistochemical DAB staining of caudal sections (Fig. 2A) and compared with similarly stained motile sperm isolated from the cauda epididymis that were fixed on poly-lysine coated slides. The quiescent sperm in caudal lumen (tissue sections) had deeply stained sperm tails (midpiece), indicating activated (phosphorylated) p70S6K. Comparatively, the motile sperm were weakly stained (Fig. 2B). However, this difference was seen more distinctly by western blotting and immunofluorescence, as demonstrated later in Fig. 3.

Figure 2
Figure 2

Immunohistochemical localization of phospho-p70S6K in rat caudal sperm in situ and in isolated quiescent/motile sperm. (Ai) Hematoxylin-and-eosin stained sections of rat cauda epididymis showing normal morphology at 10× (bar = 20 µm) and 40× (bar = 10 µm). (Aii) Immunohistochemical (DAB) staining of cauda epididymis sections for phospho-p70S6K at 10× (bar = 10 µm) and 40× (bar = 10 µm) showing deeply stained sperm tails and (B) comparison of similarly stained quiescent and motile sperm isolated from cauda epididymis and fixed on poly-lysine coated slides at 40× (bar = 10 µm) and 100× (bar =1 µm).

Citation: Reproduction 162, 5; 10.1530/REP-21-0202

Figure 3
Figure 3

Phosphorylation of p38, Akt, mTOR and p70S6K in quiescent and motile sperm, the effect of activators/inhibitors on protein phosphorylation levels and immunofluorescent localization of phosphorylated p38, Akt, mTOR and p70S6K in rat sperm. Phosphorylation of (A) p38, (B) Akt, (C) mTOR and (D) p70S6 kinases in quiescent, motile and activator/inhibitor treated cells by immunoblotting (upper panel); and statistical analysis of immunoblots (lower panel). Localization of (E) phosphorylated (activated) p38 in quiescent, motile, sorbitol (activator) and SB203580 (inhibitor) treated sperm; (F) phosphorylated Akt in quiescent, motile, SC79 and Akti-1/2 treated sperm; (G) phosphorylated mTOR in quiescent, motile, MHY1485 and rapamycin treated rat sperm; and (H) phosphorylated p70S6K in quiescent, motile, RA (retinoic acid) and LYS6K2 treated sperm. Images were captured by fluorescence microscopy at 100× (bar = 1 µm) for p70S6K, Akt and mTOR and for p38 at 60× (bar = 20 µm) (fluorescence tags: Alexafluor (red); nucleus staining by DAPI (blue)). (Quantitative assessment of fluorescent intensities by Image-J software is provided in Supplementary Fig. 3.)

Citation: Reproduction 162, 5; 10.1530/REP-21-0202

Activation by phosphorylation of p38MAPK, Akt, mTOR and p70S6K helps in maintaining quiescence of caudal sperm

Immunoblotting studies showed significantly increased phosphorylation (activation) of p38 (P < 0.01), Akt (P < 0.05), mTOR (P < 0.05) and p70S6K (P < 0.05) in quiescent sperm, as compared with motile sperm. Phosphorylation levels were found to be accordingly and significantly modulated by adding respective kinase activator or inhibitor in the caudal semen diluent (HBSS) during sperm motility activation (Fig. 3A, B, C and D). These results were in line with the signaling array (Fig. 1A) and immunofluorescence (Fig. 3E, F, G and H) results and their consequent effect on sperm motility (Fig. 4).

Figure 4
Figure 4

Effect of inhibition and activation of intracellular signaling proteins on rat sperm motility parameters during motility initiation. Sperm motility parameters analyzed by CASA in sperm isolated from cauda epididymis and diluted in vehicle HBSS (motile); p38MAPK-activator sorbitol (0.5 M), -inhibitor SB203580 (25 µM); p70S6K-activator retinoic acid (RA, 1 mM) -inhibitor LYS6K2 (100 µM); AKT-activator SC79 (50 µM), -inhibitor Akti-1/2 (50 µM) and mTOR-activator MHY1485 (50 µM), -inhibitor rapamycin (25 µM). Motility parameters were analyzed by computer-assisted sperm analyser (CASA). (A) total motility, (B) progressive motility, (C) average path velocity (VAP), (D) straight line velocity (VSL), (E) curvilinear velocity (VCL), (F) amplitude of lateral head displacement (ALH), (G) beat cross frequency (BCF), (H) straightness (STR) and (I) Linearity (LIN). Mean (±s.e.); significant difference from control (motile) is denoted as *P < 0.05; **P < 0.01; ***P < 0.001; ns (nonsignificant).

Citation: Reproduction 162, 5; 10.1530/REP-21-0202

Localization and validation of activated kinases in quiescent and motile rat sperm

The selected kinases were localized in sperm cells by immunofluorescence. Phosphorylated kinases (p-p38, p-AktSer473, p-mTOR and p-p70S6K) were mainly localized to the tail region, specifically in the mid-piece. This was further validated by use of activators and inhibitors. The phosphorylation (activation) level of p38MAPK was observed mainly in the tail region (mostly in mid-piece) with noticeably higher phosphorylation in quiescent, compared with motile. Sorbitol (p38 activator) increased phosphorylation of p38 in motile sperm mid-piece while it was reduced to negligible levels by the inhibitor SB203580 (Fig. 3E). AktSer473 phosphorylation was conspicuously higher in quiescent compared to motile. Akt specific activator SC79 further increased the level of AktSer473 phosphorylation, while Akt inhibitor Akti-1/2 mildly reduced its phosphorylation levels (Fig. 3F). Similarly, higher phosphorylation of mTOR in rat sperm mid-piece was dephosphorylated during motility activation in HBSS. The presence of its activator MHY1485 kept mTOR phosphorylated in sperm mid-piece and its inhibitor rapamycin reduced its activation to negligible levels (Fig. 3G). Expectedly, the downstream effector of these kinases, p70S6K, was highly phosphorylated in quiescent sperm mid-piece compared with motile. The presence of p70S6K activator (retinoic acid) increased the phosphorylation of p70S6K in motile sperm while its inhibitor (LYS6K2) reduced phosphorylation to negligible levels (Fig. 3H). The fluorescence levels were also quantitated by ‘Image-J’ software and the data indicated statistically significant changes (Supplementary Fig. 3).

Regulation of sperm motility by MAPK-p38, Akt, mTOR and p70S6K

To analyze the role of p38, Akt, mTOR and p70S6K signaling in sperm motility, activators/inhibitors of these targets were used. Quiescent sperm from cauda epididymis were directly diluted in media (HBSS) containing these protein activators/inhibitors and motility activation in comparison with vehicle (HBSS) was analysed using CASA. Results have indicated that the total motility (%) of vehicle (HBSS) diluted rat spermatozoa (54.33 ± 0.76%) (supporting video: https://youtu.be/sq--ZeaC6hI) was significantly decreased in presence of p38 activator sorbitol (0.5 M, 3 ± 1%, P < 0.001) (supporting video: https://youtu.be/IOhfIfD-CsY), p70S6K activator retinoic acid (13 ± 0.5%, P < 0.001), Akt activator SC79 (35.67 ± 0.29%, P < 0.001) and mTOR activator MHY1485 (10.67 ± 0.29%, P < 0.001) in HBSS. In contrast, the total motility of spermatozoa was significantly increased in presence of p38 inhibitor SB203580 (76.67 ± 0.76%, P < 0.001) (supporting video: https://youtu.be/oY5l3g7LqLU), p70S6K inhibitor LYS6K2 (74.33 ± 0.58%, P < 0.001), Akt inhibitor Akti-1/2 (61.67 ± 1.61%, P < 0.05) and mTOR inhibitor rapamycin (64 ± 1%, P < 0.01) in HBSS (Fig. 4A). Similarly, the progressive motility was also decreased significantly with these activators though among inhibitors only SB203580 (p38 inhibitor) was able to induce marked increase in progressive motility (P = 0.05) (Fig. 4B). These activators and inhibitors also altered other sperm motility parameters such as average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and Beat cross frequency (BCF) to different extents (Fig. 4C, D, E, F and G). However, straightness (STR) and linearity (LIN) remained largely unaffected (Fig. 4H and I).

The p70S6K activity is controlled by p38MAPK, Akt and mTOR signaling in rat sperm

To confirm the interconnectivity of these kinase pathways in sperm (as predicted by the ‘STRING’ interactome), we analyzed the effect of p38MAPK, Akt and mTOR activator/inhibitor on phosphorylation of the downstream effector p70S6K. Results clearly indicate activation of p70S6K in sperm mid-piece by activators of p38 (sorbitol), AKT (SC79) and mTOR (MHY1485). Likewise, there was clear inactivation of p70S6K in sperm mid-piece in the presence of inhibitors of p38 (SB203580), AKT (Akti-1/2) and mTOR (rapamycin). Hence the selected kinases act in a cohort for regulation of rat sperm motility (Fig. 5). The fluorescence intensities were also quantitated by ‘Image-J’ software and the data indicated statistically significant changes in fluorescence of p70S6K by activators/inhibitors of p38, Akt and mTOR pathways (Supplementary Fig. 4).

Figure 5
Figure 5

Effect of p38, AKT and mTOR modulation on phosphorylation of p70S6K. Phosphorylation of p70S6K in rat sperm treated with p38 activator/inhibitor: sorbitol (0.5 M), SB203580 (25 µM); AKT activatot/inhibitor: SC79 (50 µM), Akti-1/2 (50 µM) and mTOR activator/inhibitor: MHY1485 (50 µM) and rapamycin (25 µM), by fluorescence microscopy. Bar = 2 µm for sorbitol, SB203580, SC79, Akti-1/2 and bar =1 µm for MHY1485 and rapamycin (fluorescence tags: Alexafluor [red]; nucleus staining by DAPI (blue)). (Quantitative assessment of fluorescent intensities by Image-J software is provided in Supplementary Fig. 4.)

Citation: Reproduction 162, 5; 10.1530/REP-21-0202

MMP, ATP and ROS levels in quiescent and motile sperm

The investigated kinases were mainly localized in the mid-piece region of sperm tail, which houses the mitochondria; hence levels of MMP, ATP and ROS were investigated. Results indicated that quiescent sperm have the highest polarization of MMP as well as ATP level that was significantly (P < 0.001) higher than the motile sperm. All the activators and inhibitors (sorbitol, retinoic acid, SC79, LYS6K2, Akti-1/2, MHY and rapamycin) significantly reduced MMP and ATP levels. Conversely, MAPKp38 inhibitor SB203580 increased sperm MMP significantly (P < 0.01) and ATP level non-significantly, as compared with untreated motile sperm (Fig. 6A, B and C).

Figure 6
Figure 6

MMP, ATP and ROS level in quiescent and motile sperm and the effect of activators/inhibitors on these mitochondrial parameters. (A) Dot plots for changes in mitochondrial membrane potential (MMP) using JC-1 dye and flow cytometery at Ex/Em = 488/530 nm (B) Statistical data of dot plots. (C) Sperm ATP content, (D) Reactive oxygen species (ROS) levels by DCFDA fluorescence at 525 nm in the FL-1 green channel and (E) its statistical analysis in quiescent, motile and treated rat sperm. Significant difference from motile indicated as *P < 0.05; **P < 0.01; ***P < 0.001, ns, nonsignificant.

Citation: Reproduction 162, 5; 10.1530/REP-21-0202

Mitochondrial functionality may generate ROS. Significantly higher ROS level was also seen in quiescent sperm than in motile sperm (P < 0.001). Amongst activator treated motile sperm, sorbitol and retinoic acid generated maximum level of ROS (P < 0.001). Conversely, ROS was reduced by p70S6K inhibitor LYS6K2 (P < 0.05) and increased by AKT inhibitor Akti-1/2 (P < 0.01). All other compounds (SB203580, SC79, MHY1485 and rapamycin) did not affect the ROS level as compared with motile sperm (Fig. 6D and E).

Role of MAPKp38, AKT/mTOR/p70S6K pathways on sperm viability

Supravital staining with trypan blue was used to test sperm viability at 60 min. The percentage of viability was maximum in quiescent cells (80.6 ± 1.07%) that was reduced significantly (P < 0.001) on motility activation in HBSS alone to 61.15 ± 1.06%. However, AKT activator (SC79) could increase sperm viability significantly above HBSS (71.01 ± 1.31%, P < 0.001) while sorbitol (24.07 ± 0.54%) and retinoic acid (44.88 ± 0.81%) reduced it below HBSS (P < 0.001). The other inhibitors SB203580, LYS6K2, Akti-1/2, rapamycin and the mTOR-activator MHY1485 treatment did not change percent sperm viability significantly, as compared with untreated motile cells (Fig. 7).

Figure 7
Figure 7

Role of MAPKp38, AKT/mTOR/p70S6K pathways in maintaining rat sperm cell viability. Quiescent, motile and inhibitor/activator treated sperm stained with trypan blue (dead) and unstained (live) were counted under light microscope (minimum 200 cells in 10 different fields). Significant difference from quiescent is denoted as C, and from untreated motile is denoted as c, CcP < 0.001, ns, nonsignificant.

Citation: Reproduction 162, 5; 10.1530/REP-21-0202

Discussion

The two utmost vital functions of cauda epididymis are to keep the sperm completely quiescent by suppressing motility and to keep them viable till ejaculation. How these two complementary functions are achieved is still unknown. Nevertheless, the proper functioning of cauda epididymis is crucial for male fertility.

A general observation of intracellular signaling array data indicates that most of the cell-signaling pathways that promote cell survival were found activated in quiescent sperm stored in cauda epididymis as compared with motile (ejaculated) sperm, for example, AKT, mTOR, PRAS40, P70S6K, MAPKp38 and HSP27. Conversely, signaling proteins that promote cell death were inactivated, for example, BAD and GSK3β. Normally these signaling pathways activate transcription factors, which promote the transcription of specific genes for cell survival. Nevertheless in transcriptionally inactive sperm these signaling pathways may use their extra-nuclear targets in mitochondria, which play a major role in cell survival. AKT phosphorylates mitochondrial protein BAD to inhibit apoptosis (Park et al. 2015), while mTOR associated with outer mitochondrial membrane signals cell survival by phosphorylating p70S6K (Desai et al. 2002), which in turn phosphorylates (inactivates) BAD (Harada et al. 2001). Also, during spermatogenesis p70S6K is translocated from nucleus to cytoplasm (Yu et al. 2006) perhaps to support cell survival at mitochondrial level (Harada et al. 2001). Phospho-PRAS40 activates mTOR complex-1 (Nascimento & Ouwens 2009) while p53 phosphorylation can help in mitochondrial DNA damage repair (Chen et al. 2006). On the other hand, HSP27 is a survival protein which inhibits apoptotic cell death by oxidative stress (Garrido et al. 2006) and is activated by phospho-p38MAPK (Larsen et al. 1997).

Besides survival, sperm motility is also dependent on a fully functional mitochondria (Piomboni et al. 2012, Amaral et al. 2013) and incidentally phosphorylation of these signaling proteins is associated with sperm motility inhibition (quiescence) as well (Silva et al. 2015). Sperm use its flagella like a propeller, which is a unique biological motor that rotates continuously at 360° and is considered to be one of the most powerful machines, relative to its size. The rotational torque generated at the base of the flagellum is essential for motility (Subramanian & Kearns 2019), which in case of sperm is a sheath of mitochondria arranged neatly in gyres around the central axoneme complex in mid-piece, with 10‒-12 gyres in human and ~350 in rat sperm (Gu et al. 2019). The importance of mitochondria in mammalian sperm motility is evident by the fact that a strong allometric correlation exists between lengths of flagellum and mid-piece (Cardullo & Baltz 1991).

The four key kinases viz. MAPKp38, AKT, mTOR and p70S6K, playing central role in sperm interactome were selected to investigate their possible role in caudal sperm motility suppression. Interestingly, all these target proteins were found to be chiefly activated (phosphorylated) in the mid-piece region of quiescent and activator-treated rat sperm, which contains the mitochondria (immunofluorescence images) though p70S6K was also constitutively phosphorylated in the sperm acrosome. The activation and deactivation of these proteins had a direct effect on rat sperm motility. We could successfully demonstrate significant suppression of sperm motility by activators of these targets. However, most amazingly, we could also validate significant stimulation of total rat sperm motility by specific inhibitors of p38, AKT, mTOR and p70S6K, above control. Amongst these pathways, p38 and p70S6K inhibitions were most effective in stimulating different parameters of sperm motility (please see supporting live sperm videos for p38 activation and inhibition by following links provided in results section). It is well known that the distal (cauda) epididymis stores sperm in a hyperosmotic environment (Joseph et al. 2010), which may be as high as 415mOsM in mice (Si et al. 2009). Hyperosmotic stress is known to activate p38 (Aggeli et al. 2002) and thus may help in quiescence and motility activation during ejaculation. Other studies have shown that activation of p38 reduces human sperm motility, which could be corrected by p38 inhibitor SB203580 (Yu et al. 2019). As indicated by the PPI network, these proteins closely interact with each other in controlling cell signaling. This was verified by investigating the effect of the activators and inhibitors of p38, AKT and mTOR on phosphorylation of p70S6K, which is the downstream target for these proteins. It is quite apparent that these pathways work in a cohort in controlling sperm motility. MAPK-p38 gene knockout is embryonic lethal (Mudgett et al. 2000); however, germ cell specific knockout (KO) of mTOR complex1 gene in mice reduced sperm number by 50% in cauda epididymis (Wang et al. 2016). Similarly, severe oligoasthenozoospermia was seen in caudal sperm of AKT-KO mice (Kim et al. 2012). On the other hand, sperm p70S6K was found to be hyper phosphorylated in a mutant mice model with male infertility (Maekura et al. 2021).

Consequent to their localization in the rat sperm mid-piece and their role in sperm motility activation/suppression and also survival, we investigated if these signaling proteins had a role in regulating mitochondrial function by affecting mitochondrial polarization (membrane potential), sperm energetics (ATP content) and production of ROS. Highly polarized mitochondria of quiescent sperm (indicating high viability and motility potential) were significantly depolarized on motility initiation, and further depolarized significantly by different treatments during motility initiation. Only MAPK-p38 inhibitor (SB203580) kept sperm mitochondrial membrane significantly polarized above control sperm during motility initiation. It’s noteworthy to mention here that p38 inhibition also increased sperm progressive motility without changing the cell viability as compared with vehicle-treated motile sperm. Interestingly, the high ATP content of quiescent sperm was reduced during motility initiation, and was further reduced by different treatments, except after p38 inhibition by SB203580, where it remained comparable to untreated motile. (It is important to note that the ATP levels of quiescent sperm with suppressed metabolism denotes static levels with minimal consumption for maintenance of viability. Conversely, the ATP level in motile cells is a dynamic equilibrium of high rate of synthesis vs. high rate of consumption, for motility). On the other hand, cell viability increased significantly only in the presence of AKT activator (SC79), though ATP levels and cell motility remained lower in comparison to vehicle-treated motile sperm. Apparently, p38 signaling has a greater role to play in regulating cell motility while AKT signaling helps in maintaining cell viability of rat sperm in cauda epididymis. Activated (phosphorylated) p38 in ejaculated human sperm is associated with poor motility (Almog et al. 2008, Ding et al. 2011) while AKT activation has been shown to preserve the viability of stallion sperm (Gallardo Bolanos et al. 2014). ROS level of quiescent sperm was decreased on motility initiation but was not affected further in the presence of SB203580, SC79, MHY1485 and rapamycin during motility activation. Perhaps quiescent sperm with suppressed metabolism maintain the required ROS levels for activating cell-signaling. Sorbitol and retinoic acid increased ROS and decreased motility. Hyperosmotic stress (caused by sorbitol here) has been shown to increase oxidative stress in monkey sperm (McCarthy et al. 2010), while retinoic acid is reported to increase ROS in rat Sertoli cells (Conte da Frota et al. 2006). Overall, optimum ROS levels in quiescent sperm could help in activating cell signaling pathways for inhibiting sperm motility and apoptosis, though this could not be exactly recreated in vitro by pathway modulators. Retinoic acid exhibited mild toxicity and compromised the viability of about 5% sperm as compared with untreated motile sperm during 10 min exposure (Supplementary Fig. 5). This may have had some minor effect on the overall results with this compound. Other compounds did not have any significant effect on sperm viability during different experimental procedures, except in viability test, where the exposure was for 60 min.

It is thus evident that sperm survive quiescently in cauda epididymis for up to a few weeks before ejaculation by a combined action of several cell signaling proteins working chiefly at mitochondrial level. The mechanisms controlling this cell signaling is not known but could be as simple as osmoregulation and oxidative regulation. Apparently, alterations in caudal sperm cell signaling may adversely affect motility and viability (numbers) leading to oligoasthenozoospermia which is seen in many infertile patients, but this aspect needs further investigation.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/REP-21-0202.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study was funded by the BSC0101, OLP0101 and MLP2026 grants from the CSIR-CDRI under ‘Reproductive Health Research’.

Data availability

Data available on request.

Author contribution statement

G G conceived the study, designed the experiments; G G and R S analyzed and interpreted the data; A D, B K and J P M performed the experiments, generated and compiled the data. A D and G G wrote the manuscript, all authors edited and approved the final draft.

Acknowledgements

The authors are thankful to the Director CSIR-CDRI for funding of this study under ‘Reproductive Health Research’ (OLP0101) and CSIR for grants BSC0101 and MLP2026. A D would like to thank the Department of Biotechnology, Government of India, for the grant of research fellowship. The authors are grateful to the SAIF division for their help in acquisition of Flow Cytometry data.

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    Figure 1

    Alterations in rat sperm cell intracellular signaling during motility initiation. (A) Intracellular signaling array analysis of 18 key phosphorylated/cleaved targets (proteins) in quiescent and motile rat sperm cell lysates; (B) Array layout chart, (C) Target proteins, their modification sites and types; (D) Proteins–protein interaction (PPI) network of array proteins by STRING software, the color of lines denote mode of action; (E) Mutual interactions among the four key targets viz. p38MAPK, AKT, mTOR and p70S6K that apparently play the central role in the intracellular interactome.

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    Figure 2

    Immunohistochemical localization of phospho-p70S6K in rat caudal sperm in situ and in isolated quiescent/motile sperm. (Ai) Hematoxylin-and-eosin stained sections of rat cauda epididymis showing normal morphology at 10× (bar = 20 µm) and 40× (bar = 10 µm). (Aii) Immunohistochemical (DAB) staining of cauda epididymis sections for phospho-p70S6K at 10× (bar = 10 µm) and 40× (bar = 10 µm) showing deeply stained sperm tails and (B) comparison of similarly stained quiescent and motile sperm isolated from cauda epididymis and fixed on poly-lysine coated slides at 40× (bar = 10 µm) and 100× (bar =1 µm).

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    Figure 3

    Phosphorylation of p38, Akt, mTOR and p70S6K in quiescent and motile sperm, the effect of activators/inhibitors on protein phosphorylation levels and immunofluorescent localization of phosphorylated p38, Akt, mTOR and p70S6K in rat sperm. Phosphorylation of (A) p38, (B) Akt, (C) mTOR and (D) p70S6 kinases in quiescent, motile and activator/inhibitor treated cells by immunoblotting (upper panel); and statistical analysis of immunoblots (lower panel). Localization of (E) phosphorylated (activated) p38 in quiescent, motile, sorbitol (activator) and SB203580 (inhibitor) treated sperm; (F) phosphorylated Akt in quiescent, motile, SC79 and Akti-1/2 treated sperm; (G) phosphorylated mTOR in quiescent, motile, MHY1485 and rapamycin treated rat sperm; and (H) phosphorylated p70S6K in quiescent, motile, RA (retinoic acid) and LYS6K2 treated sperm. Images were captured by fluorescence microscopy at 100× (bar = 1 µm) for p70S6K, Akt and mTOR and for p38 at 60× (bar = 20 µm) (fluorescence tags: Alexafluor (red); nucleus staining by DAPI (blue)). (Quantitative assessment of fluorescent intensities by Image-J software is provided in Supplementary Fig. 3.)

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    Figure 4

    Effect of inhibition and activation of intracellular signaling proteins on rat sperm motility parameters during motility initiation. Sperm motility parameters analyzed by CASA in sperm isolated from cauda epididymis and diluted in vehicle HBSS (motile); p38MAPK-activator sorbitol (0.5 M), -inhibitor SB203580 (25 µM); p70S6K-activator retinoic acid (RA, 1 mM) -inhibitor LYS6K2 (100 µM); AKT-activator SC79 (50 µM), -inhibitor Akti-1/2 (50 µM) and mTOR-activator MHY1485 (50 µM), -inhibitor rapamycin (25 µM). Motility parameters were analyzed by computer-assisted sperm analyser (CASA). (A) total motility, (B) progressive motility, (C) average path velocity (VAP), (D) straight line velocity (VSL), (E) curvilinear velocity (VCL), (F) amplitude of lateral head displacement (ALH), (G) beat cross frequency (BCF), (H) straightness (STR) and (I) Linearity (LIN). Mean (±s.e.); significant difference from control (motile) is denoted as *P < 0.05; **P < 0.01; ***P < 0.001; ns (nonsignificant).

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    Figure 5

    Effect of p38, AKT and mTOR modulation on phosphorylation of p70S6K. Phosphorylation of p70S6K in rat sperm treated with p38 activator/inhibitor: sorbitol (0.5 M), SB203580 (25 µM); AKT activatot/inhibitor: SC79 (50 µM), Akti-1/2 (50 µM) and mTOR activator/inhibitor: MHY1485 (50 µM) and rapamycin (25 µM), by fluorescence microscopy. Bar = 2 µm for sorbitol, SB203580, SC79, Akti-1/2 and bar =1 µm for MHY1485 and rapamycin (fluorescence tags: Alexafluor [red]; nucleus staining by DAPI (blue)). (Quantitative assessment of fluorescent intensities by Image-J software is provided in Supplementary Fig. 4.)

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    Figure 6

    MMP, ATP and ROS level in quiescent and motile sperm and the effect of activators/inhibitors on these mitochondrial parameters. (A) Dot plots for changes in mitochondrial membrane potential (MMP) using JC-1 dye and flow cytometery at Ex/Em = 488/530 nm (B) Statistical data of dot plots. (C) Sperm ATP content, (D) Reactive oxygen species (ROS) levels by DCFDA fluorescence at 525 nm in the FL-1 green channel and (E) its statistical analysis in quiescent, motile and treated rat sperm. Significant difference from motile indicated as *P < 0.05; **P < 0.01; ***P < 0.001, ns, nonsignificant.

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    Figure 7

    Role of MAPKp38, AKT/mTOR/p70S6K pathways in maintaining rat sperm cell viability. Quiescent, motile and inhibitor/activator treated sperm stained with trypan blue (dead) and unstained (live) were counted under light microscope (minimum 200 cells in 10 different fields). Significant difference from quiescent is denoted as C, and from untreated motile is denoted as c, CcP < 0.001, ns, nonsignificant.

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