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. Author manuscript; available in PMC: 2025 Apr 27.
Published in final edited form as: Reproduction. 2025 Mar 3;169(4):e240448. doi: 10.1530/REP-24-0448

Potent and sustained enhancement of human sperm motility using novel cyclic AMP up-regulators

Natalia Oscoz-Susino 1, Guillermina M Luque 1, Florencia Minotti 2, Patricia Otero 2, Mariano Lavolpe 3, Mariana Ferrulli 3, Dario Krapf 4, Mariano G Buffone 1, Clara I Marín-Briggiler 1
PMCID: PMC12034240  NIHMSID: NIHMS2060586  PMID: 39932044

Abstract

Cyclic adenosine monophosphate (cAMP) plays a central role in sperm physiology. Various cAMP up-regulators, both cAMP analogs and phosphodiesterase (PDE) inhibitors, have been used in handling human sperm in vitro, though conflicting results and variable responses among patients have been reported. This study aims to evaluate the ability of two compounds - Sp-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3’-5’-cyclic monophosphothioate (cBiMPs), a cAMP analog, and TAK-063, a specific PDE10A inhibitor - to enhance human sperm motility parameters and, consequently, improve sperm preparation procedures. Our results showed that both cBiMPs and TAK-063 significantly enhanced human sperm motility and hyperactivation compared to the control (dimethyl sulphoxide (DMSO)). They also increased protein phosphorylation levels without inducing premature acrosomal exocytosis or DNA fragmentation. The enhancement of sperm motility persisted for 4 h after their removal, surpassing the effects of known cAMP analogs (8-bromo-adenosine-3’, 5’-cAMP (8-Br-cAMP) or dibutyryl cAMP (db-cAMP)) or PDE inhibitors (3-isobutyl-1-methylxanthine (IBMX) or pentoxifylline (PTX)). Furthermore, the presence of cBiMPs or TAK-063 during sperm selection resulted in higher recovery rates in comparison to the control, and these compounds effectively improved sperm motion in both fresh and cryopreserved samples with impaired motility. In conclusion, cBiMPs and TAK-063 exhibit potent and sustained effects on human sperm motility, enhancing the efficiency of sperm preparation techniques. The ability to improve sperm motility holds significant implications for male infertility treatment, facilitating the use of low complexity techniques such as intrauterine insemination or in vitro fertilization, and may also aid in selecting viable testicular sperm for intracytoplasmic sperm injection.

Keywords: Sperm motility, hyperactivation, cyclic AMP, PKA, sperm selection

IN BRIEF

The cyclic adenosine monophosphate (cAMP) pathway is essential for maintaining sperm physiology. This study examines a cAMP analog and a phosphodiesterase inhibitor that effectively enhance human sperm motility, thereby improving the efficiency of in vitro sperm selection.

INTRODUCTION

Sperm fertilizing ability depends on their capacity to navigate through the female reproductive tract and penetrate the oocytés vestments, and reduced sperm motility is a cause of male infertility (Pereira & Sousa, 2023). Assisted reproduction techniques (ART) have evolved to address this issue, incorporating various procedures and strategies to select motile sperm from the ejaculate and enhance sperm motility when it is diminished (Henkel & Schill, 2003, Oseguera-López et al. 2019). These advancements rely on a deep understanding of the mechanisms that regulate sperm motility (Cavarocchi et al. 2022).

The intracellular pathway involving cyclic adenosine monophosphate (cAMP) plays a central role in the complex mechanisms governing mammalian sperm motility and function (Buffone et al. 2014, Balbach et al. 2018). When sperm enter the female reproductive tract, they are subjected to an increase in intracellular calcium (Ca2+) concentrations (Jeschke et al. 2021) and are exposed to high bicarbonate (HCO3−) concentrations (Vishwakarma 1962). This exposure activates a soluble adenylate cyclase (sAC; aka ADCY10), resulting in cAMP synthesis (Chen et al. 2000). Sperm intracellular cAMP levels are also maintained by extrusion through specific transporters (Osycka-Salut et al. 2014), and degradation by cyclic nucleotide phosphodiesterases (PDE) (Jaiswal & Majumder 1996). Several PDE have been described in mammalian sperm (Dimitriadis et al. 2022), and particularly, there is evidence of the presence of PDE1, PDE1A, PDE3A, PDE4, PDE5 and PDE10A in human sperm (Lefièvre et al. 2002, Dimitriadis et al. 2008, Maréchal et al. 2017). Elevated cAMP levels subsequently activate protein kinase A (PKA), leading to the phosphorylation of PKA substrates (pPKAs) and tyrosine residues (pY). Such events, as well as membrane lipid remodeling, hyperpolarization and intracellular alkalinization, are part of the sperm capacitation process (Puga Molina et al. 2018, Vyklicka & Lishko 2020). Capacitation allows the transition from progressive motility to a vigorous type of motility called hyperactivation, and the release of the acrosomal content or acrosomal exocytosis (AE) in response to proper stimuli (Yanagimachi 1994, Visconti et al. 2011).

Given the importance of the cAMP/PKA pathway in sperm physiology, various cAMP up-regulators, both cAMP analogs and/or PDE inhibitors, have been used in the treatment of infertile patients and/or in sperm handling in vitro (Henkel & Schill 2003, Dcunha et al. 2022, Dimitriadis et al. 2022). Some cell-permeable, synthetic cAMP analogs and PKA activators, such as dibutyryl cAMP (db-cAMP), have been included in the management of mammalian sperm in vitro but they have shown certain drawbacks as instability or acting as AE inducers (De Jonge et al. 1989, Doherty et al. 1995). Specific PDE inhibitors, such as sildenafil and tadalafil (PDE5 inhibitors), rolipram (PDE4 inhibitor) and papaverine (PDE10A inhibitor), as well as non-selective inhibitors like pentoxifylline (PTX), caffeine, theophylline and 3-isobutyl-1-methylxanthine (IBMX), have been widely used by oral administration as therapeutic options for different pathologies of male infertility (Tan et al. 2017, Dong et al. 2021). Moreover, it has been described that certain PDE inhibitors increase sperm motility, enhance capacitation and pY, act as antioxidant agents, and improve sperm fertilizing capacity in vitro (Calogero et al. 1998, Ebner et al. 2011, Tardif et al. 2014). Consequently, they have been included to treat semen samples with reduced motility and to activate motility in testicular sperm used in intracytoplasmic sperm injection (ICSI) protocols (Mahaldashtian et al. 2021, Sharpe et al. 2022, Gala et al. 2023). However, there is scarce information about their effect on embryo development, pregnancy and neonatal outcomes (Satish et al. 2021, Dong et al. 2024). Moreover, in the case of PTX, some conflicting biological effects (including potential sperm DNA damage), inconsistent results, and variable responses among different patients have been reported (Tournaye et al. 1994, Unsal et al. 2016, Gala et al. 2023). Altogether, this evidence indicates that further studies are needed to identify effective and safe cAMP up-regulators to be used with human gametes in vitro (Sharpe et al. 2022).

The objective of our study was to determine the effect of two cAMP up-regulators on human sperm parameters: i) a cAMP analog, Sp-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3’-5’-cyclic monophosphothioate (cBiMPs), and ii) a PDE10A inhibitor, TAK-063. We investigated their effectiveness in enhancing human sperm motility compared to other cAMP up-regulators commonly used in clinical practice, even after the removal of the compounds. Additionally, we evaluated their ability to increase the yield of sperm preparation procedures using fresh and frozen-thawed samples with low motility.

MATERIALS AND METHODS

Reagents and media

Chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA), unless otherwise indicated. cBiMPs was obtained from Santa Cruz Biotechnology (Cat # sc-201566, Dallas, TX, USA; stock solution: 8 mM in 10 % (v/v) dimethyl sulphoxide (DMSO)) and TAK-063 from Cayman Chemical (Cat # 22208; Ann Arbor, MI, USA; stock solution: 2.5 mM in DMSO). The antibodies used were: anti-phospho-protein kinase A substrates (pPKAs) (Cat # 9624s; Cell Signaling Technology, Danvers, MA, USA), anti-phosphotyrosine (pY) (clone 4G10; Millipore Corporation, Temecula, USA), anti-β-tubulin (Cat # T4026; Sigma-Aldrich), anti-rabbit and anti-mouse conjugated with horseradish peroxidase (HRP) (Sigma-Aldrich and Vector Laboratories, Inc., Burlingame, CA, USA, respectively). Modified Human Tubal Fluid (mHTF) was used throughout the study, which consisted of: 101.48 mM NaCl, 4.69 mM KCl, 0.202 mM MgSO4, 0.37 mM KH2PO4, 2.04 mM CaCl2, 2.77 mM glucose, 0.33 mM Na-pyruvate, 21.4 mM Na-lactate, 4 mM NaCO3H and 20 mM HEPES, pH = 7.4 (World Health Organization 2010). For sperm cryopreservation, refrigeration medium or test-yolk buffer with gentamicin was used (Cat # 90129; FUJIFILM Irvine Scientific, Santa Ana, CA, USA).

Semen samples and sperm processing

Semen samples used in the study were provided by volunteer donors, or patients undergoing assisted reproduction treatment at In Vitro Buenos Aires and the Hospital Dr. Carlos Durand, and obtained under written consent. Protocols were reviewed and approved by the Ethics Committee of the Instituto de Biología y Medicina Experimental, Buenos Aires (Ref: CE 10755) and the Hospital Dr. Carlos Durand, Buenos Aires (Ref: CE 7662). Donors included healthy men aged ≥ 18 years-old with semen parameters exceeding the fifth percentile distribution, as defined by the World Health Organization (2021). Samples exhibiting altered viscosity, containing more than 1 × 106 round cells / mL, or displaying other alteration in macroscopic characteristics (World Health Organization 2021) were excluded from the analysis.

The semen samples were obtained by masturbation after approximately 48 h of abstinence. They were allowed to liquefy for up to 1 h at 37°C and were subjected to routine analysis (World Health Organization 2021).

Motile sperm were selected by the swim-up procedure. Briefly, 0.5 – 1 mL of semen were placed in a 15-mL sterile conical tube and 1 mL of mHTF was gently layered over it. Tubes were incubated at 45° angle, at 37°C in a 5 % (v/v) CO2 atmosphere. After 1 h, the upper layer containing highly motile sperm was recovered and 5 mL of media was added. Samples were centrifuged at 400 x g for 8 min, the supernatant was discarded, fresh media was added and the centrifugation step was repeated. The pellet was resuspended in media, sperm concentration was determined with a Neubauer hemocytometer and adjusted to 7 × 106 cells / mL. Sperm were then incubated at 37°C for up to 4 h in mHTF supplemented with 0.5 % bovine serum albumin (BSA; Cat # A7960; Sigma-Aldrich), in the presence of 0.2 % DMSO (as control), cBiMPs (10, 30, 100 μM) (Goto & Harayama 2009, Murase et al. 2010) or TAK-063 (0.01, 0.1, 1 μM) (Gruber et al. 2022). Sperm were also incubated with 8-bromo-adenosine-3’, 5’-cyclic monophosphate (8-Br-cAMP, 1 mM), db-cAMP (1 mM), 3-isobutyl-1-methylxanthine (IBMX, 0.2 mM) or pentoxifylline (PTX, 3.6 mM, 1 mg / mL), as previously used to enhance human sperm motility (Jiang et al. 1984, McKinney et al. 1994, Liu et al. 2003, Wen et al. 2021). When indicated, compounds were removed by adding fresh media, followed by centrifugation at 500 x g for 5 min. This step was repeated twice. Samples were resuspended in mHTF and incubated for additional 4 h in the absence of the compounds. Under these conditions, the compounds were diluted 1:600.

To analyze whether the cAMP up-regulators can be used to enhance sperm recovery in the selection techniques, semen samples were split into two aliquots, that were supplemented with either 0.2 % DMSO (Ctrl) or 30 μM cBiMPs, or alternatively, with either 0.2 % DMSO (Ctrl) or 1 μM TAK-063. The swim-up procedure was done as indicated above, using mHTF containing the same amount of the corresponding compound as the upper layer. After 1 h, the upper fraction was recovered, centrifuged and the sperm pellet was resuspended in mHTF. Concentration and motility of the selected cells were determined, and the percentage of sperm recovery ([volume x concentration x total motility of the recovered aliquot] / [volume x concentration x total motility of the used semen aliquot] x 100) was calculated.

In samples with < 15 × 106 total motile sperm and/or low volume (0.6 mL), the seminal plasma was removed by centrifugation. The semen sample was mixed with 5 mL of mHTF, centrifuged at 400 x g for 8 min, and the sperm pellet was resuspended in medium containing either 0.2 % DMSO (Ctrl) or 30 μM cBiMPs, and incubated for an hour. Alternatively, the sperm pellet was resuspended in medium containing either 0.2 % DMSO (Ctrl) or 1 μM TAK-063.

Other experiments were performed with cryopreserved semen samples. After complete liquefaction, samples were mixed drop by drop with an equal volume of refrigeration medium and placed in 1 mL cryovials. They were cooled at 4°C for 30 min, placed in liquid nitrogen vapor for 10 min and then stored in liquid nitrogen for at least 1 week. For thawing, cryovials were incubated at 37°C for 10 min, samples were placed in a new tube, an equal volume of mHTF was added and they were centrifuged at 400 x g for 8 min. The sperm pellet was resuspended in mHTF with either 0.2 % DMSO (Ctrl) or 30 μM cBiMPs, or with either 0.2 % DMSO (Ctrl) or 1 μM TAK-063. Samples with > 30 % motile sperm were subjected to swim-up in the presence of the compounds and the sperm recovery in each condition was calculated. In samples with < 30 % motility, the cryoprotectant was removed by centrifugation and sperm motility was recorded after 1-h incubation with the compounds.

In all cases, replicate experiments of each assay were conducted with samples from different donors.

Computer-assisted sperm analysis (CASA)

Sperm motility parameters were evaluated using the Sperm Class Analyzer® system (SCA v.6.2.0.1.; Microptic SL, Barcelona, Spain) that acquires 60 frames per second (s). Sperm suspensions (9.8 μL) were placed onto a slide, using an 18 × 18 mm2 coverslide (preparation depth of 31 μm), and temperature was maintained at constant 37°C using a temperature-controlled stage. At least 5 microscopic fields and at least 300 sperm were analyzed. The kinematic parameters assessed were: curvilinear velocity (VCL, μm/s), straight line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity (LIN: VSL/VAP x 100, %), straightness (STR x 100, %) and amplitude of lateral head displacement (ALH, μm). Sperm motility was measured and classified as follows: rapid progressive (VCL ≥ 35 μm/s; STR ≥ 80 %), medium progressive (VCL ≥ 15 μm/s; STR ≥ 80 %), in situ (VCL < 15 μm/s; VAP ≥ 5 μm/s) and immotile (VAP < 5 μm/s). Percentages of total (rapid progressive + medium progressive + in situ) and progressive (rapid + medium progressive) motility were recorded. Drifting was set in 25 μm/s. Sperm were considered hyperactivated when showing VCL ≥ 150 μm/s, LIN < 50 % and ALH ≥ 3.5 μm (modified from Mortimer et al. 1998).

Protein extracts, SDS-PAGE and Western immunoblotting

Motile sperm were incubated in the corresponding conditions for the indicated periods of time and processed as described (Marín-Briggiler et al. 2021). Samples were subjected to SDS-PAGE in 10 % polyacrylamide gels and Western immunoblotting. To analyze pPKAs, membranes were developed with anti-pPKAs (1:5000) and anti-rabbit IgG conjugated with HRP (1:3000). To evaluate pY, primary antibody (1:3000) and anti-mouse conjugated with HRP (1:2000) were used. As loading control, membranes were developed with anti-β-tubulin (1:5000) and anti-mouse conjugated with HRP (1:2000). The reactive bands were detected by enhanced chemiluminiscence (ECL) using standard procedures. The same membranes were developed with each of the 3 primary antibodies, followed by stripping with 0.2 M NaOH for 2 min. In some cases, pPKAs was first developed; in other cases, pY was detected in the first place. Western immunoblot images were analyzed with ImageJ 1.48k (National Institute of Health, USA), following the specifications of ImageJ User Guide, IJ 1.46r. The optical densities of all bands, from 250 to 25 kDa, were quantified and expressed as relative to the β-tubulin band. Results from the Ctrl condition were considered 1.

Assessment of AE

The acrosomal status of sperm incubated under different conditions for 4 h was determined. Suspensions were fixed for 4 min with 2 % para-formaldehyde (PFA) in PBS at 4°C, washed twice with PBS, placed on slides, and dried at 37°C. The slides were immersed for 20 s in methanol at 4°C for cell permeabilization. Acrosome reaction was evaluated by sperm staining with 50 μg/mL fluorescein isothiocyanate (FITC) labeled - Pisum sativum agglutinin in duplicate assays. Stained cells (at least 200 cells per treatment) were scored in a microscope equipped with epifluorescence (Primo Star iLED, ZEISS, Göttingen, Germany). The presence of a bright, uniform staining over the acrosome was indicative of intact sperm; those cells showing heterogeneous “patchy” staining, fluorescence restricted to the equatorial segment, or lack of staining were considered acrosome-reacted.

DNA fragmentation assay

The DNA integrity of sperm incubated for 4 h with the cAMP up-regulators was evaluated by the TUNEL assay. After 2 washings with PBS, sperm were fixed by incubation with 2 % PFA for 20 min at room temperature. An aliquot of 1 mL of PBS was added and they were centrifuged for 3 min at 700 x g. Supernatant was discarded and the sperm pellet was resuspended in PBS. Sperm were placed on slides, dried at 37°C, and washed twice with PBS drops (5 min each). Cells were permeabilized by a 5-min incubation on ice with a solution of 0.1 % Triton X-100 in 0.1 % sodium citrate in PBS. After 2 washings with PBS, the positive control was treated with DNAse I (RQ1 RNase-Free, PROMEGA, Madison, WI, USA) for 20 min at 37°C. The sperm DNA fragmentation was measured by the TUNEL kit (In situ Cell Death Detection Kit, Fluorescein; Cat # 11684795910; Roche, Mannheim, Germany), following the manufacturer’s instructions. At least 200 cells per treatment were scored in a microscope equipped with epifluorescence (Primo Star iLED, ZEISS, Göttingen, Germany). The presence of a bright, uniform staining on the sperm nucleus was considered as positive (fragmented DNA).

Statistical analysis

Statistical analyses were done using the GraphPad Prism program (version 6.01 for Windows; GraphPad Software, San Diego, CA, USA). To assume normal distribution, percentages were converted to ratios and subjected to the arcsine square root transformation. Results were compared by one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test, or Student’s t test, as indicated. A P value of < 0.05 was considered statistically significant.

RESULTS

Incubation with cBiMPs or TAK-063 enhances human sperm motility, hyperactivation and protein phosphorylation

In the present study, we initially incubated the human sperm for 4 h with the cAMP analog cBiMPs, and several parameters were analyzed (Fig. 1). Incubation with either 10, 30 or 100 μM cBiMPs led to a significant increase in the percentage of both total and progressive motility compared to the control (Fig. 1B). Sperm kinematic parameters such as VCL and ALH were also significantly increased with all concentrations of cBiMPs (Supplementary Fig. 1A), resulting in higher values of hyperactive motility (Fig. 1B). Under these conditions, there was a concentration-dependent enhancement of both pPKAs and pY (Fig. 1C), but the cAMP analog caused neither no induction of the AE (Fig. 1D) nor fragmentation of sperm DNA (Fig. 1E).

Figure 1: Sperm parameters after incubation with cBiMPs, a cAMP analog.

Figure 1:

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(A) Experimental design. Motile sperm were selected by swim-up and incubated for 4 h with cBiMPs (10, 30, 100 μM) or DMSO (0.2 %) as control (Ctrl). (B) Total, progressive and hyperactive motility assessed by CASA (n = 6 experiments). (C) pPKAs and pY levels. Protein signals were quantified and expressed as relative to β-tubulin (β-tub) (n = 4 experiments). (D) Acrosomal status evaluated by Pisum sativum agglutinin staining and microscopy (n = 5 experiments). (E) Sperm DNA fragmentation determined by the TUNEL assay (n = 4 experiments). Results are expressed as mean ± SEM, a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. One-way ANOVA, and Tukey’s multiple comparison test.

We also evaluated the effect of the PDE inhibitor TAK-063 (0.01, 0.1, 1 μM) on the same sperm parameters (Fig. 2). Using this compound, a significant and concentration-dependent increase in motility percentages, kinematic parameters and protein phosphorylation levels was also obtained in comparison with the control condition (Fig. 2B and C, Supplementary Fig. 1B). Similarly to cBiMPs, sperm incubation with TAK-063 did not induce acrosomal loss or affect DNA integrity (Fig. 2D and E).

Figure 2: Sperm parameters after incubation with TAK-063, a PDE inhibitor.

Figure 2:

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(A) Experimental design. Motile sperm were selected by swim-up and incubated for 4 h with TAK-063 (0.01, 0.1, 1 μM) or DMSO (0.2 %) as control (Ctrl). (B) Total, progressive and hyperactive motility (n = 6 experiments). (C) pPKAs and pY levels. Protein signals were quantified and expressed as relative to β-tubulin (β-tub) (n = 4 experiments). (D) Acrosomal status (n = 5 experiments). (E) Sperm DNA fragmentation (n = 4 experiments). Results are expressed as mean ± SEM, a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. One-way ANOVA, and Tukey’s multiple comparison test.

cBiMPs and TAK-063 are more efficient than other cAMP analogs and PDE inhibitors in enhancing human sperm hyperactivation and protein phosphorylation levels

The ability of cBiMPs or TAK-063 to enhance human sperm parameters was also compared with that of other known cAMP analogs or PDE inhibitors, respectively. This analysis was conducted over a shorter period of time (1 h), aligning more closely with clinical practice (Fig. 3A and 4A). As observed for 4 h, sperm incubated for 1 h with cBiMPs exhibited a significant increase in total, progressive and hyperactive motility, as well as in protein phosphorylation levels compared to the control (Fig. 3B and C). Total and progressive motility with all concentrations of cBiMPs were similar than those obtained with 8-Br-cAMP or db-cAMP (1 mM) (Fig. 3B), but cBiMPs was more efficient than the known analogs in enhancing sperm VCL, ALH (Supplementary Fig. 2A) and hyperactive motility (Fig. 3B). Moreover, higher pY levels were observed for 100 μM cBiMPs compared those of 8-Br-cAMP or db-cAMP (Fig. 3C).

Figure 3: Sperm parameters after incubation with cBiMPs or known cAMP analogs.

Figure 3:

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(A) Experimental design. Motile sperm were selected by swim-up and incubated for 1 h with cBiMPs (10, 30 or 100 μM), 8-Br-cAMP (1 mM), db-cAMP (1 mM), or DMSO (0.2 %) as control (Ctrl). (B) Total, progressive and hyperactive motility (n = 5 experiments). (C) pPKAs and pY levels. Protein signals were quantified and expressed as relative to β-tubulin (β-tub) (n = 4 experiments). Results are expressed as mean ± SEM, a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl, or the condition indicated with the bracket. One-way ANOVA, and Tukey’s multiple comparison test.

Figure 4: Sperm parameters after incubation with TAK-063 or known PDE inhibitors.

Figure 4:

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(A) Experimental design. Motile sperm were selected by swim-up and incubated for 1 h with TAK-063 (0.01, 0.1 or 1 μM), IBMX (0.2 mM), PTX (3.6 mM) or DMSO (0.2 %) as control (Ctrl). (B) Total, progressive and hyperactive motility (n = 6 experiments). (C) pPKAs and pY levels. Protein signals were quantified and expressed as relative to β-tubulin (β-tub) (n = 4 experiments). Results are expressed as mean ± SEM, a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl, or the condition indicated with the bracket. One-way ANOVA, and Tukey’s multiple comparison test.

Regarding the PDE inhibitors, 1-h sperm incubation with TAK-063 (0.01, 0.1 and 1 μM), IBMX (0.2 mM) or PTX (3.6 mM) also elicited a significant increase in all motility parameters (Fig. 4B and Supplementary Fig. 2B). However, hyperactivation values obtained with 1 μM TAK-063 were higher than those found with PTX (Fig. 4B). Cells incubated with 0.1 and 1 μM TAK-063, IBMX and PTX depicted a significant increase in pPKAs levels compared with the control, but a higher pY signal was only observed with 1 μM TAK-063 (Fig. 4C).

cBiMPs and TAK-063 have a long lasting effect on human sperm parameters

The former experiments showed the beneficial effects of the cAMP up-regulators to enhance sperm parameters in vitro, but it was important to determine if these responses could be sustained after their removal. Such protocol intends to follow the clinical procedures in which compounds should be washed prior to insemination. In this case, selected sperm were incubated for 1 h with the cAMP up-regulators, compounds were removed by centrifugation, and the sperm parameters were evaluated 4 h later (Fig. 5A and 5C). Results showed that cells exposed to increasing concentrations of cBiMPs, but not to 8-Br-cAMP or db-cAMP, maintained higher levels of hyperactive motility compared with the control (Fig. 5B). Under this treatment, increased percentages of total and progressive motility, kinematic parameters (Supplementary Fig. 3), as well as pPKAs and pY levels (Supplementary Fig. 5) were also observed.

Figure 5: Sperm parameters after removing the cAMP up-regulators.

Figure 5:

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(A) Experimental design. Motile sperm were selected by swim-up and incubated for 1 h with cBiMPs (10, 30 or 100 μM), 8-Br-cAMP (1 mM), db-cAMP (1 mM) or DMSO (0.2 %) as control (Ctrl). Compounds were removed by two washings, sperm were resuspended in fresh medium, incubated for additional 4 h and sperm parameters were determined. (B) Hyperactive motility (n = 8 experiments). (C) Experimental design. Motile sperm were selected by swim-up and incubated for 1 h with TAK-063 (0.01, 0.1 or 1 μM), IBMX (0.2 mM), PTX (3.6 mM) or DMSO (0.2 %) as control (Ctrl), and processed as indicated in (A). (D) Hyperactive motility (n = 8 experiments). Results are expressed as mean ± SEM, a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl, or the condition indicated with the bracket. One-way ANOVA, and Tukey’s multiple comparison test.

Similarly, sperm that had been incubated with increasing concentrations of TAK-063 exhibited higher percentages of hyperactivation (Fig. 5D), motility parameters (Supplementary Fig. 4) and protein phosphorylation levels than those incubated with IBMX or PTX (Supplementary Fig. 5).

cBiMPs and TAK-063 improve human sperm preparation procedures

Considering that the cAMP up-regulators enhance human sperm motility, we were interested in assessing their effectiveness in improving sperm preparation procedures. First, the ability of the cAMP up-regulators to increase the swim-up recovery in fresh samples was analyzed by performing the selection procedure in the presence of either 30 μM cBiMPs or 1 μM TAK-063 (Fig. 6A). Using this protocol, a significant higher yield was observed with both compounds in comparison with the control (Fig. 6B). Then, their ability to improve sperm motility in samples with diminished volume, motility and/or concentration was evaluated. As these samples cannot be processed by swim-up because of low recovery rate, seminal plasma was removed by centrifugation, 30 μM cBiMPs or 1 μM TAK-063 was added, and motility parameters were analyzed 1 h later (Fig. 6C). Under these conditions, sperm progressive motility was enhanced in most of the samples analyzed (Fig. 6D), and a 2.8 ± 0.5 and 2.6 ± 0.4-fold increase was obtained for cBiMPs and TAK-063, respectively.

Figure 6: Effect of cAMP up-regulators on the preparation of fresh human sperm samples.

Figure 6:

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(A) Experimental design. Fresh semen samples were split into two aliquots, that were supplemented with either 30 μM cBiMPs or 0.2 % DMSO (Ctrl), or alternatively, with either 1 μM TAK-063 or 0.2 % DMSO (Ctrl). The swim-up procedure was done using medium containing the same amount of the corresponding compound (as upper layer). (B) Total sperm recovery, calculated as ([volume x concentration x total motility of the recovered aliquot] / [volume x concentration x total motility of the used semen aliquot] x 100) (n = 10 experiments for cBiMPs and n = 7 experiments for TAK-063). (C) Experimental design. In fresh semen samples with diminished parameters, the seminal plasma was removed by centrifugation. The sperm pellet was resuspended in medium containing either 30 μM cBiMPs or 0.2 % DMSO (Ctrl), and incubated for 1 h. Alternatively, the sperm pellet was resuspended in medium containing either 1 μM TAK-063 or 0.2 % DMSO (Ctrl). (D) Progressive motility (n = 25 experiments for cBiMPs and n = 21 experiments for TAK-063). Results of individual samples, as well as mean values are shown. b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. Studentś t test.

Sperm cryopreservation is a widely used technique during infertility treatments, but one of its disadvantages is that sperm lose motility after thawing (Ozimic et al. 2023). In this study, our next objective was to test the effect of sperm incubation with 30 μM cBiMPs or 1 μM TAK-063 on frozen-thawed samples subjected to swim-up (Fig. 7A) or the washing procedure (Fig. 7C). The addition of cAMP up-regulators also led to a significantly higher recovery rate in comparison with the control (Fig. 7B). Moreover, they were able to significantly improve sperm motility in non-selected thawed samples (Fig. 7D). In this case, a 1.5 ± 0.1 and 1.8 ± 0.1-fold increase in progressive motility was obtained after incubation with cBiMPs and TAK-063, respectively.

Figure 7: Effect of cAMP up-regulators on the preparation of cryopreserved human semen samples.

Figure 7:

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(A) Experimental design. Frozen-thawed semen samples with > 30 % motile sperm were split into two aliquots, that were supplemented with either 30 μM cBiMPs or 0.2 % DMSO (Ctrl), or alternatively, with either 1 μM TAK-063 or 0.2 % DMSO (Ctrl). The swim-up procedure was done using medium containing the same amount of the corresponding compound (as upper layer). (B) Total sperm recovery (n = 8 experiments for cBiMPs and TAK-063). (C) Experimental design. In frozen-thawed semen samples with < 30 % motility, the cryoprotectant was removed by centrifugation. The sperm pellet was resuspended in medium containing either 30 μM cBiMPs or 0.2 % DMSO (Ctrl), and incubated for 1 h. Alternatively, the sperm pellet was resuspended in medium containing either 1 μM TAK-063 or 0.2 % DMSO (Ctrl). (D) Progressive motility (n = 12 experiments for cBiMPs and TAK-063). Results of individual samples, as well as mean values are shown. b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. Studentś t test.

DISCUSSION

Cyclic AMP orchestrates different aspects of sperm function that are necessary for the acquisition of fertilizing capacity, including the development of motility and hyperactivation by the activation of PKA, leading to protein phosphorylation on tyrosine residues (Buffone et al. 2014, Balbach et al. 2018). Such responses can be mimicked by cAMP analogs or PDE inhibitors, which have been used in the management of sperm in vitro. However, such compounds have several limitations, suggesting that novel cAMP up-regulators need to be explored in their ability to sustain human sperm function (Dcunha et al. 2022). In the present study, we assessed the effect of cBiMPs, as a cAMP analog, and TAK-063, as a PDE inhibitor, in regulating sperm motility parameters. Additionally, we explored their ability to improve sperm motion and recovery during preparation procedures in fresh and frozen-thawed semen samples.

The compound cBiMPs is a permeable, metabolically stable cAMP analog that specifically activates PKA. It has been shown as more permeable to the membrane and more potent than other cAMP analogs, such as db-cAMP or 8-(p-chlorophenylthio)-cAMP (8-CPT-cAMP) (Sandberg et al. 1991). There are several reports including the use of cBiMPs to activate the PKA/pY pathway and to promote hyperactivation, AE and other capacitation-related events in boar and bovine sperm (Adachi et al. 2008, Harayama et al. 2010, Murase et al. 2010). Moreover, there are some studies describing the effect of cBiMPs as stimulator of protein phosphorylation and enhancer of hyperactive motility in mouse sperm (Goto & Harayama 2009, Harayama et al. 2010), but it has not yet been used with human gametes.

Regarding TAK-063 (also known as balipodect), it is a highly potent and selective PDE10A inhibitor (Kunitomo et al. 2014) that increases cyclic nucleotide levels in mice striatum after oral administration (Suzuki et al. 2018). This compound had been investigated in a phase 1 study (Tsai et al. 2016) and had been under phase 2 clinical trial for the treatment of patients with schizophrenia (Macek et al. 2019). Active PDE10A has been described in human sperm (Maréchal et al. 2017) and TAK-063 has been identified as a sperm motility stimulant in a recent study using a high-throughput screening platform (Gruber et al. 2022). These authors report that human sperm incubation for a short period of time with TAK-063 enhances motility and VCL. However, the effect of this compound on other sperm parameters is unknown, and there is no evidence on its activity on samples from selected groups of patients or no comparison with other PDE inhibitors.

Our results have shown that both cBiMPs (10–100 μM) and TAK-063 (0.01–1 μM) efficiently enhance human sperm motility parameters and hyperactivation when incubated for up to 4 h. As expected, such effects were accompanied by an increase in pPKAs and pY, suggesting the activation of the cAMP/PKA pathway. Moreover, these compounds did not cause premature acrosomal loss or undesirable sperm DNA fragmentation, which would be critical for their use in the clinical practice. The participation of cAMP pathway in the induction of AE has been described (Romarowski et al. 2015, Lucchesi et al. 2016), but our results can be explained by the experimental conditions used (low bicarbonate concentration of mHTF and short incubation time) that would be insufficient to sustain complete human sperm capacitation.

The hyperactivation-stimulatory effects obtained with cBiMPs and TAK-063 were higher than those found for known cAMP analogs (8-Br-cAMP or db-cAMP) or PDE inhibitors (IBMX or PTX) at the tested concentrations. Notably, previous evidence suggests that 8-Br-cAMP concentrations above 10 μM do not induce hyperactivated motility (Allouche-Fitoussi et al. 2018), though their experimental conditions differ from ours. Given that these authors suggest that the development of hyperactivated motility is precisely regulated by cAMP, our results indicate that cBiMPs and TAK-063 at the concentrations used, effectively activate intracellular pathways leading to increased hyperactivation. However, additional studies are necessary to determine the effect of these compounds on the activation of sperm signaling pathways, such as those involving exchange proteins directly activated by cAMP (EPAC), cyclic nucleotide gated channels (CNGc), Na+/H+ exchangers (sNHE) or Ca2+-regulated pathways (Balbach et al. 2018, Puga Molina et al. 2018), which would influence sperm swimming behavior. A recent report has described that PF-2545920, a PDE10A inhibitor, caused an enhancement of sperm motility involving an increase in intracellular Ca2+ levels (Yang et al. 2023). In this regard, there is evidence indicating the activation of the specific Ca2+ channel CatSper by PKA in mouse sperm (Orta et al. 2018, Luque et al. 2021), although the regulation of human CatSper by cAMP/PKA is controversial. A direct activation of human CatSper by extracellular cAMP and cAMP analogs has been reported, and the existence of so-far-unknown cAMP binding sites in CatSper has been proposed (Wang et al. 2020).

In our study, the effect of cAMP up-regulators on sperm function was also analyzed after the agents’ removal. In this case, samples were subjected to two consecutive washing steps to effectively get rid of the compounds. This protocol tries to mimic the necessary removal of stimulants before their use in clinical procedures. Results showed that both cBiMPs and TAK-063 are able to maintain sperm motility parameters, hyperactivation and protein phosphorylation for 4 h after their removal. Such effects were also higher in comparison with those obtained for the known cAMP agonists or PDE inhibitors tested. The ability of cBiMPs and TAK-063 to sustain sperm function for several hours after removal constitutes a great advantage when planning their potential use for patientś treatment, especially considering that some of the known cAMP up-regulators, such as PTX, need to be freshly dissolved and removed immediately before insemination because they rapidly lose their effect (Kay et al. 1993).

When preparing the semen samples for ART, motile sperm need to be isolated from seminal plasma, immotile sperm and other cells, and several sperm preparation procedures have been developed (World Health Organization 2021). Among them, the swim-up is a widely used sperm preparation protocol whose advantages are its simplicity and low cost. However, its main disadvantage is the low recovery rate that restricts its use for samples with high sperm count and motility (Henkel & Schill, 2003). In semen samples with decreased, volume, sperm concentration and/or motility, the washing procedure or seminal plasma removal by centrifugation is recommended (World Health Organization 2021). Some compounds have been added during the sperm preparation techniques and have been probed to increase sperm motility and selection yield (Garbarino Azúa et al. 2017, Satish et al. 2021). In the present study, using fresh and also frozen-thawed samples we showed that the addition of both cBiMPs and TAK-063 led to improved recovery rates in the swim-up procedure, and to higher percentages of motility in washed sperm when compared to the control. Our findings are in agreement with those of reports describing that the use of PDE inhibitors enhances sperm motility after cryopreservation (Mahaldashtian et al. 2021). Such results led to consider these compounds as supplements for improving human sperm function in vitro and would allow the use of cost-effective, non-invasive techniques, as intrauterine insemination, or in vitro fertilization (that require high number of motile sperm), instead of ICSI (Sharpe et al. 2022). Moreover, these cAMP regulators would also help for selecting viable sperm after testicular biopsies.

In conclusion, our findings indicate that cBiMPs is a potent cAMP analog, and that TAK-063 is an efficient PDE inhibitor with a long lasting effect in enhancing human sperm motility and hyperactivation, without causing adverse effects either on sperm acrosome or DNA integrity. Such agents effectively improved sperm recovery in sperm preparation procedures in both fresh and cryopreserved samples, which would be very relevant for the clinical practice. However, further studies are needed to determine the efficacy and safety of cBiMPs and TAK-063 before their use in the handling of human sperm during ART.

Supplementary Material

01

Supplementary Fig. 1: Kinematic characteristics of human sperm incubated for 4 h with cBiMPs or TAK-063. (A) Motile sperm were selected by swim-up and incubated for 4 h with cBiMPs (10, 30, 100 μM) or DMSO (0.2 %) as control (Ctrl). Motility parameters were recorded by CASA. Curvilinear velocity (VCL), linearity (LIN) and amplitude of lateral head displacement (ALH) are shown (n = 6 experiments). (B) Alternatively, sperm were incubated with TAK-063 (0.01, 0.1, 1 μM) (n = 6 experiments). Results are expressed as mean ± SEM. a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. One-way ANOVA, and Tukey’s multiple comparison test.

02

Supplementary Fig. 2: Sperm parameters in sperm incubated with cBiMPs or TAK-063 and known cAMP up-modulators. (A) Motile sperm were selected by swim-up and incubated for 1 h with cBiMPs (10, 30 or 100 μM), 8-Br-cAMP (1 mM), db-cAMP (1 mM) or DMSO (0.2 %) as control (Ctrl). VCL, LIN and ALH are shown (n = 5 experiments). (B) Sperm were incubated for 1 h with TAK-063 (0.01, 0.1, 1 μM), IBMX (0.2 mM), PTX (3.6 mM) or DMSO (0.2 %) as control (Ctrl) (n = 6 experiments). Results are expressed as mean ± SEM. a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. One-way ANOVA, and Tukey’s multiple comparison test.

03

Supplementary Fig. 3: Sperm parameters after removing cBiMPs or known cAMP analogs. Motile sperm were selected by swim-up and incubated for 1 h with cBiMPs (10, 30 or 100 μM), 8-Br-cAMP (1 mM), db-cAMP (1 mM) or DMSO (0.2 %) as control (Ctrl). Compounds were removed by two washings, sperm were resuspended in fresh medium, incubated for additional 4 h and sperm parameters were determined. Total motility, progressive motility, VCL, LIN and ALH are shown (n = 8 experiments). Results are expressed as mean ± SEM. b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. One-way ANOVA, and Tukey’s multiple comparison test.

04

Supplementary Fig. 4: Sperm parameters after removing TAK-063 or known PDE inhibitors. Motile sperm were selected by swim-up and incubated for 1 h with TAK-063 (0.01, 0.1, 1 μM), IBMX (0.2 mM), PTX (3.6 mM) or DMSO (0.2 %) as control (Ctrl). Compounds were removed by two washings, sperm were resuspended in fresh medium, incubated for additional 4 h and sperm parameters were determined. Total motility, progressive motility, VCL, LIN and ALH are shown (n = 8 experiments). Results are expressed as mean ± SEM. a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. One-way ANOVA, and Tukey’s multiple comparison test.

05

Supplementary Fig. 5: Sperm protein phosphorylation after removing the cAMP up-regulators. pPKAs and pY levels of sperm treated as indicated in Supplementary Fig. 3 (A) and Supplementary Fig. 4 (B). β-tub is included as loading control. Typical results are shown.

ACKNOWLEDGEMENTS

We thank Rene Baron and Williams foundations. We also would like to thank other members of our lab for critically reading the manuscript.

FUNDING

This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 2021–2023 #1068 to CIMB), the Agencia Nacional de Promoción Científica y Tecnológica (PICD 2016–0043 to DK, and PICT 2020–00988 to MGB), and the National Institutes of Health (NIH, RO1RHD106968A to MGB).

Footnotes

DECLARATION OF INTEREST

The authors declare no conflict of interest.

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Supplementary Materials

01

Supplementary Fig. 1: Kinematic characteristics of human sperm incubated for 4 h with cBiMPs or TAK-063. (A) Motile sperm were selected by swim-up and incubated for 4 h with cBiMPs (10, 30, 100 μM) or DMSO (0.2 %) as control (Ctrl). Motility parameters were recorded by CASA. Curvilinear velocity (VCL), linearity (LIN) and amplitude of lateral head displacement (ALH) are shown (n = 6 experiments). (B) Alternatively, sperm were incubated with TAK-063 (0.01, 0.1, 1 μM) (n = 6 experiments). Results are expressed as mean ± SEM. a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. One-way ANOVA, and Tukey’s multiple comparison test.

NIHMS2060586-supplement-01.pdf (377.5KB, pdf)
02

Supplementary Fig. 2: Sperm parameters in sperm incubated with cBiMPs or TAK-063 and known cAMP up-modulators. (A) Motile sperm were selected by swim-up and incubated for 1 h with cBiMPs (10, 30 or 100 μM), 8-Br-cAMP (1 mM), db-cAMP (1 mM) or DMSO (0.2 %) as control (Ctrl). VCL, LIN and ALH are shown (n = 5 experiments). (B) Sperm were incubated for 1 h with TAK-063 (0.01, 0.1, 1 μM), IBMX (0.2 mM), PTX (3.6 mM) or DMSO (0.2 %) as control (Ctrl) (n = 6 experiments). Results are expressed as mean ± SEM. a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. One-way ANOVA, and Tukey’s multiple comparison test.

NIHMS2060586-supplement-02.pdf (245.3KB, pdf)
03

Supplementary Fig. 3: Sperm parameters after removing cBiMPs or known cAMP analogs. Motile sperm were selected by swim-up and incubated for 1 h with cBiMPs (10, 30 or 100 μM), 8-Br-cAMP (1 mM), db-cAMP (1 mM) or DMSO (0.2 %) as control (Ctrl). Compounds were removed by two washings, sperm were resuspended in fresh medium, incubated for additional 4 h and sperm parameters were determined. Total motility, progressive motility, VCL, LIN and ALH are shown (n = 8 experiments). Results are expressed as mean ± SEM. b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. One-way ANOVA, and Tukey’s multiple comparison test.

NIHMS2060586-supplement-03.pdf (261.5KB, pdf)
04

Supplementary Fig. 4: Sperm parameters after removing TAK-063 or known PDE inhibitors. Motile sperm were selected by swim-up and incubated for 1 h with TAK-063 (0.01, 0.1, 1 μM), IBMX (0.2 mM), PTX (3.6 mM) or DMSO (0.2 %) as control (Ctrl). Compounds were removed by two washings, sperm were resuspended in fresh medium, incubated for additional 4 h and sperm parameters were determined. Total motility, progressive motility, VCL, LIN and ALH are shown (n = 8 experiments). Results are expressed as mean ± SEM. a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001 versus Ctrl. One-way ANOVA, and Tukey’s multiple comparison test.

NIHMS2060586-supplement-04.pdf (236.2KB, pdf)
05

Supplementary Fig. 5: Sperm protein phosphorylation after removing the cAMP up-regulators. pPKAs and pY levels of sperm treated as indicated in Supplementary Fig. 3 (A) and Supplementary Fig. 4 (B). β-tub is included as loading control. Typical results are shown.

NIHMS2060586-supplement-05.pdf (407.7KB, pdf)

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