Mouse sperm energy restriction and recovery (SER) revealed novel metabolic pathways.

Mammalian sperm must undergo capacitation to become fertilization-competent. While working on mice, we recently developed a new methodology for treating sperm in vitro, which results in higher rates of fertilization and embryo development after in vitro fertilization. Sperm incubated in media devoid of nutrients lose motility, although they remain viable. Upon re-adding energy substrates, sperm resume motility and become capacitated with improved functionality. Here, we explore how sperm energy restriction and recovery (SER) treatment affects sperm metabolism and capacitation-associated signaling. Using extracellular flux analysis and metabolite profiling and tracing via nuclear magnetic resonance (NMR) and mass spectrometry (MS), we found that the levels of many metabolites were altered during the starvation phase of SER. Of particular interest, two metabolites, AMP and L-carnitine, were significantly increased in energy-restricted sperm. Upon re-addition of glucose and initiation of capacitation, most metabolite levels recovered and closely mimic the levels observed in capacitating sperm that have not undergone starvation. In both control and SER-treated sperm, incubation under capacitating conditions upregulated glycolysis and oxidative phosphorylation. However, ATP levels were diminished, presumably reflecting the increased energy consumption during capacitation. Flux data following the fate of 13C glucose indicate that, similar to other cells with high glucose consumption rates, pyruvate is converted into 13C-lactate and, with lower efficiency, into 13C-acetate, which are then released into the incubation media. Furthermore, our metabolic flux data show that exogenously supplied glucose is converted into citrate, providing evidence that in sperm cells, as in somatic cells, glycolytic products can be converted into Krebs cycle metabolites.

Capacitation induces changes in metabolic pathways supporting motility of epididymal and ejaculated sperm.

Mammalian sperm require sufficient energy to support motility and capacitation for successful fertilization. Previous studies cataloging the changes to metabolism in sperm explored ejaculated human sperm or dormant mouse sperm surgically extracted from the cauda epididymis. Due to the differences in methods of collection, it remains unclear whether any observed differences between mouse and human sperm represent species differences or reflect the distinct maturation states of the sperm under study. Here we compare the metabolic changes during capacitation of epididymal versus ejaculated mouse sperm and relate these changes to ejaculated human sperm. Using extracellular flux analysis and targeted metabolic profiling, we show that capacitation-induced changes lead to increased flux through both glycolysis and oxidative phosphorylation in mouse and human sperm. Ejaculation leads to greater flexibility in the ability to use different carbon sources. While epididymal sperm are dependent upon glucose, ejaculated mouse and human sperm gain the ability to also leverage non-glycolytic energy sources such as pyruvate and citrate.

In vivo characterization of sAC null sperm.

Targeted disruption of the soluble adenylyl cyclase (ADCY10; sAC) gene results in male-specific sterility without affecting spermatogenesis, mating behavior, or spermatozoa morphology and count; however, it dramatically impairs sperm motility and prevents capacitation. These phenotypes were identified in sperm from sAC null mice surgically extracted from the epididymis and studied in vitro. Epididymal sperm are dormant, and never exposed to physiological activators in semen or the female reproductive tract. To study sAC null sperm under conditions which more closely resemble natural fertilization, we explored phenotypes of ejaculated sAC null sperm in vivo post-coitally as well as ex vivo, collected from the female reproductive tract. Ex vivo ejaculated sAC null sperm behaved similarly to epididymal sAC null sperm, except with respect to the physiologically induced acrosome reaction. These studies suggest there is a sAC-independent regulation of acrosome responsiveness induced upon ejaculation or exposure to factors in the female reproductive tract. We also studied the behavior of sAC null sperm in vivo post-coitally by taking advantage of transgenes with fluorescently labelled sperm. Transgenes expressing GFP in the acrosome and DsRed2 in the mitochondria located in the midpiece of sperm (DsRed2/Acr3-EGFP) allow visualization of sperm migration through the female reproductive tract after copulation. As previously reported, sperm from wild type (WT) double transgenic mice migrated from the uterus through the uterotubular junction (UTJ) into the oviduct within an hour post-copulation. In contrast, sperm from sAC null double transgenic mice were only found in the uterus. There were no sAC null sperm in the oviduct, even 8 h after copulation. These results demonstrate that sAC KO males are infertile because their sperm do not migrate to the fertilization site.

Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic ß cells.

Male mice lacking the androgen receptor (AR) in pancreatic ß cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in ß cells to amplify glucagon-like peptide-1 (GLP-1) insulinotropic action. Here, we examined the architecture of AR targets that regulate GLP-1 insulinotropic action in male ß cells. Testosterone cooperates with GLP-1 to enhance cAMP production at the plasma membrane and endosomes via: (1) increased mitochondrial production of CO2, activating the HCO3--sensitive soluble adenylate cyclase; and (2) increased Gαs recruitment to GLP-1 receptor and AR complexes, activating transmembrane adenylate cyclase. Additionally, testosterone enhances GSIS in human islets via a focal adhesion kinase/SRC/phosphatidylinositol 3-kinase/mammalian target of rapamycin complex 2 actin remodeling cascade. We describe the testosterone-stimulated AR interactome, transcriptome, proteome, and metabolome that contribute to these effects. This study identifies AR genomic and non-genomic actions that enhance GLP-1-stimulated insulin exocytosis in male ß cells.

Carbonic Anhydrase Inhibitor Modulation of Intraocular Pressure Is Independent of Soluble Adenylyl Cyclase.

Purpose: We investigated whether a clinically used carbonic anhydrase inhibitor (CAIs) can modulate intraocular pressure (IOP) through soluble adenylyl cyclase (sAC) signaling. Methods: IOP was measured 1 h after topical treatment with brinzolamide, a topically applied and clinically used CAIs, using direct cannulation of the anterior chamber in sAC knockout (KO) mice or C57BL/6J mice in the presence or absence of the sAC inhibitor (TDI-10229). Results: Mice treated with the sAC inhibitor TDI-10229 had elevated IOP. CAIs treatment significantly decreased increased intraocular pressure (IOP) in wild-type, sAC KO mice, as well as TDI-10229-treated mice. Conclusions: Inhibiting carbonic anhydrase reduces IOP independently from sAC in mice. Our studies suggest that the signaling cascade by which brinzolamide regulates IOP does not involve sAC.

Scaffold Hopping and Optimization of Small Molecule Soluble Adenyl Cyclase Inhibitors Led by Free Energy Perturbation.

Free energy perturbation is a computational technique that can be used to predict how small changes to an inhibitor structure will affect the binding free energy to its target. In this paper, we describe the utility of free energy perturbation with FEP+ in the hit-to-lead stage of a drug discovery project targeting soluble adenyl cyclase. The project was structurally enabled by X-ray crystallography throughout. We employed free energy perturbation to first scaffold hop to a preferable chemotype and then optimize the binding affinity to sub-nanomolar levels while retaining druglike properties. The results illustrate that effective use of free energy perturbation can enable a drug discovery campaign to progress rapidly from hit to lead, facilitating proof-of-concept studies that enable target validation.

Corrigendum: Assessing potency and binding kinetics of soluble adenylyl cyclase (sAC) inhibitors to maximize therapeutic potential.

[This corrects the article DOI: 10.3389/fphys.2022.1013845.].

On-demand male contraception via acute inhibition of soluble adenylyl cyclase.

Nearly half of all pregnancies are unintended; thus, existing family planning options are inadequate. For men, the only choices are condoms and vasectomy, and most current efforts to develop new contraceptives for men impact sperm development, meaning that contraception requires months of continuous pretreatment. Here, we provide proof-of-concept for an innovative strategy for on-demand contraception, where a man would take a birth control pill shortly before sex, only as needed. Soluble adenylyl cyclase (sAC) is essential for sperm motility and maturation. We show a single dose of a safe, acutely-acting sAC inhibitor with long residence time renders male mice temporarily infertile. Mice exhibit normal mating behavior, and full fertility returns the next day. These studies define sAC inhibitors as leads for on-demand contraceptives for men, and they provide in vivo proof-of-concept for previously untested paradigms in contraception; on-demand contraception after just a single dose and pharmacological contraception for men.

Design, Synthesis, and Pharmacological Evaluation of Second-Generation Soluble Adenylyl Cyclase (sAC, ADCY10) Inhibitors with Slow Dissociation Rates.

Soluble adenylyl cyclase (sAC: ADCY10) is an enzyme involved in intracellular signaling. Inhibition of sAC has potential therapeutic utility in a number of areas. For example, sAC is integral to successful male fertility: sAC activation is required for sperm motility and ability to undergo the acrosome reaction, two processes central to oocyte fertilization. Pharmacologic evaluation of existing sAC inhibitors for utility as on-demand, nonhormonal male contraceptives suggested that both high intrinsic potency, fast on and slow dissociation rates are essential design elements for successful male contraceptive applications. During the course of the medicinal chemistry campaign described here, we identified sAC inhibitors that fulfill these criteria and are suitable for in vivo evaluation of diverse sAC pharmacology.

Assessing potency and binding kinetics of soluble adenylyl cyclase (sAC) inhibitors to maximize therapeutic potential.

In mammalian cells, 10 different adenylyl cyclases produce the ubiquitous second messenger, cyclic adenosine monophosphate (cAMP). Amongst these cAMP-generating enzymes, bicarbonate (HCO3 -)-regulated soluble adenylyl cyclase (sAC; ADCY10) is uniquely essential in sperm for reproduction. For this reason, sAC has been proposed as a potential therapeutic target for non-hormonal contraceptives for men. Here, we describe key sAC-focused in vitro assays to identify and characterize sAC inhibitors for therapeutic use. The affinity and binding kinetics of an inhibitor can greatly influence in vivo efficacy, therefore, we developed improved assays for assessing these efficacy defining features.

Strategies to safely target widely expressed soluble adenylyl cyclase for contraception.

In humans, the prototypical second messenger cyclic AMP is produced by 10 adenylyl cyclase isoforms, which are divided into two classes. Nine isoforms are G protein coupled transmembrane adenylyl cyclases (tmACs; ADCY1-9) and the 10th is the bicarbonate regulated soluble adenylyl cyclase (sAC; ADCY10). This review details why sAC is uniquely druggable and outlines ways to target sAC for novel forms of male and female contraception.

Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization.

Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkalinization, beat frequency and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and human sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in women.

Discovery of TDI-10229: A Potent and Orally Bioavailable Inhibitor of Soluble Adenylyl Cyclase (sAC, ADCY10).

Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 (12), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound.

Bicarbonate, carbon dioxide and pH sensing via mammalian bicarbonate-regulated soluble adenylyl cyclase.

Soluble adenylyl cyclase (sAC; ADCY10) is a bicarbonate (HCO3 -)-regulated enzyme responsible for the generation of cyclic adenosine monophosphate (cAMP). sAC is distributed throughout the cell and within organelles and, as such, plays a role in numerous cellular signalling pathways. Carbonic anhydrases (CAs) nearly instantaneously equilibrate HCO3 -, protons and carbon dioxide (CO2); because of the ubiquitous presence of CAs within cells, HCO3 --regulated sAC can respond to changes in any of these factors. Thus, sAC can function as a physiological HCO3 -/CO2/pH sensor. Here, we outline examples where we have shown that sAC responds to changes in HCO3 -, CO2 or pH to regulate diverse physiological functions.

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation.

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca2+, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD+] ratio, which was corroborated by using the NADH/NAD+ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD+ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD+ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.

Capacitation increases glucose consumption in murine sperm.

Mammalian sperm acquire fertilization capacity in the female reproductive tract in a process known as capacitation. During capacitation, sperm change their motility pattern (i.e., hyperactivation) and become competent to undergo the acrosome reaction. We have recently shown that, in the mouse, sperm capacitation is associated with increased uptake of fluorescently labeled deoxyglucose and with extracellular acidification suggesting enhanced glycolysis. Consistently, in the present work we showed that glucose consumption is enhanced in media that support mouse sperm capacitation suggesting upregulation of glucose metabolic pathways. The increase in glucose consumption was modulated by bicarbonate and blocked by protein kinase A and soluble adenylyl cyclase inhibitors. Moreover, permeable cyclic adenosine monophosphate (cAMP) agonists increase glucose consumption in sperm incubated in conditions that do not support capacitation. Also, the increase in glucose consumption was reduced when sperm were incubated in low calcium conditions. Interestingly, this reduction was not overcome with cAMP agonists. Despite these findings, glucose consumption of sperm from Catsper1 knockout mice was similar to the one from wild type suggesting that other sources of calcium are also relevant. Altogether, these results suggest that cAMP and calcium pathways are involved in the regulation of glycolytic energy pathways during murine sperm capacitation.

Metabolic changes in mouse sperm during capacitation†.

Mammalian sperm are stored in the epididymis in a dormant state. Upon ejaculation, they must immediately start producing sufficient energy to maintain motility and support capacitation. While this increased energy demand during capacitation is well established, it remains unclear how mouse sperm modify their metabolism to meet this need. We now show that capacitating mouse sperm enhance glucose uptake, identifying glucose uptake as a functional marker of capacitation. Using an extracellular flux analyzer, we show that glycolysis and oxidative phosphorylation increase during capacitation. Furthermore, this increase in oxidative phosphorylation is dependent on glycolysis, providing experimental evidence for a link between glycolysis and oxidative phosphorylation in mouse sperm.

Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Non-alcoholic Steatohepatitis.

Incomplete understanding of how hepatosteatosis transitions to fibrotic non-alcoholic steatohepatitis (NASH) has limited therapeutic options. Two molecules that are elevated in hepatocytes in human NASH liver are cholesterol, whose mechanistic link to NASH remains incompletely understood, and TAZ, a transcriptional regulator that promotes fibrosis but whose mechanism of increase in NASH is unknown. We now show that increased hepatocyte cholesterol upregulates TAZ and promotes fibrotic NASH. ASTER-B/C-mediated internalization of plasma membrane cholesterol activates soluble adenylyl cyclase (sAC; ADCY10), triggering a calcium-RhoA-mediated pathway that suppresses ß-TrCP/proteasome-mediated TAZ degradation. In mice fed with a cholesterol-rich NASH-inducing diet, hepatocyte-specific silencing of ASTER-B/C, sAC, or RhoA decreased TAZ and ameliorated fibrotic NASH. The cholesterol-TAZ pathway is present in primary human hepatocytes, and associations among liver cholesterol, TAZ, and RhoA in human NASH liver are consistent with the pathway. Thus, hepatocyte cholesterol contributes to fibrotic NASH by increasing TAZ, suggesting new targets for therapeutic intervention.

Optimization of lead compounds into on-demand, nonhormonal contraceptives: leveraging a public-private drug discovery institute collaboration†.

Efforts to develop new male or female nonhormonal, orally available contraceptives assume that to be effective and safe, targets must be (1) essential for fertility; (2) amenable to targeting by small-molecule inhibitors; and (3) restricted to the germline. In this perspective, we question the third assumption and propose that despite its wide expression, soluble adenylyl cyclase (sAC: ADCY10), which is essential for male fertility, is a valid target. We hypothesize that an acute-acting sAC inhibitor may provide orally available, on-demand, nonhormonal contraception for men without adverse, mechanism-based effects. To test this concept, we describe a collaboration between academia and the unique capabilities of a public-private drug discovery institute.