STUDY QUESTION:How does soluble adenylyl cyclase (sAC)-generated cyclic AMP (cAMP) control hyperactivated motility in human sperm? SUMMARY ANSWER:sAC-generated cAMP rapidly initiates and is required to maintain hyperactivated motility in human sperm. WHAT IS KNOWN ALREADY:Mouse and human sperm devoid of sAC activity (either genetically or pharmacologically) are immotile and do not undergo capacitation; thus, the HCO3--dependent stimulation of sAC and consequent increase in cAMP is responsible for activating basal motility and initiating capacitation in multiple mammalian species. Among the changes sperm undergo during capacitation is acquisition of hyperactivated motility, which is presumed to be essential for male fertility. STUDY DESIGN, SIZE, DURATION:In this study, the kinetics of cAMP generation and motility were assessed in sperm from healthy semen donors with no known fertility issues subjected to capacitating media components (HCO3- and albumin). Controls included cAMP agonists and adenylyl cyclase inhibitors. PARTICIPANTS/MATERIALS, SETTING, METHODS:The motility of sperm purified from donors' semen samples was analyzed by a Computer-Assisted Sperm Analysis (CASA) system, and the intracellular cAMP was quantified using a cAMP ELISA. MAIN RESULTS AND THE ROLE OF CHANCE:HCO3- stimulates sAC-dependent cAMP production and the transition to hyperactivated motility at the earliest times measured. Sperm hyperactivated motility seems to be a reversible process, as maintaining hyperactivated motility requires sustained sAC activation. LIMITATIONS, REASONS FOR CAUTION:The CASA system, used to measure hyperactivated motility, employs snapshot technology; sperm trajectories are observed for only short segments of time. This is an ex vivo study of sperm motility parameters in aqueous solutions. The conditions used were established for successful IVF, and the capacitation-induced hyperactivated motility studied here is proven essential for IVF and positively correlated with in vivo fertilization competence. However, in vivo, ejaculated sperm must navigate through the female reproductive tract, which is lined by viscous mucus, to reach the site of fertilization. Future studies should examine motility behaviors in solutions whose viscosity more accurately reflects the mucus-lined environment of the female reproductive tract. WIDER IMPLICATIONS OF THE FINDINGS:There are two novel findings presented here; that hyperactivation of human sperm occurs early during capacitation and that hyperactivated motility is reversible. These findings raise the possibility that the rapid, sAC-dependent hyperactivated motility allows human sperm to escape the harsh vaginal environment. Its roles modulating sperm motility define sAC as an optimal target for both male and female contraception. Additionally, sAC inhibitors with different off-rates are shown here to be useful tools enabling us to study the kinetics of sAC activation in a physiological context. STUDY FUNDING/COMPETING INTEREST(S):The research was funded by Male Contraceptive Initiative (to J.B. and L.R.L.) and National Institutes of Health via HD113015 and HD111549 (to J.B. and L.R.L.). C.R. was awarded a Male Contraceptive Initiative fellowship. L.R.L. and J.B. are co-inventors of a panel of in vivo, validated sAC inhibitors (patent PCT/US2022/02652) and are co-founders, co-owners, and members of the Board of Directors of Sacyl Pharmaceuticals Inc., which licensed the sAC inhibitors for development into on-demand male contraceptives. C.R. has been a paid consultant to Sacyl Pharmaceuticals Inc. TRIAL REGISTRATION NUMBER:N/A.

Male fertility depends on sperm motility, which enables sperm to traverse the female reproductive tract to the site of fertilization. Soluble adenylyl cyclase (sAC) is a key regulator of sperm motility, defining it as a promising non-hormonal contraceptive target for women and men. To evaluate the efficacy of sAC inhibitors under conditions mimicking the female reproductive tract, we employed a capillary assay using cervical mucus isolated from humans or cows as well as methylcellulose, a viscoelastic liquid that simulates cervical mucus. Addition of a sAC inhibitor, either directly to semen, modeling an on-demand male contraceptive, or to the cervical mucus, approximating a female contraceptive, effectively blocked sperm migration. These studies provide proof-of-concept for the efficacy of sAC inhibitors under physiologically relevant conditions.

Soluble adenylyl cyclase (sAC; ADCY10) is an evolutionarily ancient, intracellular source of cAMP that is molecularly and mechanistically distinct from the more widely studied, hormone-responsive, G protein-regulated transmembrane adenylyl cyclases. Unlike other mammalian cyclases, sAC is most abundantly expressed in male germ cells and is directly regulated by bicarbonate and calcium. Genetic and pharmacological evidence in rodents and humans establishes sAC as essential for male fertility: loss of sAC activity yields immotile sperm incapable of fertilizing the egg, resulting in male-specific infertility. These features position sAC as a promising target for developing nonhormonal, on-demand contraceptives suitable for men and women. A proof-of-concept inhibitor has demonstrated a rapid, reversible contraceptive effect in vivo in mice, but translation to a clinical product must address challenges inherent to on-demand sperm-targeted pharmacology. In addition to ensuring a high safety margin and navigating an emerging regulatory and commercial landscape, common to any male contraceptive, on-demand male contraception must define the onset/duration of efficacy while ensuring persistent inactivation of sperm function after ejaculation.

cAMP is a ubiquitous second messenger produced from ATP and involved in many cellular processes. In humans, cAMP is produced from two types of adenylyl cyclases: G protein-regulated transmembrane adenylyl cyclases (ADCY1-9) and bicarbonate- (HCO3-) and calcium- (Ca2+) regulated soluble adenylyl cyclase (sAC; ADCY10). sAC is molecularly and biochemically distinct from other mammalian nucleotidyl cyclases. In most mammals, sAC arises from a single gene which is predicted to generate multiple isoforms via alternative splicing. In rodents, there are two molecularly identified splice variants: the "full-length" isoform (sACfl) and a "truncated" isoform (sACt). To date, biochemical and structural characterization of sAC has focused almost exclusively on the sACt isoform. Longer sAC isoforms, including the longest known sACfl, contain additional presumptive regulatory domains which have not yet been functionally characterized. Thus far, studies have been limited by the inability to obtain sufficient sACfl protein to allow in vitro biochemical characterization. Here, we describe attempts to heterologously express and purify human sACfl as well as generation of a novel genetically modified mouse strain which permits biochemical separation and purification of endogenously expressed mouse sACfl and sACt. We use these heterologously expressed and endogenous proteins to compare and contrast the biochemically properties of human and mouse sACfl and sACt.

Prior to ejaculation, mammalian sperm are stored in the epididymis in a "resting" metabolic state. Upon ejaculation, sperm must alter their metabolism to generate the energy needed to support the motility and maturation process known as capacitation to reach and fertilize the oocyte. How sperm regulate the capacitation-induced increase in carbon flux is unknown. Here, we use 13C stable isotope labeling in mouse sperm isolated from the cauda epididymis to follow glucose metabolism through central carbon metabolic network before and after sperm activation. As sperm transition from resting to highly activated states, they boost energy yield by increasing flux through glycolysis at the expense of the pentose phosphate pathway. Increased glycolytic activity seems to be achieved via capacitation-induced stimulation of flux through aldolase. In the mitochondria-containing midpiece, glycolytically generated pyruvate feeds the tricarboxylic acid (TCA) cycle to further maximize energy yield via oxidative phosphorylation. In the mitochondria-free principal piece of the flagellum, pyruvate produced from glycolysis is reduced to lactate by lactate dehydrogenase, which also serves to regenerate oxidized nicotinamide adenine dinucleotide (NAD+) ensuring a sufficient supply to support glycolysis. The resultant lactate is at least partially secreted. Finally, we find evidence that there is an as yet unknown endogenous source of energy in sperm, feeding the upregulation of TCA cycle intermediates. These studies provide the most complete picture of the metabolic shift which occurs in capacitating mouse sperm in glucose.

Sperm capacitation involves a series of biochemical and physiological changes essential for fertilization. A critical regulator of capacitation, the soluble adenylyl cyclase (sAC; ADCY10)-dependent production of the second messenger cyclic AMP (cAMP), drives key downstream events such as protein kinase A (PKA) substrate phosphorylation. sAC activity is directly stimulated by bicarbonate (HCO3 -) and calcium (Ca2+). CatSper, a sperm-specific Ca2+ channel, is considered the primary pathway for Ca2+ influx during capacitation; however, emerging evidence suggests additional pathways exist. This study reveals that bovine serum albumin (BSA) influences the dynamics of intracellular Ca2+ concentration ([Ca2+]i) in CatSper1 knockout (KO) sperm and plays a novel role in sAC activation. Using single-cell live imaging and flow cytometry, we observed a rapid [Ca2+]i rise in the head of CatSper1 KO sperm under capacitating conditions, indicating an alternative Ca2+ entry mechanism. BSA alone, in the absence of HCO3 -, triggered a significant [Ca2+]i rise. Removal of extracellular Ca2+ abolished this [Ca2+]i rise, confirming the necessity of Ca2+ influx. This BSA-induced [Ca2+]i rise was upstream of sAC activation, since it was not affected by sAC inhibitors and led to increased cAMP production and PKA substrate phosphorylation. Our findings provide new insights into the regulatory mechanisms of sAC, highlighting the existence of a CatSper-independent Ca2+ entry pathway activated by BSA during sperm capacitation. This rapid [Ca2+]i rise is initiated in the sperm head and propagates throughout the cell, and is sufficient to activate sAC and stimulate cAMP synthesis independently of HCO3 -. KEY POINTS: Sperm capacitation, essential for fertilization, is regulated by sAC, which produces cAMP in response to HCO3 - and Ca2+, driving key events like protein kinase A substrate phosphorylation. We demonstrate the existence of a CatSper-independent Ca2+ entry pathway that initiates in the sperm head and propagates throughout the cell, occurring rapidly after sperm encounters albumin, a critical component of the capacitation medium used in in vitro fertilization procedures in mammals. This albumin-induced Ca2+ influx is sufficient to activate sAC and stimulate cAMP synthesis independently of HCO3 -. We further reveal a novel role for albumin, beyond its well-established function as a cholesterol acceptor, in triggering this rapid Ca2+ influx and downstream signalling events essential for sperm capacitation. By demonstrating a CatSper-independent regulatory pathway, we expand the current paradigm of Ca2+ signalling in sperm physiology.

Soluble adenylyl cyclase (sAC) is molecularly and biochemically distinct from other mammalian nucleotidyl cyclases. It is uniquely regulated directly by bicarbonate (HCO3-) and calcium (Ca2+) ions and is responsive to physiologic fluctuations in levels of its substrate, adenosine triphosphate (ATP). Our initial in vitro biochemical studies suggested two mechanisms for HCO3--dependent elevation of sAC activity: increasing catalytic rate and relieving inhibition observed in the presence of supraphysiological levels of substrate, ATP. Structural and mutational studies revealed that HCO3- increases catalytic rate via the disruption of a salt bridge that facilitates productive interactions with the substrate. Here, we demonstrate that the HCO3- stimulation observed under supraphysiological ATP concentrations is due to the mitigation of ATP-dependent acidification. Therefore, we conclude that the sole physiologically relevant mechanism of HCO3- regulation of sAC is through its pH-independent effect facilitating productive substrate binding to the catalytic site.

The second messenger cAMP plays multiple critical roles in the control of sperm functions essential for male fertility, including motility. The enzyme soluble adenylyl cyclase (sAC; ADCY10) was shown genetically and pharmacologically to be the essential source of cAMP mediating many of these functions. Male mice and men with genetic deletions of sAC are infertile, and their sperm are progressively immotile. Pharmacologically, delivery of potent and specific sAC inhibitors to male mice renders them temporarily infertile, and their sperm are similarly immotile. Here, we show that males from a second, independently derived mouse sAC knockout line are also infertile with progressively immotile sperm. We use these mouse models to determine optimal conditions for pharmacologically elevating intracellular cAMP to rescue the sAC null motility defect. We show that cell-permeable cAMP analogs, but not forskolin, rescue the motility defects of sAC deficient sperm, and we demonstrate that 8Br-cAMP is an efficient cAMP analog to rescue motility.

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.

Because nearly half of pregnancies worldwide are unintended, available contraceptive methods are inadequate. Moreover, due to the striking imbalance between contraceptive options available for men compared to the myriad of options available to women, there is an urgent need for new methods of contraception for men. This review summarizes ongoing efforts to develop male contraceptives highlighting the unique aspects particular to on-demand male contraception, where a man takes a contraceptive only when and as often as needed.

Dr. David Garbers made many impactful contributions to science and vastly improved our understanding of sperm biology. In this review, we focus on his identification of a key role for the second messenger cAMP in mammalian sperm. As a graduate student David discovered that sperm motility, which is essential for sperm to fertilize the egg, is under the control of the (at the time) recently identified, prototypical second messenger cAMP. Fast-forwarding to the present, agents which turn off sperm's ability to generate cAMP and block sperm motility are being investigated as potential nonhormonal contraceptives for men and women. Should these efforts prove successful, Dave's discoveries will prove to be the spark which ignited a revolution in human health.

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.

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.

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.

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.

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.

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

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 (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.