Transient exposure to calcium ionophore enables in vitro fertilization in sterile mouse models

Mammalian sperm acquire fertilizing capacity in the female tract in a process called capacitation. At the molecular level, capacitation requires protein kinase A activation, changes in membrane potential and an increase in intracellular calcium. Inhibition of these pathways results in loss of fertilizing ability in vivo and in vitro. We demonstrated that transient incubation of mouse sperm with Ca2+ ionophore accelerated capacitation and rescued fertilizing capacity in sperm with inactivated PKA function. We now show that a pulse of Ca2+ ionophore induces fertilizing capacity in sperm from infertile CatSper1 (Ca2+ channel), Adcy10 (soluble adenylyl cyclase) and Slo3 (K+ channel) KO mice. In contrast, sperm from infertile mice lacking the Ca2+ efflux pump PMACA4 were not rescued. These results indicate that a transient increase in intracellular Ca2+ can overcome genetic infertility in mice and suggest this approach may prove adaptable to rescue sperm function in certain cases of human male infertility.

Mammalian sperm acquire fertilizing capacity in the female tract in a process called capacitation. At the molecular level, capacitation requires protein kinase A activation, changes in membrane potential and an increase in intracellular calcium. Inhibition of these pathways results in loss of fertilizing ability in vivo and in vitro. We demonstrated that transient incubation of mouse sperm with Ca 2+ ionophore accelerated capacitation and rescued fertilizing capacity in sperm with inactivated PKA function. We now show that a pulse of Ca 2+ ionophore induces fertilizing capacity in sperm from infertile CatSper1 (Ca 2+ channel), Adcy10 (soluble adenylyl cyclase) and Slo3 (K + channel) KO mice. In contrast, sperm from infertile mice lacking the Ca 2+ efflux pump PMACA4 were not rescued. These results indicate that a transient increase in intracellular Ca 2+ can overcome genetic infertility in mice and suggest this approach may prove adaptable to rescue sperm function in certain cases of human male infertility.
In 1978, Steptoe and Edwards reported the birth of Louise Joy Brown, the first successful "Test-Tube" baby 1 . A major step toward this achievement occurred in the early 1950's, when Chang 2 and Austin 3 demonstrated independently that sperm have to be in the female reproductive tract for a period of time before acquiring fertilizing capacity, a phenomenon now known as sperm capacitation. Capacitation includes all post-ejaculation biochemical and physiological changes that render mammalian sperm able to fertilize 4 . As part of capacitation, sperm acquire the ability to undergo acrosomal exocytosis 4,5 and undergo changes in their motility pattern (i.e., hyperactivation). Molecularly, capacitation is associated with; (1) activation of a cAMP/protein kinase A pathway 6 ; (2) loss of cholesterol 7 and other lipid modifications 8 ; (3) increase in intracellular pH (pH i ) 9 ; (4) hyperpolarization of the sperm plasma membrane potential 10-12 ; (5) increase in intracellular Ca 2+ concentration [Ca 2+ ] i 13 ; and (6) increase in protein tyrosine phosphorylation 14,15 . These pathways were first identified as playing a role in capacitation using compounds that either stimulate or block the respective signaling processes. More recently, the essential roles of cAMP, Ca 2+ and plasma membrane hyperpolarization were confirmed using knock-out (KO) genetic approaches.
The role of cAMP in capacitation and fertilization was originally asserted using reagents such as cAMP agonists (dibutyryl cAMP, 8-BrcAMP) and antagonists of PKA-dependent pathways (e.g. H89, PKI, rpScAMP), as well as other conditions in which soluble adenylyl cyclase Adcy10 (aka sAC) 16,17 , the major source of cAMP in sperm, cannot be activated (e.g. HCO 3 − -free incubation media; addition of KH7, a specific sAC inhibitor) 18 . These roles of cAMP were confirmed using KO genetic mouse models lacking either the PKA sperm-specific catalytic splicing variant Cα 2 19 , or sAC 18 ; these mice are sterile and their sperm cannot fertilize in vitro. Our group has recently demonstrated that hyperpolarizing changes in membrane potential are necessary and sufficient to prepare the sperm for a physiological acrosome reaction 20 . Accordingly, sperm missing the sperm-specific K + channel SLO3 cannot hyperpolarize and are infertile 21 . Finally, Ca 2+ was shown to be essential for hyperactivation and the acrosome reaction both by removing it using Ca 2+ -free incubation media, either with or without A 23187 treatment rescues hyperactivation and fertilizing capacity of CatSper1 KO sperm. In the absence of the CatSper channel complex, sperm fail to undergo hyperactivated motility and are unable to fertilize 24 . To test whether Ca 2+ ionophore treatment can overcome the CatSper infertile phenotype, sperm from CatSper1 KO mice were incubated in conditions that support capacitation in the absence or in the presence of 20 μ M A 23187 . After 10 min, the sperm were washed twice by centrifugation in A 23187 -free media and the percentage of hyperactive sperm was measured using CASA. As expected, in the absence of A 23187, CatSper KO sperm did not undergo hyperactivation ( Fig. 2A, Supplementary Table II and Supplementary Movie 1). However, once exposed to Ca 2+ ionophore, a significant number of CatSper KO sperm exhibited hyperactivated motility ( Fig. 2A, Supplementary Table II and Supplementary Movie 2). In addition, A 23187 -treated CatSper KO sperm were competent to fertilize metaphase II-arrested eggs in vitro (Fig. 2B). In two independent experiments, fertilized eggs were allowed to develop to late morula or blastocyst stage (Fig. 2C, left panel) and ten embryos in each case were non-surgically transferred to pseudopregnant WT female mice [28][29][30] . From these experiments, five CatSper (+ /− ) mouse pups were born from two different females (Fig. 2C, right panel). These heterozygous F1 mice were fertile; mating a male and female from this heterozygous population yielded a normal litter with 1 wild type, 4 heterozygous and 3 CatSper KO F2 progeny (Fig. 2D).
A 23187 treatment rescues hyperactivation and fertilizing capacity in sperm of Adcy10 (aka sAC) KO and Slo3 KO but not in sperm from Pmca4 KO mice. Capacitation requires up-regulation of cAMP concentrations 18,19 and hyperpolarization of the sperm plasma membrane 21 . Under normal capacitation conditions, neither sAC KO nor SLO3 KO sperm undergo hyperactivation (Fig. 3B), and while SLO3 KO sperm are able to move (Supplementary Table III Fig. 1E and ref. 23), we tested whether these KO mouse models could be rescued by a Ca 2+ ionophore pulse. When treated with A 23187 for 10 min, a significant fraction of sAC KO sperm became motile and both sAC KO and SLO3 KO sperm underwent hyperactivation ( Fig. 3B and Supplementary Movies 4 and 6). Moreover, A 23187 treatment induced in vitro fertilizing capacity in sperm from both KO models (Fig. 3C).
We previously showed that the increase in intracellular Ca 2+ caused by A 23187 has to be followed by a reduction in intracellular concentrations of this ion after removal of the ionophore 23 . In sperm, two molecules are thought to mediate Ca 2+ extrusion, namely the Na + /Ca 2+ exchanger and the more efficient, sperm-specific Ca 2+ ATPase PMCA4 31 . Male Pmca4 KO mice are infertile 32 ; their sperm display poor motility and do not undergo hyperactivation (Fig. 3D,E). These data suggest this molecule is involved in regulation of normal Ca 2+ homeostasis in sperm. We hypothesized that sperm lacking PMCA4 would have diminished capacity to efflux Ca 2+ following ionophore treatment and be less susceptible to A 23187 rescue. Treatment with A 23187 rendered all Pmca4 −/− sperm motionless, and their motility was not recovered after ionophore removal (Fig. 3D). Consequently, neither their hyperactivated motility nor their fertilizing capacity was rescued (Fig. 3E).

Discussion
Capacitation encompasses a series of sequential and concomitant biochemical changes required for sperm to gain full fertilization competency. Despite the relevance of capacitation, the molecular mechanisms intrinsic to this process are not well understood. A very early event in sperm capacitation is the activation of motility by a cAMP-dependent pathway 33 . The activation of cAMP synthesis occurs immediately after sperm are released from the epididymis and come into contact with high HCO 3 − and Ca 2+ present in the seminal fluid 34,35 . Plasma membrane transport of these ions regulates sperm cAMP metabolism through stimulation of Adcy10 (aka sAC) 18 , which elevates intracellular cAMP and activates PKA. Then, PKA phosphorylates target proteins and initiates several signaling pathways. These pathways include sperm plasma membrane hyperpolarization, increase in pH i , and increase in intracellular Ca 2+ ions. Consistent with the influence of these events, KO mice models in which any of these pathways is interrupted are infertile.
Physiologically, sperm capacitation is associated with preparation for a physiological acrosome reaction and changes in their motility pattern collectively known as hyperactivation. Originally observed in hamster sperm moving in the oviduct, hyperactivated motility 36 was later described in other mammalian species including humans 37 . Hyperactivation is associated with a strong, high-amplitude asymmetrical flagellar beating that appears to be essential for the sperm to loosen their attachment to the oviductal epithelium and to penetrate the zona pellucida 38 . Consistent with an essential role of hyperactivation for fertilization competency, low motility and/or defects in hyperactivation is one of the most common phenotypes observed in sperm from many different infertile knock-out models, including those used in the present work (i.e., 18,21,24,32 . Although very little is known about the molecular pathways regulating hyperactivation, Ca 2+ ions have been shown to play roles in the initiation and maintenance of this type of movement 22 . Most of the information regarding the role of Ca 2+ in hyperactivation has been obtained using loss-of-function approaches analyzing sperm motility in media devoid of Ca 2+ ions. Gain-of-function experiments using Ca 2+ ionophores (e.g. A 23187 , ionomycin) to increase [Ca 2+ ] i have yielded unexpected results because, instead of enhancing hyperactivation, these compounds stopped sperm movement 7,23,39 . Despite being motionless, ionophore-treated sperm are alive as they recover motility after the compound is quenched with lipophilic agents 39 or removed by centrifugation 23 . The reversibility of the A 23187 effect suggests that the sperm is able to return to physiological [Ca 2+ ] i after a drop in free ionophore concentration. In our previous work, we showed that a short incubation period with A 23187 , in addition to initiating hyperactivation, accelerated the acquisition of fertilizing capacity. Most importantly, our data indicated that 10 min incubation with A 23187 induced fertilization competence even when activation of cAMP-dependent signaling pathways was blocked 23 . Considering these results, we hypothesized that a temporary elevation of intracellular Ca 2+ primes the sperm for hyperactivation and bypasses the need for other signaling pathways required to up-regulate Ca 2+ influx in sperm. To test this hypothesis, in the present work, we selected four KO models affecting independent signaling pathways involved in sperm motility. Three of these signaling molecules are believed to act upstream of the increase in Ca 2+ required for hyperactivation: CatSper, sAC and SLO3. Sperm from each of these mouse models were unable to undergo hyperactivation and are incapable of fertilizing metaphase II arrested eggs in vitro. In addition, Pmca4 KO sperm were used, which would not allow intracellular Ca 2+ lowering after saturating sperm cells with this ion. Pmca4 KO mice are sterile because their sperm are deficient in both progressive and hyperactivated motility 25,40 . PMCA4 has been shown to be an essential source of Ca 2+ clearance in sperm, and it is required to achieve a low resting [Ca 2+ ] i 31 . Consistent with our hypotheses, a short incubation of sperm with A 23187 induced hyperactivation of CatSper, Adcy10 and Slo3 KO but not of Pmca4 KO sperm.
Male factors contribute to approximately half of all cases of infertility 41 . However, in over 75% of these cases it is unusual to have a clear diagnosis of the abnormalities found in semen parameters 42,43 . Currently, assisted reproductive technologies (ART) remain the main therapy available. Recent studies using KO mouse models, including those used in the present work, revealed that loss of function of a variety of genes results in infertility. Interestingly, several of these models display normal sperm counts, and their main deficiency is found in capacitation-associated processes such as impediments to undergo hyperactivation 24 , to undergo the acrosome reaction 21 , or to go through the utero-tubal junction in vivo 44,45 . We hypothesize that strategies designed to elevate [Ca 2+ ] i such as the use of A 23187 pulse should overcome the need of upstream signaling pathways including but not limited to PKA activation. In addition, although IVF has been successfully employed in multiple species 5 , requirements of sperm for capacitation vary greatly among species and have been developed for each sperm type essentially by trial and error. In some species, such as the horse, effective methods for IVF have yet to be established despite decades of work 46 . Failure of equine IVF does not appear to be associated with oocyte characteristics 47 but with the inability of horse sperm to hyperactivate and to penetrate the egg zona pellucida (ZP), two landmarks of capacitation. A better understanding of capacitation signaling processes have the potential to generate a "universal" IVF technology that can be used in endangered/exotic species for which ART is not currently available.
Improving IVF conditions would be of great value; however, at the clinical level, ICSI has replaced IVF when confronted with cases of infertility due to unknown male factor(s). ICSI is reliable and, from the patient's point of view, more economical because of higher probability of success. Despite these advantages, ICSI bypasses certain aspects of normal fertilization and may bear effects that are not easily observed. Taking this into consideration, a method to improve IVF can be a desirable option in some male factor cases. It is worth noting that A 23187 has already been used in the clinic for patients with repeated ICSI failure 48 due to problems in egg activation. In these cases, fertilized eggs are transiently incubated with ionophore after ICSI, which exposes the zygote to high Ca 2+ . On the contrary, with the method described here, where sperm are transiently treated with A 23187 , the ionophore is washed out and does not come in contact with the embryo. More interestingly, using this methodology to overcome infertility problems related to motility and hyperactivation could be used to improve the success rate of intrauterine insemination, which is a significantly less invasive and less costly procedure than either IVF or ICSI. In experiments in which phosphorylation by PKA was investigated, C57BL/6J male mice were used. Vasectomized males were obtained from Charles River, and used to induce pseudopregnancy as previously described 49 .Non-surgical embryo transfer (NSET) was performed with an NSET device (ParaTechs, Lexington, KY) 29,30 . Media. Medium used for sperm capacitation and fertilization assays was Toyoda-Yokoyama-Hosi (standard TYH) medium 50  − , 0.51 mM Na-pyruvate, 5.56 mM glucose, and 4 mg/mL bovine serum albumin (BSA), 10 μ g/mL Gentamicin and phenol red 0.0006% at pH 7.4 equilibrated with 5% CO 2 . For capacitating conditions Ca 2+ ionophore A 23187 was used at a final concentration of 20 μ M in TYH as previously described 23 . Mouse Sperm Preparation. Cauda spermatozoa were collected from each of the mouse strains described above. Each cauda epididymis was placed in 500 μ L of TYH media. After 10 min. incubation at 37 °C (swim-out), epididymis tissue debris were removed, and the suspension adjusted to a final concentration of 1-2 10 7 cells/ml and divided into two aliquots. Aliquots were supplemented with either 20 μ M A 23187 or equivalent quantities of DMSO (for controls) and further incubated at 37 °C. After 10 min. incubation, sperm were washed with 2 rounds of centrifugations (first one at 500 × g and the second one at 300 × g for 5 min each) in A 23187 -free TYH medium. Sperm were then re-suspended in A 23187 -free TYH and capacitated in CO 2 incubator for an additional hour and 20 min. To evaluate sperm in conditions in which PKA is inactivated, H89 was used at a concentration of 50 μM for all incubation periods including those used for washing the ionophore A 23187 . After capacitation in each condition, sperm were used for the analysis of phosphorylated PKA substrates, hyperactivation and fertilizing capacity (see below).

SDS-PAGE and Immunoblotting.
After 1 hour and 20 min incubation in each condition, sperm proteins were extracted for Western blot analysis as previously described 22 . Protein extracts equivalent to 1 × 10 6 sperm were loaded per line and subjected to SDS-PAGE an electro-transferred to PVDF membranes (Bio-Rad) at 250 mA for 90 min on ice. To analyze phosphorylated PKA substrates, anti-phosphoPKA substrate (anti-pPKAS) (1/10000) Western blots were used as described 22 . Hyperactive and Motility Parameters. Sperm suspensions (25 μ l) were loaded into one pre-warmed chamber slide (depth, 100 μ m) (Leja slide, Spectrum Technologies) and placed on a microscope stage at 37 °C. Sperm movements were examined using the CEROS computer-assisted semen analysis (CASA) system (Hamilton Thorne Research, Beverly, MA). The default settings include the following: frames acquired: 90; frame rate: 60 Hz; minimum cell size: 4 pixels; static head size: 0.13-2.43; static head intensity: 0.10-1.52; static head elongation: Scientific RepoRts | 6:33589 | DOI: 10.1038/srep33589 5-100. Sperm with hyper activated motility, defined as motility with high amplitude thrashing patterns and short distance of travel, were sorted and analyzed using the CASAnova software 27 . At least 20 microscopy fields corresponding to a minimum of 200 sperm were analyzed in each experiment.
Sperm Motility Video Recordings. Sperm suspensions (25 μ l) were loaded into one pre-warmed chamber slide (depth, 100 μ m) (Leja slide, Spectrum Technologies). Videos were recorded for 15 seconds using an Andor Zyla microscope camera (Belfast, Northern Ireland) mounted on Nikon TE300 inverted microscope (Chiyoda, Tokyo, Japan) fitted with 20 times objective lenses. Sample temperatures were maintained at 37 °C using a Warm Stage (Frank E. Fryer scientific instruments, Carpentersville, Illinois).
Mouse eggs collection and IVF assays. Metaphase II-arrested mouse eggs were collected from 6-8 week-old super ovulated CD1 (ICR) female mice (Charles River Laboratories) as previously described 22 . Females were each injected with 5-10 IU equine chorionic gonadotropin and 5-10 IU human chorionic gonadotropin 48 h apart. The cumulus-oocyte complexes (COC's) were placed into a well with 500 μ l of media (TYH standard medium) previously equilibrated in an incubator with 5% CO 2 at 37 °C. Fertilization wells containing 20-30 eggs were inseminated with sperm incubated as described above in medium supporting capacitation with or without A 23187 treatment (final concentration of 1 × 10 6 cells/ml). After 4 h of insemination, eggs were washed and put in fresh media. The eggs were evaluated 24 h post-insemination. To assess fertilization the three following criteria were considered: 1) the formation of the male and female pronuclei; 2) the emission of the second polar body; and 3) two-cells stages.
Embryo Culture, Embryo transfer and Mice Genotyping. Twenty-four hours post-insemination, fertilized 2 cell embryos were transferred to drops containing KSOM media and further incubated between 3.5 and 4.1 days. At this stage, the percentage of blastocyst formation was evaluated. In some cases, 10 to 20 blastocysts were transferred to 2.5 days post coitum (dpc) pseudo-pregnant CD-1 recipient females using the non-surgical uterine embryo transfer device 28 . Pseudo-pregnant CD-1 recipient females were obtained by mating with vasectomized males (obtained from Charles River) one day after in vitro fertilization. Only females with a clear plug were chosen as embryo recipients; late morula and early stage blastocysts were chosen to be transferred. Routine genotyping was performed with total DNA from tail biopsy samples from weaning age pups as templates for PCR using genotyping primers for CatSper gene forward