Claudia Sánchez-Cárdenas

Pituitary growth hormone network responses are sexually dimorphic and regulated by gonadal steroids in adulthood

There are well-recognized sex differences in many pituitary endocrine axes, usually thought to be generated by gonadal steroid imprinting of the neuroendocrine hypothalamus. However, the recognition that growth hormone (GH) cells are arranged in functionally organized networks raises the possibility that the responses of the network are different in males and females. We studied this by directly monitoring the calcium responses to an identical GH-releasing hormone (GHRH) stimulus in populations of individual GH cells in slices taken from male and female murine GH-eGFP pituitary glands. We found that the GH cell network responses are sexually dimorphic, with a higher proportion of responding cells in males than in females, correlated with greater GH release from male slices. Repetitive waves of calcium spiking activity were triggered by GHRH in some males, but were never observed in females. This was not due to a permanent difference in the network architecture between male and female mice; rather, the sex difference in the proportions of GH cells responding to GHRH were switched by postpubertal gonadectomy and reversed with hormone replacements, suggesting that the network responses are dynamically regulated in adulthood by gonadal steroids. Thus, the pituitary gland contributes to the sexually dimorphic patterns of GH secretion that play an important role in differences in growth and metabolism between the sexes.

Compartmentalization of distinct cAMP signaling pathways in mammalian sperm.

Background: cAMP is essential for the acquisition of sperm fertilizing capacity. The presence of transmembrane adenylyl cyclases (tmACs) in sperm remains controversial. Results: tmAC activity and its activator Gs are detected in the sperm head. Conclusion: Two cAMP synthesis pathways coexist in sperm and lead to capacitation. Significance: Understanding capacitation is essential for improvement of assisted fertilization and for finding novel contraceptive targets. Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. Although the HCO3−-dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric Gs proteins; however, the presence of Gs or tmACs in mammalian sperm has been controversial. In this study, we used Western blotting and cholera toxin-dependent ADP-ribosylation to show the Gs presence in the sperm head. Also, we showed that forskolin, a tmAC-specific activator, induces cAMP accumulation in sperm from both WT and Adcy10-null mice. This increase is blocked by the tmAC inhibitor SQ22536 but not by the Adcy10 inhibitor KH7. Although Gs immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, Gs reactivity is lost in acrosome-reacted sperm, and forskolin is able to increase intracellular Ca2+ and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with Gs and tmAC in the head and Adcy10 and PKA in the flagellum.

Biphasic Role of Calcium in Mouse Sperm Capacitation Signaling Pathways

Mammalian sperm acquire fertilizing ability in the female tract in a process known as capacitation. At the molecular level, capacitation is associated with up‐regulation of a cAMP‐dependent pathway, changes in intracellular pH, intracellular Ca2+, and an increase in tyrosine phosphorylation. How these signaling systems interact during capacitation is not well understood. Results presented in this study indicate that Ca2+ ions have a biphasic role in the regulation of cAMP‐dependent signaling. Media without added Ca2+ salts (nominal zero Ca2+) still contain micromolar concentrations of this ion. Sperm incubated in this medium did not undergo PKA activation or the increase in tyrosine phosphorylation suggesting that these phosphorylation pathways require Ca2+. However, chelation of the extracellular Ca2+ traces by EGTA induced both cAMP‐dependent phosphorylation and the increase in tyrosine phosphorylation. The EGTA effect in nominal zero Ca2+ media was mimicked by two calmodulin antagonists, W7 and calmidazolium, and by the calcineurin inhibitor cyclosporine A. These results suggest that Ca2+ ions regulate sperm cAMP and tyrosine phosphorylation pathways in a biphasic manner and that some of its effects are mediated by calmodulin. Interestingly, contrary to wild‐type mouse sperm, sperm from CatSper1 KO mice underwent PKA activation and an increase in tyrosine phosphorylation upon incubation in nominal zero Ca2+ media. Therefore, sperm lacking Catsper Ca2+ channels behave as wild‐type sperm incubated in the presence of EGTA. This latter result suggests that Catsper transports the Ca2+ involved in the regulation of cAMP‐dependent and tyrosine phosphorylation pathways required for sperm capacitation. J. Cell. Physiol. 230: 1758–1769, 2015.

Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction

Background: Sperm capacitation, a process associated with phosphorylation and membrane potential changes, is required for acrosome reaction and fertilization. Results: Inducing hyperpolarization in non-capacitated sperm does not result in protein tyrosine phosphorylation but allows physiologically-induced [Ca2+]i increases and acrosome reaction. Conclusion: Sperm hyperpolarization appears to be necessary and sufficient for acrosome reaction. Significance: Advancing our understanding of capacitation, the acrosome reaction and fertilization. Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pHi) and Ca2+ ([Ca2+]i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K+ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K+ or by addition of solubilized zona pellucida (sZP). Moreover, K+ and sZP were also able to increase [Ca2+]i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K+ concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca2+]i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction.

Ca2+ ionophore A23187 can make mouse spermatozoa capable of fertilizing in vitro without activation of cAMP-dependent phosphorylation pathways

Significance Sperm capacitation enables spermatozoa to undergo the acrosome reaction and to exhibit vigorous motility called hyperactivation. At the molecular level, capacitation is associated with activation of a cAMP-dependent pathway and with the increase of intracellular pH and Ca2+ concentrations. Ca2+ ionophore A23187 elevates intracellular Ca2+ and induces the acrosome reaction but renders the spermatozoa motionless. However, when the ionophore was washed away, spermatozoa recovered motility, showed hyperactivation, and were able to fertilize cumulus-intact eggs. In these conditions, sperm acquired fertilizing capacity even when the cAMP pathway was inactivated. Fertilized oocytes with A23187-treated sperm developed into normal offspring. These data indicate that a short elevation of intracellular Ca2+ overcomes other necessary signaling pathways during capacitation and renders sperm fertile. Ca2+ ionophore A23187 is known to induce the acrosome reaction of mammalian spermatozoa, but it also quickly immobilizes them. Although mouse spermatozoa were immobilized by this ionophore, they initiated vigorous motility (hyperactivation) soon after this reagent was washed away by centrifugation. About half of live spermatozoa were acrosome-reacted at the end of 10 min of ionophore treatment; fertilization of cumulus-intact oocytes began as soon as spermatozoa recovered their motility and before the increase in protein tyrosine phosphorylation, which started 30–45 min after washing out the ionophore. When spermatozoa were treated with A23187, more than 95% of oocytes were fertilized in the constant presence of the protein kinase A inhibitor, H89. Ionophore-treated spermatozoa also fertilized 80% of oocytes, even in the absence of HCO3−, a component essential for cAMP synthesis under normal in vitro conditions. Under these conditions, fertilized oocytes developed into normal offspring. These data indicate that mouse spermatozoa treated with ionophore are able to fertilize without activation of the cAMP/PKA signaling pathway. Furthermore, they suggest that the cAMP/PKA pathway is upstream of an intracellular Ca2+ increase required for the acrosome reaction and hyperactivation of spermatozoa under normal in vitro conditions.

The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm.

Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2−/− mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro. Highlighted article: The increase in tyrosine phosphorylation seen during sperm capacitation is mediated by the FER kinase, but this does not seem to be essential for fertilisation in vivo in mice.

Are TRP channels involved in sperm development and function

Spermatozoa must translate information from their environment and the egg to achieve fertilization in sexually reproducing animals. These tasks require decoding a variety of signals in the form of intracellular Ca2+ changes. As TRP channels constitute a large family of versatile multi-signal transducers, they are interesting subjects in which to explore their possible participation in sperm function. Here, we review the evidence for their presence and involvement in sperm motility, maturation, and the acrosome reaction, an exocytotic process required for sperm–egg fusion. Since store-operated Ca2+ entry (SOCE) has been proposed to play an important role in these three functions, the main proteins responsible for this transport (STIM and ORAI) and their interaction with TRPs are also discussed. Improving our tools to solve infertility, improve animal breeding, and preserve biodiversity requires a better understanding of how Ca2+ is regulated in spermatozoa.

Acrosome Reaction and Ca2+ Imaging in Single Human Spermatozoa: New Regulatory Roles of [Ca2+]i

ABSTRACT The spermatozoa acrosome reaction (AR) is essential for mammalian fertilization. Few methods allow visualization of AR in real time together with Ca2+ imaging. Here, we show that FM4-64, a fluorescent dye used to follow exocytosis, reliably reports AR progression induced by ionomycin and progesterone in human spermatozoa. FM4-64 clearly delimits the spermatozoa contour and reports morphological cell changes before, during, and after AR. This strategy unveiled the formation of moving tubular appendages, emerging from acrosome-reacted spermatozoa, which was confirmed by scanning electron microscopy. Alternate wavelength illumination allowed concomitant imaging of FM4-64 and Fluo-4, a Ca2+ indicator. These AR and intracellular Ca2+ ([Ca2+]i) recordings revealed that the presence of [Ca2+]i oscillations, both spontaneous and progesterone induced, prevents AR in human spermatozoa. Notably, the progesterone-induced AR is preceded by a second [Ca2+]i peak and ∼40% of reacting spermatozoa also manifest a slow [Ca2+]i rise ∼2 min before AR. Our findings uncover new AR features related to [Ca2+]i.

GnRH-Induced [Ca2+]i-Signalling Patterns in Mouse Gonadotrophs Recorded from Acute Pituitary Slices in vitro

In this study we used [Ca2+]i imaging to monitor GnRH-induced intracellular Ca2+ signalling from dozens of gonadotrophs in mouse male pituitary slices. Responses of individual cells vary in magnitude, latency, duration and frequency of oscillation. Approximately 20% of gonadotrophs in situ display Ca2+ oscillations of increasing frequency at higher [GnRH] and biphasic (peak-plateau) responses at saturating [GnRH]. Nevertheless, this orderly progression, reported in cultured cells, is less well organized in 55% of cells. Furthermore, approximately 30% cells display non-oscillatory GnRH responses, reminiscent of immature gonadotrophs. Dose-response curves of slices from different animals suggest inter-individual differences in GnRH sensitivity. When the same dose of GnRH is applied repeatedly, individual cell responses are almost identical both in latency, oscillatory pattern and duration resembling the ‘Ca2+ fingerprint’ phenomenon. In addition, gonadotrophs in situare arranged in small clusters with similar GnRH-induced intracellular Ca2+-signalling patterns. Neighbouring gonadotrophs within clusters often display synchronized GnRH-induced responses with high correlation indices (>0.75). Nevertheless, synchronized responses between pairs of gonadotrophs are unaffected by incubation with blockers of gap-junction channels or P2X receptor channels, suggesting that they are not mediated by gap junctions or ATP. Alternative explanations are discussed, including pseudo-synchronization. In summary, while gonadotrophs in situ display GnRH-induced responses similar to those observed in cultured cells, different patterns and novel aspects of functional organization were found which deserve further investigation. This study on GnRH-induced Ca2+ signalling in the acute mice pituitary gland might be of potential relevance for characterizing GnRH actions in gonadotrophs in transgenic and knockout animals.

A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm

ABSTRACT During capacitation, sperm acquire the ability to undergo the acrosome reaction (AR), an essential step in fertilization. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AR. An increase in intracellular Ca2+ ([Ca2+]i) is necessary for AR to occur. In this study, we investigated the spatiotemporal correlation between the changes in [Ca2+]i and AR in single mouse spermatozoa in response to progesterone. We found that progesterone stimulates an [Ca2+]i increase in five different patterns: gradual increase, oscillatory, late transitory, immediate transitory, and sustained. We also observed that the [Ca2+]i increase promoted by progesterone starts at either the flagellum or the head. We validated the use of FM4-64 as an indicator for the occurrence of the AR by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, we have simultaneously visualized the rise in [Ca2+]i and the process of exocytosis in response to progesterone and found that only a specific transitory increase in [Ca2+]i originating in the sperm head promotes the initiation of AR.