Dual role of valosin-containing protein (VCP/p97) in mouse sperm during capacitation.

Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.

Cdc42 localized in the CatSper signaling complex regulates cAMP-dependent pathways in mouse sperm.

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.

Everything you ever wanted to know about PKA regulation and its involvement in mammalian sperm capacitation.

The 3', 5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) is a tetrameric holoenzyme comprising a set of two regulatory subunits (PKA-R) and two catalytic (PKA-C) subunits. The PKA-R subunits act as sensors of cAMP and allow PKA-C activity. One of the first signaling events observed during mammalian sperm capacitation is PKA activation. Thus, understanding how PKA activity is restricted in space and time is crucial to decipher the critical steps of sperm capacitation. It is widely accepted that PKA specificity depends on several levels of regulation. Anchoring proteins play a pivotal role in achieving proper localization signaling, subcellular targeting and cAMP microdomains. These multi-factorial regulation steps are necessary for a precise spatio-temporal activation of PKA. Here we discuss recent understanding of regulatory mechanisms of PKA in mammalian sperm, such as post-translational modifications, in the context of its role as the master orchestrator of molecular events conducive to capacitation.

CFTR/ENaC-dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC.

To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3--dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (cystic fibrosis transmembrane conductance regulator channel) activity and for the modulation of membrane potential (Em). However, the main HCO3- transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na+/HCO3- cotransporter (NBC) and epithelial Na+ channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3- influx, resulting in lower PKA activity, and that events downstream of the cAMP activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3- also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation.

Disruption of protein kinase A localization induces acrosomal exocytosis in capacitated mouse sperm.

Protein kinase A (PKA) is a broad-spectrum Ser/Thr kinase involved in the regulation of several cellular activities. Thus, its precise activation relies on being localized at specific subcellular places known as discrete PKA signalosomes. A-Kinase anchoring proteins (AKAPs) form scaffolding assemblies that play a pivotal role in PKA regulation by restricting its activity to specific microdomains. Because one of the first signaling events observed during mammalian sperm capacitation is PKA activation, understanding how PKA activity is restricted in space and time is crucial to decipher the critical steps of sperm capacitation. Here, we demonstrate that the anchoring of PKA to AKAP is not only necessary but also actively regulated during sperm capacitation. However, we find that once capacitated, the release of PKA from AKAP promotes a sudden Ca2+ influx through the sperm-specific Ca2+ channel CatSper, starting a tail-to-head Ca2+ propagation that triggers the acrosome reaction. Three-dimensional super-resolution imaging confirmed a redistribution of PKA within the flagellar structure throughout the capacitation process, which depends on anchoring to AKAP. These results represent a new signaling event that involves CatSper Ca2+ channels in the acrosome reaction, sensitive to PKA stimulation upon release from AKAP.

Role of Actin Cytoskeleton During Mammalian Sperm Acrosomal Exocytosis.

Mammalian sperm require to undergo an exocytotic process called acrosomal exocytosis in order to be able to fuse with the oocyte. This ability is acquired during the course of sperm capacitation. This review is focused on one aspect related to this acquisition: the role of the actin cytoskeleton. Evidence from different laboratories indicates that actin polymerization occurs during capacitation, and the detection of several actin-related proteins suggests that the cytoskeleton is involved in important sperm functions. In other mammalian cells, the cortical actin network acts as a dominant negative clamp which blocks constitutive exocytosis but, at the same time, is necessary to prepare the cell to undergo regulated exocytosis. Thus, F-actin stabilizes structures generated by exocytosis and supports the physiological progression of this process. Is this also the case in mammalian sperm? This review summarizes what is currently known about actin and its related proteins in the male gamete, with particular emphasis on their role in acrosomal exocytosis.

PKA-dependent phosphorylation of LIMK1 and Cofilin is essential for mouse sperm acrosomal exocytosis.

Mammalian sperm must acquire their fertilizing ability after a series of biochemical modifications in the female reproductive tract collectively called capacitation to undergo acrosomal exocytosis, a process that is essential for fertilization. Actin dynamics play a central role in controlling the process of exocytosis in somatic cells as well as in sperm from several mammalian species. In somatic cells, small GTPases of the Rho family are widely known as master regulators of actin dynamics. However, the role of these proteins in sperm has not been studied in detail. In the present work we characterized the participation of small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during mouse sperm capacitation. We observed that most of the proteins of this signaling cascade and their effector proteins are expressed in mouse sperm. The activation of the signaling pathways of cAMP/PKA, RhoA/C and Rac1 is essential for LIMK1 activation by phosphorylation on Threonine 508. Serine 3 of Cofilin is phosphorylated by LIMK1 during capacitation in a transiently manner. Inhibition of LIMK1 by specific inhibitors (BMS-3) resulted in lower levels of actin polymerization during capacitation and a dramatic decrease in the percentage of sperm that undergo acrosomal exocytosis. Thus, we demonstrated for the first time that the master regulators of actin dynamics in somatic cells are present and active in mouse sperm. Combining the results of our present study with other results from the literature, we have proposed a working model regarding how LIMK1 and Cofilin control acrosomal exocytosis in mouse sperm.

Novel, low cost, highly effective, handmade steroid pellets for experimental studies.

The basic component of Silastic® glue (Dow Corning) used to prepare Silastic® pellets is polydimethylsiloxane. This compound is also present in other commercial adhesives such as FASTIX® (Akapol SA) that are available in any store for that category. In the present study we developed low cost, easy to prepare handmade steroid pellets (HMSP) by mixing 17ß-estradiol, progesterone or other synthetic steroids with FASTIX® adhesive. We assessed serum levels of 17ß-estradiol, progesterone, prolactin and luteinizing hormone in ovariectomized mice treated for 24 and 48 h or 7, 14 and 28 days with 20 µg or 5 mg of 17ß-estradiol or 5 mg progesterone HMSP. We found a time dependent and significant increase in the levels of both natural hormones, and a downregulation of serum luteinizing hormone levels, while both 17ß-estradiol doses increased serum prolactin. Uterine weights at sacrifice and histological examination of the uteri and the mammary glands correlated with estrogen or progestin action. Finally, we evaluated the biological effects of HMSP compared to commercial pellets or daily injections in the stimulation or inhibition of hormone dependent mammary tumor growth, and found that HMSP were as effective as the other methods of hormone administration. These data show that HMSP represent a useful, low cost, easily accessible method for administering steroids to mice.

Central dopamine D2 receptors regulate growth-hormone-dependent body growth and pheromone signaling to conspecific males.

Competition between adult males for limited resources such as food and receptive females is shaped by the male pattern of pituitary growth hormone (GH) secretion that determines body size and the production of urinary pheromones involved in male-to-male aggression. In the brain, dopamine (DA) provides incentive salience to stimuli that predict the availability of food and sexual partners. Although the importance of the GH axis and central DA neurotransmission in social dominance and fitness is clearly appreciated, the two systems have always been studied unconnectedly. Here we conducted a cell-specific genetic dissection study in conditional mutant mice that selectively lack DA D2 receptors (D2R) from pituitary lactotropes (lacDrd2KO) or neurons (neuroDrd2KO). Whereas lacDrd2KO mice developed a normal GH axis, neuroDrd2KO mice displayed fewer somatotropes; reduced hypothalamic Ghrh expression, pituitary GH content, and serum IGF-I levels; and exhibited reduced body size and weight. As a consequence of a GH axis deficit, neuroDrd2KO adult males excreted low levels of major urinary proteins and their urine failed to promote aggression and territorial behavior in control male challengers, in contrast to the urine taken from control adult males. These findings reveal that central D2Rs mediate a neuroendocrine-exocrine cascade that controls the maturation of the GH axis and downstream signals that are critical for fitness, social dominance, and competition between adult males.