Immunolocalization of multiple Galpha subunits in mammalian spermatozoa and additional evidence for Galphas

Like somatic cells, mammalian spermatozoa appear to contain several different heterotrimeric G protein alpha-subunits that could mediate specialized cell responses. However, the precise Galpha subunits present, their subcellular location and their possible roles are still incompletely defined. In this study, using commercially available specific antibodies, we have shown by immunoblotting that Galpha(s) is present in human and mouse sperm lysates. Immunolocalization using intact spermatozoa from both species revealed this protein to be in the acrosomal cap region and the flagellum, particularly the principal piece. Treatment of permeabilized mouse spermatozoa with cholera toxin led to enhanced ADP-ribosylation of a protein the same size as Galpha(s), as well as an increase in cAMP, providing further proof for Galpha(s). Evidence for the presence and distinct localizations of Galpha(i2), Galpha(i3), Galpha(o), Galpha(q/11), and Galpha(olf) was also obtained. Of particular interest was Galpha(i2) which, like Galpha(s), was present in the acrosomal cap region and flagellum, the same regions where stimulatory and inhibitory adenosine receptors are localized. These observations are consistent with our hypothesis that G proteins mediate adenosine receptor modulation of adenylyl cyclase, with consequent alterations in cAMP production, apparently crucial for the spermatozoon's acquisition and maintenance of fertilizing ability.     

Particulate adenylate cyclase plays a key role in human sperm olfactory receptor-mediated chemotaxis

Human sperm chemotaxis is a critical component of the fertilization process, but the molecular basis for this behavior remains unclear. Recent evidence shows that chemotactic responses depend on activation of the sperm olfactory receptor, hOR17-4. Certain floral scents, including bourgeonal, activate hOR17-4, trigger pronounced Ca(2+) fluxes, and evoke chemotaxis. Here, we provide evidence that hOR17-4 activation is coupled to a cAMP-mediated signaling cascade. Multidimensional protein identification technology was used to identify potential components of a G-protein-coupled cAMP transduction pathway in human sperm. These products included various membrane-associated adenylate cyclase (mAC) isoforms and the G(olf)-subunit. Using immunocytochemistry, specific mAC isoforms were localized to particular cell regions. Whereas mAC III occurred in the sperm head and midpiece, mAC VIII was distributed predominantly in the flagellum. In contrast, G(olf) was found mostly in the flagellum and midpiece. The observed spatial distribution patterns largely correspond to the spatiotemporal character of hOR17-4-induced Ca(2+) changes. Behavioral and Ca(2+) signaling responses of human sperm to bourgeonal were bioassayed in the presence, or absence, of the adenylate cyclase antagonist SQ22536. This specific agent inhibits particulate AC, but not soluble AC, activation. Upon incubation with SQ22536, cells ceased to exhibit Ca(2+) signaling, chemotaxis, and hyperactivation (faster swim speed and flagellar beat rate) in response to bourgeonal. Particulate AC is therefore required for induction of hOR17-4-mediated human sperm behavior and represents a promising target for future design of contraceptive drugs.     

Plant Adenylate Cyclases

Adenylate cyclase (AC) (ATP diphosphate-lyase cyclizing; EC 4.6.1.1) is a key component of the adenylate cyclase signaling system and catalyzes the generation of cyclic adenosine monophosphate (cAMP) from ATP. This review summarizes data from the literature and the authors' laboratory on the investigation of plant transmembrane (tmAC) and soluble (sAC) adenylate cyclases, in comparison with some key characteristics of adenylate cyclases of animal cells. Plant sAC has been demonstrated to exhibit similarities with animal sAC with respect to certain characteristics. External factors, such as far-red and red light, temperature, exogenous phytohormones, as well as specific triggering compounds of fungal and bacterial origin exert a significant influence on the activity of plant tmAC and sAC.     

Soluble adenylyl cyclase (sAC) is indispensable for sperm function and fertilization

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC(-/-) spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC(-/-) spermatozoa and are undetectable after sperm purification. HCO(3)(-) is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca(2+) entry into sAC(-/-) spermatozoa. Moreover, the delayed HCO(3)(-)-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC(-/-) spermatozoa. However, sAC(-/-) sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC(-/-) sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC(-/-) spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.     

Particulate and soluble adenylyl cyclases participate in the sperm acrosome reaction

cAMP is important in sea urchin sperm signaling, yet the molecular nature of the adenylyl cyclases (ACs) involved remained unknown. These cells were recently shown to contain an ortholog of the mammalian soluble adenylyl cyclase (sAC). Here, we show that sAC is present in the sperm head and as in mammals is stimulated by bicarbonate. The acrosome reaction (AR), a process essential for fertilization, is influenced by the bicarbonate concentration in seawater. By using functional assays and immunofluorescence techniques we document that sea urchin sperm also express orthologs of multiple isoforms of transmembrane ACs (tmACs). Our findings employing selective inhibitors for each class of AC indicate that both sAC and tmACs participate in the sperm acrosome reaction.     

Inactivation of the mouse adenylyl cyclase 3 gene disrupts male fertility and spermatozoon function

Mammalian spermatids and spermatozoa express functional G protein-coupled receptors. However, bicarbonate-regulated soluble adenylyl cyclase (AC), the major AC present in these cells, is not directly coupled to G proteins. To understand how G protein-coupled receptors signal in spermatozoa, we investigated whether a conventional transmembrane cyclase is present and biologically active in these cells. Here, we provide evidence for expression of type 3 AC (AC3) in male germ cells and describe the effects of disruption of the AC3 gene on fertility and function of mouse spermatozoa. As previously reported in rat, AC3 mRNA is expressed in mouse testes and localized, together with soluble AC mRNA, mainly in postmeiotic germ cells. AC3 protein was detected by immunolocalization in round and elongating spermatids in a region corresponding to the developing acrosome and was retained in the mature spermatozoa of the epididymis. Forskolin caused a small increase in cAMP production in mouse spermatozoa, but this increase could not be detected in the AC3(-/-) mice. Inactivation of the AC3 gene did not have overt effects on spermatogenesis; however, AC3(-/-) males were subfertile with only three litters generated by 11 males over a period of 6 months. When used in in vitro fertilization, spermatozoa from these AC3(-/-) mice produced few embryos, but their fertilizing ability was restored after removal of the zona pellucida. Despite an apparently normal structure, these spermatozoa had decreased motility and showed an increase in spontaneous acrosome reactions. These data support the hypothesis that AC3 is required for normal spermatid or spermatozoa function and male fertility.     

Expression of the soluble adenylyl cyclase during rat spermatogenesis: evidence for cytoplasmic sites of cAMP production in germ cells

To gain insight into the mechanisms of cAMP signaling in germ cells, the expression and subcellular localization of the full-length form of the soluble adenylyl cyclase (sAC) was investigated during rat spermatogenesis and in spermatozoa. A full-length sAC-specific antibody was generated by using a glutathione S-transferase (GST)-sAC carboxyl-terminal region (1399aa-1608aa) fusion protein as the antigen. The selectivity of the purified antibody was confirmed by immunoblotting with lysates from HEK293 cells overexpressing full-length sAC or truncated sAC. Western blot analysis demonstrated that full-length sAC protein appeared on day 25 during testis development. The expression levels increased progressively on days 30 and 35 and remained elevated in adult testis. Full-length sAC protein is retained in spermatozoa from the cauda epididymis. Consistent with the timing of the appearance of the Western blot signal, immunohistochemistry with testis sections at different stages of development detected sAC in late pachytene spermatocytes as well as round and elongating spermatids. Further experiments on the subcellular localization of native or recombinant enzymes revealed that full-length sAC is not only recovered in soluble fractions but also in particulate fractions of testis extracts. Immunofluorescence detection showed localization of the protein in the cytoplasm as well as in organelles of pachytene spermatocytes and spermatids. These findings indicate that cAMP production in spermatids and spermatozoa may occur at sites other than the plasma membrane and suggest that full-length sAC may play a role during spermatid differentiation.     

Molecular details of cAMP generation in mammalian cells: a tale of two systems

The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.     

Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Adenylyl cyclases (ACs) synthesize cAMP and are present in cells as transmembrane AC and soluble AC (sAC). In sperm, the cAMP produced regulates ion channels and it also activates protein kinase-A that in turn phosphorylates specific axonemal proteins to activate flagellar motility. In mammalian sperm, sAC localizes to the midpiece of flagella, whereas in sea urchin sperm sAC is along the entire flagellum. Here we show that in sea urchin sperm, sAC is complexed with proteins of the plasma membrane and axoneme. Immunoprecipitation shows that a minimum of 10 proteins is tightly associated with sAC. Mass spectrometry of peptides derived from these proteins shows them to be: axonemal dynein heavy chains 7 and 9, sperm specific Na+/H+ exchanger, cyclic nucleotide-gated ion channel, sperm specific creatine kinase, membrane bound guanylyl cyclase, cyclic GMP specific phosphodiesterase 5A, the receptor for the egg peptide speract, and alpha- and beta-tubulins. The sAC-associated proteins could be important in linking membrane signal transduction to energy utilisation in the regulation of flagellar motility.     

Somatic 'soluble' adenylyl cyclase isoforms are unaffected in Sacy tm1Lex/Sacy tm1Lex 'knockout' mice

Background

Mammalian Soluble adenylyl cyclase (sAC, Adcy10, or Sacy) represents a source of the second messenger cAMP distinct from the widely studied, G protein-regulated transmembrane adenylyl cyclases. Genetic deletion of the second through fourth coding exons in Sacy^tm1Lex/Sacy^tm1Lex knockout mice results in a male sterile phenotype. The absence of any major somatic phenotype is inconsistent with the variety of somatic functions identified for sAC using pharmacological inhibitors and RNA interference.

Principal Findings

We now use immunological and molecular biological methods to demonstrate that somatic tissues express a previously unknown isoform of sAC, which utilizes a unique start site, and which ‘escapes’ the design of the Sacy^tm1Lex knockout allele.

Conclusions/Significance

These studies reveal increased complexity at the sAC locus, and they suggest that the known isoforms of sAC play a unique function in male germ cells.     

The equatorial subsegment in mammalian spermatozoa is enriched in tyrosine phosphorylated proteins

The equatorial subsegment (EqSS) was originally identified by atomic force microscopy as a discrete region within the equatorial segment of Artiodactyl spermatozoa. In this investigation, we show that the EqSS is enriched in tyrosine phosphorylated proteins and present preliminary evidence for its presence in mouse and rat spermatozoa. The anti-phosphotyrosine monoclonal antibody (McAb) 4G10 bound strongly and discretely to the EqSS of permeabilized boar, ram, and bull spermatozoa. It also bound to a small patch on the posterior acrosomal region of permeabilized mouse and rat spermatozoa, suggesting that the EqSS is not restricted to the order Artiodactyla. An anti-HSPA1A (formerly Hsp70) antibody recognized the EqSS in boar spermatozoa. Immunogold labeling with McAb 4G10 localized the tyrosine phosphorylated proteins to the outer acrosomal membrane. This was verified by freeze-fracture electron microscopy, which identified the EqSS in three overlying membranes, the plasma membrane, outer acrosomal membrane, and inner acrosomal membrane. In all five species, tyrosine phosphorylated proteins became restricted to the EqSS during sperm maturation in the epididymis. The major tyrosine phosphorylated proteins in the EqSS of boar and ram spermatozoa were identified by mass spectrometry as orthologs of human SPACA1 (formerly SAMP32). Immunofluorescence with a specific polyclonal antibody localized SPACA1 to the equatorial segment in boar spermatozoa. We speculate that the EqSS is an organizing center for assembly of multimolecular complexes that initiate fusion competence in this area of the plasma membrane following the acrosome reaction.     

Adenylyl cyclases in oocyte maturation: a characterization of AC isoforms in bovine cumulus cells

Mammalian oocytes are arrested at the G(2)/M transition in the meiotic cell cycle. It is well known that a decrease in intraoocyte cAMP concentrations accompanies resumption of meiosis, but the precise trigger of this decrease remains a mystery. Follicular somatic cells are intimately coupled to the oocyte and are thought to transmit maturation signals to the oocyte in response to hormonal stimulation. Here, we investigate the nature of the follicular somatic cell response to hormonal stimulation by identifying and characterizing the adenylate cyclase isoforms present in bovine cumulus cells. RT-PCR and Western blot analysis revealed the presence of multiple adenylyl cyclase isoforms in bovine granulosa and cumulus cells. Pharmacological manipulation of the AC isoforms showed that multiple isoforms were indeed active. Our data indicate that the PKC inhibited adenylate cyclases IV and VI and the calcium-stimulated isoform I predominate in bovine cumulus cells.     

The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.     

Functional activity of adenylyl and guanylyl cyclases in human spermatozoa with different motility

The most important functional characteristic of ejaculated spermatozoa is their ability to engage in directed sustained movement, which to a large extent determines their fertility. It is assumed that enzymes with cyclase activity—adenylyl cyclase (AC) and guanylyl cyclase (GC)—soluble and membrane-bound forms of which are found in human and mammalian sperm, play the key role in regulation of motility. However, the functional activity of the cyclases in ejaculated spermatozoa with different motilities and their contribution to the regulation of this process are virtually unexplored. The goal of this work was to determine the functional characteristics of AC and GC in ejaculates of human spermatozoa with different contents of motile forms and the study of regulation of these enzymes by hormones and nonhormonal agents. We found differences in the activity and regulatory properties of AC and GC in ejaculates differing in motile forms of spermatozoa. The basal AC activity and its sensitivity to bicarbonate anions and manganese cations, activators of cytosolic AC (cAC), were increased in ejaculates with a high proportion of motile spermatozoa. At the same time, the AC effects of forskolin, GppNHp, and adrenergic receptor agonists acting via membrane-bound AC (mAC) in this case were significantly reduced. Cytosolic GC in the ejaculates with a high proportion of motile spermatozoa was more sensitive to manganese cations, but the basal activity of GC was altered slightly. An increase in the content of motile spermatozoa in ejaculate led to a decrease in the sensitivity of CNP to receptor GC, while the sensitivity to ANP was maintained, which indicates a change in the pattern of enzyme regulation with natriuretic peptides in favor of ANP, an important regulator of sperm chemotaxis. Thus, we have concluded that the change in proportion of motile spermatozoa in ejaculate induces changes of functional activity and regulatory properties of soluble and membrane-bound forms of AC and GC, which can be used to control the motility, chemotaxis, acrosomal reaction, and other processes determining fertility of male germ cells.     

Functional role of membrane-bound adenylyl cyclases and coupled to them receptors and G-proteins in regulation of fertility of spermatozoa

The cAMP-dependent signaling cascades play the key role in regulation of fertility of spermatozoa. Synthesis of cAMP in spermatozoa is realized both by soluble, and by transmembrane (membrane-bound) forms of adenylyl cyclases (AC). For the recent years numerous data appeared about the presence in spermatozoa at different stages of their maturation of a wide spectrum isoforms of membrane-bound AC and their regulation by hormones and hormone-like substances via the coupled to B-proteins receptors (GPCR). Agonists of GPCR in spermatozoa can be adenosine, biogenic amines, peptide hormones, odorants. Study of structural-functional organization and regulatory properties of AC of the signal system in spermatozoa is of great practical significance for reproductive technologies, as via the membrane-bound AC forms and signal cascades there are controlled such processes as motility and chemotaxis of spermatozoa, their capability for capacitation acrosomal reaction. In the review there are summarized and analyzed data on functioning and role of AC of signal system in spermatozoa of human and vertebrate animals and are discussed achievements and unsolved problems in this field.     

Ultrastructure of testes and spermatogenesis in the trombiculid mite,Hirsutiella zachvatkini (Schluger)

Summary

Spermatogenesis and sperm ultrastructure of the trombiculid mite Hirsutiella zachvatkini (Schluger 1948) have been investigated using transmission electron microscopy and compared with other arachnids studied. Sperm differentiation takes place in groups of synchronously developed germ cells of the two large sac-like paired testes. Each testis is composed of a secretory epithelium, which occupies their medio-ventral regions, and of a germinative epithelium situated in the latero-dorsal parts of testes together with large somatic cells. The germ cells are represented on sections by spermatogonia, spermatocytes, early, middle and late spermatids, and mature spermatozoa. Spermatocytes and spermatids contain two centrioles, which disappear afterwards, and a small Golgi-like structure forming an acrosomal cistema. Mature spermatozoa, which lie both within the meshes of somatic cells and also free in the lumen of testes, are compact oval aflagellate cells provided with peripheral channels. They also contain an acrosome, flattened between the cell membrane and the round electron-dense chromatin body, an oval body of lesser density lying in close proximity to the chromatin body, and a group of 5–7 mitochondria with spherically arranged cristae situated immediately behind the nuclear bodies. An acrosomal filament may be sometimes seen beneath the acrosome in the middle spermatids and disappears in the mature spermatozoa. These findings show that the mode of differentiation and pattern of organization of the male sex cells in trombiculid mites are of rather primitive type compared with other acarine spermatozoa.