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Signaling events during male germ cell differentiation: bases and perspectives
No full textIn all species, reproductive function depends on the ability of the individual to produce functional differentiated gametes. Spermatogenesis is a cyclic process in which diploid spermatogonia differentiate into mature haploid spermatozoa. Thus from a genetic point of view, spermatogenesis can be divided into two phases, namely the diploid and haploid phase. Indeed, this complex
differentiation process is still more intriguing since primary spermatocytes, if genetically diploid, are functionally tetraploid, while elongating spermatids, the germ cells undergoing the most dramatic morphological changes, if genetically haploid, become functionally anucleate due to ongoing condensation of chromatin resulting in an inactive nuclear DNA. This multi-step differentiative pathway is dependent on a specific environment provided by the anatomical and cellular relationships that take place in the testis and more specifically within the seminiferous tubules.
Already, early anatomists (mind comes to Enrico Sertoli and Gustaf Retzius) were fascinated by the mixed cellular composition of the testis correctly deciphered as a whole of interacting and interdependent cell types despite the fact these belong to two well-established and different cell
lineages, i.e, the somatic and germinal line. Since their time (the XIX century) up to-day a conspicuous bulk of experimental work and a relative massive bibliographic
documentation have been provided. From this it stands out : a) a sophisticated role played by the cyclic hormonal control elicited by the hypothalamic-pituitary axis; b) the structural membrane specializations of Sertoli-germ cell communications; c) the existence and action of a paracrine
and autocrine testicular regulative secretion; d) a regulation of germ cell gene expression, highly specialized both at transcriptional, posttranscriptional, and translational level; e) an active participation of the haploid genome in the final steps of cell differentiation. Each of these points has been the matter of several more and less recent reviews to which the present author hands back in the course of this note. However all these points, although topics of separate and extensive treatises, are conceptually jointed by a ‘leitmotiv’, that is, the intracellular transduction of an
exogenous signal evoking a specific stimulatory/inhibitory, proliferative/differentiative event. The spirit with which the present author interpreted this minireview was to recall some points to which to draw attention having as a scenario the complex process of male germ cell differentiation in
mammals
Multiple forms of boar, bull, and human acrosin
No full textAnalytical disk gel electrophoresis with staining techniques for amidohydrolase activity at pH 7.6 demonstrated that partially purified acrosomal extracts of ejaculated bull, boar, and human spermatozoa contained three, apparently four, and two fractions, respectively, with acrosin-like activity. Acrosin amidohydrolase activity is present in the gels incubated in the staining medium at pH 5.0. Some methods for the extraction of human acrosin have been compared. These consist essentially of the extraction by detergent treatment and the extraction by acid procedures. Acid extraction of human spermatozoa yields a higher amount of acrosin than does detergent extraction; the acrosin specific activity, extracted by these methods, seems to be similar
Evidence of a high molecular weight form of acrosin in boar acrosomal extract
No full textAcrosomal extracts of freshly ejaculated and immediately processed boar spermatozoa were investigated to detect which and how many acrosin molecular forms were present. Electrophoretic analyses of the acrosomal extract showed the presence of only one, slowly migrating, acrosin molecular form. Enzyme-linked-immuno-electro-transfer blot revealed the molecular weight of this form to be about 66 kdalton. Preliminary electrophoretic analyses under nondenaturating conditions of the acrosomal extract previously treated with thermolysin suggested that the approximately 66 kdalton form gives rise to two comigrating acrosin molecular forms
Signaling events during male germ cell differentiation : Update, 2006
No full textThe intracellular transduction of exogenous and cell-autonomous stimuli triggers the transformation of a multipotent stem cell, the spermatogonion, into a highly differentiated, motile and fertile cell, the spermatozoon. This differentiation process is mediated by cell-cell contact and via key players including hormones, growth factors, and cytokines. Female hormones, estrogens and progestins, play a role in the production and functionality of spermatozoon. New findings, however, reconsider the direct action for estrogens on male germ cells while progestins work through non-canonical receptors. Similarly, testosterone, the male hormone, besides acting through its receptor expressed in the somatic cells of testis, seems to work by means of non-classical mechanisms. The recent identification of growth factors, transcriptional regulators, and media for in vitro growth of spermatogonial stem cells should now make it feasible to unravel the entire spermatogenic process. A peculiar feature of the meiotic cycle is the maintenance of condensed chromatin so that DNA duplication is prevented and reduction of genome is achieved. Recently, molecular mechanisms that lead to such a condensation have been discovered. Junctional intercellular complexes between Sertoli and germ cells are critical for coordinating spermatogenesis. Molecular players involved in such cell-cell communication have been identified in Sertoli cells. Now, there is also a need for unravelling the germ cell molecules involved. These issues are the major topics which are discussed here with the goal to suggest a possible answer
Evidence for calcium-mediated F-actin/phospholipid bining of human sperm calpactin II
No full textWe have previously reported the presence in human spermatozoa of calpactin II, a calcium-binding component of the membrane skeleton (Berruti, Exper. Cell Res. 179, 374, 1988). Reported here are studies which show the ability of sperm calpactin II to interact with filamentous actin and acidic phospholipids in a Ca(2+)-dependent fashion. At high Ca2+ concentrations (greater than 1 mM) sperm calpactin II binds to actin filament and it is resolubilized by EGTA. Liposome binding experiments reveal that sperm calpactin II does associate with phospholipidic vesicles at micromolar free Ca2+ levels. These interaction properties together with the cellular distribution of the protein revealed by immunofluorescence analysis in Ca2+ and ionophore A23187-treated human spermatozoa allow to hypothesize a physiological role for calpactin II in sperm Ca(2+)-mediated events
The effect of starvation on the appearance of the definitive upper jaw in Ophryotrocha puerilis (Annelida,Polychaeta)
No full texthe aim of this work was to verify if starvation influences the appearance of the definitive upper jaw in Ophryotrocha puerilis (Annelida, Polychaeta). On the basis of the data obtained from two series of experiments carried out parallelly on individuals of O. puerilis, maintained in pair culture or in isolation, the following results were obtained. Starvation did not interfere with the replacement of the juvenile upper jaw by the definitive upper jaw in the animals kept in isolation; rather, the appearance of the definitive upper jaw showed a frequency significantly lower than that shown by the controls in those maintained in pair culture and under starvation for one month. The following interpretation is suggested. Starvation by itself does not influence the replacement of the juvenile upper jaw with the definitive; nevertheless, in the presence of some other environmental factor, such as pair culture (the Paarkultur of Hartmann & Huth), starvation does exert some inhibiting influence on the replacement of the upper jaw
Calpactin-like proteins in human spermatozoa
No full textPolyclonal antibodies directed against human calpactin I (p36) and calpactin II (p35) have been employed to investigate the distribution of calpactin-like proteins in human spermatozoa. Calpactins are a family of Ca2+-regulated cytoskeletal proteins that are major substrates of oncogene and growth factor receptor protein tyrosine kinases. The existence of a Triton-soluble 37-kDa protein antigenically related to calpactin II from somatic cells was revealed by Western blot analysis of human sperm extracts. The 37-kDa protein was not released from spermatozoa after experimental induction of the acrosome reaction by A23187 and Ca2+. Treatment of sperm homogenates with an EGTA-containing buffer partially solubilized the 37-kDa protein from the corpuscolate matter. Indirect immunofluorescence microscopy showed that anticalpactin II binds specifically to the sperm tail and to a band-like structure encircling the sperm head at the equatorial segment. In contrast, antibodies to calpactin I were found to bind to the tail midpiece, but failed to bind to Western blots of sperm proteins. This is the first immunological and biochemical report on the presence of calpactin proteins in a germ cell, the human spermatozoon
Multiple forms of bovine acrosin: purification and characterization
No full text1. The preparation of highly purified bovine acrosin (acrosomal proteinase, EC 3.4.21.10) is described.
2. Three forms of acrosin (α, β, and γ) with calculated approximate mol. wt of 36,000, 34,000 and 25,000 daltons, respectively, were obtained.
3. The amino acid composition of the 36,000 mol. wt form shows a relative similarity with the molecular content of the other acrosin forms till now known, i.e. that of the human and rabbit acrosomal proteinase
A novel Rap1/B-Raf/14-3-3 theta protein complex is formed in vivo during the morphogenetic differentiation of postmeiotic male germ cells
No full textThe 14-3-3 family of proteins is expressed in a broad range of organisms and tissues. Based on data essentially obtained with tissue culture cells and yeast, 14-3-3 proteins have been implicated as potential regulators of diverse signaling pathways, in particular those involving the activity of the Raf family protein kinases. The 14-3-3 theta mouse isoform is expressed almost exclusively in testis and brain. In an effort to understand the function of 14-3-3 theta in testis, we sought to identify endogenous proteins that interact with 14-3-3 theta in spermatogenic cells. A recombinant 14-3-3 theta fusion protein was used in Far Western and GST pulldown. assays. Here we report that 14-3-3 theta interacts in vivo and in vitro with 93- to 95-kDa B-Raf, originally described as specific of neural tissues and never reported in male germ cells. Moreover, in mouse spermatids, i.e., the haploid cytodifferentiating cells, a so far unidentified protein complex formed by endogenous Rap1/B-Raf/14-3-3 theta can be coimmunoprecipitated. The intracellular localization of endogenous 14-3-3 theta, B-Raf, and Rap1 was analyzed in distinct spermatogenic cell types and a peculiar codistribution of the three proteins was immunorevealed in differentiating spermatids. Together, these data demonstrate that a protein complex formed by endogenous Rap1, 93- to 95-kDa B-Raf, and 14-3-3 theta exists in vivo and the finding that this has been detected in cytodifferentiating, not dividing cells, strengthens the hypothesis for a role of Rap1/B-Raf-mediated signaling in cell morphogenesis and differentiation
Biochemical characterization of the boar sperm 42 kDa protein tyrosine kinase: its potential for tyrosine as well as serine phosphorylation towards MAP2 and histone H2B
No full textThe majority of cellular responses to changing environmental conditions is regulated by protein kinases. Spermatozoa have many special properties, including motility with demonstrated chemotaxis, the ability to undergo capacitation, and the acrosome reaction, which are in part controlled by extracellular signals and in which sperm kinases are considered to be involved. We have previously reported that there is a protein kinase activity, which phosphorylates the synthetic substrate poly-(Glu, Tyr) with a Km value of 2.3 mu M, and is inhibited by the tyrosine kinase inhibitor tyrphostin, in the protein extract from boar spermatozoa (Berruti and Porzio, 1992: Biochim Biophys Acta 1118:149-154). Now we have demonstrated that the enzyme is cytosolic, is active as a monomer of M(r) 42,000, is stimulated by Mg2+ > Mn2+ but not by Ca2+, is renaturable, and can phosphorylate native protein substrates such as microtubule-associated protein 2 (MAP2) and histone H2B both on the tyrosine and serine residues. N-terminal sequence analysis suggests that it is a novel protein. These new findings imply that the boar sperm 42 kD kinase may be a novel member of the emerging class of dual-specificity protein kinases, and they raise the intriguing question of its function in the protein kinase network mediating signal transduction in mammalian spermatozoa
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