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After ejaculation, mammalian sperm undergo a series of molecular events conducive to the acquisition of fertilizing competence. These events are collectively known as capacitation and involve acrosomal responsiveness and a vigorous sperm motility called hyperactivation. When mimicked in the laboratory, capacitating bovine sperm medium contains bicarbonate, calcium, albumin and heparin, among other components. In this study, we aimed at establishing a new capacitation protocol for bovine sperm, using calcium ionophore. Similar to our findings using mouse sperm, bovine sperm treated with Ca 2+ ionophore A23187 were quickly immobilized. However, these sperm initiated capacitation after ionophore removal in fresh medium without heparin, and independent of the Protein Kinase A. When A23187-treated sperm were used on in vitro fertilization (IVF) procedures without heparin, eggs showed cleavage rates similar to standardized IVF protocols using heparin containg synthetic oviduct fluid (IVF-SOF). However, when A23187 pre-treated sperm were further used for inseminating eggs in complete IVF-SOF-heparin, a significantly higher percentage of embryo development was observed, suggesting a synergism between two different signaling pathways during bovine sperm capacitation. These results have the potential to improve current protocols for bovine IVF that could also be applied in other species of commercial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mammalian spermatozoa must undergo biochemical and structural changes to acquire the capacity for fertilization, in a process known as capacitation. Activation of PKA enzymes is essential for capacitation, and thus cAMP levels are tightly regulated during this process. Previously, we demonstrated that during capacitation, bovine spermatozoa extrude cAMP through multidrug resistance-associated protein 4 (MRP4, also known as ABCC4), which regulates intracellular levels of the nucleotide and provides cAMP to the extracellular space. Here, we report the presence of functional MRP4 in murine spermatozoa, since its pharmacological inhibition with MK571 decreased levels of extracellular cAMP. This also produced a sudden increase in PKA activity, with decreased tyrosine phosphorylation at the end of capacitation. Blockade of MRP4 inhibited induction of acrosome reaction, hyperactivation and in vitro fertilization. Moreover, MRP4 inhibition generated an increase in Ca 2+ levels mediated by PKA, and depletion of Ca 2+ salts from the medium prevented the loss of motility and phosphotyrosine inhibition produced by MK571. These results were supported using spermatozoa from CatSper Ca 2+ channel knockout mice. Taken together, these results suggest that cAMP efflux via MRP4 plays an essential role in mouse sperm capacitation.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Oocyte activation at fertilization is brought about by the testis-specific phospholipase C zeta (PLCZ), owing to its ability to induce oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Whereas this is a highly conserved mechanism among mammals, important species-specific differences in PLCZ sequence, activity, and expression have been reported. Thus, the objectives of this research were to clone and characterize the intracellular Ca(2+)-releasing activity and expression of equine PLCZ in sperm and testis. Molecular cloning of equine PLCZ yielded a 1914-bp sequence that translated into a protein of the appropriate size (~73 kDa), as detected with an anti-PLCZ-specific antibody. Microinjection of 1 μg/μl of equine PLCZ cRNA supported [Ca(2+)](i) oscillations in murine oocytes that were of a higher relative frequency than those generated by an equivalent concentration of murine Plcz cRNA. Immunofluorescence revealed expression of PLCZ over the acrosome, equatorial segment, and head-midpiece junction; unexpectedly, PLCZ also localized to the principal piece of the flagellum in all epididymal, uncapacitated, and capacitated sperm. Immunostaining over the acrosome was abrogated after induction of acrosomal exocytosis. Moreover, injection of either sperm heads or tails into mouse oocytes showed that PLCZ in both fractions is catalytically active. Immunohistochemistry on equine testis revealed expression as early as the round spermatid stage, and injection of these cells supported [Ca(2+)](i) oscillations in oocytes. In summary, we report that equine PLCZ displays higher intrinsic intracellular Ca(2+)-releasing activity than murine PLCZ and that catalytically active protein is expressed in round spermatids as well as the sperm flagellum, emphasizing important species-specific differences. Moreover, some of these results may suggest potential novel roles for PLCZ in sperm physiology.

Varicocele is a prevalent pathology among infertile men. The mechanisms linking this condition to infertility, however, are poorly understood. Our previous work showed a relationship between sperm functional quality and the ability of spermatozoa to respond to capacitating conditions with increased membrane fluidity and protein tyrosine phosphorylation. Given the reported association between varicocele, oxidative stress, and sperm dysfunction, we hypothesized that spermatozoa from infertile patients with varicocele might have a combined defect at the level of membrane fluidity and protein tyrosine phosphorylation. Semen samples from infertile patients with and without grade II/III left varicocele were evaluated for motion parameters (computer-assisted semen analysis [CASA]), hyperactivation (CASA), incidence and intensity of protein tyrosine phosphorylation (phosphotyrosine immunofluorescence and western blotting), and membrane fluidity (Laurdan fluorometry), before and after a capacitating incubation (6 hr at 37 degrees C in Ham's F10/BSA, 5% CO(2)). Spermatozoa from varicocele samples presented a decreased response to the capacitating challenge, showing significantly lower motility, hyperactivation, incidence and intensity of tyrosine phosphorylation, and membrane fluidity. The findings reported in this article indicate that the sperm dysfunction associated to infertile varicocele coexists with decreased sperm plasma membrane fluidity and tyrosine phosphorylation. These deficiencies represent potential new pathophysiological mechanisms underlying varicocele-related infertility.

The Authors report an interesting case of liposarcoma of the leg and review the international Literature. Moreover, a particular analysis of different aspects such as anatomy, clinic, diagnosis and treatment has been carried out.

Background: Human semen is composed of a heterogeneous population of sperm with varying degrees of structural and functional differentiation and normality, which result in subpopulations of different quality., Methods: Using a discontinuous Percoll gradient, we separated three subsets of sperm [(45%; L45), (65%; L65) and (90%; L90) fractions] from normozoospermic human semen samples from healthy donors and proceeded to characterize their morphology, motility and hyperactivation, as well as their ability to undergo tyrosine phosphorylation under capacitating conditions., Results: As expected, sperm isolated from the lowest density layer (L45) showed the poorest quality, displaying the smallest percentage of morphologically normal and motile sperm. During a capacitating incubation, this subset of cells also showed deficient capacity to undergo hyperactivation and protein tyrosine phosphorylation. Conversely, sperm isolated from the other layers (L65 and L90) showed a time-dependent progressive increment in tyrosine phosphorylation, establishing statistically significant differences with sperm from L45. The tyrosine phosphorylation deficiency of L45 sperm could be overcome when sperm from that fraction were stimulated with activators of the cAMP-dependent kinase (PKA) pathway (dbcAMP + pentoxifylline), pointing to the sperm's plasma membrane as the main site of such deficiency., Conclusions: Poor quality sperm isolated from a Percoll gradient display an intrinsic tyrosine phosphorylation deficiency, possibly caused by a plasma membrane defect, which is associated with their inability to undergo normal capacitation and, ultimately, acquire optimal fertilizing potential.

Objective: To determine extracellular calcium (Ca(2+)) requirements for the maintenance of human sperm function in vitro., Design: Prospective study., Setting: Basic research laboratory., Patient(s): Normozoospermic volunteers provided fresh semen samples; follicular fluid (human FF) and oocytes were collected from women undergoing IVF-ET., Intervention(s): Spermatozoa were incubated for /=0.1 mM of Ca(2+) were able to undergo the AR when exposed to human FF in the presence of 2.5 mM of Ca(2+). Calcium concentrations of >/=0.22 mM were sufficient to reach protein tyrosine phosphorylation levels and hyperactivated motility values similar to those of controls. Higher Ca(2+) concentrations (>/=0.58 mM) were required to produce maximum human FF-induced AR in previously capacitated cells and to obtain an adequate sperm-ZP binding., Conclusion(s): Different steps of the fertilization process have distinctive Ca(2+) requirements. Whereas 0.22 mM of Ca(2+) is sufficient for the development of some capacitation-related events, human FF-induced AR and sperm-ZP interaction require 0.58 mM of this cation.

Loss, failure and desertion are those words, which better describe the most frequent difficulties incurred by teenagers, and their relation to the school environment, and indicate, as well, the lack of connection between individual aspirations and school achievements obtained. Despite the likelihood of school difficulties throughout the entire educational career, from the kindergarten to the college, we are able to recognize certain specific "disturbances" which come out during the adolescence, basically relating to the school experiences. School failure, school desertion and school abandonment are, in fact, issues mainly discussed in coincidence with the beginning of the adolescent age and the attending of the high school. The aetiopathogenesis of school discomfort is mostly determined by more than only one factor: psychological, physical, cognitive and environmental aspects, all together, contribute in various ways, to the rise and persisting of the above described problems. Suggesting a univocal characterization of the adolescent with school difficulties, is indeed a hard task, since school failure is not an exclusive feature of only one kind of personality or the expression of only one type of conflict. Once identified the individuals more subject to risk, and the warning signs of actual uneasiness, it is important therefore to intervene timely and in different ways in order to avoid the worsening or the excessive extension of the school difficulties and of their frequent and serious consequences on the teenagers' individual and domestic well-being. Such type of prevention definitely belongs to the Adolescent Medicine, and must be developed on different levels, by involving not only the teenagers, but also their families, the school, and the whole social environment in which the teenagers live and with which they interact.

During the last years the Italian Government has taken many different initiatives to protect the adolescents rights to benefit from physical, psychical and social well-being. In particular, various projects concerning the promotion and the support of the school, the family, sport, mass-media, judicial and medical infrastructures, have been organised, promoted and financed. However, it is not always possible to assure a real safeguarding of teenagers rights; this especially happens because the problem concerning the autonomous capacity of minors to consent (or non consent) to the medical treatment is much debated. However, many contradictions still persist about the effective duration of the pediatric age. All the same, it must be noted that the Legislator apparently realised that not only the physical, psychological and social maturity proceed by steps, but also the legal capacity.

Background: In this paper we present growth height standards obtained by an auxological study on a primary school population of central Italy (Lazio), in the 1991-1992 and 1992-1993 school-years., Methods: 4,175 children were included in the study: 2,024 females and 2,151 males, aged from 6 to 11 years. The sampling rate corresponds to more than 98% of the total primary school-population of the areas mentioned. The statistical analysis of the collected data was carried out by the Dipartment of Statistics of the University of Rome, using the widely available statistics packages STAT-GRAPHICS and SAS., Results and Conclusions: The analysis of height mean and standard deviation showed an increasing trend of height related to age, without significant differences by sex. The mean heights for females and males were practically the same in each age class. Height variance increases with age, too. This fact necessitates the use of the weighted regression technique to evaluate the functions that express the height growth trend. The relationship between height and age is linearlike and almost coincides for the two sexes. The height percentiles were calculated and the relative curves were developed, based on the distribution of normalized residuals. The centile curves confirm that there are no significant differences in the growth trends for both sexes and therefore the lines are overlapping. Substantial differences exist only in the end centiles (3th and 97th) and age extremes (6 and 11 years), possibly because of the small number of subjects.

The function of sex accessory glands in 95 varicocele patients was investigated by determining the fructose, prostatic acid phosphatase (PAP), and zinc levels in the seminal plasma. These biochemical substances are, in fact, markers of the seminal vesicles and prostate, respectively. It was observed that both the PAP and zinc levels were significantly lower (P less than .002) in varicoceles than in the control group, while both fructose and PAP levels were particularly low in a small subgroup of the older patients. The authors suggest that the impaired sex accessory gland functions could arise from decreased venous drainage in the vesico-prostatic plexus. The fructose/zinc ratio appears to be negatively related to sperm motility (r = .31, P = .01); this suggests that in varicocele the impaired sex accessory gland secretions could themselves influence the motility of ejaculated spermatozoa.