We evaluated the presence and role of internal calcium stores in human uncapacitated spermatozoa by determining the effects of two inhibitors of Ca2+ ATPase of the sarco-endoplasmic reticulum (SERCA-ATPase), thapsigargin and cyclopiazonic acid (CPA) on intracellular calcium concentrations, [Ca2+](i), plasma membrane potential and acrosome reaction. Using a fluorescent conjugate of thapsigargin, we localized internal Ca2+ stores on the acrosome, post-acrosomal region and sperm midpiece. SERCA-ATPase inhibitors induced a rise in [Ca2+](i) both in Ca2+ and Ca2+-free media but under these latter conditions it was reduced with a progressive decline to baseline values; the re-addition of Ca2+-stimulated a rise in [Ca2+](i). This demonstrated that internal Ca2+ store depletion can evoke the opening of Ca2+-channels on sperm plasma membrane, thus showing the existence of "capacitative" Ca2+ entry into these specialized cells. The addition of thapsigargin to human spematozoa induced a dose-dependent increase in acrosome reaction percentages, but only when Ca2+ was present in the external medium. Plasma membrane potential monitoring showed that these inhibitors induced a depolarization dependent on Ca2+ influx from external medium and that this was preceded by a transient hyperpolarization caused by activation of Ca2+-dependent K+ channels. When K+-dependent plasma membrane hyperpolarization was inhibited, the thapsigargin- and CPA-stimulated rise in [Ca2+](i) plasma membrane depolarization and acrosome reaction were abolished. In conclusion, the present study demonstrates that human spermatozoa possess internal Ca2+ stores and that the capacitative Ca2+ entry pathway present in these cells regulates important biological processes that are fundamental for the acrosome reaction.

In this study we have investigated the arrangement of sex chromosomes in sperm from two severe oligozoospermic patients, apparently affected by the classic form of Klinefelter's syndrome (KS). Multicolor fluorescence in situ hybridization has been used to recognize chromosomes X, Y, and 8 in sperm from patients and 10 fertile men with normal 46,XY karyotype. In patients affected by KS, we detected important numerical sex chromosome abnormalities (approximately 20%). In all normal fertile men, X- and Y-bearing spermatozoa were present in a 1:1 ratio. On the contrary, in our patients the frequency of 23,Y-bearing sperm was strongly reduced compared with that of both 23,Y sperm in the controls and 23,X sperm in the same subject affected by KS, resulting in a 23,X-/23,Y-bearing sperm ratio of 2:1. Moreover, the frequency of 24,XY disomic sperm was significantly higher in the absence of the 22,0 hypoaploidy expected from a common origin from a nondysjunction during the first meiosis in a normal 46,XY cell. In conclusion, the results of the present study demonstrate a peculiar distribution of sex chromosomes in sperm from two patients with KS, in agreement with the hypothesis that 47,XXY germ cells are able to complete the meiotic process by producing mature spermatozoa.

Azoospermic subjects affected by Klinefelter's syndrome may occasionally show the presence of intratesticular residual foci of spermatogenesis, and the retrieval of mature spermatozoa from the testis may permit fertility and paternity by means of intracytoplasmic sperm injection. Previous studies have demonstrated that these subjects show the presence of an increased incidence of hyperaploid spermatozoa. Here we analyzed, by fluorescence in situ hybridization using specific probes for chromosomes 8, X, and Y, the spermatogenic process and the meiotic progression of 47,XXY germ cells retrieved by fine needle aspiration of the testis in ten azoospermic patients affected by classic Klinefelter's syndrome. All patients had lower testicular volume, higher gonadotropins, and lower testosterone plasma levels compared with control subjects. Cytological analysis of the testicular cells retrieved by fine needle aspiration showed the presence of Sertoli cells only in eight subjects, while germ cells were observed in two patients. In each patient Sertoli cells showed a 47,XXY karyotype, and the same chromosome pattern was observed in spermatogonia and primary spermatocytes of patients presenting a residual spermatogenesis. Secondary spermatocytes, spermatids, and mature spermatozoa showed different sex chromosome patterns, reflecting their origin from 47,XXY spermatogonia. In conclusion, this study demonstrated that, in subjects affected by Klinefelter's syndrome, residual germ cells may be present in the testis and that 47,XXY spermatogonia are able to undergo and complete the spermatogenic process leading to mature spermatozoa. These data further suggest the need to evaluate the sex chromosome status of sperm from patients affected by Klinefelter's syndrome undergoing assisted reproductive techniques.

Previous work from our laboratory has revealed that extracellular ATP is a rapid and potent activator of human sperm acrosome reaction and fertilizing ability. In the present study, we assessed the effects of in-vitro sperm incubation with ATP on fertilization and embryo development in couples undergoing in-vitro fertilization (IVF) for male factor infertility. Oocytes from 22 women undergoing ovulation induction were divided in two groups and inseminated in vitro either with selected spermatozoa from the corresponding partner suffering from male factor infertility pre-incubated with ATP (2.5 mM) for 1 h, or with spermatozoa incubated with 0.9% NaCl solution (control group). After insemination, fertilization was assessed by the presence of pronuclei and then by embryo cleavage. The fertilization rate in the group of oocytes inseminated with ATP-treated spermatozoa improved significantly with respect to the control group (65.7 versus 42.5%, P < 0.01). No significant differences were observed in embryo cleavage and embryo quality. Embryos from both treated and control groups were transferred together in 20 transfer procedures, and in two couples fertilization was not obtained. Nine pregnancies occurred: one biochemical, one miscarriage, and seven patients delivered 9 healthy babies. Two pregnancies were twin with an overall pregnancy rate of 40.9% per cycle and of 45% per transfer. In conclusion, the results of the present study demonstrate that, in humans, extracellular ATP induces a significant increase of sperm fertilizing potential, as these findings are a rationale for the use of ATP for in-vitro treatment of human spermatozoa during IVF.

Steroid hormones, beside their classical genomic mechanism of action, exert rapid, non genomic effects in different cell types. These effects are mediated by still poorly characterized plasma membrane receptors that appear to be distinct from the classic intracellular receptors. In the present study we evaluated the non genomic effects of estradiol (17bE 2 ) in human sperm and its effects on sperm stimulation by extracellular ATP, a potent activator of sperm acrosome reaction. In human sperm 17bE 2 induced a rapid increase of intracellular calcium (Ca 2+ ) concentrations dependent on an influx of Ca 2+ from the extracellular medium. The monitoring of the plasma membrane potential variations induced by 17bE 2 showed that this steroid induces a rapid plasma membrane hyperpolarization that was dependent on the presence of Ca 2+ in the extracellular medium since it was absent in Ca 2+ free-medium. When sperm were pre-incubated in the presence of the K + channel inhibitor tetra-ethylammonium, the 17bE 2 induced plasma membrane hyperpolarization was blunted suggesting the involvement of K + channels in the hyperpolarizing effects of 17bE 2 . Extracellular ATP induced a rapid plasma membrane depolarization followed by acrosome reaction. Sperm preincubation with 17bE 2 inhibited the effects of extracellular ATP on sperm plasma membrane potential variations and acrosome reaction. The effects of 17bE 2 were specific since its inactive steroisomer 17aE 2 was inactive. Furthermore the effects of 17bE 2 were not inhibited by tamoxifen, an antagonist of the classic 17bE 2 intracellular receptor.