Scielo RSS <![CDATA[Biological Research]]> vol. 44 num. 2 lang. en <![CDATA[SciELO Logo]]> <![CDATA[<b>A tribute to Professor Claudio Barros (1936-2008)</b>]]> <![CDATA[<strong>Gamete interactions in teleost fish</strong>: <strong>the egg envelope. Basic studies and perspectives as environmental biomonitor</strong>]]> The current knowledge about teleost fish egg envelope is summarized. The paper analyzes the organization and deposition process of the protein composition and genes involved in the synthesis of teleost fish egg envelopes and their role in gamete interaction during fertilization. Pelagic and demersal species that our research group is working with are especially considered. The vertebrate ZP family of proteins, the evolution and relationship among the different genes and their expression are taken into account. We consider fish envelope as a possible biomonitor for ecological contaminants. The biotechnological applications for aquaculture and genomic and post-genomic approaches are auspicious. <![CDATA[<strong>In vitro fertilization of the rock shrimp, <i>Rhynchocinetes typus </i>(Decapoda, Caridea)</strong>: <strong>a review</strong>]]> This review compiles all the research done on gametes and fertilization in the rock shrimp, R. typus, and describes the sequence of events from the first gamete interaction to zygote formation and the first cleavage of the embryo, with light, fluorescence confocal and electron microscopes. Early studies showed that sperm from the vas deferens have a tack-shape with a "needle-like process" or rigid spike (RS) that extends from a semi-spherical body that contains the arms with chromatin and spines. Upon contact with seawater and by action of Na +, the arms and spines extend, producing an inverted umbrella form of the spermatozoa. The first sperm-oocyte interaction occurs between protein receptors type lectins of the sperm RS and oocyte chorion sperm ligands. These ligands contain residues of a-Glu, Man (a 1-3) Man, a and p-GlcNAc and a-GalNA terminal residues. It was found that a-Man and GlcNAc residues are the ligands that are directly related to the adhesion process and further penetration of sperm. After this first interaction, the RS enters the oocyte envelope by the action of a trypsin-like enzyme, rhynchocinecine, present in the acicular process. Later, arms and spines penetrate the oocyte cytoplasm, where the chromatin of the arms begin to migrate to the central area of the sperm, condensing in a cup-shaped structure near the connecting piece, which forms the male pronucleus. <![CDATA[<strong>Cysteine-Rich Secretory Proteins (CRISP) and their role in mammalian fertilization</strong>]]> Epididymal protein CRISPI is a member of the CRISP (Cysteine-RIch Secretory proteins) family and is involved in sperm-egg fusion through its interaction with complementary sites on the egg surface. Results from our laboratory have shown that this binding ability resides in a 12-amino-acid region corresponding to a highly conserved motif of the CRISP family, named Signature 2 (S2). In addition to this, our results revealed that CRISP1 could also be involved in the previous step of sperm binding to the zona pellucida, identifying a novel role for this protein in fertilization. As another approach to elucidate the participation of CRISP1 in fertilization, a mouse line containing a targeted disruption of CRISP1 was generated. Although CRISP1-deficient mice exhibited normal fertility, CRISP1-defficient sperm presented a decreased level of protein tyrosine phosphorylation during capacitation, and an impaired ability to fertilize both zona-intact and zona-free eggs in vitro, confirming the proposed roles for the protein in fertilization. Evidence obtained in our laboratory indicated that testicular CRISP2 would also be involved in sperm-egg fusion. Competition assays between CRISP1 and CRISP2, as well as the comparison of their corresponding S2 regions, suggest that both proteins bind to common complementary sites in the egg. Together, these results suggest a functional cooperation between CRISP1 and CRISP2 to ensure the success of fertilization. <![CDATA[<strong>Acrosin release and acrosin activity during incubation in capacitating media using fresh and frozen-thawed dog sperm</strong>]]> We evaluated the effect of time and temperature on acrosin release from the acrosomal cap and the activity of this enzyme during in vitro capacitation in fresh and frozen/thawed dog sperm. Sperm-rich fractions of six ejaculates from three dogs were processed as fresh and frozen samples. Each sperm sample was incubated in canine capacitation medium (CCM) for 0, 1, 2 and 3 h at 20°C and at 37°C. After incubation, the samples were assessed by the indirect immunofluorescent staining technique. The probability of having unlabeled sperm (PUS), indicating acrosin loss, was modelled by a binomial distribution using logistic regression. There was a linear relationship between PUS and time at both temperatures (p<0.001); however, a major percentage of unlabeled sperm was observed in frozen/thawed samples soon after incubation, indicating that the release of acrosin was affected by capacitation time, mainly in frozen samples. Temperature influenced acrosin release only in cryopreserved sperm (p<0.05). Acrosin activity was measured by digestion halos on slides coated with gelatin-substrate film during each time period; a significant increase in the number of large halos was observed in fresh samples throughout the experiment, whereas frozen/thawed sperm showed a decreased rate of halo diameters during culture. Thus, there appears to differences between fresh and frozen dog sperm in terms of acrosin release and the level of acrosin activity in the course of in vitro capacitation. <![CDATA[<strong>Protease activity involvement in the passage of mammalian sperm through the zona pellucida</strong>]]> The interaction between acrosome-reacted sperm and zona pellucida proteins is not yet fully understood. Serine protease acrosin and its zymogen proacrosin have been proposed to fulfill this function due to their capacity to bind zona pellucida glycoproteins. However, the molecular mechanism underlying this interaction has been merely speculative. Here we show that fucoidan (a sulfated polysaccharide) and solubilized zona pellucida glycoproteins, but not soybean trypsin inhibitor, are able to detach bound spermatozoa, which suggests that live sperm binds to the zona pellucida in a non-enzymatical way. Interestingly, mild proteolytic digestion with acrosin or trypsin does not modify the structure of the zona pellucida, but rather results in fewer spermatozoa binding to the zona. These results agree with a model where the active site of acrosin digests the zona pellucida and binds through the polysulfate-binding domain through a three-dimensional zona structure rather than a single ligand. <![CDATA[<strong>Modulation of spermatozoon acrosome reaction</strong>]]> Spermatozoon acrosome reaction is an exocytotic event of the utmost importance for the development of mammalian fertilisation. Current evidence shows that the triggering of the acrosome reaction (AR) could be regulated by the action of diverse compounds, namely, metabolites, neurotransmitters and hormones. The aim of the present review is to describe the modulating effects of several compounds that have been classified as inductors or inhibitors of acrosome reaction. Among AR inductors, it is necessary to mention progesterone, angiotensin II, atrial natriuretic peptide, cathecolamines, insulin, leptin, relaxin and other hormones. Regarding the inhibitors, oestradiol and epidermal growth factor are among the substances that retard AR. It is worth mentioning that gamma-aminobutyric acid, a neurotransmitter known to be an inhibitor in the central nervous system, has been shown to induce AR. The multiple hormones located in the fluids of the female reproductive tract are also likely to act as subtle regulators of AR, constituting a fundamental aspect for the development of successful fertilisation. Finally, it is necessary to emphasise that the study of regulation exerted by hormones and other compounds on AR is essential for further understanding of mammalian reproductive biology, especially spermatozoon physiology. <![CDATA[<strong>Effects of hypoxia on epididymal sperm parameters and protective role of ibuprofen and melatonin</strong>]]> Hypobaric hypoxia is of interest due to an increase of human populations working at high altitude. Testicular damage is related to the physiological response (neoangiogenesis) to increased intrascrotal blood flow as temperature rises. Hypoxia is a stress factor with overproduction of reactive oxygen species (ROS). The effect of hypoxia in mice reproductive parameters is analyzed. Animals were exposed to simulated hypoxia of 4,200 meters above sea level (m.a.s.l.) in a chamber for 33.2 days, both to continuous (HH) or intermittent hypoxia (HI) with an intermittency period of 4 days hypoxia /4 days normoxia (500 m.a.s.l.). The anti-inflammatory drug Ibuprofen was administered to a group of mice to control vasodilation and increased blood flow. Melatonin was administered to another group of mice as a potent ROS scavenger. Animals in both HH and HI exposure were compared to normoxic non-treated controls. There was a hematological response in hypoxia, with an increase in hematocrit and reticulocytosis. There was also increased teratozoospermia. This damage was more pronounced in HH than HI, suggesting that alternating normoxic periods permits compensation for the effects of hypoxia. In both hypoxia systems, the level of lipoperoxidation and the instability of DNA increased. In HH, there was a reduction of teratozoospermia in melatonin-treated mice. Ibuprofen presented a protective effect on the same parameters as melatonin with both HI and HH. The quality of sperm DNA, fragmentation, unpacking and DNA stability diminished. In conclusion, reproductive damage elicited by HH or HI was partially ameliorated by simultaneous treatment with antiflogistic and/or antioxidant agents. <![CDATA[<strong>Vitamin C and oxidative stress in the seminiferous epithelium</strong>]]> In this article, we focus on the fundamental role of vitamin C transporters for the normal delivery of vitamin C to germ cells in the adluminal compartment of seminiferous tubules. We argue that the redox status within spermatozoa or in semen is partly responsible for the etiology of infertility. In this context, antioxidant defence plays a critical role in male fertility. Vitamin C, a micronutrient required for a wide variety of metabolic functions, has long been associated with male reproduction. Two systems for vitamin C transport have been described in mammals. Facilitative hexose transporters (GLUTs), with 14 known isoforms to date, GLUT1-GLUT14, transport the oxidized form of vitamin C (dehydroascorbic acid) into the cells. Sodium ascorbic acid co-transporters (SVCTs), SVCT1 and SVCT2 transport the reduced form of vitamin C (ascorbic acid). Sertoli cells control germ cell proliferation and differentiation through cell-cell communication and form the blood-testis barrier. Because the blood-testis barrier limits direct access of molecules from the plasma into the adluminal compartment of the seminiferous tubule, one important question is the method by which germ cells obtain vitamin C. Some interesting results have thrown light on this matter. Expression of SVCT2 and some isoforms of GLUT transporters in the testis have previously been described. Our group has demonstrated that Sertoli cells express functionally active vitamin C transporters. Kinetic characteristics were described for both transport systems (SVCT and GLUT systems). Sertoli cells are able to transport both forms of vitamin C. These findings are extremely relevant, because Sertoli cells may control the amount of vitamin C in the adluminal compartment, as well as regulating the availability of this metabolite throughout spermatogenesis. <![CDATA[<strong>Propylthiouracil-induced hypothyroidism delays apoptosis during the first wave of spermatogenesis</strong>]]> Mammalian germ cell apoptosis plays a key role in controlling the correct number of germ cells supported by Sertoli cells during the first wave of spermatogenesis in mammalian puberty. However, little is known about hormonal factors that could influence the rate of germ cell apoptosis during puberty or adulthood. In this work we evaluate germ cell apoptosis under hypothyroidism induced by goitrogen propylthiouracil (PTU) during the first wave of spermatogenesis. Neonatally administered PTU promoted a delay in the differentiation of Sertoli cells as evaluated by the expression of clusterin using immunohistochemistry and RT-PCR. Clusterin had different expression levels in control and PTU-treated animals, but under both conditions the highest levels were found in 35-day-old rats. In addition, clusterin displayed a cytoplasmic localization in control testes, but appeared located in the nucleus in PTU-treated animals. The wave of apoptosis (determined by caspase activity and quantification of apoptotic cells) characteristic of the first round of spermatogenesis was delayed by at least 10 days in these animals. The expression levels of proapoptotic genes like BAX or BAD were different between control and PTU-treated rats; although in both groups the highest level was found at the same age (days). Thus our results indicate that the characteristic pubertal apoptotic wave during rat spermatogenesis is delayed in neonatal hypothyroid rats. <![CDATA[<strong>The effect of cigarette smoke on fertilization and pre-implantation development</strong>: <strong>assessment using animal models, clinical data, and</strong><strong> </strong><strong>stem cells</strong>]]> Numerous studies have repeatedly shown that women who smoke experience problems establishing and maintaining pregnancies, and recent work has further demonstrated that the in utero effects of smoke may not be manifested until months or even years after birth. The purpose of this review is to examine the recent literature dealing with the effects of cigarette smoke on the earliest stages of human prenatal development. Studies in this area have included the use of animal models, patients undergoing in vitro fertilization, and embryonic stem cell models. Events leading to fertilization, such as cumulus expansion, hyperactivation of sperm motility, and oocyte pick-up by the oviduct are all impaired by smoke exposure in animal models. Steps crucial to fertilization such as the acrosome reaction and sperm binding to the zona pellucida are likewise inhibited by cigarette smoke. Preimplantation embryos and stem cells that model embryos show a number of adverse responses to smoke exposure, including poor adhesion to extracellular matrices, diminished survival and proliferation, and increased apoptosis. The current literature demonstrates that the earliest stages of prenatal development are sensitive to smoke exposure and indicates that pregnant women should be advised not to smoke during this time. <![CDATA[<b>Extended culture up to the blastocyst stage</b>: <b>a strategy to avoid multiple pregnancies in assisted reproductive technologies</b>]]> The aim of this study was to review the experience and outcomes of assisted reproduction cycles with embryos grown up to day 5 of development, comparing different parameters according to the ages of the patients. We retrospectively studied 1,874 assisted reproduction cycles where embryo culture was extended up to the fifth or sixth day of development. All IVF and ICSI cycles were included, comparing, according to patient age, the following rates: blastocyst formation, pregnancy, implantation and abortion. As control, we analyzed cycles with donated oocytes from young donors (OD). The number of embryos reaching the blastocyst stage is similar in all groups of patients. Only the OD group was different in terms of blastocyst formation, pregnancy and implantation rates. Patients over 39 years of age had an abortion rate of 59.1 %, which is significantly higher than the other groups. Extended embryo culture up to the blastocyst stage can be implemented in programs of assisted reproduction in order to increase the pregnancy rate. The potential of blastocyst implantation is high, allowing us to transfer fewer embryos and reduce the probability of multiple pregnancies. <![CDATA[<strong>The beginning of life of a new human being from the scientific biological perspective and its bioethical implications</strong>]]> The issue of when the human life begins is a very important subject since it has a significant impact on the decisions that we have to take in relation to human beings in development, particularly human embryos. In this article we discuss some of the more relevant biological evidence supporting the fact that beginning human life begins unquestionably at fertilization and the bioethical consequences.