Roberto A. Rovasio

Chemotaxis of Capacitated Rabbit Spermatozoa to Follicular Fluid Revealed by a Novel Directionality-Based Assay

Abstract Precontact communication between gametes is established by chemotaxis. Sperm chemotaxis toward factor(s) in follicular fluid (FF) has been demonstrated in humans and mice. In humans, the chemotactic responsiveness is restricted to capacitated spermatozoa. Here, we investigated whether sperm chemotaxis to factor(s) present in FF also occurs in rabbits and, if so, whether only capacitated spermatozoa are chemotactically responsive. Chemotaxis assays were performed by videomicroscopy in a Zigmond chamber. We measured chemotactic responsiveness as a function of FF dilution by means of a novel directionality-based method that considers the ratio between the distances traveled by the spermatozoa both parallel to the chemoattractant gradient and perpendicular to it. A peak of maximal response was observed at 10−4 dilution of FF, resulting in a typical chemotactic concentration-dependent curve in which 23% of the spermatozoa were chemotactically responsive. In contrast, the percentage of cells exhibiting FF-dependent enhanced speed of swimming increased with the FF concentration, whereas the percentage of cells maintaining linear motility decreased with the FF concentration. The percentages of chemotactically responsive cells were very similar to those of capacitated spermatozoa. Depletion of the latter by stimulation of the acrosome reaction resulted in a total loss of the chemotactic response, whereas the reappearance of capacitated cells resulted in a recovery of chemotactic responsiveness. We conclude that rabbit spermatozoa, like human spermatozoa, are chemotactically responsive to FF factor(s) and acquire this responsiveness as part of the capacitation process.

Lack of species-specificity in mammalian sperm chemotaxis

Attraction of spermatozoa by way of chemotaxis to substances secreted from the egg or its surrounding cells has been demonstrated in marine species, amphibians, and mammals. This process is species- or family-specific in marine invertebrates: a chemoattractant for one marine species is usually not recognized by another species or by a member of another family. It is not known whether this selectivity is also the rule in other phyla. Furthermore, it is not at all obvious that such selectivity would be advantageous to species with internal fertilization. Here, using a directionality-based assay for chemotaxis, we studied in vitro the chemotactic response of human and rabbit spermatozoa to human, rabbit, and bovine egg-related factors. We found that spermatozoa from each of the two sources responded similarly well to egg-related factors obtained from any of the three species examined. These results indicate lack of chemotaxis-related, species specificity between these species, suggesting that their sperm chemoattractants are common or very similar. The findings further suggest that mammals do not rely on species specificity of sperm chemotaxis for avoidance of interspecies fertilization.

Trophic and proliferative perturbations of in vivo/in vitro cephalic neural crest cells after ethanol exposure are prevented by Neurotrophin 3

Neural crest cells (NCCs), a transient population that migrates from the developing neural tube, distributes through the embryo and differentiates into many derivatives, are clearly involved in the damage induced by prenatal exposure to ethanol. The aim of this work was to evaluate alterations of trophic parameters of in vivo (in ovo) and in vitro NCCs exposed to teratogenic ethanol doses, and their possible prevention by trophic factor treatment. Chick embryos of 24-30h of incubation were treated during 10h with 100mM ethanol, or 40 ng/ml Neurotrophin 3 (NT3), or 10 ng/ml Ciliary Neurotrophic Factor (CNTF), or ethanol plus NT3 or CNTF, or defined medium; then the topographic distribution of NCC apoptosis was assessed using a whole-mount acridine orange supravital method. Cultures of cephalic NCCs were exposed to the same ethanol or NT3, or CNTF treatments, or ethanol plus one of both trophic factors, or N2 medium. A viability assay was performed using the calcein-ethidium test, apoptosis was evaluated with the TUNEL test, and proliferative capacity after BrdU labeling. After direct exposure of embryos to 100mM ethanol for 10h, a high level of NCC apoptosis was coincident with the abnormal closure of the neural tube. These anomalies were prevented in embryos exposed to ethanol plus NT3 but not with CNTF. In NCC cultures, high cell mortality and a diminution of proliferative activity were observed after 3h of ethanol treatment. Incubation with ethanol plus NT3 (but not with CNTF) prevented NCC mortality as well as a fall in NCC proliferation. The consequences of direct exposure to ethanol expand data from our and other laboratories, supporting current opinion on the potential risk of alcohol ingestion (even at low doses and/or during a short time), in any period of pregnancy or lactation. Our in vivo/in vitro model encourages us to examine the pathogenic mechanism(s) of the ethanol-exposed embryo as well as the use of trophic factors for the treatment and/or prevention of anomalies induced by prenatal alcohol.

Ultrastructural characterization by ruthenium red of the surface of the fat globule membrane of human and rat milk with data on carbohydrates of fractions of rat milk

SummaryThe fat globules of the cream fractions of human and rat milk were stained with ruthenium red. Under the electron microscope, discrete granules and an amorphous coat of lesser density are seen at the surface of the milk fat globules. Since ruthenium red binds anionic groups selectively, it is probable that the granules containe the greatest concentration of these groups.The cream fraction of rat milk contains hexoses, hexosamines, methylpentoses and sialic acid. Methylpentoses and hexosamines are significantly enriched in the cream fraction.It is concluded that the finding of a surface coat in milk fat globules is in keeping with the Bargmann-Knoop model and suggests a distinct mechanism for carrying certain complex carbohydrates in milk. The role of the negative charges at the outer surface of the membrane coat in maintaining fat globules in suspension and in binding certain cations such as calcium is suggested.

Prenatal and postnatal ethanol experiences modulate consumption of the drug in rat pups, without impairment in the granular cell layer of the main olfactory bulb

The effect of moderate exposure to ethanol during late gestation was studied in terms of its interaction with moderate exposure during nursing from an intoxicated dam. A further issue was whether behavioral effects of ethanol, especially the enhanced ethanol intake known to occur after moderate ethanol prenatally or during nursing, depend upon teratological effects that may include death of neurons in the main olfactory bulb (MOB). During gestational days 17-20 rats were given 0, 1 or 2g/kg ethanol doses intragastrically (i.g.). After parturition these dams were given a dose of 2.5g/kg ethanol i.g. each day and allowed to perform regular nursing activities. During postnatal days (PDs) 15 and 16, ethanol intake of pups was assessed along with aspects of their general activity. In a second experiment pups given the same prenatal treatment as above were tested for blood ethanol concentration (BEC) in response to an ethanol challenge on PD6. A third experiment (Experiment 2b) assessed stereologically the number of cells in the granular cell layer of the MOB on PD7, as a function of analogous pre- and postnatal ethanol exposures. Results revealed that ethanol intake during the third postnatal week was increased by prenatal as well as postnatal ethanol exposure, with a few interesting qualifications. For instance, pups given 1g/kg prenatally did not have increased ethanol intake unless they also had experienced ethanol during nursing. There were no effects of ethanol on either BECs or conventional teratology (cell number). This increases the viability of an explanation of the effects of prenatal and early postnatal ethanol on later ethanol intake in terms of learning and memory.

Sonic hedgehog is a chemotactic neural crest cell guide that is perturbed by ethanol exposure.

Our aim was to understand the involvement of Sonic hedgehog (Shh) morphogen in the oriented distribution of neural crest cells (NCCs) toward the optic vesicle and to look for potential disorders of this guiding mechanism after ethanol exposure. In vitro directional analysis showed the chemotactic response of NCCs up Shh gradients and to notochord co-cultures (Shh source) or to their conditioned medium, a response inhibited by anti-Shh antibody, receptor inhibitor cyclopamine and anti-Smo morpholino (MO). Expression of the Ptch-Smo receptor complex on in vitro NCCs was also shown. In whole embryos, the expression of Shh mRNA and protein was seen in the ocular region, and of Ptch, Smo and Gli/Sufu system on cephalic NCCs. Anti-Smo MO or Ptch-mutated plasmid (Ptch1(Δloop2)) impaired cephalic NCC migration/distribution, with fewer cells invading the optic region and with higher cell density at the homolateral mesencephalic level. Beads embedded with cyclopamine (Smo-blocking) or Shh (ectopic signal) supported the role of Shh as an in vivo guide molecule for cephalic NCCs. Ethanol exposure perturbed in vitro and in vivo NCC migration. Early stage embryos treated with ethanol, in a model reproducing Fetal Alcohol Syndrome, showed later disruptions of craniofacial development associated with abnormal in situ expression of Shh morphogen. The results show the Shh/Ptch/Smo-dependent migration of NCCs toward the optic vesicle, with the support of specific inactivation with genetic and pharmacological tools. They also help to understand mechanisms of accurate distribution of embryonic cells and of their perturbation by a commonly consumed teratogen, and demonstrate, in addition to its other known developmental functions, a new biological activity of cellular guidance for Shh.

Histochemistry and ultrastructure of the cell surfaces of the guinea pig kidney with quantitative data on carbohydrate components of glycosaminoglycans of kidney

SummaryThis report deals with a histochemical, electron microscopic and chemical study of carbohydrates of the guinea pig kidney. Histochemical reactions were positive for glycoproteic components in the lumenal surface of the proximal convoluted tubules as well as in the basal aspects of tubular cells. Mucosubstances containing carboxyl and sulphate groups and sialic acid were present at the cell surfaces of the Bowmans capsule, proximal and distal convoluted tubules, loop of Henle and, in greater concentrations, at the cell surfaces of the collecting tubules and papillary epithelium. Data seemed to indicate that glycosaminoglycans were present at the cell surfaces of the collecting tubules.By electron microscopy, the alcian blue stained a dense coat of granular appearance at the lumenal surface of the loop of Henle, measuring up to 100 nm in thickness. Ruthenium red stained the glycocalyx of the collecting tubules which measured up to 60 nm in thickness. The glycocalyx of the collecting tubules was made up of thin filaments and a dense component of about 10 nm thick which merged with the outer leaflet of lumenal plasma membrane, which thus appeared asymmetric. With Alcian blue, the glycocalyx of the collecting tubules, which measured 600 to 1500 nm in thickness, was made up of a meshwork of filaments containing globular or irregular structures measuring 50 to 150 nm in diameter. Chemical assays of kidney homogenates revealed carbohydrate components and sulphate of glycosaminoglycans.

Neurotrophic factor NT-3 displays a non-canonical cell guidance signaling function for cephalic neural crest cells.

Chemotactic cell migration is triggered by extracellular concentration gradients of molecules segregated by target fields. Neural crest cells (NCCs), paradigmatic as an accurately moving cell population, undergo wide dispersion along multiple pathways, invading with precision defined sites of the embryo to differentiate into many derivatives. This report addresses the involvement of NT-3 in early colonization by cephalic NCCs invading the optic vesicle region. The results of in vitro and in vivo approaches showed that NCCs migrate directionally up an NT-3 concentration gradient. We also demonstrated the expression of NT-3 in the ocular region as well as their functional TrkB, TrkC and p75 receptors on cephalic NCCs. On whole-mount embryo, a perturbed distribution of NCCs colonizing the optic vesicle target field was shown after morpholino cancelation of cephalic NT-3 or TrkC receptor on NCCs, as well as in situ blocking of TrkC receptor of mesencephalic NCCs by specific antibody released from inserted microbeads. The present results strongly suggest that, among other complementary cell guidance factor(s), the chemotactic response of NCCs toward the ocular region NT-3 gradient is essential for spatiotemporal cell orientation, amplifying the functional scope of this neurotrophic factor as a molecular guide for the embryo cells, besides its well-known canonical functions.

Distribution patterns of neural-crest-derived melanocyte precursor cells in the quail embryo.

In vertebrate embryos, migration of trunk neural crest cells (NCC) proceeds mainly in two streams: a dorsoventral path between the neural tube and somites, and a dorsolateral one between somites and ectoderm. This last pathway is taken by melanocyte precursor cells (MPC) homing the skin, while pigment cells seeding internal organs and the peritoneal wall follow the dorsoventral pathway. Early routes taken by subpopulations of NCC have been well documented using the quail‐chick chimaera system and monoclonal antibodies to NCC. However, very little is known about the advanced migratory behavior of MPC, which determines their late distribution patterns at different embryonic axial levels.

LETHAL AND TERATOGENIC EFFECTS OF LAMBDA-CARRAGEENAN, A FOOD ADDITIVE, ON THE DEVELOPMENT OF THE CHICK EMBRYO*

Lambda-Carrageenan, a food additive, injected into the yolk sac of fertile chicken eggs had teratogenic and lethal effects on chick embryo development. Anomalies were mainly localized in the cephalic end (exencephaly, abnormal beak, anophtalmy, etc.). All abnormal chicks of the group injected with carrageenan showed two or more anomalies. Growth of newborn chicks in the carrageenan-injected group was significantly diminished until the 4th day of age.