Roberto Gualtieri

Slow cooling of human oocytes: ultrastructural injuries and apoptotic status

OBJECTIVE To identify the damages caused by slow cooling human metaphase II (MII) oocytes comparing the ultrastructure, inner mitochondrial membrane potential (DeltaPsim), and apoptotic status of fresh and cryopreserved oocytes. DESIGN Experimental study. SETTING University biology research unit and private IVF unit. PATIENT(S) Fresh and cryopreserved supernumerary MII oocytes donated from women undergoing IVF cycles. MAIN OUTCOME MEASURE(S) Ultrastructure was assessed by transmission electron microscopy (TEM), mitochondrial function by means of the fluorescent DeltaPsim reporter JC-1, and apoptotic status through fluorescent labeling with the pan-caspase inhibitor fluorescein isothiocyanate conjugate (FITC)-VAD FMK, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. RESULT(S) Compared to fresh oocytes, frozen/thawed (F/T) oocytes showed reduced cortical granule densities (F/T 3.35 +/- 1.94/10 microm vs. fresh 10.30 +/- 3.9/10 microm), swelling of smooth endoplasmic reticulum (F/T 0.084 +/- 0.03 microm(2) vs. fresh 0.040 +/- 0.02 microm(2)), decreased electron density of the mitochondrial matrix and damage to the mitochondrial membranes, low DeltaPsim of pericortical mitochondria, but no signs of apoptosis. CONCLUSION(S) Slow cooling is associated with cortical granule exocytosis, swelling of smooth endoplasmic reticulum vesicles, and mitochondrial damage, but does not induce early or late apoptotic events. The observed injuries might be responsible for the reduced developmental competence of cryopreserved oocytes.

IN VITRO-CULTURED BOVINE OVIDUCTAL CELLS BIND ACROSOME-INTACT SPERM AND RETAIN THIS ABILITY UPON SPERM RELEASE

Abstract The mammalian oviduct plays a key role in sperm storage, capacitation, and selection. Specific oviduct secretions and/or binding to oviductal cells are thought to be responsible for the extension of the fertile life span of sperm. In this in vitro study, a quantitative assay for sperm binding was developed to analyze the mechanisms of sperm–oviductal cell adhesion and release in the bovine species. Distribution and acrosomal status of sperm bound to in vitro-cultured ampullary and isthmic cell monolayers were followed until the time of sperm release by means of fluorescence labeling techniques. In order to understand whether release is due to surface changes of sperm or oviductal cells, double incubation experiments with unlabeled and Hoechst-labeled sperm have been performed. Main findings demonstrate that (1) only acrosome-intact sperm bind specific bovine oviductal epithelial cells; (2) acrosomes of bound sperm are preserved intact over time; and (3) release of unreacted sperm is likely to be due to changes of the sperm surface, probably triggered by capacitation. These findings support the hypothesis that binding to oviductal cells is essential for preserving the sperm fertilization competence during the interval from the onset of estrus to ovulation.

Sulfated Glycoconjugates Are Powerful Modulators of Bovine Sperm Adhesion and Release from the Oviductal Epithelium In Vitro

Abstract The mechanisms of sperm adhesion and release within the mammalian oviduct are still poorly understood. In this in vitro study, a previously developed adhesion assay was used to analyze the effects of heparin, N-desulfated heparin, fucoidan, dextran sulfate, and dextran on bovine sperm-oviductal cell adhesion and release. Results showed that 1) all sulfated glycoconjugates were powerful inhibitors of sperm binding to oviductal monolayers in a dose-dependent manner, whereas N-desulfated heparin and dextran had no effect; 2) sperm pretreatment with heparin and fucoidan markedly inhibited adhesion; 3) treatment of oviductal monolayers with heparinase I, II, or sodium chlorate (an inhibitor of sulfation) had no effect on sperm adhesion; 4) sulfated glycoconjugates were also powerful and quick inducers of sperm release from oviductal monolayers; and 5) addition of sulfated glycoconjugates to the cocultures caused a sudden increase of bound-sperm flagellar beat frequencies, followed by a release of highly motile sperm. In conclusion, these data support the hypothesis that sulfated glycoconjugates may act as signals that induce sperm release and migration from the oviductal reservoir.

Redox control of surface protein sulphhydryls in bovine spermatozoa reversibly modulates sperm adhesion to the oviductal epithelium and capacitation.

Oviductal fluid molecules, such as sulphated glycosaminoglycans and disulphide-reductants, may represent periovulatory signals for the release of spermatozoa from the oviductal reservoir in the bovine species. Disulphide-reductants release spermatozoa through the reduction of sperm-surface disulphides to sulphhydryls (SH). Herein, we studied sperm-surface protein SH through labelling with maleimidylpropionyl biocytin in the initial sperm suspension, in the subpopulations able and unable to adhere to the in vitro cultured oviductal epithelium, and in spermatozoa released either through the disulphide-reductant penicillamine (PEN) or the sulphated glycosaminoglycan heparin (HEP). Adhesion assays were performed to study the ability of released spermatozoa to readhere to the oviductal epithelium. Results showed that the level of SH in sperm-surface proteins was: 1) low in adhering spermatozoa; 2) high in spermatozoa unable to adhere; and 3) markedly increased in released spermatozoa. Adhesion assays showed that: 1) PEN-released spermatozoa promptly recovered adhesion after removal of the disulphide-reductant and could be released again in response to PEN; 2) conversely, a limited number of HEP-released spermatozoa was able to readhere to the oviductal epithelium and this ability was not affected by HEP removal. Recovery of adhesion was associated to reoxidation of sperm-surface protein SH and to the reversal of capacitation. In conclusion, redox modulation of sperm-surface protein SH is involved in the release of spermatozoa adhering to the oviduct in vitro; the reversible action of disulphide-reductants might be responsible for intermittent phases of adhesions and releases; and the irreversible action of HEP indicates that it may represent a terminal releasing signal.

Redox Regulation of Sperm Surface Thiols Modulates Adhesion to the Fallopian Tube Epithelium

Abstract Sperm that adhere to the fallopian tube epithelium are of superior quality and adhesion extends their fertile life. It has been postulated that periovulatory signals, as yet undefined, promote sperm release. In the in vitro studies described here, we examined the effects of several antioxidants, reportedly present within oviductal fluid, on the modulation of sperm-oviduct adhesion in bovine species. Results showed that 1) the cell-permeant thiols (penicillamine, beta mercaptoethanol, cysteine, and dithiotreitol), as well as the nonpermeant thiol, reduced glutathione, cause adhering spermatozoa to release from the epithelium; 2) thiol action is exerted on spermatozoa; and 3) oxidized glutathione, as well as the non-thiol antioxidants (dimethylthiourea, trolox, superoxide dismutase, and catalase) have no effect. Sperm surface sulfhydryls labeled with iodoacetamide fluorescein showed that spermatozoa devoid of sulfhydryls on the head surface adhered to the fallopian epithelium in vitro, whereas thiol-induced release increased the exposure of sulfhydryls on the sperm head surface. Finally, analysis of capacitation status demonstrated that uncapacitated spermatozoa adhered to the oviduct, and that thiol-induced release of spermatozoa was accompanied by capacitation. In conclusion, thiol-reducing agents in the oviductal fluid may modulate the redox status of sperm surface proteins, leading to the release of spermatozoa selected and stored through adhesion to the fallopian tube epithelium in the bovine species.

Intercellular bridges between granulosa cells and the oocyte in the elasmobranch Raya asterias

In the present ultrastructural study intercellular bridges, connecting somatic granulosa cells to oocyte, have been detected for the first time and their modifications have been followed during Raja oogenesis. Intercellular bridges make their first appearance in small previtellogenic follicles as connecting devices between small cells and the oocyte. Later on, when the follicular epithelium becomes polymorphic and multilayered, for the presence of small, large, and pyriform‐like cells, intercellular bridges link the oocyte and the different granulosa cells. Intercellular bridges contain ribosomes, whorl of membranes, mitochondria and vacuoles. Such cytoplasmic components are present also in the cell apex of large and pyriform‐like cells thus suggesting, in agreement with other species (Motta et al. J. Exp. Zool., 1996;276:223–241) they may flow toward the oocyte. In this regard the presence of intercellular bridges during the oogenesis of cartilagineous fish may represent a crucial event of the active cooperation between granulosa cells and the oocyte. Anat Rec 255:180–187, 1999.

Ability of sulfated glycoconjugates and disulfide-reductants to release bovine epididymal sperm bound to the oviductal epithelium in vitro

In Bos taurus, at ejaculation, epididymal sperm acquire a number of proteins secreted in the seminal plasma that increase their ability to interact with the female reproductive tract. Sperm-oviduct interaction comprises a transient sperm adhesion to the isthmus, the lower portion of the oviduct, followed by sperm release around ovulation. Oviductal fluid molecules, such as sulfated glycoconjugates and disulfide-reductants, are able to release bovine ejaculated sperm bound to the oviductal epithelium in vitro through the reduction of sperm surface protein disulfides to sulfhydryls. To understand whether the sperm molecules sensitive to releasing signals are already exposed on the surface of epididymal sperm, we studied the ability of cauda epididymal sperm to adhere to the oviductal epithelium and to be released by sulfated glycoconjugates and the disulfide-reductant penicillamine. Surface protein sulfhydryls in cauda epididymal sperm were analyzed in the initial suspension, in sperm bound to the in vitro-cultured oviductal epithelium, and in released sperm. Results showed that epididymal sperm are able to bind the oviductal epithelium in vitro, although at a lower extent than frozen-thawed ejaculated sperm; the interaction is mediated by oviductal cell microvilli that closely bind to the plasma membrane of the sperm head rostral region, as previously shown for ejaculated sperm. The sulfated glycoconjugates heparin, fucoidan, and dextran sulfate, as well as the disulfide-reductant penicillamine, are all powerful inducers of sperm release. The level of sulfhydryls in sperm surface proteins was (1) high in the initial sperm suspension; (2) low in bound sperm; (3) markedly increased in sperm released by heparin or by penicillamine. In conclusion, epididymal sperm are already able to bind the oviductal epithelium and to respond to the inducers of release through the reduction of sperm surface protein disulfides to sulfhydryls.

Treatment with zinc, d-aspartate, and coenzyme Q10 protects bull sperm against damage and improves their ability to support embryo development

Reactive oxygen species (ROS) are physiologically generated during mitochondrial respiration and are involved in several signaling mechanisms. However, under pathological conditions, the concentration of ROS may exceed the antioxidant scavenging systems and subsequently lead to cell damage. High ROS levels have been proven to be detrimental to spermatozoa and furthermore compromise sperm function through lipid peroxidation, protein damage, and DNA strand breakage. Although the oral administration of antioxidants has been demonstrated to improve the semen quality in subfertile men, it is still a matter of debate if it can positively influence fertilization outcome and embryo developmental competence. Studies carried out in suitable animal models could resolve these fundamental questions. Hence, the main aims of the present study were to evaluate: (1) the effects of zinc, d-aspartate, and coenzyme Q10, included in the dietary supplement Genadis (Merck Serono), on bull sperm motility and DNA fragmentation; and (2) whether treated spermatozoa have a superior competence in fertilization and in supporting the development of healthy embryos. Our data indicate that this treatment prevents the loss of sperm motility and the rise in sperm DNA fragmentation over time. Moreover, blastocyst rate was found to be significantly higher in oocytes fertilized by treated spermatozoa, and these blastocysts harbored a significantly lower percentage of apoptotic cells.

Long-term viability and differentiation of bovine oviductal monolayers: Bidimensional versus three-dimensional culture

Different in vitro models have been developed to study the interaction of gametes and embryos with the maternal tract. In cattle, the interaction of the oviduct with gametes and embryos have been classically studied using oviductal explants or monolayers (OMs). Explants are well differentiated but have to be used within 24 h after collection, whereas OMs can be used for a longer time after cell confluence but dedifferentiate during culture, losing cell polarity and ciliation. Herein, OMs were cultured either in M199 plus 10% fetal calf serum or in a semidefined culture medium (Grays medium), in an immersed condition on collagen-coated coated microporous polyester or polycarbonate inserts under air-liquid interface conditions. The influence of culture conditions on long-term viability and differentiation of OMs was evaluated through scanning electron microscopy, localization of centrin and tubulin at the confocal laser scanning microscope, and assessment of maintenance of viability of sperm bound to OMs. Findings demonstrated that OMs cultured in an immersed condition with Grays medium retain a better morphology, do not exhibit signs of crisis at least until 3 wks postconfluence, and maintain the viability of bound sperm significantly better than parallel OMs cultured in M199 plus 10% fetal calf serum. OM culture with Grays medium in air-liquid interface conditions on porous inserts promotes cell polarity, ciliation, and maintenance of bound sperm viability at least until 3 wks postconfluence. In conclusion, oviduct culture in Grays medium in an immersed or air-liquid condition allows long-term culture and, in the latter case, also ciliation of bovine OMs, and may represent in vitro systems that mimick more closely the biological processes modulated by the oviduct in vivo.

Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells.