Dysregulation of GdA Expression in Endometrium of Women With Endometriosis: Implication for Endometrial Receptivity.

Glycodelin-A (GdA) has been proposed to represent a potential biomarker of endometrial function, but little is known about its expression during the different phases of the menstrual cycle and under pathological conditions. In the light of its potential importance also in embryo implantation, we aimed to evaluate the expression profile of GdA as well as the presence of different glycosylated glycoforms and the immunolocalization in endometrial tissue from women with endometriosis and in women with proven fertility, at different times during the menstrual cycle. Our results showed that GdA is synthesized by endometrial epithelial and stromal cells, both in healthy endometrium and eutopic endometrium from women with endometriosis, with a profile including several glycosylated glycoforms, differentially expressed in each phase of the menstrual cycle. During the secretory phase, a significant increase in GdA protein expression, with a different glycoforms profile, was observed in endometriotic eutopic endometrium. Protein localization in eutopic endometrial tissue resulted significantly different in comparison with endometrium from women with proven fertility. This study indicate that GdA is a complex glycoprotein including up to 6 different glycoforms specifically expressed during the different phase of the menstrual cycle; in pathologic conditions such as endometriosis, the expression profile is altered possibly related to the impaired endometrial receptivity.

Antioxidants reduce oxidative stress in follicular fluid of aged women undergoing IVF.

BACKGROUND: The status characterized by the imbalance between pro-oxidants and antioxidants molecules, defined as oxidative stress, has been suggested to be involved in the pathogenesis of subfertility in females. This study aims to evaluate the impact of a complete micronutrients supplementation on oxidative stress levels in follicular microenvironment as well as on in vitro fertilization (IVF) outcome. METHODS: This preliminary study was conducted between January 2014 and July 2015 at the Siena University Hospital Infertility Clinic. Serum and follicular fluid were collected from infertile women aged > 39 years who underwent two in vitro fertilization cycles: in the first cycle they were treated with GnRH-antagonist protocol and gonadotropins for controlled ovarian hyperstimulation, whereas in the second cycle ovarian stimulation protocol was associated to micronutrients supplementation, starting three months earlier. Protein oxidation levels and total antioxidant capacity in serum and in follicular fluid were evaluated in IVF cycles with or without micronutrients supplementation. Differences in IVF outcome parameters were statistically evaluated. RESULTS: Two-dimensional electrophoresis analyses demonstrated that when patients assumed micronutrients before IVF cycles, follicular fluid and serum proteins were protected from oxidative damage. Comparable results were obtained when total antioxidant capacity was measured. Moreover, the mean number of good quality oocytes retrieved when patients received micronutrients supplementation was significantly increased. CONCLUSION: The additional treatment with micronutrients, starting three months before IVF cycles, protects the follicular microenvironment from oxidative stress, thus increasing the number of good quality oocytes recovered at the pick up.

Protein pathways working in human follicular fluid: the future for tailored IVF?

The human follicular fluid (HFF) contains molecules and proteins that may affect follicle growth, oocyte maturation and competence acquiring. Despite the numerous studies, an integrated broad overview on biomolecular and patho/physiological processes that are proved or supposed to take place in HFF during folliculogenesis and oocyte development is still missing. In this review we report, for the first time, all the proteins unambiguously detected in HFF and, applying DAVID (Database for Annotation, Visualization and Integrated Discovery) and MetaCore bioinformatic resources, we shed new lights on their functional correlation, delineating protein patterns and pathways with reasonable potentialities for oocyte quality estimation in in vitro fertilisation (IVF) programs. Performing a rigorous PubMed search, we redacted a list of 617 unique proteins unambiguously-annotated as HFF components. Their functional processing suggested the occurrence in HFF of a tight and highly dynamic functional-network, which is balanced by specific effectors, primarily involved in extracellular matrix degradation and remodelling, inflammation and coagulation. Metalloproteinases, thrombin and vitamin-D-receptor/retinoid-X-receptor-alpha resulted as the main key factors in the nets and their differential activity may be indicative of ovarian health and oocyte quality. Despite future accurate clinical investigations are absolutely needed, the present analysis may provide a starting point for more accurate oocyte quality estimation and for defining personalised therapies in reproductive medicine.

Protein modification as oxidative stress marker in normal and pathological human seminal plasma.

OBJECTIVE: Our study aims to assess the oxidative stress status of seminal plasma from normozoospermic, azoospermic, and leukocytospermic males, since abnormal sperm and leukocytes in human ejaculates are the main source of reactive oxygen species (ROS) which lead to oxidative damages. For this purpose we applied a biochemical approach to the assessment of the oxidative stress status by using two-dimensional (2D) electrophoresis to check the level of protein oxidation after specific labeling of free thiol (-SH) groups. METHODS: Seminal plasma samples from normal and pathological males were analyzed by a luminol-based chemiluminescent assay. The same samples after specific labeling of free -SH groups with 3-N-maleimidopropionyl biocytin, were analyzed by 2D electrophoresis and computer-assisted semiquantitative determination of the amount of free -SH groups. RESULTS: Using a standard chemiluminescence assay, we demonstrated a high, low and normal level of ROS, respectively, in seminal plasma from leukocytospermic, azoospermic, and normozoospermic subjects. By 2D electrophoresis and streptavidin blotting of specifically labeled free -SH groups of proteins, we detected in the same samples a higher level of oxidated -SH groups comparable between azoospermic and leukocytospermic samples, whereas a significantly higher level of free -SH groups was detected in normozoospermic subjects. DISCUSSION: Our results demonstrated that a pathological oxidative stress status in seminal plasma may be revealed by the levels of the protein free -SH groups, both in the presence or absence of cells.

Impact of insemination technique, semen quality and oocyte cryopreservation on pronuclear morphology of zygotes derived from sibling oocytes.

Pronuclear morphology seems to be an important predictive value of zygote development and integrity. In this study we want to evaluate the effect of insemination technique, male factor and oocyte cryopreservation on pronuclear morphology of zygotes derived from sibling oocytes in our Centre of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova, Reggio Emilia, Italy. Subjects (n = 190) were submitted to IVF cycles with non-frozen and frozen sibling oocytes. Morphological evaluations were assessed using zygote pronuclear morphology (pronuclei, nucleoli and axis) in four groups: Group 1: 144 zygotes from 85 conventional IVF cycles with non-frozen oocytes; Group 2: 164 zygotes from 85 intracytoplasmic sperm injection (ICSI) cycles with Group 1 patients' sibling frozen oocytes; Group 3: 221 zygotes from 123 ICSI cycles with non-frozen oocytes; Group 4: 197 zygotes from 123 ICSI cycles with Group 3 patients' sibling frozen oocytes. No differences between Group 1 and Group 2 were seen. Group 3 was statistically different from Group 4 in relation to the nucleolar morphology. Oocyte cryopreservation procedure modified the nucleolar morphology of zygotes only in the presence of poor semen quality.

Protein profile of capacitated versus ejaculated human sperm.

Freshly ejaculated sperm acquire the fertilizing potential by a continuing process that occurs during sperm transport through the female genital tract, and it is physiologically not complete until the spermatozoon reaches the oocyte. The process termed capacitation can be mimicked in vitro by using appropriate capacitation media. Despite its importance, the molecular mechanisms underlying capacitation are poorly understood. This work deals with a proteomic approach to the analysis of protein profile variations in human normospermic samples as a consequence of three hours in vitro capacitation. 2DE gels were produced per freshly ejaculated sperm and per capacitated sperm and several quantitative and qualitative significant variations were found. Among the MS obtained identifications, proteins with a significant decrease after capacitation were found to be involved in protein fate, metabolism, and flagellar organization; on the contrary, increasing proteins were found to be related to cellular stress. Interestingly, the detected flagellar organization proteins decreased during capacitation whereas their corresponding fragments increased. A swim-up selected and three-hour capacitated sperm subpopulation has also been resolved by 2DE, and its synthetic gel has been analyzed for the variations observed in the entire sperm population. An immunofluorescence analysis of this sperm typology was carried out with antiactin and antitubulin antibodies.

A fucose-containing O-glycoepitope on bovine and human nucleolin.

In this paper, we demonstrate the existence and localization of fucosyl-containing O-glycoforms of nucleolin in cultured bovine endothelial cells (CVEC) and malignant cultured human A431 cells. The tool for this discovery was an antibody raised against gp273, a glycoprotein ligand for the sperm-egg interaction in the mollusc bivalve Unio elongatulus. The function and immunological properties of gp273 mainly depend on clustered Lewis-like, fucose-containing O-glycans. Here an anti-gp273 antibody was used to evaluate whether glycoepitopes similar to those of gp273 are part of potential ligands of selectins in endothelial cells. We found that anti-gp273 strongly and exclusively interacted with a 110 kDa protein in CVEC and A431 tumor cells. After partial purification, mass spectrometry identified the protein as nucleolin. This was confirmed by comparing anti-gp273 and anti-nucleolin antibody immunoblotting after nucleolin depletion. We confirmed that anti-gp273 binding to nuclear and extranuclear nucleolin was against a fucose-containing O-glycoepitope by immunoblot analysis of the protein after chemically removing O-glycans and by lectin-blot analysis of control and nucleolin-depleted samples. Using anti-gp273 IgG, we detected nucleolin on the plasma membrane and cytoplasm. O-Glycosylation may regulate the plethora of functions in which nucleolin is involved.

Cellular and molecular aspects of ovarian follicle ageing.

It is well established that age-related decline of the biological capacity of a woman to reproduce is primarily related to the poor developmental potential of her gametes. This renders female ageing the most significant determinant of success in IVF. Starting with a reference picture of the main molecular and cellular failures of aged oocytes, granulosa cells and follicular microenvironment, this review focuses on age-related biochemical mechanisms underlying these changes. According to the most relevant concept of ageing, age-associated malfuction results from physiological accumulation of irreparable damage to biomolecules as an unavoidable side effect of normal metabolism. More than a decade after the free radical theory of ovarian ageing, biological and clinical research supporting the involvement of oxidative injuries in follicle ageing is discussed. Looking for the aetiology of oxidative stress, we consider the effect of ageing on ovarian and follicular vascularization. Then, we propose a potential role of advanced glycation end-products known to be involved in the physiological ageing of most tissues and organs. We conclude that future investigation of age-related molecular damage in the different ovarian components will be imperative in order to evaluate the possibility to save or rescue the developmental potential of aged oocytes.

The GPI-anchored CD52 antigen of the sperm surface interacts with semenogelin and participates in clot formation and liquefaction of human semen.

CD52 is a human glycosylphosphatidylinositol (GPI)-anchored antigen exclusively expressed in leukocytes and epididymal cells. It is also present in sperm, being inserted in their plasma membrane as they pass through the epididymis. In a previous paper we identified a new CD52 form without GPI anchor by fast performance liquid chromatography (FPLC) fractionation of semen components. The form has a lower negative charge than the GPI-anchored form and occurs as the only CD52 form in prostasome-free seminal plasma. It was also found associated with the ejaculated sperm, but in contrast to the GPI-anchored one, it is lost during the capacitation process. In this paper we indicate that (1) the GPI-anchored CD52 of the sperm surface serves as receptor for semenogelin I during clot formation, (2) liquefaction involves cleavage of the GPI anchor from certain CD52 molecules, releasing sperm from the clot and the soluble antigen bound to semenogelin fragments into the seminal plasma and (3) the clot is a sponge-like structure housing sperm. Soluble CD52 was immunopurified from the soluble CD52-containing FPLC fraction using CAMPATH-1G and was found to be complexed with a semenogelin-derived peptide of the carboxyl terminal portion of semenogelin I, having the sequence SQTEKLVAGKQI and starting from amino acid 376. Immunoprecipitation and immunoblot analyses using CAMPATH-1G and anti-semenogelin as immunoprecipitating antibodies and anti-gp20 and anti-semenogelin as immunoblot detectors of the corresponding antigens, confirmed that the soluble CD52 formed a complex with semenogelin. The semenogelin-CD52 soluble form was found to be a direct consequence of the liquefaction process since only the GPI-anchored CD52 was recovered in uniquefied semen after recovering sperm and seminal plasma by urea solubilization of the clot.

Identification and characterization of a 38 kDa glycoprotein functionally associated with mating activity of Paramecium primaurelia.

In Paramecium primaurelia mating interactions take place immediately after mixing mating-competent cells of opposite mating types. The cells clump in clusters (mating reaction) and then separate in pairs. Previous results have shown that sialic acid-containing glycoconjugates are present on the cell surface and are involved in mating-cell pairing. In order to identify the sialic acid-containing glycoprotein(s), we first metabolically radiolabelled non-mating-competent cells with D-[6-(3)H]galactose, and then analyzed the radiolabelled proteins by anion exchange chromatography. We characterized a 38 kDa (gp38) sialic acid-containing glycoprotein and raised the corresponding polyclonal antibody by means of which we localized the antigen at the level of the oral region of non-mating-competent cells and on the ciliary surface of mating-competent cells. Immunoblot analysis of the ciliary protein fraction showed that the anti-gp38 serum interacted with a 38 kDa protein in both mating types I and II cells. We also demonstrated the functional activity of gp38 in the mating reaction by means of anti-gp38 antibody competition assays.

Menstrual cycle-related sialidase activity of the female cervical mucus is associated with exosome-like vesicles.

OBJECTIVE: To study endogenous sialidase activity in genital tract secretions of pregnant and nonpregnant women. DESIGN: Laboratory study. SETTING: Department of Evolutionary Biology and Department of Obstetrics and Reproductive Medicine, University of Siena, Siena, Italy. INTERVENTION(S): Vaginal and cervical mucus samples were obtained from pregnant and nonpregnant women in different phases of the menstrual cycle and in different weeks of pregnancy. MAIN OUTCOME MEASURE(S): Sialidase activity was assessed by fluorimetric assay and localized by transmission electron microscopy and differential centrifugation. RESULT(S): Sialidase activity in cervical mucus of healthy women reaches a maximum in the ovulatory phase. Cervical mucus from pregnant and nonpregnant women had significant sialidase activity that was associated with membranous vesicles having an exosome-like structure. CONCLUSION(S): Female cervical mucus contains an endogenous menstrual cycle-related sialidase that could be involved in modifying the rheologic properties of mucus to favor sperm progression at fertilization. Its association with exosome-like vesicles also suggests a role in intercellular communication before and after fertilization.

Sperm quality and pregnancy rate after COX-2 inhibitor therapy of infertile males with abacterial leukocytospermia.

BACKGROUND: Leukocytes are a frequent finding in seminal plasma of infertile males with abacterial inflammation. We evaluated the effects of treatment with rofecoxib, a cyclooxygenase-2 inhibitor, on sperm quality and pregnancy rate after intrauterine insemination (IUI) or monitored intercourse. METHODS: We selected 47 infertile patients referred to our sterility centre for semen analysis. Sperm evaluation was performed by light microscopy with Papanicolau and eosin staining, before and 1 month after therapy. Swim-up selection was carried out in two steps. Starting 6-8 weeks after the end of therapy, couples underwent different procedures of assisted fertilization according to their semen parameters. RESULTS: Semen analysis 30 days after the end of therapy showed a significant reduction in leukocyte concentrations with respect to baseline, an improvement of sperm motility and morphology, particularly the presence and shape of the acrosomal complex and tail structure. After monitored intercourse and IUI, pregnancy rate was 15.8 and 11.3%, respectively. CONCLUSIONS: Our results suggest that a decrease in leukocytospermia after rofecoxib therapy was associated with recovery of all seminal characteristics in basal and swim-up selected samples. This general improvement could justify the positive outcome of ART after anti-inflammatory therapy.

Glycoconjugate profiles of the lancelet (Branchiostoma lanceolatum) ovary: a lectin histochemical study by laser confocal microscopy.

The presence and the distribution of carbohydrate moieties in ripe lancelet (Branchiostoma lanceolatum) oocytes (mean diameter 130 microm) was studied by lectin histochemistry in combination with enzyme and chemical treatments. Binding sites for eight lectins with specificities towards different glycan moieties were studied on sections of the whole body of mature female lancelets. Only three of the lectins tested reacted positively. Concanavalin-A (ConA)-binding glycoconjugates were localized in the cytoplasm, namely in yolk granules, whereas Artocarpus integrifolia (AIA) and Ricinus communis (RCA) agglutinins bound strongly to extracellular coats of the oocyte identified as the jelly coat and vitelline layer. No other tissues of the lancelet body were found to be positive to any lectin tested, except gut enterocytes which reacted strongly with AIA. Reactivity to ConA was abolished by pretreatment of sections with N-glycosidase F but not by mild alkaline hydrolysis, confirming that the glycoconjugates were of the N-linked type. On the contrary, chemical removal of O-linked chains by mild alkaline hydrolysis abolished AIA and RCA reactivity but had no effect on ConA positivity.

Envelope glycosylation determined by lectins in microscopy sections of Acinetobacter venetianus induced by diesel fuel.

It was suggested in a previous study that cells of Acinetobacter venetianus VE-C3 adhere to diesel fuel by synthesizing a capsular polysaccharide containing glucose and/or mannose. To study the fine structure of cells and localization of bacterial polysaccharide in the presence of diesel fuel, two lectins were used: ConA, an agglutinin from Canavalia ensiformis specific for mannose and/or glucose residues, and PNA, an agglutinin from Arachis hypogaea, for terminal galactose residues. The lectins were conjugated with electron dense ferritin for transmission electron microscopy (TEM) and with fluorescein isothiocyanate (FITC) for scanning confocal laser microscopy (SCLM). Samples were prepared by freeze substitution, which allows glycosylation to be determined in situ in thin sections of specimens. The distribution of glycosylation was imaged with and without treatment of specimens with their specific hapten (glucose and galactose). The glycosylation activity produced a polysaccharide capsule. Emulsified diesel fuel nanodroplets were observed at the cell envelope perimeter. Fine structure of vesicles consisted of polysaccharide and diesel fuel nanodroplets. Lectin blotting analysis showed ConA-positive glycoprotein with an apparent molecular mass of 22 kDa in the outer membrane. Its production was induced by diesel fuel. This glycoprotein was probably responsible for bioemulsifying activity at the cell envelope. Several other glycoproteins were positive for PNA lectin, the main constituent migrating with an apparent molecular weight of 17.8 kDa. However, they were all constitutive and probably involved in cell biofilm formation at the oil surface.

Immunoglobulins against gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus, are directed against charged O-linked oligosaccharide chains bearing a Lewis-like structure and interact with epitopes of the human zona pellucida.

In oocytes of the mollusc bivalve Unio elongatulus, gp273 is the ligand molecule for sperm-egg interaction and binding is mediated by its O-glycans. A serum raised against this protein enabled its localization in the crater region, the area of the vitelline coat where sperm recognition occurs, and showed that after cyanogen bromide fragmentation, the anti-gp273 epitope(s) was retained by a peptide where the O-glycans are localized. In this article, we utilized purified anti-gp273 immunoglobulins to characterize the corresponding epitope by: (i) immunoblotting analysis of the protein after removal of O- and N-glycans; (ii) solid phase binding analysis of anti-gp273 IgG to gp273 N- and O-glycans; and (iii) binding analysis of the same antibody to commercially available oligosaccharides. The results showed that the epitope consists of O-glycans and contains a Lewis-like structure with fucose as determinant. Anti-gp273 IgG were then used to investigate human zona pellucida by immunoelectronmicroscopy and immunoblotting. Epitopes recognized by the antibody were demonstrated on the outer surface of the zona pellucida and shown to belong to a zona pellucida protein having electrophoretic mobility similar to human ZP3. Since human sperm specifically bind to gp273, and anti-gp273 interferes with this binding a functional role for these epitopes is suggested.