Biomedical subjects

G F Doncel

Publications and source records attributed to G F Doncel.

30 records · Page 2Linked to original sources

PubMed1996

Mannose-binding sites on human spermatozoa and sperm morphology.

OBJECTIVE: To verify a possible association between the expression of sperm head mannose-binding sites and sperm morphology. DESIGN: Prospective in vitro study. SETTING: University-based sperm biology and andrology laboratories. PARTICIPANTS: Twenty-seven fertile donors and 45 patients consulting for infertility. INTERVENTIONS: D-mannose-binding sites were identified using a fluoresceinated mannose-enriched bovine serum albumin. Hoechst 33258 was included to confirm sperm viability and hypo-osmotic swelling test to assess plasma membrane integrity. Sperm morphology was judged by strict criteria and semen samples were classified into three groups: normal (group N, > 10% morphologically normal sperm, n = 27), good prognosis (group G, 5% to 10%, n = 23), or poor prognosis (group P, < or = 4%, n = 22). RESULTS: Only viable (Hoechst 33258 negative) spermatozoa with intact plasma membrane (hypo-osmotic swelling test positive)were considered in the evaluation of mannose-binding sites. The incidence of spermatozoa showing surface mannose-binding sites was higher in the N group (48.1% +/- 1.9%; mean +/- SEM) then in the G (17.6% +/- 4.4%) or P (7.6% +/- 2.3%) groups. Total mannose-binding site expression and percentage of morphologically normal sperm showed a high positive correlation (r = 0.80). CONCLUSION: Altered expression of mannose-binding sites, putative zona receptors, may be one of the molecular defects causing decreased fertility in morphologically abnormal human sperm.

Binding Sites↗

PubMed1994

Intravaginal inoculation of rhesus macaques with cell-free simian immunodeficiency virus results in persistent or transient viremia.

The simian immunodeficiency virus (SIV)-rhesus macaque model of heterosexual human immunodeficiency virus transmission consists of atraumatic application of cell-free SIVmac onto the intact vaginal mucosa of mature female rhesus macaques. This procedure results in systemic infection, and eventually infected animals develop the clinical signs and pathologic changes of simian AIDS. To achieve 100% transmission with the virus stocks used to date, multiple intravaginal inoculations are required. The current titration study utilized two stocks of SIVmac and demonstrated that a single intravaginal dose of cell-free SIV can reliably produce infection in rhesus macaques. This study also demonstrated that some animals intravaginally inoculated with cell-free SIVmac develop transient viremia characterized by a limited ability to isolate virus from peripheral blood mononuclear cells and lymph node mononuclear cells and no seroconversion to SIV antigen. SIV could be isolated from the peripheral lymph nodes of transiently viremic animals only during periods of viremia and not at times when SIV was not detected in circulating mononuclear cells. Thus, peripheral lymphoid tissues were not reservoirs of infection in the transiently viremic animals. Taken together, these results suggest either that the SIV infection was cleared in the transiently viremic animals or that SIV infection is limited to a compartment of the genital mucosal immune system that cannot be assessed by monitoring SIV infection in peripheral blood mononuclear cells and peripheral lymphoid tissue.

Animals↗

PubMed1993

Phenotypic characterization of lymphocytic cell infiltrates into the testes of rats undergoing autoimmune orchitis.

Experimental autoimmune orchitis (EAO) was induced in adult Wistar rats by active immunization with a testicular homogenate (TH) and adjuvants. Fifty per cent of the immunized rats developed EAO. Testicular damage became evident at 50 days after the primary immunization and increased in severity at 80 days. Phenotypic characterization of T-cell subsets (CD4+ and CD8+) and Ia+ cells was performed on cryostat sections of testis obtained from normal rats, rats immunized with adjuvant (control group) and rats immunized with TH and adjuvants (experimental group) at 50 and 80 days. Labelled cells were only detected in the interstitial area; no labelled cells were observed inside the seminiferous tubules with any of the monoclonal antibodies used (W3/25, OX-8, OX-6). A significant increase in the numbers of CD4+ and CD8+, as well as of Ia+ cells, were observed in the testis of rats with severe EAO at 80 days after the first immunization. Rats of the same experimental group without testicular damage showed no major differences compared to rats from the control group, with the exception of a lower number of CD8+ cells. Variations in the lymphocyte subsets in lymph nodes draining the site of immunization showed the opposite pattern to that observed in the testis. In conclusion, these data suggest the traffic of specifically sensitized lymphocytes from lymph nodes to the testis and an active role of CD4+, CD8+ and Ia+ cells in the pathogenesis of EAO in the rat.

Animals↗

PubMed1991

Testicular lesions and lymphocyte subpopulations in rats immunized with a soluble fraction of testicular homogenate.

A soluble fraction obtained from a testicular homogenate by precipitation with ammonium sulphate (ASPM) was emulsified with Freund's complete adjuvant (CFA) and injected into Wistar rats. At 50 days after the first immunization (total of three injections) the animals had developed moderate and multifocal testicular damage, characterized mainly by sloughing of the seminiferous epithelium. A delayed-type hypersensitivity response and circulating antibodies to ASPM were detected at different times with maximum levels at 50 days. The addition of Bordetella pertussis to the immunization did not increase the severity of the lesion but augmented the cellular and humoral immune response to ASPM. The phenotypic characterization of cells present in the lymph nodes draining from the site of immunization in animals injected with CFA alone (control group) revealed an increase in CD8+ T-cells and a low CD4/CD8 ratio. Conversely, rats immunized with CFA plus ASPM (experimental group) exhibited testicular damage and showed a significant decrease in CD8+ cells with a normal CD4/CD8 ratio. In conclusion, rats immunized with a testicular antigen developed focal aspermatogenic lesions and a concomitant specific immune response as well as lymph-node cell variations focused apparently on the CD8+ T-cell subpopulation.

Ammonium Sulfate↗

PubMed1991

Lymphocyte subsets in the lymph nodes of rats with autoimmune orchitis.

We determined temporal variations of cell subpopulations in the immunization draining lymph nodes during the development of an experimental autoimmune orchitis (EAO) induced in Wistar rats. A phenotypic characterization of T cells and their subsets (CD4+ and CD8+), B, and Ia+ cells was performed by immunofluorescent technique. At the end of the immunization period (30 days), rats injected with testicular homogenate plus adjuvants presented a considerable increase in absolute cell number but normal lymphocyte subset percentages. Testicular damage became evident at 50 days after the first immunization and increased its severity at 80 days: animals that developed EAO presented a lower number of CD8+ cells as compared with undamaged rats. This latter group showed a low CD4/CD8 ratio due to the high proportion of CD8+ cells, which could probably have a suppressor function. At 80 days massive testicular infiltration and decreased absolute cell number in lymph nodes suggest the possible migration of specific lymphocytes to the target organ.

Adjuvants, Immunologic↗

PubMed1989

Sequential study of the histopathology and cellular and humoral immune response during the development of an autoimmune orchitis in Wistar rats.

Wistar rats immunized with an homologous testicular homogenate (TH) and complete Freund's adjuvant, followed by i.v. injection of Bordetella pertussis, developed an autoimmune orchitis (EAO). Animals were studied at 7, 16, 30, 50, and 80 days (d) after the first immunization. An important lesion of the testis only appeared at 50 d, increasing in severity and incidence (77%) at 80 d. Lesions were characterized by a prevalent aspermatogenesis with tubular atrophy and mild interstitial mononuclear infiltrates. Delayed-type hypersensitivity response (DTH) against TH was detected early at 7 d and, except for 16 d, it increased with time, reaching a maximum at 80 d. A good temporal relationship between DTH and histopathology was found. Circulating antibodies to TH, detected by ELISA, were only present in 64% of the animals with testis lesion, while no deposits of IgG or C3 in the seminiferous tubules were seen. We describe a sequence of immunological events, concomitant with pathological changes of the testis, during the development of a severe EAO in Wistar rats.

Animals↗

PubMed1989

[Experimental autoimmune orchitis].

Experimental autoimmune orchitis (EAO) has been extensively studied in spite of which its pathogenic mechanisms are still poorly understood. It has been mostly induced in guinea pigs using spermatozoa or a testicular homogenate plus adjuvants. Initially, the aim of our work was to establish if non-spermatic antigens, such as extracellular components of the walls of seminiferous tubules, were able to induce an autoimmune orchitis. For this purpose, we obtained from rat testes: a) a preparation rich in basement membranes of seminiferous tubules (STBM) and b) a soluble fraction of STBM, non-related to collagen (D-STBM), presenting common antigenic determinants with laminin, the main non-collagen glycoprotein of basement membranes. Fifty per cent of rats immunized with STBM, D-STBM or a murine laminin, developed a multifocal and moderate damage of the testes characterized by mild interstitial cell infiltrates, alterations of the basement membranes of seminiferous tubules and Sertoli cells, sloughing of the germinal epithelium and tubular atrophy. Circulating antibodies and a specific cellular immune response were also detected. Moreover, rats passively injected with an heterologous anti-D-STBM serum developed a similar testicular lesion and showed Ig deposits on the basement membranes of seminiferous tubules. In relation to the pathogenic mechanisms of EAO, we studied the variations of T and B cell populations, at the immunization draining lymph nodes, during the development of orchitis. A severe EAO was induced in Wistar rats by immunization with an homologous testes homogenate plus adjuvants.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Testis lesions and cellular and humoral immune responses induced in rats by immunization with laminin.

Sixty-six percent of rats immunized with laminin isolated from a mouse Engelbreth-Holm-Swarm (EHS) sarcoma developed moderate lesions in the testis characterized by multiple foci of seminiferous tubules with different degrees of sloughing of the germinal epithelium or atrophy intermingled with normal histological areas. Interstitial mononuclear cell infiltrates were seen in the epididymis. By electron microscopy, pathological changes in the basement membranes of the seminiferous tubules, such as splitting and focal thickenings of knob-like projections toward the epithelium, were observed. Moreover, Sertoli cell cytoplasm showed dilated smooth endoplasmic reticulum and large vacuoles. By electron microscopy with the immunoperoxidase technique, staining for in vivo-bound rat IgG was detected along the walls of the seminiferous tubules as a bright linear immunofluorescence and as a dense reaction product on the basal lamina. High titers of circulating antilaminin antibodies were detected by ELISA in all the rats immunized with laminin. As revealed by the skin test, a delayed type hypersensitivity reaction to laminin was observed in these rats.

Animals↗

PubMedDate not supplied

Effect of tyrosine kinase inhibitors on tyrosine phosphorylation and motility parameters in human sperm.

Tyrosine phosphorylation has recently been associated with capacitation and suggested as a regulator of sperm movement, especially characterizing hyperactivation. The objective of this study was to verify if tyrosine phosphorylation of human sperm proteins was essentially required for the maintenance of motility as well as the development of hyperactivation. Washed sperm were incubated for 6 h in Ham's F10 + 0.35% HSA at 37 degrees C in 5% CO(2), with and without the tyrosine kinase inhibitors genistein, tyrphostin, erbstatin, or herbimycin A and the wide-spectrum kinase inhibitor staurosporin. The concentrations of the inhibitors used in the experiments did not induce sperm toxicity, as measured by membrane integrity and mitochondrial function assays. Samples incubated without the inhibitors (control), increased their tyrosine kinase activity (ELISA), the number and intensity of tyrosine-phosphorylated (PY) protein bands (Western blot), the incidence of PY-immunoreactive sperm (immunofluorescence), and some of the sperm motion characteristics (CASA), such as velocity (VEL), amplitude of lateral head displacement (ALH), and hyperactivation. Among the selective protein tyrosine kinase inhibitors, genistein was the most active and consistent, inhibiting sperm tyrosine kinase activity, PY proteins, incidence of PY sperm, and sperm motility and motion parameters, such as VEL, ALH, and hyperactivation. The rest of the kinase inhibitors decreased motion characteristics to a varied extent and had different effects on phosphorylation parameters. In general, they decreased PY phosphorylation of 2 proteins (83 and 54 kDa) present in whole sperm extracts, and two sets of proteins of low (39-49 kDa) and medium (55-87 kDa) molecular weight present in the Triton X-100-solubilized sperm protein fraction. This inhibition was evident regardless of the total tyrosine kinase activity of the samples or the incidence of PY-immunoreactive sperm. The described findings further support the association between motility and protein tyrosine phosphorylation in human sperm and point to certain proteins as the main linkers.

Amino Acid Sequence↗

PubMedDate not supplied

Effect of sperm viability, plasmalemma integrity, and capacitation on patterns of expression of mannose-binding sites on human sperm.

The objective of this study was to characterize patterns of surface expression of mannose-binding sites (MBS) on human spermatozoa while evaluating the influence of sperm viability, plasma membrane integrity, and capacitation, D-Mannose binding sites were visualized by fluorescence microscopy using fluoresceinated mannose-enriched bovine serum albumin (FITC-DMA). To verify the probe specificity, 200 mM D-mannose and D-mannosylated albumin 200 micrograms/mL (DMA) were used as competitive inhibitors. Fluoresceinated bovine serum albumin (FITC-BSA) was used as control. Sperm membrane integrity was checked with a hypoosmotic swelling test (HOST) and sperm viability with Hoechst 33,258 at 1 microgram/mL. Viable spermatozoa with intact plasma membrane presented two main patterns: light bar (weak labeling of the equatorial segment) and slot (labeling of the pre- and postequatorial areas with a negative band in between). These patterns were significantly inhibited when unlabeled D-mannose or DMA were included in the medium. The percentages of spermatozoa displaying these two patterns increases significantly during capacitation. Nonviable spermatozoa with altered plasma membrane integrity presented multiple fluorescent patterns, all of which were present when FITC-BSA was used as the marker. None of them could be suppressed by unlabeled D-mannose or DMA. Viable spermatozoa displayed two main patterns which increased their incidence with capacitation and may be the only specific patterns for surface MBS. Other patterns detected in spermatozoa bearing altered plasma membranes may be due to nonspecific BSA binding or intracellular MBS recognition.

Animals↗

PubMedDate not supplied

Evaluation of poly(styrene-4-sulfonate) as a preventive agent for conception and sexually transmitted diseases.

A commercial preparation of a sodium polystyrene sulfonate (designated as N-PSS; its molecular weight is 500000 daltons) was tested as an inhibitor of sperm function and as a preventive agent for conception and the transmission of sexually transmitted diseases. The polymer is an irreversible inhibitor of hyaluronidase and acrosin; its IC50 values are 5.7 microg/mL and 0.5 microg/mL, for hyaluronidase and acrosin, respectively. N-PSS is also a stimulus of human sperm acrosomal loss. It produces maximal acrosomal loss at 2.5 microg/mL. Contraception in rabbits is nearly complete when rabbit spermatozoa are pretreated with 0.5 mg/mL of N-PSS before artificial insemination; however, N-PSS does not immobilize spermatozoa at concentrations as high as 50 mg/mL. N-PSS has broad spectrum antiviral and antibacterial activities. Infection by human immunodeficiency virus and herpes simplex virus are inhibited by N-PSS; 3-log reductions are produced by 7 microg/mL and 3 microg/mL, respectively. N-PSS is active against Chlamydia trachomatis and Neisseria gonorrhoeae. At 1 mg/mL, N-PSS inhibits chlamydial infectivity by more than 90%. N-PSS produces a 3-log reduction in gonococcal growth at 15 microg/mL. In contrast, N-PSS (5 mg/mL) does not affect the growth of Lactobacillus (normal component of the vaginal flora). N-PSS can be classified as a noncytotoxic contraceptive antimicrobial agent. These properties justify bringing a polystyrene sulfonate into clinical trials for its evaluation as a preventive agent for conception and several sexually transmitted diseases.

Animals↗

PubMedDate not supplied

Expression of mannose-binding sites on human spermatozoa and their role in sperm-zona pellucida binding.

A D-mannosylated albumin (DMA) neoglycoprotein was assessed to validate experimentally a probe capable of detecting mannose-binding sperm receptors involved in human sperm-egg interaction. DMA specifically blocked zona binding of swim-up human spermatozoa in a concentration-dependent manner. While no considerable effect was observed on sperm-zona initial contact, almost 50% of spermatozoa bound to the zona during a 2-hour period detached from it when DMA was introduced in the incubation medium. DNA inhibition was evident when 10% fetal bovine serum, but not 3.5% human serum albumin (HSA), was used as Ham's F10 medium supplementation. This may be due to the amount of free calcium in the medium since addition of 40 mM CaCl2 to F10-HSA restored DMA inhibition. Furthermore, the higher the calcium concentration in the incubation buffer, the greater the DMA blockage of sperm-zona binding. Unfixed sperm presented fluorescent DMA label over the entire acrosomal area (cap pattern), or concentrated at the equatorial segment (bar pattern). These patterns increased during capacitation, appearing on an average of 20% of the sperm after overnight incubation. They also increased, especially the bar pattern, following calcium ionophore treatment. Nearly all of methanol-fixed spermatozoa displayed the fluorescent label at the head level. Concomitant assessment of sperm membrane integrity and DMA fluorescent patterns revealed that DMA fluorescence coincided mostly with permeabilized or altered sperm plasma membrane. In conclusion, DMA is a suitable probe to identify human sperm mannose-binding sites crucially involved in sperm-zona interaction. These sites appear to require free calcium concentrations to operate, and their expression changes with capacitation and acrosome reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrosome↗