scholarly journals Glycoconjugates on the surface of epididymal spermatozoa in a marsupial, the brushtail possum, Trichosurus vulpecula

Reproduction ◽  
2001 ◽  
pp. 165-176 ◽  
Author(s):  
NJ Cooper ◽  
RV McClean ◽  
CM Leigh ◽  
WG Breed
Keyword(s):  
Fluorescein Isothiocyanate ◽  
Surface Proteins ◽  
Brushtail Possum ◽  
Trichosurus Vulpecula ◽  
Western Blots ◽  
Sperm Surface ◽  
Lectin Staining ◽  
Epididymal Spermatozoa ◽  
Cauda Epididymidis ◽  
Caput Epididymidis

Variation in localization and distribution of saccharides on the sperm surface of a marsupial, the brushtail possum, Trichosurus vulpecula, was compared between spermatozoa from the caput and cauda epididymides. Spermatozoa were subjected to the following treatments: (i) unfixed and fixed spermatozoa were stained with fluorescein-labelled lectins; (ii) unfixed spermatozoa were incubated with lectins for determination of agglutination; and (iii) spermatozoa were incubated with detergent to remove the plasmalemma, the glycoproteins were separated on SDS-PAGE and western blots were stained with biotinylated lectins. Many of the fluorescein isothiocyanate (FITC)-labelled lectins bound selectively to the sperm surface, and marked differences were found in lectin staining affinity between caput and cauda epididymal spermatozoa. Incubation of spermatozoa from the cauda epididymidis with neuraminidase reversed many of the differences in staining of the cauda epididymal spermatozoa, indicating masking of some terminal saccharides by sialic acid. Agglutination of spermatozoa from the caput epididymidis occurred after incubation with Concanavalin A (ConA) and soybean agglutinin (SBA), but agglutination was less extensive for spermatozoa from the cauda epididymidis. Western blot analysis indicated several ConA-positive bands in caput sperm extracts, but fewer positive bands in the cauda sperm extracts, whereas SBA stained four bands from caput but none from the cauda epididymal spermatozoa. These results demonstrate extensive glycosylation of the surface proteins of spermatozoa from the caput epididymidis and significant differences in spermatozoa from the cauda epididymidis. In general, the findings indicate similar glycosylation of the surface of marsupial spermatozoa to those from eutherian mammals despite marked differences in their morphology and early divergence of marsupials from eutherian mammals. It would appear that this situation differs markedly from that in sub-mammalian vertebrates.

scholarly journals Regional differentiation of the sperm surface as studied with 125I-diiodofluorescein isothiocyanate, an impermeant reagent that allows isolation of the labeled components.

1979 ◽  
Vol 82 (3) ◽  
pp. 742-754 ◽  
Author(s):  
C A Gabel ◽  
E M Eddy ◽  
B M Shapiro
Keyword(s):  
Sea Urchin ◽  
Specific Activity ◽  
High Specific Activity ◽  
Fluorescein Isothiocyanate ◽  
Surface Proteins ◽  
Regional Differentiation ◽  
Specific Cell ◽  
Sperm Surface ◽  
Erythrocyte Surface ◽  
Triton X

The regional differentiation of the sperm surface has been studied with the aid of a novel covalent labeling technique that permits concurrent cytological, biochemical, and immunological analyses. For these studies isothiocyanate derivatives of fluorescein (FITC) and diiodofluorescein (IFC) were employed: the latter can be prepared with radioiodine to high specific activity (125IFC) and is an impermeant reagent for the erythrocyte surface. Sperm of sea urchin (Strongylocentrotus purpuratus), medaka )Oryzias latipes), and golden hamster bind the fluorescent chromophores with a nonuniform distribution, most of the fluorescence being associated with the midpiece. The radioactive derivative 125IFC permits an analysis of the proteins that are responsible for most of the binding. Additionally, 125 IFC-labeled sperm are capable of fertilizing eggs, as assessed by autoradiography. That IFC labels the surface of the sperm was inferred from the following: (a) the labeling of the surfaces of other cells by fluorescein isothiocyanate and its derivatives; (b) the agglutination of labeled sperm by antibodies directed against IFC; (c) the use of peroxidase-dependent immunocytochemical reaction using anti-IFC antibodies, with analysis by electron microscopy; and (d) extraction of labeled sea urchin sperm with Triton X-100 under conditions that preferentially solubilize the plasma membrane. The antiserum directed against IFC was used to isolate the labeled surface components from Triton X-100 extracts of whole sperm, by immunoprecipitation, with Staphylococcus-A protein serving as a coprecipitant. The results support previous data showing that the sperm surface is a heterogeneous mosaic of restricted domains, one notable zone being the midpiece, where common molecular properties may be shared by sperm with distinctly different morphologies. In addition, IFC-mediated covalent alteration of specific cell surface proteins may be used to label, to identify, and, with the use of anti-IFC antibodies, to isolate such proteins from other cellular constituents.

scholarly journals β-Defensin 22 is a major component of the mouse sperm glycocalyx

Reproduction ◽  
2008 ◽  
Vol 136 (6) ◽  
pp. 753-765 ◽  
Author(s):  
Ashley I Yudin ◽  
Theodore L Tollner ◽  
Cathy A Treece ◽  
Robert Kays ◽  
Gary N Cherr ◽  
...  
Keyword(s):  
Vas Deferens ◽  
Ultrastructural Level ◽  
Protein Band ◽  
Seminal Vesicles ◽  
Major Site ◽  
Western Blots ◽  
Sperm Surface ◽  
Single Protein ◽  
Cauda Epididymidis ◽  
Purified Antigen

Surface components of sperm isolated from the cauda epididymides were stabilized by whole sperm fixation for immunization of rabbits. The resulting immunoglobulins (Igs) recognized a single protein of 130 kDa (non-reduced) or 54–57 kDa (reduced) on western blots of cauda sperm. Igs recognized the same 54–57 kDa protein band on whole tissue blots of the corpus and cauda epididymidis and vas deferens. No immunoreactive bands were detected on blots of the prostate, seminal vesicles, testes, caput epididymis, or any of various non-reproductive tissues. Removal of sperm from the vas deferens prior to blotting eliminated the detection of the sperm antigen. Antibodies raised to synthetic peptides, identical in amino acid sequence to two unique spans of DEFB22, recognized the same 130/54–57 kDa antigen on western blots of both caudal sperm and the purified antigen isolated with the anti-sperm Ig. From indirect immunofluorescence, both the anti-sperm and anti-peptide Igs appeared to localize to the entire sperm surface, a pattern confirmed at the ultrastructural level. Real-time PCR identified the corpus epididymides as the major site of expression of DEFB22, with negligible expression in the testes, caput epididymides, and vas deferens. Immunostaining of epididymal sections showed DEFB22 being released into the lumen at the distal caput/proximal corpus, with sperm becoming intensely coated with DEFB22 as they reached the distal corpus. Most uterine sperm recovered from mice 4 h following copulation exhibited DEFB22 coating the entire sperm surface. By contrast, some sperm recovered from the oviduct and cumulus extracellular matrix showed loss of DEFB22 from the sperm head.

229. Identification of transforming growth factor beta2 (TGF-β2) and its receptors TGF-βRI and TGF-βRII in the possum (Trichosurus vulpecula) prostate: evidence of seasonal changes

2008 ◽  
Vol 20 (9) ◽  
pp. 29
Author(s):  
H. Martyn ◽  
K. Pugazhenthi ◽  
B. McLeod ◽  
H. D. Nicholson
Keyword(s):  
Growth Factor ◽  
Epithelial Cells ◽  
Stromal Cells ◽  
Seasonal Changes ◽  
Transforming Growth Factor ◽  
Brushtail Possum ◽  
Trichosurus Vulpecula ◽  
Western Blots ◽  
Prostate Growth

Benign Prostatic Hyperplasia is an enlargement of the prostate affecting the ageing male population. The common Brushtail possum (Trichosurus vulpecula) has been identified as a possible model to study factors regulating prostate growth because its prostate grows and regresses seasonally. Transforming growth factor Beta 2 (TGF-β2) is present in human prostatic tissue. In vitro, TGF-β inhibits epithelial cell, but stimulates stromal cell proliferation (Mori et al. 1990). TGF-β2 binds to TGF-β receptor II (TGF-βRII), which then recruits the type 1 receptor (TGF-βRI) (Saez et al. 1998) The aim of this study was to identify any seasonal changes in expression of TGF-β2 and its receptors in the possum prostate. Six wild-caught possums were sacrificed in each of the months of January, March, May, July, September and November. The prostates were divided into a cranial and caudal region and immunohistochemistry and Western Blot analysis performed. In each animal the glandular and periurethral areas of the caudal and cranial prostates were examined separately. Immunohistochemistry identified the presence of TGF-β2 in both the stromal and epithelial cells of the glandular and periurethral areas of the cranial and caudal regions. In the cranial tissue, more immuno-positive stromal cells than epithelial cells were present, whereas in the caudal tissue immuno-reactivity was predominantly localised to the epithelial cells. Analysis of the western blots suggested that TGF-β2 expression was lowest immediately before and during the breeding season (March, May). Both TGF-βRI and TGF-βRII were identified in all regions of the prostate. Furthermore, immunohistochemistry revealed that the receptors were co-localised in the epithelial and stromal cells in all areas. TGF-β2 and its receptors are present in the possum prostate. TGF-β2 localisation varies between the caudal and cranial regions and as predicted from in vitro experiments TGF-β2 expression decreases during prostate growth. (1) Mori H. et al. (1990). The Prostate, 16, 71 - 80. (2) Saez C. et al. (1998). The Prostate, 37, 84 - 90.

scholarly journals Sperm surface proteins persist after fertilization.

1984 ◽  
Vol 99 (4) ◽  
pp. 1343-1353 ◽  
Author(s):  
G G Gundersen ◽  
B M Shapiro
Keyword(s):  
Sea Urchin ◽  
Limited Proteolysis ◽  
Fluorescein Isothiocyanate ◽  
Surface Proteins ◽  
Gastrula Stage ◽  
Sperm Surface ◽  
Sperm Proteins ◽  
Image Intensification ◽  
Sea Urchin Sperm

Certain sperm components labeled with fluorescein isothiocyanate or its radioactive derivative, 125I-diiodofluorescein isothiocyanate (125IFC), are transferred at fertilization to the egg, where they persist throughout early cleavage stages at a localized site in the embryo cytoplasm (Gabel, C. A., E. M. Eddy, and B. M. Shapiro, 1979, Cell, 18:207-215; Gundersen, G. G., C. A. Gabel, and B. M. Shapiro, 1982, Dev. Biol., 93:59-72). By using image intensification we have extended these observations in the sea urchin to the pluteus larval stage, in which greater than 60% of the embryos have localized fluorescent sperm components. Because of the unusual persistence of the sperm components in the embryo, a characterization of the nature of the labeled species in sea urchin sperm was undertaken. Approximately 10% of the 125IFC was in sperm polypeptides of Mr greater than 15,000. These proteins were on the sperm surface as shown by their sensitivity to externally added proteases. The remainder of the 125IFC in sperm was in several low-molecular-weight species, none of which was 125IFC-derivatized phospholipid. To determine if any labeled sperm polypeptides remained intact in the embryo after fertilization, 125IFC-labeled sperm proteins were recovered from one-cell and late gastrula stage embryos by using an anti-IFC immunoadsorbent. Most of the labeled sperm proteins were degraded shortly after fertilization; however, distinct sets of labeled polypeptides were recovered from both one-cell and gastrula stage embryos. Six of the labeled polypeptides recovered from both embryonic stages had identical SDS gel mobilities as labeled sperm polypeptides. Other polypeptides in the embryos appeared to arise from limited proteolysis of sperm proteins. Thus, in this physiological cell fusion system, individual sperm proteins are transferred to the egg at fertilization, and some persist intact or after specific, limited degradation long after gamete fusion, until at least the late gastrula stage.

scholarly journals Buffalo sperm surface proteome profiling reveals an intricate relationship between innate immunity and reproduction

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Vipul Batra ◽  
Vanya Bhushan ◽  
Syed Azmal Ali ◽  
Parul Sarwalia ◽  
Ankit Pal ◽  
...  
Keyword(s):  
Male Fertility ◽  
Female Reproductive Tract ◽  
Surface Proteins ◽  
Glycosylation Pattern ◽  
Sperm Surface ◽  
Beta Defensins ◽  
Surface Proteome ◽  
Related Proteins ◽  
Sperm Surface Proteins

Abstract Background Low conception rate (CR) despite insemination with morphologically normal spermatozoa is a common reproductive restraint that limits buffalo productivity. This accounts for a significant loss to the farmers and the dairy industry, especially in agriculture-based economies. The immune-related proteins on the sperm surface are known to regulate fertility by assisting the spermatozoa in their survival and performance in the female reproductive tract (FRT). Regardless of their importance, very few studies have specifically catalogued the buffalo sperm surface proteome. The study was designed to determine the identity of sperm surface proteins and to ascertain if the epididymal expressed beta-defensins (BDs), implicated in male fertility, are translated and applied onto buffalo sperm surface along with other immune-related proteins. Results The raw mass spectra data searched against an in-house generated proteome database from UniProt using Comet search engine identified more than 300 proteins on the ejaculated buffalo sperm surface which were bound either by non-covalent (ionic) interactions or by a glycosylphosphatidylinositol (GPI) anchor. The singular enrichment analysis (SEA) revealed that most of these proteins were extracellular with varied binding activities and were involved in either immune or reproductive processes. Flow cytometry using six FITC-labelled lectins confirmed the prediction of glycosylation of these proteins. Several beta-defensins (BDs), the anti-microbial peptides including the BuBD-129 and 126 were also identified amongst other buffalo sperm surface proteins. The presence of these proteins was subsequently confirmed by RT-qPCR, immunofluorescence and in vitro fertilization (IVF) experiments. Conclusions The surface of the buffalo spermatozoa is heavily glycosylated because of the epididymal secreted (glyco) proteins like BDs and the GPI-anchored proteins (GPI-APs). The glycosylation pattern of buffalo sperm-surface, however, could be perturbed in the presence of elevated salt concentration or incubation with PI-PLC. The identification of numerous BDs on the sperm surface strengthens our hypothesis that the buffalo BDs (BuBDs) assist the spermatozoa either in their survival or in performance in the FRT. Our results suggest that BuBD-129 is a sperm-surface BD that could have a role in buffalo sperm function. Further studies elucidating its exact physiological function are required to better understand its role in the regulation of male fertility.

scholarly journals Preovulatory follicle development and ovulation in the brushtail possum (Trichosurus vulpecula) monitored by repeated laparoscopy

Reproduction ◽  
1997 ◽  
Vol 110 (2) ◽  
pp. 361-370 ◽  
Author(s):  
J. L. Crawford ◽  
G. H. Shackell ◽  
E. G. Thompson ◽  
B. J. McLeod ◽  
P. R. Hurst
Keyword(s):  
Brushtail Possum ◽  
Follicle Development ◽  
Trichosurus Vulpecula ◽  
Preovulatory Follicle
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