Influence of arginine-glycine-aspartic acid (RGD), integrins (αV and α5) and osteopontin on bovine sperm–egg binding, and fertilization in vitro (original) (raw)
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The use of IVF in horses has a limited efficiency, reflecting low oocyte developmental competence and inadequate sperm capacitation procedures. In a preliminary study, using carboxyfluorescein diacetate/propidium iodide staining, we determined that the freezing-thawing procedure left only 56.6 ± 3.4 % of the sperm cells with an intact membrane. The following incubation in TALP-IVF induced membrane damage at high rates with only 9.58 ± 1.8 % of them intact after 18 h. However, the presence of at least four cumulus-enclosed oocytes (CEO) in the medium significantly increased the number of membrane-intact spermatozoa at the end of incubation (53.87 ± 1.99%). This indicated that the sperm thawing and capacitating procedures can damage the cell membrane but the presence of four or more CEO in TALP-IVF could prevent further damages. The aim of the study was to investigate in detail the membrane damages and to analyze the differences induced by the presence of CEO. Spermatozoa were thawed in water at 37 • C, and centrifuged for 30 minutes at 600g in a 45-90% Percoll gradient made with modified Tyrode's medium. The sperm pellet was washed once in the same medium and diluted to a final concentration of 1 × 10 6 spermatozoa/ml TALP supplemented with 0.6% (w/v) BSA fatty acid free and 12 µg mL −1 heparin (TALP-IVF). Sperm cells were incubated with 0 or 4 in vitro-matured CEO. Sperm cells were examined after thawing, 0, 2 and 18 h from the beginning of incubation in TALP-IVF. Each experiment was replicated at least 3 times. Both scanning and transmission electron microscopy were performed on sperm samples fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, using standard procedures. Specimens for scanning electron microscopy were examined under a field emission gun JEOL JSM 6301 microscope. For transmission electron microscopy the samples were examined with a JEOL JEM 100 SX. A minimum of 25 cells were analyzed for each group. Immediately after thawing, damaged spermatozoa showed, on the surface of their heads, small vesicles correlated to a progressive process of vacuolisation and degeneration of membrane integrity. The same lesions were visible at all the successive time points taken into account. Moreover, a loss of the acrosome integrity with acrosomal swelling and a decrease of content homogeneity were observed particularly in the spermatozoa cultured for 18 h without CEO. When CEO were present in the IVF medium lesions were visible in a lower percentage of spermatozoa but the type of lesions did not differ from those observed in their absence. These observations confirmed our previous data and gave more details on the lesions that occur during the IVF procedures in the horse. Supported by MURST COFIN grant n. 2001078849.
Expression of α6 integrin subunit in bovine oocyte and its potential role during fertilization
Advances in Bioscience and Biotechnology, 2013
Fertilization in mammals requires the successful completion of a sequence of steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion to produce a zygote. Although some integrin subunits are known to be associated with the plasma membrane of some mammalian oocytes and spermatozoa, the presence of α 6 integrin on bovine oocytes with intact zona pellucida has not been reported. The present study was undertaken to evaluate the expression of α 6 integrin subunit in bovine oocyte and to determine if in vitro binding to the zona pellucida and fertilization were affected by treating oocytes with α 6 integrin subunit antibody. The α 6 integrin subunit was identified on the bovine oocyte by immunocytochemistry. In vitro fertilization was significantly decreased when in vitro matured bovine oocytes were pre-incubated with α 6 integrin subunit antibody at concentration 5 and 20 µg/mL, and spermoocyte binding increased. These studies demonstrated the presence of α 6 integrin subunit on bovine oocyte, and its importance in fertilization and polyspermy.
Reproduction, 2009
Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm-egg interaction in human. Recently, it has been demonstrated that Fn -when present during bovine IVF -strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (a 5 b 1 ) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin a 5 on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin a 5 at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm-oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these results, the inhibitory effect of exogenously supplemented Fn seemed to be exerted on the male gamete by binding to the exposed integrin a 5 b 1 receptor after acrosome reaction. The presence of endogenous Fn underneath the ZP together with integrin a 5 expression on oolemma and acrosome-reacted (AR) sperm cell surface suggests a 'velcro' interaction between the endogenous Fn ligand and corresponding receptors on both (AR) sperm cell and oolemma, initiating sperm-egg binding.
REPRODUCTION, 2009
Fibronectin (Fn) is a 440 kDa glycoprotein assumed to participate in sperm–egg interaction in human. Recently, it has been demonstrated that Fn – when present during bovine IVF – strongly inhibits sperm penetration. The present study was conducted firstly to evaluate the expression of Fn and its integrin receptor (α5β1) on male and female bovine gametes using indirect immunofluorescence and secondly, to determine the function of Fn during bovine IVF. Endogenous Fn was detected underneath the zona pellucida (ZP) and integrin α5on the oolemma of cumulus-denuded oocytes. Bovine spermatozoa displayed integrin α5at their equatorial segment after acrosome reaction. We established that the main inhibitory effect of exogenously supplemented Fn was located at the sperm–oolemma binding, with a (concurrent) effect on fusion, and this can probably be attributed to the binding of Fn to spermatozoa at the equatorial segment, as shown by means of Alexa Fluor 488-conjugated Fn. Combining these resu...
Influence of osteopontin, casein and oviductal fluid on bovine sperm capacitation
Anim. Reprod, 2007
Sperm must undergo a process termed capacitation in the female reproductive tract before they are capable of fertilization. Previous studies strongly suggest that capacitation of sperm occurs in the oviduct. The secreted extracellular matrix phosphoprotein osteopontin (OPN) has been positively correlated to fertility in Holstein bull seminal plasma and identified in the bovine oviduct, where we hypothesized it plays a role in sperm capacitation. To determine the effect of OPN on sperm capacitation, sperm were incubated in OPN purified from bovine milk in concentrations ranging from 1 to 20 µg/ml and subjected to dual fluorescence acrosomal and viability staining. Sperm were also incubated in casein (CSN; 1 to 20 µg/ml) and isthmic non-luteal oviductal fluid (INLODF), ampullary non-luteal oviductal fluid (ANLODF) and skim milk. Flow cytometry analysis showed that OPN induced capacitation at low concentration; the largest percentage of capacitated sperm followed after incubation in INLODF. Sperm intracellular calcium levels, an indicator of sperm capacitation, were increased by OPN and INLODF, and OPN had an overall positive effect on sperm viability. There was, however, no effect of any treatment on sperm mitochondrial activity. Experiments utilizing biotinylated OPN and CSN demonstrated that CSN, but not OPN, bound to sperm. The results presented here offer the first direct evidence of the capacitating effect of OPN on bovine sperm and provide confirmation of the increasing number of roles this fascinating protein plays in reproductive biology.
Reproduction, 2007
Osteopontin (OPN) is a secreted extracellular matrix phosphoprotein identified in various tissues and fluids including those of the male and female reproductive tracts. OPN was previously identified as a 55 kDa high fertility marker in Holstein bull seminal plasma, produced by the ampulla and the vesicular gland. The objectives of this study were to characterize OPN on ejaculated and cauda epididymal sperm using immunofluorescence and western blot analysis, and to assess the role of sperm OPN in fertilization. Solubilized sperm membrane proteins from ejaculated and cauda epididymal sperm were separated by 1D SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to bovine milk OPN. A 35 kDa protein was detected by this antibody in both ejaculated and cauda epididymal sperm membranes. Analyses also recognized OPN at 55 and 25 kDa in cauda epididymal fluid and testicular parenchyma homogenates respectively. Immunofluorescent analysis of ejaculated and cauda epididymal sperm showed OPN localization in a well-defined band in the postacrosomal region of the sperm head and also on the midpiece. Results of in vitro fertilization experiments showed that sperm treated with an antibody to OPN fertilized fewer oocytes than sperm treated with control medium while increasing incidence of polyspermy, suggesting a role of sperm-associated OPN in fertilization and a block to polyspermy. These studies demonstrate that OPN exists at multiple molecular weight forms in the bull reproductive tract and its presence on ejaculated sperm may signal its importance in fertilization by interacting with integrins or other proteins on the oocyte plasma membrane. Reproduction (2007) 133 909-917 q 2007 Society for Reproduction and Fertility