1 Department of Biological Sciences, University of New Orleans, New Orleans, Louisiana 70148, 2 Audubon Institute Center for Research of Endangered Species, New Orleans, Louisiana 70131 and 3 Colorado Center for Reproductive Medicine, Englewood, Colorado 80110 USA 4 To whom correspondence should be addressed at: Audubon Center for Research of Endangered Species, 14001 River Rd, New Orleans, LA 70131, USA. e-mail: bbavister{at}acres.org
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Abstract |
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Key words: acrosome reaction/albumin/capacitation/defined culture medium/fertilization
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Introduction |
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Almost without exception, it has been necessary to include some kind of protein in the culture medium to support sperm capacitation and/or fertilizing ability. Initially, follicular fluid was used because it contains factors that maintain sperm motility in vitro as well as components that support capacitation and stimulate the acrosome reaction, which are prerequisites for sperm penetration of the zona pellucida (Yanagimachi, 1969a,b). Later, it was discovered that follicular fluid could be replaced by serum albumin (Bavister, 1969
, 1981a, 1982, 1989; Miyamoto and Chang, 1973a
,b,c; Hoppe and Whitten, 1974
; Davis, 1976
), and the latter protein source has been used universally for sperm capacitation in IVF ever since, with bovine serum albumin (BSA) being preferred for animal IVF and human serum albumin (HSA) for human IVF.
Despite these advances, the use of commercial serum albumin preparations is not without problems because these products are not pure, but contain other compounds. The concentrations and types of contaminants vary widely and range from fatty acids (Chen, 1967) to other proteins, some of which are potent activators of biological pathways (Tigyi et al., 1991
). BSA can contain up to 23 moles of fatty acids per mole of protein (Chen, 1967
), which could represent the introduction of as much as 15 µg of fatty acid per mg of BSA used in preparing the culture medium. Some contaminants co-purify with albumin during its extraction from serum (Tigyi et al., 1991
), others no doubt reflect the major physiological role of albumin as a low affinity, high capacity carrier for innumerable small molecules and as a chelator of heavy metals (Maurer, 1992
).
In view of the chemical heterogeneity of serum albumin preparations, it is not surprising that their biological activities, including their effectiveness in supporting sperm capacitation, acrosome reactions or embryo development (Batt et al., 1991; McKiernan and Bavister, 1992
), also vary considerably. As a result, in practice it is usually necessary to test several batches or lots of serum albumin in order to find one that works well in the chosen application. Although commercial serum albumin preparations are available in different forms or purities, none of them is free from all contaminating molecules; moreover, the biological activity of these preparations does not correlate with the claimed purity or grade of serum albumin. Commercial preparations of HSA may also contain deliberately introduced compounds, such as stabilizers that maintain the proteins conformation and solubility. For therapeutic use such as in human IVF, it is mandatory in the USA that HSA is heat-treated to inactivate viruses, which therefore necessitates the use of stabilizers that may affect the proteins biological activity or otherwise influence cell functions.
In addition, the chemical and biological variability of serum albumin preparations makes it difficult to elucidate how they stimulate sperm-fertilizing ability, which in turn hinders attempts to find chemically defined alternatives. It is not clear to what extent the capacitation-inducing properties of these preparations result from, or are only modified by, the various contaminating molecules. Yet another problem is that serum derivatives can be contaminated with pathogenic agents, such as viruses or the prions that cause CreuzfeldtJacob disease and possibly those responsible for bovine spongiform encephalopathy. Such pathogenic contamination of some commercial serum albumin preparations is a serious problem for human IVF, and perhaps also for animal IVF when embryos are transferred to recipients.
In view of all the drawbacks associated with serum albumin, it would be ideal if the chemically defined protein, i.e. recombinant albumin, was available for use in IVF. This would simultaneously eliminate the problems of batch variability and contamination by chemical and pathogenic agents. However, because the mechanism by which commercial serum albumin preparations act on spermatozoa is uncertain, it is not known if recombinant albumin would support sperm capacitation and acrosome reactions in vitro or not. This study was designed to test the hypothesis that recombinant human albumin (rHA) will support sperm fertilizing ability in a chemically defined culture system. The test system used was hamster IVF, because a highly reproducible protocol has been described for this species and sperm motility can be sustained at high levels in the absence of protein, which allows a negative control to be provided (Bavister, 1981a, 1982, 1989).
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Materials and methods |
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Gamete collection
Experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of New Orleans and animal care was consistent with the National Institute of Health Guide to Animal Care and Use.
Spermatozoa were obtained from excised epididymides of adult golden hamsters (Mesocricetus auratus) as previously described (Bavister, 1989). Epididymal contents were collected under mineral oil (Sigma) and held at
25°C until use (within 10 min), when they were diluted 1/1000 into 2 ml volumes of TLP-PVA that had been equilibrated with 5% CO2 in air, under oil in 30 mm Petri dishes (Falcon Plastics; Becton Dickinson, NJ, USA). Immediately before adding spermatozoa, 20 µl of a 100x stock solution of PHE (Bavister, 1989
) was added to each dish to maintain sperm motility. These sperm suspensions were incubated for 4 h at 37°C under 5% CO2 in air, before they were used to inseminate oocytes.
Ovulated oocytes were collected 16 h after hCG injection from female hamsters that had been previously injected with a single dose of 1525 IU equine chorionic gonadotrophin, depending on their body weight. Oocyte collection was begun
3060 min before the end of the 4 h sperm incubation period. The oviducts were excised and immersed in HEPES-buffered TLP medium. The swollen ampullae were then torn with forceps and the cumulus-enclosed oocyte mass was extruded. The total cumulus-enclosed oocyte mass from one female was immediately placed into a 100 µl drop of equilibrated (5% CO2 in air) TLP-PVA medium containing 1 mg/ml hyaluronidase and 1 mg/ml soybean trypsin inhibitor (Sigma) to remove cumulus cells. After a few minutes in the incubator at 37°C (5% CO2 in air), the dish was removed and the oocytes were completely denuded of remaining cumulus cells by pipetting. Oocytes were washed through a series of four 50 µl drops of equilibrated TLP-PVA, finally being placed into 98 µl drops of the same culture medium, with or without albumin for fertilization, under mineral oil in a 60 mm plastic Petri dish (Falcon Plastics). Care was taken to avoid cross-contamination of media among the different treatments; a fresh pipette was used for each transfer of oocytes into albumin-containing fertilization drops. Finally, 1 µl of PHE was added to each fertilization drop, using a fresh pipette tip for each drop.
IVF and evaluation of fertilization
After incubating the sperm suspension for 4 h, a 1 µl aliquot was used to inseminate oocytes in fertilization drops to produce a final sperm concentration of 2x104/ml (Bavister, 1974
). Spermatozoa and oocytes were co-incubated for 4 h at 37°C under 5% CO2 in air. Then, the oocytes were mostly cleaned of attached spermatozoa by pipetting and fixed in glutaraldehyde/formaldehyde (Bavister, 1989
). The oocytes were later examined microscopically using differential interference optics and were considered to be undergoing fertilization when all of the following signs were observed: two pronuclei, two polar bodies and a sperm tail within the oocyte cytoplasm (Bavister, 1981b
). These structures are all very prominent in fertilized hamster ova and can be seen without staining the oocytes.
Experimental design
The procedures described above were replicated five times across days. Spermatozoa from a single male were used for each replicate. On each experiment day, cumulus-free oocytes from each female were processed separately and then randomly assigned to one of the four treatment groups. One of the replicates used one female, two replicates used two females (two duplicate fertilization dishes) and two replicates used three females (three dishes).
Differences in fertilization frequencies were assessed using linear logistic regression where the error distribution was assumed to be binomial. The null hypothesis of no treatment effect against a treatment effect was tested using the log-likelihood ratio statistic. Differences were determined using the General Linear Models (GLIM) statistical package (Numerical Algorithms Group, Oxford, UK).
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Results |
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Discussion |
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Taken together, the data from the present study demonstrate that support of sperm capacitation and fertilizing ability during in-vitro culture of gametes is dependent on the presence of the native albumin molecule. The alternative result, which was not found, would have been the absence of fertilization in the rHA treatment; this would have indicated that other compounds present as contaminants of serum-derived albumin play an essential role in supporting sperm-fertilizing ability. Thus, we conclude from this study that the activity in albumin responsible for in-vitro sperm capacitation and the acquisition of fertilizing ability resides within the properties of the protein itself. Furthermore, our data indicate that the myriad of contaminants in commercial serum albumin preparations are not required, or responsible, for the acquisition of sperm-fertilizing ability. Neither is this activity particularly altered by the presence of such contaminants in the batches tested, because there was no difference in the results using impure BSA versus rHA.
Contaminating molecules, some of which are always present in varying amounts in different commercial serum albumin preparations, may detract from the biological activities of these preparations, rather than having an essential role in these activities. We suggest that the different levels and kinds of contaminants in these preparations might account for the wide variations in activity that are known to occur among different batches of serum albumin.
It was previously inexplicable that the ability of commercial serum albumin preparations to support sperm capacitation and fertilizing ability in vitro varied greatly among different batches (lot numbers), but much less so among preparation types of serum albumin, e.g. crystalline versus fatty-acid free BSA, or among different commercial suppliers. Neither is there a notable species-specificity of serum albumin. Serum albumin from a variety of species can be effective in supporting sperm capacitation and fertilizing ability in another species. For example, serum albumin from hamsters, human and cattle can all support IVF in the hamster (Bavister, unpublished data). The present study is consistent with the idea that the purity of serum albumin, i.e. the extent to which certain contaminating molecules are present or absent, determines the biological activity of commercial serum albumin preparations. In support of this, rHA has been successfully used in both human and mouse IVF (Gardner and Lane, unpublished data). Furthermore, rHA is an effective replacement for serum albumin in both oocyte maturation (Gardner et al., 2001) and embryo culture (Gardner and Lane, 2000
; Hooper et al., 2000
).
We therefore suggest that these results obtained using hamster IVF may be extrapolated to IVF in other species. This assumption is warranted by the universality of the requirement for albumin to support in-vitro sperm capacitation and the acrosome reaction, which has been found across all species. In one study, even when the need for serum albumin to support sperm capacitation was replaced by a chemically defined compound that mimicked some of the unusual properties of this protein, the addition of serum albumin was still required to stimulate capacitated spermatozoa to undergo functional acrosome reactions (Andrews and Bavister, 1989).
There are two important potential practical implications of the data reported here. First, the ability to use rHA for human IVF would eliminate the introduction of dangerous pathogens, which are sometimes associated with commercial serum-derived preparations, into the culture milieu. Secondly, if contaminants are responsible for reducing or counteracting the biological activity of the native protein, then using rHA could help to make IVF results more consistent. In addition to these practical considerations, the use of rHA could assist efforts to elucidate the mechanism of sperm capacitation and the acrosome reaction, in which the native albumin molecule appears to play an essential role. Such efforts are seriously undermined when commercial serum albumin preparations are used, partly because of the high variability in their biological activities, and partly because it was not previously possible to discount a potential active role for contaminating molecules in supporting sperm capacitation and fertilizing ability. For example, such preparations may contain a protein that co-purifies with serum albumin and activates the phosphatidylinositol system in oocytes (Tigyi et al., 1991). It would be very difficult to discount the activity of such bioactive contaminants when investigating the mechanism of albumin-induced sperm capacitation and/or acrosome reactions. Freed from such constraints by the use of recombinant albumin, research into the precise nature of the interactions between albumin and spermatozoa will be greatly facilitated.
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Acknowledgements |
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References |
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Submitted on May 30, 2002; accepted on September 5, 2002