Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Reexamination Certificate
1999-10-25
2001-07-10
Smith, Lynette R. F. (Department: 1645)
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
C435S007210, C435S001300, C435S806000, C435S906000
Reexamination Certificate
active
06258525
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for determining the distribution of mannose lectins on the surface of mammalian sperm cells. More specifically, this invention relates to a method of quantitating the distribution of mannose lectins on mammalian sperm cells wherein the relative number of sperm cells that are labeled in distinctive patterns on their surfaces is determined. In addition, this invention relates to a method for detecting whether or not a mammalian male patient has mannose lectin-correlated infertility, which is determined on the basis of the relative numbers of sperm cells labeled with particular labeling patterns.
This invention further relates to a method for detecting capacitation-induced changes in the distribution of mannose lectins on mammalian sperm cells. It also relates to a method for detecting whether or not a mammalian male patient has mannose lectin-correlated infertility, wherein the relative percentage of cells having particular mannose lectin labeling patterns both before and after capacitation is determined, and the comparative relative labeling in said capacitated and non-capacitated sperm cells is used as a means of determining fertility or infertility.
This invention also relates to a kit for determining the distribution of mannose lectins on mammalian sperm cells, and also to a method of screening candidate compounds for possible effects on mammalian sperm cell mannose lectin distribution and fertility.
BACKGROUND OF THE INVENTION
1. Clinically Used Methods for Detecting Sperm Fertility Problems
The incidence of male infertility in couples desiring conception has been estimated to be as great as 15% to 40% (11). Despite the great strides that have been made in understanding and treating infertility, one of the greatest difficulties has been the lack of adequate means to diagnose the existence, and potentially the cause, of the male contribution to such infertility.
Methods for evaluating male infertility are currently limited to the assessment of a few general aspects of function (7,8) and these largely depend upon determining whether the sperm meets certain descriptive criteria. The most commonly relied-upon “classical” parameters of semen analysis are sperm number, motility and morphology, and ability to penetrate the cervical mucus (14). Unfortunately, when such parameters are analyzed by different laboratories, there are substantial variations in the results obtained (12,18,19). In addition, if samples of the same patient are tested repeatedly, the results can be substantially different, and the results can also vary considerably when tests from multiple samples of known fertile sperm are compared (20,21).
Even though such analysis has its problems, human male infertility can often be correlated with specific derangements in sperm characteristics. However, there is a subset of infertile men who have normal sperm parameters, yet repeatedly fail to fertilize oocytes in vitro. The lack of a method to detect such infertility leads to much frustration and expense, as such patients can only learn about it by repeated failures in efforts to undergo in vitro fertilization (IVF).
The need to find a more reliable indicator of male infertility has been a long-felt one, and has even encouraged some to evaluate the use of extremely complex methods of analysis. For example, some investigators have attempted to measure sperm movement characteristics using computerized systems that can reconstruct how sperm move (reviewed in Ref. 8). Investigators have also tried to apply an animal model that examines the motility of hyper-activated sperm as an indicator of fertilizing ability (1). Unfortunately, no more than 24% of all human spermatozoa incubated 3 to 24 hours conform to this animal model (22).
That even sophisticated methods of observing sperm numbers or mobility do not yield suitable tests for sperm fertility is not all that surprising, since more than 80% of infertile males have sperm counts which meet or exceed the “normal standards” (20), and since fertility only requires that a small fraction of sperm be functional (22,23). Observations of the sperm as a group is therefore not likely to be accurate.
2. Cellular Events Leading to Fertilization
Efforts to understand the process of fertilization has led to the possibility that distinct molecular markers might be evaluated to determine if sperm can undergo the fertilization process properly.
Before fertilization can occur, human sperm must undergo a special pre-conditioning series of maturation steps that together are known as “capacitation”; only then can they recognize and fertilize human eggs (reviewed in 26). The stages of a sperm cell's capacitation are:
1. The sperm cell develops a vigorous nonlinear flagellar motility, which is known as “hyper-activation”;
2. The sperm cell then binds to the proteinaceous layer surrounding the egg (the zona pellucida), in a species-specific manner;
3. The sperm cell then ejects from the head portion, by the process of exocytosis, certain materials that have been accumulated into the head of the sperm into a region called the “acrosome” (this exocytosis of the acrosome is called the “acrosome reaction”); and finally,
4. The sperm fuses with the oolemma and fertilizes the egg.
Although the process of capacitation is understood morphologically, the ultrastructural and biochemical changes that are involved are only beginning to be understood (1). For example, in animals, it has been demonstrated that the expression of sperm membrane proteins which recognize specific glycoconjugates on the zona is altered during capacitation (reviewed in Ref. 2), and that interaction of these sperm proteins with zona ligands triggers the acrosome reaction (3,4). There is fragmentary evidence suggesting that analogous processes occur in man (5-7). However, no study has conclusively established the molecular nature of human sperm zona binding proteins, nor has there been a demonstration of the time course of appearance on the sperm surface of putative zona receptors.
3. Theoretical Foundation for the Invention
The basis for this invention is the hope that if sperm membrane proteins involved in capacitation could be identified and characterized, it might be possible to establish criteria that would distinguish the identity and character of such proteins in normal, fertile cells as opposed to infertile sperm cells. Such a method might also provide a better understanding of biochemical abnormalities involved in human sperm dysfunction, a subject of increasing interest to many clinicians (8,9).
Unfortunately, research directed at identifying and characterizing proteins on the human sperm surface that mediate capacitation in general, and the zona penetration step in particular, have suffered from the limited availability of human oocytes. Recently, however, Mori and co-workers examined the role of monosaccharides in human fertilization (10). Utilizing sugar competition in human sperm-zona binding/penetration experiments, they identified mannose as a saccharide critical for human zona recognition. Sperm binding and penetration occurred when zonae pellucidae were pre-treated with D-mannose. In contrast, when human oocytes were pre-treated with Concanavalin A, a mannose-binding lectin, no human sperm bound to or penetrated the zona. Pretreatment of sperm and co-incubation of sperm and zonae with D-mannose markedly reduced zona binding and completely inhibited zona penetration. The inhibition of sperm binding by D-mannose pretreatment was considerably stronger than that observed with any other monosaccharide or complex sugar tested.
4. Prior Methods Using Mannose Lectin Labeling
Other investigators have attempted to apply these observations to the study of male infertility, but their results have not been clinically useful. In one such effort, Tesarik and co-workers (7) attempted to surface label sperm with bovine serum albumin (BSA) that had been coated with mannose and labeled with a fluorescent tag, fluorescein isothiocyanate (FITC), in order to distinguish ferti
Kenyon & Kenyon
North Shore University Hospital
Portner Ginny Allen
Smith Lynette R. F.
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