WO1995001799A1

WO1995001799A1 - Compositions and method for modulating fertility

Info

Publication number: WO1995001799A1
Application number: PCT/US1994/007510
Authority: WO
Inventors: Nadav Zamir; Michael Eisenbach; Zvi Naor
Original assignee: Ramot University Authority For Applied Research And Industrial Development Ltd.; Yeda Research And Development Company Limited; Shoshan, Herbert, Z.
Priority date: 1993-07-06
Filing date: 1994-07-06
Publication date: 1995-01-19
Also published as: IL106246A0; AU7396994A

Abstract

A method and pharmaceutical composition for modulating sperm cell fertility. The modulating can be enhancement of sperm cells' fertility by contacting them with atrial natriuretic peptide (ANP), an agonist thereof, cGMP or a permeable analogue thereof or agents which raise the level of cGMP in cell. The modulation can be inhibition of sperm cell fertility and in this case the sperm cells are contacted with an antagonist of ANP or an agent which lowers the levels of, or inhibits formation of cGMP in cells.

Description

DESCRIPTION COMPOSITIONS AND METHOD FOR MODULATING FERTILITY

FIELD OF THE INVENTION

The invention is generally in the field of fertility, and more particularly is concerned with composition methods for the manipulation of movement, direction and acrosome reaction of the sperm cell (spermatozoa).

BACKGROUND OF THE INVENTION

Infertility is a problem afflicting approximately 10% of all couples. Of these, about 40-50% are related to female factors, about 40% to male factors, while the remaining 5-20% remain unexplained. While an ever growing number of means for the treatment of female sterility are available, means for the treatment of male sterility or of sterility caused by defects in the sperm-egg interactions are quite limited.

It has been estimated that the motility of spermatozoa is an important factor in determining its fertility properties even more than the ejaculate volume or the number of spermatozoa per unit volume of the ejaculate (Motility Parameters and Fertility, R.J. Aitken, In "Controls of Sperm Motility, Biological and Chemical Aspects", ed: Claud Gafnon, CRC PRES, BOCARATOR, 1990). In addition to the degree of motility per se of the spermatozoa, other dominant motility related factors which have an effect on the sperm cell's fertility include speed of travel, direction of travel, manner of move¬ ment, ability undergo an acrosome reaction and kinetics of this reaction. In the following the term "chemokinesis" will be used to denote a response in which the steady state speed of a cell is governed by the chemical concentration; the term "chemotaxis" will be used to denote the response of motile cells to a gradient of chemical stimuli, resulting in modulation of the direction of travel so as to approach an attractant or to get away from a repellant; and the term "acrosome reaction" will be used to denote the exocytotic process of fusion and vesiculation of the outer acrosomal membrane with the plasma membrane of the spermatozoa (Gregory S. Kopf and George L. Gerton, The Mammalian Sperm Acrosome membrane and the Acrosome Reaction. Elements of Mamma - lian Fertilization, Nol. I: 155-203, 1992, CRC Press, Bocarator, 1992).

Sperm cell chemotaxis is known to occur in species with external fertilization, e.g. metazoa, whose female spawn their eggs into sea water before fertilization. Contrary to the striking necessity of chemotaxis in cases of external fertilization, the necessity of the involvement of spermatozoa chemotaxis in mammalian fertilization is not self-evident. It was assumed that since a very large number of spermatozoa (about 10⁷ to 10⁹) are ejaculated directly into the female reproductive tract, a sufficient number of spermatozoa will reach the egg simply by coincidence with no need for a mechanism of chemotaxis. However, recently several experimental results indicate that sperm chemotaxis is also present in mammals.

It has been found that small synthetic Ν-formylmethyl peptides can serve as attractants in a receptor-mediated chemotaxis of human spermatozoa (Gnessi et al, Exp. Cell Res., 161, 219-230 (1985)). However, the physiological significance of spermatozoa chemotaxis in response to N-formyl peptides, which are known to promote leukocyte accumulation at damaged tissue sites, is currently unclear. In addition, it has recently been found that human spermatozoa are attracted to an unknown factor or factors in the follicular fluid which contains factor(s) released by the egg or its surrounding cells (Rait et al, Proc. Natl. Acad. Sci. USA 88, 2840-2844 (1991)).

The ability of the spermatozoa to fertilize the oocyte depends on its motility behavior, i.e. its ability to reach the oocyte, which largely depends on the spermatozoa's chemotaxis and chemokinetic behavior, as well as on its acrosome reaction. Impairment of any of these functions may lead to failure in the fertilization process. Accordingly, it would have been desired to have agents which could improve the spermatozoa's motility behavior as well as enhance the acrosome reaction. Such agents could be useful for increasing the fertilization success rate in both artificial inse ina- tion as well as in vitro fertilization (INF).

Atrial natriuretic peptide (AΝP) is known to be involved in the control of volume homeostasis in humans and to possess natriuretic, diuretic and vasorelaxant activities, (Brenner B.M. et al., Adverse Biological Action of Atrial Natriuretic Peptides: Physi. Rev., 70, 665-699, (1990)). AΝP also exerts endocrine function including the modulation of steroid synthesis in a variety of steroidogenic tissues. Immunoreactive AΝP as well as AΝP receptors have been identified in the corpus luteum. Detectable AΝP immunoreactivity was found in ovarian follicular fluid and a role for AΝP in ovarian steroidogenesis, follicular volume regulation and oocyte develop- ments has been hypothesized (Steegers et al, Gynecol. Obstet. Invest, 29, 185-187, (1990)).

Quite recently it has been found that human spermatozoa contain AΝP receptors but the physiological role of these receptors has never been determined (Silverstroni, L. et al, Archives of Andrology, 28: 75-82 (1992).

SUMMARY OF THE INVENTION It has been surprisingly found, in accordance with the present invention, that ANP has an attracting chemotaxis effect on spermatozoa as well as a chemokinetic effect in increasing the speed of movement of the spermatozoa. In addition, it has been found, in accordance with the present invention, that ANP enhances the acrosome reaction of human spermatozoa after capacitation. ANP thus appears to influence a whole gamut of spermatozoa-related functions which play a key role in the fertilization process and accordingly may be very useful in modulating the fertilization process, i.e. increasing the fertilization rate. The ANP receptor in other cellular systems is known to increase the activity of particulate guanylate cyclase whereby the cGMP level inside the cell increases. It was found in accordance with the invention that a permeable analog of cGMP has a similar effect to that of ANP on spermatozoa. Thus, permeable analogs of cGMP or substances which are capable of stimulating the particulate guanylate cyclase, e.g. ANP analogs, and substances which are capable, by another mechanism, of increasing the level of cGMP inside cells, are also useful, in accordance with the invention, in facilitating fertilization.

It has further been found that ANP-antagonists block the ANP- induced acrosome reaction. Thus, the present invention also opens the way for new contraceptive agents which function by blocking ANP-activated responses in spermatozoa. These contraceptive agents may be ANP antagonists or substances which inhibit cGMP formation.

The present invention provides by one of its aspects a method for modulating sperm fertility comprising contacting sperm with an effective amount of an active agent being a compound selected by the group consisting of: (i) ANP;

(ii) an ANP agonist which can bind to and activate the ANP respon- sive receptor;

(iii) cGMP or a membrane permeable analogue thereof; (iv) agents that induce a rise in the cGMP levels in cells; (v) a combination of two or more members of groups (i) to (iv); (vi) an antagonist of ANP; (vii) an agent which lowers the level of cGMP in cells or inhibiting its formation; and (viii) a combination of members of groups (vi) and (vii).

The present invention further provides use of an active agent being a compound selected by the group consisting of: (i) ANP;

(ii) an ANP agonist which can bind to and activate the ANP respon¬ sive receptor; (iii) cGMP or a membrane permeable analogue thereof; (iv) agents that induce a rise in the cGMP levels in cells; (v) a combination of two or more members of groups (i) to (iv);

(vi) an antagonist of ANP; (vii) an agent which lowers the level of cGMP in cells or inhibiting its formation; and (viii) a combination of members of groups (vi) and (vii); for the preparation of a pharmaceutical composition for modulating sperm fertility. Thus, the present invention still further provides a composition for modulating fertility, comprising a pharmaceutically acceptable carrier and an active agent selected from the group consisting of: (i) ANP; (ii) an ANP agonist which can bind and activate the ANP responsive receptor; (iii) cGMP or a membrane permeable analogue thereof; (iv) agents that induce a rise in the cGMP levels in cells; (v) a combination of two or more members of groups (i) to (iv)

(vi) an antagonist of ANP; (vii) an agent which lowers the level of cGMP in cells or inhibits its formation; and (viii) a combination of members of groups (vi) and (vii). In accordance with a further aspect of the present invention, there is provided use of said active agents (i) to (v) in the preparation of a composition for enhancement of fertility for either INF or artificial insemination application; or use of said active agents (vi) to (viii) for the preparation of a contraceptive composition. In accordance with one embodiment of the present invention, to be referred to herein as the "facilitatoiy embodiment", the composition or the method are used for enhancing fertility. This comprises use of any of the active agents of groups (i) - (iv) or any combination thereof.

The active agent in the compositions in accordance with the facilitatory embodiment of the present invention may be purified native AΝP or synthetically prepared AΝP. In addition, various agonists of AΝP which can bind and activate the AΝP receptors, may also be used, which include, for example: derivatives of AΝP, being AΝP in which one or more amino acids has been added, replaced, deleted or chemically modified; fragments of AΝP, e.g. AΝP₅_₂₈ or human AΝP _^, or derivatives of such fragments in which one or more amino acids has been added, replaced, deleted or chemically modified; various known agonists of AΝP such as brain natriuretic peptide (BΝP), C-type natriuretic peptide (CΝP), CΝP-22, CΝP- 53, Atriopeptin III as well as various active fragments of these proteins or _ _

peptides and derivatives thereof in which one or more amino acids has been added, replaced, deleted or chemically modified. The choice of the respective agonists for use in accordance with the invention is no doubt within the reach of the artisan. For example, such fragments or derivatives may be chosen by testing their activity in an assay system comprising a cell with ANP receptors on its surface. The activity in such a system may be determined, for example, by testing the change in the level of cGMP in the cell in response to the tested agonist.

As binding of ANP to its receptor (particulate guanylate cyclase) is known to trigger the formation of the cGMP, the active agent in accordance with the facilitatory embodiment, can be a compound which brings about formation of cGMP in cells either by stimulating the particulate guanylate cyclase such as the abovementioned agonists of ANP or by stimulating the soluble guanylate cyclase such as nitrous oxide (NO) or nitroprusside. The active agent, in accordance with this embodiment, can also be a substance which induces a rise in cGMP level by inhibiting its degradation, e.g. a phosphodiesterases inhibitor; in addition said active agent can also be a membrane permeable analog of cGMP such as 8- bromo-cGMP, dibutyryl cGMP, etc. It has been found in accordance with the invention that ANP has both a chemotactic and a chemokinetic effect on spermatozoa. The combined chemotactic and chemokinetic effect is the enhancement of the swimming activity of spermatozoa towards the oocyte. For the sake of convenience, the combined positive effect of ANP on the spermatozoa's chemotaxis and chemokinesis will be referred to at times as "improvement in motility properties".

One application of the active agent in accordance with the facilitatory embodiment of the invention would be in improving the motility properties of spermatozoa, particularly of low quality spermatozoa having defective motility properties. An additional application of the facilitatory embodiment would be in the improvement of the motility properties of thawed spermatozoa which were previously stored frozen in a sperm bank. Particularly preferred applications of the facilitatory embodiment of the present invention are in artificial insemination of both humans and domestic animals as well as in in vitro fertilization (INF). In artificial insemination, the active agent of the present invention can be mixed with the sperm to be injected into the female reproductive tract. In artificial insemination of domestic animals this may potentially increase the rate of conception, which for example, in milking herds, is of a high commercial significance. In IVF, the active agents should be placed in proximity to the oocyte, whereby it facilitates fertilization by either one or more of the following mechanisms: attracting the spermatozoa to the oocyte; improve¬ ment in motility properties of the spermatozoa; and enhancing the acrosome reaction after capacitation.

Compositions in accordance with the facilitatory embodiment of the present invention in which said active agent is a peptide and particularly such intended to be used in INF, preferably comprise also an anti-endopep- tidase. Peptidases present around the oocyte may decrease the effective concentration of the AΝP around the oocyte and thus reduce its effect. The inclusion of an anti-peptidase in the composition of the present invention will thus enhance the effect of AΝP in increasing fertility. It has been found in accordance with the invention that in compositions comprising only AΝP, the concentration thereof should be at least 10^"5M in order to achieve an effect in increasing sperm cells' mobility or to enhance acrosome reaction; where the composition comprises also an anti-peptidase an AΝP effect is observed in concentrations as low as lO^M. An example of such an anti-peptidase is phosphoramidon. In accordance with another embodiment of the present invention, to be referred to herein as the "inhibitory embodiment", an active agent or combination of agents of any of the above groups (vi) to (viii) is used for contraception. An example of an ANP antagonist is HS-142-1 (Imura et al., Molecular Pharmacology, 42, 982-990 (1992)); anantin which is an antagonist to the ANP receptor (Weber et al., J. Antibiotics, 44, 164-171 (1991); Wyss et al., J. Antibiotics, 44, 172-180 (1991)); and the antagonist A71915 manufactured by Abbot Park, II Trachte, J. of Pharmacol. Experimental Therapeutics, 264(3), 1227-1233 (1993)). In accordance with the inhibitory embodiment, any of these active agents may suitably be combined with a contraceptive device, such as a condom, diaphragm or an IUD.

In accordance with another aspect of the present invention, there is provided an assay for determining the fertilization quality of either the oocyte or the spermatozoa. The fertilization qualities of the oocyte, in the context of the present invention, can be determined by measuring the ANP level in the follicular fluid by any means known per se. The fertilization properties of the spermatozoa can be assessed by measuring the spermatozoa's reaction to ANP, e.g. by determining motility factors, for example, by the capillary assay or by measuring the change in the intracel- lular cGMP levels in response to ANP application.

In accordance with a further aspect of the present invention, there is provided a method for enriching sperm samples with high quality spermatozoa. Sperm samples used in either INF or artificial insemination, contain both high and low quality spermatozoa as regards their motility properties. In accordance with this aspect of the invention, a sperm sample can be enriched by selecting spermatozoa on the basis of their migration along an AΝP gradient. In accordance with such a sperm enriching method, the spermatozoa are allowed to migrate for a certain time in an AΝP gradient and only those sperm cells which migrated more than a predeter¬ mined distance are then selected in order to obtain a sperm sample containing the most motile sperms and thus only high quality spermatozoa.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the density of sperm cells as a function of time in various regions of a circular test chamber. A schematic representation of the test chamber is shown in the insert in each of Figs. 1(A) - 1(D); the shaded area in the insert shows the region where the density of a sperm cell was measured. In Figs. 1(A) -1(D), the filled circles (•) are results of measure¬ ments made closer to the well filled with ANP, and the empty circles (o) are results from measurements made closer to a buffer containing well (without ANP);

Fig. 2 shows the results of a capillary assay with the well containing either buffer or buffer supplemented with ANP. Grey column - capillary filled with buffer; black column - capillary filled with an ANP solution; Fig. 3 shows the acrosome reaction of human spermatozoa with a control solution and with various concentrations of ANP;

Fig. 4 shows the acrosome reaction of bull spermatozoa in a control solution and with various concentrations of ANP;

Fig. 5 shows the acrosome reaction of human spermatozoa with a control solution and with two concentrations of 8-brono cCMP;

Fig. 6 shows the acrosome reaction of bull spermatozoa with 8-bromo cGMP; Fig. 7 shows the effect of the ANP-antagonist anantin on the acrosome reaction; and

Fig. 8 shows the effect of ANP on cGMP accumulation in bull spermatozoa. DETAILED DESCRIPTION OF THE INVENTION Experimental Procedures:

I. (a) Preparation of human sperm cells

Human ejaculates were collected by masturbation from normal healthy donors. Each ejaculate was allowed to liquify at room temperature and was then washed twice (using centrifugation at 120 g for 30 min) with biggers, Whitten and Whittingham medium (Briggers et al, "The Culture of Mouse Embryos in vitro", In: Daniel J.P. (ed) Methods in Mammalian Embryolo¬ gy, Freeman, San Francisco 86-116 (1971)) supplemented with HEPES (10 mM, pH 7.4) and 0.1% polyvinylpyrrolidone 40 (M_r 40,000 Sigma) to reduce sperm adsorption. (This solution will be denoted below as "buffer"; when supplemented with ANP it will be denoted as "ANP buffer"). The sperm cells were resuspended in this buffer to a concentration of l-2xl0⁷ cells/ml (for capillary assays) or l-5xl0⁸ cells/ml (for microscopic assays) and incubated for 2 hours at 37°C under 5% C0₂. (b) Preparation of bull sperm cells

Frozen bull sperm cells were thawed and diluted at 39°C in a medium comprising 150 mM NaCL, 10 mM histidine pH 7.4, were washed by three centrifugations at 780 xg for 10 mins. Samples with a minimum of 70% motile spermatozoa were used for experimentation. Capacitation was accomplished as follows:

Sperm pellets were resuspended to reach a final concentration of 10^s cells/ml in glucose free TALP (100 mM NaCl, 3.1 mM₈ KC1, 25 M NaHC0₃, 0.29 mM KH₂P0₄, 21.6 mM lactic acid, 1.5 mM MgCl₂, 0.1 mM pyruvate, 50 μg/ml bovine serum albumin, 10 mM HEPES, pH 7.4), containing 20 μg/ml heparin. The cells were incubated in capacitation medium for 4 hours at 39°C with shaking. //. Microscopic assay

Microscopic assays were carried out in sealed chambers having a depth of lOμm, thus confining the ability of the sperm cells to swim in only two dimensions within the focal plane of a microscope throughout the observa- tion period. The behavior of the sperm cells chamber was recorded on video and then analyzed (Rait et al, supra). The video recording com¬ menced within 1 min. after the chamber had been sealed and continued for 15 min.

III. Capillary assay

Capillary assays were carried out in a system consisting of a series of Teflon wells and polyethylene tubes (Intramedic, Parsippany, N.J., PE-50, I.D. 0.058 mm), denoted below as "capillaries" (Rait et al, supra). The wells were filled with 100 μ\ of sperm cells at a concentration of l-2xl0⁷ cells/ml suspended in buffer or in ANP buffer. The capillaries were filled with either the buffer or the ANP buffer and sealed at one end with a clamp. The open side of each capillary was inserted into the well and the capillaries were incubated for a period of 20-30 min. in a 37°C incubator under 5% CO,. At the end of the incubation period the total content of the capillary was transferred into an Eppendorf tube containing 3 μ\ of glutaraldehyde in water and the average concentration of the spermatozoa in each capillary was determined by direct counting under the microscope by a hemocytome- ter.

IV. Endopeptidase assay

For measuring the activity of neutral endopeptidase (EC 3.4.24.11), 10 μ\ of 10-fold diluted sperm suspension were transferred into a 96 wells microplate, to which 200 μ\ of the endopeptidase substrate was added. Neutral endopeptidase substrate (3-carboxypropanoyl-alanyl-alanyl- leucine 4-nitroanalide) at a concentration of 0.4 mM was prepared in a buffer solution containing Tris-HCl (50 mM), Nacl (100 mM) and CaCl₂ (1 mM), pH 7.5, supplemented with Streptomyces griseus aminopeptidase (6-7 g/ml) (Indig. F.E., et al, FEBS Lett., 255, 237-240 (1989)). The plate was incubated overnight at 35°C and the absorbance at 405 nm (due to the release of 4-nitroaniline) was monitored by a bio-rad microplate reader (model 450).

V. (a) Acrosome reaction - Experimental procedure by triple -stain technique

To sperm cells prepared as described above were added to varying concentrations of ANP together with 1.7 μM of phosphoramidin and the acrosome reaction was determined. The percentage of cells that underwent acrosome reaction was determined by the Triple-stain technique, described in detail in Talbot P, et al, The Journal of Experimental Zoology, 215: 201-208 (1981).

(b) Acrosome reaction experimental procedure - by measurement of acrosin release

Occurrence of acrosome reaction can also be determined by measuring the activity of released acrosin in the supernatent as described in

Lax et al, FEBS LETTERS, 339, 234-238 (1994) and Breitbart et al,

Biochem. J., 281, 471-476 (1992). It has been verified, by morphological procedures that the release of acrosin does indeed reflect the physiological acrosome reaction (Cross et al, Genetic Research, 15, 213-226 (1986);

Mendoza et al, J. Reprod. Fert., 95, 755-763 (1992)).

VI. Cyclic GMP measurement After capacitation, the sperm cells (10^s cells/tube) were further incubated for 10 min. at 39°C with shaking in the presence of ANP, or combination of ANP and anantin. ANP-induced acrosome reaction was stopped by boiling the samples for 10 mins. then samples were sonicated, centrifuged (10,000 xg, 10 min. at 4°C) and supernatant fluids were collected and dried down in Speed Vac. cGMP was acetylated. Anti cGMP serum, (DK 763/092889,

1:3000 final dilution), iodinated cyclic GMP, standards and samples were mixed with 50 mM sodium acetate buffer pH 6.0. After incubation for 18- 24 hours a6 4°C the free iodine precipitated by adding dextran-coated charcoal. Tubes were centriguged (2000 xg for 20 mins. at 4°C) and radioactivity in supernatant fluid was counted. Each sample was analyzed in triplicate. The cross reactivity of the anti cGMP serum for cAMP was 0.8%. Intra assay variability was 4-6% and interassay variability was 10%.

VII. Data Analysis The statistical significance of the results in the capillary assays, expressed as the mean ± S.E.M., was calculated by one way analysis of variance (ANON A) followed by Fisher test.

Results I. AΝP effects on sperm cells motile behavior

In order to evaluate the effect of AΝP on sperm cells' motile activity, the response of the cells to a spatial gradient of AΝP was monitored in a special constructed sealed chamber (Maker et al., Fertil Steril 57, 1066-1074 (1992)). A circular chamber was constructed which contained four wells: the top and bottom wells were filled with sperm cells, the left well was filled with buffer and the right well with 10^"5M AΝP diluted in buffer. Thus, a gradient of AΝP was established by diffusion between the left and right wells. The behavior of sperm cells in response to this gradient was recorded on video and then analyzed. The results are shown in Fig. 1. Fig. 1A to Fig. ID each show the distribution of sperm cells in another region of the test chamber. Fig. 1A in the region close to the wells (• - near the ANP containing well; and o - near the buffer (without ANP) containing well), Fig 1(B), 1(C) and 1(D) in the regions of increased distance from the well and closer to the center of the test chamber, respectively. (The shaded areas in the Fig. indicate the zones in which the measurement was made).

The result clearly show that the sperm cell have a preferential migration tendency towards the ANP source as can be seen from the comparison of the filled circles and the empty circles.

As can be seen in Fig. 1(A), and to a lesser extent in Fig. 1(B), there was a much higher cell count in regions with higher ANP concentra¬ tion, which are close to the ANP chamber, than in regions with a low ANP concentration, close to the buffer (without ANP) chamber. Against this, in regions close to the center Fig. 1(D) and to a lesser extent Fig. 1(C), which did not differ significantly in their ANP concentration, there was no significant difference in cell count.

In order to gain further understanding on the nature of the ANP effect on spermatozoa, the spermatozoa's migration in the capillary assay was determined under four different combinations of solutions in the well and in the capillary, and the results are shown in Fig. 2:

Combination 1 - ANP buffer in the well and buffer in the capillary (column 1);

Combination 2 - ANP buffer in the well and ANP buffer in the capillary (column 2);

Combination 3 - buffer in the well and buffer in the capillary (column

3);

Combination 4 - buffer in the well and ANP buffer in the capillary (column 4). In the presence of a descending gradient of ANP (combination 1), the number of sperm cells that migrated down the gradient into the capillary was significantly (P<0.01) lower than in the absence of a gradient (combination 2). However, in the presence of an ascending gradient of ANP (combination 4), the number of sperm cells that migrated up the gradient into the capillary was significantly (P<0.01) higher than in the absence of a gradient (combination 3). This dependence of the sperm migration on the ANP gradient is indication of a chemotactic effect. Furthermore, in the absence of a gradient, when both the well and the capillary contained the same ANP solution (combination 2), the number of sperm cells that migrated into the capillary was significantly (P<0.01) higher than the number that migrated when both the well and the capillary contained just buffer (combination 3) which is indicative of a chemokinetic effect of ANP.

II. Effect of inhibition of endopeptidase activity

The capillary assay was carried out as described above where each well contained 1.5xl0⁷ sperm cells/ml. ANP at a concentration of 10^"9 was added either to the well only (combination 1) or to both the well and the capillary (combination 2) either in the presence (+) or absence (-) of phosphramide 1.7 μM which is an inhibitor of endopeptidase activity. The number of sperm cells which accumulated in the capillary as a result of migration from the well was determined by ANONA followed by a statistical Fisher test and the results are shown in Table 1.

Table 1

Phosphoramidon effects on sperm attraction to ANP

Combination ANP in Sperm concentration (xlO^-4) in capillary well capillary -phosphoramidon +phosphoramidon

1 + 156+22 76±20

2 + + 129+15 198+6

As can be seen in the presence of phophoramiden, the differences between combinations no. 1 and no. 2 were more significant (P<0.01). Thus, it is clear that in the presence of an endopeptidase inhibitor ANP induced chemotaxis and chemokinesis could be detected at concentrations as low as 10^"9 and 10^"8M.

II. (a) ANP effects on human sperm cells' acrosome reaction

As can be seen in Fig. 3, increasing the concentration of ANP from none (control) to 10^"11, 10^"10 and 10^"9 increased the percentage of cells that have undergone an acrosome reaction tested by the triple-stain technique. However, an additional increase in the ANP concentration to 10^" ⁸M decreased the percentage of cells that have undergone acrosome reaction, which results signify a "bell-shaped" response of the cells to varying concentration of ANP showing an optimal concentration for response. (b) ANP effects of bull sperm cells' acrosome reaction

As can be seen in Fig. 4, increasing the concentration of ANP from none (control) to 1 and 3 mM increased the percentage of cells that have undergone an acrosome reaction tested by acrosin release (BAEE stands for the substrate of the acrosin enzyme benzoylarginin ethyl ester). As in human sperm cell, additional increase in the ANP concentration decreases the acrosome reaction signifying a "bell-shaped" response. III. (a) 8-bromo cGMP effects on human sperm cells' acrosome reaction

The acrosome reaction was also determined with varying concentration of 8-bromo cGMP which is a permeable analogue of GMP.

The results are shown in Fig. 5. The concentration of ImM 8-bromo cGMP was most effective in enhancing the acrosome reaction tested by the triple stain technique, while a further increase to 3mM decreased the acrosome reaction. From the above results it is clear that there is an optimal concentration of ANP or one of its effector, 8-bromo-cGMP which significantly raises the percentage of cells that underwent acrosome reaction.

(b) 8-bromo cGMP effects on bull cells' acrosome reaction

The acrosome reaction was determined with 1 mm of 8-bromo cGMP, by measuring acrosin release. As can be seen in Fig. 6, 1 mM 8-bromo cGMP was able to raise the acrosin release by about 6 fold.

IV Effect of ANP-antagonist anantin on acrosome reaction in bull spermatozoa

Acrosome reaction was determined, by measuring acrosin release in four preparations: control solution; solution of ANP 10^"8 M; solution of ANP 10^"8 M and anantin 10^"7 M; and solution of anantin 10^"7 M. As can be seen in Fig. 7, anantin was able to reduce the ANP-induced acrosome reaction back to control levels. These results indicate the ANP-antagonists are able to block acrosome reaction and thus can be used effectively as contraceptives.

V. Effect of ANP on cGMP accumulation in bull spermatozoa The x-fold stimulation of cGMP was measured in response to four solutions: control solution (which served as a base line against which changes of cGMP were measured and therefore in the control x fold = 1); ANP^"8 M; ANP 10^"8 M and anantin 10^"7; and anantin 10^"7 M.

As can be seen in Fig. 8, ANP at a concentration of 10^"8 M was able to increase cGMP levels about 2 fold. The ANP-antagonist anantin was able to decreaes cGMP levels both solely and together with 10^"8 M ANP about 0.5 and 0.75 fold respectively. Thus, it seems that anantin which is an antagonist of the ANP receptor, particulate guanylate cyclase, is able to inhibit formation of even basal levels of cGMP, and thus decrease the levels of cGMP produced even in the absence of ANP.

Claims

CLAIMS:

1. A method for modulating sperm fertility comprising contacting sperm with an effective amount of an active agent being a compound selected by the group consisting of: (i) ANP;

(ii) an ANP agonist which can bind to and activate the ANP respon¬ sive receptor; (iii) cGMP or a membrane permeable analogue thereof; (iv) agents that induce a rise in the cGMP levels in cells;

(v) a combination of two or more members of groups (i) to (iv); (vi) an antagonist of ANP;

(vii) an agent which lowers the level of cGMP in cells or inhibiting its formation; and (viii) a combination of members of groups (vi) and (vii).

2. A method according to Claim 1 for enhancing sperm fertility by contacting sperm with an effective amount of an agent of any one of groups

(i) " (v).

3. A method according to Claim 2, wherein the ANP agonist is a member selected from the group consisting of ANP in which one or more amino acids has been added, replaced, deleted or chemically modified which can bind and activate ANP receptors in spermatozoa's membranes and fragments of ANP which can bind and activate the ANP receptors.

4. A method according to Claim 3, wherein said agonist is ANPs.^ or ANP₄_₂₈.

5. A method according to Claim 2, wherein the ANP agonist is a member selected from the group consisting of brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), CNP-22, CNP-53, Atriopeptin III, and active fragments and derivatives of these proteins or peptides which can bind and activate ANP receptors in spermatozoa's membranes.

6. A method according to Claim 2, wherein the active agent further comprises an anti-endopeptidase.

7. A method according to Claim 2, wherein the cGMP membrane permeable analogue is 8-bromo-cGMP or dibutyryl cGMP.

8. A method according to Claim 2, wherein the agents which induce rise in the cGMP levels are selected from the group of nitrous oxide (NO), nitroprusside and inhibitors of phosphodiesterases.

9. Use of an active agent being a compound selected by the group consisting of: (i) ANP;

(ii) an ANP agonist which can bind to and activate the ANP respon¬ sive receptor; (iii) cGMP or a membrane permeable analogue thereof;

(iv) agents that induce a rise in the cGMP levels in cells; (v) a combination of two or more members of groups (i) to (iv); (vi) an antagonist of ANP;

(vii) an agent which lowers the level of cGMP in cells or inhibiting its formation; and

(viii) a combination of members of groups (vi) and (vii); for the preparation of a pharmaceutical composition for modulating sperm fertility.

10. Use according to Claim 1, wherein the active agent is of any of groups (i) - (v) for the preparation of a pharmaceutical composition for enhancing sperm fertility.

11. Use according to Claim 10, wherein the ANP agonist is a member selected from the group consisting of ANP in which one or more amino acids has been added, replaced, deleted or chemically modified which can bind and activate ANP receptors in spermatozoa's membranes and fragments of ANP which can bind and activate the ANP receptors.

12. Use according to Claim 11, wherein said agonist is ANP₅__M or

ANP₄.₂₈.

13. Use according to Claim 10, wherein the ANP agonist is a member selected from the group consisting of brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), CNP-22, CNP-53, Atriopeptin III, and active fragments and derivatives of these proteins or peptides which can bind and activate ANP receptors in spermatozoa's membranes.

14. Use according to Claim 10, wherein the active agent further comprises an anti-endopeptidase.

15. Use according to Claim 10, wherein the c-GMP permeable analogue is 8-bromocGMP or dibutyryl cGMP.

16. Use of an agent according to Claim 10, wherein the agents which induce rise in the cGMP levels are selected from the group of nitrous oxide

(NO), nitroprusside and inhibitors of phosphodiesterases.

17. A pharmaceutical composition for modulating sperm fertility comprising a pharmaceutically acceptable carrier and an active agent being a compound selected by the group consisting of: (i) ANP;

(ii) an ANP agonist which can bind and activate the ANP responsive receptor; (iii) cGMP or a membrane permeable analogue thereof; (iv) agents that induce a rise in the cGMP levels in cells; (v) a combination of two or more members of groups (i) to (iv);

(vi) an antagonist of ANP; (vii) an agent which lowers the level of cGMP in cells or inhibiting its formation; and (viii) a combination of members of groups (vi) and (vii).

18. A composition according to Claim 17 for enhancing sperm fertility, comprising an active agent of any one of groups (i) - (v).

19. A composition according to Claim 18, wherein the ANP agonist is a member selected from the group consisting of ANP in which one or more amino acids has been added, replaced, deleted or chemically modified which can bind and activate ANP receptors in spermatozoa's membranes and fragments of ANP which can bind and activate the ANP receptors.

20. A composition according to Claim 19, wherein said agonist is ANP₅_₂₈ or ANP^.

21. A composition according to Claim 18, wherein the ANP agonist is a member selected from the group consisting of brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), CNP-22, CNP-53, Atriopeptin III, and active fragments and derivatives of these proteins or peptides which can bind and activate ANP receptors in spermatozoa's membranes.

22. A composition according to Claim 18, further comprising an anti-endopeptidase.

23. A composition according to Claim 18, wherein the c-GMP permeable analogue is 8-bromo-cGMP or dibutyryl cGMP.

24. A composition according to Claim 18, wherein the agents which induce rise in the cGMP levels are selected from the group of nitrous oxide

(NO), nitroprusside and inhibitors of phosphodiesterases.

25. A method for in vitro fertilization comprising contacting the sperm cells, the oocyte or both with an active agent as defined in Claim 2.

26. A contraceptive composition, comprising as an active agent a compound selected from the group consisting of:

(i) an antagonist of ANP;

(ii) an agent which lowers the level of cGMP in cells or inhibiting its formation; and (iii) a combination of members of groups (i) and (ii).

27. A contraceptive composition according to Claim 25, wherein the active agent is anantin.

28. An assay for determining the fertilization quality of the follicular fluid comprising determining the level of ANP therein.

29. An assay for determining the fertilization quality of sperm comprising:

(a) contacting spermatozoa with an effective concentration of ANP or an ANP agonist;

(b) determining the change in the level of cGMP in the spermatozoa or determining the effect of ANP or the agonist thereof on the spermatozoa's chemokinetic and chemotactic properties.

30. A method for enriching sperm with spermatozoa having improved motility properties comprising introducing sperm into an ANP gradient and selecting spermatozoa having travelled along the gradient at a distance which is above a predetermined distance.