WO1983002057A1

WO1983002057A1 - Contraceptive method and composition

Info

Publication number: WO1983002057A1
Application number: PCT/AU1982/000213
Authority: WO
Inventors: COMMONWEALTH OF AUSTRALIA care of THE ... THE
Original assignee: Quinn, Patrick, James; Stanger, James, David
Priority date: 1981-12-17
Filing date: 1982-12-17
Publication date: 1983-06-23

Abstract

A method of preventing or inhibiting the fertilization of mammalian ova by mammalian spermatozoa comprises contacting the spermatozoa with trichloroacetic acid (TCA) -extracted serum albumin to bind the modified serum albumin to the spermatozoa before the spermatozoa contact the ova to effect fertilization thereof. The method can be applied to prevention or inhibition of conception in a female mammal by intravaginal administration or application of the modified serum albumin. Compositions comprising the modified serum albumin are also disclosed.

Description

"CONTRACEPTIVE METHOD AND COMPOSTTION"

This invention relates to a method of and composition for preventing or at least inhibiting the fertilization of mammalian ova by mammalian spermatozoa, and thus relates to a method and composition^" which may be used for contraceptive purposes.

Currently used spermicidal contraceptive agents are spermicidal in the true sense of the word, i.e. they disrupt the metabolism of spermatozoa and thereby kill them. A commonly used test of spermicidal effectiveness is the ability of the compound to immobilise spermatozoa. Csee Population Reports, Series H, No.5, Sept. 1973 and Series H, No. 3, Jan. 1975; published by the Population Information Program, The Johns Hopkins University, 624 North Broadway, Baltimore, Maryland 21205, U.S.A.).

It has now been discovered that one particular proteinaceous material, a preparation of modified serum albumin (a blood protein) , may be effective to completely prevent mammalian spermatozoa from fertilizing mammalian ova. The modified albumin has no other deleterious effects on the spermatozoa, it is not toxic to the ova, and does not inhibit the motility of spermatozoa or prevent them from binding to the surface of the ova. The effectiveness of this

f OMP modified serum albumin, appears to lie in its ability to "prevent spermatozoa from penetrating the zona pell cida membrane (/the final membranous layer) around the egg. 5

According to a first aspect of the present invention, there is provided a method of preventing or inhibiting the fertilization of mammalian ova by mammalian spermatozoa, characterised in that said _ spermatozoa are contacted with trichloroacetic acid- extracted serum albumin to bind said modified serum albumin to said spermatozoa before said spermatozoa contact said ova to effect fertilization thereof.

_j_5 In one specific embodiment of this aspect of the invention there is provided a method of preventing or inhibiting conception in a female mammal which comprises administering or applying trichloroacetic acid-extracted serum albumin intravaginally to said

20 female mammal so that mammalian spermatozoa introduced into the vagina of said female mammal contact said TCA- extracted serum albumin before contacting ova produced by said female mammal to effect fertilization of said ova.

^'25

In another aspect, this invention provides a composition for preventing or inhibiting the fertilization of mammalian ova by mammalian spermatozoa, which comprises trichloroacetic acid-extracted serum

30 albumin and a physiologically acceptable carrier therefor.- Such a composition has particular application as a contraceptive composition. According to this latter aspect of the invention, modified serum albumin is provided as the active fertilization-preventing or -inhibiting agent in, for example, a vaginal contraceptive composition or a lubricant composition for condoms. In particular, a vaginal contraceptive composition of the present invention may be formulated as a cream Cin an oil/water base) as a foam Cin a known foaming basel or as a jelly (.in a known gel basel , buffered to the normal vaginal - . acidity ( H 4.5). In addition to the modified serum albumin, the composition may also include one or more known spermicidal agents such as, for example, Octoxynol, Nonoxynol or p-diisobutylphenoxypolyethoxyethanol.

The general formulations for the compositions as described above are well known and do not require specific description. By way of example, however, a suitable cream base may comprise purified water, propylene glycol, stearic acid, sorbitan stearate, polysorbate 60, boric acid and fragrance, pH 4.5.- A suitable gel base may comprise propylene glycol, cellulose gum, boric acid, sorbitol, starch, simethicone, purified water and fragrance, pH 4.5.

The modified serum albumin which characterises the method and composition of this invention has been found to preferentially and irreversibly bind to spermatozoa, rendering them unable to pass through the zona pellucida membrane around the ovum. The modification of serum albumin by extraction with tri-

OM chloroacetic acid CTCA) has been published by Lui, C.W., Cornett, L.E. & Meizel, S. C1977J. "Identification of the bovine follicular fluid protein involved in the in vitro induction of the hamster sperm aerosome reaction". Biology of Reproduction, Volume 17, pp.34-41.

In general terms, the modification comprises the steps of extracting the serum albumin with TCA, resolubiliz- ation with ethanol and dialysis against distilled wate . ' This reference discloses that the modified albumin does not affect the motility of the spermatozoa, however the modi ied albumin has never been used to test whether spermatozoa which have been exposed to it are capable of fertilizing ova in vitro and in vivo.

In the following Example, detailed discussion is included by way of elucidation of the present invention of the effectiveness of modified bovine serum albumin (BSA) in inhibiting the ability of mouse spermatozoa to fertilize mouse ova in vitro. It will be appreciated that as a result of the similarity in the basic physiology of spermatozoa from a range of mammalian species, similar results can be expected using human spermatozoa and human ova.

E.:X A M P L E

In this Example, the effect of three different preparations of bovine serum albumin (BSA) - normal BSA, trichloroacetic acid extracted BSA, and heated BSA - on fertilization of mouse ova in vitro is demonstrated

A. MATERIALS AND METHODS The basic techniques are described in detail elsewhere (Quinn, 1979; Quinn and Stanger, 1980, 1981; Quinn et al, 1981a) .

Fertilization Medium Modified Tyrode's Medium (.Quinn et al, 1981b) or modified Whitten's Medium (.Quinn and Stanger, 1981) containing 15 g/ml BSA (.fraction V, Sigma Chemical Co.) was used in all experiments. Similar results were obtained with both media. The BSA was modified by extraction with trichloroacetic acid CTCA) , resolubilization with ethanol and dialysis against distilled water or by heating at 90 C for 30 min (Quinn and Stanger, 1980) . After the addition of BSA,

PI the medium was adjusted to pH 7.4 - 7.6 with IN NaOH and sterilized by filtration. Aliquots of 0.5 ml of medium were placed in 30 mm plastic culture dishes (Falcon No. 2001) , overlaid with equilibrated paraffin oil (BDH, lightweight) and incubated at 37°C in a humidified atmosphere of 5% C0₂ in air for at least 30 min before use.

Gametes Mouse ova were obtained from superovulated

CC57BL/6 x CBA) F, donors 14-16 h after the injection of human chorionic gonadotrophin and freed from cumulus cells by hyaluronidase treatment. Spermatoz^'oa were extruded from the caudae epididymides of two mature (3-6 months old) F_χ CC57BL/6 x CBA) males into 0.5 ml medium in each experiment and after allowing 10 min for dispersion, an aliquot was added to the fertiliz¬ ation dishes so that the final concentration of spermatozoa was 1-2 x 10 /ml. The diluted spermatozoa were usually preincubated for 1-2 h before the addition of ova. Cumulus-fre^'e ova were placed with the pre¬ incubated spermatozoa for 4-5 h. Overall, the sperma¬ tozoa were incubated for a total of 6 h. Zona-free ova were prepared by a combined pronase and mechanical treatment (.Quinn and Stanger, 1981) .

Assessment of Fertilization

At the completion of incubation with spermatozoa, the ova were collected and washed thoroughly through two changes of fresh medium to remove excess and loosely adherent spermatozoa. The ova were then fixed overnight with 10% (v/v) formalin in 0.1 M phosphate buffer (pH 7.4) before being mounted and stained with lacto-aceto-orcein (Toyoda and Chang, 1974) . The ova were only considered to be penetrated when they contained a decondensing sperm head or male pronucleus with accompanying sperm tail and the female metaphase II chromosomes had resumed meiosis or had progressed to the pronuclear stage.

Replicate experiments were tested for homogeneity by chi-squared analysis. Differences between pairs of treatments were analysed by 2 x 2 contingency tables using the combined homogeneous totals for each treatment.

B. RESULTS The necessity for the presence of BSA to permit mouse spermatozoa to penetrate through the zona pellucida and fuse with the vitelli of mousa ova is demonstrated by the results given in Table 1. Sperma¬ tozoa Cunwashed or washed by centrifugation) failed to fertilize zona-intact or zona-free ova in the absence of BSA but when BSA was present, both samples of spermatozoa were equally effective in- fertilizing a high proportion (5- 80%) of both zona-intact and zona- free ova.

The results of the effect of different BSA preparations on the fertilization of cumulus-free mouse ova are given in Table 2. Over 90% of the ova incubated with spermatozoa in medium containing normal untreated BSA were fertilized and nearly all of the ova had well developed male pronuclei 5 h after insemination. On some occasions ova were placed in culture after removal from the fertilization dishes and over 90% (put o;ξ.20Q.L cleaved to the 2-cell stage 24 h later and over 70% of them formed normal expanded blastocysts after a further 4 days of culture (P.Quinn, unpublished observations) . In medium containing heated BSA, only 20% of the zona-intact ova were fertilized and the decondensation of the sperm head into a fully formed male pronucleus was delayed with over half of the fertilized ova having swollen sperm heads with no nucleoli-like structures present. The TCA-extracted BSA did not allow any spermatozoa to pass through the zona pellucida and effect fertilization. When the zona pellucida was absent high fertilization rates (> 95%) were obtained with both the heated and TCA-extracted BSA and the fertilizing spermatozoa had-decondensed to a similar degree to those in the normal control ova.

When 2 mM caffeine was present during the pre- incubation of spermatozoa and the insemination period, the fertilization rate was considerably increased in the medium containing the heated BSA but did not reach the .control values (Table 2) . The progress of fertilization was slower in the ova in medium containing heated BSA and caffeine compared to those in medium containing normal BSA since in over half of the former ova, the fertilizing spermatozoon had only progressed to the swollen sperm head stage 5 h after insemination. Fertilization failed to take place in medium containing the TCA-extracted BSA and caffeine.

In order to obtain information on the temporal aspects of spermatozoal penetration through the zona pellucida and whether the type of BSA affected the completion of fertilization after spermatozoa had attached to the zona, a transfer study was undertaken. The results are given in Table 3. When ova were inseminated with spermatozoa preincubated for 1 h in medium containing normal BSA, transfer to medium containing heated BSA 1 and 2 h after the addition of ova resulted in a 70% fertilization rate after 1 h which increased to 80% after the 2 h transfer. The inadequacy of the heated BSA to support the completion of fertilization was still evident, however, as the. -.. fertilization rate after the 2 h transfer was still significantly lower ? < 0.05) than when the gametes were maintained in normal BSA for the full 5 h period. A similar trend but with lower values was obtained when the ova were transferred to medium containing TCA- extracted BSA after 1 h and 2 h in the normal BSA. The inadequacy of the heated and the TCA-extracted BSA to support the spermatozoa during penetration of the zona pellucida was also evident when the transfers were done in the reverse order i.e. 1 or 2 h in medium containing treated BSA and then transfer to normal BSA for the remainder of the 5 h period. As the period of exposure of the ova to heated BSA increased before transfer, the rate of fertilization decreased. Incubation of sperma¬ tozoa and ova in medium containing the TCA-extracted BSA for as little as 1 h before transfer to the^' normal BSA almost completely inhibited fertilization.

In the next series of experiments, the inhibitory effects of TCA-extracted BSA on the fertilization of zona-intact ova was studied in more detail. The results in Table 4 show that even a 10 min incubation of sperma¬ tozoa in medium containing TCA-extracted BSA significantly lowers (P < 0.05) the fertilizing capacity to a similar value obtained with continuous incubation in this type of BSA (see also Tables 2 and 3) . When varying amounts of medium containing normal or TCA- extracted BSA were mixed, even a 10% proportion of the latter BS significantly decreased (P_ < 0.001) the fertilization rate of zona-intact ova (Table 5) . This decline became progressively -greater as the proportion of TCA-extracted BSA in the medium increased. When the medium contained 40% Cv/v) TCA-extracted BSA, fertiliz atiόn was almost completely .abolished as it was when the medium contained only TCA-extracted BSA, indicating that this BSA irreversibly inhibits the capacity^'of the spermatozoa to pass through the zona pellucida and effect fertilization.

To ensure that the TCA-extracted BSA did not reduce the fertilizing ability of the spermatozoa by inhibiting their motility or capacity to bind to the zona pellucida, these properties were measured and the results are given in Tables 6 and 7. In medium containing the TCA-extracted BSA or heated BSA, neither the motility or zona-binding ability of the spermatozoa was significantly different from spermatozoa incubated in medium containing normal, untreated BSA. The motility of the spermatozoa in Suspension was also not affected by the absence of BSA CTable 6). , although very few spermatozoa bound to the zona pellucida when no exogenous protein was present in the medium (Table 7) TABLE 1

Effect of BSA on the Fertilization of Zona-intact and Zona-free Mouse Ova in vitro

Treatment

± Zona Centrifugation BSA pellucida of Spermatozoa n

N (.%)

-BSAt Present No 0/42 (0)

Absent No 0/21 (0)

Present Yes 0/38 (.0)

Absent Yes 0/20 (.0)

+BSA Present No ^'62/69 (90)

Absent No 32/38 (84)

Present Yes 48/54 (88)

Absent Yes 28/32 (88)

Replicated 2-3 times. Final sperm concentration 2 x 10⁶/ml.

* n = number of ova fertilized:

N = total number of ova inseminated.

t - BSA = medium with 1 mg/ml polyvinyl pyrrolidine. pyp) + BSA = medium with 15 mg/ml normal BSA.

(Control of + BSA and PVP gave 43/47 (.91%! of zona- intact ova fertilized by noncentrifuged spermatozoa.) TABLE 2. Effect of Treated-BSA on the Fertilization of Zona-intact and Zona-free Mouse Ova in vitro.

Type of BSA in Zona Caffeine medium pellucida 2 rru

Normal (untreated) Present Absent 75/82 (91) 71/75 (95)

Present Present 41/46 (.89) 37/41 (90) Heated Present Absent 9/47 (19) 4/9 (44)

Absent Absent 30/31 (97) 29/30 (97) Present Present 35/47 (74) 14/35 (40)

TCA-e traσted Present Absent 0/38 (0) 0/0 (0)

Absent Absent 67/68 (99) 66/67 (99)

Present Present 0/26 (0) 0/0 (0)

Replicated 3 times. Final sperm concentration 1.0 - 1.8 x 10 /ml.

* n = number of ova fertilized; N = total number of oVa inseminated, t P = number of fertilized ova with fully formed male pronucleus.

TABLE 3

Effect of Transfer Between Media Containing Different BSA's on the Fertilization of Zona-intact Mouse Ova in vitro.

Length of incubation Fertilized ova/Total ova (%) in different BSA (h)

Type of treated BSA

Initial Final Heated TCA-extracted.

Normal BSA Treated BSA

1 4 42/59 (71) 16/55 (29)

2 3 49/60 (80) . 29/54 (54)

5 0 56/56 (100) 35/40 (88)

Treated BSA Normal BSA

1 4 29/56 (52) 1/55 (2)

2 3 23/57 (40) 1/56 (2)

5 0 15/55 (27) 0/40 (0)

Normal BSA Normal BSA Normal BSA (control)

1 4 48/50 (96)

2 3 44/49 (90)^" ^•5 0 42/45 (93)

Replicated 3 times. Final sperm concentration 1.0 - 2.0 x 10 /ml. Ova and attached spermatozoa were picked up with a pipette from the initial treatment dish and transferred without washing to the final treatment medium at the times indicated.

- yimE TABLE 4.

Effect of Preincubating Spermatozoa in TCA-extracted BSA on Their Subsequent Ability to Fertilize Zona- intact Mouse Ova in vitro

Treatment n/*(%)

Normal BSA no centrifugation 64/67 (S6.) Normal BSA 1 h →- normal BSA 137/182 (75) TCA-extracted BSA 10 min →- normal BSA 7/123 C6) TCA-extracted BSA 1 h. →- normal BSA 1/101 CD. TCA-extracted BSA 1 h →- TCA-extracted BSA 3/145 (2)

Replicated 4 times.

Final sperm concentration 2 x 10 /ml.

* n = number of ova fertilised;

N = total number of ova inseminated,

TABLE 5

Effect of Varying Ratios of Normal and TCA-extracted BSA on the Fertilization of Zona-intact Mouse Ova in vitro.

Ratio of BSA

Normal n/_N (%)

: TCA-extracted

1 _, 0 89/95 (94)

9 • 1 27/92 (30)

8 : 2 9/93 (10)

6 : 4 1/64 (2)

4 : 6 1/67 (2)

2 : 8 0/55 (0)

0 _: 1 1/70 (1)

Replicated 3 times. "

Final sperm concentration 2 x 10 /ml.

* n = number of ova fertilized;

N = total number of ova inseminated,

TABLE 6. Effect of BSA on Spermatozoal Motility In Vitro

Time After Percent motility commencement of incubation Type of BSA ^un reated₎ TCA-extracted Heated PVP only (hr)

1 66±6 65±7 62±6 69±8 2 55±6 60±5 51±7 54±8 3 49±4 44±6 47±7 47±6 8 31±5 27±4 28+6 29+5

Mean values ± SEM of four replicates/treatment.

TABLE 7 Effect of BSA on the Number of Mouse Spermatozoa Bound to the Zona Pellucida of Ova in Vitro*

Type of BSA Number of ova Sperm bound/ present examined. zona-intact ovum

Normal (untreated) 25 6.3±2.5 TCA-extracted 24 6.9+2.4 Heated BSA 32 5.9+2.0 PVP only 30 0.4±0.1

*Mean values ± SEM of four replicates 1 hr after insemination with 2 x 10 spermatozoa/ml.

C. DISCUSSION

The results set out above confirm that serum albumin is an obligatory component of the incubation medium for the fertilization of mouse ova. Normal, untreated bovine serum albumin supports high rates of fertilization of cumulus-free ova both with and without their zonae pellucidae, whereas heat treated or trichloroacetic acid extracted bovine serum albumin is unable to support the fertilization of a majority of zona-intact va but fertilization of zona-free ova is unimpaired. Trichloroacetic acid extracted BSA preferentially and irreversibly inhibits zona pene¬ tration by spermatozoa but this effect is not mediated by an inhibition of spermatozoal motility or zona- binding ability. The TCA extracted BSA may bring about its effect by inactivating acrosin and/or other acrosomal enzymes. This effect occurs after only a

10 min preincubation of the spermatozoa in the modified

BSA or when the medium contains only a 10% Cv/ J. proportion of this albumin.

From these results, it c^n also be concluded that serum albumin has at least two separate roles in the fertilization of cumulus-free mouse ova in vitro. Firstly, to maintain the motility of spermatozoa during penetration of the zona pellucida and secondly to maintain the- activity of acrosomal enzymes for zona penetration.

D. REFERENCES

Quinn, P. (1979) ;

Failure of human spermatozoa to penetrate zona free mouse and rat ova in vitro.

J. Exp. Zool. 210:497-506. Quinn, P., Stanger, J.D. (1980):

Effect of purification of bovine serum albumin on the interaction of human semen with mouse ova in vitro. Biol. Reprod. 22:134-140.

Quinn, P., Stanger, J.D. Q.981) :

Fertilization of pronase-treated mouse ova in vitro. Aust. J. Biol. Sci. 34:245-248. ^•

Quinn, P., Whittingham, D.G., Stanger, J.D. (1981a): Interaction of semen with ova in vitro. Arch. Androl. Cin press) .

Quinn, P., Barros, C. , Whittingham, D.G. Q.981b) : Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. J. Reprod. Fert. Csubmitted for publication) .

Toyoda, Y. , Chang, M.C, (19.741:

Fertilization of rat eggs in vitro by epididymal spermatozoa and the development of eggs following transfer. J. Reprod. Fert. 36:9-22.

It will be appreciated that the specific Example set out in detail above is given by way of exemplification and not limitation of the present invention. Accordingly, many modifications and variations, may be made thereto without departing from the spirit and scope of the invention as broadly described above.

Claims

CLAIMS :

1. A method of preventing or inhibiting the fertilization of mammalian ova by mammalian spermatozoa, characterised in that said spermatozoa are contacted with trichloroacetic acid CTCA) -extracted serum albumin to bind said modified serum albumin to said spermatozoa before said spermatozoa contact said ova to effect fertilization thereof.

2. A method according to claim 1, characterised in that said TCA-extracted serum albumin is TCA-extracted bovine serum albumin.

3. A method according to claim 1, characterised in that said TCA-extracted serum albumin is prepared by the steps of extracting the serum albumin with TCA, resolubilisation with ethanol and dialysis against distilled water.

4. A method according to claim 1, characterised in that said spermatozoa are contacted with said TCA- extracted serum albumin for a period of at least ten minutes.

5. A method according to claim 1, characterised in that said spermatozoa are contacted with said TCA- extracted serum albumin in a medium containing at least 10% fr/v). of said TCA-extracted serum albumin.

6. A method according to claim 1, characterised in that said spermatozoa are contacted with TCA-extracted serum albumin which is in composition with a physiologic¬ ally acceptable carrier therefor.

7. A method according to claim 6, wherein said composition comprises a, vaginal contraceptive composition with, said TCA-extracted serum albumin as the active fertilization-preventing or -inhibiting agent therein.

8. A method according to claim 6, wherein said composition further comprises a spermicide. _ . ..

9. A method of preventing or inhibiting conception in a female mammal which comprises administer¬ ing or applying trichloroacetic acid CTCA) -extracted serum albumin intravaginally to said fema e mammal so that mammalian spermatozoa introduced into the vagina of said female mammal contact said TCA-extracted serum albumin before contacting ova produced by said female mammal to effect fertilization of said ova.

1Q. A method according to claim 9, wherein said TCA-extracted serum albumin is TCA-extracted bovine serum albumin.

11. A method according to claim 9, wherein said TCA-extracted serum albumin is prepared by the steps of extracting the serum albumin with TCA, resolubilis- ation with ethanol and dialysis against distilled water.

12. A method according to claim 9, wherein said TCA-extracted serum albumin is administered or applied intravaginally in composition with a physiologically acceptable carrier therefor.

13. A method according to claim 12, wherein said composition comprises a vaginal contraceptive composition.

^J E

14. A method according to claim 12, wherein said composition further comprises a spermicide.

15. A method according to claim 12, wherein said composition is in the form of a cream, a foam, or a jelly, buffered to the normal vaginal acidity (pH 4.5) .

16. A composition for preventing or inhibiting - - the fertilization of mammalian ova by mammalian spermatozoa, which, composition comprises trichloro¬ acetic acid CTCA). -extracted serum albumin and a physiologically acceptable carrier therefor.

17. A composition according to claim 16, wherein said TCA-extracted serum albumin is TCA-extracted bovine

,» serum albumin.

18. A composition according to. claim 16, wherein said TCA-extracted serum albumin is prepared by the steps of extracting the serum albumin with TCA, resolubilisation with ethanol and dialysis against distilled water.

19. A composition according to claim 16, which comprises a vaginal contraceptive composition buffered to the normal vaginal acidity C H 4.5J_.

20. A composition according to claim 16, further comprising at least one known spermicidal agent.

21. A composition according to claim 20, wherein said at least one spermicidal agent is selected from the group consisting of Octoxynol, Nonoxynol or p-dϋsobutylphenoxypolyethoxyethanol.

22. ^• A composition according to claim 16, wherein said physiologically acceptable carrier is selected from the group consisting of an oil/water cream base known per se, an aerosol or foaming base known per se, or a gel base known per se.

23. ^■ A composition according to claim 16 , wherein said physiologically acceptable carrier comprises a_ lubricant base known per se.