Effect of SF-50, a partially purified protein from a marine Gastropoda mollusc Telescopium telescopium on female reproduction

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1 Indian Journal of Marine Sciences Vol. 30, March, 200, pp Effect of SF-50, a partially purified protein from a marine Gastropoda mollusc Telescopium telescopium on female reproduction Uttam Datta, Herambananda Roy & Anita Pakrashi* Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Calcutta , India Received 7 February 2000; revised 22 November 2000 The protein SF 50, isolated from crude extract of spermatheca gland of a marine Gastropoda mollusc, Telescopium telescopium, has been found to prevent entry of human spermatozoa at the dose of 6 and 5 mg/ml. No implantation site was observed in rats when 0 mg of SF 50 was administered in the uterus and the sites were significantly less (P< 0.00) in the group treated with 5 mg than those in BSA control. Continuous diestrous phase up to 23 to 50 days were observed in the actively immunized female rats with SF 50. There was no degenerative change or any significant variation in the number of corpus luteum, implantation sites or litter size in actively immunized rats as compared to the controls. The anti SF 50 rat serum showed homogeneity and identity with the corresponding antigen. No toxic effect of SF 50 was observed in ocular irritation test in rabbits. Present findings indicated a plausible topical vaginal contraceptive from marine bio-active substances. Sperm motility is known to be pre-requisite to penetrate sperm through cervical mucus, the cumulus 2, and zonapellucida 3. Again, the quality of actively motile spermatozoa is an important factor for fertility potential 4. Antibodies of the sperm immobilizing, sperm agglutinating and spermatoxic varieties also impair sperm penetration through human cervical mucus 5. It has been reported that some sperm-specific surface antigenicity from invertebrates are common even with a phylogenetic distant species which has a broad implication with regard to the development of immunocontraceptive methods 6. Therefore, a common marine snail, Telescopium telescopium under the class Gastropoda was chosen for this investigation. The protein SF 50, obtained by 50% ammonium sulfate precipitation from the crude extract of spermatheca gland of Telescopium telescopium was found to have spermicidal and sperm agglutinating properties on mammalian sperm cells 7. The present study has been undertaken to observe the potentiality of SF 50 as a topical contraceptive agent and its effect on the reproductive milieu in female rats when administered parenterally. Materials and Methods Proven (first weaning) female Sprague-Dawley rats * For correspondence. Present address : Dr. B.C. Roy Post-graduate Institute of Basic Medical Sciences, University College of Medicine, Calcutta University, 244B, A.J.C. Bose Road, Calcutta (206 ± 0 g of body weight) were selected randomly from the breeding colony. The rats, five in each group, were housed in polystyrene stainless steel cagesin a room kept at 22.5 ±.5ºC with relative humidity at around 60% provided with 4 h light and 0 h darkness throughout the study. Diet (dry pellet) and water were given ad libitum. The vaginal smears were examined at 9 a.m. every day by light microscopy and recorded to monitor the cyclicity of each animal. Collection of snail (Telescopium telescopium) and preparation of organ lysates have already been reported 8. About 20 m of lyophilized powder (CSPT) extracted from spermatheca gland was dissolved in 9 ml of 0.5 M phosphate buffer saline (PBS), ph 7.2 and the protein (SF 50) from the CSPT was obtained by precipitation with 50% ammonium sulfate 9. Precipitated protein was dialyzed against double distilled water for overnight at 4ºC. Total protein content of the SF 50 was estimated by Lowry et al. 0 method. Human semen samples were collected from fertile donors into sterile glass containers and kept at 37ºC in an incubator for 45 min. Only those samples which liquified completely within this period were assessed for their concentration, motility and progressively motile spermatozoa. These semen samples were then diluted with 500 μl of Baker s buffer (Na 2 HPO 4.4 g/l, KH 2 PO g/l, NaCl 0.8 g/l, glucose 2 g/l, distilled water up to lit.), to a concentration of sperm/ml. Only those samples having an

2 DATTA et al. : EFFECT OF SF-50 ON RERPRODUCTION 45 overall sperm motility of >70% and forward progression about 50% were used for in vitro test. The cervical mucus penetration test (CMPT) was performed by using Penetrak kit, (Serono Diagnostic, USA). The flat capillary tubes, filled with bovine cervical mucus, sealed and stored at 20ºC, were thawed at room temperature and scored with a diamond pencil above the mucus meniscus and broken carefully avoiding any bubble. The cut end of the tube was inserted vertically in small size round bottom test tubes containing sperm suspension. The prepared set was incubated at 37ºC for 90 min. The capillary tubes were then cleaned by tissue paper and placed on a measuring slide (provided with the kit) under phase-contrast microscope and the distance from the semen reservoir to the foremost spermatozoon in each capillary tube i.e. highest migration distance by a single sperm was measured and the penetration density of sperm cells within the mucus column was measured ( 00 and 400). The sets were used in duplicate for each solution of SF 50 (experimental) and bovine serum albumin (BSA; control) respectively. The mean number of each set was considered. For the test, 40 μl of liquified semen sample and 200 μl of SF 50 solution (:5), containing 6 mg, 5 mg, 2.5 mg, 0.62 mg and 0.3 mg per ml in Baker s buffer were taken into different round bottom glass tubes. The control set was maintained with 200 μl of BSA solution (6 mg/ml) in Baker s buffer. For intrauterine test, proven female rats were divided into twelve groups and eight in each group. Prior to intra-uterine inoculation, the females in proestrus were anaesthetized and their uterine horns were opened by mid-ventral incision of the abdomen. Lyophilized SF 50 and BSA 0, 5, 2.5,.25, 0.62 and 0.3 mg each were dissolved separately in 00 μl of normal saline solution into different Eppendorf tubes. Different concentration of SF 50 and BSA solutions were inoculated separately by tuberculin syringe with a 30 gauge needle into each uterine horn at the cervical end (e.g. 50 μl into each horn) of eight rats in each group as per doses received. Similarly, BSA administered rats were considered as control group and all the operated rat s abdomen were sutured. Each treated female rats were paired and caged with two proven males (:2). The next morning, vaginal smears were checked for the presence of spermatozoa and positive mating was recorded as Day of the pergnancy. All the mated rats were operated again on Day 0 to observe presence of any uterine inflammation, number of corpus luteum, implantation and resorbed sites. The animals were sutured again and kept for full term to observe the total number of corpus luteum, implantation sites and resorbed sites per rat : No.of pregnant animal Quantal pregnancy = No.of mated animal Total no. of littered pups Litter index = Total no. of implantation Pre implantaton loss Total no. of corpus luteum total no. of implantation = Total no. of corpus luteum Post-implantation loss and also for the presence of abnormality, if any, to the new born pups. Normal cycling proven female rats were taken in four groups of ten animals in each group for active immunization. A dose of 0.2 ml emulsified antigen ( SF 50 dissolved in 0.5 M phosphate buffer saline : paraffin oil :2) was inoculated intramuscularly to the two groups of experimental animals in (Gr.-I and II) for consecutive five days, (i.e. total 40 mg of SF-50 per animal). Similarly, emulsion of BSA and paraffin oil (:2) was administered (total ml/rat) to the animals of groups Gr.-III and IV and kept as controls. Vaginal smears were observed routinely after the treatment. The rats of each group were allowed to mate when they came into proestrus with proven males. On the 0th day of pregancy, mated females from experimental Group-I, and control Group-III, were anaesthetized and laparotomized to observe the number of corpus luteum and implantation sites in both the uterine horns and sutured again. These two groups (Gr.-I and III) were considered as operated group. The animals were allowed to complete the term. After parturition, litter size was counted and examined for the presence of any abnormality. Blood was drawn from each rat (Gr.-I to IV) by heart puncture 2, 7 days after inoculation. The individual serum samples were decomplemented at 37ºC for 5 min., aliquoted and kept at 20ºC. To detect the immune response of the antigen, spermatheca gland was dissected carefully from the body of Telescopium telescopium and minced carefully in a polystyrene petridish containing ml of 0.5 M PBS (ph 7.2). The suspension was filtered

3 46 INDIAN J. MAR. SCI., VOL 30, MARCH 200 through a double glass wool column to avoid any debris and collected into sterile glass tube. Filtered suspension was centrifuged twice at 500 g for 0 min. discarding the supernatant each time. Thereafter 0.5 M PBS (0.5 ml) was carefully added to the pellet and kept for 30 min. at 37ºC in an incubator allowing the sperm cells to swim up into the medium. After incubation, 20 μl sperm suspension was aspirated from the upper layer of the solution by an automatic pipette and placed on two clear glass slides. About 20 μl of the pulled anti SF 50 rat serum and anti BSA rat serum were added individually with each sperm suspension and mixed thoroughly by separate glass rods and covered by cover slips. Observations were made under phase contrast microscope ( 000) for the presence of any agglutination. For local (ocular) irritation test, one drop of the solution prepared by disolving 0 mg of lyophilized SF 50 in ml of normal saline solution was applied thrice daily to the right eye of white Newzealand rabbits for seven days consecutively. Left eye as control was treated with normal saline solution only. Three rabbits were used for the experimental purpose. Results and Discussion The amount of protein in SF 50 was estimated to be 5.5 μg/ml. The lowest effective dose of SF 50 to block the penetration of spermatozoa through cervical mucus was found to be 5 mg/ml (Table ). In intrauterine test, vaginal smears from mated females of the SF 50 treated group contained agglutinated sperms but was absent in the BSA treated groups. Gross examination of whole rat reproductive tract showed absence of uterine inflammation and virtually no variation in the number of corpus luteum in both SF-50 and BSA treated groups.the implantation sites and litter size were absent in the group treated with 0 mg, significantly less (P< 0.00) with 5 mg and had more resorption with 2.5 mg of SF 50 (Tabel 2). The potential of a spermicidal agent as an effective barrier contraceptive in vivo depends on its ability to debar either the entry or penetration of spermatozoa through cervical mucus. The ability of SF 50 treated spermatozoa to penetrate the cervical mucus column in vitro was measured by using Penetrak kit containing bovine cervical mucus which is known to have physical characteristics remarkably similar to that of human cervical mucus and extremely receptive Table Effect of SF 50 on cervical mucus penetration of human spermatozoa Treatment (mg/ml) Migration distance of spermatozoa Penetration density (cm) (cm) BSA SF n = 6 tubes; =absent to human sperm 3,4. The ability of SF 50 treated spermatozoa (6 and 5 mg/ml) to enter into the capillary tube in the cervical mucus penetration test clearly indicated sperm immobilization. In lower doses, however, the reduced penetration was due to agglutination of spermatozoa that hindered the forward progression of the sperm cells. Moreover, follicular development, ovulation, libido and mating behaviour remained unaffected in the intrauterine treated animals. In actively immunized non-operated groups, inoculated with BSA : paraffin oil (Gr.-IV) had no change in their estrus cycle or in vaginal smear. However, SF-50 treated group (Gr.-II) exhibited diestrus phase from the 4th day of inoculation and marked increase in infiltration of leukocytes, macrophages, neutrophils and lymphocytes were observed. The animals returned to their normal cycle after 23 to 50 days. The operated control group (Gr.- III) also showed normal cyclicity and vaginal smears and the number of corpus luteum, implanation sites and the litter size were.4 ± 0.47, 0.8 ± 0.38 and 0.4 ± 0.45 respectively. The cyclicity of the animals of SF 50 treated operated group (Gr.-I) was similar to that of the non-operated SF 50 teated group (Gr.-II) and there was no significant change in the number of corpus luteum (0.3 ± 0.30) though implantation sites (9.5 ± 0.63) or litter size (8.7 ± 0.65) were less. The general appearance on autopsy of non-estrus rats treated with SF 50, showed no significant reduction or enlargement of the ovaries but the uterine horns were flaccid. The alteration of estrus cycle to a continuous diestrus phase, maintenance of corpora lutea in the

4 DATTA et al. : EFFECT OF SF-50 ON RERPRODUCTION 47 Table 2 Effect of intra-uterine inoculation of SF 50 on fertility of rats (Values expressed as mean ±SE) Treatment* SF 50 (mg) Corpora lutea (per rat) 9.5± ± ± ± ± ±0.82 Implantation sites (per rat).50± ± ±.05 0± ±0.42 Resorbed sites (total no.) No. of animals mated but not pregnant 6 rats mated and not All mated but 3 except one 6 rats mated and except one Mean no. of pups born Quantal pregnancy Litter index Pre-implantaion loss Post-implantation loss *No. of rats 8 per group. Not found Not found 0.87± ± ± ± ± ovary and the presence of leukocytic infiltration in the vaginal smear indicated the immunological reactions mediated by SF 50. Microscopic examination of the pulled anti-sf 50 rat serum with sperm cells of the snail exhibited agglutination but not with control (BSA) serum. This immune response was possibly due to precipitation of antigenic particles by cross-linking with antibodies directed to their antigenic determinants. No irritation or inflammation was observed in the rabbit s eyes treated with SF 50 which clearly indicated that the protein is devoid of any local toxicity. Thus, presence of sperm agglutinating and immobilizing properties, continuous diestrus phase of the estrus cycle, loss of conceptus and absence of any local toxicity indicated that SF 50 deserves to be considered as a potential candidate for local barrier contraceptive or as an immunocontraceptive. References Mortimer D, Pandey I J & Sawers R S, Relationship between human sperm motility characteristic and sperm penetration into human cervical mucus in vitro., J Reprod Fertil, 78 (986) Tesarik J, Oltras C M & Testort J, Effect of the human cumulus oophorus on movement characteristics of human capacitated spermatozoa, J Reprod Fetil, 88 (990) Green D P L, Sperm thrusts and the problem of penetration, Biol Rev, 63 (988) Macleod J & Gold R Z, The mode factor in fertility and infertility IV. Sperm morphology in fertile and infertile marriage. Fertil Steril, 2 (95) Davajan V, Nakamura R M, The cervical factor, in Progress in infertility. 2nd Edn edited by Beharman S J and Kistner R W, (Little Brown Co, Boston), 975, pp Lopo A C & Vacquier V O, Sperm specific surface antigenicity common to several animal phyla, Nature (London), 288 (980) Pakrashi A & Datta U, In vitro sperm agglutination and spermicidal activity of protein isolated from a marine mollusc Telescopium telescopium. Indian J Mar Sci, 200 (in press). 8 Pakrashi A, Dutta U & Chowdhury A, A search for immunocontraceptive agent from marine source-role of antispermatheca globulin of Telescopium telescopium on fertility regulation of male rat Indian J Exp Biol, 30 (992) Scopes R K, Protein purification Principles and practice, 2nd Edn (Springer-Verlag, New York), 987 pp Lowry O H, Rosenbrough N J, Farr A L & Randall R J, Protein measurement with folin phenol reagent, J Biol Chem, 93(95)

5 48 INDIAN J. MAR. SCI., VOL 30, MARCH 200 Kremer J, In vitro sperm penetration in cervical mucus and AIH. in Homologus artificial insemination (AIM), Emperaire J E Auderber A, Hafez E S E & Martinus Highnoff, (The Hague, Boston), 980, pp Herbert W J, Handbook of experimental immunology, Application of immunological methods, (Blackwell Scientific Publications, Oxford), 979, p Alexander N J, Evaluation of male infertility with an in vitro cervical mucus penetration test, Fertil Steril, 36 (98) Lee Wt, Gaddum-Rosse P & Blandau R J, Sperm penetration into cervical mucus, in vitro III. Effect of freezing on estrus bovine cervical mucus. Fertil Steril, 36 (98)