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delta 9-Tetrahydrocannabinol (THC) and two other major cannabinoids derived from marihuana--cannabidiol (CBD) and cannabinol (CBN)--inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of sperm (Schuel et al., 1987). Sperm fertility depends on their motility and on their ability to undergo the acrosome reaction upon encountering the egg's jelly coat. Pretreatment of S. purpuratus sperm with THC prevents triggering of the acrosome reaction by solubilized egg jelly in a dose (0.1-100 microM) and time (0-5 min)-dependent manner. Induction of the acrosome reaction is inhibited in 88.9 +/- 2.3% of sperm pretreated with 100 microM THC for 5 min, while motility of THC-treated sperm is not reduced compared to solvent (vehicle) and seawater-treated controls. The acrosome reaction is inhibited 50% by pretreatment with 6.6 microM THC for 5 min and with 100 microM THC after 20.8 sec. CBN and CBD at comparable concentrations inhibit the acrosome reaction by egg jelly in a manner similar to THC. THC does not inhibit the acrosome reaction artificially induced by ionomycin, which promotes Ca2+ influx, and nigericin, which promotes K+ efflux. THC partially inhibits (20-30%) the acrosome reaction induced by A23187, which promotes Ca2+ influx, and NH4OH, which raises the internal pH of the sperm. Addition of monensin, which promotes Na+ influx to egg jelly or to A23187, does not overcome the THC inhibition. Inhibition of the egg jelly-induced acrosome reaction by THC produces a corresponding reduction in the fertilizing capacity of the sperm. The adverse effects of THC on the acrosome reaction and sperm fertility are reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse ova with or without follicular cells (denuded) were treated with wheat germ agglutinin (WGA) in concentrations ranging from 5-500 mug/ml and fertilized in vitro. Ova subjected to concentrations greater than 10 mug/ml WGA were incapable of fertilization in vitro, although sperm were observed adhering to the zona pellucida after treatment in concentrations as high as 500 mug/ml WGA. At concentrations of 50 mug/ml or higher, WGA adversely affected the shape of the vitellus. Of various saccharides employed to block the effects of WGA D-glucose was the most effective. Injections of 0.03 ml of 100 mug/ml WGA into the ovarian bursa two to three hours before the expected time of ovulation had little or no effect on fertilization in vivo.

Rabbit antiserum against hamster ovary was examined on agargel diffusion plates against several hamster tissues, and also against rat and mouse ovarian extracts. Unabsorbed anti-hamster ovary antiserum showed eight to nine precipitin bands for hamster ovary and four to eight bands for other tissue extracts, but no bands against sperm antigens. Anti-hamster ovary antiserum also showed three to four bands for rat and one to two bands for mouse ovarian extracts. The present experiments confirmed previous reports for the hamster and mouse that treatment of eggs with anti-ovary antiserum blocked in vitro fertilization and that the extent of the inhibition was related to the formation of a precipitate on the zone pellucida. A single injection of anti-hamster ovary antiserum inhibited fertilization in mice but not in rats. In vitro fertilization of mouse eggs was also inhibited in the presence of such antiserum.

Zona-free eggs were introduced to fresh or preincubated sperm suspensions and the penetration of eggs by foreign spermatozoa was examined, as evidenced by enlargement of the sperm head and formation of the male pronucleus. It was found that zona-free hamster eggs can be penetrated by guinea-pig, deer mouse and rabbit spermatozoa but zona-free rat, mouse and rabbit eggs cannot be penetrated by guinea-pig spermatozoa. Furthermore, zona-free rat and mouse eggs cannot be penetrated by spermatozoa from two species of deer mice and the Mongolian gerbil. The zona pellucida of a few intact rat eggs can be penetrated by mouse (6%) and by P. leucopus spermatozoa (14%) but enlargement of the sperm head and formation of pronuclei were observed in the former but not in the latter. It seems that (1) sperm capacitation is required for the penetration of zona-free eggs, (2) the attachment of foreign spermatozoa to eggs may indicate their potential ability of penetration in some cases, (3) there is a certain affinity between the vitellus of one species and spermatozoa from another species, (4) the block to the entry of foreign spermatozoa is not only in the zona pellucida but also in the vitelline membrane, (5) zona-free hamster eggs can be penetrated by spermatozoa of six species, (6) mouse spermatozoa can penetrate zona-free eggs of three species, and (7) fertilization of intact P. maniculatus eggs can be achieved in vitro.

Spermatozoa in the vas deferens of the hamster lose their fertilizing capacity 3 days after ligation of the initial part of the duct and after 2 days if the testes are removed at the time of ligation. Sham-ligation had no effect on the fertilizing life of vasal spermatozoa on the contralateral side even 3 days after bilateral castration. Unilateral castration for 3 days had no effect on the fertilizing capacity of spermatozoa from the ipsilateral unobstructed duct, whereas no eggs were fertilized by spermatozoa from the contralateral ligated duct associated with the remaining testis. Unlike testosterone, 5alpha-dihydrotestosterone injected daily or implanted subcutaneously in Silastic tubes maintained normal fertilizing capacity for 2 days in castrates and for 3 days in intact males. Within each group, ligation had no effect on the level of fructose in the seminal vesicle on that side compared with the level in the gland on the other side.

Hamster eggs with follicular cells were fertilized by epididymal spermatozoa in two chemically defined media. The proportion of penetrated eggs was significantly higher in a medium for rabbit (16%) than in a medium for rat eggs (6%), but much lower than in Tyrode's solution containing follicular fluid or blood serum as reported by others. The optimal sperm concentration for sperm penetration ranged from 0.5 to 5 X 10(6)/ml but penetration of denuded eggs failed in these media. When exposed to hamster spermatozoa in the rabbit medium containing living or dead spermatozoa of guinea-pig, rat, mouse or hamster, high proportions of denuded eggs (24-96%) and eggs with follicular cells (93%) were penetrated. By exposure of denuded hamster eggs to hamster spermatozoa in supernatant fluid of frozen-thawed guinea-pig spermatozoa, 97% of eggs were penetrated in 8 hr compared to 0% in the control group. Sperm capacitation was also efficiently induced by preincubation of hamster spermatozoa in the supernatant fluid. The fertilizing capacity of hamster spermatozoa was maintained for 12 hr during incubation with frozen-thawed guinea-pig spermatozoa when the concentration of hamster spermatozoa ranged between 10 and 20 X 10(6)/ml. The beneficial factor of guinea-pig spermatozoa appeared to be from spermatozoa themselves, not from the vasal or epididymal fluids. The presence of follicular cells, blood serum, bovine serum albumin, or even polyvinylpyrrolidone in the media is essential for the capacitation and acrosome reaction of hamster spermatozoa. The components of guinea-pig spermatozoa appear to maintain the fertilizing capacity of hamster spermatozoa and stimulate the process of capacitation.

Mature female rabbits were given subcutaneous injections of 20 or 50 mg Depo-Provera (Upjohn Company, Kalamazoo, MI) (medroxyprogesterone acetate). Ovulation by mating was inhibited for 40 to 65 days. Although ovulation could be induced by injection of human chorionic gonadotropin, fertilization of eggs failed in all females 15 to 83 days after treatment. When treated and untreated females were inseminated with a definite number of spermatozoa and given human chorionic gonadotropin, fertilized eggs and spermatozoa were found in the oviducts of untreated, but not in the treated, females. Although spermatozoa were found in the uterine horns of treated rabbits, the number was much lower than in the untreated females. It is concluded that long after injection of Depo-Provera, not only was ovulation inhibited, but also fertilization, due to the suppression of sperm transport in the female tracts. The effects of progestins on various aspects of animal reproduction are discussed, stressing the effectiveness and efficiency of their contraception.

In vitro fertilization of rat and mouse eggs by ejaculated or epididymal spermatozoa in chemically defined media was studied. Penetration rates by ejaculated sperm was very low (0 to 8%) in the rat, but 11 to 41% of eggs were penetrated by ejaculated sperm in the mouse. The optimal concentration of sperm for in vitro fertilization appears to be similar whether ejaculated or epididymal sperm were used. The time of sperm penetration in the mouse eggs, however, was delayed for one-half to one hour when ejaculated sperm were used. The importance of sodium pyruvate, sodium lactate and glucose in the medium containing bovine serum albumin for in vitro fertilization of rat eggs was examined. When rat eggs in cumulus clot were exposed to epididymal sperm preincubated for five hours, the presence of sodium pyruvate, sodium lactate and glucose was found to play an important role. When exposed to non-incubated epididymal sperm sodium pyruvate could be omitted without much decline of the fertilization rate. When the denuded eggs were exposed to non-incubated sperm, penetration rates were very low (0 and 5%) in the absence of pyruvate. It appears that although lactate, pyruvate and glucose are all important for in vitro fertilization of rat eggs, pyruvate can be supplied by the follicular cells surrounding the eggs.

In vitro fertilization of hamster eggs by ejaculated or epididymal spermatozoa in the presence of seminal plasma or male accessory gland secretions was examined. There was no difference in the penetration rates, the time of sperm penetration and the optimal sperm concentration between ejaculated and epididymal spermatozoa. The swelling of the zona pellucida, a high incidence of polyspermy, distortion of the vitellus and the degeneration of eggs were observed after incubation of ejaculated or epididymal spermatozoa in the presence of seminal plasma or accessory gland secretions. Using ejaculated spermatozoa, the fertilization rates of eggs with or without follicular cells were similar but fertilization by epididymal spermatozoa was inhibited by secretion of the seminal vesicle or ventral prostrate gland but not by that of the coagulating gland or the dorsal prostrate.

Oocytes recovered at various times from immature rats treated with PMSG and HCG were incubated with capacitated epididymal spermatozoa of mature rats. In the presence of follicular cells, sperm penetration was not observed 4 hr after incubation in the oocytes at stages from the intact germinal vesicle to the chromatin mass, but 7 to 55% of oocytes were penetrated at stages from the condensed germainal vesicle to metaphase II. After the removal of follicular cells, 15 to 72% of the oocytes at any stage were penetrated. After further incubation for 15 hr, the proportion of penetrated oocytes increased from 8 to 98% from early to late stages and that of penetrated oocytes with a male and female pronucleus increased from 9 to 100% as maturation progressed. Although the average number of spermatozoa/oocyte was not correlated with its maturation, transformation of the sperm head into a male pronucleus was retarded or failed, especially in the younger oocytes. Following incubation in a defined medium for 13 hr, 85% of oocytes at the intact germinal vesicle stage matured to the stage of the first polar body formation, but only 18 to 22% of these mature oocytes were penetrated by spermatozoa and only a few of the penetrated oocytes cleaved into normal two-cell eggs. When eggs recovered from oviducts 14 to 20 hr after ovulation were exposed to capacitated spermatozoa, the proportion of penetrated eggs (86 to 98%) and that of polyspermic eggs (11 to 27%) were not related to the ages of the eggs, but failure of transformation of the sperm head and the proportion of abnormal eggs increased 14 to 20 hr after ovulation.

Newly ovulated eggs from immature deer mice (Peromyscus maniculatus and P. polionotus) and mature laboratory mice (Mus musculus) treated with PMSG and HCG were inseminated in vitro with spermatozoa recovered from the cauda epididymidis of mature males. The time required for capacitation of deer mouse sperm in culture was estimated to be about two to five hours based on the dispersal of sperm agglutination and increase of sperm motility. The rate of sperm penetration through the zona pellucida of deer mouse eggs by homologous or heterologous sperm was relatively high (72-91%) but that of laboratory mouse eggs by deer mouse sperm was low (20-21%). After penetration through the zona pellucida, a high proportion of deer mouse eggs (79-93%) were fertilized by homologous or heterologous deer mouse sperm but no laboratory mouse eggs were fertilized by sperm of two species of deer mice. The zona pellucida was dissolved in a higher proportion of laboratory mouse eggs cultured with P. maniculatus (45%) than with P. polionotus sperm (3.4%), but this did not happen by incubation of deer mouse eggs with homologous or heterologous sperm. It seems that there is little difference in sperm penetration and fertilization between these two closely related species of deer mice but the reactions between the mouse eggs and deer mouse sperm are quite different.

Mouse eggs (12-14%) and hamster eggs (87-94%) appear normal upon thawing after having been stored for 30 min to 25 h at -70 degrees C and -196 degrees C. While 89-98% of the normal appearing hamster eggs are capable of fertilization in vitro, only 4-13% of the mouse eggs can be fertilized. The ability of fertilized eggs to develop in culture and their viability upon transfer to pseudopregnant recipients is under investigation.

A single intraperitoneal injection of female mice with rabbit antisera against denuded mouse eggs and isolated zonae pellucidae inhibited fertilization for at least 25--30 days. Intraperitoneal injection appeared to be more effective and lasted longer than subcutaneous or intramuscular injection but oral administration had no effect. Such a passive immunization of females given after fertilization had no effect on embryonic development or the sex ratio of fetuses. The fertilizing capacity of mouse spermatozoa was suppressed by treatment of the sperm suspension with anti-egg but not with anti-zona serum. Treatment of mouse eggs with these antisera did not affect their development to blastocysts in culture but did inhibit the shedding of the zona pellucida and attachment to the dish.