M. C. Chang

Effects of Gossypol on the fertility of male rats, hamsters and rabbits.

Oral administration of Gossypol acetic acid induced sterility in male hamsters and rats when given at a dose level of 5 or 10 mg/kg daily for 12 weeks. This was shown by the appearance of dead and abnormal sperm in the male tract and the failure of pregnancy or decrease in implantation sites in the females mated to the Gossypol-treated males. Similar treatment of male rabbits at dose levels varying from 1.25 to 10 mg/kg and given for 5 to 14 weeks did not affect the average number of sperm per ejaculate, although the motility of sperm was poor during treatment in some bucks. The pregnancy rates and the proportion of implantation sites were also not affected by insemination of female rabbits with sperm from males during treatment with Gossypol.

Effect of Estrogens and Other Compounds as Oral Antifertility Agents on the Development of Rabbit Ova and Hamster Embryos

A-norsteroid (H241) estradiol cyclopentylpropionate (ECP) a derivative of diphenyl-dihydronaphthalene (U-11100A) and estrone were fed to rabbits 1 2 and 3 days after insemination in order to test antifertility activities. When examined 6 days after mating estrone H241 and ECP in the dose of .5 or 1 mg per rabbit induced 98-100% 90- 100% and 83-97% degeneration of ova respectively. The results of administration of U-11100A were inconsistent; 5 to 10 mg per rabbit induced 82-97% degeneration of ova. Feeding of ECP .5 mg per rabbit once on Day 1 or Day 3 was less effective than feeding 3 times on Days 1 2 and 3. Except for estrone and ECP feeding these compounds on Day s 6 7 and 8 at the time of implantation had no effect on implantation. Feeding the compounds 3 times before insemination did not inhibit ovulation or fertilization and did not indicate their antifertility activity. When the animals were fed on Day 1 and examined on Day 2 a certain percentage of ova was found in the uterus sooner than expected although the morphology (not necessarily the physiology) of the ova recovered from the tube or the uterus was normal. When hamsters were fed with these compounds (with the exception of U-11100A) 3 times on Days 1 2 and 3 after mating and examined on Days 12-13 a high percentage of embryonic degeneration was observed at a dose level of 1 mg per hamster indicating that a much higher dose level is required for the hamsters than for the rabbits. It is concluded that natural estrogen or any other compounds that have the activity of causing retention of ova in the tube expulsion of ova and fast transportation of ova into the uterus would be effective as an antifertility agent if administered soon after mating.(AUTHORS MODIFIED)

EFFECT OF INDOMETHACIN ON BLOOD PLASMA LEVELS OF LH AND TESTOSTERONE IN MALE RATS

Treatment of adult male rats with indomethacin, an inhibitor of prostaglandin (PG) synthesis, caused a significant decrease in LH and testosterone levels in the blood plasma and in the weight of the seminal vesicles, but the weight of the testes and ventral prostate, the levels of FSH in the plasma and fertility were not affected. The concentration of PGF in the blood plasma of the treated animals was reduced, even though measurable amounts of PGs were present in every group. The results of this study, together with the known effects of PG administration on LH release, suggest that the reduction of plasma LH levels in rats injected with indomethacin was due to decreased PG synthesis. It appears that PGs are normally involved in the regulation of LH release.

A-nor steroids as post-coital contraceptives in the hamster with special reference to the transport and degeneration of eggs.

Two A-nor steroids, Anordrin and H241, showed a marked antifertility effect when given orally to hamsters at 10 mg/kg/day for three or four days after mating. Further study indicated that the antifertility effect was due to a disturbance of egg transport, retarded development and degeneration of fertilized eggs.

Induction of Blastokinin by Oral Contraceptive Steroids: Implications for Fertility Control *

An investigation designed to examine whether or not various oral contraceptive steroids can induce blastokinin (BKN) a protein specific to the preimplantation uterus in rabbits is described. BKN was introduced in rabbits treated with oral contraceptive steroids such as chlormadinone acetate dimethisterone medroxyprogesterone acetate or 20 alpha-dihydroproges- terone. All these contraceptive steroids contain the carbon-19 methyl group. BKN induction was not accomplished by administration of norethindrone acetate norethynodrel estranol or mestranol all of which are 19-nor steroids. Results of the study suggest a new mode of action for certain oral contraceptives that is the establishment of asynchony between embryonic development and an altered uterine environment as a consequence of treatment with certain synthetic steroids during the follicular phase. It is proposed that a new class of steroidal contraceptives need not possess either progestational or estrogenic activities and would therefore be free of the associated side effects.

Termination of pregnancy in rabbit and mouse by Trichosanthin.

The effects of a single intraperitoneal injection of Trichosanthin in 6-, 10-, 17- or 22-day pregnant rabbit (2 mg/rabbit) or in 11-day pregnant mouse (50 micrograms/mouse) were studied. Trichosanthin induced abortion in 100% of the 17- or 22-day pregnant rabbits within 48-72 hours and decreased circulating progesterone (delta 4P)concentrations with 24 hrs. On the other hand, the same dose failed to terminate pregnancy in 6- or 10-day pregnant rabbits and caused no significant changes in circulating delta 4P levels. Exogenous delta 4P or prolactin + human chorionic gonadotropin given twice daily failed to reverse the Trichosanthin-induced termination of pregnancy. However, this resulted in a delay of fetal expulsion. It is concluded that Trichosanthin-induced termination of pregnancy is not solely a result of luteolysis but is likely to be due to its toxic effects on placenta, embryo or both. A dose of 50 microgram Trichosanthin given to 11-day pregnant mice resulted in a termination of pregnancy within 96 hours and also a significant decrease in delta 4P levels in 24 hrs. The ratio of delta 4P to 20 alpha-dihydroprogesterone was also decreased steadily after Trichosanthin administration.

FURTHER STUDIES ON THE TRICHOSANTHIN-INDUCED TERMINATION OF PREGNANCY

Trichosanthin (TCS), a protein from the root extract of Trichosanthis kirilowii, terminated pregnancy when injected once in 15-day pregnant rabbits (2 mg/doe) but failed to interrupt pregnancy in 12-day pregnant rabbits even at higher doses. In vitro release of progesterone (delta 4P) from the maternal or fetal placental tissue into the incubation medium was not affected by TCS. When the distribution of 125I-TCS was traced in 12-day pregnant mice, persistently higher concentration of 125I-radioactivity was detected in the kidney. By contrast, in other organs, including the reproductive organs, blood serum and amniotic fluid 125I-radioactivity declined between 12-48 h after treatment. The low 125I-radioactivity in both the reproductive organs and amniotic fluid suggests a possible barrier between the embryo and maternal blood. It is suggested that TCS might be acting directly on the placental unit, causing fetal death and dislodging of the placenta. Administration of TCS to PD-19 mice or PD-28 rabbits resulted in premature delivery. This effect of TCS in pregnant mice was comparable to the administration of PGF2 alpha.

Suppression of Pregnancy in the Rabbit by Subcutaneous Implantation of Silastic Tubes Containing Various Estrogenic Compounds

This study attempts to find out: 1) whether normal fertility can be resumed after the removal of subcutaneous implants containing various estrogenic compounds; 2) whether other compounds with estrogenic activities have such effects; and 3) whether such effect is on the embryo or the female tract. 10-mm long sealed silastic tubes were used to implant .5 or 2 mg of ethinyl estradiol (EE) diethylstilbestrol or quinestrol (ethinyl estradiol 3-cyclopentyl ether) under the skin in the neck region of adult rabbits. Subsequently the rabbits were bred or inseminated. Fertility reverted to normal after removal of .5 or 2 mg of ethinyl estradiol implanted for 42 days. After 10 to 25 days of implantation of .5 mg stilbestrol 17-21% of eggs developed into normal blastocysts and embryos but none developed after implantation of 2 mg of stilbestrol. When animals were inseminated 31 days after stilbestrol implantation no eggs developed into embryos although 14% developed into blastocysts. Insemination 10-25 days after implantation of 2 mg quinestrol resulted in a complete degeneration of eggs but after 33 days 47% of the eggs developed into normal blastocysts and 61% into normal embryos. When early blastocysts recovered from estrogen-treated rabbits were transferred to the uteri of untreated rabbits development of embryos occurred; when transferred from untreated to treated rabbits none developed into embryos. Compounds with estrogenic activity are thought to cause degeneration of eggs by too fast or too slow transport from the tube to the uterus and also to cause a hostility of the uterus to early blastocysts. The effectiveness of estrogens to prevent pregnancy is dependent upon the compound the dosage and the absorption rate of the compound.

Effects Of Estriol Treatment of Male Rats and Mice on the Fertility of Mated Females

To determine the antifertility effect on female rats and mice of mating with estrogen-treated males 2 studies with each animal were organized. In the first study 31 Holtzman rats 7-8 months old received daily subcutaneous injections of 3.2 or 4.0 mg estriol in corn oil. 13 rats served as controls. On the fourth eighth and twelfth day of injection they were mated with proestrous females. All females were killed 9 days after mating and the number of implantation sites were recorded. Body testicular and accessory gland weights were determined by autopsies in the males. In addition 8 younger rats 75 days old were treated similarly with 2 mg estriol mated and examined. 3 rats of this age were used as controls. In the second study 23 male mice received daily subcutaneous injections of 0.25 or 1 mg estriol mated and examined. 20 mice were used as controls. Results revealed no significant decrease in the fertility of male rats treated with 3.2 mg estriol after 8 days of treatment but complete sterility resulted after 12 days. Similar results resulted from the 4 mg treated rats who also showed a decrease in sexual activity. 7 of the 8 younger rats did not mate but the eighth proved fertile. The treated mice showed greatly reduced libido and sterility after 10 days of treatment. The weights of ventral prostate seminal vesicles and coagulating glands were significantly reduced in the treated rats (p less than .01) and mice (p less than .05). It is concluded that age may be an important factor in determining the antifertility effect of estriol in rats. Evidence of seminal transfer of estrogen by this treatment was not found. The estrogen did produce sterility after 10 days or more of treatment possibly by reducing the secretory activity of the seminal vesicles affecting the cauda epididymidis or preventing sperm maturation.

Fertilizing Ability of Hamster Spermatozoa Following Subcutaneous Implantation or Insertion into Seminal Vesicles of Silastic Tubes Containing Estradiol Benzoate

A comparison of the effects of estradiol benzoate(EB) on the fertilizing ability of hamster epididymal spermatozoa when Silastic tubes containing estradiol benzoate were either implanted subcutaneously or inserted bilaterally into seminal vesicles was undertaken. In control animals empty tubes were either implanted or inserted. Fertility tests were conducted with females in estrus. Fertilizing ability showed a complete loss after 12 days while control animals with empty tubes retained their normal fertilizing ability. 10 weeks after removal of the estrogen implants the animals regained normal fertilizing ability. No increase occurred in the uterine weights of females bred with estrogen-treated males 24 or 36 hr. previously. 4 days following insertion of EB implants into seminal vesicles or subcutaneous implantation the weights of the testes and caudae epididymides decreased significantly (p less than .05); further regression occurred after 8 and 12 days of estrogen treatment.