Effects of cadmium chloride on ovulation and on induction of sterility in the female golden hamster.

The effects of single subcutaneous injections of cadmium chloride (CdCl2) on ovulation, egg transport and early pregnancy in the golden hamster were studied. While a single dose of 1.25 or 2.5 mg/kg of CdCl2 imposed none to marginal effects, hamsters treated with 5 or 10 mg/kg CdCl2 experienced a period of sterility ranging from 11-69 (5 mg/kg) or 46-71 (10 mg/kg) days, followed by a normal pregnancy. Administration of CdCl2 also induced ovulation inhibition which was dose-and time-dependent. A minimum dose of 5 mg/kg CdCl2 was needed to inhibit ovulation. When CdCl2 was given closer to the time of the luteinizing hormone (LH) surge on the day of proestrus, a more pronounced effect on ovulation was recorded. The incidence of failure of ovulation was associated with decreased progesterone levels in serum and inflammation, hemorrhages and necrosis in the ovary. However, the ovarian lesions lasted less than 4 days. The results indicate that CdCl2 inhibits ovulation when administered close to the time of ovulation, whereas its influence on pregnancy is pronounced but temporary.

Maintenance of pregnancy in rabbits treated with long-lasting progestins.

Fertilized eggs from rabbits 1 or 2.5 days after insemination were transferred to the oviduct or uterine horn of recipients that had received a single subcutaneous injection of 20 or 30 mg of one of seven long-acting progestins. The rabbits were observed daily, and the number of implantation sites was determined 10 or 11 days after egg transfer. No implantation sites were recorded in the recipient does treated with progesterone or ZK-5623 (Schering AG, Berlin, West Germany), a nor-steroid compound. Thirty-nine percent and 51% of the transferred eggs implanted in the recipient does treated with ZK-5410 (Schering AG) and chloromadinone acetate (Eli Lilly & Company, Indianapolis, IN), respectively. However, most of the pregnant animals aborted 14 to 20 days after egg transfer. The pregnancy was either maintained to term or was prolonged beyond the normal gestation length in the does treated with other compounds, ZK-53915, ZK-9349 (Schering AG) or Depo-Provera (Upjohn Company, Kalamazoo, MI). The young delivered after a subcutaneous injection of 17 beta-estradiol and oxytocin were found normal and active.

Cadmium: its effects on ovulation, egg transport and pregnancy in the rabbit.

The subcutaneous administration of cadmium chloride (CdCl2) 24 h before hCG-induced ovulation in rabbits sacrificed 14 h after induction of ovulation leads to a dose-dependent decrease in the number of oviductal eggs and the number of eggs shed. While a dose of 1.25 mg/kg CdCl2 imposed no effect, only 50-67% of the dogs treated with 2.5 or 5 mg/kg CdCl2 ovulated and of those ovulated eggs 35.6 and 45%, respectively, were found in the oviducts. At a dose of 7.5 mg/kg CdCl2, a higher proportion (57.1%) of the does failed to ovulate and only 16% of the eggs were recovered from the oviducts. Ligature of the uterotubal junction failed to increase the rate of eggs recovered from the oviducts. A sharp increase in serum progesterone (delta4 P) level within 2 h after induction of ovulation suggests that the reduction in the percentage of eggs recovered from the oviducts probably occurred due to altered fimbrial functions. On the other hand, in does treated with 5 mg/kg CdCl2 1 or 7 days before mating, pregnancy was interrupted in 60 and 75% of the does together with reduced delta4 P concentration and the conception rate (no. of implantations/CL) was significantly reduced. CdCl2 given 21 days before or 3 days after mating did not affect pregnancy and delta4 P concentration remained unaltered. Results suggest that the developing or mature follicles are sensitive to CdCl2 treatment. Once the CL is formed, the effect of CdCl is reduced. The receptivity of CdCl2-treated females indicates that probably the circulating estrogens were not affected and the ability to ovulate in response to hCG and mating reflects that, at least at the doses used, the hypothalamic-pituitary gonads functions are functional in the rabbit.

Antifertility effects of gossypol in male hamsters.

In the male hamster, administration of 10 or 15 mg/kg/day of gossypol for 5 weeks did not alter the serum concentration of testosterone (T), prolactin (PRL), and luteinizing hormone (LH) or fertility. However, the total sperm population was significantly reduced in the males treated with 15 mg/kg/day of gossypol, and total sterility occurred within 8 weeks. On the other hand, males treated with 10 mg/kg/day of gossypol were sterile after 10 weeks of treatment. The T and total sperm population along the reproductive tract in these infertile males was significantly reduced. During the treatment period no change in body weight or weight of testes or seminal vesicles was observed. A full regaining of fertility, serum T level, and sperm population to the normal range were recorded within 8 nd 14 weeks (15 and 10 mg/kg/day, respectively) after the cessation of treatment.

The concentration of progesterone, 20 alpha-dihydroprogesterone, testosterone, oestrone and oestradiol-17 beta in serum, amniotic fluid and placental tissue of pregnant rabbits.

The concentrations of progesterone (delta 4P), 20 alpha-dihydroprogesterone (20 alpha-DHP), testosterone (T), oestrone (E1) and oestradiol-17 beta (E2 beta) in peripheral blood serum (PBS), amniotic fluid (AF) and placental tissue of rabbits during gestation were determined by radioimmunoassay. The placenta of the 10-day pregnant rabbit was fragile and composed mainly of maternal tissue. By the 12th day of pregnancy it was separable into maternal and foetal placentae. The mean concentration of delta 4P in PBS rose from 200 pg/ml (day 1 pregnancy) to 17--21 ng/ml (days 10--15) and decreased gradually to 1 ng/ml a few hours before parturition. The 20 alpha-DHP in PBS also showed an increase from 1.5 ng/mg (day 1) to 12 ng/ml (day 6) but fluctuated thereafter. The concentration of 20 alpha-DHP in the PBS tended to be lower than that of delta 4 P during pregnancy until the regression of the corpus luteum. An interesting observation was an increase of T on days 6--8 of pregnancy, the time when implantation occurs. The concentrations of E1 and E2 beta in PBS remained very low throughout pregnancy. delta 4P and 20 alpha-DHP in AF ranged between 25 pg to 1 ng/ml and in no case during the course of pregnancy were the levels of T, E1 and E2 beta in AF higher than in PBS. Where the maternal placental delta 4P content remained between 1--2 ng/placenta, the foetal placenta delta 4P rose to a level of 15 ng/placenta by day 31 of pregnancy. A similar trend was recorded for 20 alpha-DHP content. It is concluded that although a parallelism between PBS and myometrial steroid concentration was observed, no relationship could be drawn between the concentrations of steroid in PBS and those of the placental tissue and AF.

The pituitary-ovarian complex in the aged anoestrous golden hamster.

Plasma and pituitary hormones of young (3--5 months of age) dioestrous hamsters with normal cycles and aged (13--17 months of age) anoestrous hamsters were compared. The anoestrous hamsters exhibited lower plasma values of progesterone (P less than 0.001), oestradiol-17 beta (P less than 0.005) and prolactin (P less than 0.001) and higher levels of plasma gonadotrophins (P less than 0.001) than did the dioestrous animals. Pituitary concentrations of LH were higher (P less than 0.005) in anoestrous hamsters, but pituitary FSH and prolactin values did not differ. In another series of experiments three groups of hamsters (3--5- and 13--17-month-old with normal cycles and 13--17-month-old in anoestrus) were ovariectomized. Blood samples were taken by cardiac puncture every 3--4 weeks after receiving s.c. injections of oestradiol-17 beta (1 or 10 micrograms/100 g body wt) for 2 or 9 consecutive days. The markedly lower levels of gonadotrophins in aged anoestrous hamsters indicated that the hypothalamic-hypophysial complex was incapable of responding to the same degree as it did in young and aged cyclic animals. Prolactin values were similarly depressed in all 3 groups. Oestradiol-17 beta treatment caused reduced gonadotrophin and increasing prolactin concentrations in all 3 groups. These results indicate that the ovaries of the senescent anoestrous hamster produce less steroids and suggest that age-related changes in the hypothalamic-hypophysial complex are largely responsible for the cessation of regular oestrous cycles.

Effects of 17 beta-hydroxy-7 alpha-methylandrost-5-en-3-one on early pregnancy in the rat.

A single sc injection of 1 mg 17-beta-hydroxy-7 alpha-methylandrost-5-en-3-one (RMI-12,936) given a few hours after mating interrupted pregnancy in all the treated rats. Circulating progesterone (delta 4P) levels were higher in RMI-12,936 treated females than in controls on the corresponding days during the course of termination of pregnancy. Higher levels of delta 4P were recorded on day-4 (P less than 0.01) and day-6 (P less than 0.05) of pregnancy. In addition to the changes in serum delta 4P, an acceleration of egg transport was encountered. The eggs were prematurely expelled from the uterus within 48 h of the treatment. Although the oestrous cycle of the RMI-12,935 treated females was disturbed, they were found sexually receptive. Successful matings resulting in normal gestation and morphologically normal foetuses were recorded 20-26 days after RMI-12,936 induced pregnancy termination. These results suggest that in addition to its mid-pregnancy terminating effect, RMI-12,936 is capable of interrupting early pregnancy when given soon after mating in the rat. The safety and efficacy of this compound as a post-coital contraceptive deserves further investigation.

Effects of Colprone on in vitro release of androgens from the reproductive organs of the male rat.

Effects of medrogestone (6,17 alpha-dimethyl-4-,6-pregnadiene-3, 20-dione; Colprone) on the release of testosterone (T) and 5 alpha-dehydrotestosterone (5 alpha-DHT) in the reproductive organs of the male rat were tested in vitro. The minced testes released twice as much T into the medium when incubated in the presence of 50 mIU/ml of hCG. The release of T was inhibited significantly when 10 or 50 micrometers Colprone was added to the incubation medium. More 5 alpha-DHT was released into the medium from the testes mince in the presence of hCG, while the addition of 10 or 50 micrometers of Colprone inhibited 5 alpha-DHT release as compared to hCG controls. On the other hand, hCG failed to stimulate the release of T and 5 alpha-DHT from the minced caput and cauda epididymis and ventral prostate. The addition of Colprone (10--50 micrometers/ml) to the medium containing caput epididymis or ventral prostate resulted in a significant inhibition of T and 5 alpha-DHT. However, no significant change in the rate of T or 5 alpha-DHT release was recorded in medium containing cauda epididymis. In spite of its anti-androgenic effects reported by several investigators, Colprone in the present study has shown differential effects on T and 5 alpha-DHT release from different reproductive tissues of the male rat.

Effects of prostaglandin-F2 alpha and a plant protein "Trichosanthin", on 10-day pregnant rabbits.

The effect of Trichosanthin (TCS), a protein obtained from the roots of Trichosanthis kirilowii, alone or in combination with prostaglandin-F2 alpha (PGF2 alpha; Tromethamine salt) on the termination of pregnancy in rabbits was investigated. Intraperitoneal injection of 1 or 2 mg TCS/10-day pregnant rabbits neither altered the serum progesterone (delta 4P) level nor interrupted pregnancy. Doses of 0.5 or 0.25 mg PGF2 alpha incorporated in a Silastic-PVP tube and inserted intravaginally, terminated pregnancy within 72 h of treatment in 75 and 16% of the treated does, respectively. By contrast, does treated on day-10 of pregnancy with a combination of a non-effective dose of TCS (1 mg) and a sub-effective dose of PGF2 alpha (0.25 mg) terminated pregnancy in all the treated animals as no live embryos were found within 3 days of treatment. Pregnancy interruption was associated with a significant reduction of serum delta 4P and delta 4P/20 alpha-dihydroprogesterone (20 alpha-DHP) ratio. The present study indicates that TCS and PGF2 alpha act synergistically, rendering the termination of 10-day pregnancy possible in the rabbit. It was also noted that pregnancy cannot be maintained when the serum delta 4 P level drops below 4 ng/ml and delta 4P/20 alpha-DHP falls lower than 0.6 in the 10-day pregnant rabbit.

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.

Temporal relationship between progestin and luteinizing hormone concentrations in pseudopregnant rats treated with prostaglandin-F2 alpha.

The temporal changes in progesterone (delta 4P), 20 alpha-dihydroprogesterone (20 alpha-DHP) and luteinizing hormone (LH) concentrations in pseudopregnant (PSP) rats after treatment with a single subcutaneous Silastic-PVP tube containing 600 micrograms PGF2 alpha were correlated. Progesterone levels fell and LH levels rose significantly 2h after initiation of treatment, while 20 alpha-DHP levels were found to increase significantly 12h after treatment. Since the changes in delta 4P and LH concentrations occurred concurrently, it seems that the increase in LH levels could have been due to a direct effect of PGF2 alpha on the ovary causing a reduction in delta 4P and thus a negative feedback effect on LH release. Alternatively, PGF2 alpha might exert a direct effect on LH secretion at the hypothalamic-pituitary level.

Effects of prostaglandin F2 alpha and an antiprogestational steroid, RMI 12,936, on rat pregnancy.

The abortifacient efficacy of RMI 12,936 (17 beta-hydroxy-7 alpha-methyl androst-5-en-3-one), a synthetic steroid, alone or in combination with prostaglandin-f2 alpha (PGF2 alpha), was studied in the rat. Administration on day-8 of pregnancy (PD-8) of 0.25 mg RMI 12,936/rat neither altered the progesterone (delta 4P) levels, the relative ovarian weight (ROW) nor terminated pregnancy. Doses of 2.0, 1.0 or 0.5 mg RMI 12,936/rat, when given on PD-8, terminated pregnancy in 100% of the animals. Highly significant increases in ROW and serum alpha 4P level were recorded on PD-14. However, delta 4P/20 alpha-dihydroprogesterone ratio remained unaltered. These results suggest that RMI 12,936 increased luteal activity which contributed to the enhanced serum delta 4P levels and ROW. Bilateral insertion of Silastic-PVP tubes containing 75 micrograms PGF2 alpha/tube into each uterine horn on PD-10 did not disturb pregnancy, whereas similar tubes given on PD-10 to rats treated with 1.0 mg RMI 12,936 on PD-8 expelled the embryos and placental tissue on PD-11. This phenomenon was attributed to a decreased serum delta 4P caused by PGF2 alpha, allowing the expulsion of uterine contents. A time lapse of 17--35 days was needed for the rats to regain fertility in terms of normal gestation period and offsprings. Although expulsion of conceptus was facilitated by PGF2 alpha in the RMI 12,936 treated rats, PGF2 alpha treatment did not alter the time lapse between RMI 12,936 treatment and subsequent conception. It is concluded that RMI 12,936 is feto-toxic; it possesses high contraceptive activity and its abortifacient efficacy can be improved by PGF2 alpha.

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.

Effects of 17 beta-hydroxy-7 alpha-methylandrost-5-en-3-one (RMI 12,936) on pregnant rabbits.

A single subcutaneous injection of RMI 12,936 interrupted 8- and 15-day pregnancy in the rabbit. A dose dependent loss of embryos was observed. Higher doses of RMI 12,936 were needed to terminate pregnancy in 8-day pregnant (74% efficacy with 10 mg/kg) than in 15-day pregnant rabbits (100% efficacy with 8 mg/kg). Termination of pregnancy by RMI 12,936 was accompanied by a significant increase of progesterone (delta 4 p) in rabbits receiving 10 mg/kg RMI 12,936 on day 8 of pregnancy. On the other hand, treatment with 8 mg/kg RMI 12,936 on day 15 of pregnancy did not alter the delta 4 p levels, but induced a significant decrease in 20 alpha-dihydroprogesterone. In these two treatment groups, a consistent and highly significant increase of serum testosterone (T) was observed. The increase of T value possibly was due to a decrease in aromatizing ability of the follicles after RMI 12, 936 treatment and might have contributed towards the interruption of pregnancy. The failure of RMI 12,936-treated rabbits to ovulate even after hCG administration suggests that his compound might have lowered the sensitivity of the mature follicles to LH stimulation.

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.

Temporary sterility induced by intrascrotal deposition of silastic-polyvinylpyrrolidone-prostaglandin F2alpha tubes in the rabbit: effect on fetal survival after regain of fertility.

In male rabbits of proven fertility, the intrascrotal deposition of two Silastic-polyvinylpyrrolidone tubes containing 3 mg of prostaglandin F2alpha (PGF2alpha)/tube induced within 2 to 4 weeks temporary sterility which lasted for 10 to 14 weeks. Associated with induced sterility were reduction in testicular weight, increase in abnormal spermatozoa (8% to 78% versus 0 to 3.7%), and reduction in sperm motility along the reproductive tract and in the semen for a period of 6 to 7 weeks. During the period of temporary sterility the weight of the epididymis, the sexual drive, and semen volume remained unaltered. Normal fertility was associated with an increase in testicular weight, reduction in the proportion of abnormal sperm, and improved sperm motility. In addition to an altered spermatogenesis, the integrity of mature spermatozoa seemed to be severely affected after PGF2alpha treatment. The reduced number of viable young sired by males that recovered from temporary sterility (pregnancy wastage 35%) as compared with sham-treated controls (pregnancy wastage 3%) suggests that a small percentage of spermatozoa might still be defective at the time of testing. A longer waiting period might be needed to ensure a completely normal reproductive process.

Age dependent changes in the sperm population and fertility in the male rat.

The entire reproductive tract was devoid of spermatozoa during the first 42 days of life. The first appearance of spermatozoa was detected in the caput epididymidis of 45-day old rats and in the cauda epididymidis of 52-day or older rats. The number of spermatozoa in the reproductive tract increased with age. The sperm population reached its maximum in the caput epididymidis by day-72 and in the caud epididymidis by day-100. These high levels were maintained beyond 450 days of age. Fifty-two day old male rats failed to impregnate the female. Although male rats of 62-500 days were able to sire litters, the peak reproductive period was found to be between days 100-270, during which the number of young per litter ranged between 11-16. Towards advanced age the litter size dropped to 8. The lower fertility in the males of advanced age might be due to a lower proportion of fertile spermatozoa, a change in the physiology of epididymis or an altered sperm transport in the reproductive tract.

Variations in serum androgens, estrogens, progestins, gonadotropins and prolactin levels in male rats from prepubertal to advanced age.

The concentrations of blood serum steroids from 12 to 450 days old male rats were determined by radioimmunoassay. Testosterone (T) was low (270 pg to less than 1 ng/ml) until day 42; adult levels (3--4 ng/ml) were attained by day 62 and declined tradually with advanced age. 5 alpha-dihydrotestosterone (5 alpha-DHT) did not change markedly (90--160 pg/ml) from prepubertal to advanced age. Except for a small peak on day 22, androstenedione (delta 4 A) levels ranged between 400-500 pg/ml in the adult but declined in older males. Progesterone (delta 4 P) rose steadily to a mean of 5.46 ng/ml at 52 days of age and dropped thereafter. High levels of estrone (268 +/- 38 pg/ml) and estradiol-17 beta (2.76 +/- 0.28 ng/ml) in 12 days old males are in contrast to the low estrogens (20-35 pg/ml) in adult animals. Both T/5 alpha-DHT and total T/estrogen ratios were low before puberty, increased in adults and decreased towards old age. The interplay between gonadotropin and prolactin, which exhibited reciprocal changes in the regulation of steroid production by the gonads with age, are discussed.