J. B. Thiersch

Therapeutic Abortions with a Folic Acid Antagonist, 4-Aminopteroylglutamic Acid (4-Amino P. G. A.) Administered by the Oral Route

1. Oral doses of 6 to 12 mg. 4-amino P. G. A. induced fetal death in the first trimester of pregnancy followed by spontaneous delivery of the products of conception in 10 out of 12 cases treated. 2. The doses lethal to the embryos had only a slight and transitory depressing effect on the hemoglobin and white blood counts of the mothers. 3. In three instances, a second course of the drug was given needlessly because of persistent positive “pregnancy tests.” 4. The lesions found in the younger fetuses were depression of hematopoiesis, necrosis of the liver, adrenals, and intestinal epithelia. 5. In three older fetuses the drug failed to produce immediate death, but apparently induced malformations of the cranium. One of these fetuses died later and was delivered spontaneously. The other two were alive when surgically removed. 6. The study shows that usage of the drug to induce abortions should in the absence of reliable pregnancy tests be limited to patients in whom surgical intervention is possible to avoid malformations. 7. The action of the drug is regarded as entirely “antifolic,” indicating the importance of folic acid in the early embryonic life. 8. The probable role of a folic acid deficiency in certain spontaneous abortions, in the development of malformations, and the importance of folic acid in seasonal breeding in some species is suggested by this study.

Male Reproductive Tract, Spermatophores and Spermatophoric Reaction in the Giant Octopus of the North Pacific, Octopus dofleini martini

The male reproductive tract of Octopus dofleini martini lies enclosed in the genital bag, inside the mantle cavity. It consists of the testis, vas deferens proximale, spermatophoric gland system I (seminal vesicle), spermatophoric gland system II (prostate), vas deferens distale, spermatophoric sac (Needham’s sac) and the terminal spermatophoric duct. The spermatozoa, which upon leaving the testis are as yet not encased, pass first into the vas deferens proximale, which in its gross appearance resembles the epididymis; they then traverse the two tubular spermatophoric gland systems I and II, where they become encased into the spermatophores. Subsequently the spermatophores pass through the vas deferens distale into the spindle-shaped spermatophoric sac, and from there they enter singly the terminal spermatophoric duct consisting of the diverticulum and terminal organ or ‘penis’. The slender cylindrical body of the spermatophore is about 1 m long and consists of two parts, approximately equal in length. The thicker ‘proximal’ ‘male-oriented’ portion, which emerges first from the orifice of the ‘penis’, contains the tightly coiled sperm rope suspended in a viscous and transparent fluid, the spermatophoric plasma; the thinner ‘distal’ ‘femaleoriented’ half of the spermatophore is taken up by the rod-shaped, hyaline core of the ejaculatory apparatus. In between is located the cement body, and the amber-coloured cement liquid. At the distal end of the spermatophore the outer coating forms a cap and a filamentous appendage, the cap-thread. Chemical analyses were performed on spermatozoa, spermatophoric plasma, cement liquid, outer tunic and the hyaline core of the ejaculatory apparatus, obtained from freshly recovered spermatophores. Glycogen was identified as a major constituent of spermatozoa. The extraordinarily high dry-weight content of spermatophoric plasma (nearly 30%) was shown to be largely due to bound amino sugar, carbohydrate, peptide and protein. A peptide separated from the spermatophoric plasma by ultrafiltration was found to be made up to a great extent of aspartic acid and serine. The outer tunic, a tough and elastic membrane, which envelops the body of the spermatophore, was shown to consist mostly of a protein which is rich in proline, lysine, aspartic acid and threonine. The mechanics of the spermatophoric reaction in vitro have been studied in spermatophores extracted manually from the male octopus and placed in sea-water. The complete spermatophoric reaction under such conditions lasted 1 to 2 h. During that interval the sperm rope gradually advanced a distance of about 1 m, from the proximal towards the distal end of the spermatophore. The terminal phase of this process involved an evagination of the ejaculatory apparatus, followed by a rapid movement of the sperm rope; as the sperm rope entered the end portion of the spermatophore, the latter ballooned out into an egg-shaped bladder. Among the factors which contribute to the formation of the spermatophoric bladder, the most important ones are (i) the elasticity of the membranes of the spermatophore, (ii) the extrusion, and subsequently evagination, of the ejaculatory apparatus, and (iii) the influx of sea-water into the spermatophore’s body which causes an approximately fivefold increase in the volume of spermatophoric plasma. Concomitantly with the uptake of sea-water, the dry weight of the spermatophorie plasma declines but the sodium chloride concentration increases. However, the osmolality of the spermatophoric plasma, as assessed by freezingpomt depression, is not altered during the spermatophoric reaction. Events at copulation, that is under conditions in vivo, closely resembled those observed in spermatophores undergoing a spermatophoric reaction in vitro. An interval of 2 to 3 h usually elapsed from the time when the male, using his hectocotylized arm, mserted mto the female the distal (female-oriented) end of a spermatophore, to the moment of the males withdrawal. After accomplished copulation two spermatophores were usually found firmly lodged in the two oviducts. The sperm-free remnants of the spermatophore bodies dangled free from the orifices of the oviducts. Upon dissection of recently mated females the spermatophoric bladder was usually found within the oviduct, held firmly in position by the evaginated ejaculatory apparatus.

Studies on the mucosa of postmenopausal oviducts: surface appearance ciliary activity and the effect of estrogen treatment.

The epithelial lining of the human oviduct is known to be responsive to the fluctuating hormonal levels of the normal menstrual cycle, but its response to the changes in hormonal climate at the time of the menopause is not clearly defined. In this study the oviducts of nine postmenopausal patients were obtained at the time of abdominal hysterectomy, and the lining epithelium was studied by scanning electron microscopy. The activity of cilia on the fresh tissue was assessed by their ability to transport particulate matter applied to the epithelial surface. The fimbriae of oviducts from women who had received little or no estrogen treatment before surgery showed a significant deciliation of the epithelium, compared with specimens from premenopausal patients, and even showed some sloughing of cells from the surface. The secretory cells appeared inactive. However, the specimens from patients who had been treated with estrogen for periods of 1 year or more showed a remarkable maintenance of the epithelium, with the proportion of ciliated cells remaining almost as high as in premenopausal oviducts, even as late as 25 years after the menopause. The ampullar and isthmic portions showed less obvious changes. Cilia in oviducts from the former group (short-term or no treatment) were incapable of transporting 15-mum microspheres or lycopodium spores applied to the epithelial surface, whereas the oviductal cilia obtained from patients under long-term estrogen therapy showed efficient transport of particulate matter. The results are discussed in relation to earlier conflicting reports on the postmenopausal oviduct.

D(—)-Lactic Acid and D(—)-Lactate Dehydrogenase in Octopus Spermatozoa

The spermatozoa of Octopus dofleini martini produce anaerobically D(—)-lactic acid and possess a very active D(—)-lactate dehydrogenase. In this respect, while resembling certain microorganisms, they differ strikingly from mammalian spermatozoa which produce L(+)-lactic acid and contain L(+)-lactate dehydrogenase.

Effect of O-Diazo Acetyl-L-Serine on Rat Litter.∗

Summary 1. The observation that AZS affects the rat fetus in utero is confirmed and greatly extended. 2. AZS acts on the fetus directly—and not on the ovary, placenta or pituitary. 3. AZS efficiently destroys the entire litter of rats in doses of: (a) 5 mg/kg at the time following implantation (7th and 8th days of gestation); (b) 10 mg/kg at midterm (11th and 12th days of gestation); without affecting the mothers adversely. 4. This drug effect is sharply limited in time. 5. Combination of 6MP and AZS was effective in litter destruction in smaller doses than either compound alone. 6. Neither progesterone nor adenine protected the litter against AZS. 7. AZS given before mating did not delay it, nor did it affect the subsequent litter. 8. Rats aborted 4 and 5 consecutive times with AZS showed no accumulative toxicity nor impairment of their fertility. 9. Litters raised from rats aborted 4 or 5 times were normal in all respects and produced normal litters.

Effect of 2,4,6, Triamino-“S”-Triazine (TR), 2,4,6 “Tris” (Ethyleneimino)-“S”-Triazine (TEM) and N, N′, N″-Triethylenephosphoramide (TEPA) on Rat Litter in Utero.

Summary 1. The effect of TR, TEM and TEPA on rat fetus and litter in utero was studied with 2 doses of the 5 LD50 on 4th and 5th, or 7th and 8th, or 11th and 12th days of gestation. 2. TR had no effect on the rat litter and can be regarded as a control series to TEM. 3. TEM had its maximum effect at the time of implantation, but failed to destroy the entire litters. An increase in dosage from .3 mg/kg to .5 mg/kg given at the time of implantation led to 100% litter destruction. 4. Progesterone administration protected 40% of the fetuses against otherwise lethal doses of TEM. 5. Rats treated with TEM displayed uniformly smaller placentas than normal. 6. TEM in combination with AZS given on the 15th and 16th days of gestation led to much stunting of the fetuses, to 32% of fetal death, but in no instance to complete litter destruction. 7. Freshly prepared TEPA destroyed all litters when given on the 7th and 8th, or 11th and 12th days of gestation. 8. The malformations observed with TEM and TEPA consisted of cranial defects and abnormalities of facial structures. 9. TEM and TEPA in doses used affected adversely the bone marrow and lymphoid system of the mother animals.

Effect of Podophyllin (P) and Podophyllotoxine (PT) on the Rat Litter in utero.

Summary The single LD50 for the adult rat was, for P or PT, approximately 15 mg/kg i.p. Toxicity for the rat fetus was tested in groups of pregnant rats injected with varying doses and at different gestation days. The compounds were found to be more toxic to the fetuses than to the mothers, with a peak of fetus sensitivity around midterm (11th and 12th g.d.). Doses of 5 mg/kg i.p., administered on the 11th and 12th g.d. destroyed more than 90% of all fetuses. Doses of 0.5 mg/kg or 1 mg/kg i.p., given daily over longer periods of gestation, led to a loss of 18% to 75% of all fetuses. General stunting, but no gross malformation, was observed in surviving fetuses.

Effect of 6 Diazo 5 Oxo L-Norleucine (DON) on the Rat Litter in Utero.

Summary DON affects adversely the fetus when given from time of implantation to mid-term, and two doses of .5 mg/kg during this time will result in complete litter destruction. The action of DON appears to be directly on the fetus and not on placenta, ovary or pituitary. Progesterone given prior to DON administration did not protect the litters. Large doses of adenine sulfate given prior to DON administration protected the litters to a large degree. Repeated complete litter destruction in the same animals gave no evidence of cumulative toxicity, nor did DON adversely affect the fertility of the mother rats, or subsequent offspring.

Spermatozoa of the Giant Octopus of the North Pacific Octopus dofleini martini

Spermatozoa of the giant octopus, as obtained from ruptured metre-long spermatophores, are only feebly motile. The total length of a spermatozoon is 1/2 mm, most of it due to the tail. The sperm-head, slim and oblong, is composed of a highly condensed nucleus and a cork-screw shaped acrosome. A striking feature, which distinguishes octopus spermatozoa from those of mammals, is the presence of a large amount of glycogen, concentrated mainly around the sperm-head. This, together with the occurrence of high phosphorylase and phosphoghlcomutase activity, indicates that glycogenolysis represents a pathway of carbohydrate metabolism in octopus spermatozoa. Both glucose-6-phosphate isomerase and glucose-6-phosphate dehydrogenase were demonstrated in extracts of prepared sperm- homogenates, which suggests that octopus spermatozoa may be capable of metabolizing glucose-6-phosphate along the oxidative as well as the glycolytic pathway.

Effect of 2-6 Diaminopurine (2-6 DP) 6 Chlorpurine (CIP) and Thioguanine (ThG) on Rat Litter in Utero.

Summary 1. 2-6-DP, CIP and ThG were given to groups of pregnant rats in 2 doses of 5 LD50, not toxic to the mother animals. The compounds were given on the 4th and 5th, or on the 7th and 8th, or on the 11th and 12th days of gestation. 2. 2-6-DP showed a peak of action on the fetuses and litters when given before implantation, destroying 75% of all the fetuses, but only 57% of all the litters. 3. CIP showed a peak of action on the 7th and 8 th days, destroying all fetuses and litters. Treatment on days 4 and 5 of gestation destroyed 69% of the fetuses, but only 32% of all litters. At midterm, no litter was completely destroyed, and only 10% of all the fetuses were resorbed. However 50% of all survivors were stunted and malformed. 4. ThG was the most toxic compound to the fetuses. At the time of implantation it destroyed all fetuses. On days 4 and 5 of gestation, it led to resorption of 75% of the fetuses, but only to 10% complete litter destruction. At midterm it still destroyed 61% of all fetuses, and 43% of all litters, while stunting 37% of all survivors. 5. The lethal action of CIP and ThG on the rat litter at implantation time could not be prevented by progesterone administration.