Showing papers by "Worcester Foundation for Biomedical Research published in 1968"

Ultrastructural changes in the sperm head during fertilization in the rabbit

Abstract: Spermatozoa have been observed with the electron microscope at various stages of their approach to and penetration of the rabbit ovum. No significant change in fine structure is observed in uterine sperm, or in many of the sperm at the periphery of the granulosa investment around the ovum. By contrast, a majority of sperm lying between the granulosa cells or on the surface of the zona pellucida display various stages of the “acrosome reaction”; this involves fusion and vesicle formation between the plasma membrane and the outer membrane of the acrosome. Loss of these vesicular elements, and content of the acrosome cap, takes place before sperm begin to penetrate the substance of the zona. The constricted posterior “equatorial” segment of the acrosome cap does not take part in the acrosome reaction and remains with its content intact during penetration of the zona; neither does the content of the apical sub-acrosomal region (perforatorium) or post-acrosomal region appear to change in traversing the zona. The hypothetical zona lysin is thus presumed to be closely associated with the persistent inner membrane of the acrosome, which now becomes the limiting membrane around the anterior part of the sperm nucleus. No “penetration filament” has been observed, but sperm within the zona pellucida of ageing eggs are often preceded by a straight or curved fissure in the substance of the zona. The possible nature of capacitation and its relation to the acrosome reaction and to the process of penetration, are discussed briefly.

Metabolic clearance rates and interconversions of estrone and 17β-estradiol in normal males and females

Abstract: The continuous infusion of (3)H-6,7-estrone and (3)H-6,7-estradiol has been used to study the metabolic clearance rate (MCR), the interconversions, and the red cell uptake of these steroids in normal males and females The whole blood MCR of estrone is 1,990 +/- 120 liters per day/m(2) (SE) in males and 1,910 +/- 100 liters per day/m(2) in females The whole blood MCR of estradiol is 1,600 +/- 80 liters per day/m(2) in males and 1,360 +/- 40 liters per day/m(2) in females The values in females do not vary significantly when studied in the follicular or luteal phase of the cycle At least 35% of the total estrone metabolism in both sexes is extrasplanchnic and at least 25% of the total estradiol metabolism in males, and 15% in females is extrasplanchnic The [rho](BB) (2,1) [transfer constant of estradiol to estrone, which is equivalent to the fraction of the precursor (estradiol) converted to the product (estrone) when both the infusion of the precursor and the measurement of the product are in peripheral blood] is 15%; and the [rho](BB) (1,2) [transfer constant of estrone to estradiol, which is equivalent to the fraction of the precursor (estrone) converted to product (estradiol) when both the infusion of the precusor and the measurement of the product are in peripheral blood] is 5% in both males and females Our findings concerning the radioactivity in whole blood, as measured by our procedure, were the following: 15-20% of estrone in both sexes and 15% of estradiol in males is associated with red cells Only 2% of the whole blood radioactivity of estradiol in females is associated with red cells Changes in the distribution of radioactivity between plasma and red cells will influence the MCR as calculated from plasma, but not as calculated from whole blood

Metabolism of 11-oxygenated steroids. Metabolism in vitro by preparations of liver

Abstract: 1. The isolation and partial purification of 11β-hydroxy steroid dehydrogenase from rat and guinea-pig liver microsomes has been achieved by conventional methods. 2. The efficiency of different 11-oxygenated steroids as substrates has been examined. The relative efficiencies confirm in the main the stereochemical theory of the enzyme–coenzyme–substrate complex that was proposed earlier on the basis of studies in vivo. Δ4-3-Ketones and 5α-hydrogen steroids are readily metabolized by the enzyme. 5β-Hydrogen steroids and Δ4-3-ketones with certain large α-substituents are metabolized to a limited extent or not at all. Halogen substitution in the 9α-position enhances the rate of reduction of 11-ketones but blocks the oxidation of the related 11β-ols. 3. 9α-Fluorocortisol is a competitive inhibitor of the oxidation of cortisol, but 9α-fluorocortisone is reduced at five to ten times the initial velocity of cortisone. 4. 11β-Hydroxy steroid dehydrogenase activity has been found in liver microsomes of rat, guinea pig, rabbit and calf. 5. Relative substrate efficiencies and Km values are similar in whole (debris-free) homogenates, washed microsomes and acetone-dried powders of washed microsomes. 6. A variety of conditions have been examined for the observation of 11β-hydroxy steroid dehydrogenase activity. NADP(H) is an efficient and NAD(H) a very poor coenzyme for the reaction.

Effect of posture on the metabolic clearance rate, plasma concentration and blood production rate of aldosterone in man.

Abstract: A method for the simultaneous determination of the metabolic clearance rate (MCR) and plasma concentration of aldosterone is described. From these 2 parameters the blood production rate (secretion rate) of aldosterone was calculated. Using this approach the effect of posture on aldosterone secretion and metabolism was studied in 18 normal male subjects, 3 patients with primary aldosteronism and 1 anephric subject. After sitting, there was no significant change in the MCR. The plasma concentration of aldosterone increased 6-fold and the blood production rate increased 5-fold. After standing, the MCR decreased significantly by 23%. The plasma concentration increased 10-fold and the blood production rate increased 8-fold. These results indicate that the increase in secretion rate rather than the decrease in MCR is the major factor regulating plasma aldosterone concentration. All 3 patients with primary aldosteronism had an elevated plasma aldosterone concentration in the supine state, and a correspo...

Reciprocal insemination and egg transfer between ferrets and mink.

Abstract: Epididymal mink sperm were injected into the uteri of 12 ferrets. On various days after insemination, 80 fertilized eggs and implantation sites including three living embryos in 11 ferrets were observed. No embryos, however, survived beyond 26 days. Ferret sperm were injected into the uteri or ovarian capsules of 22 mink; none of 165 eggs were fertilized. Ferret and mink eggs recovered at different times and from different parts of the genital tract were measured and recorded. When 77 ferret eggs were transferred into the uteri of nine mink, the majority of them developed at a slower rate than normal for a few days and then degenerated, but none implanted. Most of the 67 mink eggs transferred to the uteri of nine ferrets developed and implanted, but all degenerated after implantation. Fifty-one ferret eggs were transferred to the uteri of eight ferrets; 13 living fetuses and four young were obtained. Eleven mink eggs were transferred into the uteri of two mink; only one implantation site was observed. It is concluded that a high proportion of ferret eggs can be fertilized if a large number of mink sperm are injected, but mink eggs cannot be fertilized by ferret sperm. Ferret blastocysts can develop for a few days in the uterus of mink, but implantation fails. Although mink blastocysts can develop and implant in the uterus of ferrets, they degenerate after implantation.

A Method for the Measurement of Estrone and Estradiol-17β in Peripheral Human Blood and Other Biological Fluids Using 35S Pipsyl Chloride

Abstract: A double-isotope derivative method suitable for the estimation of estrone and estradiol–17β in human peripheral blood and ovarian or adrenal venous blood of sheep is described. 3H-estrone or estradiol-17β (42.4 c/ mmol) was added as indicator to the biological sample before extraction to allow for the calculation of losses. The phenolic extract of blood was reacted with 35S p.-iodobenzene sulfonyl chloride (150–200 me/mmol), and the derivatives purified by 4 chromatography and the 2 additional derivative steps. About 10 % of both steroids was present in the final sample which was used for counting. Specificity was indicated by the fact that the nonspecific blank of the method, 0.26 ±0.10 (sd) ng for estrone and 0.24±0.16 (sd) ng for estradiol, was the same for water and plasma from adrenalectomized-oophorectomized sheep or women. There was no statistically significant difference in the value calculated from aliquots taken before the acetylation and chromatography, the final stage and following an...

In Vivo Interaction of Endotoxin with a Plasma Lipoprotein Having Esterase Activity

Abstract: Circulating endotoxin was specifically precipitated from plasma samples withdrawn from three different animal species subsequent to parenteral injection of the toxin. Lipoprotein-positive staining and esterase activity were demonstrated on the precipitation lines formed in immunodiffusion, thus establishing the in vivo interaction of endotoxin with a plasma lipoprotein having esterase activity. Evidence was given to show that the intensity of this interaction in circulating plasma increased gradually with time. The concordance of this in vivo inter-action with the in vitro degradation and inactivation of endotoxin by plasma esterases is discussed. Images

Sperm penetration of rat eggs in vitro after dissolution of zona pellucida by chymotrypsin.

Abstract: SUCCESSFUL in vitro fertilization of mammalian eggs has been achieved in the rabbit and golden hamster1–4, but authentic reports of success for rat eggs are still lacking. In an attempt to fertilize rat eggs in vitro by the procedures of Yanagimachi and Chang3, we used various media with added heated rat serum, follicular, bursal or uterine fluid. After many trials, none of 844 eggs from seventy-six rats was found to have been penetrated, confirming Austin's early statement5. Various proteolytic enzymes were added to the medium to facilitate sperm penetration, and we found that, when the zona pellucida was dissolved, epididymal sperm as well as sperm recovered from the uterus could penetrate the vitelline surface, activate the eggs and lead to the formation of pronuclei. This communication reports the procedures and results of this experiment.

5-Hydroxytryptamine metabolism in rat brain and liver homogenates

Abstract: 1. The metabolism of 5-hydroxyindoleacetaldehyde derived from 5-hydroxytryptamine incubated with tissue homogenates was studied as an indicator of aldehyde dehydrogenase and alcohol dehydrogenase activities.2. In liver and brain from rats, there were indications of the presence of one or more aldehyde dehydrogenases which were stimulated by NAD(+) to a greater extent than by NADP(+).3. In liver from rats, there were indications of the presence of one or more alcohol dehydrogenases, which were stimulated by NADH to a greater extent than by NADPH.4. In brain from rats, there were indications of the presence of one or more alcohol dehydrogenases which were stimulated by NADPH to a greater extent than by NADH.

The role of deoxycorticosterone in the biosynthesis of cardenolides in Digitalis lanata

Abstract: The role of deoxycorticosterone in the biosynthesis of digitoxigenin was investigated by the simultaneous administration of deoxy[1,2-(3)H(2)]corticosterone and [4-(14)C]progesterone to a Digitalis lanata plant. The biosynthetically formed [(3)H,(14)C]digitoxigenin and deoxy[(3)H,(14)C]corticosterone were isolated and the distribution of the two isotopes in these products was determined. The transformation of progesterone into deoxycorticosterone in vivo was established. The biosynthetic route from progesterone via deoxycorticosterone to cardenolides was found to be of little significance.

Fertilization and early development in the Chinese hamster, Cricetulus griseus.

Abstract: Eggs from 79 Chinese hamsters, Cricetulus griseus, were examined at various times from before ovulation on the day of mating (Day 0) to implantation (Days 5–6). The animals were kept under reversed lighting (dark period 8 AM to 8 PM). Ovulation occurred between approximately 4 and 6 PM; mean number of eggs recovered per female was 7.6 ± 0.2. Sperm penetration was almost complete by midnight on Day 0. Of 131 eggs collected on Days 1–4, 102 (78%) were fertilized; (females without fertilized eggs excluded). The midpiece and tail of the fertilizing sperm were found in the cytoplasm of only 9% of pronuclear or cleaved eggs, and remained in the perivitelline space of 74%; in the other 17% eggs the sperm tail and most of the midpiece were outside the zona pellucida or were missing at recovery. No polyspermic eggs were found. The first three cleavages took place at 20–26 hour intervals after ovulation. Eggs reached the blastocyst stage in the oviduct and entered the uterus more than 96 hours post-ovulation. Implantation occurred between Days 5 and 6.

Interruption of pregnancy in the rat and hamster by administration of PMS or HCG.

Abstract: Intraperitoneal injection of 50 IU PMS on Days 1–3 after mating resulted in the early transport of ova from the rat tube into the uterus. This effect was less markedly shown in the hamster but retardation of cleavage was observed in a few cases. When 100 IU HCG was injected, tubal transport was not markedly accelerated in either the rat or the hamster. Following treatment with 50 IU PMS on Days 1–3, no living embryos were found in rats and hamsters killed on Days 16 and 13, respectively. Implantation was inhibited in the rat, though not in the hamster. It also failed when normal blastocysts were transferred synchronously into the PMS-treated rats. Implantation did occur in rats treated with various doses of PMS on Days 5–7, but fetal mortality (54–100%) increased proportionately with the dose. A single dose of 100 IU PMS on one of Days 5, 7, 9 and 11 in the rat induced 67, 92, 75 and 56% fetal mortality, respectively. Administration of 100 IU HCG on Days 1–3 had a slight effect on preand post-implantation...

Serotonin Metabolism in Pineal Homogenates

Abstract: Publisher Summary The conversion of 5-HT to 5-methoxytryptamine in pineal and subsequent metabolic transformations to 5-methoxyindoleacetic acid and 5-methoxy tryptophol is in vivo metabolic pathway The analogous pathway from 5-HT to 5-HIAA in pineal homogenates is demonstrated in this chapter 5-Methoxytryptophol is isolated from pineal The analogous pathway from 5-HT to 5-hydroxytryptophol by way of alcohol dehydrogenase predominates if NADPH2 is added to the pineal homogenates It has now been established that 5-hydroxyindoleacetaldehyde may be metabolized not only to 5-hydroxyindoleacetic acid and 5-hydroxytryptophol but also to 5-hydroxyindolecarboxaldehyde and the corresponding 5-hydroxyindolecarboxylic acid Further work is essential to validate this pathway both in vitro and in vivo

Interference with early pregnancy in rats by estrogen: Mechanism of action of dienestrol

Abstract: Rats were given single doses of an orally-active estrogen by mouth on days 1–5 of pregnancy. Examination of uteri on day 8 for implantations revealed that the median effective doses for preventing 50% of the eggs shed from implanting were 0.047, 0.050, 0.035, 0.145 and 0.380 mg/kg for days 1, 2, 3, 4 and 5 respectively. It was found that failure of pregnancy after treatment with the minimal 100% effective dose (ED100) on days 1, 2 or 3 was due to expulsion of eggs from the reproductive tract by day 5. Treatment of rats with the ED100 on day 4 caused expulsion of some eggs from the tract by day 5, but also had effects on the uterus: the position of the blastocysts within the lumen was abnormal and their orientation relative to the mesometrial-anti-mesometrial axis disturbed. No decidual reaction was observed on day 6 in such rats or on day 9 in pseudopregnant rats treated similarly and subjected to uterine traumatization on day 5. Treatment with ED100 on day 5 had no effect on egg transport or on the positioning of blastocysts within the lumen on day 6. A decidual reaction was seen around the blastocyst on day 6, but in pseudopregnant rats treated similarly and subjected to uterine traumatization on day 5 no decidual reaction was found on day 9. The pontamine blue reaction was negative in nearly all rats dosed on day 4 and examined on days 6–8. In rats dosed on day 5, however, the blue reaction was negative on day 6, but positive on day 7, i.e., delayed by 24 hours compared to controls and appearing later than the decidual reaction. The blastocysts were unattached in these rats on day 6, but may have attached briefly on day 7 (suggested by the positive blue reaction) and subsequently failed to develop further owing to disappearance of the decidual tissue.

Septal ablation and CAR acquisition in the golden hamster

Abstract: Bilateral septal lesions produced a significant improvement in the acquisition of a two-way active avoidance response in the hamster in comparison to the performance of operated controls and normal animals. A significant negative correlation was found between trials to criterion and intertrial spontaneous crossings for all groups combined. It appeared that normal hamsters have difficulty learning a two-way locomotor avoidance response.

Effects of maternal essential fatty-acid deficiency on neonatal rat brain

Abstract: Female weanling rats were raised to sexual maturity on either a normal stock diet or an essential fatty-acid (EFA) deficient diet, and then bred. Brain weight and lipid composition of the offspring from these animals were measured as part of an investigation into factors contributing to the early death of EFA-deficient progeny. Lactation failure does not seem to be an adequate explanation of the early deaths since the dead EFA-deficient offspring frequently have milk in their stomachs. Maternal behavior including nesting was lacking, however, in EFA-deficient dams. The EFA-deficient brains weighed less than normal neonatal brains, and frequently had subdural hemorrhaging. Relative to body weight, however, some “sparing” of the brain was apparent. Total brain sterol concentration was not measurably different, but fatty-acid composition of total brain lipid and phospholipid was. The EFA-deficient offspring had relatively less arachidonate and other EFA-related fatty acids, while the proportion of trienoic acids, principally 20:3 and 22:3 was increased considerably. In phosphatidyl ethanolamine, the proportion of 16:0 was particularly elevated in the EFA-deficient brains. The results are discussed in relation to the integrity of myelin and the synthesis of prostaglandins, as possible factors in the premature death of EFA-deficient progeny.