Showing papers in "Basic life sciences in 1974"

Capacitation of mammalian spermatozoa.

Abstract: The need for a physiological change in mammalian spermatozoa in the female genital tract as a prerequisite for fertilization was first recognized by Austin (1951) and Chang (1951) in the rabbit, and the change was termed “capacita­tion” (Austin, 1952). They found that if epididymal or ejaculated spermatozoa were introduced into the oviduct of the female rabbit before ovulation, eggs were fertilized. However, when the spermatozoa were introduced into the female after ovulation, few-if any-eggs were fertilized. In other words, the eggs lost the ability to be fertilized before the spermatozoa became capacitated.

The Mechanisms of Ejaculation

Abstract: Ejaculation is a complex process which occurs in a series of chronologically dependent phases in coordination with several organ systems. In spite of the biological importance of ejaculation for mankind and especially for the human psyche, very little is known of its mechanisms in humans. Most present knowledge is based on investigation in animals. So far, our knowledge of ejaculation in man is based on study of diseased patients in whom sexual dysfunction is usually only one symptom among many. The human being is understandably a difficult and inaccessible animal for studies of ejaculation. Studies of sex life, orgasm, and the like have long been taboo, and so barely available for evaluation.

Chromosome Abnormalities and Abortion

Abstract: Although spontaneous abortions represent one of the most frequent pathological entities, their causes have remained relatively obscure until the last decade. However, the work carried out at the Carnegie Institution at the beginning of the century and published in 1921 by Mall and Meyer had demonstrated the importance of abnormalities in the development of the zygote. In 1943, Hertig and Sheldow reported the results of anatomical and pathological examinations of 1000 abortuses. In 617 cases, they found obvious abnormalities of which 489 cases represented either the total absence of an embryo or an important disorder of embryogenesis. In 1957, Javert examined 892 abortuses. In 173 of these, the development was arrested before day 21 after conception, and 99% were morphologically abnormal.

Mechanisms involved in penetration of spermatozoa through the vestments of the mammalian egg.

Abstract: In the past, the mammal has been something of a poor relation among the various metazoa used by students of fertilization. Failure to recognize the need for capacitation of spermatozoa and the complicating factors of the internal site of fertilization, the somewhat rigorous environmental requirement for consistent success in fertilization in vitro, and the very few eggs ovulated at one time have all been at least partly responsible for our relatively poor knowledge of the physiology of mammalian fertilization. The last aspect-egg numbers-still presents a problem for many who are interested in pursuing the biochemistry of egg activation and early development in mammals.

Parturition in the sheep and the human.

Abstract: The physiology of pregnancy and parturition in sheep and humans has features of a broad character common to both species. In matters of detail, however, the dissimilarities are such as to discourage attempts to look for common ground. Ryan (1971) condemned ‘any insistence that all influences on pregnancy length or initiation of labor such as ruptured membranes, hydramnios, multiple pregnancy, fetal death, neurohypophyseal hormones, catecholamines, and others too numerous to mention can all be reconciled into a holistic theory’. This wise counsel can be interpreted too literally if the word ‘holistic’ is taken to exclude the possibility of a common final pathway by which these various factors exert their influence on the time of parturition. Clearly, all of them have in common an effect, direct or indirect, on the contractile protein of the smooth muscle of the uterus. Furthermore, it seems likely that they all ultimately express their effects through an alteration in the intracellular binding of Ca2+ (Carsten, 1973), and possibly in the concentration of cyclic AMP (Bhalla and Korenman, 1973). Taken together, the smooth muscle cell, Ca2+ flux, and cyclic AMP could be said to constitute a common pathway. Presumably, Ryan had in mind a comprehensive mechanism which could be applied in toto to many species and, within a given species, to all known influences on gestation length. If so, one can only agree with him. Yet elucidation of fundamental processes having no regard for species would seem to deserve the greatest urgency in our efforts to extend the understanding of the physiological determinants of gestation length and the initiation of labor.

Endocrine control of spermatogenesis.

Abstract: Literature on the endocrine control of spermatogenesis is reviewed. The control of spermatogenesis may begin with the stimulation of testosterone production in the Leydig cells by ICSH. Testosterone or its metabolites are then transported into the seminiferous tubules and the Sertoli cells. There testosterone possibly interacts with the germinal cells to initiate a wave of spermatogenesis. There is a possible direct action of testosterone on spermatocytes to promote meiotic division. As a result of follicle stimulating hormone (FSH) binding and stimulation of adenylate cyclase cyclic AMP (cAMP) is produced which in turn activates cAMP-dependent protein kinase. cAMP or another FSH "messenger" then increases RNA and protein synthesis by a variety of mechanisms. This increased synthetic activity increases androgen receptor protein activity permitting a complex formation betwe en testosterone and the receptor protein. The progression of spermatoge nesis is thus determined by a direct interaction of the hormone receptor complex with germinal cells or an indirect interaction through a "message" generated as a result of events in the Sertoli cells controlled by this complex.

Direct evidence that pyrimidine dimers in DNA result in neoplastic transformation

Abstract: A specific test for the biological role of UV-induced pyrimidine dimers in DNA is photoreactivation (PR). Fish contain large amounts of the PR enzyme. Portions of cell suspensions of tissue from various organs of the fish Poecilia formosa were exposed to UV radiation (254 nm), then injected into isogenic recipients. An incident fluence of 20 J/m2 resulted in 10% of the fish with large granulomas and 100% with thyroid carcinomas. If the irradiated cell suspension was illuminated with PR light before injection, the yields of both types of lesion were reduced approximately 10-Fold. If the PR light was given before the UV exposure, there was no reduction in the numbers of growths. These experiments show that pyrimidine dimers in DNA can lead to neoplastic transformation.

Cellular aspects of ovum implantation and decidualization in the rat

Abstract: Ovum implantation and decidualization, which is an integral part of the implan­tation, are complex phenomena governed by numerous endocrine as well as cell biological factors. Many reviews and treatises are available in the literature dealing with different aspects of these two important episodes in mammalian reproduction (e.g., Amoroso, 1952; Shelesnyak, 1957; Enders and Schlafke, 1965; De Feo, 1967; Potts, 1969; S. Tachi et al., 1970; Finn, 1971; McLaren, 1972). In this chapter, attention is focused on the cell biological aspects of endometrial adaptation during decidualization in the rat.

Inhibition of spermatogenesis.

Abstract: The effect upon spermatogenesis of subdermal silastic capsules containing testosterone in combination with various oral drugs was studied in groups of subfertile men Group 1 received 25 mg norethisterone daily by mouth in conjunction with 6 norethindrone implants and 3 testosterone implants (The release of testosterone per capsule was about 55 mg/24 hours) 2 of the 5 patients reached azoospermia within 6 weeks; the others showed lowered sperm counts but it is possible they took the tablets irregularly 9 weeks after discontinuing oral norethisterone all had an increasing number of sperm Group 2 contained 4 men aged 25-32 and 3 aged 66-73 In addition to 6 sc implants of megestrol acetate and 4 sc implants of testosterone each received 25 mg norethisterone daily 3 of the 4 young men had a drop in sperm count 6 weeks after treatment started while the 4th had an increase from 71 million to 126 million In the 3 older men results were assessed by testicular biopsies which showed normal wide not sclerosed tubules with all steps of spermatogenesis before treatment but reduced diameter of the tubules with tubular epithelium apparently devoid of germinal elements after treatment Also there was disorganization and depopulation of the seminal epithelium Leydig cells seemed reduced and atrophic Group 3 consisted of 6 men aged 26-39 with fertility problems They received 4 sc testosterone implants plus 100 mg medroxyprogesterone acetate depot (MPA) Between Weeks 6-12 azoospermia was found in 4 and reduced in all except 1 Group 4 consisted of 5 men aged 63-74 with benign prostatic hypertrophy treated with MPA plus 6 implants of megestrol acetate and 4 of testosterone Posttreatment biopsy showed a lack of germinal elements in the tubules the diameter of the tubules unchanged and Leydig cells partially atrophic and diminished All patients affirmed normal sexual activity and noted no changes of libido or potency Group 5 consisted of 5 men aged 32-62 treated with R2323 orally 5 mg twice a week for 6 weeks and then 25 mg twice a week plus 4 testosterone implants 3 of the men showed azoospermia between 6-12 weeks the 4th had lowered sperm count but did not take all the tablets The older man showed arrested spermatogenesis on biopsy However libido and errections decreased appreciably 2 complained of intense perspiration at night and 1 complained of heartburn These men also showed alterations in the blood levels of liver enzymes and an increased secretion of urobilinogen in the urine even within the 1st weeks of treatment

The Duration of Egg Transport and Its Regulation in Mammals

Abstract: The literature dealing with egg transport has grown considerably during the last decade and excellent recent reviews are available (Blandau, 1969; Pauerstein et al., 1974). Rather than cover the subject extensively, an attempt will be made here to discuss a single aspect of egg transport in mammals: its duration.

Progesterone-Progestin receptors.

Abstract: A correlation between binding to the progestin-specific uterine cytosol receptor and with well-defined facets of progesterone action was established. Structurally similar derivatives of norprogesterone and nortestosterone were compared. Binding was measured in vitro by determining their competitive effect on the progestin-tagged uterine cytosol receptor of the rabbit and mouse. Activity in vivo was measured by determining their ability to induce endometrial proliferation in the rabbit and their inhibitory effect on the estradiol-induced uterine weight increase in the mouse. Immature female Normandy rabbits Hartley guinea pigs immature female Swiss SPF mice and Sprague-Dawley SPF rats were used. Groups of 5 rabbits were primed from Days 1 to 5 by daily injections of 5 mcg estradiol. Progesterone or a test compound was injected from Days 6 to 10. On Day 11 the rabbits were sacrificed and the endometrium prepared for histological examination. The relative potency of each steroid was compared with that of progesterone for the rabbit. Groups of 4 mice received daily injections of .09 mcg estradiol and various doses of progesterone or test compounds. These animals were sacrificed 24 hours after the last doses and their uteri excised and weighed. Antiestrogenic activity was indicated by the inhibition of uterine weight increase. The progestin specificity of the 7-8S receptor was established. The weakest competitor was progesterone. Progestins R5020 H3510 and norgestrel were highly potent. Labeled cortisol bound in the 4-S region and served as a marker. Competition studies established that 19-nor derivatives also compete for the progesterone receptor. Structural modifications of the norprogesterone and nortestosterone molecules modified results. The in vivo biological activity of all compounds was greater than the of progesterone. The introduction of 1 or 2 double binds into the 19-nor molecule decreased this somewhat. The addition of a 17 alpha-methyl substituent (H3510) to norprogesterone also decreased activity. Other modifications of the molecules also changed activity. Results of antiestrogenic activity in the mouse showed that norprogesterone was markedly more active than progesterone. Antiestrogenic activity and binding were correlated in the case of all the monoenes (norprogesterone and H3510) and denies (H3163 and R2453). A perfect correlation was observed between binding in the rabbit and the mouse for the 12 test compounds. The search for highly potent progestins involves as an initial step the search for a compound that binds tightly to the 7-8S receptor. In vivo extraneous factors may also become critical. Only by measuring antiestrogenic activity and progestomimetic activity in the same species can the existence or lack of existence of a correlation be established.

The Stringent Response—21 Years On

Abstract: During normal growth, bacteria synthesize no more ribosomes than can be engaged with high efficiency in protein synthesis (1). The molecular mechanism maintaining this fine balance is clearly of crucial importance in determining the growth characteristics of the cell. The first inkling that the synthesis of an essential component of the ribosome, the ribosomal RNA, might be directly coupled to protein synthesis was provided in 1952 by Sands and Roberts (2). They observed that when a particular bacterial strain was deprived of a required amino acid, not only did protein synthesis stop but also the net synthesis of RNA was substantially reduced. This phenomenon, subsequently christened the “;stringent response,” has recently been the subject of several excellent reviews (1,3,4). In this chapter, I argue that the stringent response is an extreme manifestation of the normal processes of the gross control of RNA synthesis in the bacterial cell. I further suggest that a major mode of effecting the selective coupling of stable RNA species to protein synthesis is mediated by EF-TuTs, a protein synthesis elongation factor, and ppGpp, a metabolic product of the ribosome (5).

Neurohormonal Control of Epididymal Contractions

Abstract: Moore and Quick (1924) were the first to suggest that spermatozoa are propelled in the epididymis by a neuromuscular mechanism. Later it was shown (Muratori and Contro, 1951; Muratori, 1953; Cross, 1955; Risley and Turbyfill, 1957) that the caput and body of the epididymis have spontaneous contractions of the peristaltic type and that the cauda exhibits sporadic segmentation movements.

Inhibition of Fertilization with Specific Antibodies

Abstract: Interest in using immunological procedures to study events and components in reproduction has increased recently, as indicated by the number of research papers published on the subject. From several review articles (Piko, 1967; Tyler et al, 1967; Edwards, 1970; Kirby, 1970; Shulman, 1971; Fox and Shivers, 1972), workshops (Society for the Study of Reproduction, Boston, Massachusetts, 1971), and international symposia (Geneva, Switzerland, 1968, and Varna, Bulgaria, 1971) dealing with the subject of immunoreproduction, one is led to conclude that many macromolecular components of reproductive tissues serve as antigens when exposed to the immune system of the same or different mammalian species and that specific antibodies produced against these antigens are useful for a variety of diagnostic purposes including studies on fertility and sterility.

Regulation of uterine activity during gestation and parturition in rabbits and rats.

Abstract: Uterine muscular activity is influenced by ovarian steroid hormones, by nervous activity, and by endogenous, humoral or locally produced, uterus-activating agents such as the neurohypophyseal peptides, the prostaglandins, and various biogenic amines. Purely physical factors, such as stretch, also play a part in the regulation of myometrial function. While the neural control of uterine function is more intimately linked to uterine vascular supply than to its muscular elements, at least in rats and rabbits, the hormonal factors exert a profound influence directly on the contractile components of the uterus. However, in spite of much progress in recent years in the understanding of the mechanism of action of steroid hormones, the integration of biochemical events at the molecular level into various contractile responses at the tissue level remains to be elucidated. The possible interrelationship between endocrine factors and the nervous system in integrating the total activity of the whole organ has also to be clarified, but all evidence points to the hormonal and humoral agents as the dominating factors in the regulation of uterine contractility.

Contractility of myometrial transplants.

Abstract: Long-term observation of activity of the myometrium during pregnancy, parturition, and Puerperium in the woman is for obvious reasons difficult to carry out. The individual factors that might influence the initiation of parturition are not possible to study in situ, as the tissue is continuously subject to changes of mechanical, hormonal, and nervous character. In vitro studies of isolated strips of myometrium have rendered information of great importance, but this technique gives only an impression of the immediate situation and never of the preceding or following states of the same tissue. Therefore, the ideal system would permit in vivo study of individual factors having myometrial influence. The technique of grafting seems to have been overlooked for its possibilities in the study of the physiology of the myometrium.

The Corpus Luteum Studied In Vitro

Abstract: In spite of the fact that the existence of the corpus luteum has been known for almost four centuries, relatively little is known about the basic mechanism controlling its formation, function, and regression. The current status of knowledge of the corpus luteum has been reviewed in a 1973 symposium volume of Biology of Reproduction as well as in the present volumes and by Nalbandov (1973). The formation of the corpus luteum from granulosa and thecal cells appears to have a similar mechanism in most mammalian species; however, the control of its life span varies from species to species. Part of this complex nature of control of luteal life span is due to fact that there are both intrinsic mechanisms within the gland and extragonadal factors such as a possible uterine luteolysin (for review, see Anderson, 1973), prostaglandins, and hormones which can shorten or prolong luteal life span. The problem addressed in this chapter is what are the intrinsic factors which control the formation and functional life span of the corpus luteum; particular reference will be made to the primate and the pig. Once the intrinsic mechanisms are understood, a proper basis will exist for examining and understanding the action of extragonadal influences. By necessity, an in vitro method of study of these intrinsic mechanisms must be employed, without the complicating variables encountered in the in vivo situation.

Effect of a vasopressin analogue on myometrial activity in nonpregnant women.

Abstract: Every muscle must work; otherwise, it atrophies and disappears. Thus even before studying the uterine activity we know that the myometrium of the nonpregnant woman contracts. However, many unanswered questions still exist: what is the activity pattern, how is it controlled, and, finally, does the activity play physiological and pathological roles in the reproductive cycle?

The mode of action of steroid hormones: Some recent findings.

Abstract: The author described results of the "forward approach" of studying the action of steroid hormones from their transport in blood to binding by target tissues uptake into the nucleus and possible mode of action on the protein synthetic machinery of the target cells. Steroids are found dissolved in plasma nonspecifically bound to plasma proteins and specifically bound to high affinity plasma proteins which exhibit however some overlapping recognition for steroids. Steroid hormones enter into target cells by protein-facilitated diffusion. Once incide some e.g. estradiol in rat uterus excert effects unchanged; others p articularly androgens are metabolized locally where the metabolites are more active than the original steroid "prehormones." Very specific binding proteins have been isolated from the cytosol of all steroid hormone systems and they are known to be under physiological control in some systems e.g. estrogen and progestin receptors in guinea pig uterus. The steroid-receptor complex then migrates to the nucleus as shown by autoradiography and by cell-free systems. A nonhistone chromatin binding protein with virtually covalent binding affinity and another specific protein distinct from the cytosol-to-nucleus receptor protein have been implicated in steroid action. Current research is centering on the nature of early messenger-RNA and protein synthesis that initiates the response to steroid hormones. The author postulates early synthesis of a "key intermediary protein" (KIP). To support this idea he cites as evidence the "IP" or induced protein made in the first hour and susceptible to inhibition by actinomycin D and alpha-amanitin and a 15 S RNA fitting the description of mRNA. In general responses to steroid hormones are due to intracellular uptake of the steroid; perhaps modulated by but not necessarily requiring cyclic nucleotides PGs neurotransmitters or peptide hormones; probably entailing allosteric conformational changes in noncovalently bound specific receptors and positive feedback control at the transcriptional level.

Termination and antitermination in transcription: control of gene expression.

Abstract: The expression of many, if not all, genes in microbial systems is regulated according to the “operon” model proposed by Jacob and Monod (1). Regulation occurs at the step of initiation of transcription of genes or operons, as was originally outlined by these authors (2). In known cases, this is accomplished by a specific repressor molecule interacting at the operator site to inhibit transcription from the cognate promoter locus (negative control) or a positive factor(s) stimulating transcription (positive control) or both (3,4). The in vitro controlled transcription of certain specific operons has already been demonstrated using purified RNA polymerase holoenzyme. The essential feature of these systems is that transcription is initiated by the correct initiating signals, i.e., promoters. For the lac and gal operons of Escherichia coli, correct initiation depends on the presence of additional positive control elements: cyclic AMP and its recepter protein (CRP) (5–7). In case of the immediate early operons of bacteriophage λ, however, correct transcription occurs just with RNA polymerase holoenzyme alone (8,9). In all of the above three systems, purified lac gal or λ repressor specifically inhibits the transcription of its respective operon (8–11).

Synthesis by reverse transcriptase of DNA complementary to globin messenger RNA.

Abstract: RNA tumor viruses contain a DNA polymerase that can synthesize a faithful DNA copy of viral RNA (1,5,23,25,26,31,32). This enzyme is easily released and purified from virions and can utilize a wide variety of polymers as templates (6,7,13,14,18,35,38). In order for a template to be copied, a primer or initiator that binds to the template by hydrogen bonds is required (3). The 3′-OH end of the primer is then covalently attached to the newly synthesized DNA (29). When the 60–70S tumor viral RNA is transcribed, the primer is apparently a short polyribonucleotide that is found attached to the DNA product (10,18,35,37).

Species Specificity in Fertilization

Abstract: In his classical monograph, Problems of Fertilization, Lillie (1919) devoted a chapter to the problem of specificity in fertilization which, nearly 40 years later, Rothschild (1956) commended to all students of fertilization. In summing up his own chapter on “Specificity”, Rothschild considered the only advances to have been made since Lillie’s contribution were (1) more information about the immunological nature of the reactions between the gametes, (2) more information about the nature of immunological reactions in general, and, possibly, (3) the development of techniques for the quantitative study of interspecific and intergeneric cross–fertilization. Over the past few years, however, notable advances have been made in this field, particularly with reference to mammals, and it is with these that this chapter will be concerned. First, though a few general comments will be made.

Hormone patterns in early human gestation.

Abstract: Accurate measurement of the low concentration of gonadotropins and steroid hormones present in human serum has been made possible by the development of sensitive radioimmunoassay (RIA) techniques; With the use of RIA FSH and LH, progesterone and 170H-progesterone have been previously measured in early normal pregnancy (Jaffe et al., 1969; Parlow et al., 1970; Wide, 1969). In order to determine the daily pattern of hormone levels in early normal pregnancy, gonadotropins as well as steroid hormone levels were measured in serum samples obtained daily from three women from the time of the last menstrual period prior to conception throughout the first few months of gestation.