Joseph T. Velardo

Histochemical demonstration of succinic dehydrogenase activity in tissue sections by a modified technique.

A method for the histochemical demonstration of succinic dehydrogenase was first described by Seigman and Rutenburg in 1951 (7). Their technique utilized ditetrazolium chloride (BT) as an indicator dye, and the detection of reduced BT as diformazan constituted a positive test for succinic dehydrogenase activity. This initial report has been followed by two subsequent papers describing the distribution of succinic dehydrogenase in various tissues and organs of several mammals (5, 7). Two modifications of the original method have been employed in these later studies; the use of Al , Ca , and HCO3activating ions and of anaerobic conditions (nitrogen bubbling, etc.) have been found to be beneficial and more critical in demonstrating sites of low succinic dehydrogenase activity. Of further interest is the paper of Shelton and Schneider showing that among several of the tetrazoles used, neotetrazolium was the most efficacious as an indicator of dehydrogenase activity (9). The latter is in accord with the findings of Padykula (5), Malone (4) and the present workers. As a result of the difference of opinion regarding the choice of the most critical indicator dye and the consequent lack of agreement in the qualitative detection of succinic dehydrogenase activity in a number of tissues and organs previously described as having questionable or a low degree of activity, the present study was undertaken in an effort to: (1) critically evaluate the existing differences regarding this technique, and, (2) outline a method which is simple, direct, and efficient in demonstrating succinic dehydrogenase activity in many tissues preP viously described as negative for this test. Various tissues were removed from albino rats and frozen immediately on a block of dry ice. Tissues in this frozen state may be stored for long periods of time without any noticeable loss of enzyme activity. In preparation for the sectioning procedure, the tissues were trimmed and mounted by freezing to the stage of a carbon dioxide freezing microtome. Fresh frozen sections were cut at 10 to 20 and mounted upon slides by drying in air at room temperature for a short period of time (5 to 10 minutes). Pending the sectioning of other tissues, the mounted sections were stored at 4#{176}C. in 0.1 M sodium phosphate buffer contain-

Histochemical localization of vaginal oxidative enzymes and mucins in rats treated with estradiol and progesterone.

Any treatment of the basic biology of the vagina eventually must define the physicochemical mechanisms that underlie the various levels of functions in this organ structure. Also, because of the demonstrated dependence of the vaginal structural and functional components upon a rigorously defined yet varying level of circulating steroid hormones, a consideration of the interplay of such hormones and their subsequent manifestations on these mechanisms is essential. Very recently biochemical endocrinology has elucidated some of the initial interactions between ovarian steroids and certain critical enzymes, the transdehydrogenases (Talalay and Williams-Ashman, 1958). The net effect of these studies has been to indicate the most probable means by which cells in target structures, such as the uterus and vagina, initiate enzymatic mechanisms that involve an increase in available high-energy phosphate esters. The presence of these organic phosphate compounds is imperative for the expression of the induced changes in these organs by the steroid hormones and their metabolites acting in concert (Velardo, 1958a, b ; 1959). Enzymes such as the diphosphopyridine-nucleotideand triphosphopyridine-nucleotide-linked dehydrogenases, succinic dehydrogenase, and their respective systems are part of a group that effects certain specific biological oxidation-reduction reactions, the most distinguishing feature of which is the transfer of parceled energy into the phosphate bonds of compounds such as adenosine triphosphate (ATP) and creatine phosphate. The convergence of such disciplines as histochemistry and experiment a1 endocrinology on problems related to the effects of hormones on target oxidative enzyme systems has resulted in the demonstration within the uterus and the vagina of “pattern distributions” that are characteristic for a given enzyme and for specific organ regions (Rosa and Velardo, 1958). These studies, made with the use of the light microscope, have permitted the identification of active and relatively inactive regions within the vaginal epithelium. In addition, other investigations have indicated that certain histochemically demonstrable oxidative enzymes in the uterus and the vagina of the rat are apparently under thc control of circulating titers of ovarian hormones and their metabolites during the estrous cycle (Rosa and Velardo, 1959). I t is the purpose of this presentation to present. further experimental evidence to this effect. However, * The work rcportcd in this paper was supported in part by Research Grants RG 6008 and RG 4577 C2 from the National Institutes of Health, Public Health Service, Bethesda, Md. t Studies carried out during the tenure of a Lederle Medical Faculty Award.

Induction of Ovulation in Immature Hypophysectomized Rats

Immature rats given minute doses of highly purified pituitary gonadotropins (follicle-stimulating hormone and luteinizing hormone) 7 to 100 days after hypophysectomy ovulated and formed corpora lutea. Neither hormone alone was effective. Luteinizing hormone repaired in part the atrophied theca interna and interstitial tissue, and follicle-stimulating hormone stimulated the development of the granulosa cells.

Histochemical observations of oxidative enzyme systems in the uterus and vagina of the rat.

The problems that beset investigators of uterine and vaginal growth and metabolism are many and varied. I t has been and still is our conviction that cytochemical and histochemical studies particularly guided toward elucidating patterns of oxidative enzymatic activity in the uterus eventually will provide information necessary for a thorough understanding of quantitative biochemical analyses. In the following studies it has been our intention to indicate that the separate histological structures of the uterus show discrete enzymatic responses by such enzymes as the succinic dehydrogenase (SDH) and DPNdiaphorase (DPN) systems during the normal changing hormonal environment. In addition, each of these systems appears to have its own pattern of reactivity, sometimes distinct or otherwise overlapping with the other. Although precise interpretations of these results are not yet possible, histochemical observations of these enzyme systems, which are associated intimately with the synthesis of high energy phosphate compounds (adenosine triphosphate), indicate those areas in the organ wherein the most probable effective hormonal stimulation of metabolic activity occurs.

Hormonal actions of chorionic gonadotropin.

Many students of the evolution of viviparity and of the endocrinology of reproduction have become impressed with what I term Amoroso’s basic law of the comparative physiology of reproduction: “With the transition from oviparity to viviparity in mammals and also in fishes and reptiles, there have occurred profound changes in the nature of the egg and in the structure and physiology of the female animal, particularly of the reproductive tract. I t would be difficult to imagine that these complex and highly integrated changes and events, observed in vertebrates, particularly in reptiles, could have come about otherwise than through many separate lines of evolutionary specialization. Nevertheless, it is clear that none of these evolutionary innovations could have occurred in the complete absence of various interactions between tissues and hormones.”2 During the past ten years I have pursued the several metabolic alterations of the reproductive tract as they are influenced by the hormones and/or their metabolites acting in concert. 39 Specifically, I have become interested in the interactions between tissues and hormones. Although we have all learned more of the physiological actions of the estrogens, progestogens, and androgens, much less is known of the adenohypophysial and placental gonadotropins. The present report is concerned directly with a series of related experiments that have been devised with the purpose of elucidating the physiological role of a placental gonadotropin of the human, chorionic gonadotropin (HCG) on the reproductive system. Giving added impetus to this study are the numerous lamentable lacunae in our understanding of the real purpose of this placental gonadotropin in the body fluids of the pregnant human female, as well as its role during several of the known pathological variants.

Uterine Growth Promoting Action of Relaxin

Summary Experiments were performed to determine response of uteri of immature albino rats to subcutaneous injections of varying dosages of relaxin. Saline and 0.0002, 0.001, 0.01, 0.1, 1, 5, and 10 mg relaxin were injected into female rats 28 and 33 days old by dividing the dose into thirds and administering each third on 3 consecutive days. Necropsies were performed on the fourth day at ages 31 and 36 days, respectively, and the uteri weighed, dried and reweighed. Wet uterine weights, i.e. mg % of body weight, increased along a sigmoid pattern from 45 for saline to 67 for 10 mg in younger group, and from 47 for saline to 70 for 10 mg in older group. Water concentration remained relatively constant, varying between 82 and 87% of wet weights, with no correlation as to amount of relaxin received. Nitrogen content showed a similar response, ranging between 12 and 14% for 36-day-old animals, and 12 and 16% for 31-day-old animals.