Conservation of RNA polymerase during maturation of the Rana pipiens oocyte.

DNA-dependent RNA polymerase was extracted from oocytes of the frog, Rana pipiens. The bulk of the enzyme activity was present in the germinal vesicle and the amounts of each major form of such activity did not significantly change during oocyte maturation. Therefore, either nuclear polymerase activity is conserved after breakdown of the oocyte nucleus during maturation or, alternatively, de novo synthesis of the enzymes must occur during oocyte maturation concomitant with degradation. We have measured rates of protein synthesis in oocytes and determined a maximum rate of synthesis for RNA polymerases. Our kinetic studies show that no more than 20, 10, and 5% of RNA polymerases type I, IIa, and IIb, respectively, could be synthesized during steroid-induced oocyte maturation. These results thus show that the bulk of RNA polymerase accumulates in the germinal vesicle during oogenesis, is dispersed into the cytoplasm during maturation, and, since only limited synthesis seems to be occurring, the polymerase is available during embryogenesis.     

The fate of oocyte nuclear proteins during early development ofXenopus laevis

Summary The localization and movements of four nuclear proteins, originally contained in the germinal vesicle ofXenopus oocytes, were followed through early development from cleavage to late neurula. The study made use of monoclonal antibodies directed against germinal vesicle proteins. Biochemical methods showed that all proteins persist in the embryo without a change in molecular size or gross concentration. At early stages the proteins are localized preferentially in the cytoplasm of the animal hemisphere. They shift from the cytoplasm to the nucleus at stages specific for the individual proteins. During mitosis the proteins are released from the nucleus into the cytoplasm.     

Spontaneous oocyte maturation in Rana pipiens: Estrogen and follicle wall involvement

Immature (germinal vesicle stage) Rana pipiens oocytes typically remain arrested in prophase I of meiosis even after extended periods of in-vitro culture, if not stimulated with hormones. We have, however, sporadically observed “spontaneous” occurrences of oocyte maturation in vitro without the addition of hormones. This study documents some of our observations on this phenomenon and presents experimental results concerning the effects and possible involvement of estrogen and follicle wall components in regulating spontaneous oocyte maturation. Estrogen was found to inhibit spontaneous oocyte maturation (GVBD) in a dose-dependent fashion. Follicles in which spontaneous maturation was inhibited by estrogen retained their responsiveness (GVBD) to both frog pituitary homogenate (FPH) and progesterone stimulation. Inhibitory effects of estrogen on spontaneous maturation, however, were not reversed following incubation of washed follicles in plain culture medium without added hormones. Possible involvement of progesterone synthesis in spontaneous oocyte maturation was ascertained by simultaneously monitoring endogenous progesterone synthesis and the occurrence of spontaneous GVBD over the course of the maturation process. In spontaneous maturing follicle there was a gradual increase in basal levels of progesterone synthesis that preceded GVBD. Significantly, addition of estrogen abolished both the spontaneous progesterone production and spontaneous oocyte maturation. When FPH was added to follicles exhibiting spontaneous oocyte maturation, progesterone production was augmented and the time course of oocyte maturation was greatly accelerated. Involvement of ovarian components in the maturation process was investigated by selective removal of various follicle layers by microdissection. Removal of follicle epithelium and theca layer (defolliculation) markedly decreased spontaneous and FPH-induced maturation, whereas removal of the entire follicle wall (denudation) completely blocked it. Our results suggest that both spontaneous and FPH-induced maturation involve an estrogen sensitive process in the follicle wall. Thus, somatic follicle cells appear to serve as a common mediator for both types of maturation, which are linked by some intrafollicular mechanism involving steroidogenesis. Hence, estrogen may play an important role as an endogenous intrafollicular regulator of oocyte meiotic maturation.     

The ultimobranchial body in Rana pipiens

Summary Autoplastic transplants of ultimobranchial glands of male Rana pipiens were bilaterally or unilaterally placed in a homeotopic or heterotopic site. Serum calcium levels were maintained at normal values in bilateral autotransplants, while total ultimobranchialectomy resulted in hypercalcemia. Electron microscopy verified the viability and functional state of transplanted, denervated glands. During the periods of denervation, ergastoplasm and Golgi membranes exhibited hypertrophy which was reversed when unmyelinated nerves reappeared in the pericapillary space. Autotransplants under hypercalcemic conditions indicated that the process of secretion is primarily an intrinsic cellular activity and independant of innervation. The present evidence suggests that the sympathetic axons which innervate the parenchyma probably are inhibitory in nature and may allow depression of glandular functions during periods of hypercalcemia.The technical assistance of Mrs. Lilly Weeks is gratefully acknowledged. This project was supported by N. I. H. Grant No. AM-11795; The National Institutes of Arthritic and Metabolic Diseases.     

The ultimobranchial body in Rana pipiens

Summary Hypercalcemia was induced in male frogs by injection of Vitamin D₂ and maintaining animals in calcium chloride water. The fine structure of the Ultimobranchial gland was examined 3, 7 and 14 days after the initial injection. The initial response observed after the third day was a depletion of secretory granules in addition to an alteration of nuclear shape and cytoplasmic hypertrophy. After seven days secretory granule depletion continued and early cell types occurred which indicated an increase in mitotic activity. There was also a demonstrable increase in the amount of ergastoplasm and hypertrophy of the Golgi apparatus. On the fourteenth day, the height of the epithelium was markedly increased while the underlying vascular network was enlarged and more intimately associated with the secretory parenchyma. The homeostatic mechanisms of the Ultimobranchial gland appear to include both a rapid secretory response upon stimulation and a cellular renewal system to replace exhausted cells. This suggests that such a glandular system provides a mechanism to supply a rapidly expanding cell population to meet the demands of an excessive depletion of secretory materials. The response of this gland to hypercalcemia supports previous studies which suggest that the Ultimobranchial gland is the probable source of the hypocalcemic hormone, calcitonin.This project was supported in part by funds provided by the Department of Anatomical Sciences and National Institutes of Health, Grant No. AM-11795.     

The synthesis and activity of tyrosinase during development of the frog Rana pipiens

We have established by radioimmunoprecipitation that tyrosine-DOPA oxidase (TDO, tyrosinase) [EC 1.14.18.1] is first synthesized by frog embryos at the early neurula stage soon after embryonic induction of the neural plate by the underlying chordamesoderm. The DOPA moiety of the enzyme, at the time of its first appearance, is almost inactive enzymatically and can be activated by mild proteolysis (with trypsin). A very large increase in the amount of active DOPA oxidizing enzyme (without trypsinization) is observed at hatching (stage 21), and this is accompanied by melanin deposition in pigment cells. The tyrosine moiety of the enzyme is also partially inactive at the time of first synthesis, but the ratio of active to inactive enzyme remains approximately constant throughout early development. DOPA decarboxylase enzymatic activity is first detected at neurula stage, and this activity is accompanied by the first appearance of catechol amines.     

Calcium effects on progesterone accumulation and oocyte maturation in cultured follicles of Rana pipiens

Pituitary homogenates (FPH) provoke a cascade of responses in the amphibian ovarian follicle, culminating in progesterone biosynthesis and oocyte maturation (GVBD). Calcium may play an important role as an intracellular second messenger in regulating these physiological responses. Experiments were carried out on cultured, isolated follicles of Rana pipiens to assess the effects of varying extracellular calcium on follicular progesterone accumulation and oocyte maturation. In hormonally unstimulated follicles, an increase in extracellular Ca2+ alone produced a significant increase in progesterone in methanol extracts of follicles after 4 hours of culture, and in some cases also provoked oocyte maturation assessed after 24 hours of culture. In no case did elevated Ca2+ alone stimulate maximal progesterone accumulation as compared with FPH-stimulated follicles, although the time-course of accumulation was similar. The calcium ionophore, A-23187, similarly increased progesterone accumulation in a dose-dependent manner when introduced in amphibian Ringer's (1.35 mM Ca2+), but inhibited progesterone elevation caused by increasing calcium concentrations in the culture media and FPH stimulation. Depleting free calcium from the culture medium with graded doses of the chelator EGTA decreased FPH-induced progesterone accumulation and inhibited FPH- and progesterone-induced GVBD. The calcium channel blocker, verapamil, also inhibited FPH-induced progesterone accumulation and GVDB in a dose-dependent manner, while having no effect on progesterone-induced meiotic resumption. These data strongly implicate intracellular calcium levels regulating progesterone production by ovarian follicle cells and subsequent oocyte maturation.     

Influence of exogenous ions on the events of maturation in Rana pipiens oocytes

Full-grown ovarian oocytes removed from non-hormone-treated Rana pipiens females exhibit a low level of protein synthesis, the rate of which is dependent upon the ionic environment. The highest rates of protein synthesis in these oocytes are obtained in media containing either a divalent cation (Ca⁺⁺ or Mg⁺⁺) or high levels of K⁺. The dependence of protein synthesis on ionic environment persists through about the first 18-24 hours of maturation (at 18°C). Normal maturation of oocytes in vitro also has specific ionic requirements for the first 24 hours. In this case, the process requires high ionic strength (T/2 = 1.0-1.2) and divalent cations. The kinetics of K⁺ exchange suggest that K⁺ exists in the ovarian oocyte in two compartments; one in equilibrium with the exogenous medium and freely exchangeable; the other in equilibrium with the exogenous medium and freely exchangeable; the other in equilibrium with the first internal compartment and only very slowly exchangeable. The slowly exchangeable (bound) compartment contains about 95% of all endogenous K⁺. In hormone stimulated oocytes, the kinetics of K⁺ exchange are essentially the same. Oocyte adaptation to ionic environment is discussed as a possible regulatory mechanism during maturation.     

Estradiol synergism and inhibition of insulin-induced meiotic maturation in Rana pipiens oocytes in vitro

Estradiol 17-β (E2) was found to either inhibit or synergize Na-insulin (Ins)-induced meiotic maturation of Rana oocytes. Inhibition of Ins activity occurred in the presence of the follicular investments of the oocyte; synergism with Ins occurred in oocytes denuded of the follicle wall. Similarly, co-incubation of E2 with frog pituitary homogenate (FPH) or pregnenolone (Pe) significantly decreased meiotic reinitiation as determined by germinal vesicle dissolution (GVD) in follicle-enclosed oocytes. By contrast, E2 had no consistently significant effect on progesterone (P)-induced meiosis in follicle-enclosed oocytes. Furthermore, E2 had no significant effect, either inhibitory or synergistic, on Pe- or P-induced GVD of denuded oocytes. Thus, of the meiotogens tested (Ins, P, Pe, FPH), all but P were consistently inhibited by E2 in the presence of the follicle wall. Na-insulin was the only meiotogen tested (Ins, P, Pe) which was potentiated by E2 in denuded oocytes, However, when E2 and Ins were spatially separated on the surface of individual intact follicles, the result was synergism of Ins-induced GVD rather than inhibition. These results suggest that Ins acts to induce GVD in the denuded oocyte through a mechanism distinct from that used by P (ie, Ins mechanism allows E2 synergism while the P mechanism does not). The E2 inhibitory effect on Ins-induced GVD appears to be dependent upon simultaneous exposure of follicle wall tissue to mixtures of E2 and Ins. The synergistic effect of E2 on Ins-induced GVD is dependent upon the simultaneous exposure of the oocyte surface to Ins and E2, either as a homogenous mixture in the case of denuded oocytes or as single substances at independent sites, for follicle-enclosed oocytes.