Allen W. Schuetz

Local Control Mechanisms During Oogenesis and Folliculogenesis

In most animal species a single cell, the female gamete, is the primary or only cellular link between the present and successive generations; the female gamete constitutes an irreplaceable element of life. Thus, the origin, differentiation, qualities, and functions of this essential cell are of fundamental interest and importance to developmental biology and are related directly or indirectly to multiple human activities—social, personal, scientific, medical, and agricultural.

Intrafollicular action of estrogen in regulating pituitary-induced ovarian progesterone synthesis and oocyte maturation in Rana pipiens: temporal relationship and locus of action.

Previous studies demonstrated that estrogen inhibited frog pituitary homogenate (FPH) and progesterone-induced oocyte maturation. In order to determine whether estrogen interfered with intrafollicular progesterone synthesis, experiments were designed to study the effect of exogenous estrogen (estradiol-17 beta) on FPH-induced progesterone production in amphibian (Rana pipiens) ovarian follicles cultured in vitro. Intrafollicular progesterone concentrations were monitored directly using radioimmunoassay and the occurrence of germinal vesicle breakdown (GVBD) was used as a biological indicator of the action of steroids on oocyte maturation. FPH elicited a rapid and dramatic increase in follicular progesterone concentration (1000-3000 pg per follicle) which preceded germinal vesicle breakdown. Addition of estrogen to the culture medium inhibited FPH-induced progesterone production and the accompanying GVBD in a dose-dependent fashion. The presence of estrogen did not enhance the degradation of preloaded progesterone, suggesting estrogen impeded progesterone synthesis rather than enhanced progesterone metabolism. The temporal relationship between estrogen and FPH interaction was assessed by varying the relative time of hormone addition, after which intrafollicular progesterone concentration and GVBD were monitored. Progesterone production and GVBD were drastically inhibited when estrogen was added before or simultaneously with FPH. However, when addition of estrogen was delayed until after FPH simulation, a progressive loss of the inhibitory effect of the steroid on progesterone accumulation and GVBD was observed. Thus, the estrogen-sensitive phase was confined to the early portion of FPH stimulation. Continuous presence of estrogen in the culture system was not required to inhibit FPH-induced events. A short exposure (15 min) of follicles to estrogen was sufficient to inhibit oocyte maturation, whereas progesterone synthesis was not significantly affected. With longer exposure, however, FPH-induced progesterone production was impeded. Washing estrogen-treated follicles did not reverse the inhibitory effect of estrogen, however the follicles remained responsive to exogenous progesterone stimulation and exhibited GVBD. Results suggest that the inhibitory effects of estrogen on FPH action and progesterone production were not reversible under the in vitro culture conditions. To determine whether specific follicular components were involved in estrogen inhibition, progesterone production was assessed following selective removal of different follicle components by microdissection prior to being treated with FPH and estrogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Precocious induction of activation responses in amphibian oocytes by divalent ionophore A23187

Abstract Temporal relationships between maturational events and the onset of activation in response to divalent ionophore and to pricking were examined following in vitro exposure of Rana pipiens oocytes to desoxycorticosterone acetate (DOCA). Activation was evaluated on the basis of vitelline envelope elevation and cortical granule breakdown. Ionophore-induced activation was first observed after 18 hr of DOCA incubation, coincident with the time of separation of the vitelline envelope from the oocyte surface and 2–3 hr after breakdown of the germinal vesicle. Activation in response to pricking was not observed until 30 hr of DOCA incubation. Neither ionophore treatment nor pricking resulted in activation of oocytes that had not been incubated with DOCA. These results indicate that oocytes can be activated many hours earlier than previously demonstrated. The time of onset of the capacity for activation appears to be related to germinal vesicle breakdown and vitelline envelope separation.

Chemical properties and physiological actions of a starfish radial nerve factor and ovarian factor.

Abstract Incubation of starfish ovarian tissues with radial nerve factor (RNF) results in the appearance of a second biologically active substance (s) (ovarian factor) in the incubation media. Studies were conducted to eluciate the chemical and physiological properties of these two substances. The two substances could be physically separated by gel filtration following elution from columns of Sephadex G-25. The RNF and ovarian factor were both relatively heat stable. RNF but not ovarian factor was inactivated following enzymatic digestion with pronase. The ovarian factor appears to be a small molecular weight, heat stable, non proteinaceous material. The physiological effects of a starfish radial nerve factor (RNF) and the ovarian factor were compared. The actions of these substances were evaluated on the basis of changes in various components and responses in starfish ovarian fragments maintained in vitro . Both RNF and ovarian factor induced shedding of oocytes and a concomitant decrease in ovarian fragment weight. The magnitude of weight decrease was related to the dose of ovarian factor. The extent of oocyte shedding was similar in fragments treated with RNF and the highest doses of ovarian factor. RNF and ovarian factors produce oocyte germinal vesicle breakdown and dispersion of follicular cells from around oocytes within ligated and unligated ovarian fragments. Increased levels of calcium in seawater did not produce any of these effects; however, washing ovarian fragments in calcium-free seawater partially inhibited the action of RNF and ovarian factor. Oocytes matured within ligated ovarian fragments, following exposure to RNF or ovarian factor, and subsequently released, formed polar bodies. Upon addition of sperm, these eggs formed fertilization membranes and developed in a normal manner to gastrula. These experiments demonstrate that the ovarian factor has several biological effects or initiates a series of interrelated events. All biological activities previously attributed only to the RNF are apparently shared by this ovarian factor.

Estrogens and ovarian follicular functions in Rana pipiens

Abstract Ovulation and oocyte meiotic maturation (germinal vesicle breakdown) occur following the in vitro exposure of amphibian ovarian follicles to progesterone or gonadotropic hormones. The effects of estrogens on these two processes, and the separation of the follicular membranes from the oocyte, alone or in combination with these stimulatory hormones, were simultancously assessed. Estrogenic hormones did not initiate ovulation, maturation, or membrane separation and exhibited no inhibitory effects on ovulation, maturation, or membrane separation induced with progesterone. Estrone was inhibitory to pituitary-stimulated ovulation, maturation, and membrane separation when added to the incubation media at the same time as the pituitary hormone. Ovarian follicles were insensitive to the inhibitory effects of estrone if the addition of the steroid was delayed several hours after exposure to pituitary hormones. Follicular insensitivity to estrone occurs prior to germinal vesicle breakdown. These data suggest that estrogens act to prevent the synthesis or release of pituitary-stimulated ovarian intermediate(s). The ovarian folliele wall rather than the oocyte appears to be the site of estrogen inhibition of gonadotropic hormones.

Protein synthesis requirement for maturation promoting factor (MPF) initiation of meiotic maturation in Rana oocytes

Abstract Mechanisms controlling disintegration or breakdown of the germinal vesicle (GVBD) in Rana oocytes were investigated. A secondary cytoplasmic maturation promoting factor (MPF), produced in response to steroid stimulation, was shown to induce maturation when injected into immature recipient oocytes. Exposure of immature Rana oocytes to cycloheximide following injection of MPF or steroid treatment completely inhibited such maturation. Results indicate that injected MPF required protein synthesis for germinal vesicle breakdown and thus acted at some translational level. These results contrast with data obtained in Xenopus oocytes where injected MPF induced maturation in the presence of cycloheximide. Cytoplasmic MPF was also produced in Rana oocytes following treatment with lanthanum salts. This activity was similarly inhibited by cycloheximide. Time course studies conducted to compare the onset of cycloheximide insensitivity in steroid-treated and MPF-injected oocytes demonstrated that MPF-injected oocytes become insensitive to cycloheximide prior to steroid-treated germ cells. These results suggest that MPF acts as an intermediary in progesterone-induced maturation. Insensitivity to cycloheximide occurred several hours prior to the onset of germinal vesicle breakdown in both MPF-injected and steroid-treated oocytes. The data indicate that injected MPF in Rana does not induce nuclear disintegration directly, but rather requires amplification and/or autocatalytic synthesis of additional MPF or other factors for maturation to be induced. Molecular mechanisms involved in nuclear disintegration are discussed in relation to these species differences.

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian (Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0-3 microM), cAMP (0-3 mM), or dibutyryl cAMP (dbcAMP, 0-2.5 mM) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 mM) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 mM) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0-3 microM) and IBMX (0-0.09 mM) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 mM) and IBMX (0.09 mM). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.

MEIOTIC MATURATION AND THE CORTICAL GRANULE REACTION IN STARFISH EGGS

Correlative light and electron microscopic studies of immature and maturing starfish (Asterias forbesi) eggs have been carried out demonstrating(l) morphological alterations attending meiotic maturation induced by 1-methyladenine and (2) the structure of the egg cortex and cortical granule reaction. Because cortical granule components, are structurally recognizable, their fate and relation to the development of the fertilization membrane could be determined. One and possibly more of the cortical granule components become an integral part of the fertilization membrane. Comparison of maturing and immature ova indicate that germinal vesicle-containing oocytes (immature) are capable of undergoing a cortical granule reaction morphologically similar to that of eggs having undergone germinal vesicle breakdown (maturing).

Interaction of progesterone with all or isolated portions of the amphibian (Rana pipiens) oocyte surface: Physical and biological characteristics☆

Progesterone induces the resumption of meiotic maturation of fully grown oocytes of Rana pipiens both in vivo and in vitro. The nature of the interaction of progesterone with the oocyte was investigated using a technique which allowed the application of steroid to a portion of the oocyte surface. Uptake of [3H]progesterone from the incubation media with time and with varying concentrations of steroid was approximately proportional to the surface area exposed. After 1.5 or 24 hr of continuous exposure of a portion of the oocyte surface to [3H]progesterone, greater than 90% of the radioactivity was associated with the hemisphere exposed. Restriction of the portion of oocyte surface exposed reduced the biological potency of progesterone in the induction of maturation as assessed by germinal vesicle breakdown. Decrease in hormone effectiveness was not due to direct physical effects of the technique. Removal of the surface restriction resulted in an increase in biological activity of the steroid; this change in steroid potency was correlated with an increase in steroid distribution over the cell. Oocytes continuously exposed over a restricted part of their surface to high levels of progesterone (10 μg/ml) matured to a limited extent. After 24 hr of incubation, 55% of the oocytes exposed to 10 μg/ml of progesterone over the animal pole matured as compared to 0% of those oocytes exposed over the vegetal pole. Using [3H]progesterone, no difference was detected in the amount of steroid taken up or retained by the two polar regions. These investigations suggest that the amount of progesterone required to induce maturation is related to its distribution over the oocyte and that the animal and vegetal hemispheres differ in their ability to respond to progesterone.

Regulation of nuclear membrane assembly and maintenance during in vitro maturation of mouse oocytes: role of pyruvate and protein synthesis

SummaryIn the absence of a suitable energy source, mouse oocytes cultured in vitro resume, but fail to complete, meiotic maturation. However, little is known about the underlying mechanisms leading to this meiotic failure. We utilized pyruvate-deficient medium to test for the role of pyruvate throughout the meiotic maturation process. Germinal vesicle-stage (GV) oocytes underwent germinal vesicle breakdown (GVBD), but failed to form a polar body when cultured continuously in pyruvate-free medium. However, when GV oocytes were preincubated for 4 h in pyruvate-free medium containing dibutyryl cyclic adenosine monophosphate (dbcAMP) and then cultured in pyruvate-free medium, GVBD was markedly inhibited. Preincubation of GV oocytes in dbcAMP and cycloheximide, followed by culture in cycloheximide only, also inhibited GVBD. A longer preincubation period was required in the cycloheximide-dbcAMP case (12 h) than in pyruvate-free-dbcAMP medium situation (4 h). Strikingly, reassembly of the nuclear membrane without polar body formation was observed following GVBD in oocytes continuously cultured in pyruvate-free medium. The reassembled nuclear membrane increased in size with continued culture, and it surrounded partially-decondensed chromatin. Nuclear membrane reassembly also occurred in oocytes which had undergone GVBD during continuous culture in medium containing only cycloheximide. Reformation of nuclear membranes after GVBD was confirmed by electron-microscopic analyses of oocytes cultured in pyruvate-free medium or in the presence of cycloheximide. We conclude that both pyruvate and protein synthesis are required for nuclear membrane disassembly, whereas lack of pyruvate or protein synthesis is associated with interruption of the metaphase state and reassembly of the nuclear membrane. The evidence suggests that assembly and maintenance of an intact nucleus and its disintegration are all amenable to regulation by pyruvate, possibly via mechanism(s) involving protein synthesis.