CAC3(MSI1) suppression of RAS2(G19V) is independent of chromatin assembly factor I and mediated by NPR1.

Cac3p/Msi1p, the Saccharomyces cerevisiae homolog of retinoblastoma-associated protein 48 (RbAp48), is a component of chromatin assembly factor I (CAF-I), a complex that assembles histones H3 and H4 onto replicated DNA. CAC3 overexpression also suppresses the RAS/cyclic AMP (cAMP) signal transduction pathway by an unknown mechanism. We investigated this mechanism and found that CAC3 suppression of RAS/cAMP signal transduction was independent of either CAC1 or CAC2, subunits required for CAF-I function. CAC3 suppression was also independent of other chromatin-modifying activities, indicating that Cac3p has at least two distinct, separable functions, one in chromatin assembly and one in regulating RAS function. Unlike Cac1p, which localizes primarily to the nucleus, Cac3p localizes to both the nucleus and the cytoplasm. In addition, Cac3p associates with Npr1p, a cytoplasmic kinase that stablizes several nutrient transporters by antagonizing a ubiquitin-mediated protein degradation pathway. Deletion of NPR1, like overexpression of Cac3p, suppressed the RAS/cAMP pathway. Furthermore, NPR1 overexpression interfered with the ability of CAC3 to suppress the RAS/cAMP pathway, indicating that extra Cac3p suppresses the RAS/cAMP pathway by sequestering Npr1p. Deletion of NPR1 did not affect the quantity, phosphorylation state, or localization of Ras2p. Consistent with the idea that Npr1p exerts its effect on the RAS/cAMP pathway by antagonizing a ubiquitin-mediated process, excess ubiquitin suppressed both the heat shock sensitivity and the sporulation defects caused by constitutive activation of the RAS/cAMP pathway. Thus, CAC3/MSI1 regulates the RAS/cAMP pathway via a chromatin-independent mechanism that involves the sequestration of Npr1p and may be due to the increased ubiquitination of an Npr1p substrate.

Endocrine and morphological maturation of the fetal and neonatal adrenal cortex in baboons.

Developmental changes in basal and circadian fluctuations in plasma dehydroepiandrosterone sulfate (DHEAS) and cortisol (F) from birth until 3 months of life were studied in conjunction with morphological characteristics of the fetal and neonatal adrenal cortex in baboons (Papio anubis). These studies were complimented by measurements of in vitro production of DHEAS and F (basal and ACTH stimulated) by adrenal tissue slices. Cortisol, DHEAS, estrone, estradiol, and progesterone were determined in plasma and incubation medium by specific RIA. After delivery, an initial rise in plasma DHEAS was sustained for 4 days, followed by a precipitous decline, which reached a nadir between days 10-12 postpartum. Thereafter, plasma DHEAS and F concentrations stabilized with minor fluctuations. A significant (P less than 0.05) and persistent diurnal rhythm in DHEAS and F secretion was evident by the end of the first week after birth. Administration of estrone acetate and progesterone in oil maintained neonatal plasma concentrations of estrone, estradiol, and progesterone at levels comparable to those in utero, but had no quantitative or qualitative effect on the pattern of plasma DHEAS or F in the neonate. The relative production of F to DHEAS by tissue slices in vitro (the calculated F/DHEAS ratios) indicates that DHEAS secretion by the fetal adrenal is 10-fold higher than that of F near parturition; ACTH stimulation does not alter this relationship. By postnatal day 10, the basal production rates of F and DHEAS are equivalent, and the response to ACTH stimulation favors production of F. With advancing neonatal age (at 30 and 100 days), there is an increase in the F/DHEAS secretion ratio both during the basal state and in response to ACTH. The baboon adrenal glands increased in weight during the last month of gestation and then stabilized during the early postnatal period; a gradual increase in weight was observed after 30 days postpartum. Within 2 weeks after parturition, the relative width of the fetal zone decreased dramatically to occupy less than one third of the total cortex. During involution, we observed a decrease in cell size and a reduction in cytoplasmic vacuolation. A zone of closely packed cells with numerous areas of cell death (the dense band) separated the zona fasciculata from the fetal zone. Cell proliferation was observed in the upper regions of the definitive cortex. We conclude the following. 1) The hypothalamic-hypophyseal mechanisms that regulate the diurnal adrenal secretory rhythm are established in the early neonatal period in the baboon.(ABSTRACT TRUNCATED AT 400 WORDS)

Estrogen receptors, progestin receptors and DNA synthesis in the macaque endometrium during the luteal-follicular transition.

We have suggested that in the nonhuman primate endometrium, stromal cells might play a role in mediating the effects of estrogen on the epithelium, especially during the luteal-follicular transition (LFT) when target cells normally escape from the inhibitory influence of progesterone (P). We now report that like estrogen receptors (ER), endometrial progestin receptors (PR) are detectable only in stromal cells until the fifth day of the LFT. With a technique that combined immunocytochemistry and autoradiography on the same sections, we characterized the cellular distribution of ER or PR coincidentally with the localization of [3H]thymidine taken up in vitro by endometria from monkeys undergoing an LFT. DNA synthesis in the glands of the upper endometrium was E2-dependent, but the distribution of [3H]thymidine was not positively correlated with the presence of ER or PR. Readministration of P to animals on days 3 or 4 of the LFT significantly reduced the [3H]thymidine labeling index of the glandular epithelium and caused stromal ER to decline, but P did not block the eventual appearance of ER in epithelial cells on day 5 of the LFT. Thus, E2 stimulated DNA synthesis in epithelial cells that lacked ER, and P suppressed DNA synthesis in these cells even though PR was only detected in the stroma when P treatment began. These data are consistent with a role for endometrial stromal cells in mediating the effects of E2 and P on the epithelium during the LFT.

Estrogen and progestin receptors in the reproductive tract of male and female primates.

With the aid of monoclonal antibodies specific to the estrogen and progestin receptors, we have examined the cellular localization of these proteins in the reproductive tract of male and female macaques. Two striking findings have resulted from our work with these new reagents. First, these receptors are detectable only in cell nuclei, regardless of hormonal treatment, and second, they are often detectable in stromal, but not epithelial cells when the epithelial cells undergo various estrogen or progestin-dependent events. The latter observation has led us to conclude that stromal cell-epithelial cell interactions may play previously unappreciated roles in the hormonal control of the primate reproductive tract. The lines of evidence that have drawn us to this conclusion will be reviewed.

Immunocytochemistry versus binding assays of the estrogen receptor in the reproductive tract of spayed and hormone-treated macaques.

We used immunocytochemistry (ICC) with monoclonal antibodies to the estrogen receptor (ER) to localize ER in the oviducts, uteri, and cervix of untreated, estrogen-treated, and estrogen-progestin-treated spayed macaques. We also used binding assays with labeled estrogens to quantify nuclear and cytosolic ER levels in parallel samples of the same tissues. In untreated spayed animals, cytosolic ER levels were much higher than nuclear ER levels, but all specific staining was nuclear. After treatment for 14 days with estradiol (E2), the degree of staining for ER in cell nuclei in the oviduct, cervix, and endometrium had increased, and there were significant increases in both nuclear and cytosolic ER levels. In the myometrium, ER levels and ICC staining of nuclei increased minimally with E2 treatment. In animals treated for 2 weeks with E2 followed by 2 weeks with E2 and progesterone (P; sequential P treatment) the degree of nuclear ER staining in the oviduct, endometrium, and cervix greatly decreased, and cytosolic and nuclear levels of ER declined significantly. In the myometrium of such animals there was a minimal decrease in the degree of staining and a nonsignificant decline in cytosolic and nuclear ER levels. Sequential P treatment reduced the degree of nuclear staining in the oviduct and endometrium below that found in spayed animals; however, such treatment only lowered the amount of cytosolic, not nuclear, ER significantly below spayed levels in those same tissues. Some animals were treated sequentially with P and sampled 1, 3, 12, and 24 h after the onset of P treatment. By 1 h, nuclear ER levels in the endometrium were significantly suppressed, but cytosolic levels were not lowered until 3 h of treatment; ICC staining was also not substantially reduced until 3 h of P treatment. In the oviduct, nuclear ER levels were significantly reduced by 1 h of P treatment, but cytosolic levels were not lowered until after 12-24 h of P treatment; the degree of nuclear staining in the oviduct was also not substantially reduced until 12-24 h of P treatment. In myometrium, there was no significant decline in ER in nuclear or cytosolic fractions or any substantial decrease in the degree of nuclear staining at any time during this treatment. These observations suggest that the ER detected by ICC in the nuclei of target cells in frozen sections represents the total ER detectable by binding assays in cytosolic and nuclear fractions.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunocytochemical localization of estrogen receptors in the macaque reproductive tract with monoclonal antiestrophilins.

Monoclonal antibodies to the estrogen receptor (anti-ER) were used to develop an immunocytochemical method to detect ERs in frozen sections of the macaque reproductive tract. Specific nuclear, but not cytoplasmic, staining occurred with two different methods: direct, in which an antiestrophilin -horseradish peroxidase conjugate was used as the first antibody, and indirect, in which a mixture of antiestrophilins was used in the first incubation step. Nuclear staining was absent when various control antibodies replaced the anti-ER. In uteri from spayed monkeys treated with estradiol (E2) for 14 days, nuclear staining was always present. In uteri from similar animals treated for an additional 14 days with E2 and progesterone, nuclear staining was almost completely absent. Mean endometrial nuclear ER levels, measured by an exchange assay, were 5-fold greater in the E2-treated than in the E2-plus progesterone-treated group. In addition, when samples of estrogenized uterus and oviduct were incubated for 60 min in vitro with 100 nM E2, the intensity of nuclear staining increased in parallel with an increase in the concentration of nuclear ER. The nuclei of stromal, smooth muscle, and epithelial cells of the estrogenized oviduct, cervix, and vagina as well as smooth muscle cells of the estrogenized myometrium were also receptor positive. Nontarget tissues, such as duodenum, colon, esophagus, and skeletal muscle, contained no cells that showed specific nuclear staining. Some staining of cytoplasmic and extracellular components occurred in all preparations. These latter reactions were nonspecific, because they were present in many nontarget tissues or when control antibodies replaced the anti-ER. With current methods, only nuclear ERs can be reliably localized in frozen sections of monkey tissues with monoclonal antiestrophilins .

Estradiol synthesis by fetal monkey ovaries correlates with antral follicle formation.

We compared the morphology of ovaries from fetal rhesus monkeys at two different stages of gestation with the ability of ovarian tissue to synthesize androgens and estrogens in vitro. Ovaries collected between 80 and 104 days of gestation contained single-layered primordial follicles but no multilayered or antral follicles. Within these ovaries we found theca-like interstitial cells which contained abundant smooth endoplasmic reticulum and lipid droplets. These ovaries produced androstenedione and dehydropiandrosterone (but not estradiol) in vitro in the absence of exogenous substrates. Ovaries collected between Days 124 and 153 of gestation, however, contained numerous, well-developed multilayered and antral follicles. The ultrastructural characteristics of the thecal and granulosa cell layers were similar to those of adult ovarian follicles. These ovaries synthesized large amounts of androstenedione, dehydroepiandrosterone, and estradiol in vitro. Identity of the estradiol was confirmed by incubating homogenates of late gestation ovaries with [14C]progesterone, by isolation of estradiol and formation of its derivative, and by recrystallization to constant specific activity. In vitro cyclic AMP synthesis was stimulated by Pergonal (luteinizing hormone [LH] + follicle stimulating hormone [FSH] ) only in ovaries which contained multilayered and antral follicles, an indication that fetal ovaries contain gonadotropin receptors during late stages of development. These data indicate that fetal ovaries of rhesus monkeys attain the capacity for de novo estrogen biosynthesis and to respond to gonadotropins during late gestation, when multilayered and antral follicles have developed.

Secretory granules and progesterone secretion by ovine corpora lutea in vitro.

To study the relationship between formation and release of Golgi-derived secretory granules and progesterone secretion, slices of ovine luteal tissue were incubated in the presence of LH and/or calcium ionophore A23187. Increases in progesterone secretion in response to LH and/or ionophore were accompanied by a concomitant release of secretory granules. In contrast, in the presence of colchicine, LH-stimulated progesterone secretion was significantly reduced (P less than 0.01), granule formation appeared to be blocked, and there was little evidence of exocytosis. In addition, unusual pleomorphic membrane-bounded saccules containing an electron-dense material were abundant throughout the centrospheric region of cells treated with colchicine. Because of the close parallelism between formation and release of Golgi-derived secretory granules and progesterone secretion, it appears that progesterone secretion may be coupled to exocytosis of secretory granules. Although the exact content and function of the secretory granules described remains to be elucidated, the data obtained are compatible with the notion that they may contain a progesterone carrier protein.

Luteinizing hormone, progesterone and the morphological development of normal and superovulated corpora lutea in sheep.

The development of granulosa-lutein cells was studied in 27 normal and 32 superovulated ewes between days 0-4(day 0 began with the preovulatory LH peak in normal animals and the HCG injection in superovulated ewes). The pattern of differentiation was similar in both groups. Following initial hormonal stimulation (0-12 hours after LH or HCG), granulosa cells were approximately 100 mu2 and contained small, pleomorphic nuclei with large amounts of clumped chromatin. Elongate cells lining the basement membrane possessed large, heterogeneous dense bodies, and a well-developed Golgi apparatus. Mitotic figures were observed up to 6 hours prior to ovulation. Sixteen to 20 hours following the LH surge or HCG injection, hypertrophy of granulosa cells was evident. Nuclei contained definitive nucleoli. Blood vessels in the theca interna were abundant and highly dilated. Ovulation occurred approximately 24 hours after the LH peak or HCG injection. Visible signs of luteinization were evident 6-12 hours after ovulation. A slight increase in serum progesterone levels was detected. The second post-ovulatory day was characterized by continuing hypertrophy of granulosa cells and extensive proliferation of smooth endoplasmic reticulum and mitochondria. Nuclei of granulosa cells were larger and possessed extremely large nucleoli. Numerous mitotic figures were apparent within the corpus luteum. Serum progesterone concentrations began increasing at 60-72 hours after hormone stimulation. By the end of the third post-ovulatory day, the corpus luteum consisted of large, pleomorphic, parenchymal cells, interspersed between capillaries and connective tissue elements. Only an occasional mitotic figure was apparent within the corpus luteum at 100 hours. Light microscopic autoradiography of 5, 10, and 15 day corpora lutea taken from ewes pulsed with 3H thymidine at specific times before and after ovulation revealed that granulosa cells did not undergo secondary mitoses following ovulation. In contrast, thecal, mesenchymal and endothelial cells did mitose on day 3.

Ultrastructural analysis of the granulosa--luteal cell transition in the ovary of the dog.

The transition from ovarian granulosa to lutein cell during the estrus cycle of 60 pregnant and non-pregnant beagle bitches was analyzed by light and electron microscopy (both 100 and 1000 KV). Early proestrus was characterized by a gradual rise in serum estrogen levels, hyperplasia of the granulosa cells, the accumulation of follicular fluid, and the development of tortuous intercellular channels. During the second half of proestrus, serum estrogen levels continued to rise, but growth, division, and differentiation of the granulosa cells was minimal. Estrus was marked by the first acceptance of the male and a well-defined LH peak In the subsequent 24 hour period, the granulosa-lutein cells hypertrophy rapidly and develop a large Golgi apparatus, small profiles of granular endoplasmic reticulum, numerous microfilaments, and large gap junctions between the cells. Mitochondria also proliferate, enlarge, and elongate, but retain lamelliform cristae. Luteinization of the cells and progesterone secretion begin just after ovulation which in turn occurs about 24 hours after the LH peak. On the third and fourth day of estrus, numerous small vesicles of agranular endoplasmic reticulum fill the extoplasm and the mitochondria swell up and round off. The vesicles rapidly fuse into whorled and flattened cisternae or anastomosing tubules of agranular endoplasmic reticulum, while the mitochondria develop tubulovesicular cristae. These structures gradually become organized with respect to the basal lamina. The Golgi apparatus is centered over the pole of the nucleus that faces the pericapillary space. Stacked and whorled cisternae of agranular ER develop in the lateral margins and avascular end of the cell while mitochondria and tubular elements of agranular ER predominate in the central medial and most basal portions of the cytoplasm. Microfilaments are ubiquitous and appear to be instrumental in this orientation process. The cell surface develops three distinct regional specializations that coincide with the underlying cellular compartments: interconnecting pleomorphic folds fill the pericapillary space; long tenous microvilli project from the lateral cell surface and form tortuous intercellular channels and canaliculi; and large gap junctions form along the margins of the cell furthest removed from the basal lamina. By the sixth day of estrus, the granulosa-luteal cell transition is nearly complete and serum progesterone levels are on the rise.

Subcellular compartmentalization of the luteal cell in the ovary of the dog.

The compartmentalization of the parenchyma of the corpus luteum in the dog was studied by both 100 and 1000 KV electron microscopy. The organells within the luteal cell are oriented with a high degree of consistency towards the pericapillary space. Characteristically, the avascular pole and the lateral margins of the cell posses predominantly stacked and whorled cisternae of agranular ER. In the central medial portions of the cell, pleomorphic mitochondria with tubulo-vesicular cristae and anastomosing tubules of agranular ER predominate. However, the distribution of organelles in this compartment is graded. Mitochondria predominate in the central medial areas while tubular ER is more dominant peripherally. Microfilaments are ubiquitous in this compartment and run a longitudinal course between and around the subcellular components towards the pericapillary space. The Golgi apparatus is large and prominent and is positioned over the pole of the nucleus that faces the basal lamina. Coated vesicles are abundant in the Golgi regions and along the lateral surface of the cell. Three distinct regional specializations of the cell surface exist. The basal surface contains long pleomorphic cytoplasmic folds that fill the pericapillary space, are interconnected by small gap junctions and contain abundant multivesicular bodies. The lateral cell surface is covered with microvilli and is organized into tortuous intercellular channels and canaliculi. These are interrupted at intervals by cytoplasmic protrusions that extend from one cell well into the cytoplasm of the next. Large, well-developed gap junctions line the margins of the cells furthest removed from the pericapillary space. Finally, the individual cells exhibit heterogeneity with respect to the amount one subcellular organelle or compartment is expressed relative to another. These observations are discussed in relation to the subcellular compartmentalization of progesterone synthesis and release.