Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer.

Calmodulin-dependent protein kinase III (CaM kinase III, elongation factor-2 kinase) is a unique member of the Ca2+/CaM-dependent protein kinase family. Activation of CaM kinase III leads to the selective phosphorylation of elongation factor 2 (eEF-2) and transient inhibition of protein synthesis. Recent cloning and sequencing of CaM kinase III revealed that this enzyme represents a new superfamily of protein kinases. The activity of CaM kinase III is selectively activated in proliferating cells; inhibition of the kinase blocked cells in G0/G1-S and decreased viability. To determine the significance of CaM kinase III in breast cancer, we measured the activity of the kinase in human breast cancer cell lines as well as in fresh surgical specimens. The specific activity of CaM kinase III in human breast cancer cell lines was equal to or greater than that seen in a variety of cell lines with similar rates of proliferation. The specific activity of CaM kinase III was markedly increased in human breast tumour specimens compared with that of normal adjacent breast tissue. The activity of this enzyme was regulated by breast cancer mitogens. In serum-deprived MDA-MB-231 cells, the combination of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulated cell proliferation and activated CaM kinase III to activities observed in the presence of 10% serum. Inhibition of enzyme activity blocked cell proliferation induced by growth factors. In MCF-7 cells separated by fluorescence-activated cell sorting. CaM kinase III was increased in S-phase over that of other phases of the cell cycle. In summary, the activity of Ca2+/CaM-dependent protein kinase III is controlled by breast cancer mitogens and appears to be constitutively activated in human breast cancer. These results suggest that CaM kinase III may contribute an important link between growth factor/receptor interactions, protein synthesis and the induction of cellular proliferation in human breast cancer.

The different forms of the prolactin receptor in the rat corpus luteum: developmental expression and hormonal regulation in pregnancy.

The corpora lutea of pregnancy in the rat are highly dependent on the action of PRL and PRL-like hormones to hypertrophy and to produce progesterone needed for the maintenance of gestation. Two forms of the PRL receptor (PRL-R), designated as long (PRL-RL) and short (PRL-RS), have been described in rat tissues. To determine whether both forms are present in the corpus luteum during pregnancy and to examine the developmental and hormonal regulation of their expression, total RNA isolated from corpora lutea at different stages of pregnancy and from highly luteinized granulosa cells subjected to different hormonal treatments were analyzed by semiquantitative RT-PCR. Immunoblotting of luteal proteins from early and late pregnancy was also performed to determine if the pattern of PRL-R proteins follows that of PRL-R messenger RNA (mRNA) expression. In addition, the correlation between the well characterized PRL-regulated gene, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), and PRL-R gene expression was investigated during the time of luteolysis. Both PRL-RL and PRL-RS mRNA and protein were expressed in corpora lutea of pregnancy, with the long form being the most dominant at all stages. Whereas no changes in mRNA level of either PRL-RL or PRL-RS were found until day 20 of gestation, a profound decline in PRL-R mRNA and protein for both receptor types occurred at the end of pregnancy. This drop in PRL-R expression was accompanied by a sharp and abrupt expression of 20alpha-HSD mRNA. Studies performed in vivo and in luteinized cells in culture indicate that PRL can up-regulate the expression of the PRL-RL mRNA, an effect prevented by the tyrosine kinase inhibitor, genistein. PRL-RL mRNA was also selectively increased by cAMP. In summary, the results of this investigation have established that: 1) the corpus luteum of pregnancy expresses both the short and long forms of the PRL-R with the long form being more abundant; 2) the mRNA for both forms of the PRL-R remains at constant levels throughout pregnancy but drops before parturition; 3) the decline in PRL-R mRNA at the end of pregnancy is accompanied by a dramatic rise in 20alpha-HSD; 4) PRL is able to increase the expression of PRL-R mRNA; and that 5) both A kinase and tyrosine kinase mediated pathways appear to participate in the up-regulatory mechanism involved in PRL-R mRNA expression.

PRAP, a prolactin receptor associated protein: its gene expression and regulation in the corpus luteum.

We have recently identified, characterized, and cloned a luteal microsomal 32-kDa phosphoprotein that we named PRAP (for PRL-receptor associated protein), and we have demonstrated that PRAP binds to the intracellular domain of the short but not the long form of the PRL receptor. In this study, we used PRAP cDNA to examine the tissue specificity, the developmental expression, and the hormonal regulation of PRAP gene expression. Northern blot analysis revealed that in the corpus luteum, PRAP cDNA hybridized to multiple transcripts (5.5 kb, 4.3 kb, and 1.8 kb), with the smallest transcript (1.8 kb) corresponding to the size of the cDNA clone. However, none of these transcripts were detected in any other tissues examined. PRAP appears to be tightly regulated by steroids and PRL. When pregnant rats were treated with aminoglutethimide, a steroid synthesis inhibitor, all three PRAP transcripts became barely detectable. Similar results were obtained when all luteotropic support was removed by hypophysectomy and hysterectomy. Estradiol up-regulated PRAP expression and, more specifically, the two lower transcripts. PRL had no stimulatory effect on PRAP messenger RNA (mRNA) expression but caused a substantial increase in the level of PRAP protein when administered to hypophysectomized pregnant rat, suggesting that PRL may stabilize this protein. Similar dissociation between levels of mRNA and protein were observed during luteal development. Although both PRAP mRNA and protein were barely detectable in early pregnancy, their expression increased abruptly from midpregnancy; however, whereas levels of PRAP mRNA declined from day 18, those of the protein remained elevated until parturition. In summary, results of this study have defined the tissue specificity and developmental expression of PRAP mRNA during pregnancy. The data have also revealed that the gene expression of this protein is up-regulated by estradiol, suggesting a pivotal role for PRAP in the synergistic action of estradiol and PRL on the function of the rat corpus luteum.

Prolactin-mediated inhibition of 20alpha-hydroxysteroid dehydrogenase gene expression and the tyrosine kinase system.

The rat luteal 20alpha-hydroxysteroid dehydrogenase plays a key role at catabolizing progesterone and at decreasing the level of this steroid secreted by the ovaries. Throughout pregnancy and before parturition neither the mRNA nor the protein for this enzyme could be detected. In this investigation we set to examine whether PRL and PRL-like hormone from placental origin silence the expression of this gene and whether PRL action involves tyrosine kinase activity and/or de novo protein synthesis. The results revealed that PRL and PRL-like hormone from rat placental origin (rPL-1 and rPL-2), but not rat growth hormone, caused a rapid and profound inhibition of 20alpha-HSD mRNA expression in highly luteinized granulosa cells. Immunoprecipition and western blot analysis indicate that PRL-R associates with JAK2 and Stat5, and this association is increased within 30 seconds with PRL treatment. Although both JAK2 and Stat5 were phosphorylated on tyrosine upon PRL treatment, the PRL mediated inhibition of 20alpha-HSD was not reversed by either tyrosine kinase inhibitors, AG18 and genistein, but was largely reversed by the protein synthesis inhibitor cycloheximide. In summary, results of this investigation indicate that although PRL can activate the JAK2/Stat5 system in the corpus luteum, the down regulation of 20alpha-HSD mRNA by PRL does not appear to involve tyrosine kinase activity but depends on de novo synthesis of protein(s).

Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase.

The several hundred members of the eukaryotic protein kinase superfamily characterized to date share a similar catalytic domain structure, consisting of 12 conserved subdomains. Here we report the existence and wide occurrence in eukaryotes of a protein kinase with a completely different structure. We cloned and sequenced the human, mouse, rat, and Caenorhabditis elegans eukaryotic elongation factor-2 kinase (eEF-2 kinase) and found that with the exception of the ATP-binding site, they do not contain any sequence motifs characteristic of the eukaryotic protein kinase superfamily. Comparison of different eEF-2 kinase sequences reveals a highly conserved region of approximately 200 amino acids which was found to be homologous to the catalytic domain of the recently described myosin heavy chain kinase A (MHCK A) from Dictyostelium. This suggests that eEF-2 kinase and MHCK A are members of a new class of protein kinases with a novel catalytic domain structure.

Effects of rottlerin, an inhibitor of calmodulin-dependent protein kinase III, on cellular proliferation, viability, and cell cycle distribution in malignant glioma cells.

Calmodulin-dependent protein kinases phosphorylate certain substrates that have been implicated in regulating cellular proliferation. For example, upon mitogenic stimulation, there is a rapid activation of calmodulin-dependent protein kinase III (CaM kinase III), which leads to the phosphorylation of elongation factor 2. Recently, our laboratory demonstrated that the activity of CaM kinase III is increased in glioma cells following exposure to mitogens and is diminished or absent in nonproliferating glial tissue. Rottlerin, a 5,7-dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzy l)-8-cinnamoyl-1,2-chromene isolated from the pericarps of Mallotus phillippinensis, has been shown to be an effective CaM kinase III inhibitor. Therefore, we evaluated the effects of rottlerin on the growth and viability of glioblastoma cell lines. Rottlerin decreased growth and induced cytotoxicity in rat (C6) and two human gliomas (T98G and U138MG) at concentrations that inhibited the activity of CaM kinase III in vitro and in vivo. Far less demonstrable effects were observed on other Ca2++/CaM-sensitive kinases. Incubation of glial cells with rottlerin produced a block at the G1-S interface and the appearance of a population of cells with a <2N complement of DNA. In addition, rottlerin induced changes in cellular morphology such as cell shrinkage, accumulation of cytoplasmic vacuoles, and packaging of cellular components within membranes. These data suggest that CaM kinase III may be an important link between the activation of CaM-dependent signaling, proliferation, and viability in malignant cells, and that inhibition of CaM kinase III may represent an interesting pharmacological target in malignant gliomas.

Growth hormone (GH) modulates insulin-like growth factor I (IGF-I) and type I IGF receptor mRNA levels in the ovary of prepubertal GH-deficient rats.

In order to explore the potential role of growth hormone (GH) in modulating insulin-like growth factor I (IGF-I) gene expression in the prepubertal rat ovary, female rats were rendered GH deficient by neonatal administration of monosodium glutamate (MSG). One group of rats received vehicle and served as the control. At 21 days of age, MSG-treated rats received either GH or vehicle for 2 weeks. On days 21, 24, 28 and 31 animals were weighed and subsets were sacrificed for liver RNA extraction. The remaining animals were sacrificed at day 35 when livers and ovaries were collected, and serum was obtained for GH determinations. The IGF-I mRNA levels were estimated by Northern blots and corroborated further by slot-blot analysis. The MSG-treated rats had lower body weights (p < 0.01) and GH levels (p < 0.05) than controls. Growth hormone replacement significantly accelerated the weight gain of MSG-treated rats. At day 24 and thereafter, three RNA IGF-I species (7.5, 1.8 and 0.8-1.2 kB) were seen in the liver. In the ovary, at age 35 days, two major IGF-I mRNA species (7.5 and 0.8-1.2 kb) were seen. The MSG treatment consistently reduced the levels of both IGF-I mRNA species in the ovary. Growth hormone administration partially restored their expression, both in the liver and in the ovary. In addition, ovarian type IIGF receptor mRNA levels were increased in the MSG-treated rats when compared to controls. This trend was reversed by GH replacement.(ABSTRACT TRUNCATED AT 250 WORDS)

Does the rat corpus luteum express the progesterone receptor gene?

To determine whether the progesterone receptor (PR) gene is expressed in rat corpora lutea (CL), PR messenger RNA (mRNA) and protein levels were examined in CL of both cycling and pregnant rats using in situ hybridization, reverse transcription-polymerase chain reaction, and Western blotting analyses. During the estrous cycle, levels of luteal PR mRNA were below the sensitivity of in situ hybridization. Although no signal could be detected in luteal cells, granulosa cells of preovulatory follicles of the same ovary expressed PR mRNA at high levels during the evening of proestrus. Likewise, CL of pregnant rats expressed undetectable levels of PR mRNA, which remained unchanged throughout pregnancy, as determined by in situ hybridization and reverse transcription-polymerase chain reaction. In addition, no PR protein could be detected in rat CL by Western analysis, indicating that luteal expression of the PR gene, if any, is negligible. To determine whether the lack of PR mRNA in the rat CL is due to progesterone-induced down-regulation of PR mRNA or to low levels of estrogen, aminoglutethimide was used to block the synthesis of progesterone in the presence and absence of exogenous estrogen, and PR mRNA levels were examined in the CL as well as the placenta. Inhibition of progesterone synthesis did increase PR mRNA levels in the placenta, and additional estrogen treatment further increased PR mRNA levels in this tissue. In contrast, neither aminoglutethimide alone nor aminoglutethimide plus estrogen induced PR mRNA expression in CL of the same rats. The temperature-sensitive luteal cell line derived from the rat CL, which produces very low levels of progesterone, also did not express PR mRNA. These results indicate that the rat corpus luteum expresses undetectable levels of PR mRNA and protein, which is not attributable to progesterone-induced down-regulation of PR mRNA or to a lack of estrogen-induced up-regulation of PR mRNA in this tissue.

Isolation and characterization of a rat luteal cDNA encoding 20 alpha-hydroxysteroid dehydrogenase.

We report the isolation and characterization of a full length cDNA encoding rat 20 alpha hydroxysteroid dehydrogenase derived from rat corpus luteum RNA. The predicted amino acid sequence of the protein encoded by the 20 alpha HSD clone is composed of 323 amino acids possessing an approximate molecular weight of 37 kDa. The sequence of peptides derived from the purified protein was found in the translated sequence of the open reading frame. cDNA and amino acid sequence indicate that the rat ovarian 20 alpha HSD belongs to the aldo-keto reductase family of enzymes. Northern analysis revealed a 1.2 Kb 20 alpha HSD mRNA in corpora lutea undergoing luteolysis. Prolactin reduced markedly 20 alpha HSD mRNA expression. No signal was detected in other tissues examined, demonstrating the specific expression of this enzyme in the corpus luteum.

Identification of a major prolactin-regulated protein as 20 alpha-hydroxysteroid dehydrogenase: coordinate regulation of its activity, protein content, and messenger ribonucleic acid expression.

We have previously reported that an abundant 37,000 mol wt protein with a pI of 6.15 (37K) is expressed specifically in the corpus luteum and is markedly inhibited by PRL. To identify the 37K, amino acid sequence analysis of the protein was performed. The 37K protein showed sequence similarity with rabbit 20 alpha-hydroxysteroid dehydrogenase (20 alpha HSD), chlordecone reductase, prostaglandin synthase, and 3 alpha-hydroxysteroid dehydrogenase, which are members of the aldo-keto reductase group of enzymes that catalyze the NADPH-dependent reduction of carbonyl compounds. Comparison of 20 alpha HSD activity with the level of 37K in the corpus luteum throughout pregnancy demonstrated a close correlation between enzyme activity and luteal levels of the protein. Both protein and enzyme activity were low early in pregnancy, reached a nadir between days 5-19, and reappeared abruptly between days 19-21 of pregnancy. To establish that the enzyme activity is intrinsic to the 37K, the protein was purified from sodium dodecyl sulfate-polyacrylamide electrophoresis gels (SDS-PAGE), renatured, and assayed for 20 alpha HSD activity. The renatured protein exhibited substantial 20 alpha HSD activity. As 20 alpha HSD is known to play a major role in the termination of pregnancy in the rat, it was of interest to examine whether the rapid appearance of the 37 K protein at the end of pregnancy is accompanied by the induction of 20 alpha HSD gene expression. Northern blot analysis using a rabbit cDNA for 20 alpha HSD indicated that the pattern of 20 alpha HSD mRNA expression in the corpus luteum closely paralleled the ontogeny of 20 alpha HSD enzyme activity as well as 37K protein levels. Our studies demonstrated that 20 alpha HSD protein and mRNA levels are coordinately regulated, and that the profound inhibitory effect of PRL on 20 alpha HSD activity is apparently due to inhibition of 20 alpha HSD gene expression, leading to the disappearance of the protein from the corpus luteum.

Hormonal and immunological characterization of the 32 kilodalton ovarian-specific protein.

Recent studies from this laboratory have shown that the large luteal cell of the pregnant rat contains an abundant 32 kilodalton (32K) phosphoprotein which is up-regulated by estradiol. In order to assess the potential importance of this protein and to more fully understand its function, a specific polyclonal antibody was produced against the 32K and was used to examine its intraovarian localization, its tissue specificity, and its developmental regulation. Immunocytochemical localization of the 32K in the ovary of the pregnant rat found this protein to be selectively and abundantly expressed in the corpus luteum. Immunofluorescence study of small and large luteal cell populations clearly revealed an extensive localization of the 32K in the large luteal cells. Western blot analysis revealed that the 32K was absent from all steroidogenic and nonsteroidogenic tissues. Whereas this protein was absent from all other tissues examined in the rat, it was clearly expressed in corpora lutea of different animal species, including the mouse, hamster, cow, human, and pig. Although undetectable by immunohistochemistry, Western blot analysis showed this protein to be present in the follicle but at levels markedly lower than in the corpus luteum. Analysis of theca and granulosa cells revealed the presence of the 32K in both cell types. To further examine the developmental expression of this protein throughout gestation, Western blot analysis of microsomal fractions isolated from rat corpora lutea on days 3-21 of pregnancy was performed. The 32K was detected at low levels in early pregnancy, increased markedly on day 11, reached a peak on days 14-15, and remained elevated through day 21. Treatment with estradiol and PRL increased the content of the 32K in the corpus luteum. Human CG, known to cause follicular development to the preovulatory stage and to enhance luteal estradiol synthesis, also increased levels of the 32K in the corpus luteum, while it concomitantly decreased this protein in the follicle. In summary, the presence of a unique ovarian-specific 32 kilodalton protein has been established. This protein, which is present in low abundance in theca and granulosa cells, is localized to the large luteal cell and becomes abundantly expressed during midpregnancy, a time when there is a remarkable increase in luteal cell size and activity. Results of this study also demonstrate a multihormonal regulation of the 32K by tropic hormones. Thus, because of its apparent uniqueness and its timely and highly regionalized expression, the 32K may play a central role in the regulation of corpus luteum growth and function.

Alpha 2-macroglobulin expression in the mesometrial decidua and its regulation by decidual luteotropin and prolactin.

During decidualization, cells of the endometrium grow and differentiate giving rise to two different decidual tissues located in either the antimesometrial or mesometrial site of the uterus in the rat. These tissues have different functions in pregnancy. The antimesometrial decidua is an endocrine gland that secretes hormones, whereas the mesometrial decidua appears to play an important role in limiting trophoblast invasion. Since the decidual tissue of the rat produces alpha 2-macroglobulin (alpha 2MG), we examined whether this potent protease inhibitor is specifically expressed by the mesometrial tissue, the site of trophoblast invasion, and whether the alph 2MG gene is regulated by decidual luteotropin (DLt), the PRL-like hormone secreted by the neighboring antimesometrial cells. To determine the secretory proteins of the rat decidua, antimesometrial and mesometrial tissues were dissected out from pseudopregnant rats and cultured with [35S]methionine. The major protein secreted by the antimesometrial cell was the 29-kilodalton decidual luteotropin, whereas a 180-kilodalton protein was predominantly secreted by the mesometrial tissue. Immunoprecipitation studies of 35S-radiolabeled proteins revealed that this high mol wt protein is alpha 2MG and that it is secreted exclusively by the cells forming the mesometrial tissue. To examine whether the alpha 2MG gene was also expressed specifically in the mesometrial decidua, total RNA was isolated from both mesometrial and antimesometrial tissues of day 9-12 pseudopregnant rats and hybridized with alpha 2MG cDNA. Northern blot analysis revealed a 5.4-kilobase message, which was abundantly expressed in the mesometrial decidua. Little, if any, alpha 2MG mRNA was detected in antimesometrial decidua. The ontogeny of the message in the decidua correlated well with the development of the mesometrial tissue. To examine whether alpha 2MG expression is regulated by DLt and/or PRL, a highly specific polyclonal antibody to DLt was generated and decidual tissues were cultured in the presence or absence of DLt antibodies with or without PRL. Neutralization of DLt caused a marked decrease in alpha 2MG mRNA levels. This down-regulation was totally reversed by the addition of PRL and was not affected by alpha 2MG antibodies. In summary, the results of this investigation revealed a compartmentalized gene expression, synthesis and secretion of alpha 2MG in the decidua. The secretion of this protease inhibitor, specifically by the mesometrial tissue which is the site of trophoblast invasion, may be the reason for the minimal amount of tissue damage that occurs during placentation.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression, action, and steroidal regulation of insulin-like growth factor-I (IGF-I) and IGF-I receptor in the rat corpus luteum: their differential role in the two cell populations forming the corpus luteum.

The overall aim of this investigation was to examine the expression and steroidal regulation of insulin-like growth factor-I (IGF-I) and the IGF-I receptor in the rat corpus luteum and to examine the specificity of IGF-I action in the two luteal cell populations. We first examined whether the corpus luteum expresses the IGF-I and IGF-I receptor genes. Using a solution hybridization/RNase protection assay, IGF-I and IGF-I receptor mRNAs were represented by protected bands 224 and 265 bases in length, respectively. In addition, Northern blot analysis showed that, as in liver, rat IGF-I and IGF-I receptor cDNAs hybridized with 7.5-, 1.8-, and 0.8- to 1.2-kilobase transcripts and an 11-kilobase transcript, respectively. Both IGF-I and IGF-I receptor mRNAs were detected on all days of pregnancy tested (days 5-21). Since the rat corpus luteum increases remarkably in size and steroidogenic capacity at midpregnancy due to estradiol stimulation, we determined whether these developmental changes are accompanied by an increased expression of the IGF-I and/or IGF-I receptor genes. Total RNA was isolated from corpora lutea of day 12 hypophysectomized-hysterectomized rats treated with or without estradiol for 3 days. Estradiol caused a clear and marked reduction in IGF-I and IGF-I receptor mRNA. [125I]IGF-I bound with high specificity and affinity to luteal cell membranes. Large and small cell populations forming corpora lutea of day 3 and 14 pregnant rats were separated by elutriation and used for the determination of binding activity and for cell culture, respectively. IGF-I receptors were found to be localized principally in the large luteal cell population. The small luteal cells had approximately 6.5-fold less IGF-I-binding activity. The difference in binding activity in both cell populations was reflected in the ability of both cell types to respond to IGF-I. IGF-I (25 ng/ml) had a profound effect on the production of progesterone by the large luteal cells. No stimulatory effect of IGF-I on the small luteal cells was observed. Addition of estradiol (10 ng/ml) to the cell culture remarkably enhanced IGF-I stimulation of progesterone biosynthesis by the large luteal cells. In summary, the results of this investigation have revealed that the corpus luteum of the pregnant rat is a major site of expression of both the IGF-I and IGF-I receptor genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Desensitization to follicle-stimulating hormone in cumulus cells is coincident with hormone induction of oocyte maturation in the rat follicle.

The objective of the present studies was to assess whether hormone induction of oocyte maturation in isolated intact follicles may be linked to desensitization of follicle-stimulating hormone (FSH) in the oocyte-cumulus complex (OCC). Incubation of follicles with chorionic gonadotropin (hCG), FSH or epidermal growth factor (EGF) produced a marked inhibition of FSH-dependent cyclic AMP accumulation in OCC with a time-course coincident with the onset of germinal vesicle breakdown (GVBD). These effects were evident within 3 h for both hCG and FSH, but with EGF a reduced response to FSH was seen within 1 h of treatment followed by an increase in GVBD. In contrast, no inhibition of cyclic AMP accumulation was seen in response to cholera toxin, forskolin or LH in OCC derived from follicles incubated with hCG for 3 h. The time-course for induction of oocyte maturation by incubation of the intact follicle with hCG was also coincident with production of prostaglandin (PG) F2 alpha, an indirect marker of cyclooxygenase induction. No effect on metabolic coupling between the oocyte and cumulus cells was seen until 9 h after hCG treatment. Retinoic acid caused a marked decrease in metabolic coupling between the oocyte and cumulus cells but inhibited oocyte maturation both in denuded oocytes and OCC. Since FSH desensitization in OCC, the resumption of meiosis, and production of arachidonic acid-derived products were coincident, it is suggested that abrogation of FSH action in cumulus cells by the ovulatory surge of gonadotropins may initiate oocyte maturation.

Morphologic and functional studies of immature rat oocyte-cumulus complexes after cryopreservation.

The effects of freezing and thawing (F/T) on functional activity of immature rat oocyte-cumulus complexes (OCC) were studied. The OCC were divided into three groups according to the number of cumulus-cell layers surrounding them. The OCC were then frozen and thawed (F/T), with dimethyl sulfoxide (DMSO) as cryoprotectant. The survival rates after thawing increased significantly (P less than 0.001) as the number of cumulus-cell layers increased. Germinal vesicle breakdown (GVBD) was evaluated in F/T oocytes. After 2 hours, significantly (P less than 0.05) fewer oocytes demonstrated GVBD than did those in the control group. There was no difference, however, after 4 hours of culture. A significant (P less than 0.05) decrease in follicle-stimulating hormone (FSH)-dependent cyclic adenosine monophosphate (cAMP) accumulation was observed in the F/T group. However, the amount of cAMP produced was sufficient to maintain the oocyte in meiotic arrest. There was a borderline significant decrease of the coupling between cumulus cells and the oocyte in F/T OCC, as evaluated by the transport of 3H-uridine into the oocyte. It is concluded that immature rat oocytes can be successfully cryopreserved when they are surrounded by five or more layers of cumulus cells. FSH responsiveness and intercellular communication were essentially maintained. There was a slight delay in GVBD, which needs further clarification.

Apolipoprotein gene expression in the rat is regulated in a tissue-specific manner by thyroid hormone.

We have studied the regulation of rat intestinal and hepatic apolipoprotein gene expression, in vivo, after alterations in thyroid hormone status. When compared to those of chow-fed controls, rates of synthesis of intestinal apoA-I and apoB-48 decreased 60-66% in hypothyroid animals and increased three- to fourfold after triiodothyronine (T3) administration. These changes were not accompanied by changes in mRNA abundance. By contrast, intestinal apoA-IV synthesis rates and mRNA abundance were both unaltered over the range of thyroid hormone manipulations tested. Hepatic apoA-I and apoA-IV synthesis rates decreased by 70-80% in hypothyroid animals, while synthesis rates and mRNA abundance increased coordinately six- to eightfold when hypothyroid rats were made hyperthyroid. Hepatic apoE synthesis rates increased twofold in hypothyroid rats and decreased sevenfold in hyperthyroid animals. ApoE mRNA abundance, however, was comparable in all groups. Hypothyroid animals had reduced synthesis rates of hepatic apoB-100 and apoB-48. After induction of hyperthyroidism, apoB-100 synthesis (studied from 5 to 60 min) was undetectable (less than 0.01%) without further change in apoB-48 synthesis and without alterations in either apoB mRNA abundance or transcript size. Despite undetectable hepatic apoB-100 synthesis rates in hyperthyroid animals, total plasma triglyceride secretion rates (after Triton WR-1339 injection) were normalized compared to a 50% decrease in hypothyroid rats. Taken together, the data provide evidence for tissue-specific, independent regulation of apolipoprotein gene expression in vivo. Furthermore, the data suggest that aspects of hepatic triglyceride assembly and secretion and apolipoprotein gene expression may be coordinately responsive to alterations in thyroid hormone status.

Oocyte maturation is regulated by modulation of the action of FSH in cumulus cells.

A biochemical and cellular model is described for the regulation of oocyte maturation by modulation of FSH-dependent accumulation of cyclic AMP (cAMP) in the rat oocyte-cumulus complex (OCC). We propose that accumulation of cAMP in cumulus cells is specifically stimulated by FSH and modulation of this response to FSH is a primary site for the control of oocyte maturation. On the one hand, adenosine amplifies inhibition of oocyte maturation by up-modulation of FSH-sensitive accumulation of cAMP. In contrast, calcium ionophores induce oocyte maturation by down-modulation of FSH-dependent accumulation of cAMP. We suggest that adenosine and calcium interact at a similar site because adenosine reverses the calcium-dependent inhibition of the action of FSH. This model appears to have physiological relevance since induction of oocyte maturation by gonadotropin in the intact follicle occurs in parallel with inhibition of the FSH-sensitive accumulation of cAMP in the OCC. Moreover, gonadotropin-induced desensitization of the OCC to the action of FSH appears to precede the loss of intercellular junctional processes, as measured indirectly by uptake of radiolabelled uridine into the oocyte. Thus, a decrease in the FSH-sensitive accumulation of cAMP in cumulus cells, produced either by direct elevation of the intracellular concentration of calcium or by treatment of the intact follicle with gonadotropin, results in oocyte maturation. On the other hand, an increase in the cAMP response to FSH, produced by agents such as adenosine, results in inhibition of oocyte maturation. Thus, responsiveness of the cumulus cells to FSH appears to be acutely modulated and this site may be a major determinant for the regulation of oocyte maturation.

Thyroid function, growth hormone, and organ growth in broilers deficient in phosphorus.

The effects of a dietary P deficiency on thyroid function, serum growth hormone, and growth parameters in 10 to 29-day-old broiler cockerels was determined. Chicks fed severely P-deficient diets (.05% or .10% available P) grew more slowly and ate less feed than controls fed .65% P. The deficiency was also accompanied by hypercalcemia, hypophosphatemia, and decreases in percent bone ash, fat-free tibial weight, and tibial length and width. Increases in the relative weights of kidneys, hearts, and pituitary glands (.05% P only) occurred as well. Most of these changes occurred to a lesser extent or not at all in pair-fed controls, showing that they resulted specifically from the P deficiency and were not simply a result of reductions in feed intake. Phosphorus deficiency also was accompanied by peripheral edema and hydropericardium. Relative thyroid weight was unaffected. Serum triiodothyronine was consistently lower in the P-deficient chicks, although effects were significant only in one experiment. Thyroxine levels tended to be low also, but not consistently so. Serum growth hormone in P-deficient chicks in both studies was consistently lower than that in pair-fed controls, but this was significant only when .10% but not .05% available P was fed. The findings suggest that serum levels of both thyroid hormone and growth hormone are altered by P deficiency, but the results were not clearly definitive.

Adenosine reverses calcium-dependent inhibition of follicle-stimulating hormone action and induction of maturation in cumulus-enclosed rat oocytes.

Previous studies indicate that an increase in calcium in the oocyte-cumulus complex (OCC) appears to be associated with induction of oocyte maturation. In contrast, we previously showed that adenosine augments FSH inhibition of maturation in isolated rat OCC. The objective of the present studies was to assess whether adenosine may inhibit calcium-induced oocyte maturation in rat OCC. Two calcium ionophores (A23187 and ionomycin) were used for these studies. In the intact OCC, both ionophores inhibited FSH-stimulated cAMP accumulation and stimulated germinal vesicle breakdown when spontaneous maturation was inhibited by FSH, but the ionophores had no effect when maturation was inhibited by Bu2 cAMP. In contrast, adenosine amplified FSH-sensitive cAMP accumulation and augmented FSH inhibition of oocyte maturation in the intact OCC. Moreover, adenosine reversed ionophore-dependent inhibition of cAMP accumulation and ionophore induction of oocyte maturation in the intact OCC incubated with FSH. Adenosine, ionophore, or FSH had no effect on oocyte maturation in denuded oocytes. Based on these results we conclude that the ionophores induced oocyte maturation by inhibition of FSH-dependent cAMP accumulation in the cumulus compartment, an effect that was probably calcium mediated. Since adenosine reversed the ionophore effects, we suggest that adenosine antagonizes the inhibitory effect of calcium on cAMP accumulation in cumulus cells and by this mechanism amplifies FSH inhibition of oocyte maturation.