Evidence for luteal cell hyperplasia during pregnancy

The pronounced increase in luteal size and weight that occurs during rat pregnancy has been largely attributed to luteal cell hypertrophy. It is generally believed that hyperplasia does not play a role in luteal growth, since it is thought that luteal cell division in vivo does not occur. Recent data suggest that this may not be the case. Thus, to determine whether luteal cell hyperplasia occurs during rat pregnancy, osmotic minipumps filled with 3H-thymidine were implanted in timed-pregnant rats on Day 6 or Day 11 of pregnancy. These pumps provided a continuous infusion of 10 microCi 3H-thymidine per hour for up to 7 days. Seven days later (Day 13 and Day 18 respectively), rats were killed, and the ovaries were removed and prepared for autoradiography. Labeled cells, which have the morphological characteristics of luteal cells, were clearly observed in autoradiographs of ovaries exposed to 3H-thymidine. The labeling index of these cells from ovaries exposed to 3H-thymidine on Days 6-13 of pregnancy was 6.0% and from ovaries exposed to 3H-thymidine on Days 11-18 of pregnancy was 1.2%. Whether the presence of labeled cells signifies proliferation of luteal cells or whether these cells are derived from another cell type that develops into cells morphologically similar to luteal cells is not known at present. However, regardless of origin, these data clearly demonstrate that the number of parenchymal cells in the corpus luteum does increase during pregnancy.     

Evidence that basal secretion of relaxin by individual cultured large luteal cells is influenced by mobilization of intracellular calcium: analysis by a reverse hemolytic plaque assay.

Ca2+ redistribution from an intracellular site(s) is a key biochemical event associated with relaxin (RLX) secretion by large luteal cells (LLCs) of porcine origin. However, the functional significance of internal stores of Ca2+ to basal rates of RLX secretion is not well understood. In addition, the identity of the intracellular storage site(s) for Ca2+ within LLCs is not known, nor is it clear if all RLX-releasing LLCs are equally dependent on this pool. In the present study, release of RLX from 24 h cultured luteal cells derived from early pregnant swine was monitored by a reverse hemolytic plaque assay (RHPA). Incubation of cultures in the presence of graded concentrations of thapsigargin (1 nM-1 microM), a plant sesquiterpene lactone that inhibits endoplasmic reticulum Ca(2+)-ATPase and thereby increases cytosolic Ca2+ concentrations, resulted in a dose-related increase in basal RLX secretion. The stimulatory effect of thapsigargin on RLX production was not abrogated by culture in Ca(2+)-free medium. Suppression of Ca2+ release from the endoplasmic reticulum of LLCs, achieved by incubating monolayers in medium containing dantrolene (1-100 microM), resulted in dose-related inhibition of basal RLX release. Taken together, these results suggest that the endoplasmic reticulum serves as a major storage site for Ca2+ redistribution within LLCs and, furthermore, that mobilization from this site is functionally coupled to basal secretion of RLX.     

Morphometric quantification of mitochondria in the two steroidogenic ovine luteal cell types

Progesterone secretion is regulated by different mechanisms in large and small steroidogenic ovine luteal cells. Large cells secrete approximately 7-fold more progesterone in an unstimulated state than small cells. Since cholesterol side-chain cleavage, which is catalyzed by an inner mitochondrial membrane enzyme complex, is a major rate-limiting step in progesterone synthesis, mitochondrial components were quantified in the two steroidogenic cell types throughout the estrous cycle. Corpora lutea collected on Days 4 (n = 4), 8 (n = 4), 12 (n = 5), and 16 (n = 6) of the estrous cycle were prepared for electron microscopy. Volume densities of cell types within corpora lutea and mitochondrial densities within cell types were estimated by point-counting; nuclear and cytoplasmic volume densities were estimated by planimetric analysis. A total of 570 micrographs (magnification 5300 X) were analyzed. Large cell volume density was unchanged during the cycle (35 +/- 1%) while small cell volume density increased (p less than 0.05) from 13 +/- 1% on Day 4 to 20 +/- 3% on Day 12. Large cell mitochondrial volume density increased (p less than 0.05) from 13 +/- 1% on Day 4 to 23 +/- 1% on Day 16 accompanied by an increase in cytoplasmic volume density such that nuclear to cytoplasmic ratio increased (p less than 0.05) from 1:14 to 1:34 between Days 4 and 16. Small cell mitochondrial volume density increased from 11 +/- 1% on Day 4 to 14 +/- 1% (p less than 0.05) for the rest of the cycle while the nuclear to cytoplasmic ratio remained at 1:14.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that type II 5'-deiodinase is not a selenoprotein.

Brain type II 5'-iodothyronine deiodinase and liver type I 5'-iodothyronine deiodinase activities are decreased in rats fed a Se(2+)-deficient diet suggesting that both enzymes are Se(2+)-dependent proteins. Since serum thyroxine (T4) concentrations are twice normal in the Se(2+)-deficient animals, it is unclear whether the Se2+ deficiency or the increased circulating T4 account for the decrease in the brain enzyme. In order to separate these two possibilities, the effects of Se2+ on 5'-deiodinase in glial cells (type II) and LLC-PK1 cells (type I) were examined. LLC-PK1 and glial cells were grown in serum-free defined medium containing 0, 1 pM, 10 nM, and 40 nM Se2+ for 3-5 days or in medium containing 75Se2+ for 24 h. Deiodinase isozymes were determined by measuring catalytic activity and by quantification of the BrAc[125I]T4 affinity-labeled substrate binding subunits. Se2+ deficiency was confirmed by measuring the activity of the selenoprotein, glutathione peroxidase. Se2+ caused a concentration-dependent increase in glutathione peroxidase activity in both cell types, as well as in the type I enzyme, but had no effect on the type II enzyme. LLC-PK1 cells contained multiple 75Se(2+)-labeled proteins including the 27-kDa substrate binding subunit of the type I 5'-deiodinase. Glial cells contained seven 75Se(2+)-labeled proteins ranging in size from 12 to 62 kDa, none of which corresponded to the type II substrate binding subunit. these data show that, unlike the type I enzyme, the type II enzyme does not contain a selenocysteine or selenomethionine, further emphasizing the differences between these two isozymes.     

Evidence supporting the existence of a host cell surface receptor for Trypanosoma cruzi

Membrane fragments from trypomastigote forms of Trypanosoma cruzi inhibited the association of intact trypomastigotes with rat heart myoblasts whereas a similar preparation from non-invasive epimastigotes did not. Furthermore, killed trypomastigotes bound to the host cell surface and prevented the attachment of living organisms. Conversely, the extent of association of killed parasites with the host cells was reduced by the presence of living flagellates. These results suggest the presence of a distinct structure(s) on the surface of rat heart myoblasts to which infective forms of T. cruzi can bind.     

Evidence for a switch in the site of relaxin production from small theca-derived cells to large luteal cells during early pregnancy in the pig

The presence of immunoreactive relaxin was studied in corpora lutea of sows during the oestrous cycle and early pregnancy by immunohistochemistry and radioimmunoassay using three different anti-relaxin sera. Sections were immunostained using the peroxidase-anti-peroxidase or the immunogold-silver technique. Before Day 14, staining in corpora lutea from non-pregnant and pregnant animals was indistinguishable. With all antisera, no immunostaining was seen on Day 3, but was detected on Days 5-7 in cells from the theca interna. In non-pregnant animals, this immunostaining decreased and by Day 15 only an occasional large cell in the centre of the corpus luteum was stained. No staining was seen by Day 22. The relaxin content of corpora lutea measured by radioimmunoassay remained low throughout the luteal phase. In contrast, the amount of immunoreactive relaxin in corpora lutea rose dramatically (140-fold) between Days 11 and 14 of pregnancy and by Day 14 of pregnancy immunostaining was seen in the majority of large luteal cells. By Day 20 of pregnancy the concentrations of immunoreactive relaxin had further increased. Histochemical staining for alkaline phosphatase suggested that, while the relaxin-immunoreactive cells seen in the early luteal phase may be theca-derived, those during early pregnancy may be derived from the granulosa. The results are compatible with the suggestion that relaxin is produced by theca-derived cells during the early luteal phase and that between Days 11 and 14 there is a switch in the site of relaxin synthesis from theca-derived cells to granulosa-derived large luteal cells. In the absence of luteolysis, as during pregnancy, this switch is accompanied by a dramatic increase in relaxin synthesis.     

Evidence that the phosphatidylinositol cycle is linked to cell motility

Transmembrane signaling via specific ligand/receptor interactions induces the immediate polymerization of actin and formation of microfilament assemblies close to the plasma membrane. The profilin:actin complex appears to provide the actin for this filament formation. A clue to the nature of the regulatory mechanism involved was recently found in that phosphatidylinositol 4,5-bisphosphate can bind to profilin, dissociate the profilactin complex, and thus liberate actin for polymerization. This suggests that the phosphatidylinositol (PI) cycle, which plays important roles in cellular regulation, also might control microfilament-based motility. We show here that neomycin, a drug which has a high affinity for phosphoinositides and in vivo interferes with the PI cycle, inhibits the polymerization of actin in platelets induced either by thrombin or by ADP. When ADP was used as agonist (but not in the case of thrombin) the induction of actin polymerization could also be blocked by the addition of aspirin. Introduction of Ca2+ into platelets by the use of the ionophore A23187 or stimulation of protein kinase C (PkC) by the phorbol ester TPA did not induce actin polymerization; neither did the addition of a combination of these two agents. Retinoic acid which inhibits PkC was also without effect on thrombin-induced actin polymerization.     

Evidence for the existence of a new receptor for CGRP,which is not CRLR

Receptors for calcitonin gene-related peptide (CGRP), a neuropeptide known to be the most potent vasodilator, are abundantly expressed in cerebellum. A monoclonal antibody to cerebellar CGRP receptors specifically detects a 66 kDa protein from rat cerebellum and other rat and human tissues, but not from SK-N-MC cells which express calcitonin receptor-like receptor (CRLR), a recently described component of CGRP receptors. In contrast, mRNA expression for CRLR was abundant in SK-N-MC cells, but it was undetectable in rat cerebellum. Furthermore, the antibody could not detect any immunoreactive protein in HEK 293 cells transiently transfected with CRLR and receptor activity-modifying protein 1 (RAMP(1)) indicating the possible existence of another CGRP receptor, which does not involve CRLR. Due to the absence of biochemical or structural data on the existence of a CGRP(2) receptor and the new data provided in this paper, we suggest to identify the two CGRP receptors as CGRP-A and CGRP-B.     

Evidence that the Bacillus subtilis SpoIIGA protein is a novel type of signal-transducing aspartic protease

The bacterium Bacillus subtilis undergoes endospore formation in response to starvation. sigma factors play a key role in spatiotemporal regulation of gene expression during development. Activation of sigma factors is coordinated by signal transduction between the forespore and the mother cell. sigma(E) is produced as pro-sigma(E), which is activated in the mother cell by cleavage in response to a signal from the forespore. We report that expression of SpoIIR, a putative signaling protein normally made in the forespore, and SpoIIGA, a putative protease, is necessary and sufficient for accurate, rapid, and abundant processing of pro-sigma(E) to sigma(E) in Escherichia coli. Modeling and mutational analyses provide evidence that SpoIIGA is a novel type of aspartic protease whose C-terminal half forms a dimer similar to the human immunodeficiency virus type 1 protease. Previous studies suggest that the N-terminal half of SpoIIGA is membrane-embedded. We found that SpoIIGA expressed in E. coli is membrane-associated and that after detergent treatment SpoIIGA was self-associated. Also, SpoIIGA interacts with SpoIIR. The results support a model in which SpoIIGA forms inactive dimers or oligomers, and interaction of SpoIIR with the N-terminal domain of SpoIIGA on one side of a membrane causes a conformational change that allows formation of active aspartic protease dimer in the C-terminal domain on the other side of the membrane, where it cleaves pro-sigma(E).     

Age-dependent changes in myogenic precursor cell compartment sizes. Evidence for the existence of a stem cell

Individual myogenic cells were isolated from the pectoralis muscles of chick embryos from days 8-14 of embryogenesis. When separately cloned, these cells produced three types of colonies in culture: (1) Positive: all cells in the clone were terminally differentiated muscle cells; (2) negative: no cells in the clone were terminally differentiated muscle; (3) mixed: some cells in the clone were terminally differentiated muscle. Positive clones from all ages tended to contain 2n cells (n = 0, 1, 2, 3, 4). Negative clones were found in all sizes and did not cluster around powers of 2 in cell number. Mixed clones were, by far, the most common type among those clones larger than 24 in cell number. Estimates of cell numbers in embryonic muscle tissue revealed that, while the numbers of cells in all myogenic compartments increased steadily with embryonic age, the number and percentage of precursor cells that produced large mixed clones increased dramatically. Subclones, prepared from populations of cells equivalent to large mixed clones, yielded both small positive and large mixed colonies. This indicated that the precursors to the large mixed clones were also precursors to the smaller positive clones. These observations suggest a model for the myogenic lineage in which there exists a stem cell that can generate, by a series of asymmetric divisions, cohorts of terminally differentiated muscle cells. The model can explain the asynchrony of production of terminally differentiated muscle cells both in vitro and in vivo.     

Evidence for the existence of a soybean resistant protein that captures bile acid and stimulates its fecal excretion

Feeding HMF, an insoluble "high-molecular-weight fraction" from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestible protein or peptide that can be called a "resistant protein" in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestible fraction of HMF.     

Evidence that tamoxifen is a histamine antagonist

Recently we reported that both the triphenylethylene antiestrogen tamoxifen, and the novel compound N,N-diethyl-2-[(4 phenylmethyl)-phenoxy]-ethanamine. HCl (DPPE), which is selective for the antiestrogen binding site, may be histamine antagonists and have suggested that the antiestrogen binding site may be a growth-promoting histamine receptor different from H1 and H2 (?H3). We now show that along with established H1-antagonists, tamoxifen and DPPE specifically block the histamine-induced (H1) contraction of canine tracheal smooth muscle in the order: pyrilamine = hydroxyzine greater than tamoxifen = 4-hydroxytamoxifen greater than DPPE. The H1-antagonist hydroxyzine, which competes about equally with DPPE for the antiestrogen binding site, is up to 10(3) times stronger than DPPE in blocking histamine-induced muscle contraction. This shows that H1 antagonism is distinct from binding to the antiestrogen binding site and suggests that if the latter is a histamine receptor, it is not H1; presumably tamoxifen and DPPE compete for this novel site in addition to, and with greater affinity than, H1.     

Evidence for a Sertoli cell, FSH-suppressible inhibiting factor(s) of testicular steroidogenic activity

By using a model of immature porcine Leydig and Sertoli cells cultured in serum free defined medium, we evidenced a paracrine control of Leydig cell steroidogenic activity by Sertoli cells via a secreted inhibiting protein(s). This protein(s), partially purified using gel filtration (M.W. 20,000-30,000) suppresses the steroidogenic responsiveness to LH/hCG by decreasing the specific LH/hCG binding (52% decrease) and hormone steroid biosynthesis (73% decrease) at a level(s) located between cAMP production and pregnenolone formation. The suppression of this inhibitor(s) by FSH, in a dose dependent manner, is one mechanism by which FSH "sensitizes" Leydig cell response to LH/hCG stimulation.