Follicular Cell Function Research Articles

1,25-Dihydroxycholecalciferol modulates 3H-thymidine incorporation in FRTL5 cells.

1,25-dihydroxycholecalciferol (1,25(OH)2D3) possesses proliferation and differentiation modulating effects in many cell types in vitro. We studied the effect of 1,25(OH)2D3 on 3H-thymidine incorporation in FRTL5 cells, a cultured rat thyroid follicular cell line. 1,25(OH)2D3 alone at 10(-11) and 10(-9) M exerted no effect on 3H-thymidine incorporation. However, at 10(-7) M, 1,25(OH)2D3 slightly enhanced 3H-thymidine incorporation. In the presence of 5% calf serum, 1,25(OH)2D3 increased 3H-thymidine incorporation induced by calf serum in a dose-dependent manner. 1,25(OH)2D3 also enhanced 3H-thymidine incorporation induced by PMA, an extrinsic stimulator of protein kinase C, without directly affecting PMA-induced protein kinase C translocation. In contrast to the stimulatory effects of 1,25(OH)2D3 on the calf serum and PMA-induced 3H-thymidine incorporation, 1,25(OH)2D3 inhibited the increase in 3H-thymidine incorporation induced by TSH in a dose-dependent manner. This effect of 1,25(OH)2D3 on TSH-induced 3H-thymidine incorporation may be, in part, due to post-cAMP pathways since 1,25(OH)2D3 also inhibited the increase in 3H-thymidine incorporation induced by Bu2cAMP without affecting the TSH-induced increase in cAMP. The stimulatory effect of insulin on 3H-thymidine incorporation, a cAMP-independent process, was also inhibited by 1,25(OH)2D3. We conclude that 1,25(OH)2D3 affects 3H-thymidine incorporation in FRTL5 cells raising the possibility of a physiologic role for 1,25(OH)2D3 in the growth and function of thyroid follicular cells.

Characterization of a 1,25-dihydroxy-vitamin D 3 receptor in FRTL-5 cells. Evidence for an inhibitory effect of 1,25-dihydroxy-vitamin D 3 on thyrotropin-induced iodide uptake

When FRTL-5 cell cytosol was incubated with increasing amounts of [ 3H]1,25-dihydroxy-vitamin D 3 ([ 3H]1,25-(OH) 2D 3), saturation of specific hormone binding occurred. Scatchard analysis of specific binding of [ 3H]1,25-(OH) 2D 3 to the macromolecule yielded an apparent K dvalue of 0.41 ± 0.08 × 10 −10 M and a single maximum binding capacity of 42.8 ± 8.8 fmol/mg protein. Sucrose gradient analysis revealed substantial [ 3H]1,25-(OH) 2D 3 association with a macromolecule sedimentating slightly faster than ovalbumin (3.7 S). [ 3H]1,25-(OH) 2D 3 was completely displaced by excess 1,25-(OH) 2D 3. The 1,25-(OH) 2D 3-receptor complex bound to DNA cellulose columns in low salt buffer, and eluted as a single peak at 0.15–0.20 M KCl. Thus, we have shown for the first time the existence of a functional 1,25-(OH) 2D 3 receptor in thyroid follicular cells. Furthermore, 1,25-(OH) 2D 3 inhibited the thyrotropin (TSH)-stimulated iodide uptake in a dose-dependent manner, indicating that 1,25-(OH) 2D 3 has an effect on the physiological function of rat thyroid follicular cells in culture.

Platelet-activating factor induces progesterone secretion and changes in morphological appearance in luteinizing granulosa cells in vitro

Recent evidence indicates the involvement of platelet-activating factor (PAF) in ovarian periovulatory processes. The objective of this study was to investigate the influence of PAF on ovarian follicular cellular function. We examined the effects of PAF on progesterone (P) secretion by cultured human luteinizing granulosa cells obtained from women undergoing in vitro fertilization. Follicular cells were cultured in serum-free conditions for 12 to 36 hours with and without PAF (at dosages of 10(-8) to 10(-14) M). Progesterone secretion was significantly increased at 12 and 36 hours after treatment with 10(-8) M PAF (170% +/- 40% and 210% +/- 40%, respectively) and with 10(-10) M PAF (170% +/- 30% and 180% +/- 10%, respectively). Smaller increases were seen with 10(-12) and 10(-14) M PAF. Maximal increases in P secretion in 10(-10) M PAF-treated wells occurred at 3 to 12 hours. At 36 hours, distinct morphological differences between cells in control wells and those containing PAF were noted. These results indicate that PAF may influence follicular cell function during ovulation and corpus luteum formation.

Erythroid differentiation factor can modulate follicular granulosa cell functions

The action of human erythroid differentiation factor (EDF) on the functions of rat granulosa cells cultured in a chemically defined medium was investigated. In the presence of FSH that induced LH receptor expression and progesterone synthesis during culture of the cells, EDF augmented both responses in a dose- and time-dependent manner. Unlike FSH, EDF itself did not have such an inducing effect at all. Furthermore, in the absence of FSH, EDF was found to strongly enhance the ability of granulosa cells to produce inhibin. Thus, EDF may play an important role in the regulation of granulosa cell function and differentiation during follicle development.

Characterization of insulin-like growth factor binding to human granulosa cells obtained during in vitro fertilization.

Insulin and IGF-I affect in vitro ovarian stromal and follicular cell function in several species. We previously characterized insulin receptors on human granulosa cells obtained from in vitro fertilization procedures but were unable to demonstrate specific binding of IGF-I. Following modification of the assay conditions, we now report specific, high affinity IGF-1 binding sites on human granulosa cells. Substitution of equimolar concentrations of sucrose for sodium chloride in the buffer solution increased binding of IGF but not insulin in equilibrium assays. Maximal specific IGF-I binding was 2.69 +/- 0.30%/10(5) cells (SEM, n = 9) with half-maximal inhibition of binding at 2 ng/ml IGF-I. Unlabeled insulin recognized the type I IGF receptor with low affinity. An IGF-I receptor monoclonal antibody (alpha IR-3) inhibited 125I-IGF-I but not 125I-insulin binding. Affinity crosslinking followed by SDS/PAGE under reducing conditions revealed IGF-I binding at a molecular weight compatible with the alpha subunit of the type I IGF receptor and with a pattern of inhibition by various ligands that paralleled the equilibrium binding assays. IGF-I receptors are present on freshly isolated human ovarian granulosa cells obtained following pharmacologic stimulation with gonadotrophin according to the protocols of in vitro fertilization. The biologic function of these receptors currently is being investigated.

Pathogenesis of heterogeneity in human multinodular goiter. A study on growth and function of thyroid tissue transplanted onto nude mice.

Functional and morphologic heterogeneity of human multinodular goiters was investigated in 300 samples from "cold" and "hot" regions of 20 goiters transplanted onto nude mice. Transplants were labeled with [3H]thymidine and radioiodine, while the host's thyroid-stimulating hormone (TSH) secretion was either stimulated or suppressed. Proliferation and function of follicular cells were assessed in whole follicles reconstructed from autoradiographs of serial sections. Hot transplants had a higher autonomous iodine uptake than those of cold tissue in TSH-suppressed hosts. Functional autonomy widely varied among the follicles, but even more so among individual cells. Hot grafts differed from cold ones only by a comparatively larger fraction of autonomous cells. Intercellular differences of iodinating activity were not abolished by TSH. Grafts faithfully reproduced the individual growth pattern of the original tissue. Between 0.5% and 7% of all follicular cells replicated despite suppression of TSH. Up to 70% of these cells were clustered, forming scattered foci of autonomously growing tissue. Other cells only started replicating after long-term TSH stimulation. Thus, goiters contained subsets of cells with high and others with low growth response. Progenies of replicating cells remained clustered, sometimes budding outwards to form new follicles. Autonomy of growth and autonomy of function are independent traits of epithelial cells. Epithelial cells have their individual growth pattern, replication rate, and functional capacity. These traits are passed on from a mother cell to its progeny during follicle neogenesis. To this main mechanism accounting for the morphologic and functional heterogeneity of human goiters, inheritable modifications of gene expression must probably be added.

Specific epidermal growth factor receptors on porcine and human thyroid membranes.

Specific, high affinity, saturable receptors for epidermal growth factor (EGF) have been demonstrated both on porcine and on human thyroid membranes. The binding affinities of porcine (Ka 3.0 X 10(-9) M) and human thyroid EGF receptors (Ka 1.75 X 10(-9) M) are very similar. TSH does not inhibit the binding of 125I-EGF to either membrane. These results suggest the possibility that EGF may be involved in the regulation of human as well as porcine thyroid follicular cell growth and function.

The function and origin of follicular dendritic cells.

Follicular dendritic cells (dendritic reticular cells) in germinal centres bind antigen-antibody complexes via C3 receptors and retain the complexes at their surface for long periods of time. The follicular dendritic cells (FDC) are distinct from macrophages and from dendritic cells found in T-dependent areas, and are not derived from bone marrow stem cells. On histological evidence it has been proposed that they are derived from reticulum cells. Complexes are probably transported to FDC by a subpopulation of B cells in the marginal zone. Binding of complexes to FDC causes germinal centre enlargement and is a very efficient, and possibly essential stimulus to the generation of B memory cells which recognize epitopes on antigen or antibody in the complexes. An hypothesis is discussed which draws together these observations and suggests that antigen on FDC plays a central role in control of humoral immunity.

Specific epidermal growth factor receptors on porcine thyroid cell membranes

Specific saturable receptors for epidermal growth factor (EGF) of high affinity ( K a 1.7 × 10 9 M −1) have been demonstrated on porcine thyroid membranes. Optimal conditions for EGF binding have been determined. TSH and other peptide hormones do not inhibit the binding of 125I-EGF and EGF does not inhibit 125I-TSH binding to thyroid membranes. The results suggest that EGF may be involved in the regulation of thyroid follicular cell growth and function.

Structure-function relationships in the wall of the ovarian follicle.

This paper reviews current knowledge of the light and electron microscopic structure of the three layers of the mammalian follicular wall-follicular epithelium (membrana granulosa), theca interna and theca externa--and discusses correlations between structure and function. The ultrastructure of follicular epithelial cells in growing follicles emphasized their protein synthetic and secretory functions; features suggestive of a major steroidogenic function appear only at later stages. Regional differences in follicular epithelial cell function are probably important, although structurally these cells are relatively homogeneous. Structural diversity is more marked in the thecal layers: differentiation in the theca interna is towards fibrocytic and steroidogenic cell types, while that in the theca externa is towards fibrocytic and myoid types. Adherens and gap junctions are present between cells in all layers; however, tight (occludens) junctions have not been convincingly demonstrated between the cells in any of the three layers. Blood and lymph vessels are confined to the thecal layers. However, follicles possess no structural barrier comparable to that associated with the "blood-testis barrier', and show a correspondingly greater permeability to large molecules than seminiferous tubules. Interactions between the layers of the follicular function. To date, the best-documented interaction between layers is that described in the "two-cell hypothesis' of oestrogen production. some potentially useful directions for future research are proposed.

Evidence that changes in tonic luteinizing hormone secretion determine the growth of preovulatory follicles in the rat.

Physiological concentrations of progesterone (20-100 ng/ml), maintained by the insertion of implants into 30-day-old rats, delayed first ovulation, and withdrawal of progesterone on day 47 of age synchronized first ovulation in rats. Inhibition of ovulation involved negative feedback regulation of tonic LH and FSH secretion, blockage of gonadotropin surges, and suppression of preovulatory, but not antral, follicular growth. Removal of implants resulted in a rapid decline in serum progestrone from 100 to 5 ng/ml within 0-12 h. Between 0-36 h there were progressive increases in serum concentrations of LH and FSH, enhanced accumulation of estradiol by individual follicles incubated in vitro with or without exogenous substrate, and marked progressive increases in the content of LH (but not FSH) receptors in both thecal and granulosa cells. These events were followed by gonadotropin surges at 48 h (1800 h on day 49), ovulation, and morphological and biochemical signs of luteinization, including decreases in follicular gonadotropin receptor content and estradiol accumulation, evident by 60 h. With the exception of changes in basal LH, this sequence of events is remarkably similar in time and pattern to that after the decline of progesterone on diestrous day 2 and ovulation on proestrus of a 5-day cycle. Although a direct effect of progesterone on ovarian follicular cell function cannot be excluded, the data suggest that subtle but sustained increases in LH (and possibly FSH) are required for the enhanced follicular accumulation of estradiol and LH-binding activity occurring between diestrus and proestrus of the rat estrous cycle. Thus, perhaps some of the mystery surrounding the endocrine events between diestrus and proestrus can be ascribed to changes in serum LH that have been too small and/or variable for current nonserial sampling methods and RIAs to detect reliably.

Culture of hormone-dependent functional epithelial cells from rat thyroids.

Primary cultures of rat thyroid cells were made in medium supplemented with 0.1--0.5% calf serum and containing six hormones or growth factors: insulin, thyrotropin, transferrin, hydrocortisone, somatostatin, and glycyl-L-histidyl-L-lysine acetate. The FRTL strain was purified by successive colonial isolations and was found to maintain highly differentiated features (secretion into the culture medium of physiological amounts of thyroglobulin and concentration of iodide by 100-fold). The FRTL strain has been observed for more than 3 years in continuous culture. It has maintained the same biochemical and morphological characteristics that typified the primary cultures of thyroid follicular cells immediately after their enzymatic release from the rat thyroid. Thyroid epithelial cells that were grown under more conventional cell culture conditions failed to retain these specialized characteristics. We show that maintenance in vitro of these specialized functions of rat thyroid follicular cells is dependent on low serum concentrations and supplementation with hormones in the primary cultures. Our observations indicate that this culture strategem may be aplicable to the general problem of maintenance of differentiated characteristics in cultures of other epithelial cells.

Granulosa Cell Deficient Follicles: Occurrence, Structure, and Relationship to Ovarian Teratocarcinogenesis in Strain LT/Sv Mice

The diameters of oocytes in follicles having a single layer of granulosa cells were measured hi four week old mice of various strains. There is a unique population of these follicles hi strains LT/Sv and C58/J in which the oocytes are significantly larger than the oocytes in single granulosa cell layered follicles of other common strains (C57BL/6J, BALB/cJ, and DBA/2J). These unique follicles are referred to as granulosa cell deficient (GCD) follicles since oocytes of these sizes are usually found in follicles with more than a single layer of granulosa cells. The parthenogenetic embryos that give rise to ovarian teratomas in strain LT/Sv are usually found in GCD-follicles. Some of the ova of strains LT/Sv and LTXBP, but not the ova of the other strains, are capable of spontaneous parthenogenetic activation after meiotic maturation. Although the ovulated ova of strain LTXBP are capable of spontaneous parthenogenetic development, the frequency of GCD-follicles and teratocarcinogenesis is low. Therefore, the frequency of ovarian teratocarcinogenesis is correlated with the simultaneous occurrence of two atypical conditions: first, the capability of the matured ova to undergo spontaneous parthenogenetic activation and, second, the high frequency of GCD-follicles. GCD-follicles containing oocytes with a diameter greater than 65 μm were studied by electron microscopy. The follicles are usually enclosed within a layer of flattened theca-like cells. A basal lamina separates these cells from a single layer of cuboidal granulosa cells. Granulosa cell processes traverse the zona pellucida to contact the oocyte which shows ultrastructural characteristics typical of oocytes in the final growth stages. It is proposed that the GCD-follicles are competent to participate in the normal functions of follicular cells relating to oocyte growth and meiotic maturation.

Effects of diuretics on thyroid function of guinea pigs

Uptake of radioiodide by the isolated thyroid and serum concentrations of thyroxine (T 4) and tri-iodothyronine (T 3) were determined in guinea pigs pretreated with ethacrynic acid (20 mg/kg), furosemide (40 mg/kg) or hydrochlorothiazide (40 mg/kg). It was found that both ethacrynic acid and furosemide suppressed the 131I − uptake by the isolated thyroid tissues. In addition, thyroid weight and serum T 3 concentration were lower in ethacrynic acid-treated animals. It seems that some diuretics, particularly ethacrynic acid, depressed the function of thyroidal follicular cells.

Ultrastructure of Quali Thyroid Gland

An electronmicroscopic study was made on the thyroid gland of quail given the diet con tainning 0.1% thyouracil or not (a control), with special regard to the correlation between fine structures and function of the glandular cells.Follicular epithelial cells of the thyroid consisted of columnar, cuboidal and squamous cells, which were transformable from one to another according to their functional states. The low cuboidal and squamous cells were the main epitherial constituents in the nomal group, while the columnar cells were usually encountered in the group treated with thyouracil.The cokmnar cells in the group treated with thyouracil were characterized by the presence of abundant microvilli, rough endplasmic reticulum, large mitochondria and well-developed Golgi apparatus.These results suggested that the function of follicular epithelianl cells of the thyroid gland was activated in the quail by thyouracil administration.

Ovarian structures of the adult alderfly, Sialis mitsuhashii Okamoto (Megaloptera : Sialidae)

The ovary of Sialis mitsuhashii consists of about 100 telotrophic ovarioles. No transverse septum was observed at the boundary between the terminal filament and the germanium. The germarial region is composed of the extensive syncytial nutritive tissue or core, the peripheral cells, and the interstitial cells. The cells of the nutritive tissue and the peripheral cell layer seem to originate from the same mother cells, because each peripheral cell is connected to the cytoplasm of the nutritive tissue by a cytoplasmic bridge (ca. 1.5 μm in diameter). Some of the peripheral cells situated in the posterior region of the germanium develop into young oocytes, surrounded by the several prefollicular cells. Clear nutritive cords are seen connecting the nutritive tissue to the developing oocytes. In the nutritive cord connecting with the previtellogenic oocytes, a large number of ribosomes and mitochondria are distributed evenly throughout the cytoplasm. This pattern seems to indicate that the flow of these organelles from the nutritive tissue to oocytes occurs mainly during the previtellogenic stage. The degree of development of the follicular cell structure and function does not seem to be as advanced as that of the telotrophic ovaries in hemipteran and polyphagous coleopteran insects.