N. Chegini

Binding of 125I-epidermal growth factor in human uterus

SummaryQuantitative light-microscopic autoradiography was used on five human uteri at two different phases of the menstrual cycle to ascertain the cell types with binding sites for epidermal growth factor (EGF). The results revealed that stromal cells, glandular epithelium of endometrium, elongated and circular muscle cells of myometrium, smooth muscle and endothelial cells of arterioles in the basal endometrium and myometrium contained numerous silver grains following incubation with 125I-EGF. Coincubation with 100-fold excess unlabeled EGF resulted in a complete disappearance of silver grains from all cell types. Quantitative grain analysis indicated that stromal cells contained the highest number of EGF-binding sites (P<0.05) with no significant differences among the others (P>0.05). There was no cyclic variation of EGF-binding to any of the uterine cell types. The present data demonstrate that all the cell types of human uterus, including arterioles, contain EGF-binding sites. This suggests that all the cells in human uterus subserving different functions are targets of EGF action.

Prostaglandin binding to different cell types of human uterus: Quantitative light microscope autoradiographic study

Five human uteri of the menstrual cycle were analyzed by quantitative light microscope autoradiography to determine which uterine cell types contain prostaglandin (PG) binding sites. The results showed that stromal cells, glandular epithelium, elongated and circular smooth muscles, arterioles and erythrocytes in lumen of arterioles contained numerous PGE and very few or no detectable PGF2 alpha binding sites. The grains which were present when incubated with [3H]PGE2 alone completely disappeared following coincubation with excess unlabeled PGE2 but not with excess unlabeled PGA1. Analysis of grain count data revealed that PGE binding sites in arterioles were lower than those in other uterine cell types and PGE binding sites in endometrial cells of proliferative phase uteri were higher than those in secretory phase uteri (P less than 0.05). Thus the present results demonstrate that all the cell types, including arterioles of human uterus of the menstrual cycle, contain PGE and minimal PGF2 alpha binding sites. This suggests that PGE binding sites in different cell types of human uterus subserve diverse effects of PGEs.

A quantitative electron microscope autoradiographic study on 125I-human choriogonadotropin internalization in bovine luteal slices

Very few silver grains were seen on the cell surface and none intracellularly after incubation for 2 h at 4 degrees C. However, numerous grains were seen in various subcellular organelles when the tissues were incubated for 2 h at 22 degrees or 38 degrees C. The grain distribution was qualitatively similar, but quantitatively, there were fewer grains at 22 degrees than at 38 degrees C. Co-incubation of 125I-hCG with excess unlabelled hCG resulted in the virtual disappearance of silver grains from all the subcellular organelles. Excess unlabelled human luteinizing hormone (but not follicle-stimulating hormone or prolactin) inhibited the appearance of silver grains in luteal tissue. There were no silver grains in bovine liver slices incubated with 125I-hCG. The plasma membrane-associated grains progressively decreased, while intracellular organelle-associated grains increased with time at 38 degrees C. There were no grains in nuclei at 5 min, but they appeared at 10 min and increased until 120 min. After correction for radiation spread by three-step mask analysis, smooth endoplasmic reticulum and mitochondria did not contain any grains. The grain density was the highest in Golgi, followed by lysosomes, rough endoplasmic reticulum, nuclei, and plasma membranes after incubation for 2 h at 38 degrees C. Thus, the electron microscope autoradiography approach confirmed our biochemical data in the preceding paper (Chegini et al., Exp cell res 151 (1984) 466 [5]) on time, temperature dependency and specificity of 125I-hCG internalization, association of internalized hormone with a variety of intracellular organelles, and the highest uptake in Golgi.

Light and electron microscope immunocytochemical localization of 5- and 12-lipoxygenases and cyclooxygenase enzymes in human granulosa cells from preovulatory follicles

Cellular and subcellular distribution of 5- and 12-lipoxygenases and cyclooxygenase enzymes were investigated in human granulosa cells from preovulatory follicles using light and electron microscope immunocytochemistry. The results demonstrated that all three enzymes are present in granulosa cells but not in minor contaminating red blood cells. While the distribution of cyclooxygenase and 12-lipoxygenase was relatively uniform among the granulosa cells, 5-lipoxygenase was not uniformly distributed among these cells. All three enzymes are present in microvillus plasma membranes, rough endoplasmic reticulum, cytoplasm, nuclear membranes and chromatin. In summary, 5- and 12-lipoxygenases and cyclooxygenase enzymes, which catalyze the transformation of arachidonic acid into different eicosanoids, are present in several subcellular organelles including nuclei of granulosa cells from preovulatory follicles.

Light and Electron Microscope Immunocytochemical Localization of Epidermal Growth Factor Receptors in Bovine Corpora Lutea of Pregnancy

Both luteal and nonluteal cells, i.e., macrophages, fibroblasts, vascular smooth muscle and endothelium, contain EGF receptors. Small luteal and nonluteal cells, however, contain more receptors than large luteal cells. The nuclei of both luteal and nonluteal cells also seem to contain EGF receptors. However, the pattern of nuclear receptor distribution differed with the receptor antibodies specific to the carbohydrate moiety and the binding sites on the extracellular domain of EGF receptor molecules. At the electron microscope level using the receptor antibody specific to the carbohydrate moiety, EGF receptors were found not only in plasma membranes, but also in rough endoplasmic reticulum, lysosomal vesicles, mitochondria and nuclei. The nuclear receptors are associated with nuclear membranes, condensed chromatin and the border between the condensed and dispersed chromatin. In conclusion, our results suggest that EGF may have steroidogenic and nonsteroidogenic roles in bovine corpora lutea and that transduction of EGF signals may also involve nuclear receptors.

A quantitative electron microscope autoradiographic study on [3H]prostaglandin E1 and F2α internalization in bovine luteal slices

Very few silver grains were seen on the luteal cell surface and none intracellularly after incubation for 2 h at 4 degrees C with 10 nM [3H]prostaglandin (PG) E1 or [3H]PGF2 alpha. However, incubation at 22 degrees C or 38 degrees C for 2 h resulted in association of numerous grains on the luteal cell surface as well as in several intracellular organelles. Qualitatively, the grain distribution was similar at 22 degrees C and 38 degrees C, but quantitatively there were fewer grains at 22 degrees C than at 38 degrees C. In addition to luteal cells, the grains were also found on erythrocytes, platelets, fibroblasts and capillary endothelial cells in luteal tissue incubated with [3H]PGE1. Grain association with non-luteal cells was never seen with [3H]PGF2 alpha. Coincubation of [3H]PGE1 or [3H]PGF2 alpha with only corresponding excess unlabeled PGs resulted in complete disappearance of silver grains from all the subcellular organelles. In autoradiographs, grains were seen on plasma membranes, nuclei, lysosomes, mitochondria, rough and smooth endoplasmic reticulum and Golgi. Three-step mask analysis, which effectively corrects for radiation spread, revealed that the grains found on mitochondria and rough endoplasmic reticulum in the case of [3H]PGE1, and those found on mitochondria, rough and smooth endoplasmic reticulum and Golgi in the case of [3H]PGF2 alpha, were due to the radiation spread. For both [3H]PGE1 and [3H]PGF2 alpha the plasma membrane associated grains progressively decreased, while the intracellular organelle associated grains increased with time at 38 degrees C. There were no grains in nuclei at 5 min but they appeared at 10 min and increased until 60 or 120 min. The optimized source density following 2 h of incubation at 38 degrees C with [3H]PGE1 was Golgi greater than plasma membranes greater than lysosomes = nuclei, and for [3H]PGF2 alpha, lysosomes greater than plasma membranes greater than nuclei. In summary, the present studies demonstrate for the first time that exogenously added [3H]PGE1 and [3H]PGF2 alpha internalize in bovine luteal cells in a ligand-specific, time- and temperature-dependent manner. The observations that the internalized [3H]PGE1 does not associate with rough endoplasmic reticulum and that [3H]PGF2 alpha does not associate with rough endoplasmic reticulum and Golgi, even though they contain the binding sites, suggest the presence of mechanism(s) to direct the internalized [3H]PGs to only certain intracellular organelles of luteal cells.

Cellular distribution of prostacyclin synthase and specific prostacyclin binding sites in bovine corpora lutea of pregnancy

The cellular distribution of prostacyclin (PGI2) synthase and specific PGI2 binding sites was investigated by light microscope immunocytochemistry and quantitative autoradiography. The immunostaining for the enzyme was found in small (15-18 microns) and large (18-45 microns) cells as well as in non-luteal cells, arteriole smooth muscle and endothelium. There was no consistent difference between small and large luteal cells and luteal vs. non-luteal cells in the immunostaining. Vascular smooth muscle and endothelial cells, on the other hand, contained less immunostaining than the other cells. Contrary to wide-spread distribution of PGI2 synthase, specific PGI2 binding sites were only found in the luteal cells. On a per cell basis, large luteal cells contained more sites than small luteal cells and vice versa when expressed per unit cell area. The [3H]PGI2 binding to the luteal cells was time and temperature dependent and was inhibited by excess unlabeled PGI2 but not by its metabolite, 6-keto-PGF1 alpha or other eicosanoids which bind to their own receptors. In conclusion, while PGI2 synthase is widely distributed among different cell types in bovine corpora lutea, specific binding sites which may mediate luteotropic actions of PGI2 are only present in small and large luteal cells.

The presence of prostacyclin binding sites in nonpregnant bovine uterine tissue

Myometrium of various animal species makes a considerable amount of prostacyclin (PGI2) which is a potent myometrial and uterine vascular smooth muscle relaxing agent. This action of PGI2 is perhaps mediated by binding to specific receptors, which have never been demonstrated in uterine tissue of any animal species until very recently. The quantitative light microscopic autoradiographic approach used in the present studies demonstrated that while bovine myometrial smooth muscle and uterine vascular smooth muscle contained PGI2 specific binding sites, endometrial and perimetrial cells contained few or no binding sites. The number of binding sites in circular and elongated myometrial smooth muscle and in arteriolar smooth muscle were similar (P greater than 0.05). The PGI2 binding to the uterine cells was greatly reduced (P less than 0.001) following coincubation with excess unlabeled PGI2, but not with its stable metabolite, 6-keto PGF1 alpha, PGE2, PGF2 alpha and leukotriene C4 which bind to nonpregnant bovine uterine tissue, also had no effect of PGI2 binding. In conclusion, nonpregnant bovine uterine tissue contain specific PGI2 binding sites which may mediate its potent relaxing effect on myometrium and uterine vasculature.

Nuclear volume and chromatin conformation of small and large bovine luteal cells: effect of gonadotropins and prostaglandins and dependence on luteal phase.

SummaryChange in nuclear volume and chromatin conformation are generally considered to reflect altered gene expression in eukaryotic cells. The present studies were undertaken to investigate whether these nuclear parameters of luteal cells can be altered by hormone treatment in vitro or change during the estrous cycle. The nuclear volume of small luteal cells was significantly lower than that of large luteal cells during the cycle and pregnancy. The nuclear volumes of small and large luteal cells from pregnancy did not change during incubation without any hormone or with 10 nM prostaglandin (PG)F2α. However, incubation with 1 nM human chorionic gonadotropin (hCG) or 10 nM PGE1 resulted in a significant increase of nuclear volume of small luteal cells by 4 h and that of large luteal cells by 6 h. Small cells were more responsive to hCG than large luteal cells. The nuclear volumes of small and large luteal cells also significantly increased from early to mid luteal phase with no further change in late luteal phase. hCG and PGE1, as well as PGF2α, treatment resulted in a change of chromatin conformation of small and large luteal cells. Dibutyryl cyclic AMP (10 mM) mimicked the hormones by increasing nuclear volumes and changing the chromatin conformation of small and large luteal cells. Chromatin conformation of small and large luteal cells also changed from early to mid luteal phase and mid to late luteal phase. In conclusion, in vitro, hCG and PGs can regulate nuclear volume and/or chromatin conformation of small as well as large bovine luteal cells. In vivo, these nuclear changes occur during the periods of luteal growth, development and regression in the estrous cycle.

Role of luteal cell nucleus in the expression of gonadotropin action

Gonadotropin receptors are not only present in cell membranes, but also in nuclei of bovine and human luteal cells. hCG/hLH can directly regulate several nuclear functions. To further investigate the role of luteal cell nucleus in the expression of gonadotropin action, the effect of enucleation of luteal cells on gonadotropin receptors and gonadotropin response was studied. Luteal cytoplasts were prepared by colchicine treatment of purified whole luteal cells followed by centrifugation at 37 C in a Percoll gradient. The cytoplasts were 85 to 90% pure with a recovery of about 57%. Cytoplasts were viable as determined by trypan blue exclusion (87%) and metabolically competent as determined by 3H-leucine incorporation into proteins. On the day of preparation, the viability and metabolic competency of cytoplasts were similar to control cells, i.e. untreated and colchicine treated whole luteal cells. In addition, cytoplasts and control cells showed a similar decline in number and viability during storage at 4 C. While control cells continue to be metabolically competent, cytoplasts showed a dramatic decline by 48 h of storage at 4 C. Neither the cytoplasts nor control cells degraded 1251-hCG. The kinetics of 1251-hCG association and dissociation, specificity and affinity of binding to cytoplasts were similar to control cells. However, the number of available gonadotropin receptors in cytoplasts was significantly lower than in control cells. Cytoplasts contained lower progesterone levels and more importantly, they could not be stimulated by 10 nM hCG or 10 mM dibutyry1 cyclic AMP to produce more progesterone. Controls cells, on the other hand, contained higher progesterone levels and responded to hCG and dibutyryl cyclic AMP stimulation. In summary, removal of nuclei from luteal cells results in a partial loss of gonadotropin receptors and complete loss of steroidogenic response to hCG and dibutyryl cyclic AMP. Whether the loss of steroidogenic response was related or coincidental to partial gonadotropin receptors loss is unknown.