👤 Nikolaos G Papadopoulos

Recent studies indicate that the immune system adaptation during pregnancy could play a significant role in the pathophysiology of perinatal depression. The aim of this study was to investigate if inflammation markers in a late pregnancy plasma sample can predict the presence of depressive symptoms at eight weeks postpartum. Blood samples from 291 pregnant women (median and IQR for days to delivery, 13 and 7-23days respectively) comprising 63 individuals with postpartum depressive symptoms, as assessed by the Edinburgh postnatal depression scale (EPDS≥12) and/or the Mini International Neuropsychiatric Interview (M.I.N.I.) and 228 controls were analyzed with an inflammation protein panel using multiplex proximity extension assay technology, comprising of 92 inflammation-associated markers. A summary inflammation variable was also calculated. Logistic regression, LASSO and Elastic net analyses were implemented. Forty markers were lower in late pregnancy among women with depressive symptoms postpartum. The difference remained statistically significant for STAM-BP (or otherwise AMSH), AXIN-1, ADA, ST1A1 and IL-10, after Bonferroni correction. The summary inflammation variable was ranked as the second best variable, following personal history of depression, in predicting depressive symptoms postpartum. The protein-level findings for STAM-BP and ST1A1 were validated in relation to methylation status of loci in the respective genes in a different population, using openly available data. This explorative approach revealed differences in late pregnancy levels of inflammation markers between women presenting with depressive symptoms postpartum and controls, previously not described in the literature. Despite the fact that the results do not support the use of a single inflammation marker in late pregnancy for assessing risk of postpartum depression, the use of STAM-BP or the novel notion of a summary inflammation variable developed in this work might be used in combination with other biological markers in the future. Show less

To determine what factors regulate gonocyte proliferation in newborn rats, we first examined the expression of several signal transduction molecules by immunocytochemistry in 3-day-old rat testis sections. We found that gonocytes specifically expressed the iota and zeta isoforms of protein kinase (PK) C (PKC) and the phosphatidylinositol 3-kinase (PI3-K). Because both the zeta PKC and PI 3-K have been shown to play a role in platelet-derived growth factor (PDGF)-induced cell proliferation, we examined the effects of PDGF on gonocytes. For this, we developed a method to obtain highly purified and viable gonocytes in culture. After enzymatic digestion, differential adhesion, and two successive gradient fractionations, the gonocyte suspension obtained was over 90% pure, as assessed by light microscopy. The viability of cultured gonocytes exceeded 90% after 48 h in the presence of 2.5% FBS used as a survival factor. Immunodetection studies showed that isolated gonocytes expressed zeta PKC, PI 3-K, and the PDGF receptor. Treatment with 10 ng/ml PDGF induced a 4-fold increase of bromodeoxyuridine incorporation into gonocytes (from 5% proliferative gonocytes under basal conditions to 20% in the presence of PDGF). Because neonatal Sertoli cells secrete high levels of the growth promoting steroid, 17 beta-estradiol, we also tested its effect and found that it induced gonocyte proliferation at a level comparable with that of PDGF and that this effect was blocked by the estrogen receptor antagonist, ICI 164384. The combination of PDGF and estradiol, however, was not additive, suggesting that their effects were mediated by common molecular target(s). These results demonstrate that PDGF and estradiol activate gonocyte proliferation in vitro, suggesting that they may act as the physiological regulators of gonocyte development in vivo. Show less

The role of second messenger pathways, cyclic AMP, calcium, and protein kinase C (PKC) in the transcriptional regulation of c-fos protooncogene expression in rat Sertoli cells was investigated. c-fos expression was monitored by Northern blot analysis. Although the action of FSH on Sertoli cells is considered to be mediated by cAMP, dibutyryl cAMP (dbcAMP), a potent membrane permeable analog of cAMP, induced much less c-fos mRNA expression than FSH ( < 50%) suggesting that additional cAMP-independent mechanisms may mediate the effect of FSH on c-fos. Specific intracellular inhibitors of PKC decreased c-fos induction in response to FSH by more than 50%. Ionomycin, which increases intracellular free calcium concentration, induced c-fos expression significantly. These data demonstrate that Sertoli cell c-fos mRNA expression is under multifactorial regulation by cAMP, calcium, and PKC. Show less

Sertoli cells cultured on basement membrane substrates differentiate morphologically into polarized cells and exhibit an enhanced responsiveness to FSH. The signal transduction mechanisms by which the extracellular matrix induces changes in the morphology and function of Sertoli cells are not known. Since calcium has been implicated in mediating changes in cytoskeletal assembly and organization, we investigated to see if basement membrane can modulate cytosolic free calcium concentrations during the process of adhesion and spreading of Sertoli cells. A direct quantification of the intracellular free cytosolic calcium concentration [Ca2+]i in freshly isolated immature rat Sertoli cells plated on laminin was performed by digital imaging microscopy using the fluorescent probe Fura-2 AM. [Ca2+]i levels rose by 1.5-2-fold within 1 h after plating on laminin, suggesting that calcium may be involved in adhesion and spreading of the cells on basement membrane. Furthermore, the possibility that matrix influences [Ca2+]i levels upon stimulation with FSH was examined by adding FSH directly to the cells spreading on laminin. A dramatic decrease in [Ca2+]i was observed compared to the level in untreated cells. Similarly, a significant decrease in [Ca2+]i in response to FSH was observed in cells already spread on laminin or Matrigel. Addition of dibutyryl cAMP did not significantly alter the basal calcium levels. Long-term exposure of Sertoli cells cultured on either laminin or Matrigel to FSH was studied by incubating the cells with 45CaCl2 in the presence or absence of FSH for 24 h. FSH induced a decrease or no change in 45Ca concentration in cells cultured on basement membrane. Addition of dibutyryl cAMP, instead of FSH, did not alter the basal 45Ca concentrations. In cells cultured on the peptides derived from laminin (RGD and SIKVAV), FSH increased the uptake of 45Ca significantly, whereas on YIGSR, also a laminin-derived peptide, it did not have any effect. Thus, basement membrane induces an early increase in [Ca2+]i in cultured Sertoli cells during spreading, and FSH appears to significantly decrease [Ca2+]i levels. Show less

When Sertoli cells of the seminiferous epithelium in the testis were cultured on Matrigel (a reconstituted basement membrane), laminin, or one of the biologically active laminin-derived peptides (YIGSR, SIKVAV, or RGD), they exhibited dramatic changes in morphology accompanied by changes in protein secretion and gene expression, including a rapidly induced stimulation of c-fos mRNA. To examine the role of c-fos in Sertoli cell attachment, spreading, and differentiation on extracellular matrix, we constructed sense and antisense c-fos phosphorothioate oligodeoxynucleotides (ODNs). Sertoli cells, in small clumps of 10-20 cells, cultured on Matrigel or laminin in the presence of ODNs antisense to c-fos (30 micrograms/ml) did not spread for the first 10-20 hr. After that time, normal spreading occurred, probably as a result of ODN degradation. Cells cultured in medium supplemented with ODNs sense to c-fos (30 micrograms/ml), or without ODNs, spread within 30 min to 1 hr, and after 12 hr a monolayer was established. Furthermore, incubation of Sertoli cells with ODNs antisense to c-fos resulted in a significant reduction of c-fos protein levels, whereas treatment with ODNs sense to c-fos barely affected c-fos protein expression. These data indicate that c-fos may mediate the events involved in Sertoli cell attachment and spreading upon contact of the cells with extracellular matrix. Show less

Despite the important role of calcium in the growth and differentiation of a variety of cell types, its exact location and function in the somatic and germ cells of the testis remain to be determined. In the present study, we examined the subcellular distribution of calcium in the immature and adult rat testis. Calcium was localized at the electron microscopic level by ion-capture cytochemistry using combined oxalate and pyroantimonate procedures. Calcium-containing precipitates localized primarily within the nuclei, mitochondria, and cytosol of somatic and germ cells. Differences in the size and quantity of the calcium precipitates were observed among the various cellular compartments. In the somatic cells (Sertoli, Leydig, and myoid), the nuclei exhibited large round-shaped calcium-containing precipitates, whereas the mitochondria in these cell types contained numerous smaller precipitates. The cytoplasmic vesicles possessed single precipitates. These vesicles could be calciosomes, which have been described in other non-muscle cell types. Among germ cells, round spermatids exhibited a large number of vesicular, calsiosome-like structures in the cytoplasm containing single precipitates. The elongating spermatids from adult testis showed calcium localization within the nuclear matrix unassociated with the nuclear envelope, or in a peripheral alignment of precipitates along the nuclear envelope. Calciosome-like structures were also seen in round spermatids. Spermatogonia and spermatocytes exhibited calcium in nuclei, mitochondria, and cytoplasmic vesicles. These results demonstrate a differential distribution of calcium within the various cell types of the testis. The presence of calcium in the nucleus may suggest a role in cell growth and differentiation; calsiosome-like structures may represent the active exchangeable pool of calcium, and the differential type of distribution of calcium in elongating spermatids suggests a role for calcium in spermatid differentiation. Show less

Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development. Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells. When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively. When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed. However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes. These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb. However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS) Show less

The expression of guanine nucleotide-binding proteins (G proteins) during the development of rat testes was investigated. Immunohistochemical studies on frozen sections and isolated testicular cells demonstrated that the expression of the GTP-binding proteins was developmentally regulated and specific for different cell types. The alpha subunit of the cholera toxin-sensitive stimulatory G protein (Gs alpha) was first detected in testes from 7-day-old rats; its value reached a maximum at 23 days and then decreased to very low or undetectable amounts in testes of 45-day-old and adult rats (60-90 days of age). The Gs alpha subunit appears to be expressed by Sertoli, peritubular myoid and interstitial cells. The common beta subunit (G beta) was present at all ages during development and was more prominent around the periphery of the tubules in younger animals but then became more evident in the cytoplasm of germ cells with increasing age. The pertussis toxin-sensitive inhibitory G proteins, Gi1/2 and Gi3, showed a similar pattern of expression. Sertoli cells and peritubular cells expressed Gi1/2 and Gi3 at very low levels at all ages, whereas pachytene spermatocytes and round spermatids expressed the inhibitory binding proteins only at later ages of development (45-day-old and adult testis). Northern blot analysis showed that with increasing age the Gs alpha mRNA in the testis decreased and this was confirmed by in situ hybridization. These latter studies showed localization of the transcripts to somatic cells but not to germ cells.(ABSTRACT TRUNCATED AT 250 WORDS) Show less

A number of years ago we reported that tight junctions between adjacent Sertoli cells subdivide the seminiferous epithelium into two compartments, basal and adluminal, thus forming the morphological basis of the blood-testis barrier. It is now generally believed that the special milieu created by the Sertoli cells in the adluminal compartment is essential for germ cell differentiation. In order to duplicate the compartmentalization that occurs in vivo, Sertoli cells were cultured in bicameral chambers on Millipore filters impregnated with a reconstituted basement membrane. Confluent monolayers of these cells were tall columnar (40-60 microns in height) and highly polarized. These Sertoli cell monolayers established electrical resistance that peaked when the Sertoli-Sertoli tight junctions developed in culture. In addition, the monolayers formed a permeability barrier to 3H-inulin and lanthanum nitrate. The bicameral chambers were utilized in a number of studies on protein secretion, and it was revealed that numerous proteins are secreted in a polarized manner. In another study, hormone-stimulated aromatase activity was measured in Sertoli cells grown on plastic culture dishes, plastic dishes coated with laminin or Matrigel, and in the bicameral chambers. Cell culture on basement membrane substrates decreased the FSH-dependent estrogen production. No estrogen production was observed when the Sertoli cells were cultured in the bicameral chambers. These results are in accord with the hypothesis that differentiated Sertoli cells lose their ability to metabolize androgen to estrogen in an hormone-dependent manner, whereas undifferentiated cells in culture, or in vivo, have a very active FSH-dependent aromatase activity.(ABSTRACT TRUNCATED AT 250 WORDS) Show less

Laminin, a major component of basement membrane extracellular matrices, promotes differentiation in a number of cell types, including Sertoli cells. We have identified and characterized Sertoli cells. We have identified and characterized Sertoli cell surface molecules which interact with laminin. Using laminin-Sepharose affinity chromatography and [125I]laminin binding to Sertoli cell plasma membranes, binding proteins have been identified with the Mr 110,000, 67,000, 55,000, 45,000, 36,000, and 25,000. In addition, the Mr 110,000 and 67,000 laminin binding proteins were phosphorylated. The 67,000, 45,000, and 36,000 react with antibodies to the previously characterized laminin receptor and these antibodies stain the basolateral surface of Sertoli cells in vivo. Cultured Sertoli cells stain for laminin receptor both on the cell surface and within the cells. Antiserum to the 32,000 and 67,000 laminin binding proteins partially inhibited spreading of Sertoli cells on a laminin-coated culture dish, suggesting a functional importance of those proteins in Sertoli cell differentiation. The 25,000 and 45,000 laminin binding proteins reacted with integrin antibodies, but no high-molecular-weight forms could be detected. Integrin was localized to the cell surface and intracellularly but antibodies did not block Sertoli cell spreading on laminin. This work represents the first identification and characterization of extracellular matrix binding proteins in an endocrine organ and suggests an important role for the nonintegrin 32/67 laminin binding proteins. Show less

Laminin is a potent stimulator of neurite outgrowth. We have examined the signal transduction events involved in the neuronal cell response to laminin. Cyclic nucleotides, calcium, and sodium-proton exchange do not appear to be required for the transduction of the laminin signal during neurite outgrowth. Direct measurement of cAMP and cGMP levels shows no changes in NG108-15 cells when cultured on laminin. Exogenous cAMP alone had no effect on either the rate of process formation or process length, but did alter the morphology of laminin-induced neurites. A four-fold increase in the number of branches per neurite and a two-to-three-fold increase in the number of neurites per cell were observed in both NG108-15 and PC12 cells cultured on laminin when either 8-BrcAMP or forskolin was added. The cAMP-induced branching was also observed when PC12 cells were cultured on a laminin-derived synthetic peptide (PA22-2), which contains the neurite-promoting amino acid sequence IKVAV. By immunofluorescence analysis with axonal or dendritic markers, the PC12 processes on laminin and PA22-2 were axonal, not dendritic, and the cAMP-induced morphological changes were due to axonal branching. These data demonstrate that changes in cAMP are not involved in laminin-mediated neurite outgrowth, but cAMP can modulate the effects of laminin. Show less

On a basement membrane substrate, Sertoli cells in culture have been shown to assume a phenotype similar to that of the in vivo differentiated cells. Sertoli cells from 10-day-old rats were cultured on plastic and on different extracellular matrix substrates [laminin, a reconstituted basement membrane (Matrigel), and a synthetic laminin peptide containing the arginine-glycine-aspartic acid (RGD) tripeptide sequence] to investigate the effects of the extracellular matrix on FSH responsiveness. Both laminin and Matrigel markedly enhanced the cAMP response to FSH and cholera toxin, indicating modifications at the level of guanine nucleotide-binding regulatory (G) proteins. Furthermore, Sertoli cell grown on either of these two substrates responded to physiological levels of FSH (25-50 ng/ml), whereas pharmacological levels of FSH (500 ng/ml) were required for cells grown on either plastic or on the RGD-containing laminin peptide. Immunoblotting of Sertoli cell plasma membranes with antibodies directed against the alpha-subunit of the stimulatory G-protein (Gs alpha) of adenylyl cyclase indicated that Sertoli cell culture on either laminin or Matrigel increased the amounts of Gs alpha. These results were further confirmed by immunoprecipitating the Gs alpha protein from the particulate fraction of [35S]methionine metabolically labeled Sertoli cells. However, Northern blot analysis using a cDNA probe for Gs alpha did not demonstrate changes in gene expression when Sertoli cells were grown on the various substrates. Immunofluorescent studies revealed that the Gs complex of adenylyl cyclase was preferentially located at the base of the Sertoli cells at the site of contact with the extracellular matrix. These data suggest that culture of epithelial Sertoli cells on basement membrane substrates enhances the Gs complex of adenylyl cyclase and the cAMP response to FSH, consistent with the more differentiated morphology and function of the cells. Show less

We studied expression of laminin, fibronectin, and Type IV collagen in the testis by means of immunofluorescence and immunoblot analysis and also examined gene expression of fibronectin using the ribonuclease protection assay. By immunofluorescence on sections from 20-day-old rats, laminin, fibronectin, and Type IV collagen were found in the basement membrane of the seminiferous tubules and in the interstitial regions of the testis. No localization of any extracellular matrix components was found inside the sectioned cells. However, when Sertoli cells were cultured on glass coverslips, laminin and Type IV collagen were both found inside the cells, suggesting new synthesis. In cultured peritubular cells, Type IV collagen, laminin, and fibronectin were found within the cells. When examined by immunoblot analysis, freshly isolated Sertoli and peritubular cells from 20-day-old rats did not demonstrate production of laminin or fibronectin. After 5 days in culture, peritubular cells produced both laminin and fibronectin, whereas cultured Sertoli cells produced only laminin. In contrast, freshly isolated and cultured Sertoli and peritubular cells all produced Type IV collagen. Moreover, the ribonuclease protection assay indicated that the bulk of fibronectin gene expression occurs within the first 10 days of postnatal development, with lower maintenance levels occurring thereafter. These results indicate that in the testis the highest levels of expression of laminin and fibronectin occur during development and in primary cell culture, whereas expression of Type IV collagen is higher at later stages. Show less

We have examined the effects of Sertoli cell-secreted proteins (SCSP) on [3H]thymidine incorporation by purified preparations (greater than 96%) of rat Leydig cells to determine whether Sertoli cells influence DNA synthesis in these cells in vitro. Incubation of Leydig cells isolated from testes of rats of ages 16 to 90 days with SCSP (Mr greater than 10,000) induced significant dose-, time- and age-related increases in [3H]thymidine incorporation by the cells. A dose-response curve to SCSP showed that as little as 0.2 micrograms SCSP/ml consistently induced a small but significant increase (31% and 10% above control; P less than 0.001) in [3H]thymidine incorporation by Leydig cells isolated from immature (26 days) and mature (70 days) rats, respectively. The maximum response (230% and 48% above control) was obtained with a concentration of 18 micrograms SCSP/ml in cells isolated from immature and mature rats, respectively. Hydroxyurea, a specific inhibitor of replicative DNA synthesis, significantly (P less than 0.001) inhibited both basal and SCSP-induced [3H]thymidine incorporation in Leydig cells from immature and adult rats without affecting the viability of the cells. Incubation of immature rat Leydig cells in SCSP for 48 h also stimulated a 3-fold increase in cell number. The component of the crude SCSP which stimulated Leydig cell [3H]thymidine incorporation is trypsin-sensitive, heat-stable, and adsorbs to a heparin-agarose affinity column but not to concanavalin A-Sepharose. The secretion of this factor(s) by Sertoli cells is stimulated independently by FSH and testosterone. These results demonstrate for the first time that cultured Sertoli cells secrete a protein(s) which, in vitro, stimulates rat Leydig cell replicative DNA synthesis. Show less