Spexin role in human granulosa cells physiology and PCOS : expression and negative impact on steroidogenesis and proliferation

International audienceAbstract Spexin (SPX) is a novel neuropeptide and adipokine negatively correlated with obesity and insulin resistance. A recent study investigated expression and regulatory function of SPX in the hypothalamus and pituitary; however, the effect on ovarian function is still unknown. The aim of this study was to characterize the expression of SPX and its receptors, galanin receptors 2 and 3 (GALR2/3), in the human ovary and to study its in vitro effect on granulosa cells (GC) function. Follicular fluid (FF) and GC were obtained from normal weight and obese healthy and diagnosed with polycystic ovarian syndrome (PCOS) women. Expression of SPX and GALR2/3 in the ovary was studied by qPCR, western blot, and immunohistochemistry. The level of SPX in FF was assessed by enzyme-linked immunosorbent assay. The in vitro effect of recombinant human SPX on GC proliferation, steroidogenesis, and signaling pathways (MAP3/1, STAT3, AKT, PKA) was analyzed. Moreover, GC proliferation and estradiol (E2) secretion were measured with and without an siRNA against GALR2/3 and pharmacological inhibition of the above kinases. The results showed that both the SPX concentration in FF and its gene expression were decreased in GC of obese and PCOS women, while the protein expression of GALR2/3 was increased. We noted that SPX reduced GC proliferation and steroidogenesis; these effects were mediated by GALR2/3 and kinases MAP3/1, AKT, and STAT3 for proliferation or kinases MAP3/1 and PKA for E2 secretion. The obtained data clearly documented that SPX is a novel regulator of human ovarian physiology and possibly plays a role in PCOS pathogenesis

The impact of isolated gastric cancer oncosomes on activity of peripheral blood human monocyte subsets

Pęcherzyki zewnątrzkomórkowe (EVs, ang. extracellular vesicles) stanowią zróżnicowaną grupę, zazwyczaj kulistych struktur uwalnianych przez niemal wszystkie typy komórek. Onkosomy są to pęcherzyki generowane przez komórki nowotworowe do środowiska zewnątrzkomórkowego. Materiał („cargo”) przenoszony przez onkosomy w postaci białek, materiału genetycznego, a także receptory/ligandy występujące na błonie onkosomów mogą modyfikować funkcje prawidłowych komórek organizmu, w tym również MO.Celem niniejszej pracy była próba zbadania aktywności wydzielniczej oraz cytotoksycznej względem komórek nowotworowych MO krwi obwodowej człowieka i ich subpopulacji po stymulacji onkosomami komórek linii raka żołądka. Aby określić wpływ badanych onkosomów na aktywność wydzielniczą MO i ich subpopulacji oceniano zarówno poziom uwalnianych cytokin (TNF-α, IL-12p40, IL-10), jak i poziom kodującego je mRNA. Aktywność cytotoksyczną MO określano wobec komórek nowotworowych linii raka żołądka, z których uzyskiwano onkosomy.Otrzymane wyniki wskazują na zróżnicowanie onkosomów komórek raka żołądka pod względem wielkości. Zróżnicowanie to występuje zarówno pomiędzy różnymi liniami komórek raka żołądka, jak i w obrębie populacji onkosomów pochodzącej z tej samej linii nowotworowej. Wykazano, że onkosomy komórek linii raka żołądka mogą wpływać stymulująco zarówno na produkcję IL-10, TNF-α oraz IL-12 przez MO i ich subpopulacje, jak i na poziom mRNA dla badanych cytokin w MO i ich subpopulacjach. Ponadto stwierdzono, że odpowiedź MO na stymulację onkosomami różni się pomiędzy subpopulacjami MO. Przeprowadzone badania wskazują, że MO i ich subpopulacje stymulowane onkosomami wykazują zwiększoną aktywność cytotoksyczną względem komórek nowotworowych.Reasumując, uzyskane wyniki wskazują na możliwość modyfikacji funkcji efektorowych MO krwi przez onkosomy, co może zostać wykorzystane w opracowaniu nowych, skuteczniejszych form terapii przeciwnowotworowych.Extracellular vesicles (EV) are heterogeneous group of typically spherical structures released by almost all cell types. Oncosomes or tumor-derived microvesicles (TMV), are membrane-bound sacks, shed from the surface of tumor cells into the extracellular environment. The oncosome cargo, which includes proteins, nucleic acids and surface receptors/ligands may affect the function of normal cells, including monocytes (MO).The aim of the study was to investigate secretory function and cytotoxic activity against tumor cells of peripheral blood human MO and their subsets, after stimulation with oncosomes from gastric cancer cell lines. In order to assess the impact of the oncosomes on MO secretory activity, both the release of TNF-α, IL-10, and IL-12p40 cytokines and their respective mRNA level was evaluated.The obtained results showed size-related heterogeneity of analysed oncosomes. This variation occurs both between oncosomes originated from different gastric cancer cell lines, as well as within the population of TMV from the same line of cancer. It has been documented that oncosomes from gastric cancer cell lines can stimulate MO production of IL-10, TNF-α and IL-12, as well as respective mRNA level in MO and their subpopulations. It was also demonstrated that monocytes’ response to TMV stimulation varies between subpopulations. Moreover, the study showed that MO and their subpopulations exhibit increased cytotoxic activity against tumour cells after TMV stimulation.In summary, the results suggest that MO effector functions can be modulated by oncosomes what may provide possibilities for development of new, more effective forms of antitumor therapy

Apelin, APJ, and ELABELA : role in placental function, pregnancy, and foetal development - an overview

The apelinergic system, which includes the apelin receptor (APJ) as well as its two specific ligands, namely apelin and ELABELA (ELA/APELA/Toddler), have been the subject of many recent studies due to their pleiotropic effects in humans and other animals. Expression of these factors has been investigated in numerous tissues and organs—for example, the lungs, heart, uterus, and ovary. Moreover, a number of studies have been devoted to understanding the role of apelin and the entire apelinergic system in the most important processes in the body, starting from early stages of human life with regulation of placental function and the proper course of pregnancy. Disturbances in the balance of placental processes such as proliferation, apoptosis, angiogenesis, or hormone secretion may lead to specific pregnancy pathologies; therefore, there is a great need to search for substances that would help in their early diagnosis or treatment. A number of studies have indicated that compounds of the apelinergic system could serve this purpose. Hence, in this review, we summarized the most important reports about the role of apelin and the entire apelinergic system in the regulation of placental physiology and pregnancy

Does 4-tert-octylphenol affect estrogen signaling pathways in bank vole Leydig cells and tumor mouse Leydig cells in vitro?

Primary Leydig cells obtained from bank vole testes and the established tumor Leydig cell line (MA-10) have been used to explore the effects of 4-tert-octylphenol (OP). Leydig cells were treated with two concentrations of OP (10(-4) M, 10(-8) M) alone or concomitantly with anti-estrogen ICI 182,780 (1 µM). In OP-treated bank vole Leydig cells, inhomogeneous staining of estrogen receptor alpha (ER alpha) within cell nuclei was found, whereas it was of various intensity among MA-10 Leydig cells. The expression of ER alpha mRNA and protein decreased in both primary and immortalized Leydig cells independently of OP dose. ICI partially reversed these effects at mRNA level while at protein level abrogation was found only in vole cells. Dissimilar action of OP on cAMP and androgen production was also observed. This study provides further evidence that OP shows estrogenic properties acting on Leydig cells. However, its effect is diverse depending on the cellular origin

Hydroxyflutamide affects connexin 43 via the activation of PI3K/Akt-dependent pathway but has no effect on the crosstalk between PI3K/Akt and ERK1/2 pathways at the Raf-1 kinase level in primary rat Sertoli cells

We investigated the effects of 2-hydroxyflutamide (HF), an active metabolite of the anti-androgen flutamide, on the activation of the phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) in rat Sertoli cells. Sertoli cells, isolated from 20-day-old rat testes, were cultured in vitro and treated with HF, testosterone, or HF + testosterone. Studies by western blotting demonstrated that HF inhibited the testosterone-mediated increase in c-Src activity (p \u3c 0.05). In contrast, Akt phosphorylation was augmented within 5 min after HF treatment (p \u3c 0.01). This effect was accompanied by a rapid upregulation in PTEN phosphorylation (p \u3c 0.001). Despite no changes in Raf-1 phosphorylation, HF increased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation (p \u3c 0.001), indicating that the effect of the anti-androgen on ERK1/2 was independent of PI3K/Akt-pathway activation at this level. Since HF inhibited the testosterone-mediated increase in c-Src activity, it is likely that activation of both Akt and ERK1/2 occurred in a p-Src independent manner. Activation of PI3K/Akt-pathway by HF resulted in the reduced level of Sertoli cell functional marker, connexin 43 (p \u3c 0.01). Collectively, these data provide evidence that HF rapidly and transiently affects the protein kinase-dependent signaling pathways, acting both as an antagonist and agonist. Moreover, using testes of flutamide-treated rats for 7 days, we demonstrated that the anti-androgen can modulate the protein kinase-dependent pathways in long term by enhancing Akt and ERK1/2 protein expression (p \u3c 0.05)

Mutation c.256_257delAA in RAG1 Gene in Polish Children with Severe Combined Immunodeficiency : diversity of Clinical Manifestations

Mutations in RAG1 gene may result in different types of severe combined immunodeficiencies. In this study, we compare clinical symptoms and laboratory findings in four children with identical mutation in RAG1 gene. All of analyzed patients presented symptoms of severe combined immunodeficiencies associated or not with Omenn syndrome (OS) features. In our patients two different types of variants in RAG1 gene were detected. The first of the mutation was the deletion of AA dinucleotide at position c.256_257 (p.Lys86ValfsTer33), the second gene variant was substitution c.2867T>C (p.Ile956Thr). In Patient 1 we detected that compound heterozygous mutations involved both of the mentioned variants. Whereas, in Patients 2, 3 and 4, we confirmed the presence of the dinucleotide deletion but in a homozygous state. In all described patients, sequence analysis of RAG2 gene did not reveal any nucleotide changes. Our data show that mutation c.256_257delAA in RAG1 gene seems to occur quite frequently in the polish patients with severe combined immunodeficiency and may result in classical OS as well as in severe combined immunodeficiency without clinical and laboratory features of OS when occurred in homozygous state. The same mutation but in heterozygous state, in combination with other mutation in RAG1 gene, may result in incomplete OS