Change in the levels of adenine-related compounds in starfish ovarian follicle cells following treatment with gonad-stimulating substance

The resumption of meiosis in starfish oocytes is induced by 1-methyladenine (1-MeA), which is produced by ovarian foilicle cells under the influence of a gonad-stimulating substance (GSS). It has been reported that the 1-MeA produced is newly synthesized via a process of methylation, rather than being pre-stored within follicle cells or a breakdown product of some 1-MeA-containing substance. The present study examined a possible substrate for 1-MeA biosynthesis stored in follicle cells of the starfish Asterina pectinifera . Analyses using high-performance liquid chromatography indicated a large source of ATP among the adenine-related compounds in these follicle cells. When follicle cells were incubated in seawater in the presence of GSS, 1-MeA production was stimulated significantly. GSS also caused a reduction in intracellular levels of ATP. There was no change in the levels of either ADP or AMP. The amount of ATP consumed under the influence of GSS was similar to the amount of 1-MeA produced. Methionine and selenomethionine enhanced both 1-MeA production and ATP consumption by GSS in follicle cells. In contrast, ethionine and selenoethionine, competitive inhibitors of methionine, inhibited these processes. These results suggest that ATP is a possible substrate in the biosynthesis of 1-MeA by starfish ovarian follicle cells.     

A relaxin‐like gonad‐stimulating peptide identified from the starfish Astropecten scoparius

A relaxin‐like gonad‐stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B‐chain (21 aa), C‐peptide (47 aa), and A‐chain (24 aa). There are three putative processing sites (Lys‐Arg) between the B‐chain and C‐peptide, between the C‐peptide and A‐chain, and within the C‐peptide. This structural organization revealed that the mature AscRGP is composed of A‐ and B‐chains with two interchain disulfide bonds and one intrachain disulfide bond. The C‐terminal residues of the B‐chain are Gln‐Gly‐Arg, which is a potential substrate for formation of an amidated C‐terminal Gln residue. Non‐amidated (AscRGP‐GR) and amidated (AscRGP‐NH₂) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP‐GR and AscRGP‐NH₂ induced oocyte maturation and ovulation in similar dose‐dependent manners. This is the first report on a C‐terminally amidated functional RGP. Collectively, these results suggest that AscRGP‐GR and AscRGP‐NH₂ act as a natural gonadotropic hormone in A. scoparius.     

DC‐CIK cells derived from ovarian cancer patient menstrual blood activate the TNFR1‐ASK1‐AIP1 pathway to kill autologous ovarian cancer stem cells

Ovarian cancer stem cells (OCSCs) are highly carcinogenic and have very strong resistance to traditional chemotherapeutic drugs; therefore, they are an important factor in ovarian cancer metastasis and recurrence. It has been reported that dendritic cell (DC)‐cytokine‐induced killer (CIK) cells have significant killing effects on all cancer cells across many systems including the blood, digestive, respiratory, urinary and reproductive systems. However, whether DC‐CIK cells can selectively kill OCSCs is currently unclear. In this study, we collected ovarian cancer patient menstrual blood (OCPMB) samples to acquire mononuclear cells and isolated DC‐CIK cells in vitro. In addition, autologous CD44+/CD133+ OCSCs were isolated and used as target cells. The experimental results showed that when DC‐CIK cells and OCSCs were mixed and cultured in vitro at ratios of 5:1, 10:1 and 50:1, the DC‐CIK cells killed significant amounts of OCSCs, inhibited their invasion in vitro and promoted their apoptosis. The qPCR and Western blot results showed that DC‐CIK cells stimulated high expression levels and phosphorylation of TNFR1, ASK1, AIP1 and JNK in OCSCs through the release of TNF‐α. After the endogenous TNFR1 gene was knocked out in OCSCs using the CRISPR/Cas9 technology, the killing function of DC‐CIK cells on target OCSCs was significantly attenuated. The results of the analyses of clinical samples suggested that the TNFR1 expression level was negatively correlated with ovarian cancer stage and prognosis. Therefore, we innovatively confirmed that DC‐CIK cells derived from OCPMB could secret TNF‐α to activate the expression of the TNFR1‐ASK1‐AIP1‐JNK pathway in OCSCs and kill autologous OCSCs.     

TGF‐β1 sustains germ cell cyst reservoir via restraining follicle formation in the chicken

The transforming growth factor β (TGF‐β) superfamily members are important molecules that regulate many ovarian functions under normal physiological and pathological conditions. TGF‐β1 and its receptors are highly expressed in the ovarian cells of many species. However, the effect of TGF‐β1 on the capacity of the avian germ cell reservoir remains unknown. In this study, 5‐day‐old chicks were injected with TGF‐β1 (2.5, 12.5, and 62.5 μg/kg body weight) for 3 days to assess the effect of TGF‐β1 on early follicle development. Morphological analysis showed that treatment with TGF‐β1 (12.5 μg/kg) increased the number of germ cell cysts and reduced the number of primordial and growing follicles. The diameter and area of oocytes and follicles were decreased after TGF‐β1 treatment. Immunohistochemical staining of the proliferating cell nuclear antigen revealed that the ratios of the positive somatic and granulosa cells were decreased by 16.2% and 2.48%, respectively. Furthermore, more apoptotic cells were observed in the TGF‐β1 group than those of the control by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. In addition, we cultured the 5d chicken ovaries for 3 days in vitro and found that treatment with TGF‐β1 (10 ng/mL) manifested similar results as the in vivo experiment. However, the negative effect of TGF‐β1 on early ovary development was rescued by treatment with a TGF‐βR1 inhibitor SD208, resulting in increased expression of steroidogenic enzymes and cell cycle‐regulating proteins. In conclusion, TGF‐β1 could maintain the germ cell reservoir by restraining follicle activation involving reduced cell proliferation and steroidogenic enzymes gene expression at the early stage of ovarian development.     

Interleukin‐6 contributes to chemoresistance in MDA‐MB‐231 cells via targeting HIF‐1α

Chemoresistance is a critical challenge in the clinical treatment of triple‐negative breast cancer (TNBC). It has been well documented that inflammatory mediators from tumor microenvironment are involved in the pathogenesis of TNBC and might be related to chemoresistance of cancer cells. In this study, the contribution of interleukin‐6 (IL‐6), one of the principal oncogenic molecules, in chemoresistance of a TNBC cell line MDA‐MB‐231 was first investigated. The results showed that IL‐6 treatment could induce upregulation of HIF‐1α via the activation of STAT3 in MDA‐MB‐231 cells, which consequently contributed to its effect against chemotherapeutic drug‐induced cytotoxicity and cell apoptosis. However, knockdown of HIF‐1α attenuated such effect via affecting the expressions of apoptosis‐related molecules as Bax and Bcl‐2 and drug transporters as P‐gp and MRP1. This study indicated that targeting at IL‐6/HIF‐1α signaling pathway might be an effective strategy to overcome chemoresistance in TNBC therapy.     

The Skp1‐like protein SSK1 is required for cross‐pollen compatibility in S‐RNase‐based self‐incompatibility

The self‐incompatibility (SI) response occurs widely in flowering plants as a means of preventing self‐fertilization. In these self/non‐self discrimination systems, plant pistils reject self or genetically related pollen. In the Solanaceae, Plantaginaceae and Rosaceae, pistil‐secreted S‐RNases enter the pollen tube and function as cytotoxins to specifically arrest self‐pollen tube growth. Recent studies have revealed that the S‐locus F‐box (SLF) protein controls the pollen expression of SI in these families. However, the precise role of SLF remains largely unknown. Here we report that PhSSK1 (Petunia hybrida SLF‐interacting Skp1‐like1), an equivalent of AhSSK1 of Antirrhinum hispanicum, is expressed specifically in pollen and acts as an adaptor in an SCF(Skp1‐Cullin1‐F‐box)^SLF complex, indicating that this pollen‐specific SSK1‐SLF interaction occurs in both Petunia and Antirrhinum, two species from the Solanaceae and Plantaginaceae, respectively. Substantial reduction of PhSSK1 in pollen reduced cross‐pollen compatibility (CPC) in the S‐RNase‐based SI response, suggesting that the pollen S determinant contributes to inhibiting rather than protecting the S‐RNase activity, at least in solanaceous plants. Furthermore, our results provide an example that a specific Skp1‐like protein other than the known conserved ones can be recruited into a canonical SCF complex as an adaptor.     

Follicle‐stimulating hormone impairs dental pulp stem cells odontogenic differentiation

In addition to bone, the dentin‐pulp complex is also influenced by menopause, showing a decreased regenerative capacity. High levels of follicle‐stimulating hormone (FSH) during menopause could directly regulate bone metabolism. Here, the role of FSH in the odontogenic differentiation of the dentin‐pulp complex was investigated. Dental pulp stem cells (DPSCs) were isolated. CCK‐8 assays, cell apoptosis assays, Western blotting (WB), real‐time RT‐PCR, alkaline phosphatase activity assays, and Alizarin Red S staining were used to clarify the effects of FSH on the proliferation, apoptosis and odontogenic differentiation of the DPSCs. MAPK pathway‐related factors were explored by WB assays. FSH and its inhibitor were used in OVX rats combined with a direct pulp‐capping model. HE and immunohistochemistry were used to detect reparative dentin formation and related features. The results indicated that FSH significantly decreased the odontogenic differentiation of the DPSCs without affecting cell proliferation and apoptosis. Moreover, FSH significantly activated the JNK signalling pathway, and JNK inhibitor partly rescued the inhibitory effect of FSH on DPSC differentiation. In vivo, FSH treatment attenuated the dentin bridge formation and mineralization‐related protein expression in the OVX rats. Our findings indicated that FSH reduced the odontogenic capacity of the DPSCs and was involved in reparative dentinogenesis during menopause.     

Co‐ordinated expression of the VEGF system components in granulosa cells to develop a proangiogenic autocrine milieu during ovarian follicle development

In the present study, we investigated the temporal relationship between angiogenic and antiangiogenic vascular endothelial growth factor isoforms (VEGFxxxa and VEGFxxxb, respectively), the receptors VEGFR1 and VEGFR2, their soluble forms, and the kinases and the splicing factors regulating the synthesis of VEGF isoforms in healthy and atretic antral follicles. The results show a higher (p < 0.05) messenger RNA (mRNA) expression of VEGF120a, VEGF164a, and VEGF120b in healthy than in atretic follicles, but the mRNA expression of VEGF164b was not detected. The mRNA of serine–arginine protein kinase 1 ( SRPK1) was higher ( p < 0.05) in large healthy follicles than in large atretic follicles. In contrast, atretic follicles had higher mRNA expression of a soluble form of the receptor 2 of VEGF ( sVEGFR2) than healthy follicles ( p < 0.05). Additionally, we observed a positive relationship ( p < 0.05) between SRPK1 and serine–arginine‐rich splicing factor 1 ( SRSF1) with the angiogenic isoforms VEGF120a and VEGF164a and between CDC‐like kinases‐1 ( CLK1) and SRSF6 with the antiangiogenic VEGF120b isoform. Principal components analysis (PCA) resulted in two PC explaining 71% of the variation, which was formed by the VEGF isoforms, the kinases and the splicing factor (PC1) and by the VEGF receptors (PC2). When PC analysis was carried out within follicular health status, there were no differences for PC1 between follicular status, whereas PC2 differed between healthy and atretic follicles. In conclusion, the higher mRNA expression for VEGF120a and VEGF164a, the low expression of sVEGFR2, and absent expression of mRNA for VEGF164b provide evidence of a proangiogenic autocrine milieu to support granulosa cells during follicle development.