Biology subjects

Poutanen, M.

Publications and source records attributed to Poutanen, M..

8 recordsLinked to original sources

bioRxiv2026

Measurement of a panel of 21 steroids in a quantitative assay in human plasma, adipose tissue, and fecal samples using ultra-high-performance liquid chromatography-tandem mass spectrometry

Comprehensive detection of steroids, beyond the limited panels typically analyzed in clinical chemistry laboratories, has become increasingly important given their pivotal roles in diverse biological processes. However, steroid quantification poses several analytical challenges, including differences in ionization efficiency and structural similarities across the entire steroid metabolic network. To address these challenges, we developed a targeted ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay to analyze 21 steroids using reverse-phase chromatography combined with rapid polarity switching. Mass spectrometry (MS) analysis was performed in scheduled multiple reaction monitoring (sMRM) mode. Depending on the steroid and matrix, the validated lower limits of quantitation (LLOQ) ranged from 12.0 pM to 1216 pM in plasma and 41.1 pM to 384 pM in fecal sample homogenates. In adipose tissue, it was from 0.01 pmol/g to 9 pmol/g. Measured steroid concentrations obtained from the commercial control samples (MassTrak Steroid Serum QC Set 1 and the MassCheck(R) Steroid Panel 1 Serum Control) showed close agreement with the reference values. As a proof of concept, the method was successfully applied to 469 plasma samples in several projects, 15 adipose tissue samples, and 332 fecal samples, demonstrating its applicability to large-scale studies. In conclusion, the method enables sensitive, derivatization-free quantification of an expanded steroid panel in plasma and complex biological matrices, including adipose tissue and fecal samples, representing a significant advancement in comprehensive steroid profiling. Graphical abstractFor Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/737297v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@807582org.highwire.dtl.DTLVardef@1a2cbe9org.highwire.dtl.DTLVardef@f96f76org.highwire.dtl.DTLVardef@4b8c9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Testosterone exposure during fetal masculinization programming window determines the kidney size in adult mice.

Kidney size is sex-dimorphic and regulated by androgens in adult humans and mice. However, the effects of developmental androgen deficiency on kidneys remain elusive. We hypothesized that androgens program future kidney growth during a fetal development. Male mice lacking main testosterone-producing enzyme HSD17B3 had reduced testosterone at embryonic day 15.5, but the concentrations increased by E18.5, creating a short time window of androgen deficiency resulting in reduced kidney size in adult males. In male Hsd17b3-/-kidneys, nephron development was qualitatively normal, but number of glomeruli and proliferation of proximal tubules was reduced. Testosterone supplementation at E14.5-17.5 normalized the renal size in adult Hsd17b3-/-males. Our data suggests that androgen receptor and Hnf4a jointly regulate Igfbp5, FOXO1 and mTOR signaling to convey male-specific kidney growth. In conclusion, we have identified a novel developmental programming effect on male kidneys, where fetal androgen deficiency reduces kidney growth and androgen responsiveness in adult males.

developmental biology↗

bioRxiv2024

Functionality of BRCA1 supports the survival of prostate cancer cells during the development of castration resistance

Androgen deprivation therapy (ADT; castration) is the main treatment option for metastatic prostate cancer (PCa), but eventually, castration-resistant prostate cancer (CRPC) develops with no curative treatments. In CRPC, more than 20% of men carry mutations in DNA damage response (DDR) genes, including BRCA1/2. In this study, we elucidated the prostate tissue-specific functional role of BRCA1 protein. Our results indicate that DDR is dynamically regulated by androgen receptor (AR) signaling, and AR activation by the natural ligand dihydrotestosterone strongly downregulates the expression of BRCA1 in multiple cell lines. Consistent with these findings, our analyses of patient samples and mouse xenografts showed that DNA damage and BRCA1 expression were sustained after ADT. With unbiased mass spectrometry and bioinformatics approaches as well as experimentally, we found that BRCA1 interacts with Raptor, an mTORC1 component, and regulates the mTOR signaling pathway and PCa growth in vitro. Furthermore, we found that mTOR inhibition reduced the recruitment of DDR proteins, BRCA1 and Rad51, to DNA damage sites, creating a vulnerability towards DNA damage-inducing androgen deprivation. Moreover, we observed that BRCA1 supported ADT-induced activation of the oxidative stress sensor NRF2. Our findings shed further light on the complex DDR-AR interplay in PCa and suggest that, during PCa progression, BRCA1 expression may be retained due to the beneficial modulation of mTORC1 signaling in the AR environment by BRCA1. SignificanceAndrogen receptor activation acts as a strong suppressor of BRCA1. Consequently, androgen deprivation activates BRCA1, which in turn promotes survival in castration resistance by supporting mTOR signaling and NRF2-mediated antioxidant processes.

cancer biology↗

bioRxiv2022

Dissecting the KNDy hypothesis: KNDy neuron-derived kisspeptins are dispensable for puberty but essential for preserved female fertility and gonadotropin pulsatility

Kiss1 neurons in the hypothalamic arcuate-nucleus (ARC) play key roles in the control of GnRH pulsatility and fertility. A fraction of ARC Kiss1 neurons, termed KNDy, co-express neurokinin B (NKB; encoded by Tac2). Yet, NKB- and Kiss1-only neurons are also found in the ARC, while a second major Kiss1-neuronal population is present in the rostral hypothalamus. The specific contribution of different Kiss1 neuron sub-sets to reproductive control remains unfolded. To tease apart the physiological roles of KNDy-born kisspeptins, conditional ablation of Kiss1 in Tac2-expressing cells was implemented in vivo. Mice with Tac2 cell-specific Kiss1 KO (TaKKO) displayed reduced ARC kisspeptin content and Kiss1 expression, with greater suppression in females, which was detectable at infantile-pubertal age. In contrast, Tac2/NKB levels were fully preserved. Despite the drop of ARC Kiss1/kisspeptin, pubertal timing was normal in TaKKO mice of both sexes. However, young-adult TaKKO females displayed disturbed LH pulsatility and sex steroid levels, with suppressed basal LH and pre-ovulatory LH surges, early-onset subfertility and premature ovarian insufficiency. Conversely, testicular histology and fertility were grossly conserved in TaKKO males. Ablation of Kiss1 in Tac2-cells led also to sex-dependent alterations in body composition, glucose homeostasis and locomotor activity. Our data document that KNDy-born kisspeptins are dispensable/compensable for puberty in both sexes, but required for maintenance of female gonadotropin pulsatility and fertility, as well as adult metabolic homeostasis. Significance StatementNeurons in the hypothalamic arcuate nucleus (ARC) co-expressing kisspeptins and NKB, named KNDy, have been recently suggested to play a key role in pulsatile secretion of gonadotropins, and hence reproduction. However, the relative contribution of this Kiss1 neuronal-subset, vs. ARC Kiss1-only and NKB-only neurons, as well as other Kiss1 neuronal populations, has not been assessed in physiological settings. We report here findings in a novel mouse-model with elimination of KNDy-born kisspeptins, without altering other kisspeptin compartments. Our data highlights the heterogeneity of ARC Kiss1 populations and document that, while dispensable/compensable for puberty, KNDy-born kisspeptins are required for proper gonadotropin pulsatility and fertility, specifically in females. Characterization of this functional diversity is especially relevant, considering the potential of kisspeptin-based therapies for management of human reproductive disorders. Disclosure StatementThe authors have nothing to disclose in relation to the contents of this work.

physiology↗

bioRxiv2021

Embigin deficiency leads to delayed embryonic lung development and high neonatal mortality

Embigin (gp70), a transmembrane glycoprotein, has been shown to regulate hematopoietic stem cell and progenitor cell niche. Still, little is known about its expression and function in other organ systems during development or adulthood. By combining immunofluorescence, RNA sequencing, and in vivo mouse models, we show that embigin is highly expressed during development and in adult lung, kidney, epididymis, skin, and testis. Adult Emb-/- mice have a normal lifespan and fertility without apparent pathologies. In contrast, the Emb-/- embryos are significantly smaller than their WT littermates. Markedly increased mortality of the Emb-/- embryos is seen especially during the neonatal period. Embigin is present in the placenta, but placental morphology and gene expression patterns stay unaltered. At E17.5, Emb-/- mice show defective morphogenesis of the lung, low alkaline phosphatase activity in amniotic fluid, and remarkable activation of genes involved in cell proliferation in the lungs. Thus, lung underdevelopment explains the high neonatal mortality. Our work demonstrates the crucial role of embigin during development, and it paves the way to further characterization of embigin in specific organ systems in development and homeostasis. Summary statementEmbigin is a basigin-group transmembrane glycoprotein. In vivo mouse model shows that embigin is crucial for embryonic lung development and neonatal survival.

developmental biology↗

bioRxiv2021

Akr1d1-/- mice have a sexually dimorphic metabolic phenotype with reduced fat mass, increased insulin sensitivity and hypertriglyceridemia in males

BackgroundSteroid 5{beta}-reductase (AKR1D1) plays important roles in hepatic glucocorticoid clearance and bile acid synthesis. Glucocorticoids and bile acids are potent metabolic regulators, but whether AKR1D1 controls metabolic phenotype in vivo is unknown. MethodsAkr1d1-/-mice were generated on a C57BL/6 background. Liquid chromatography / mass spectrometry, metabolomic and transcriptomic approaches were used to determine effects on glucocorticoid and bile acid homeostasis. Metabolic phenotypes including body weight and composition, lipid homeostasis, glucose tolerance and insulin sensitivity were evaluated. Molecular changes were assessed by RNASeq and western blotting. Male Akr1d1-/-mice were challenged with a 60% high fat diet. ResultsAkr1d1-/-mice had a sex specific metabolic phenotype. At 30-weeks of age male, but not female, Akr1d1-/-mice were more insulin sensitive and had reduced lipid accumulation in the liver and adipose tissue, concomitant with hypertriglyceridemia and increased intramuscular triacylglycerol. This phenotype was underpinned by sexually dimorphic changes in bile acid metabolism and composition, but without overt effects on glucocorticoid action. Male Akr1d1-/-mice were not protected against diet induced obesity and insulin resistance. ConclusionThis study shows that AKR1D1 controls bile acid homeostasis in vivo and that altering its activity can affect insulin sensitivity and lipid homeostasis in a sex dependent manner.

physiology↗

bioRxiv2020

Imaging mass spectrometry and shotgun proteomics reveal dysregulated pathways in male infertility

Spermatogenesis is a complex multi-step process involving intricate interactions between different cell types in the male testis. Disruption of these interactions results in infertility. Combination of shotgun tissue proteomics with MALDI imaging mass spectrometry is markedly potent in revealing topological maps of molecular processes within tissues. Here, we use a combinatorial approach on a characterized mouse model of hormone induced male infertility to uncover misregulated pathways. Comparative testicular proteome of wildtype and mice overexpressing human P450 aromatase (AROM+) with pathologically increased estrogen levels unravels gross dysregulation of spermatogenesis and emergence of proinflammatory pathways in AROM+ testis. In situ MS allowed us to localize misregulated proteins/peptides to defined regions within the testis. Results suggest that infertility is associated with substantial loss of proteomic heterogeneity, which define distinct stages of seminiferous tubuli in healthy animals. Importantly, considerable loss of mitochondrial factors, proteins associated with late stages of spermatogenesis and steroidogenic factors characterise AROM+ mice. Thus, the novel proteomic approach pinpoints in unprecedented ways the disruption of normal processes in testis and provides a signature for male infertility.

biochemistry↗

bioRxiv2019

Transcriptomic responses to hypoxia in endometrial and decidual stromal cells

Human reproductive success depends on a properly decidualized uterine endometrium that allows implantation and the formation of the placenta. At the core of the decidualization process are endometrial stromal fibroblasts (ESF) that differentiate to decidual stromal cells (DSC). As variations in oxygen levels are functionally relevant in endometrium both upon menstruation and during placentation, we assessed the transcriptomic responses to hypoxia in ESF and DSC. In both cell types hypoxia upregulated genes in classical hypoxia pathways such as glycolysis and the epithelial mesenchymal transition. In DSC hypoxia restored an ESF like transcriptional state for a subset of transcription factors that are known targets of the progesterone receptor, suggesting that hypoxia partially interferes with progesterone signaling. In both cell types hypoxia modified transcription of several inflammatory transcription factors that are known regulators of decidualization, including decreased transcription of STATs and increased transcription of CEBPs. We observed that hypoxia upregulated genes had a significant overlap with genes previously detected to be upregulated in endometriotic stromal cells. Promoter analysis of the genes in this overlap suggested the hypoxia upregulated Jun/Fos and CEBP transcription factors as potential drivers of endometriosis-associated transcription. Using immunohistochemistry we observed increased expression of JUND and CEBPD in endometriosis lesions compared to healthy endometria. Overall the findings suggest that hypoxic stress establishes distinct transcriptional states in ESF and DSC, and that hypoxia influences the expression of genes that contribute to the core gene regulation of endometriotic stromal cells.

physiology↗