Retinoic acid and meiosis induction in adult versus embryonic gonads of medaka

In vertebrates, one of the first recognizable sex differences in embryos is the onset of meiosis, known to be regulated by retinoic acid (RA) in mammals. We investigated in medaka a possible meiotic function of RA during the embryonic sex determination (SD) period and in mature gonads. We found RA mediated transcriptional activation in germ cells of both sexes much earlier than the SD stage, however, no such activity during the critical stages of SD. In adults, expression of the RA metabolizing enzymes indicates sexually dimorphic RA levels. In testis, RA acts directly in Sertoli, Leydig and pre-meiotic germ cells. In ovaries, RA transcriptional activity is highest in meiotic oocytes. Our results show that RA plays an important role in meiosis induction and gametogenesis in adult medaka but contrary to common expectations, not for initiating the first meiosis in female germ cells at the SD stage

Crosstalk Between Retinoic Acid and Sex-Related Genes Controls Germ Cell Fate and Gametogenesis in Medaka

Sex determination (SD) is a highly diverse and complex mechanism. In vertebrates, one of the first morphological differences between the sexes is the timing of initiation of the first meiosis, where its initiation occurs first in female and later in male. Thus, SD is intimately related to the responsiveness of the germ cells to undergo meiosis in a sex-specific manner. In some vertebrates, it has been reported that the timing for meiosis entry would be under control of retinoic acid (RA), through activation of Stra8. In this study, we used a fish model species for sex determination and lacking the stra8 gene, the Japanese medaka (Oryzias latipes), to investigate the connection between RA and the sex determination pathway. Exogenous RA treatments act as a stress factor inhibiting germ cell differentiation probably by activation of dmrt1a and amh. Disruption of the RA degrading enzyme gene cyp26a1 induced precocious meiosis and oogenesis in embryos/hatchlings of female and even some males. Transcriptome analyzes of cyp26a1–/–adult gonads revealed upregulation of genes related to germ cell differentiation and meiosis, in both ovaries and testes. Our findings show that germ cells respond to RA in a stra8 independent model species. The responsiveness to RA is conferred by sex-related genes, restricting its action to the sex differentiation period in both sexes

The Transcriptional Profile of Mesenchymal Stem Cell Populations in Primary Osteoporosis Is Distinct and Shows Overexpression of Osteogenic Inhibitors

Primary osteoporosis is an age-related disease characterized by an imbalance in bone homeostasis. While the resorptive aspect of the disease has been studied intensely, less is known about the anabolic part of the syndrome or presumptive deficiencies in bone regeneration. Multipotent mesenchymal stem cells (MSC) are the primary source of osteogenic regeneration. In the present study we aimed to unravel whether MSC biology is directly involved in the pathophysiology of the disease and therefore performed microarray analyses of hMSC of elderly patients (79-94 years old) suffering from osteoporosis (hMSC-OP). In comparison to age-matched controls we detected profound changes in the transcriptome in hMSC-OP, e.g. enhanced mRNA expression of known osteoporosis-associated genes (LRP5, RUNX2, COL1A1) and of genes involved in osteoclastogenesis (CSF1, PTH1R), but most notably of genes coding for inhibitors of WNT and BMP signaling, such as Sclerostin and MAB21L2. These candidate genes indicate intrinsic deficiencies in self-renewal and differentiation potential in osteoporotic stem cells. We also compared both hMSC-OP and non-osteoporotic hMSC-old of elderly donors to hMSC of similar to 30 years younger donors and found that the transcriptional changes acquired between the sixth and the ninth decade of life differed widely between osteoporotic and non-osteoporotic stem cells. In addition, we compared the osteoporotic transcriptome to long term-cultivated, senescent hMSC and detected some signs for pre-senescence in hMSC-OP. Our results suggest that in primary osteoporosis the transcriptomes of hMSC populations show distinct signatures and little overlap with non-osteoporotic aging, although we detected some hints for senescence-associated changes. While there are remarkable inter-individual variations as expected for polygenetic diseases, we could identify many susceptibility genes for osteoporosis known from genetic studies. We also found new candidates, e.g. MAB21L2, a novel repressor of BMP-induced transcription. Such transcriptional changes may reflect epigenetic changes, which are part of a specific osteoporosis-associated aging process

1,25-Dihydroxyvitamin D3 Treatment Delays Cellular Aging in Human Mesenchymal Stem Cells while Maintaining Their Multipotent Capacity

<div><p>1,25-dihydroxyvitamin D3 (1,25D3) was reported to induce premature organismal aging in <em>fibroblast growth factor-23</em> (<em>Fgf23</em>) and <em>klotho</em> deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC). Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, β-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence- and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to β-galactosidase staining and <em>P16</em> expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105) and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging.</p> </div

Morphological characteristics of 1,25D3 cultured hMSC and ß-galactosidase quantification.

<p><b>A</b> Morphology of hMSC in P1 (upper row) and P3 (lower row) treated with 1,25D3 over three passages compared to control cells. 1,25D3 incubated hMSC lost their typical fibroblast-like features and showed a broadened and extended cellular morphology. Scale bar = 50 µm. <b>B</b> The induction of ß-galactosidase was significantly reduced in 1,25D3 cultured cells compared to control hMSC. Cells from three different donors were used and were cultured up to three passages. The results are shown as mean+SEM of three independent experiments, each normalized to its control. Each time five pictures of ß-galactosidase staining were analyzed using the AutMess tool of AxioVision Rel. 4.6 software. (***, p<0.001, student's t-test).</p

1,25D3 effects on proliferation, apoptosis and cumulative population doublings.

<p><b>A/B</b> Measurement of proliferation capacity and apoptosis induction of hMSC from three different donors after stimulation with 1,25D3 for 24, 48 and 72 h. The proliferation rate was decreased time-dependently after 1,25D3 treatment (gray bars) compared to control cells (black bars), with significant effects after 48 h and 72 h (A). 1,25D3 stimulation showed the strongest reduction of apoptosis rates after 72 h 1,25D3 treatment (gray bars) compared to control cells (black bars, B). The results are shown as mean+SEM of three independent experiments, each normalized to its control and performed in triplicates. Cells from three different donors were used. (*, p<0.05; **, p<0.01;***, p<0.001, student's t-test). <b>C</b> The growth rates of cells were determined by population doublings at each subcultivation. Cumulative population doubling (CPD) was first determined for P2. Population doublings were observed for up to six cell passages. Persistent 1,25D3 supplementation resulted in a reduction of population doublings (gray bar), but the difference was not statistically significant. Independent experiments were performed using hMSC from several human donors. Control: P1, P2, n = 6; P3, P4, n = 5; P5, n = 3; P6, n = 2. 1,25D3: P1, P2, n = 6; P3, P4, n = 4; P5, P6, n = 1. <b>D</b> A representative example of CPDs of cells from one donor. 1,25D3 cultured cells exhibited lower CPDs compared to control hMSC (**, p<0.01, student's t-test). <b>E</b> The time required to reach subconfluence was determined. 1,25D3 treated cells (gray bars) needed more days until they reached subconfluence compared to control hMSC (black bars) (**, p<0.01, student's t-test). Independent experiments were performed using hMSC from several human donors. Control: P1, P2, P3, P4, n = 6; P5, n = 5; P6, n = 3. 1,25D3: P1, P2, P3, n = 6; P4, n = 5; P5, n = 4; P6, n = 1.</p