The Pancreas Is Altered by In Utero Androgen Exposure: Implications for Clinical Conditions Such as Polycystic Ovary Syndrome (PCOS)

Using an ovine model of polycystic ovary syndrome (PCOS), (pregnant ewes injected with testosterone propionate (TP) (100 mg twice weekly) from day (d)62 to d102 of d147 gestation (maternal injection – MI-TP)), we previously reported female offspring with normal glucose tolerance but hyperinsulinemia. We therefore examined insulin signalling and pancreatic morphology in these offspring using quantitative (Q) RT-PCR and western blotting. In addition the fetal pancreatic responses to MI-TP, and androgenic and estrogenic contributions to such responses (direct fetal injection (FI) of TP (20 mg) or diethylstilbestrol (DES) (20 mg) at d62 and d82 gestation) were assessed at d90 gestation. Fetal plasma was assayed for insulin, testosterone and estradiol, pancreatic tissue was cultured, and expression of key β-cell developmental genes was assessed by QRT-PCR. In female d62MI-TP offspring insulin signalling was unaltered but there was a pancreatic phenotype with increased numbers of β-cells (

Effects of CreERT2, 4-OH Tamoxifen, and Gender on CFU-F Assays

Gene function in stem cell maintenance is often tested by inducing deletion via the Cre-loxP system. However, controls for Cre and other variables are frequently not included. Here we show that when cultured in the presence of 4-OH tamoxifen, bone and marrow cells containing the CreERT2 construct have a reduced colony forming ability. Inactive CreERT2 recombinase, however, has the opposite effect. Young female marrow cells containing the inactive CreERT2 construct grew more colonies than cells lacking the construct altogether. Young female control marrow cells (i.e., negative for CreERT2) also produced significantly greater colony numbers when cultured with 4-OH tamoxifen, compared with the ethanol vehicle control. In conclusion, we report that the use of the Cre-loxP system is inadvisable in combination with CFU-F assays, and that appropriate controls should be in place to extend the future use of Cre-loxP in alternate assays

Colony forming abilities are reduced when adult CreER^T2 positive cells are cultured with 4-OH tamoxifen.

<p>(A) Brightfield images of representative colonies stained with Toluidine Blue from male marrow cells cultured under hypoxia. (B) The colony forming assays were performed by culturing male cells from central marrow and enzymatically digested flushed long bone for 10 days following culture in media with 4-OH tamoxifen (1μM) or vehicle control ethanol. Colonies were then stained with Toluidine Blue to assess cartilaginous matrix content. Mean (±SEM) Toluidine Blue positive CFU-F assay colony numbers are reduced following CreER^T2 activation with 4-OH tamoxifen in all Cre⁺ normoxic and hypoxic bone cells and hypoxic marrow cells, compared to culture with ethanol. Mean (±SEM) Toluidine Blue positive CFU-F assay colony numbers are also reduced following CreER^T2 activation with 4-OH tamoxifen in Cre⁺ normoxic bone cells, compared with Cre^- cells cultured with 4-OH tamoxifen. (C) Brightfield images of representative colonies stained with Alkaline Phosphatase from male marrow cells cultured under hypoxia. (D) Colonies were then stained with ALP to assess the degree of bone formation. Mean (±SEM) ALP positive CFU-F assay colony numbers are reduced following CreER^T2 activation with 4-OH tamoxifen in Cre⁺ hypoxic bone cells, compared to culture with ethanol. Mean (±SEM) ALP positive CFU-F assay colony numbers are also reduced following CreER^T2 activation with 4-OH tamoxifen in Cre⁺ normoxic and hypoxic bone cells and hypoxic marrow cells, compared with Cre^- cells cultured with 4-OH tamoxifen. *p<0.05 **p<0.01 ***p<0.001. (Cre^-: n = 3 experiments. Cre⁺: n = 4 experiments. All experiments were performed in technical triplicate). (y axis = mean number of colonies with diameter greater than 1mm).</p

Colony forming abilities are reduced when CreER^T2 positive cells from young mice are cultured with 4-OH tamoxifen. Young female CreER^T2 positive marrow cells form more colonies than CreER^T2 negative cells in the absense of 4-OH tamoxifen, and young female marrow CreER^T2 negative cells cultured with 4-OH tamoxifen give rise to more colonies than when cultured with the ethanol vehicle control.

<p>(A) Brightfield images of representative colonies from female marrow cells cultured under hypoxia (scale bar = 5mm). (B) The colony forming assays were performed by culturing cells from central marrow and enzymatically digested flushed long bone for 10 days following culture in media with 4-OH tamoxifen (1μM) or vehicle control ethanol. Mean (±SEM) CFU-F assay colony numbers are reduced following CreER^T2 activation with 4-OH tamoxifen in all Cre⁺ marrow and bone cells, irrespective of sex or culture conditions, compared to culture with ethanol. Mean (±SEM) CFU-F assay colony numbers are also reduced following CreER^T2 activation with 4-OH tamoxifen in Cre⁺ male marrow and bone cells, and female bone cells, compared to Cre^- culture with 4-OH tamoxifen. Mean (±SEM) CFU-F assay colony numbers are higher from female Cre⁺ marrow cells cultured under both normoxia and hypoxia with ethanol, compared to Cre^- marrow cells with ethanol. Mean (±SEM) CFU-F assay colony numbers are increased in female Cre^- marrow cells cultured with 4-OH tamoxifen compared to ethanol, when cultured under both normoxia and hypoxia. *p<0.05 **p<0.01 ***p<0.001. (n = 3 experiments. All experiments were performed in technical triplicate). (y axis = mean number of colonies with diameter greater than 1mm).</p

Effects of CreER^T2, 4-OH Tamoxifen, and Gender on CFU-F Assays

<div><p>Gene function in stem cell maintenance is often tested by inducing deletion via the Cre-<i>loxP</i> system. However, controls for Cre and other variables are frequently not included. Here we show that when cultured in the presence of 4-OH tamoxifen, bone and marrow cells containing the CreER^T2 construct have a reduced colony forming ability. Inactive CreER^T2 recombinase, however, has the opposite effect. Young female marrow cells containing the inactive CreER^T2 construct grew more colonies than cells lacking the construct altogether. Young female control marrow cells (i.e., negative for CreER^T2) also produced significantly greater colony numbers when cultured with 4-OH tamoxifen, compared with the ethanol vehicle control. In conclusion, we report that the use of the Cre-<i>loxP</i> system is inadvisable in combination with CFU-F assays, and that appropriate controls should be in place to extend the future use of Cre-<i>loxP</i> in alternate assays.</p></div

Colony forming abilities are reduced when adult CreER^T2 positive cells are cultured with 4-OH tamoxifen.

<p>(A) Brightfield images of representative colonies from male bone cells cultured under hypoxia. (B) The colony forming assays were performed by culturing cells from central marrow and enzymatically digested flushed long bone for 10 days following culture in media with 4-OH tamoxifen (1μM) or vehicle control ethanol. Mean (±SEM) CFU-F assay colony numbers are reduced following CreER^T2 activation with 4-OH tamoxifen in all Cre⁺ marrow and bone cells, irrespective of sex or culture conditions, compared to culture with ethanol. Mean (±SEM) CFU-F assay colony numbers are also reduced following CreER^T2 activation with 4-OH tamoxifen in Cre⁺ male marrow and bone cells, female bone cells, and female hypoxic marrow, compared to Cre^- culture with 4-OH tamoxifen. *p<0.05 **p<0.01 ***p<0.001. (Cre^-: n = 3 experiments. Cre⁺: n = 4 experiments. All experiments were performed in technical triplicate). (y axis = mean number of colonies with diameter greater than 1mm).</p

Fetal pancreatic expression of key developmental genes and <i>in vitro</i> insulin secretion is dependent upon fetal sex and timing of exposure to excess androgens.

<p><b>A: </b><b><i>Female fetuses</i></b>: At d90, (vehicle n = 5, MI-TP n = 8), d62 MI-TP significantly increased expression of <i>IGF1R</i> (<i>P</i><0.05), <i>PDX1</i> (<i>P</i><0.01), <i>INSR</i> (<i>P</i><0.05) and <i>INS</i> (<i>P</i><0.05) mRNA. <b><i>Male fetuses</i></b>: Whilst there were differences in expression attributable to the sex of the fetuses (see results section for full description), there were no differences in expression of any genes studied attributable to <i>in utero</i> treatment in male fetuses studied at d90 (vehicle n = 6, MI-TP n = 6) of gestation. Different superscripts denote significant differences (P<0.01–0.05, see results sections for full details).</p

Comparison of direct, fetal administration of TP and DES: effects on pancreatic gene expression, β-cell counts and <i>in vitro</i> insulin secretion.

<p>To delineate androgenic effects of MI-TP administration from potential estrogenic effects attributable to metabolism of applied androgen to estrogen, TP or the estrogen agonist DES were directly applied to female fetuses (FI-TP/FI-DES) and tissue collected at d90 of gestation. Genes whose expression was significantly altered in response to MI-TP (Panel A) were analysed (vehicle n = 6, FI-TP n = 7, FI-DES n = 5), <i>in vitro</i> insulin secretion in response to euglycaemic conditions (5.5 mM glucose) was determined (Panel C) (vehicle n = 5, FI-TP n = 7, FI-DES n = 4). <i>INS</i>, <i>PDX1</i> and <i>INSR</i> were significantly elevated by FI-TP (<i>P</i><0.05), but not FI-DES. As regards <i>IGF1R</i>, there was a trend towards increased expression associated with FI-TP but not FI-DES (<i>P</i> = 0.06). FI-TP, but not FI-DES was associated with elevated insulin secretion <i>in vitro</i> in response to euglycaemic culture conditions as compared to <i>in vivo</i> vehicle treated cultures (<i>P</i><0.05).</p

Ovine fetal pancreatic androgen receptor localization, <i>AR</i> expression and β-cell content.

<p>Panel A: (i) Fetal pancreas showing nuclear AR protein (brown) in multiple cell types including glandular tissue (black arrows) and presumed islets (red arrows). (<b>ii</b>) Fluorescent immunohistochemical staining for insulin (green) in a (vehicle) d90 fetal pancreas. (<b>iii</b>) Fluorescent immunohistochemical staining for AR (red) in a (vehicle) d90 fetal pancreas and (<b>iv</b>) represents a dual merge of insulin (green) and AR (red) confirming that β-cells express AR. (<b>v</b>) and (<b>vi</b>) show negative controls for insulin and androgen receptor respectively. Scale bar is 100 µm (<b>i</b>) and 25 µm (<b>ii–vi</b>). MI-TP had no effect on either <i>AR</i> abundance (n = 5, n = 8, vehicle and TP fetuses respectively) (Panel B) or fetal β-cell numbers (Panel C).</p