Use of a prolactin-Cre/ROSA-YFP transgenic mouse provides no evidence for lactotroph transdifferentiation after weaning, or increase in lactotroph/somatotroph proportion in lactation

Abstract

In rats, a shift from somatotroph dominance to lactotroph dominance during pregnancy and lactation is well reported. Somatotroph to lactotroph transdifferentiation and increased lactotroph mitotic activity are believed to account for this and associated pituitary hypertrophy. A combination of cell death and transdifferentiation away from the lactotroph phenotype has been reported to restore non-pregnant pituitary proportions after weaning. To attempt to confirm that a similar process occurs in mice, we generated and used a transgenic reporter mouse model (prolactin (PRL)-Cre/ROSA26-expression of yellow fluorescent protein (EYFP)) in which PRL promoter activity at any time resulted in permanent, stable, and highly specific EYFP. Triple immunochemistry for GH, PRL, and EYFP was used to quantify EYFP+ve, PRL-ve, and GH+ve cell populations during pregnancy and lactation, and for up to 3 weeks after weaning, and concurrent changes in cell size were estimated. At all stages, the EYFP reporter was expressed in 80% of the lactotrophs, but in fewer than 1% of other pituitary cell types, indicating that transdifferentiation from those lactotrophs where reporter expression was activated is extremely rare. Contrary to expectations, no increase in the lactotroph/somatotroph ratio was seen during pregnancy and lactation, whether assessed by immunochemistry for the reporter or PRL: findings confirmed by PRL immunochemistry in non-transgenic mice. Mammosomatotrophs were rarely encountered at the age group studied. Individual EYFP+ve cell volumes increased significantly by mid-lactation compared with virgin animals. This, in combination with a modest and non-cell type-specific estrogen-induced increase in mitotic activity, could account for pregnancy-induced changes in overall pituitary size.

Figure 1

Examples of monodispersed pituitary cells from PRL-Cre/ROSA26-EYFP mice double immunostained for prolactin (panel A) and EYFP (panel B) with superimposed images of the same field in panel C. It can be seen that with few exceptions (arrows with open heads), the EYFP+ve cells are also positive for prolactin, but several prolactin+ve cells are negative for EYFP (examples of which are shown by the arrows with solid heads). Panels D and E show the sample field of cells triple immunostained for prolactin (panel D), GH (panel E), and EYFP (not shown), with panels D and E merged in panel F. The entirely distinct cell populations shown illustrate the rarity of mammosomatotrophs. Panels G, H, and I show the same field triple immunostained for EYFP, GH, and prolactin, with panel J showing merged fields with occasional prolactin to GH transdifferentiated (i.e. EYFP+ve and GH+ve but prolactin−ve) cells (arrowed).

Figure 2

Change in pituitary wet weights of virgin 12-week-old PRL-Cre/ROSA26-EYFP mice at term, at the end of the first, second, and third weeks of lactation, and one week, two weeks, and three weeks after weaning. For each group, n=8. Means and s.e.m. are shown. *P<0·05, **P<0·01, and ***P<0·001 compared with virgin controls (panel A). Pituitary weights increased from virgin to term by 25%, from 1·82±0·057 to 2·28±0·12 mg. Changes in the proportion of lactotrophs assessed in single cell dispersals of pituitary glands from virgin C57BL/6 and PRL-Cre/ROSA26-EYFP mice at the same time points are shown in panel B (n=16 for each time point) using double (PRL and EGFP) and triple (PRL, GH, and EGFP) immunochemistry reactions. Panel C shows concurrent changes in the proportion of GH-positive cells and transdifferentiating cells (i.e. EYFP and GH+ve, and prolactin negative). Means and s.e.m. are shown. *P<0·05, **P<0·01, and ***P<0·001 compared with virgin animals. With the exception of the significant drop in number of prolactin-immunopositive cells between term and the end of the first week of lactation (P<0·05) (panel B), all the significance markers refer to comparisons with virgin animals.

Figure 3

Changes in average size of EYFP+ve and EYFP−ve pituitary cells (solid lines and dashed lines in panel A respectively) derived from virgin and lactating PRL-Cre/ROSA26-EYFP mice using FACS (panels A and B). The increase in the size of the EYFP+ve population is shown by the open arrow (panel B). Panel C shows changes in mean forward scatter in comparison to a set of six polystyrene bead standards (open square symbols) plotted as cross-sectional areas. The extrapolated (dashed) line was used to derive approximations of changes in cross-sectional areas of EYFP+ve and EYFP−ve cells from virgin and end of first week of lactation PRL-Cre/ROSA26-EYFP mice (open circles). Differences in refractive index and resistance to deformation between pituitary cells and beads, and the effects of variable cytoplasm to nuclear ratio as well as characteristics of the laser significantly limit derivation of quantitative information. Despite these caveats, qualitatively, the mean cell volume of the EYFP+ve lactotroph population unequivocally increased from virgin to end of first week of lactation compared with the EYFP−ve population, which remained relatively unchanged in size.

Figure 4

Panel A shows the rapid decline in circulating estrogen levels in PRL-Cre/ROSA26-EYFP males after 15 days of pre-treatment with s.c. injections of 100 μg 17β-estradiol dissolved in DMSO and sesame oil, or vehicle every third day (n=2–3). Corresponding body weight changes are shown in panel B (mean±s.e.m. of the mean), and pituitary weight changes are shown in panel C. Significance markers are relative to pituitary wet weights at time zero. n=at least 5 for all groups.

Figure 5

Changes in PRL-Cre/ROSA26-EYFP male mouse prolactin+ve cells (panel A), EYFP+ve cells (panel B), GH+ve cells (panel C), and transdifferentiating cells (i.e. EYFP+ve, GH+ve, and prolactin−ve: panel D) in response to estrogen withdrawal after high-dose estrogen exposure for 15 days (100 μg 17β-estradiol dissolved in DMSO and sesame oil, or vehicle every third day). Means±s.e.m. are shown. *P<0·05, **P<0·01, and ***P<0·001: n=8 throughout. The data are summarized in panel E, where the control and vehicle-only groups are combined, and the means of cell populations during the first and second weeks after estrogen withdrawal are also combined. Transdifferentiating cells are shown as a percentage of total cells, and as a percentage of EYFP+ve cells. Panel F shows a further summary in which the mean proportions of EYFP+ve, prolactin+ve, GH+ve, and transdifferentiating cells after estrogen treatment from all time points are combined. *P<0·05, **P<0·01, and ***P<0·001.