Dataset DOI: 10.5061/dryad.bzkh189qm

Description of the data and file structure

Body mass, body composition measurements, and estrous cycle staging are shared in CSV files from control and RERKO mice (Reprimo-specific Esr1 KO). We also share supplementary figures and statistical tables. 

Files and variables

File: Table_1._Statistical_details_of_all_tests.csv (on Zenodo)

Description:  results of statistical tests for each figure in the manuscript.  

File: Supplementary_Figures_and_Legends.docx (on Zenodo)

Supplemental Figure 1: Rprm lineage tracing reveals robust expression in the brain and peripheral tissues. Heterozygous Rprm^Cre mice were bred with Ai14^f/f mice to produce Rprm^Cre;Ai14 ^f/+ mice expressing tdTomato in Rprmlineage tissues. Cre-negative littermates (WT*;Ai14* ^f/+ mice) were used to establish baseline tdTomato levels. (A-C) Representative images of tissues harvested from adult (13-15 weeks old) Rprm^Cre;Ai14 ^f/+ and WT*;Ai14* ^f/+ mice. (A) Left – images highlighting tdTomato expression throughout the whole brain in male mice. Right – 30 µm DAPI-stained coronal brain sections emphasize the expression of tdTomato in the cortex, thalamus, and hypothalamus across representative bregma levels. Scale bars = 1 mm. (B) Peripheral tissues exhibiting tdTomato expression include the uterus, ovary, pancreas, and pituitary in female mice. (C) Peripheral tissues that do not express tdTomato include adipose tissue depots (BAT, iWAT, and gWAT) in female mice. Brightfield images displayed on top, fluorescent images below. Scale bars = 200 µm. BAT = brown adipose tissue, iWAT = inguinal white adipose tissue, gWAT = gonadal white adipose tissue.

Supplemental Figure 2:  Knock-in construct does not alter Rprm expression in the MPO and VMH. Rprm+ cell counts were compared between Rprm^Cre^^ and wildtype C57BL/6J female mice (n=5-9 per group) in the medial preoptic area (MPO) (A) and ventromedial hypothalamus (VMH) (B). Statistical analysis performed using Welch’s t-test.

Supplementary Figure 3. Longitudinal body weight analysis of RERKO mice. (A) Body mass measurements of control (black) and RERKO (blue) male mice from 5 to 16 weeks of age (n = 16 WT, n = 18 RERKO). (B) Body mass measurements of control (black) and RERKO (pink) female mice from 5 to 16 weeks of age (n = 13 WT, n = 13 RERKO). In females, we only detected an effect of age (F_11,238 = 61, p < 0.0001). Mean ± SEM is plotted at each time point. A mixed-effects model using Restricted Maximum Likelihood (REML) estimation was used for statistical analysis due to missing values. Pairwise comparisons at each hourly timepoint were not significant using Šídák's multiple comparisons test.

Supplementary Figure 4. RERKO female mice have disrupted reproduction. (A) Images depict vaginal lavages from WT (left) and RERKO (right) mice stained with Giemsa. While the WT mice display expected cellular morphological changes across the estrous cycle, swabs from RERKO mice mostly contain leukocytes. (B) The percent of days across 14 days spent in diestrus [D], proestrus [P], estrus [E], and metestrus [M] in WT (left) and RERKO (right) mice. C) Dissected ovarian and uterine mass from control (black) and RERKO (gray) mice. (D) Ovarian histology visualized with hematoxylin & eosin stain. Follicular cysts are present and the corpus luteum is absent in the ovary from RERKO mice. CL = corpus luteum, F = ovarian follicles, Cy = cyst. Scales are 100 µm (ovary) and 500 µm (uterus). T-test used to compare groups. The mean ± SEM are depicted. Individual data points are shown in Figure D.  p < 0.05, p < 0.0001.

Supplementary Figure 5. The estrous cycle in controls and mice with Reprimo**+ cells in the MBH ablated. The percent of days across 7 days spent in diestrus [D], proestrus [P], estrus [E], and metestrus [M] in controls expressing GFP (left) or caspase (right). n = 2 GFP, n = 6 caspase.

File: RERKO_longitudinal_bodymass_males_females.csv

First column contains the mouse ID. 

Second column contains the sex of the mouse (Male or female; M or F) and Genotype (control or RERKO; WT or KO). 

Rest of the columns contains body mass (in grams) for each mouse for that week of age (starting from 5 weeks until 16 weeks of age). 

Empty cells indicate measurements were not taken at that time point for these mice. 

File: RERKO_EchoMRI_data.csv

A separate cohort of mice were weighed and placed into an EchoMRI^tm (Houston, TX, USA) to record fat and lean mass at 16 weeks of age.

First column contains "Label" (mouse ID). 

Second column reports fat mass (grams), third column reports lean mass (grams), fourth reports "free water" (grams), and fifth "total water" (grams). Weight of mouse is reported in the sixth column (grams). "Gene" column contains the sex of the mouse (Male or female; M or F) and Genotype (control or RERKO; WT or KO).  Last two columns show percent fat and lean mass per mouse ([depot mass / total body mass] x 100). 

File: RERKO_estrouscycle.csv

For estrous cycling, vaginal lavages were deposited onto slides, stained with Giemsa (0.6% in 1x phosphate buffered saline;) to visualize nucleic acid and distinguish cell morphology, and imaged using light microscopy. The estrous stage was determined by the relative amounts of leukocytes and cornified and nucleated epithelial.

First column contains the mouse ID. Rest of the columns estrous stage calls for each day for 14 consecutive days. D = diestrus, P = proestrus, E = estrus, M = metestrus.