EKAREV–NLS Biosensor Influences Mammary Gland Morphology and Reproductive Fitness

Given the absence of prior reports on the use of the EKAREV-NLS (hereafter referred to as “EKAREV”) strain in mammary gland research, we aimed to characterize and assess its suitability for studying ERK activity dynamics in mammary epithelial morphogenesis. To this end, we investigated mammary gland morphology in EKAREV positive (EKAREV+, hemizygous) and EKAREV negative (EKAREV−, wild-type) nulliparous female mice. At 10 weeks of age, all analyzed EKAREV+ mice showed significant alterations in epithelial morphology, characterized by markedly reduced epithelial outgrowth and a simplified branching pattern (Fig. 1a–c). Further examination of the mammary glands at later time points (10 to 19 weeks of age) revealed conservation of the impeded epithelial outgrowth and branching phenotype in the EKAREV+ mice, with occasionally increased fat pad penetration (Supplementary Fig. 1). Moreover, EKAREV+ females were smaller and had significantly lower body weight than the EKAREV− females (Fig. 1d, e), low visceral adipose tissue deposits and poor coat quality, which together with the mammary phenotype suggested a systemic effect.

Fig. 1

EKAREV+ female mice exhibit retarded mammary epithelial outgrowth, lower body weight, and reduced reproductive fitness. (a) Carmine-stained mammary glands of EKAREV+ and EKAREV− 10-week-old mice. The white dashed line outlines the epithelial ductal tree area while the insert shows part of the EKAREV+ epithelial tree structure; FP, fat pad; LN, lymph node. Scale bar: 1 mm. (b and c) Quantification of mammary epithelial morphological descriptors. The plots show the number of branching points (sum of primary and secondary) (b) and the length of epithelial outgrowth from the center of the lymph node to the distal part of the gland (c) as mean ± SD; N = 3 EKAREV+, 3 EKAREV−; **p ≤ 0.01, ***p ≤ 0.001 (Student’s t-test). (d) Macroscopic comparison of 10-week-old nulliparous EKAREV+ (left) and EKAREV− (right) female mice. Scale bar: 1 cm. (e) Post-mortem whole-body weights of EKAREV+ and EKAREV− female mice. The plot shows mean ± SD; N = 3 EKAREV+, 3 EKAREV−; ***p ≤ 0.001 (Student’s t-test). (f) Hematoxylin- and eosin-stained sections from ovaries of EKAREV+ and EKAREV− 10-weeks-old females. Scale bar: 100 μm. (g) Quantification of the number of corpora lutea, counted in serial sections of ovaries (taken every 25 μm within the first half of the ovary). The plot shows mean ± SD; N = 3 EKAREV+, 3 EKAREV−; ns p > 0.05 (Student’s t-test). (h, i) Level of reproductive hormones (h) estrogen, and (i) progesterone in circulating blood serum. The plots show mean ± SD. N = 3 EKAREV+, 3 EKAREV−; ns p > 0.05, ***p ≤ 0.001 (Student’s t-test). (j) The plot shows the total number of pups born from the breeding of EKAREV+ or EKAREV− females with wild-type ICR male as mean ± SD; n = 3 litters from EKAREV+ mother, 3 litters from EKAREV− mother; ns p > 0.05 (Student’s t-test). (k, l) Weight of offspring from panel (j). The plots show body weight of 3-week-old (k) and 6-week-old (l) female (pink) and male (blue) pups as mean ± SD; N = 17 born pups of 3 EKAREV+ and 3 EKAREV− mothers; ns p ≥ 0.05, **p ≤ 0.01 (Kruskal-Wallis test for 3 w females, Mann-Whitney test for 6 w females, Student’s t-test for both 3 w and 6 w males)

Because the phenotype of EKAREV+ mammary glands resembled the mammary gland of mice ovariectomized before puberty [20], we suspected that it could be caused by dysregulation of female sex hormones. Therefore, we measured estrogen and progesterone levels in blood serum and examined ovaries and estrous cycle in the EKAREV mice. Interestingly, the level of freely circulating estrogen was significantly higher in EKAREV+ animals compared to the EKAREV− control, and a similar trend was apparent in progesterone levels (Fig. 1f, g). The ovaries of the majority (approx. 90%) of EKAREV+ mice were significantly smaller, with a reduced number of late-stage follicles and mostly undetectable corpora lutea, indicating disrupted estrous cycles and impaired ovulation (Fig. 1h, i; Supplementary Fig. 2). Moreover, when bred with wild-type males, EKAREV+ females frequently failed to produce offspring (Supplementary Fig. 3b). When EKAREV+ mothers produced offspring, the litters were smaller, with reduced viability and lower body weight despite normal maternal behavior of EKAREV+ mice (Fig. 1j). EKAREV+ offspring showed the lowest body weight (Fig. 1k, l), incomplete fur development, and poor viability. Further, EKAREV+ mothers showed delayed involution following the weaning of the pups at 3 weeks of age (Supplementary Fig. 3d). Collectively, this data suggests a profound disruption of female sex hormone signaling, which negatively impacts mammary gland development, follicular maturation, and reproductive capability.

Estradiol Supplementation Results in Partial Mammary Gland Phenotype Rescue

Given the well-established roles of estrogen and progesterone in mammary gland development and female reproductive system regulation [20,21,22], we hypothesized that supplementing these hormones might stimulate reproductive system function and mammary epithelial morphogenesis in EKAREV+ mice. We implemented a hormonal supplementation regimen via drinking water, as described in a previous study [23], with non-treated, estradiol-only, and combined estradiol-progesterone groups. Analysis of mammary gland morphology revealed a significant increase in epithelial penetration and branching index in both estradiol-only and estradiol-progesterone treated groups compared to non-treated EKAREV+ mice, indicating a positive impact of the hormonal treatment on epithelial tree development and mammary gland morphology (Fig. 2a–c). Moreover, although epithelial branching and penetration improved in both treatment groups, the mammary branching complexity of the hormone-treated EKAREV+ mice did not reach the branching complexity of the EKAREV− mammary glands (Fig. 2a,b), suggesting only a partial rescue of the phenotype. The dual supplementation group showed an increased number of terminal end buds (TEB) at the time of sample collection, suggesting a greater growth potential in this group (Fig. 2d). Hormonal treatment of EKAREV− control animals had no significant impact on mammary gland morphology, suggesting a preserved signaling regulation in these animals (Supplementary Fig. 4).

Fig. 2

Hormonal supplementation rescues the mammary gland phenotype of the EKAREV+ females. (a) Carmine-stained mammary glands of 14-week-old female mice after 5 weeks of hormonal supplementation. Scale bar: 1 mm. The inserts show details of the distal ends of the epithelial ductal tree. Scale bar: 500 μm. (b, c) Quantification of mammary epithelial morphological descriptors. The plots show the number of branching points (sum of primary and secondary) (b) and the length of epithelial outgrowth from the center of the lymph node to the distal part of the gland (c) as mean ± SD. N = 10 EKAREV+ NT, 10 EKAREV+ E2, 11 EKAREV+ E2P4, 17 EKAREV− NT; * p ≤ 0.05, **** p ≤ 0.0001 (Kruskal-Wallis test in (b) one-way ANOVA in (c). (d) The plot shows the number of TEBs as mean ± SD. N = 10 EKAREV+ E2, 11 EKAREV+ E2P4; **p ≤ 0.01 (Mann-Whitney test). (e) Estrogen receptor (ER), progesterone receptor (PR), and Ki67 expression in mammary gland epithelium of 14-week-old female mice after 5 weeks of hormonal supplementation. The inserts show details of epithelial areas. Scale bar: 100 μm. (f) Quantification of ER+ cell proportion in mammary epithelium. The plots show mean ± SD. N = 17 EKAREV+ NT, 21 EKAREV+ E2, 28 EKAREV+ E2P4, 13 EKAREV− NT; ns p > 0.05 (Student’s t-test). (g) Quantification of PR+ cell proportion in mammary epithelium. The plots show mean ± SD. N = 9 EKAREV+ NT, 8 EKAREV+ E2, 9 EKAREV+ E2P4, 10 EKAREV− NT; ***p ≤ 0.001 (one-way ANOVA). (h) Quantification of Ki67+ cell proportion in mammary epithelium. The plots show mean ± SD. N = 10 EKAREV+ NT, 9 EKAREV+ E2, 22 EKAREV+ E2P4, 12 EKAREV− NT; *** p ≤ 0.001 (Student’s t-test). (i) Hematoxylin- and eosin-stained sections of ovaries from 14-week-old females after 5 weeks of hormonal supplementation. Scale bar: 100 μm. (j) Quantification of a number of corpora lutea, counted in serial sections of ovaries (taken every 25 μm within the first half of the ovary). The plot shows mean ± SD. N = 8 EKAREV+ NT, 7 EKAREV+ E2, 11 EKAREV+ E2P4, 9 EKAREV− NT; ** p ≤ 0.01, *** p ≤ 0.001 (Kruskal-Wallis test). (k) The plot shows whole-body weights of hormone-supplemented females as mean ± SD. N = 10 EKAREV+ NT, 10 EKAREV+ E2, 11 EKAREV+ E2P4, 17 EKAREV− NT; * p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (Kruskal-Wallis test). (l) Estrous cycle stage distribution in the cohort of hormone-supplemented females at the time of mammary and ovarian tissue collection. NT, non-treated; E2, 17β–estradiol-treated; E2P4, 17β–estradiol- and progesterone-treated; TEB, terminal end bud; PR, progesterone receptor

To investigate the underlying mechanism of EKAREV+ phenotype and its rescue by hormonal supplementation, we investigated estrogen receptor (ER) expression in mammary epithelium by immunohistochemistry. Interestingly, we found no significant differences in ER expression levels between EKAREV+ non-treated animals, EKAREV+ hormone-treated and EKAREV− non-treated (control) animals (Fig. 2e, f). In pubertal and adult mice, estradiol signaling upregulates progesterone receptor (PR) expression, whose signaling exerts proliferative effects on mammary epithelium [24]. To determine whether this signaling axis was affected, we assessed PR and Ki67 expression. Importantly, we found significantly decreased expression of PR in EKAREV+ non-treated animals (Fig. 2e, g). Hormonal supplementation increased PR levels in both treatment groups (Fig. 2e, g) to levels comparable to the EKAREV− mice and thus partially restoring the signaling regulation necessary for normal epithelial development. Similarly, an increased proportion of proliferative (Ki67+) cells was detected in both treatment groups compared to EKAREV+ non-treated animals (Fig. 2e, h), indicating a restored PR signaling network in mammary epithelium.

Estradiol and progesterone, alongside gonadotropins, play crucial roles in ovarian follicle development, maturation, and ovulation [25]. We hypothesized that hormonal supplementation might enhance follicle content, which would further confirm the restoration of the regulatory axis. As expected, hormonal supplementation increased ovary size and the number of late-stage follicles (Fig. 2i, j; Supplementary Fig. 5) in both groups. However, unlike the dual estradiol-progesterone group, estradiol-only supplementation did not result in detectable corpus luteum formation in EKAREV+ mice, suggesting that a more robust stimulus is required to induce successful follicle maturation and/or ovulatory signals. Hormonally supplemented mice exhibited normal behavior and showed a trend of increased body weight (Fig. 2k). Furthermore, increased visceral fat deposits as well as improved coat quality were observed in both treatment groups. The end-point estrous cycle staging showed improved stage distribution more similar to the EKAREV− mice (Fig. 2l).

Change of Genetic Background Alleviates Biosensor Effect on Mammary Gland Morphology and Reproductive Fitness

Given that our 5-week hormonal supplementation experiments did not yield mammary epithelial three with full branching and fat pad penetration and have therefore only partially rescued the mammary gland phenotype, we sought to explore an alternative approach. Noting the reported significant effect of genetic background on mammary gland development and tumorigenesis [26, 27], we questioned whether the observed phenotype was genetic background dependent. To investigate this, we outcrossed EKAREV+ C57BL/6J mice with wild-type ICR mice (Fig. 3a), an outbred strain known for its robust reproductive performance and rapid growth [28]. We then analyzed overall fitness, mammary gland morphology, ovarian follicle content, and reproductive performance. All analyzed outcrossed C57BL/6J×ICR 14-week-old EKAREV+ offspring showed no signs of compromised well-being, as they maintained good fur quality and showed no reduction in visceral body fat. However, they did have somewhat lower body weights compared to their EKAREV− littermates (Fig. 3b, c). The end-point estrous staging revealed similarities in the stage proportions between the groups (Fig. 3d).

Fig. 3

Change of genetic background rescues the mammary gland phenotype of EKAREV+ females. (a) Schematic representation of outcross experiment and subsequent breeding experiment to assess reproductive fitness. Outcross was achieved by mating male EKAREV+ C57BL/6J with wild-type ICR female. Female offspring was then used in a breeding experiment when it was bred at 14 weeks of age with 20-week-old ICR males. Their offspring was analyzed at 3 and 6 weeks of age. (b) Macroscopic comparison of 10-week-old outcrossed (C57BL/6J×ICR) EKAREV+ (left) and EKAREV− (middle) females to EKAREV− ICR (right) female. Scale bar: 1 cm. (c) The plots show whole-body weights of outcrossed EKAREV+ and EKAREV− 14-week-old females shown as mean ± SD. N = 13 EKAREV+, 14 EKAREV−; **** p ≤ 0.0001 (Student’s t-test). (d) Estrous cycle stage distribution in the cohort of outcrossed EKAREV+ and EKAREV− females at the time of mammary and ovarian tissue collection at 14 weeks of age. (e) Carmine-stained mammary glands of outcrossed 14-week-old EKAREV+ and EKAREV− mice. Scale bar 1 mm. (f, g) Quantification of mammary epithelial morphological descriptors. The plots show the number of branching points (sum of primary and secondary) (f) and the length of epithelial outgrowth (measured from the center of the lymph node to the distal part of the gland) (g) as mean ± SD. N = 13 EKAREV+, 14 EKAREV−; ns p ≥ 0.05, * p ≤ 0.05 (Student’s t-test). (h) Estrogen receptor (ER), progesterone receptor (PR), and Ki67 expression in the mammary glands of 14-week-old outcrossed EKAREV+ and EKAREV− females. The inserts show details of epithelial areas. Scale bar: 100 μm. (i) Quantification of ER+ cells in mammary epithelium. The plot shows mean ± SD. N = 20 EKAREV+, 28 EKAREV−; ns p > 0.05 (Student’s t-test). (j) Quantification of PR+ cells in mammary epithelium. The plot shows mean ± SD. N = 25 EKAREV+, 32 EKAREV−; * p ≤ 0.05 (Student’s t-test). (k) Quantification of Ki67+ cells in mammary epithelium. The plot shows mean ± SD. N = 9 EKAREV+, 24 EKAREV−; *** p ≤ 0.001 (Student’s t-test). (l, m) Level of reproductive hormones (l) estrogen and (m) progesterone in circulating blood serum. The plots show mean ± SD. N = 5 EKAREV+, 7 EKAREV−; ns p > 0.05 (Student’s t-test). (n) Hematoxylin- and eosin-stained sections of ovaries from 14-week-old outcrossed EKAREV + and EKAREV− mice. Scale bar: 100 μm. (o) Quantification of the number of corpora lutea, counted in serial sections of ovaries (taken every 25 μm within the first half of the ovary). The plot shows mean ± SD. N = 13 EKAREV+, 14 EKAREV−; * p ≤ 0.05 (Mann-Whitney test). (p) A total number of pups born from the breeding of outcrossed EKAREV+ or EKAREV− females with wild-type ICR male, shown as mean ± SD. N = 7 litters from EKAREV + mothers, 5 litters from EKAREV- mothers; *p ≤ 0.05 (Student’s t-test). (q, r) Weight of offspring born from the breeding of outcrossed EKAREV+ or EKAREV− females with wild-type ICR male. The plots show the body weight of 3-week-old (q) and 6-week-old (r) female (pink) and male (blue) pups as mean ± SD. N = 107 born pups from 7 EKAREV+ and 5 EKAREV− mothers. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ****p ≤ 0.0001 (one-way ANOVA for 3 w females and 6 w males, Kruskal-Wallis test for 3 w males and 6 w females)

Strikingly, we observed no differences in the appearance of the epithelial ductal tree in the outcrossed EKAREV+ and EKAREV− mice (Fig. 3e). Both groups displayed fully developed epithelial structures with well-formed primary and secondary branches extending into the distal mammary fat pad (Fig. 3f, g). Moreover, both EKAREV+ and EKAREV− outcrossed mice exhibited similar ER, PR, and Ki67 expression patterns, with a high percentage of receptor expressing and proliferative cells in the mammary epithelial compartment (Fig. 3h, k) and similar estrogen and progesterone levels (Fig. 3l-m). Aside from a slight reduction in ovary size in EKAREV+ outcrossed females, no significant differences in follicle counts were observed (Fig. 3n; Supplementary Fig. 6). Both EKAREV+ and EKAREV− outcrossed mice showed evidence of proper hormonal cycling and ovulation, as indicated by the presence of corpora lutea and a substantial number of late-stage follicles (Fig. 3o). When EKAREV+ and EKAREV− outcrossed mice were bred with ICR males, no significant differences were found in litter sex ratio or viability, but the EKAREV+ females produced fewer pups (Fig. 3p-r; Supplementary Fig. 7b, c). Pups from EKAREV+ mothers exhibited, in general, lower body weights than pups from EKAREV− mothers, and in particular EKAREV+ pups were lighter than their EKAREV− littermates. Nevertheless, none of the pups showed the developmental issues observed in EKAREV+ pups from C57BL/6J mothers. Both EKAREV+ and EKAREV− mothers showed normally involuting glands following the weaning of the pups (Supplementary Fig. 7d). These data suggest that the change of genetic background fully rescues mammary phenotype and restores reproductive system functionality in EKAREV+ mice.

Biosensor Function is Maintained Following Hormone Treatment and Background Change

Our results suggest that to use the EKAREV-NLS biosensor to study ERK signaling dynamics in mammary epithelial tissue, the EKAREV+ C57BL/6J mice need to be supplemented by female sex hormones or outcrossed to a mixed C57BL/6J×ICR background to develop a properly branched mammary epithelial ductal structure. To determine whether hormonal supplementation or change of genetic background influence biosensor function in the target tissue, we set out to compare ERK activity patterns in primary mammary epithelial cells (MECs) isolated from non-treated, hormone-supplemented, and outcrossed mice.

First, we assessed overall EKAREV-NLS biosensor expression in primary MECs cultured in 2D. We observed stable biosensor expression in all nuclei in the cells from all experimental animals throughout the imaging period (Fig. 4c). Immunofluorescence staining analysis revealed that the cultured MECs predominantly comprised luminal cells (KRT8+), with less than 1% MECs positively staining for basal cell marker (KRT5+) (Fig. 4c). Thus, further analysis was conducted cumulatively without segregating MEC types.

Fig. 4

Primary mammary epithelial cells (MECs) obtained from hormonally supplemented and outcrossed EKAREV+ mice exhibit distinct signaling responses. (a) Schematic representation of the working principle of the EKAREV-NLS biosensor. The biosensor is located in the nucleus and senses ERK phosphorylation activity. Both reporter fluorescent proteins are detected and FRET to donor ratio is calculated resulting in FRET index values over time. (b) Schematic representation of ERK activation wave analysis within individual cells. The FRET index was computed for the baseline and FGF2 response time points. For each wave a fitted sine curve was computed and descriptive wave parameters (basal activity, wave amplitude, wave duration, and pulse frequency) were extracted. (c) Representative images of primary MECs with donor (CFP) and FRET (YFP) channels at the first time point, with corresponding endpoint detection of basal (KRT5) and luminal (KRT8) lineage markers. Scale bar: 100 μm. (d) Population averages of ERK activity dynamics in response to stimulation with 2.5 nM FGF2, shown as FRET index mean ± SD. The gray line at t = 0 marks the point of FGF2 addition, the area left of the line represents baseline measurement, and the area right of the line represents the response to FGF2 addition. N = 449 for C57BL/6J NT, 391 for C57BL/6J E2, 626 for C57BL/6J E2P4, 484 for C57BL/6J×ICR NT. (e) Heatmaps of 50 representative single-cell tracks per experimental variant from panel B. Each row corresponds to a time series of a single cell, the gray line at t = 0 marks the point of FGF2 addition. (f–i) ERK activation wave analysis, including ERK basal activity (f), pulse frequency (g), wave maximal amplitude (h), and wave maximal duration (i) before (up) and after (down) FGF2 addition. The plots show FRET index mean ± SD; ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (Kruskal-Wallis test). NT, non-treated; E2, 17β–estradiol treated; E2P4, 17β–estradiol and progesterone treated; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; FRET, Förster resonance energy transfer; FGF2, fibroblast growth factor 2; C×I, C57BL/6J×ICR

To analyze ERK activity patterns across different groups, we conducted time-lapse imaging of the EKAREV+ MECs for 6 h. For the first hour, the MECs were imaged in a medium without growth factor supplementation to establish baseline ERK activity. Afterwards, fibroblast growth factor 2 (FGF2) was added, and the MECs were imaged for an additional 5 h. The images were processed, segmented, and tracked, and the FRET index was calculated for each individual cell trajectory (Fig. 4b, Supplementary Video 1–4). Individual cell trajectories as well as population averages of ERK activity showed a similar activation pattern in response to FGF2 stimulation across the groups, with most cells reaching maximal activation 20 min after FGF2 addition and returning to baseline activity within 90 min. In contrast, cells from E2-treated mice responded more rapidly, with maximal activity at 10 min, and after the initial peak, they dropped to a slightly higher baseline activity (Fig. 4d, e).

Next, we explored ERK activation patterns in individual cells by single-cell ERK activation wave analysis (Fig. 4b, f-i), assessing basal activity, pulse frequency, wave amplitude, and wave duration before and after FGF2 addition. We found significant differences in basal ERK activity across all groups before (Fig. 4f, top) and after FGF2 addition (Fig. 4f, bottom). ERK activation pulse frequency showed no significant differences between control (C57BL/6J, NT) and hormonally supplemented groups before (Fig. 4g, top) or after (Fig. 4g, bottom) FGF2 addition, but differed significantly between control cells and cells from outcrossed mice. Maximal wave amplitude and duration, key characteristics relevant to cell fate decisions [29, 30], showed slight to no significant differences between groups before FGF2 addition (Fig. 4h, i, top). However, after FGF2 addition, more significant changes in both amplitude and duration were detected in cells from E2P4-treated mice, and in wave duration in cells from outcrossed mice (Fig. 4h, i, bottom), indicating a potential impact on ERK response and possibly changed cellular behavior. Assessment of intervention effects indicated a higher level of intervention-induced variability following the FGF2 addition, especially between C57BL/6J NT and C57BL/6J E2P4, C57BL/6J NT and C57BL/6J×ICR NT, and C57BL/6J E2 or E2P4 and C57BL/6J×ICR NT (Supplementary Fig. 8). Together, these findings demonstrate EKAREV-NLS biosensor functionality but altered MEC responses to external stimuli conveyed via ERK between non-treated, hormone-supplemented and outcrossed mice.