Intramammary rapamycin administration to calves induces epithelial stem cell self-renewal and latent cell proliferation and milk protein expression

Anna Kosenko; Shamay Jacoby; Tomer-Meir Salame; Maya Ross; Itamar Barash

doi:10.1371/journal.pone.0269505

. 2022 Jun 22;17(6):e0269505. doi: 10.1371/journal.pone.0269505

Intramammary rapamycin administration to calves induces epithelial stem cell self-renewal and latent cell proliferation and milk protein expression

Anna Kosenko ^1,², Shamay Jacoby ¹, Tomer-Meir Salame ³, Maya Ross ¹, Itamar Barash ^1,^*

Editor: Sina Safayi⁴

PMCID: PMC9216576 PMID: 35731738

Abstract

Mammary epithelial stem cells differentiate to create the basal and luminal layers of the gland. Inducing the number of differentiating bovine mammary stem cells may provide compensating populations for the milk-producing cells that die during lactation. Inhibition of mTOR activity by rapamycin signals self-renewal of intestinal stem cells, with similar consequences in the mouse mammary gland and in bovine mammary implants maintained in mice. The implementation of these results in farm animals for better mammary development and production was studied in 3-month-old calves. mTOR activity decreased by ~50% in mammary epithelial cells subjected to 3-week rapamycin administration, with no negative consequences on mammary morphology or β-casein expression. Subsequently, stem cell self-renewal was induced, reflected by a higher propagation rate of cultures from rapamycin-treated glands compared to respective controls and higher expression of selected markers. Followed by 4-day estrogen and progesterone administration, rapamycin significantly induced proliferation rate. Higher numbers of basal and luminal PCNA⁺ cells were detected in small ducts near the elongating sites as compared to large ducts, in which only luminal cells were affected. Rapamycin administration resulted in induction of individual milk protein genes’ expression, which was negatively correlated to their endogenous levels. The inductive effect of rapamycin on luminal cell number was confirmed in organoid cultures, but milk protein expression decreased, probably due to lack of oscillation in rapamycin levels. In conclusion, intramammary rapamycin administration is an effective methodology to reduce mTOR activity in bovine mammary epithelial cells and consequently, induce stem cell self-renewal. The latent positive effect of rapamycin on epithelial cell proliferation and its potential to improve milk protein expression in calves may have beneficial implications for mature cows.

Introduction

Two major mammary characteristics: milk secretion and susceptibility to breast cancer, have paved the way for separate research avenues into improved farm animal production and human health, respectively. Interestingly, both involve a common effector—the mammary epithelial stem cell at the top of the mammary cell hierarchy. Despite a lack of definite morphological identification and debated multipotency of the entire stem cell population in the postpartum mouse mammary gland [1–4], which has not been confirmed in the human breast [5], these cells undoubtedly give rise to the basal and luminal layers that compose the functional mammary epithelium. They also renew the mammary gland between consecutive lactation periods.

Notable progress has been achieved in mouse mammary stem cell characterization since breakthrough studies were published, demonstrating reconstitution of a functional mammary gland by transplantation of a single or several stem cells into the de-epithelized mammary stroma [6, 7]. Better separation from progenitors has been performed [8], new effectors, markers and subpopulations have been identified [Reviewed by [4, 9]] and most importantly, unipotent luminal and basal stem cell populations with self-renewing potency have been identified in the postpartum mammary gland [1]. Together with their bipotent counterparts, these cells maintain the morphogenesis and homeostasis of the adult gland [2].

In contrast, only modest progress has been achieved in bovine mammary stem cell studies. This understudied population has been enriched by flow cytometry [10] and the cells’ differentiation status has been followed with identified markers [11], but not by lineage tracing. It is only very recently that these stem cells’ ability to generate a representative bovine mammary morphology has been demonstrated in bovine stroma pre-transplanted into the mammary stroma of immunocompromised mice [12].

Focusing on improved bovine milk synthesis, it has been suggested that the mammary gland’s ability to replace the differentiated milk-producing luminal cells that die during lactation is key to maintaining persistent milk production [13]. Accordingly, a higher number of mammary epithelial stem cells that continuously differentiate into milk-producing luminal cells and their basal myoepithelial counterparts may provide a replacement for the apoptizing milk-producing cells, thus moderating the decrease in milk production.

An initial attempt was made to induce bovine mammary epithelial stem cell number in 3-month-old calves by intramammary xanthosine infusion for 5 consecutive days [14]. An elevated number of retained BrdU-labeled epithelial cells and induced telomerase activity were noted. Later, induced expression of stem cell gene markers by xanthosine was also observed in goats [15]. Nevertheless, xanthosine’s self-renewing effect could not be reproduced in stem cells of transplanted bovine parenchyma implants, which were analyzed for BrdU-cell retention and by flow cytometry [16]. Moreover, xanthosine administration suppressed bovine mammary cell proliferation by 50%, due to inhibition of inosine-5′-monophosphate dehydrogenase activity, a rate-limiting enzyme in guanine synthesis.

Calorie restriction (CR) induced self-renewal of intestinal mouse stem cells [17]. These cells do not directly sense the reduced caloric intake [18]; the respective self-renewing signal is delivered by paracrine cADP ribose secreted from neighboring Paneth cells that are subjected to decreased mammalian/mechanistic target of rapamycin (mTOR) activity. Pharmacological inhibition of mTOR activity by daily rapamycin injections reproduced the inducing effect of CR on intestinal stem cell self-renewal [17]. Interestingly, oral administration of rapamycin by gavage blocked this CR effect, probably due to endogenous inhibition of mTOR activity in the intestinal stem cells [18].

Pursuing a reliable approach for inducing stem cell number in the mammary gland of farm animals, the aforementioned studies were initially reproduced in intact mice and those carrying bovine mammary implants in their mammary stroma that were subjected to CR for 6–8 weeks [19]. In both models, CR induced a shift from mammary stem cell differentiation to self-renewal, which was reproduced by a shorter, 3-week period of rapamycin administration. Moreover, latent effects of rapamycin administration on improved mammary development and production were noted.

The model-based positive effect of rapamycin on mammary epithelial stem cell self-renewal was further investigated here in bovine. It was hypothesized that skip-a-day intramammary rapamycin administration at a defined concentration will cause oscillating mTOR activity in mammary parenchymal cells. During intervals of low mTOR activity, paracrine signaling for stem cell self-renewal will ensue from an as-yet undefined niche population. Higher mTOR activity will enable internal self-renewal of the stem cells. It was anticipated that increasing the epithelial stem cell reservoir will latently improve mammary activities associated with development and production.

We report that rapamycin, administered via the nipple into the mammary ductal system of 3-month-old calves, penetrates the lining epithelial structures and suppresses S6 phosphorylation with no effect on β-casein synthesis or mammary morphology. Subsequently, enhanced stem cell self-renewal was reflected by the longer propagation of cultures prepared from rapamycin-treated glands compared to their respective controls. Latent rapamycin effects included induced luminal and basal cell proliferation in a location- and cell type-dependent manner, as well as induced milk protein gene expression that was negatively correlated to the endogenous milk protein expression potential of the individual calf.

Materials and methods

Ethics statement

All animals used in this study were treated humanely. Study protocols were in compliance with the regulations of the Israeli Ministry of Health and the Animal Experimentation Ethics Committee of the Agricultural Research Organization, Volcani Center.

Animals

Three-month-old female Holstein calves (~90 kg body weight [BW]) that were raised on the Volcani Center’s experimental farm were used in these experiments. During the experimental periods, each mammary gland was administered via the teat canal with 2 mL of vehicle, composed of sterile PBS containing 10% polyethylene glycol (Sigma, St. Louis, MO) and 10% Tween 80 (Sigma). In individual experiments, the vehicle was supplemented with Evans blue to analyze the penetration of this solution into the mammary epithelium, or with rapamycin to analyze its effect on cellular activities. The vehicle was administered using a sterile 22G IV cannula (Deltaven, Viadana, (MN) Italy), cut to ~1.5 cm and inserted (without the needle) into the teat canal. This procedure did not involve any sedation or anesthesia. In the relevant experiments, estrogen (0.1 mg/kg per day, Sigma) and progesterone (0.25 mg/kg per day, Sigma) were administered following the rapamycin treatment by daily subcutaneous injections for 4 consecutive days [20]. The design of the studies was based on the distinct morphology and activity of the individual “quarters” (i.e., glands) of the bovine udder [21], which can be individually affected [14]. Thus, in studies analyzing the rapamycin effects, front or rear glands of each side of the calf served as internal controls for the other two rapamycin-administered glands.

At the end of the experimental period, calves were heavily sedated with intravenous xylazine (20 mg/mL; 0.1 mg/kg, Sedaxylan Veterinary, Abic Phibro, Bet Shemesh, Israel) and ketamine (1 g/10 mL; 0.5 mg/kg, Clorketam, Vetoquinol, Lure, France) and sacrificed with intravenous 20% embutramide, 5% mebezonium iodide and 0.5% tetracaine hydrochloride (T-61; 0.15 mL/kg, Intervet Canada Corp., Quebec, Canada). The complete udder was then removed for further analyses.

Morphological analysis by whole-mount examination

For whole-mount examination, bovine mammary parenchymal tissue pieces (~0.5 cm³) were excised from the udder, fixed, and stained with Carmine red as previously described [22]. Stained whole mounts were visualized and photographed using a binocular equipped with cellSens standard 1.4 software (Olympus Scientific Solutions, Tokyo, Japan).

Histological analysis, immunohistochemistry and immunofluorescence

Bovine mammary samples were fixed in Bouin’s solution, dehydrated in a graded ethanol series (50% to 100%), cleared in xylene and embedded in paraffin. Paraffin sections (5 μm) were stained with hematoxylin and eosin (H&E; Sigma) to visualize the morphology of the mammary layers.

Immunohistochemistry was performed on the 5-μm paraffin-embedded sections after antigen retrieval. The reaction with primary antibodies was initiated by blocking the sections with 10% bovine serum albumen and 5% goat serum in 0.1% PBST for 1 h prior to overnight incubation with the first antibody at 4°C. Sections were washed and incubated with N-Histofine (Nichirei Biosciences, Tokyo, Japan) for 30 min at room temperature. Signals were generated with 3,3-diaminobenzidine (DAB) substrate (Vector Laboratories, Burlingame, CA). For immunofluorescence analyses, the sections were incubated with secondary antibodies for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Qbiogen, Irvine, CA). The antibodies and their dilutions are listed in S1 Table. In all of the analyses, stained tissues were visualized and photographed under an inverted fluorescence microscope (Eclipse Ti, Nikon Instruments, Melville, NY) equipped with NIS-Elements AR 3.2 imaging software (Nikon Instruments), or an Olympus IX 81 inverted laser scanning confocal microscope (FluoView 500, Tokyo, Japan).

Flow cytometry

Bovine mammary tissue pieces were harvested from the parenchymal region of each gland, digested into organoids and kept at -80°C as previously described [10]. For flow cytometry, organoids were washed and further digested into dispersed mammary cells. Lin⁻ (CD45, TER119, CD31 and BP-1) cell suspension was prepared using the EasySep mouse epithelial enrichment kit according to the manufacturer’s protocol (STEMCELL Technologies, Vancouver, Canada), labeled with PE-conjugated anti-CD24 and FITC-conjugated anti-CD49f antibodies, and sorted as previously described [10, 11, 23]. The list of antibodies is provided in S1 Table. Cell viability was tested by staining with BD Horizon Fixable Viability Stain 450 diluted 1:500 (BD Biosciences, San Jose, CA). Cell sorting was performed in a FACSAria III cell sorter (BD Biosciences) at the Department of Biological Services of the Weizmann Institute of Science (Rehovot, Israel).

RNA extraction and real-time PCR analyses

RNA was extracted from the parenchymal region of each mammary gland using TRIzol reagent and reverse-transcribed with the qScript Synthesis Kit (Quanta BioSciences, Beverly, MA). RNA quality and quantity were determined in a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). Quantitative real-time PCR analyses were performed in a StepOnePlus instrument (Applied Biosystems, Foster City, CA) in a 10-mL reaction volume containing 1 μL cDNA, 5 μL PerfeCta SYBR Green FastMix, Rox (2X) (Quanta BioSciences) and 0.5 μL of the primers listed in S2 Table. The thermal cycling conditions consisted of 20 s at 95°C followed by 40 cycles of 3 s at 95°C and 30 s at 60°C. The amplification curves for the selected genes were parallel. The gene coding for eIF4E or ribosomal S16 was used as the endogenous control. Fold change (relative expression) was calculated using StepOne v2.1 and DataAssist v2.0 software (Applied Biosystems).

Immunoblot analysis

Proteins from the parenchymal region of each mammary gland were extracted with RIPA buffer containing 10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate and 5 mM EDTA (Sigma), protease inhibitor cocktail and phosphatase inhibitor cocktail II (both from Sigma). Equal amounts of protein, determined by QPRO-BCA Kit Standard (Cyanagen, Bologna, Italy) according to the manufacturer’s protocol, were subjected to SDS–PAGE. Fractionated proteins were transferred to nitrocellulose filters and the uploading of equal amounts of protein per sample was confirmed by staining the blot with Ponceau S (Sigma). The expression levels of phosphorylated S6 (pS6) and β-actin were determined according to the signals generated with their respective antibodies. Signals were generated with WESTAR Supernova (Cyanagen, Bologna, Italy) combined with Super Signal chemiluminescent substrate (Thermo Fisher Scientific), and detected in a Syngene analyzer (Cambridge, UK).

Analysis of propagation rate

For propagation rate, cells from each treatment were seeded in 96-well cell-culture plates (Corning, Corning, NY) at a density of 10,000 cells/well and cultured in mammary medium composed of DMEM-F12 medium containing 5% FBS, hydrocortisone (0.5 mg/ml, Sigma), insulin (5 mg/ml, Sigma), gentamicin (50 mg/ml, Biological Industries), streptomycin and penicillin (100 mg/ml and 100 U/ml, respectively), hEGF (10 ng/ml, Merck, Darmstadt, Germany), hFGF2 (10 ng/ml, Merck), heparin (4 mg/ml, Merck), cholera toxin (10 ng/ml, Sigma) and B27 (4 ml stock/ml, Invitrogen, Carlsbad, CA) (10) for the indicated period.

Organoid cultures

Partially digested pieces of parenchymal tissue were cultured on inserts (40 μm Falcon nylon cell strainers, Corning) in 6-well plates and supplemented with mammary medium [10]. Rapamycin (10 ng/mL) was administered to selected wells every other day. After a 3-week period, rapamycin-treated organoids were washed and all cultures were administered mammary medium for a week to avoid any further direct rapamycin effect. Medium was replaced every day. Eventually, the serum- and growth factor-containing medium was washed and milk protein gene expression was induced during 5 consecutive days of culture in DMEM/F12 medium containing (only) insulin (5 μg/mL), hydrocortisone (1 μg/mL) and prolactin (3 μg/mL). At the end of the culture period, organoids were frozen in liquid nitrogen and kept at -80°C.

Statistics

Unless otherwise indicated, t-test was performed for statistical analyses. Correlations were calculated using the Excel calculation formula.

Results

Establishment and calibration of rapamycin intramammary administration methodology

Three-month-old female calves were selected to analyze rapamycin’s effect on mammary epithelial stem cell self-renewal and the potential consequences for development and production. The infrequently used intramammary drug-delivery method that engages the milk-mobilizing ductal system was preferred over systemic administration, to selectively confine rapamycin’s effect to the mammary parenchyma (Fig 1A). To monitor vehicle mobility and penetration into the mammary epithelium, Evans blue (1%) was dissolved in the lipophilic solution and administered via the nipple to each of the four glands. Three hours later, the calf was sacrificed, the udder was removed and blue-stained parenchyma was revealed under the skin (Fig 1B). Note that in that short time interval, about half of the injected volume (~1 mL) was still located in the cisterna and did not penetrate the ductal system. Nevertheless, analysis of Carmine-red-counterstained mammary whole mounts revealed penetration of the blue stain into the ductal system and its translocation in the mammary epithelial terminal ductal lobuloalveolar units (TDLUs)—the ductal elongation region (Fig 1C).

Fig 1 — A. Mammary gland morphology. P, parenchyma; S, stroma. Bar = 2 cm. B. Evans blue penetration of parenchyma, 2 h after administration. Bar = 1.25 cm. C. Mammary whole mount showing penetration of Evans blue into Carmine red-stained mammary epithelial structures. Bar = 1 mm. D. Immunoblot analysis showing a decrease in S6 phosphorylation (pS6) 24 and 48 h after rapamycin administration. E–G. Immunohistochemical analysis of S6 phosphorylation, 24 h after treatment, in regions close to the nipple: E. Vehicle administration; F. rapamycin administration (4.16 mg); G. rapamycin administration (8.32 mg). H–L. Immunohistochemical analysis of S6 phosphorylation, 48 h after treatment, in regions close to and far from the nipple: H. Vehicle administration; I. Rapamycin administration (4.16 mg), close to nipple; J. rapamycin administration (8.32 mg), close to nipple; K. Rapamycin administration (4.16 mg), far from nipple; L. Rapamycin administration (8.32 mg), far from nipple. Bar = 50 μm.

To determine an effective concentration and administration interval, mTOR responsiveness to rapamycin administration was studied in parenchymal regions that were close to and far from the teat. Rapamycin was delivered to the parenchymatic region of two calves at two concentrations that were scaled up relative to the daily rapamycin administration to mice [[19] and S1A Fig]. The calves were sacrificed 24 h or 48 h later and the udders were removed. Rapamycin administration did not seem to have any effect on tissue morphology in the various parenchymatic regions tested (S1B–S1F Fig). S6 phosphorylation was analyzed in the mammary parenchyma by immunoblot and immunohistochemistry (Fig 1D–1L), respectively. These figures show that suppression of S6 phosphorylation following rapamycin administration at a level of 8.28 mg/2 mL in regions close to and far from the teat was maintained for at least 48 h. Note that complete repression of mTOR activity was avoided in these experiments to preserve the self-renewing capability of the epithelial stem cells.

Rapamycin administration decreases mammary S6 phosphorylation and induces indicators of stem cell self-renewal

Based on the obtained information, we then investigated the effect of 3 weeks of skip-a-day rapamycin administration (8.32 mg/gland per day) on stem cell self-renewal (Fig 2). Here, a significant decrease of 53% in S6 phosphorylation was observed by immunoblot analysis, 48 h after termination of the experiment (Fig 2A and 2B), which was not associated with any detectable effect on β-casein synthesis (Fig 2C) or mammary gland morphology (Fig 2D). Immunohistochemical analyses localized the decreased pS6 expression exclusively to the mammary epithelial cells (Fig 2E) and confirmed comparable β-casein synthesis in vehicle- and rapamycin-treated glands (Fig 2F).

Fig 2 — A. Immunoblot analysis of phosphorylated S6 (pS6) and β-casein. One of the nipples in calf #2 was inaccessible. B. Rapamycin administration causes a decrease in pS6 expression relative to β-actin expression (see A). C. Rapamycin administration does not affect β-casein expression relative to β-actin expression (see A). Bar graphs represent mean ± SEM of 3–4 replications. D. H&E-stained mammary parenchymal sections demonstrating no effect of rapamycin on mammary morphology. Insets: higher magnifications. E. Representative immunohistochemical analysis demonstrating lower S6 phosphorylation in rapamycin-treated vs. vehicle-treated glands. Bar = 50 μm. F. Representative immunohistochemical analysis demonstrating no effect of rapamycin administration on β-casein expression in the mammary epithelial cells. Bar = 50 μm.

It was not technically feasible to perform flow cytometry analysis of rapamycin’s effect on bovine stem cell self-renewal immediately after sacrificing the animals. Therefore, the parenchyma tissue of the individual mammary glands was partially digested into organoids [10] that were kept frozen at -80°C. The organoids were later dissociated into a suspension of single cells for flow cytometry analysis using CD24 and CD49f antibodies (S1 Table and S2 Fig). A relatively low rate of cell survival and great diversity among samples were detected. Combined with the low (<1%) proportion of stem cells in the epithelial cell population, this hindered accurate detection of differences in stem cell number between rapamycin-treated glands and their respective controls. Therefore, cell-propagation rate was analyzed in cultures from individual vehicle- and rapamycin-treated glands (Fig 3A). This method produces a clear and significant distinction of the bovine mammary epithelial stem cell population from the more differentiated ones [10]. It has also served as a reliable tool to analyze the effects of CR and rapamycin on mammary epithelial stem cell self-renewal in the endogenous mouse mammary gland and in bovine mammary implants, maintained in immunocompromised mice [19].

Fig 3 — A. Rapamycin administration induces propagation rate of cultured mammary epithelial cells. Dispersed cells isolated from individual vehicle- and rapamycin-treated mammary glands were seeded in 96-well plates and cultured in mammary medium. At the indicated time points (arrows), cells were trypsinized, counted and reseeded. Inset: cell number at the end of the culture period. Bars represent mean ± SEM. B. Relative gene expression analysis of luminal progenitor marker (Stat5) and stem cell markers (Notch1, Jagged1, Delta1, LGR4, LGR5). N = 4 for vehicle-treated and 3 for rapamycin-treated glands. Six wells of cells from each gland were analyzed.

Here, a significant induction in cell number was initially detected in cultures from the vehicle-treated glands, but not in their rapamycin-treated counterparts. This probably represented propagation of partially differentiated epithelial cells with a relatively short survival rate, as evidenced by the consequent decrease in cell number. Indeed, by day 30 of culture, the number of cells from the vehicle-treated and rapamycin-treated glands equalized. In the long-term, higher propagation rate was observed for cultures from the rapamycin-treated gland, starting from 1.15-fold and 1.4-fold differences on days 37 and 52 of culture, respectively, and progressing to a 2.6-fold difference at the end of the culture period (Fig 3A inset).

There is no individual marker that specifically identifies bovine mammary stem cells [10, 11]. Nevertheless, a limited collection of genes that are highly expressed in mouse and human mammary stem cells has been analyzed in extracts of bovine mammary parenchyma [8, 24]. All of these markers were highly expressed in the rapamycin-treated glands, by 8–20% more than in their vehicle-treated counterparts (Fig 3B). In contrast, Stat5, which serves as a reliable reference for luminal progenitors [10, 11, 19, 24], was expressed at lower levels in the rapamycin-treated glands. Due to the high variability in expression among mammary glands, none of these results were significant at P < 0.05.

Higher cell proliferation in rapamycin-treated glands depends on proximity to the growing site and studied cell layer

We then studied the consequences of rapamycin administration and induced stem cell self-renewal on mammary cell proliferation and milk protein expression. Front and rear mammary glands of three calves (six for each treatment) were infused with either vehicle or rapamycin for 3 weeks (S3 Fig). Five days after the last rapamycin administration, calves were subjected to daily estrogen and progesterone administration for 4 consecutive days to reproduce mammary cell proliferation during pregnancy [20]. At the end of the experimental period, animals were sacrificed, udders were removed and mammary parenchymal tissue pieces (~0.5 cm³) were subjected to H&E staining and immunofluorescence analyses (Fig 4A and 4B). Ducts of various diameters were identified that penetrated the stroma and encompassed a multilayered luminal epithelium stained with CK18, lined by a single layer of basal myoepithelial cells stained with alpha smooth muscle actin (αSMA) (Fig 4B). There were no apparent differences in morphology between the vehicle- and rapamycin-treated glands. Tukey–Kramer (means comparison) statistical analysis ruled out any preferential effect on ductal cell number or diameter for an individual animal or gland (S3 Table). This enabled a more detailed characterization of the experimental system, by analyzing ducts in all vehicle-treated glands with a diameter of 51.35–191.81 μm (larger sizes did not enable distinguishing branching ducts from small independent ones; in smaller ducts, distinction of basal cells from stromal and immune cells was challenging). A cross section of an average-sized duct in a 3-month-old calf appeared to be comprised of 84 cells that interact to create a perimeter of ~350 μm (Fig 4C). Luminal cells represented most of the epithelial cell population, whereas the single-layered basal/myoepithelial cells made up only ~25% of the total cell number. Morphologically, these basal cells were significantly (P < 0.05) more densely packed in the perimeter of the large ducts (diameter >100 μm) compared to the small ones (diameter <100 μm) (Fig 4D).

Fig 4 — A. Representative H&E staining of vehicle- and rapamycin-treated glands. Bar = 50 μm. B. Immunofluorescence analysis demonstrating multilayered luminal cells stained with CK18 (green) and a single layer of basal myoepithelial cells stained with αSMA (red). Bar = 20 μm. C-E. Mammary ductal cell characteristics of the vehicle-treated gland. C. Diameter size distinguishes small from large ducts. D. Density of basal cells in the ductal perimeter is higher in large vs. small ducts. E. Number of PCNA⁺ cells is higher in small vs. large ducts, mainly due to difference in luminal cell proliferation. Six independent microscopic fields (10X magnification) from each of the six glands treated with vehicle were analyzed. Overall, 127 ducts and 10,619 cells were counted. *P < 0.05, **P < 0.01, ****P <* 0.001.

To monitor cell proliferation, parenchymal sections from the individual mammary glands were stained with proliferating cell nuclear antigen (PCNA; Fig 5A and 5B). About 45% of the total number of cells in the vehicle-treated glands stained PCNA⁺ (Fig 4C). The relative rate of basal cell proliferation was 1.26-fold higher (P < 5.1 x 10⁻⁶) than that of the luminal ones (Fig 4E).

Fig 5 — Following estrogen and progesterone administration, mammary tissues were stained with PCNA and counterstained with hematoxylin. A, B. Lower (A) and higher (B) magnifications of PCNA-stained parenchymal cells. S, small ducts; L, large ducts. Bar = 50 μm. C. PCNA⁺ cells were analyzed in basal and luminal cell populations of the small and large ducts. Six independent microscopic fields (10X magnification) from each of the six glands treated with vehicle and six glands treated with rapamycin were analyzed. Overall, 127 ducts and 10,619 cells, and 122 ducts and 9999 cells were analyzed in the vehicle-treated and rapamycin-treated glands, respectively. **P < 0.01, ***P < 0.001.

Separate analyses were conducted for ducts with small and large diameters (Fig 4E), representing regions close to and far from the TDLUs, respectively [12, 19]. General proliferation rate was 28% higher in the small vs. large ducts, mainly due to 31% higher proliferation of luminal cells. Interestingly, in small ducts, no significant (P < 0.05) differences could be detected between the proliferation rates of basal and luminal cells, whereas in the large ducts, basal cell proliferation was 37% higher than that of luminal cells.

Rapamycin administration increased the total number of PCNA⁺ cells by 20%, mainly due to a significant effect on cells composing the large ducts (Fig 5C). A more detailed analysis indicated that basal cell proliferation was induced by rapamycin in both small and large ducts, whereas a markedly higher rate of PCNA⁺ luminal cells was detected only in the large ducts of the rapamycin-treated glands.

Milk protein gene expression is induced in the rapamycin-treated glands, and is negatively correlated to endogenous milk protein expression potential

The latent effect of rapamycin administration on milk protein gene expression and synthesis was measured in the calves’ mammary glands (Fig 6). The detectable expression of β-casein and β-lactoglobulin (BLG) in luminal mammary epithelial cells (Fig 6A) warranted more accurate gene and protein analyses of mammary productivity. Thus, expression of αS1-casein, β-casein, BLG and α-lactalbumin was analyzed by comparing the relevant gene expression in the rapamycin-treated glands of each animal to that in the vehicle-treated ones. In calf #2, rapamycin administration resulted in a latent 4.5- to 6-fold induction in the expression of all four milk protein genes tested, with no difference between the responses of casein and whey protein genes (Fig 6B–6E). This positive effect was confirmed by immunohistochemical analysis of β-casein, localized in the luminal epithelial cells (Fig 6G), and by immunoblot analysis (Fig 6F and S4 Fig). A modest effect of rapamycin on αS1-casein and BLG expression was observed in calf #3, but not in calf #1. Importantly, the various milk protein genes were expressed by mammary glands of calves #2 and #3 at levels of 0.4–12%, as compared to mammary glands of calf #1, which maintained much higher expression levels. Associating the average endogenous expression levels of the individual milk protein genes in the two vehicle-treated glands of each of the three calves to the inductive rapamycin effect (fold change), measured by comparing average gene expression in the two rapamycin-treated glands to their respective controls, presented a negative correlation of up to 99% for each gene (Fig 6H). Taken together, these results suggest that the positive rapamycin effect is limited by the endogenous expression potential of the mammary gland.

Mammary organoids preserve some, but not all interactions between epithelial cells and their surrounding stroma. By reproducing the procedure of the in-vivo study (except for steroid administration), we tested in organoid culture the latent animal-independent effect of 3-week rapamycin administration on milk protein gene expression in differentiating, lactogenic hormone-treated epithelial cells (S5 Fig). Significant latent induction of cytokeratin 18 (CK18) was observed by both gene expression and immunofluorescence analyses (S5A and S5B Fig), indicating a higher number of differentiated epithelial cells. However, in contrast to the in vivo finding, this was associated with a significant decrease in the expression of genes encoding all four milk proteins tested (S5C Fig). In fact, there was almost no β-casein synthesis or secretion in epithelial cells treated with rapamycin (S5B Fig).

Discussion

Pharmacological studies in farm animals are often limited by the animals’ relatively large size. The 3-month-old calf appears to have mammary parenchyma that is sufficiently well developed to scale up earlier findings in mice—associating decreased mTOR activity with induced epithelial stem cell self-renewal [19]—to the farm animal level. Using Evans blue marker dissolved in a lipophilic vehicle and counterstaining with Carmine red, intraductal delivery of material to the epithelium was established as an alternative to systemic administration. Targeting mammary epithelial cells around the ductal lumen, including those that are relatively far from the nipple, requires much less material compared to systemic administration and eliminates side effects resulting from the involvement of other organs. Indeed, the rapamycin delivered in this study to suppress mammary mTOR activity is an FDA-approved immunosuppressive drug, used to treat several types of cancer [25, 26] and recognized for its longevity-inducing effect [27]. However, long-term rapamycin administration disrupts mTOR complex 2 and subsequently results in hepatic insulin resistance leading to impairment of glucose homeostasis [28].

Intramammary rapamycin administration was calibrated according to S6 phosphorylation in the epithelial cells. A skip-a-day dose of 8.16 mg/gland per day was selected to satisfy three, somewhat contradictory, criteria: (i) providing a sufficient level of mTOR inhibition to induce putative paracrine secretion of stem cell self-renewal agent from as-yet unreported niche cells [[17, 19] and Kosenko et al. in preparation]; (ii) allowing sufficient mTOR activity in stem cells for self-renewal [18]; (iii) minimizing the risk of mammary infection during rapamycin administration. Indeed, by day 21 of the experiment, treated glands maintained about 47% of the mTOR activity in their vehicle-treated counterparts. This decrease was not associated with changes in mammary morphology or expression of the milk protein β-casein. In mice, inhibition of mTOR activity during the transition between pregnancy and lactation impairs terminal mammary differentiation, and β-casein synthesis is impaired in mammary HC11 cells [29]. A similar effect was seen with mTOR hyperactivity in tuberous sclerosis complex 1-knockout (TSC1^-/-) mice, which could be reversed by rapamycin administration [30]. Hence, the amount and methodology chosen here for rapamycin administration did not have pathological consequences, but was able to induce stem cell self-renewal as depicted by the longer propagation rate of cultures originating from rapamycin-treated glands compared to their respective controls, and the collective increase in stem-cell marker expression. These results are in accordance with the rapamycin-induced stem cell self-renewal effect reported in the endogenous mouse mammary gland and in mature parenchymal bovine mammary implants maintained in the rapamycin-treated mouse mammary stroma [19]. They suggest a conserved mTOR-sensitive, age-independent effect among species.

Four days of estrogen and progesterone administration are sufficient for initial induction of bovine mammary cell proliferation [20]. Temporally constrained by ethical regulations, this steroid-administration procedure was reproduced here to follow the consequences of rapamycin administration during late pregnancy (Fig 7). Morphologically, the distance from the TDLU, depicted by increased ductal diameter, was negatively associated with proliferation rate as reflected by the significant decrease in luminal cell proliferation along the forming ducts. In the developing mouse mammary gland, most cell divisions occur in luminal progenitors and basal cells [31]. If this hierarchical definition applies to bovines, it suggests that actively proliferating basal/basal progenitor cells are more evenly distributed along the developing ducts compared to luminal progenitors, which are strongly accumulated at the TDLU sites [(see [5] for more information]. This finding is also in accordance with previous reports in humans, indicating that breast epithelial cells with a capacity for self-renewal, bipotency and clonal growth (i.e., stem cells) are derived from ducts, while active proliferating progenitors concentrate at the lobes [32]. The difference between the rates and locations of the luminal and basal cell proliferation may result from the higher proximity of the basal cells to external niche signals originating, for example, from ductal macrophages [33], as compared to their luminal counterparts.

Fig 7 — A. Representative small and large ducts in the developing mammary architecture are boxed. B. Basal and luminal cell proliferation in small and large ducts is presented in the outer three cell circles, where the relatively lower occupancy of basal cells in the small vs. large ducts can be visualized. Also demonstrated is rapamycin-induced cell proliferation in basal and luminal cells of the small ducts and only in luminal cells of the large ducts. The scheme is a precise representation of the relevant analyses. Milk protein gene expression in the mammary glands of the individual calves is presented in pie chart format, inserted inside the circles showing the proliferation analysis of the large ducts. Presented are Log 2 (N+1) values of milk protein gene expression in the individual calves, calculated to accommodate and visualize the highly different values, some of which were lower than 1. The combined expression values of the four examined milk proteins in the mammary gland of rapamycin-treated calf #1 are highest and represent the maximum potential milk protein gene expression. Respectively, the pie chart demonstrates that rapamycin’s inductive effect on milk protein gene expression is limited by the endogenous potential of the individual animal. Thus, almost no improvement in milk protein gene expression is detected in calf #1 with the highest level of endogenously expressed milk proteins. A stronger effect of rapamycin on milk protein gene expression is demonstrated calf #3, and even more so in calf #2 which expressed the lowest level of milk proteins (two orders of magnitude lower than calf #1), and maintained nonutilized expression potential.

Rapamycin administration resulted in latent induction of cell proliferation. Considering the unaffected β-casein expression at the end of rapamycin administration period, and the 9-day period separating the termination of rapamycin and the subsequent steroid administration, this positive effect probably does not represent short-term compensation, but rather reflects the consequences of the higher ratio of undifferentiated stem cells in the rapamycin-treated gland. Nevertheless, rapamycin induced cell proliferation selectively, discriminating large ducts with basal and luminal cell responses from small ducts in which only luminal cell proliferation is induced. This selectivity may be partly associated with the higher density of basal myoepithelial cells in the large ducts (i.e., far from the TDLUs) which may tightly control their proliferation.

The bovine mammary parenchyma seems to contain sufficiently differentiated epithelial cells at the age of 3 months to allow milk protein gene expression and protein synthesis. Indeed, expression of milk proteins was also observed in prepubertal buffalo mammary glands [34]. This suggests that the full repertoire of systemic and niche-dependent stimuli necessary for full differentiation of the epithelial stem cell toward a functional luminal cell already exists at this early developmental stage. Their activity in prepuberty dictates future productivity of the mammary gland [35, 36].

Rapamycin’s positive effect on milk protein gene expression varied among calves; this was related to the gland’s expression potential, determined by the relative number of epithelial cells in the parenchyma. Indeed, subjecting organoid cultures—which are insulated from the full repertoire of tissue and systemic effects—to rapamycin confirmed the latent induction of differentiated epithelial cell numbers observed in vivo. However, the lack of in-vivo oscillation in rapamycin levels resulted in a continuous negative effect on milk protein expression, as was found in HC11 cultures [29].

Conclusions

Intramammary administration of pharmaceuticals dissolved in lipophilic vehicle is an effective and efficient method to target mammary epithelial/myoepithelial cells in 3-month-old bovine mammary glands with a potential effect in the adult cow as well. A decrease of ~50% in mTOR activity by skip-a-day, 3-week intramammary rapamycin administration is sufficient to induce mammary epithelial stem cell self-renewal with no impairment of mammary morphology or milk protein production. The consequent improvements are induced cell proliferation and milk protein expression and synthesis in glands with relatively low milk protein gene expression. It reflects a genuine latent consequence of the rapamycin administration, followed by a higher number of self-renewed stem cells in the gland.

Supporting information

S1 Raw images. Complete blot for Figs 1D, 2B and 2C, and for Fig 6F.

(TIF)

Click here for additional data file.^{(30.6MB, tif)}

S1 Table. List of antibodies used in this study.

(DOCX)

Click here for additional data file.^{(13.7KB, docx)}

S2 Table. List of primers used in this study.

(DOCX)

Click here for additional data file.^{(14.8KB, docx)}

S3 Table. Tukey–Kramer (means comparison) analysis of the average cell number/ duct and average ductal diameter in mammary glands treated with vehicle or rapamycin for 3 weeks followed by estrogen and progesterone.

The data indicate no significant effect for individual heifer or gland.

(DOCX)

Click here for additional data file.^{(13.9KB, docx)}

S1 Fig. H&E analysis demonstrating no effect after 48 h of intramammary rapamycin administration on mammary morphology.

A. Designation of the bovine mammary glands for intramammary vehicle and rapamycin administration. B–F. Morphology of parenchymal regions close to and far from the nipple is not affected by rapamycin administration. Bar = 50 μm.

(TIF)

Click here for additional data file.^{(18.6MB, tif)}

S2 Fig. Flow cytometry analysis of dispersed CD24- and CD49f-labeled mammary cell population from 3-month-old calves.

Stem cells (MRU) were sorted from suspensions of single cells of individual vehicle- and rapamycin-treated glands.

(TIF)

Click here for additional data file.^{(15.8MB, tif)}

S3 Fig. Scheme demonstrating allocation of the mammary gland for vehicle and rapamycin administration.

(TIF)

Click here for additional data file.^{(14.3MB, tif)}

S4 Fig. Immunoblot analysis of β-casein and β-actin expression in mammary glands treated with vehicle or rapamycin for 3 weeks followed by estrogen and progesterone administration.

(TIF)

Click here for additional data file.^{(14.6MB, tif)}

S5 Fig. Latent inductive effect of rapamycin administration on CK18 expression in mammary organoids contrasts with its negative effect on milk protein expression.

Mammary organoids were cultured for 3 weeks in mammary medium supplemented, or not, with rapamycin. Rapamycin was omitted from the medium for an additional week, then the medium was changed to DMEM/F12 containing insulin, hydrocortisone and prolactin. A. Latent induction of CK18 relative expression by rapamycin administration. B. Immunofluorescence analysis demonstrating induced CK18 expression and decreased β-casein expression in rapamycin-treated organoids. Green: CK18 and β-casein. Red: αSMA. Bar = 20 μm. Inset: higher magnification. C. Negative effect of rapamycin on milk protein gene expression. Bars represent mean ± SEM of 4 replications. *P < 0.05, **P < 0.01, ***P < 0.001.

(TIF)

Click here for additional data file.^{(17.4MB, tif)}

Acknowledgments

We acknowledge Dr. Hen Hana Honig Felman for her contribution to the initial stages of the study and Dr. Tatiana Kisliouk for technical help.

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

This study was supported by the Chief Scientist of the Ministry of Agriculture and Rural Development (Grant number 20-04-0060) and the Israeli Milk Marketing Board (Grant number 362-0574). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0269505.r001

Decision Letter 0

Sina Safayi

7 Apr 2022

PONE-D-22-05135Intramammary rapamycin administration to calves induces epithelial stem cell self-renewal and latent cell proliferation and milk protein expressionPLOS ONE

Dear Dr. Barash,

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Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Overall this is a very well written manuscript that explores an interesting method for stimulating mammary stem cell renewal in a bovine model. This investigation is well-supported by previous literature and has considerable implications for the dairy industry. My reason for recommending minor revisions primarily pertains to the organization of the materials and methods. Particularly for the animal treatment section, it was difficult to understand how the animals were treated before they were sacrificed and the mammary glands removed. Providing a written version of the schema displayed in Figure S3 would help to clarify this. This manuscript also lacks a clear hypothesis. Please see my full review points below:

Abstract

• Well written, clear, no revisions necessary

Introduction

• Line 50 � “They also renew it between consecutive lactation periods”

o Is the “it” referring to the mammary epithelium or basal/luminal layer – please clarify

• Line 55 � add additional parentheses to (Reviewed by (4,9))

• Line 59-60 � “This rare population”

o Consider changing for clarity, bovine mammary cells are not rare but are seriously understudied

o Perhaps change to “This understudied population”

• Line 65 � add “the” between “that” and “mammary”

• Line 87 � change “to” to “for”

• Lacking hypothesis

Materials and Methods

• Lines 113-117 � unclear what was done, it is mentioned that a solution of sterile PBS containing

o Consider moving some of the information contained in lines 211-223 to the methods section

• 10% PEG and 10% Tween 80 was administered with or without the indicated supplements

o what supplements are you referring to?

o When were these supplements given?

o Overall, it is somewhat unclear what was done in the animals before they were sacrificed and the udder was removed – please clarify

• Line 131 � please add manufacturer information about cellSens software

• Line 192 � “mammary medium” – what is this?

o Also mentioned on lines 199-200

• Statistics � please indicate how correlations were analyzed

Results

• Lines 211-223 � most of this information is better suited in the materials and methods

• Lines 224-235 � same, consider moving most of this information into the materials and methods

o Perhaps just keep the information starting on line 229

• Lines 281-288 � consider moving this to the materials and methods

• Line 291 � change “difference” to “differences”

• Line 333 � remove “and that was”

• Line 339 � how were the correlations performed?

• Overall, very well written results section, figures are clear and explanations of what are presented are well done. Please consider moving some of the detailed methodology into the materials and methods section

Discussion

• Line 398 � add “which” between “progenitors” and “are”

• Very well written discussion. Usage of references was properly done. Implications of the data are clear

Conclusions

• Line 428 � remove ”the”

Supporting information

• Line 444 � change “Tuckey” to “Tukey”

Figure Legends

• Figures and figure legends were clear and easy to understand

• Figure 6 � whether in the caption, results, materials, and methods or all, a further explanation of how the correlations presented in Figure 6H were analyzed is needed

Reviewer #2: See attached.

**********

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Reviewer #1: Yes: Hunter Ford

Reviewer #2: Yes: Ratan K Choudhary

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PLoS One. 2022 Jun 22;17(6):e0269505. doi: 10.1371/journal.pone.0269505.r002

Author response to Decision Letter 0

26 Apr 2022

Reviewer #1

“Overall this is a very well written manuscript that explores an interesting method for stimulating mammary stem cell renewal in a bovine model. This investigation is well-supported by previous literature and has considerable implications for the dairy industry.”

Response: We thank the reviewer for expressing interest in the manuscript's topic and data.

“My reason for recommending minor revisions primarily pertains to the organization of the materials and methods. Particularly for the animal treatment section, it was difficult to understand how the animals were treated before they were sacrificed and the mammary glands removed. Providing a written version of the schema displayed in Figure S3 would help to clarify this”.

Response: We thank the reviewer for this suggestion. The section related to animal treatment in Materials and Methods has been expanded to provide more details on how the experiments were conducted. It now includes a written version of the scheme displayed in Fig S3.

“This manuscript also lacks a clear hypothesis”.

Response: The leading hypothesis has been added to the Introduction section.

Abstract

“Well written, clear, no revisions necessary”.

Response: Thank you

Introduction

Line 50. “They also renew it between consecutive lactation periods. Is the “it” referring to the

mammary epithelium or basal/luminal layer – please clarify”.

Response: “It” refers to the mammary gland. The sentence has been revised accordingly.

Line 55. Add additional parentheses to (Reviewed by (4,9))”.

Response: Done

Line 59-60 “This rare population”. “Consider changing for clarity, bovine mammary cells are not rare but are seriously understudied”. Perhaps change to “This understudied population”

Response: The sentence has been revised as suggested.

Line 65 add “the” between “that” and “mammary”

Response: Done

Line 87 change “to” to “for””.

Response: Done

“Lacking hypothesis.”

Response: A clear hypothesis has been added toward the end of the Introduction section.

Materials and Methods

Lines 113-117 "unclear what was done, it is mentioned that a solution of sterile PBS containing…Consider moving some of the information contained in lines 211-223 to the methods section.

Response: This section has been expanded to clarify the procedure. We carefully considered the reviewer’s suggestion to move part of, or the full section describing development, calibration, morphological verification and characterization of the intramammary methodology of rapamycin administration to the Materials and Methods. However, we feel that this section, including the associated figures, is an integral part of the research and supports the following experiments. Therefore, for the convenience of the reader and the integrity of the manuscript, we believe that it should remain in the Results section. Some of the data have already been included in Supporting Information. An example of integrating the development of experimental methodology in the Results section may be found in a previous study by Reichstein et al. “Conditional repression of STAT5 expression during lactation reveals its exclusive roles in mammary gland morphology, milk-protein gene expression, and neonate growth” Mol. Reprod. Dev. 78:585–596 (2011), where the section describing the development of the conditional Stat5 expression methodology, including the genetically modified mice, appears in the Results section, thereby providing a better understanding of the experiments that follow it.

“10% PEG and 10% Tween 80 was administered with or without the indicated supplements. What supplements are you referring to? When were these supplements given? Overall, it is somewhat unclear what was done in the animals before they were sacrificed and the udder was removed – please clarify.

Response: A more detailed description of the methodology is now provided.

Line 131. Please add manufacturer information about cellSens software.

Response: Done

Line 192 “mammary medium” – what is this? “Also mentioned on lines 199-200”.

Response: The composition of the mammary medium and a reference is now provided.

Statistics. Please indicate how correlations were analyzed.

Response: The technical aspect is now indicated in Materials and Methods. Further description is provided in the Results section in the paragraph referencing Fig 6H.

Results

Lines 211-223. Most of this information is better suited in the materials and methods.

Lines 224-235. “Same, consider moving most of this information into the materials and methods. Perhaps just keep the information starting on line 229”.

Response: Please refer to our explanation above as to why we prefer to keep this paragraph in the Results section.

Lines 281-288. Consider moving this to the materials and methods.

Response: This paragraph describes the treatment of the animals and is essential, in our opinion, for understanding the following evaluation of the consequences of rapamycin administration. For a smooth narrative flow, we prefer to keep it as an introductory paragraph in the relevant subsection of the Results.

Line 291, Change “difference” to “differences”.

Response: “There was no apparent difference between the morphology of the vehicle- and rapamycin-treated glands”. The “difference” is associated with the “morphology” and seems correct. Done

Line 333. Remove “and that was”.

Response: Done

Line 339. How were the correlations performed?

Response: A more detailed description is now provided. The correlations were performed between two variables for each milk protein: (i) average milk protein gene expression level calculated for the two vehicle-treated (control) glands in each of the 3 analyzed calves; (ii) the effect of rapamycin on that particular gene's expression. The effect of rapamycin is the fold change obtained by comparing the average gene expression level in the two rapamycin-treated glands to its respective, vehicle-treated, control in the individual calf.

Overall, very well written results section, figures are clear and explanations of what are presented are well done. Please consider moving some of the detailed methodology into the materials and methods section.

Response: Thank you. Please see our explanation above for wanting to keep some of the methodology in the Results section.

Discussion

Line 398. Add “which” between “progenitors” and “are”.

Response: Done

Very well written discussion. Usage of references was properly done. Implications of the data are clear.

Response: Thank you

Conclusions

Line 428. Remove” the”

Response: Done

Supporting information

Line 444. Change “Tuckey” to “Tukey”.

Response: Done

Figure Legends

Figures and figure legends were clear and easy to understand.

Response: Thank you

Figure 6. Whether in the caption, results, materials, and methods or all, a further explanation of how the correlations presented in Figure 6H were analyzed is needed.

Response: The technical information is now provided in Materials and Methods. A more detailed explanation of how the correlations were performed is also provided (as mentioned above) in the Results section, in the paragraph referencing Fig 6H.

Reviewer #2

“…This is an exciting study and worthy of being considered for publication upon making suggestive changes”.

Response: Thank you

1. “Line 72-73: A study suggesting the role of xanthosine in increasing stem cell population is not limited to bovine only but has similar effects in the goat mammary gland (Choudhary et al., 2018; https://pubmed.ncbi.nlm.nih.gov/29804277/). Reference should be mentioned here”.

Response: A comment and the reference are now included in the text.

2. “Line 183: Please elaborate on how protein concentration in the sample was determined to have an equal amount of protein per sample loaded for SDS-PAGE”.

Response: The information is now provided.

3. Line 416-17: The presence of differentiated mammary epithelial cells in 3- months old calf is engaging. This information should be discussed a bit more here. This result is consistent with an earlier study where expression of milk protein genes was evident in the RNAseq study in the prepubertal buffalo mammary gland (Choudhary et al. 2019; https://pubmed.ncbi.nlm.nih.gov/30467802/). Please discuss this point here.

Response: The presence of differentiated mammary epithelial cells in prepubertal calves was touched upon briefly so as not to divert the reader from the main focus of the manuscript. The reference has been included.

4. Figure 2E: Please provide a representative picture of S6P staining of mammary tissue under the “Rapamycin” group. It is harder to visualize any cellular architecture of the tissue.

Response: The relatively low expression of pS6 caused by rapamycin administration indeed resulted in decreased DAB staining and visibility of the gland’s architecture. We have attempted to sharpen the figure to slightly improve visibility. We deliberately did not use hematoxylin counterstaining here so as not to mask the significant decrease in pS6 expression caused by rapamycin treatment. The architecture of the gland can be seen in Fig 2D. Please also consider the lower resolution of the figures in PDF format.

5. “Figure 5B: Please provide a representative picture of the under Rapamycin group showing a comparatively high number of PCNA-positive epithelial cells. It is challenging to visualize the increased cell proliferation rate in the rapamycin group”.

Response: A more representative figure is now provided to demonstrate the higher number of PCNA-positive cells in the rapamycin-treated glands. Please note that the average difference between the total PCNA-stained nuclei in the control and rapamycin-treated glands is ~20% (Fig 5C).

Minor comments:

Line 638-639 and line 648-649: Please check if the information (duct no, cell no) is not the same in the legend of fig 4 and fig 5.

Response: In Fig 4C,D, only vehicle-treated “control” glands were analyzed for endogenous morphological and layer-associated aspects that characterize the 3-month-old calf mammary gland. Overall, 127 ducts and 10,619 cells were counted. In Fig 5, the rapamycin effect on latent induction of ductal cell proliferation was analyzed. For this, the same slides of the control mammary gland were used and the data were compared with the indicated and different number of 122 ducts and 9999 cells that were analyzed in the rapamycin-treated glands.

We thank the reviewers for their comments and suggestions.

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PLoS One. doi: 10.1371/journal.pone.0269505.r003

Decision Letter 1

Sina Safayi

23 May 2022

Intramammary rapamycin administration to calves induces epithelial stem cell self-renewal and latent cell proliferation and milk protein expression

PONE-D-22-05135R1

Dear Dr. Barash,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

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Reviewer #2: Yes

**********

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Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Thank you for addressing all comments. I appreciate your explanation and justification for the information included in the materials and methods vs. the results section. After reading through the manuscript again, I understand your reasoning and agree with the decision to integrate some of the methodology into both sections.

Reviewer #2: Indeed this is good work performed by Barash group and has been doing a great work in the area of mammary gland and stem cell biology of ruminants, in particular bovines. Thank you for submitting your response and addressing our concern in revised version of manuscript. Wishing you and your team a great success!

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Reviewer #1: Yes: Hunter R. Ford

Reviewer #2: Yes: Ratan K Choudhary

PLoS One. doi: 10.1371/journal.pone.0269505.r004

Acceptance letter

Sina Safayi

30 May 2022

PONE-D-22-05135R1

Intramammary rapamycin administration to calves induces epithelial stem cell self-renewal and latent cell proliferation and milk protein expression

Dear Dr. Barash:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Raw images. Complete blot for Figs 1D, 2B and 2C, and for Fig 6F.

(TIF)

Click here for additional data file.^{(30.6MB, tif)}

S1 Table. List of antibodies used in this study.

(DOCX)

Click here for additional data file.^{(13.7KB, docx)}

S2 Table. List of primers used in this study.

(DOCX)

Click here for additional data file.^{(14.8KB, docx)}

The data indicate no significant effect for individual heifer or gland.

(DOCX)

Click here for additional data file.^{(13.9KB, docx)}

S1 Fig. H&E analysis demonstrating no effect after 48 h of intramammary rapamycin administration on mammary morphology.

(TIF)

Click here for additional data file.^{(18.6MB, tif)}

S2 Fig. Flow cytometry analysis of dispersed CD24- and CD49f-labeled mammary cell population from 3-month-old calves.

Stem cells (MRU) were sorted from suspensions of single cells of individual vehicle- and rapamycin-treated glands.

(TIF)

Click here for additional data file.^{(15.8MB, tif)}

S3 Fig. Scheme demonstrating allocation of the mammary gland for vehicle and rapamycin administration.

(TIF)

Click here for additional data file.^{(14.3MB, tif)}

S4 Fig. Immunoblot analysis of β-casein and β-actin expression in mammary glands treated with vehicle or rapamycin for 3 weeks followed by estrogen and progesterone administration.

(TIF)

Click here for additional data file.^{(14.6MB, tif)}

S5 Fig. Latent inductive effect of rapamycin administration on CK18 expression in mammary organoids contrasts with its negative effect on milk protein expression.

(TIF)

Click here for additional data file.^{(17.4MB, tif)}

Attachment

Submitted filename: PONE-D-22-05135_reviewers comment.pdf

Click here for additional data file.^{(79.1KB, pdf)}

Attachment

Submitted filename: Responses to the reviewers.docx

Click here for additional data file.^{(17.9KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting information files.

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PERMALINK

Intramammary rapamycin administration to calves induces epithelial stem cell self-renewal and latent cell proliferation and milk protein expression

Anna Kosenko

Shamay Jacoby

Tomer-Meir Salame

Maya Ross

Itamar Barash

Roles

Abstract

Introduction

Materials and methods

Ethics statement

Animals

Morphological analysis by whole-mount examination

Histological analysis, immunohistochemistry and immunofluorescence

Flow cytometry

RNA extraction and real-time PCR analyses

Immunoblot analysis

Analysis of propagation rate

Organoid cultures

Statistics

Results

Establishment and calibration of rapamycin intramammary administration methodology

Fig 1. Intramammary rapamycin administration reduces S6 phosphorylation in parenchymal cells.

Rapamycin administration decreases mammary S6 phosphorylation and induces indicators of stem cell self-renewal

Fig 2. Intramammary rapamycin administration for 3 weeks significantly reduces S6 phosphorylation in the mammary gland with no effect on mammary gland morphology or β-casein synthesis.

Fig 3. Intramammary rapamycin administration induces self-renewing cell properties.

Higher cell proliferation in rapamycin-treated glands depends on proximity to the growing site and studied cell layer

Fig 4. Morphological and proliferative characteristics of 3-month-old vehicle-treated and rapamycin-treated bovine mammary gland after 4-day treatment with estrogen and progesterone.

Fig 5. Rapamycin administration results in latent induction of ductal cell proliferation in layer- and region-dependent manners.

Milk protein gene expression is induced in the rapamycin-treated glands, and is negatively correlated to endogenous milk protein expression potential

Fig 6. Unequal effects of intramammary rapamycin administration on milk protein gene expression in individual calves.

Discussion

Fig 7. Scheme summarizing the latent effects of intramammary rapamycin administration on cell proliferation and milk protein gene expression.

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Author response to Decision Letter 0

Decision Letter 1

Sina Safayi

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Acceptance letter

Sina Safayi

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Associated Data

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Data Availability Statement

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