Pubertal Mice Research Articles (Page 1)

BackgroundCD36, a vital fatty acid translocase, has been reported to participate in multiple physiological functions through palmitoylation mediated by zinc finger Asp–His–His–Cys-type palmitoyltransferases (DHHCs). This study aimed to investigate the possible involvement of DHHC-mediated CD36 palmitoylation in high-fat diet (HFD)-induced impairment of pubertal mammary gland development and explore the underlying mechanisms involved.MethodsPalmitic acid (PA)-treated HC11 cells were used as the in vitro high-fat model, and the cell proliferation was examined by 5-Ethynyl-2′-deoxyuridine (EdU) incorporation assay. The palmitoylation of CD36 was determined by the acyl–biotin exchange (ABE) method. The expression of CD36, proliferative genes, and signaling molecules was detected by immunoblotting. The cellular localization of CD36 was determined by immunofluorescence. The bindings of CD36 with zinc finger DHHC-type palmitoyltransferases 9 (DHHC9) or Fyn/Lyn were detected by co-immunoprecipitation (Co-IP). The palmitoylation inhibitor 2-bromopalmitate (2BP), DHHC9 knockdown, and point mutation of CD36 cysteine residues were applied to construct a CD36 palmitoylation deficiency model in vitro to investigate the effects of CD36 palmitoylation on HC11 proliferation. In vivo, the pubertal mice were treated with HFD and/or 2BP. Mammary gland morphology was determined by whole mount staining, and the underlying mechanisms were verified by the methods used in the in vitro system.ResultsIn vitro, the palmitoylation inhibitor 2BP eliminated PA-inhibited HC11 proliferation and inhibited CD36 palmitoylation and localization on the plasma membrane. Meanwhile, the binding of DHHC9 and CD36 in PA-treated HC11 cells was repressed by 2BP. In addition, both knockdown of DHHC9 and point mutation of CD36 cysteine residues suppressed the membrane palmitoylation and localization of CD36 and stimulated the proliferation of PA-treated HC11 cells. Furthermore, in PA-treated HC11 cells, the inhibition of CD36 palmitoylation, the knockdown of DHHC9, and the mutation of CD36 cysteine residues resulted in decreased formation of the CD36/Fyn/Lyn complex. Correspondingly, the downstream c-jun n-terminal kinase 1 (JNK1) pathway was inhibited, and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway was activated. Moreover, inhibition of the JNK pathway with SP600125 promoted the proliferation of PA-treated HC11 cells via activation of the ERK1/2 pathway. In vivo, the palmitoylation inhibitor 2BP ameliorated HFD-induced impairment of mammary gland development in pubertal female mice, which was associated with a decrease in DHHC9-mediated CD36 palmitoylation in the plasma membrane, a reduction in the CD36/Fyn/Lyn complex, inhibition of the JNK1 pathway, and activation of the ERK1/2 pathway.ConclusionsThis study revealed that inhibition of DHHC9-mediated CD36 palmitoylation mitigated HFD-induced impairment of pubertal mammary gland development via the JNK1-ERK1/2 pathway.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s11658-025-00799-3.

The mouse brain is masculinized by postnatal testicular androgens, which are active after conversion to estrogens and modulate gene expression epigenetically, at least in part. The preoptic area contains a sexually dimorphic nucleus (SDN) comprising calbindin D‐28K (Calb) neurons with a male‐biased sex difference in cell number (Calb‐SDN), although the mechanisms responsible for the sex difference are not fully understood. We have previously demonstrated that Calb neurons expressing the androgen receptor (AR) are a male‐dominant cell group of the Calb‐SDN in pubertal mice, while Calb neurons without AR exist in both sexes with equal cell numbers. In this study, we investigated the mechanisms by which more Calb/AR neurons emerge in the male Calb‐SDN than in the female one. Immunohistochemistry for Calb and AR was performed using the brain sections from pubertal male mice subjected to sham surgery or neonatal orchidectomy, from pubertal female mice treated with vehicle, testosterone, or estradiol during the postnatal period, and from pubertal male mice whose brains were treated with trichostatin A, a histone deacetylase inhibitor, during the postnatal period. Immunostained brain sections were analyzed stereologically to determine the numbers of Calb‐immunopositive and AR‐immunopositive cells (Calb+/AR+ cells) and Calb‐immunopositive and AR‐immunonegative cells (Calb+/AR− cells) in the Calb‐SDN. The number of Calb+/AR+ cells in the Calb‐SDN during the pubertal period was significantly decreased in neonatally orchidectomized males compared with sham males and increased in testosterone‐ or estradiol‐treated females compared with vehicle‐treated females; however, the number of Calb+/AR− cells remained unchanged. Trichostatin A treatment significantly reduced the number of Calb+/AR+ cells, but not the number of Calb+/AR− cells, in the Calb‐SDN of males. These findings suggest that estrogens synthesized from postnatal testicular androgens act selectively on the AR‐expressing subpopulation of Calb neurons, contributing to the sex difference in the number of Calb neurons in the mouse Calb‐SDN. Epigenetic regulation of gene expression, possibly mediated by histone deacetylation, may be involved in the emergence of the AR‐expressing subpopulation of Calb neurons.

Sex differences in resilience at puberty depend upon divergent effects of a stress steroid at α4βδ GABAA receptors.

Fibroblasts are stromal cells found in connective tissue that are critical for organ development, tissue homeostasis and pathology. Single-cell transcriptomic analyses have revealed a high level of inter- and intra-organ heterogeneity of fibroblasts. However, the functional implications and lineage relations of different fibroblast subtypes remained unexplored, especially in the mammary gland. Here, we provide a comprehensive characterization of pubertal mouse mammary fibroblasts, through single-cell RNA sequencing, spatial mapping, functional assays, and in vivo lineage tracing. We unravel a transient niche-forming population of specialized contractile fibroblasts that exclusively localize around the tips of the growing mammary epithelium and are recruited from preadipocytes in the surrounding fat pad stroma. Using organoid-fibroblast co-cultures we reveal that different fibroblast populations can acquire contractile features when in direct contact with the epithelium, promoting organoid branching. The detailed in vivo characterization of these specialized cells and their lineage history provides insights into fibroblast heterogeneity and implicates their importance for creating a signalling niche during mouse mammary gland development.

Vaspin, a novel adipokine expressed in the testis and ovary, is known to regulate ovarian steroidogenesis in rats, porcine, and humans. Recently, adipokines like visfatin have been shown to modulate testicular steroidogenesis in early pubertal and adult mice, however, effects of vaspin on testicular steroidogenesis in early pubertal is still unknown. Male mice at postnatal day 21 were selected and divided into three groups: control (n = 6), vaspin low (0.1µg/kg) (n = 6), and vaspin high (1µg/kg) (n = 6). Control group received saline as a vehicle, and other two groups were administered with vaspin at a dose of 0.1µg/kg and 1µg/kg (IP). The treatment was given for 15 days (PND21 to PND35). Vaspin treatment at a dose of 0.1 and 1µg/kg elevated the circulating testosterone, androstenedione, and estrogen, with no change in progesterone levels. StAR and CYP11A1 expression showed a significant increase at both doses of vaspin, while 3β-HSD immunostaining showed a decline only at higher doses of vaspin. 17β-HSD and CYP17A1 showed increased expression at lower doses of vaspin, and aromatase showed a significant decrease at higher doses. AR expression was unaffected; moreover, ERα and β showed a dose-dependent decreased expression in testis. The results indicate that elevated levels of vaspin enhance the secretion of testicular steroids by differential regulation of steroidogenic markers. In conclusion, excess of vaspin stimulates the testicular testosterone, estrogen, and androstenedione secretion in early pubertal mice. However, its functional significance on the testicular functions needs further investigation.

The expression of vaspin and GRP78 has been shown in the testis and ovary. The postnatal testis undergoes several changes in the expression of different proteins. The expression of vaspin and GRP78 has not been shown in the postnatal testis. It has also been shown that modulation of adipokine function could affect testicular germ cell proliferation and apoptosis. Whether vaspin regulates testicular proliferation and apoptosis in the early pubertal stage is still unknown. The aim of this study was to determine the expression of vaspin/GRP78 in postnatal testes of mice. Next, we investigated the effects of vaspin on cell proliferation and cell death (apoptosis, ferroptosis, and autophagy) in the pubertal testis. Immunohistochemistry and western blot analyses revealed that vaspin and GRP78 exhibit dynamic expression levels through developmental stages. In the testis, both proteins showed mild to moderate immunostaining in Leydig cells at early stages (PND7 and 14), with increasing intensity at PND21 and 42 in Leydig cells and spermatocytes, and round and elongated spermatids. The expression of vaspin and GRP78 was significantly down-regulated at postnatal day 21 (PND21). Moreover, exogenous vaspin treatment (PND21 to PND35) suppressed germ cell proliferation (BrdU labelling, PCNA, and GCNA) and apoptosis (decreased expression of active caspase-3 and TNFα) in the testis. The marker of autophagy, LAMP2, was elevated by vaspin treatment. Furthermore, vaspin treatment showed both stimulatory and inhibitory effects on markers of ferroptosis. In conclusion, vaspin/GRP78 could be a new regulator of cell proliferation and cell death in pubertal mouse testes.

Impact of the environmental endocrine disruptor 4-octylphenol on reproductive function in pubertal male mice.

Atrazine (ATZ), a commonly used triazine herbicide, is known to impair male reproductive function. Melatonin (MLT) is an endogenous hormone synthesized by the pineal gland. Its lipophilic nature enables it to freely penetrate the blood-testis barrier (BTB) and exert direct effects. However, it remains unclear whether MLT can prevent ATZ-induced impairment of BTB function. Pubertal male mice were randomly assigned to four groups and treated via gavage for 21 days: control group (Con), 5 mg/kg melatonin group (MLT), 170 mg/kg atrazine group (ATZ), and cotreatment group (ATZ+MLT). Furthermore, BTB-associated proteins, transepithelial electrical resistance, and cell adhesion assays were performed in TM4 cells treated with siClusterin, pcDNA3.1-Clusterin, and AKT inhibitor VIII to evaluate BTB function in vitro. Our findings demonstrate that MLT effectively restores ATZ-induced reductions in BTB protein levels and function in both in vivo and in vitro models. Moreover, MLT mitigated ATZ-induced BTB damage by reactivating the Clusterin/AKT signaling pathway.

Childhood obesity, especially in girls, often correlates with advanced puberty and long-term comorbidities. Among the central circuits controlling energy homeostasis, hypothalamic lipid sensing pathways, involving free fatty-acid receptors (FFARs), peroxisome proliferator-activated receptors (PPARs), and the bile-acid (BA) receptor, Takeda G protein-coupled receptor 5 (TGR5), have been recognized as major players, with putative pathogenic roles in obesity and its complications. However, their contribution to pubertal regulation and obesity-induced pubertal alterations remains largely unexplored. We describe herein changes in the hypothalamic profiles of specific lipid species, including certain fatty-acyls, BA derivatives, and several glycerolphospholipids, during the juvenile-pubertal transition and conditions of overweight linked to precocious puberty in female rats. Hypothalamic expression of the FFAR, Gpr84, as well as Ppar-γ and Tgr5 gradually increased during the infantile-prepubertal transition, whereas early overfeeding increased hypothalamic mRNA levels of the FFARs, Gpr43, and Gpr84. Expression of Gpr84, Ppar-α, and Tgr5 was documented in FACS-isolated Kiss1 neurons from juvenile and pubertal female mice. Central pharmacological gain- and loss-of-function manipulations of GPR84-, PPAR-, or TGR5-signaling in prepubertal lean and early overfed female rats resulted in specific changes in pubertal timing. In lean rats, central blockade of PPAR-γ/α delayed puberty onset, whereas in early overfed rats, central stimulation of TGR5 signaling partially prevented obesity-induced advanced puberty; effects that were also marginally observed after GPR84 inhibition. Our results disclose the role of brain lipid-sensing pathways in the control of puberty, with a variable contribution of central FFAR-, PPAR-, and TGR5-signaling depending on the maturational and nutritional status.NEW & NOTEWORTHY Puberty is highly sensitive to body energy status, and child obesity is often linked to perturbed puberty. However, whether this comes from excessive energy stores or specific nutrient signals altered in obesity remains largely unexplored. Using suitable preclinical models of early obesity and accelerated puberty, we disclose herein conclusive evidence for altered hypothalamic lipid profiles and the roles of specific lipid-sensing pathways in pubertal control, with a variable contribution depending on the maturational and nutritional status.

Lactational period is of extreme importance for nourishing and fostering growth in neonates. Bisphenol S (BPS) a congener of bisphenol A (BPA) is an emerging environmental toxicant reported to have deleterious effects on reproductive health. Indirect exposure of BPS to the suckling infants via breastmilk is less explored although it can lead to various public health issues. Therefore, we investigated harmful effects of lactational BPS exposure on pre-pubertal and pubertal testicular functions of the offspring and its possible amelioration by melatonin. Lactating dams were divided into 4 groups: control, melatonin treated (3mg/kg BW), BPS treated (150mg/kg BW) and BPS + melatonin co-treatment; the male offspring were evaluated at pre-pubertal (PND 22) and pubertal (PND 42) testicular developmental stages. Lactational BPS exposure affected testicular physiology, led to histological abnormalities, hormonal imbalance, alters blood-testis-barrier (E-cadherin/connexin-43), redox modulators (SIRT-1/FOXO-1/PGC-1α; Nrf2/HO-1/pSTAT-3) and germ cell dynamicity (PCNA/TUNEL positive cells) in both pre-pubertal and pubertal mice. However, melatonin supplementation to BPS exposed lactating mothers improved testicular histoarchitecture in offspring, enhanced testicular antioxidant status, modulated expression of redox/survival and BTB markers that promoted germ cell proliferation. In conclusion, our study shows that lactational BPS exposure could be deleterious to testicular physiology that may result in male infertility/subfertility in later life while melatonin supplementation improves the reproductive health compromised by lactational BPS exposure.

Conjugated linoleic acid (CLA) is known for antiobesity. However, the role of CLA in regulating high-fat diet (HFD)-impaired pubertal mammary gland development remains undefined. Here, pubertal female mice and HC11 cells were treated with HFD or palmitic acid (PA), supplemented with or without CLA, respectively. We found that CLA prevented impaired mammary gland development in pubertal mice exposed to HFD. In vitro, c9, t11-CLA, but not t10, c12-CLA, promoted PA-suppressed HC11 proliferation, accompanied by hindered CD36 palmitoylation and localization on the plasma membrane. Moreover, c9, t11-CLA reduced the formation of the CD36/Fyn/Lyn complex and inhibited the JNK pathway while activated the ERK pathway in PA-treated HC11. In mechanism, the activation of the JNK pathway and the inhibition of ERK abolished the c9, t11-CLA-stimulated proliferation of PA-treated HC11. In vivo verification, CLA reduced the total and cell membrane CD36 palmitoylation, suppressed the formation of the CD36/FYN/LYN complex, and inhibited the JNK pathway but activated the ERK pathway in the mammary gland of HFD-fed mice. In conclusion, CLA mitigated HFD-impaired mammary gland development of pubertal mice and PA-suppressed HC11 proliferation via CD36 palmitoylation and the downstream JNK-ERK pathway. These data suggested the potential application of CLA in ameliorating obesity-impaired pubertal mammary gland development.

This study elucidates the significant impact of alcohol exposure on the gut microbiota and metabolic pathways in mice, highlighting the differential responses between adolescent and adult stages. Alcohol exposure was found to damage the intestinal barrier, alter the microbial composition by decreasing beneficial bacteria like Lactobacillus, and increase harmful bacteria such as Alistipes. The study also discovered unique microbial changes and resilience in pubertal mice. Species-level metagenomic analysis revealed specific microbial taxa and metabolic functions affected by alcohol. Metagenome-assembled genomes (MAGs) found many species that could not be annotated by conventional methods including many members of Lachnospiraceae, greatly expanding our understanding of the gut microbiota composition. These findings underscore the need for further research on alcohol's effects on various organs and the implications of microbial metabolites on disease progression.

The “gut-brain axis” is involved in many physiological processes. However, its role in regulating mammary gland (MG) development remains unknown. In this study, we established the mice model of bilateral subdiaphragmatic vagotomy (Vago) to clarify the effects of “gut-brain axis” on MG development in pubertal mice. The results showed that Vago reduced the ratio of Lactobacillus and Bifidobacterium, neuronal excitability in the nucleus of solitary tract (NTS), and synthesis and secretion of BDNF, thereby slowing MG development. Transplanting the gut microbiota of Vago mice to recipient mice replicated these effects, and transplanting the gut microbiota of Control mice to Vago mice did not alleviate these effects. Galacto-Oligosaccharide (GOS), which up-regulates the ratio of Lactobacillus and Bifidobacterium, supplementation elevated NTS neuron excitability, synthesis and secretion of BDNF, and MG development, but Vago reversed these benefits. In conclusion, GOS enhances BDNF-mediated mammary gland development in pubertal mice via the “gut-brain axis”.

The early exposure of nutrients during pubertal mammary gland development may reduce the risk of developing breast cancer later in life. Anticancer n-3 polyunsaturated fatty acids (n-3 PUFA) are shown to modulate pubertal mammary gland development; however, the mechanisms of action remain unclear. Prior work focused on effects at the whole tissue level, and little is known at the cellular level, such as at the level of mammary epithelial cells (MECs), which are implicated in cancer development. This pilot study examined the effects of lifelong n-3 PUFA exposure on the transcriptome by RNA-Seq in the isolated MECs of pubertal (6-8-week-old) female fat-1 transgenic mice capable of de novo n-3 PUFA synthesis. edgeR and DESeq2 were used separately for the differential expression analysis of RNA sequencing data followed by the Benjamani-Hochberg procedure for multiple testing correction. Nine genes were found concordant and significantly different (p ≤ 0.05) by both the DESeq2 and edgeR methods. These genes were associated with multiple pathways, suggesting that n-3 PUFA stimulates estrogen-related signaling (Mlltl0, Galr3, and Nrip1) and a glycolytic profile (Soga1, Pdpr, and Uso1) while offering protective effects for immune and DNA damage responses (Glpd1, Garre1, and Rpa1) in MECs during puberty. This pilot study highlights the utility of RNA-Seq to better understanding the mechanistic effects of specific nutrients such as n-3 PUFA in a cell-specific manner. Thus, further studies are warranted to investigate the cell-specific mechanisms by which n-3 PUFA influences pubertal mammary gland development and breast cancer risk later in life.

Melatonin protects spermatogenic cells against DNA damage and necroptosis induced by atrazine

Hepatic-derived extracellular vesicles in late pregnancy promote mammary gland development by stimulating prolactin receptor-mediated JAK2/STAT5/mTOR signalling

Abstract N.D. Mansano: None. J. Kearney: None. S. Wray: None. GnRH neurons are essential for fertility, a process modulated by numerous extrinsic and intrinsic cues. As such, GnRH neurons integrate incoming signals from a wide range of systems to ensure reproductive success. GnRH neurons express receptors related to food intake, such as, the orexigenic hormone Neuropeptide Y (NPY) and anorexigenic hormone proopiomelanocortin (POMC), potentially linking metabolic function and reproduction. Although it has been reported that GnRH neurons respond independently to NPY and α-melanocyte-stimulating hormone (α-MSH, product from POMC) in adult mice, it’s unclear how these circuit might interact. This study examines the relationship between α-MSH and NPY signaling in GnRH neurons using calcium imaging of GnRH cells maintained in explants and in situ loose-cell patch-clamp recordings in brain slices from GnRH-GFP mice at different reproductive stages: prepubertal (PN10), pubertal (PN35) and adult male and female mice (PN 60-90). In addition, immunocytochemical analysis will be performed at the same stages to determine the expression of NPY receptors (Y1R, Y4R) and α-MSH receptors (MC4, MC1) in GnRH cells, receptors so far identified in GnRH neurons. For both calcium imaging electrophysiological experiments, an amino acid blocker (AAB) was added after the first serum-free media or artificial cerebrospinal fluid period to help delineate direct from indirect responses via interneurons. The AAB contains inhibitors to ionotropic glutamatergic receptors (-Cyano-7-nitroquinoxaline-2,3-dione (CQNX, 10µM), D-2-Amino-5-phospho-nopentanoic acid (D-AP5, 10µM)) and GABAA receptors (-Bicuculline methiodide (BIC, 10µM). To date, results indicate that calcium oscillations decreased in the majority of GnRH cells in explants, devoid of brain, consistent with the literature. Notably, after 5 min washout ∼22% of the GnRH cells responded with an increase in activity to α-MSH (50nM, n=90 cells/2 explants). Electrophysiological recordings show that in slices from pubertal mice α-MSH increases activity (♀n=12 cells/3mice); ♂n=10 cells/3 mice) but not in slices from adult males (♂n=10 cells/4 mice). Suggesting that pubertal mice are more sensitive to the anorexigenic hormone α-MSH, consistent with the fact that puberty is accompanied by an increase in food intake and change in body weight. Currently, we are delineating the effects of α-MSH in adult females at different stages of the estrous cycle and will use receptor agonists and antagonists to determine the inter-play between metabolic cues on GnRH neuronal activity. Saturday, June 1, 2024

Abstract Disclosure: K.D. Wiggins: None. Z. Del Mundo: None. J.A. Ayala Angulo: None. M. Lopez: None. C. Garcia: None. C. Nguyen: None. J. De La Torre: None. A. Saba: None. D. Trinh: None. D. Nicholas: None. Polycystic Ovarian Syndrome (PCOS), characterized by disruptions in both reproductive and metabolic functions, is associated with abnormal immune system activity and chronic inflammation. Although the precise mechanistic drivers of chronic inflammation in PCOS are not fully elucidated, elevated serum levels of lipopolysaccharide (LPS) have been observed. We propose that Toll-like receptor 4 (TLR4) signaling, influenced by dysregulated LPS levels, is a central driver of chronic inflammation affecting both reproductive and metabolic pathways. To investigate this hypothesis, we subcutaneously implanted a nonsteroidal aromatase inhibitor, Letrozole (LET), in pubertal TLR4 knockout mice (TLR4KO) and C57BL/6J (wild type) mice. Our five-week LET treatment revealed that TLR4KO mice successfully entered all phases of the estrous cycle, with normalized Luteinizing Hormone (LH) and increased Follicle Stimulating Hormone (FSH) levels. These findings highlight TLR4's role in shaping cycling patterns and regulating fundamental hormonal pathways vital for reproductive health. Despite the genetic manipulation of the inflammatory pathway leading to a reduction in overall body weight gain, it had no discernible impact on the overall body composition of fat and lean mass. Additionally, our study found that dysregulation of blood glucose levels over time was independent of reduced body weight. TLR4KO mice demonstrated inefficient glucose clearance compared to wild-type mice, hinting at potential increases in insulin resistance, imbalances in adipose tissue inflammation, or possible alterations in the gut microbiota. Our study underscores the significance of disrupting the pro-inflammatory TLR4 pathway in shaping the reproductive and metabolic phenotypes in Letrozole-induced PCOS. The investigation into TLR4 becomes pivotal for comprehending the role of chronic inflammation in PCOS development or progression, offering valuable insights into potential therapeutic targets. Presentation: 6/2/2024

Asprosin-mediated regulation of ovarian functions in mice: An age-dependent study

Introduction: Empathy is the ability of an individual to present and respond to the emotions of others and is thought to originate from parental behavior. Testosterone could promote aggression and inhibit biparental behavior and vasopressin (AVP) could promote aggression. Given levels of aggression and parental care are closely associated with levels of empathy, we hypothesized that testosterone may influence empathetic behavior via the AVP system. Methods: We examined testosterone levels and tested social, empathic, and anxiety-like behaviors after castration surgery to pubertal mice, and subsequently examined the molecular levels of AVP, V1aR in different brain regions. Finally, pharmacological experiments were used to test the effects on empathic behavior by injecting testosterone in combination with V1aR antagonist. Results: Here, we show that pubertal castration reduced serum testosterone levels, increased empathetic behavior and sociality, and reduced anxiety-like behaviors in male C57 mice. The pubertal castration also reduced AVP and vasopressin receptor (V1aR) protein levels, and AVP mRNA levels in the PVN. It also reduced the number of AVP-positive neurons in the PVN. In addition, pubertal subcutaneous injection of testosterone reduced emotional contagion and consolation of castrated mice, while concomitant injection of V1aR antagonists into the anterior cingulate cortex (ACC) reversed the downregulation of emotional contagion and consolation induced by testosterone. Conclusion: It is suggested that testosterone in puberty regulates empathetic behavior in C57 mice possibly via the AVP system in the ACC. These findings help us to understand the neuroendocrine mechanisms underlying empathetic behavior and provide potential targets for the treatment of psychiatric disorders associated with low empathy.