AMH protects the ovary from doxorubicin by regulating cell fate and the response to DNA damage.

Anti-Müllerian hormone (AMH) protects the ovarian reserve from chemotherapy, and this effect is most pronounced with Doxorubicin (DOX). However, the mechanisms of DOX toxicity and AMH rescue in the ovary remain unclear. Herein, we characterize these mechanisms in various ovarian cell types using scRNAseq. In the mesenchyme, DOX activates the intrinsic apoptotic signaling pathway through p53 class mediators, particularly affecting theca progenitors, while co-treament with AMH halts theca differentiation and reduces apoptotic gene expression. In preantral granulosa cells, DOX upregulates the cell cycle inhibitor Cdkn1a and dysregulates Wnt signaling, which are ameliorated by AMH co-treatment. Finally, in follicles, AMH induces Id3 , a protein involved in DNA repair, which is necessary to prevent the accumulation of DNA lesions marked by γ-H2AX in granulosa cells. Altogether this study characterizes cell, and follicle stage-specific mechanisms of AMH protection of the ovary, offering promising new avenues for fertility preservation in cancer patients undergoing chemotherapy. Highlights: Doxorubicin treatment induces DNA damage that activates the p53 pathway in stromal and follicular cells of the ovary.AMH inhibits the proliferation and differentiation of theca and granulosa cells and promotes follicle survival following Doxorubicin insult.AMH treatment mitigates Doxorubicin-induced DNA damage in the ovary by preventing the accumulation of γ-H2AX-positive unresolved foci, through increased expression of ID3, a protein involved in DNA repair.

Autologous cell transplantation for treatment of colorectal aganglionosis in mice.

Neurointestinal diseases cause significant morbidity and effective treatments are lacking. This study aimes to test the feasibility of transplanting autologous enteric neural stem cells (ENSCs) to rescue the enteric nervous system (ENS) in a model of colonic aganglionosis. ENSCs are isolated from a segment of small intestine from Wnt1::Cre;R26iDTR mice in which focal colonic aganglionosis is simultaneously created by diphtheria toxin injection. Autologous ENSCs are isolated, expanded, labeled with lentiviral-GFP, and transplanted into the aganglionic segment in vivo. ENSCs differentiate into neurons and glia, cluster to form neo-ganglia, and restore colonic contractile activity as shown by electrical field stimulation and optogenetics. Using a non-lethal model of colonic aganglionosis, our results demonstrate the potential of autologous ENSC therapy to improve functional outcomes in neurointestinal disease, laying the groundwork for clinical application of this regenerative cell-based approach.

Durable contraception in the female domestic cat using viral-vectored delivery of a feline anti-Müllerian hormone transgene.

Eighty percent of the estimated 600 million domestic cats in the world are free-roaming. These cats typically experience suboptimal welfare and inflict high levels of predation on wildlife. Additionally, euthanasia of healthy animals in overpopulated shelters raises ethical considerations. While surgical sterilization is the mainstay of pet population control, there is a need for efficient, safe, and cost-effective permanent contraception alternatives. Herein, we report evidence that a single intramuscular treatment with an adeno-associated viral vector delivering an anti-Müllerian hormone transgene produces long-term contraception in the domestic cat. Treated females are followed for over two years, during which transgene expression, anti-transgene antibodies, and reproductive hormones are monitored. Mating behavior and reproductive success are measured during two mating studies. Here we show that ectopic expression of anti-Müllerian hormone does not impair sex steroids nor estrous cycling, but prevents breeding-induced ovulation, resulting in safe and durable contraception in the female domestic cat.

Calpain modulates cyclin-dependent kinase inhibitor 1B (p27(Kip1)) in cells of the osteoblast lineage.

The ubiquitously expressed calpains-1 and -2 belong to a family of calcium-dependent intracellular cysteine proteases. Both calpains are heterodimers consisting of a large catalytic subunit and a small regulatory subunit encoded by the gene Capn4. Ablation of the calpain small subunit eliminates calpain activity and leads to embryonic lethality. We previously created osteoblast-specific Capn4 knockout mice to investigate a physiological role for the calpain small subunit in cells of the osteoblast lineage. Deletion of Capn4 reduced trabecular and cortical bone, mainly due to impaired proliferation and differentiation of cells of the osteoblast lineage. To further investigate an underlining mechanism by which osteoblast-specific Capn4 knockout mice develop an osteoporotic bone phenotype, we established osteoblastic cell lines stably expressing either control or Capn4 RNA interference for this study. Capn4 knockdown cells showed reduced cell proliferation, accumulation of total and phosphorylated cyclin-dependent kinase inhibitor 1B (p27(Kip1)) on serine 10, and reduced phosphorylation of retinoblastoma protein on threonine 821. Moreover, ablation of Capn4 increased 27 ( Kip1 ) mRNA levels, likely due to stabilized binding of Akt to protein phosphatase 2A, which presumably results in reduced phosphorylation of Akt on S473 and forkhead Box O (FoxO) 3A on T32. Collectively, calpain regulates cell proliferative function by modulating both transcription and degradation of p27(Kip1) in osteoblasts. In conclusion, calpain is a critical modulator for regulation of p27(Kip1) in cells of the osteoblast lineage.

A high fat diet-induced impaired glucose metabolism in mice with targeted deletion of calpain in osteoblasts.

The ubiquitously expressed Calpains 1 and 2 belong to a family of calcium-dependent intracellular cysteine proteases. Both calpains are heterodimers consisting of a large subunit and a small regulatory subunit encoded by the gene Capns1. To investigate a role for the calpain small subunit in cells of the osteoblast lineage in vivo, we previously generated osteoblast-specific Capns1 knockout mice and characterized their bone phenotype. In this study, we further examined effects of low calcium and high fat diets on their bone, fat, and glucose homeostasis. Osteoblast-specific Capns1 knockout mice showed significantly reduced serum levels of total and uncarboxylated osteocalcin, and this was presumably due to their impaired bone formation and bone resorption. The reduced bone resorptive function of the mutant mice was also significant under a low calcium diet. Thus, these results suggest that reduced uncarboxylated osteocalcin levels of mutant mice were, at least in part, due to their osteoporotic bone with impaired bone resorptive function. Interestingly, unlike osteocalcin knockout mice, mutant mice on a normal chow diet were leaner than control littermates; this was likely due to their reduced food intake and overall lower energy homeostasis. To test this hypothesis, we next provided mutant mice with a high fat diet and further examined an effect of their reduced uncarboxylated osteocalcin levels on body composition and glucose metabolism. The average mean body weight of mutant mice became indistinguishable with that of controls after 2 weeks on a high fat diet, and continued to show an upward trend, at least, up to 6weeks. Moreover, mutant mice on a high fat diet exhibited a significant increase in serum levels of leptin and resistin, adipocyte-specific adipokines, and developed impaired glucose tolerance. Collectively, mice with osteoporosis and reduced bone resorptive function showed reduced serum uncarboxylated osteocalcin levels and were susceptible to increase body adiposity and develop impaired glucose tolerance under a high fat diet.

Targeted deletion of Capn4 in cells of the chondrocyte lineage impairs chondrocyte proliferation and differentiation.

Calpains are calcium-dependent intracellular cysteine proteases, which include ubiquitously expressed mu- and m-calpains. Both calpains are heterodimers consisting of a large catalytic subunit and a small regulatory subunit. The calpain small subunit encoded by the gene Capn4 directly binds to the intracellular C-terminal tail of the receptor for the parathyroid hormone (PTH) and PTH-related peptide and modulates cellular functions in cells of the osteoblast lineage in vitro and in vivo. To investigate a physiological role of the calpain small subunit in cells of the chondrocyte lineage, we generated chondrocyte-specific Capn4 knockout mice. Mutant embryos had reduced chondrocyte proliferation and differentiation in embryonic growth plates compared with control littermates. In vitro analysis further revealed that deletion of Capn4 in cells of the chondrocyte lineage correlated with impaired cell cycle progression at the G(1)/S transition, reduced cyclin D gene transcription, and accumulated cell cycle proteins known as calpain substrates. Moreover, silencing of p27(Kip1) rescued an impaired cell growth phenotype in Capn4 knockdown cells, and reintroducing the calpain small subunit partially normalized cell growth and accumulated cyclin D protein levels in a dose-dependent manner. Collectively, our findings suggest that the calpain small subunit is essential for proper chondrocyte functions in embryonic growth plates.

Induction of proapoptotic gene expression and recruitment of p53 herald ovarian follicle loss caused by polycyclic aromatic hydrocarbons.

Activation of the aryl hydrocarbon receptor (AHR) by polycyclic aromatic hydrocarbons (PAH), a ubiquitous class of environmental and occupational biohazards, accelerates germ cell depletion in female mice during prenatal and postnatal life. Like AHR, BAX is also functionally required for PAH to kill oocytes. Here, we show that PAH upregulates ovarian expression of not just Bax but a large cassette of proapoptotic genes that function at multiple steps of the cell death signaling pathway. We further show that ovarian expression of p53 and several proapoptotic genes that are known transcriptional targets of p53 are increased by PAH treatment, and that mice lacking functional p53 are resistant to the ovotoxic effects of in vivo PAH exposure. This study provides further mechanistic insights into how PAH accelerate oocyte depletion in females and adds p53 to the list of genes whose functional importance to PAH-induced ovotoxicity has been demonstrated by gene knockout technology.

The postimplantation embryo differentially regulates endometrial gene expression and decidualization.

Transcriptomal changes in the uterine endometrium induced in response to the implanting embryo remain largely unknown. In this study, using Affymetrix mRNA expression microarray analysis, we identified genes differentially expressed in the murine endometrium in the presence or absence of the embryo. Compared with the pseudopregnant deciduoma induced by a mechanical stimulus in the absence of an embryo, approximately 1500 genes (753 up-regulated, 686 down-regulated; P < 0.05) were differentially expressed by at least 1.2-fold in the uterine decidua of pregnancy. Most of these genes fall into five major biological categories that include binding (45%), catalysis (24%), signal transduction (10%), transcriptional regulators (5%), and transporters (5%). This strong, embryo-induced transcriptomal impact represented approximately 10% of the total number of genes expressed in the decidualizing endometrium. Validation studies with mRNA and protein confirmed existence of the phylogenetically conserved, embryo-regulated genes involved in the following: 1) hemostasis and inflammation; 2) interferon signaling; 3) tissue growth and remodeling; and 4) natural killer cell function. Interestingly, whereas expression of many growth factors and their cognate receptors were not different between the decidual and deciduomal endometria, a number of proteases that degrade growth factors were selectively up-regulated in the decidual tissue. Increased expression of IGF and activin A neutralizing factors (i.e. HtrA1 and Fstl3) correlated with reduced stromal cell mitosis, tissue growth, and mitogenic signaling in the decidual endometrium. These results support the hypothesis that the implanting murine embryo takes a proactive role in modulating endometrial gene expression and development during early gestation.