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Canine mammary tumours (CMTs) are the most common neoplasms in intact bitches, and few chemotherapeutic options are available for highly invasive and metastatic tumours. Recent studies have shown the potential involvement of dysregulated Hippo signalling in CMT development and progression. Statins can activate the Hippo pathway by blocking protein geranylgeranylation (GGylation), resulting in decreased expression and activity of the transcriptional co-activators YAP and TAZ. In this study, we therefore sought to determine if statins could exert anti-cancer effects in CMT cells. Our results demonstrate that Atorvastatin and Fluvastatin are cytotoxic to two CMT cell lines (CMT9 and CMT47), with ED50 values ranging from 0.95 to 23.5 μM. Both statins acted to increase apoptosis and promote cell cycle arrest. Both statins also decreased YAP and TAZ expression and reduced the mRNA levels of key Hippo transcriptional target genes known to be involved in breast cancer progression and chemoresistance (CYR61, CTGF and RHAMM). Moreover, both statins effectively inhibited cell migration and anchorage independent growth, but did not influence matrix invasion. Taken together, our results demonstrate for the first time that statins act upon the Hippo pathway in CMT cells to counteract several molecular and cellular hallmarks of cancer. These findings suggest that targeting the Hippo pathway with statins represents a novel and promising approach for the treatment canine mammary gland cancers.

Recent reports suggest that the Hippo signaling pathway influences ovarian follicle development; however, its exact roles remain unknown. Here, we examined the ovarian functions of the Hippo kinases large tumor suppressors (LATS)1 and 2, which serve to inactivate the transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Inactivation of Lats1/2 in murine granulosa cells either in vitro or in vivo resulted in a loss of granulosa cell morphology, function, and gene expression. Mutant cells further underwent changes in structure and gene expression suggestive of epithelial-to-mesenchymal transition and transdifferentiation into multiple lineages. In vivo, granulosa cell–specific loss of Lats1/2 caused the ovarian parenchyma to be mostly replaced by bone tissue and seminiferous tubule-like structures. Transdifferentiation into Sertoli-like cells and osteoblasts was attributed in part to the increased recruitment of YAP and TAZ to the promoters of sex-determining region Y box 9 and bone γ-carboxyglutamate protein, key mediators of male sex determination and osteogenesis, respectively. Together, these results demonstrate for the first time a critical role for Lats1/2 in the maintenance of the granulosa cell genetic program and further highlight the remarkable plasticity of granulosa cells.—Tsoi, M., Morin, M., Rico, C., Johnson, R. L., Paquet, M., Gévry, N., Boerboom, D. Lats1 and Lats2 are required for ovarian granulosa cell fate maintenance.

Canine mammary tumours (CMTs) are common neoplasms in dogs that feature many of the clinical, genetic and molecular characteristics of human breast cancer. Despite their high metastatic potential, few adjuvant chemotherapeutic treatment options exist for malignant CMTs, and the development of novel, targeted pharmacological approaches will require a better understanding of their pathogenesis. As recent evidence suggests that dysregulated Hippo signalling is involved in the development and progression of breast cancer, we sought to determine if this pathway could also play a role in CMT. The expression of the Hippo signalling effectors YAP and TAZ was analysed by immunoblotting and immunohistochemistry in samples including normal mammary gland, lobular hyperplasia, benign tumours and malignant tumours of all grades. We found a significant increase in TAZ (but not YAP) expression occurred in lobular hyperplasia relative to normal mammary gland, suggesting a role for TAZ in non-neoplastic epithelial proliferation. Nuclear expression of both TAZ and YAP were significantly higher in malignant tumours than in benign ones, suggesting that Hippo dysregulation could play a role in CMT malignant transformation. No differences in YAP or TAZ expression were detected between grades of malignant tumours. Together, our results indicate that alterations in Hippo signalling may play a role in the pathogenesis of CMT, in a manner similar to breast cancer. Hippo pathway components may therefore represent targets for the development of novel chemotherapeutic agents that could be useful for the treatment of both the human and canine diseases.

Valosin-containing protein (VCP), through its critical role in the maintenance of protein homeostasis, is a promising target for the treatment of several malignancies, including canine lymphoma. CB-5083, a first-in-class VCP inhibitor, exerts cytotoxicity through the induction of irreversible proteotoxic stress and possesses a broad spectrum of anticancer activity. Here, we determined the cytotoxicity CB-5083 in canine lymphoma cells and its mechanism of action in vitro. Canine lymphoma cell lines were treated with varying concentrations of CB-5083 and assessed for viability by trypan blue exclusion and apoptosis by caspase activity assays. The mechanism of CB-5083 action was determined by immunoblotting and RT-qPCR analyses of Lys48 ubiquitination and markers of ER stress (DDIT3), autophagy (SQSTM1, MAP1LC3A) and DNA damage (γH2AX). Unfolded protein response markers were also evaluated by immunoblotting (eIF2α, P-eIF2α) and RT-qPCR (ATF4). CB-5083 treatment resulted in preferential cytotoxicity in canine lymphoma cell lines over control peripheral blood mononuclear cells. CB-5083 rapidly disrupted the ubiquitin-dependent protein degradation system, inducing sustained ER stress as indicated by a dramatic increase in DDIT3. Activation of the unfolded protein response occurred through the increase eIF2α phosphorylation and increased transcription of ATF4, but did not re-establish protein homeostasis. Cells rapidly underwent apoptosis through activation of the caspase cascade. These results further validate VCP as an attractive target for the treatment of canine lymphoma and identify CB-5083 as a novel therapy with clinical potential for this malignancy.

WNT signaling regulates a variety of ovarian processes, including follicle development, granulosa cell (GC) proliferation and differentiation, steroidogenesis, and ovulation. The secreted frizzled-related proteins (SFRPs) comprise a family of WNT signaling antagonists. Sfrp4 expression was previously reported to be induced in ovarian GCs and cumulus cells in vivo following human chorionic gonadotropin treatment, suggesting that it may play key roles in cumulus expansion, ovulation/luteinization, and corpus luteum (CL) function. In this study, we aimed to define the physiological roles of Sfrp4 in the ovary by gene targeting. Sfrp4-null female mice were found to produce larger litters than did their wild-type littermates. Although previous studies had suggested roles of Sfrp4 in luteal cell survival, no differences in CL formation, morphology, steroidogenesis, involution, or luteal cell apoptosis were found in Sfrp4-null mice. Likewise, cumulus expansion occurred normally in Sfrp4-null mice, with minimal changes in cumulus cell gene expression. Hyperfertility in the Sfrp4-null model was ultimately attributed to decreased antral follicle atresia, leading to an enhanced ovulatory rate. Increased expression of FSH- and LH-responsive genes was found in GCs from Sfrp4-null mice, and GCs isolated from Sfrp4-null mice were found to be hyperresponsive to FSH and LH in vitro. Although Sfrp2 was found to be overexpressed in the GCs of Sfrp4-null mice (suggesting a compensatory mechanism), Sfrp2-null mice had normal fertility and ovulatory rates, and Sfrp2/4 double knockout mice did not differ from Sfrp4-null mice. Taken together, our results suggest that SFRP4 acts to attenuate GC responsiveness to gonadotropins, thereby decreasing follicle survival, ovulatory rate, and fertility.

WNT signaling plays essential roles in the development and function of the female reproductive tract. Although crosstalk with the Hippo pathway is a key regulator of WNT signaling, whether Hippo itself plays a role in female reproductive biology remains largely unknown. In this report, we show that conditional deletion of the key Hippo kinases Lats1 and Lats2 in Müllerian duct mesenchyme cells caused them to adopt the myofibroblast cell fate, resulting in profound reproductive tract developmental defects and sterility. Myofibroblast differentiation was attributed to increased YAP and TAZ expression (but not to altered WNT signaling), leading to the direct transcriptional up-regulation of Ctgf and the activation of the myofibroblast genetic program. Müllerian duct mesenchyme cells also became myofibroblasts in male mutant embryos, which impeded the development of the male reproductive tract and resulted in cryptorchidism. The inactivation of Lats1/2 in differentiated uterine stromal cells in vitro did not compromise their ability to decidualize, suggesting that Hippo is dispensable during implantation. We conclude that Hippo signaling is required to suppress the myofibroblast genetic program and maintain multipotency in Müllerian mesenchyme cells.

The aim of the present study was to establish the presence of BMP type I and II receptor presence in preantral follicles and to investigate the effect of BMP4 supplementation on preantral follicle activation and development during organotypic culture of prepubertal ovine ovarian cortex pieces. Ovine ovarian fragments were cultured with varying concentrations (0, 25, 50 or 100 ng/ml) of BMP4 in the presence or absence of FSH in the culture media to determine the optimal minimum dose for preantral follicle activation and development. Follicular morphometry, immunohistochemistry for BMPR-IA, BMPR-IB, BMPR-II, proliferating cell nuclear antigen and TUNEL progesterone and 17β-oestradiol production were assessed. Follicle and oocyte diameter were positively influenced by the addition of BMP4 to culture. However, there was no effect of BMP4 on primordial follicle activation. Treatment with BMP4 reduced progesterone synthesis but had no effect on oestradiol. The percentage of primordial follicles stained with TUNEL was significantly greater in the culture without BMP4 compared to other culture systems after 9 days of culture. There was an increase in PCNA immunoreactivity in all follicles regardless of treatment. Treatment with BMP4 increases the size of oocytes and follicles but has minimal effects on follicular dynamics during in vitro culture of ovarian cortical tissue of sheep.

Few targeted therapies have been developed for ovarian granulosa cell tumor (GCT), even though it represents 5% of all malignant ovarian tumors in women. As misregulation of PI3K/AKT signaling has been implicated in GCT development, we hypothesized that the AKT signaling effector mammalian target of rapamycin (mTOR) may play a role in the pathogenesis of GCT and could represent a therapeutic target. Analyses of human GCT samples showed an increase in protein levels of mTOR and its downstream effectors RPS6KB1, RPS6, eIF4B and PPARG relative to normal granulosa cells, suggestive of an increase in mTOR pathway activity and increased translational activity and/or protein stability. We next sought to evaluate mTOR as a GCT therapeutic target using the Pten (tm1Hwu/tmiHwu);Ctnnb1 (tm1Mmt/+);Amhr2 (tm3(cre)Bhr/+) (PCA) mouse model, in which mTOR, RPS6KB1, eIF4B and PPARG are upregulated in tumor cells in a manner similar to human GCT. Treatment of PCA mice with the mTOR-specific inhibitor everolimus reduced tumor growth rate (1.5-fold; P < 0.05) and also reduced total tumor burden (4.7-fold; P < 0.05) and increased survival rate (78 versus 44% in the vehicle group) in a PCA surgical model of GCT peritoneal carcinomatosis. Everolimus decreased tumor cell proliferation and tumor cell volume relative to controls (P < 0.05), whereas apoptosis was unaffected. Phosphorylation of RPS6KB1 and RPS6 were decreased (P < 0.05) by everolimus, but RPS6KB1, RPS6, eIF4B and PPARG expressions were not affected. These results suggest that mTOR is a valid and clinically useful pharmacological target for the treatment of GCT, although its inhibition does not reverse all consequences of aberrant PI3K/AKT signaling in the PCA model.

Anti-Mullerian hormone (AMH) is an endocrine marker that can help predict superovulatory responses to treatments administered to cows for embryo production. However, the optimal time of the estrous cycle at which a blood test should be performed for a highly reliable prognosis has not yet been established. Moreover, little is known about the regulation of AMH production. To answer these questions, a study was designed to investigate the regulation of AMH production in cows selected for their high or low ovulatory responses to superovulation. At the granulosa cell level, AMH production was inhibited by follicle-stimulating hormone but enhanced by bone morphogenetic proteins. At the follicular level, the expression of AMH within the follicle was dependent on the stage of follicular development. At the ovarian level, the size of the pool of small antral growing follicles determined ovarian AMH production. At the endocrine level, AMH followed a specific dynamic profile during the estrous cycle, which occurred independently of the follicular waves of terminal follicular development. Cows selected for their high or low responses to superovulation did not differ in the regulation of AMH production, but cows with higher responses had higher plasma AMH concentrations throughout the cycle. The optimal period of the estrous cycle at which to measure AMH concentrations with the aim of selecting the best cows for embryo production was found to be at estrus and after Day 12 of the cycle. Based on this multiscale study, we propose a model that integrates the different regulatory levels of AMH production.

Although classically considered a WNT signaling intermediary, β-catenin (CTNNB1) can also mediate GnRH induction of gonadotropin β-subunit (Fshb and Lhb) transcription in the murine gonadotrope-like cell line LβT2. Here, we assessed CTNNB1's role in gonadotropin synthesis in vivo. We used a Cre/lox approach to introduce both gain- and loss-of-function mutations in the murine Ctnnb1 gene in gonadotrope cells. Gonadotropin production and fertility were normal in Ctnnb1 knockout mice. Similarly, females harboring a deletion of exon 3 of Ctnnb1, which stabilizes the resulting CTNNB1 protein, showed normal fertility and gonadotropin synthesis. Interestingly, males with the activating CTNNB1-Δexon 3 mutation exhibited 50% reductions in FSH synthesis and secretion, without a corresponding change in LH. This selective regulation of FSH suggested an alteration in the activin/inhibin/follistatin system. Indeed, CTNNB1-Δexon 3 males showed a 60% increase in serum inhibin B levels, and in culture, their pituitaries exhibited a greater sensitivity to exogenous inhibin than controls. At the same time, pituitary, but not testicular, follistatin (Fst) expression was increased significantly in these mice. Castration normalized FSH levels in CTNNB1-Δexon 3 males to those seen in castrated controls. Paradoxically, pituitaries from CTNNB1-Δexon 3 males exhibited greater basal and activin-stimulated FSH synthesis in vitro. Similarly, CTNNB1-Δexon 3 overexpression potentiated activin A-induced murine Fshb promoter activity in LβT2 cells. Together, these results indicate that CTNNB1 is dispensable for gonadotropin synthesis in vivo. However, sustained CTNNB1 signaling potentiates activin-induced Fshb expression in gonadotropes, but this effect is overcome in vivo by enhanced inhibin feedback sensitivity and Fst expression.

Recent evidence has suggested that vascular endothelial growth factor A (VEGFA) is an important regulator of ovarian follicle development and survival. Both LH and FSH regulate Vegfa expression in granulosa cells and signal via the transcription factor hypoxia inducible factor 1 (HIF1). To further study the mechanism of action of HIF1 in the regulation of Vegfa, we studied Vegfa(delta/delta) mice, which lack a hypoxia response element in the Vegfa promoter. Granulosa cells from Vegfa(delta/delta) mice failed to respond to FSH or LH with an increase in Vegfa mRNA expression in vitro, and granulosa cells isolated from eCG-treated immature Vegfa(delta/delta) mice had significantly lower Vegfa mRNA levels compared to controls. However, normal Vegfa mRNA levels were detected in the granulosa cells from immature Vegfa(delta/delta) mice following hCG treatment. Vegfa(delta/delta) females produced infrequent litters, and their pups died shortly after birth. Ovaries from Vegfa(delta/delta) mice were much smaller than controls and contained few antral follicles and corpora lutea. Antral follicles numbers were decreased by nearly 50% in ovaries from Vegfa(delta/delta) mice relative to controls, and 74% of antral follicles in Vegfa(delta/delta) ovaries were atretic. Serum progesterone levels in adult Vegfa(delta/delta) females were significantly lower, apparently reflecting reduced numbers of corpora lutea. This study demonstrates for the first time the requirement of HIF1 for FSH-regulated Vegfa expression in vivo and that HIF1 acts via a single hypoxia response element in the Vegfa promoter to exert its regulatory functions. Our findings also further define the physiological role of VEGFA in follicle development.

Remerciements : Laboratoire des Dosages Hormonaux UMR PRC Physiologie de la Reproduction et des Comportements, INRA Centre de Tours Chantier qualité GA; In mammals, anti-Mullerian hormone (AMH) expression is detected in the granulosa cells of all growing follicles and is highest in healthy small antral follicles, which contribute most significantly to AMH endocrine levels. AMH is a reliable endocrine marker of this population of gonadotrophin-responsive follicles in ruminants and, over the longer term, plasma AMH concentrations are characteristic of individual animals. In the cow, plasma AMH concentrations follow specific dynamic profiles throughout the prepubertal period, the oestrous cycle and the change from gestation to the post partum period, with the alterations most likely reflecting numerical changes in the population of high AMH producing follicles. In granulosa cells, bone morphogenetic proteins (BMP) enhance AMH gene expression and AMH synthesis, with these effects antagonised by FSH. BMP could both support follicular growth and contribute significantly to the induction and/or maintenance of AMH expression in small growing follicles. AMH expression decreases sharply in large follicles when they become oestrogenic, suggesting a role for FSH and/or oestradiol in these changes, but the underlying mechanisms remain hypothetical. A better understanding of the factors and mechanisms regulating AMH production is needed to propose new strategies for managing the reserve of primordial and small growing follicles, as well as for improving embryo production.

WNT4 is required for normal ovarian follicle development and female fertility in mice, but how its signal is transduced remains unknown. Fzd1 encodes a WNT receptor whose expression is markedly induced in both mural granulosa cells and cumulus cells during the preovulatory period, in a manner similar to Wnt4. To study the physiological roles of FZD1 in ovarian physiology and to determine whether it serves as receptor for WNT4, Fzd1-null mice were created by gene targeting. Whereas rare Fzd1(-/-) females were sterile because of uterine fibrosis and ovarian tubulostromal hyperplasia, most were subfertile, producing ≈1 fewer pup per litter on average relative to controls. Unlike WNT4-deficient mice, ovaries from Fzd1(-/-) mice had normal weights, numbers of follicles, steroid hormone production, and WNT4 target gene expression levels. Microarray analyses of granulosa cells from periovulatory follicles revealed few genes whose expression was altered in Fzd1(-/-) mice. However, gene expression analyses of cumulus-oocyte complexes (COCs) revealed a blunted response of both oocyte (Zp3, Dppa3, Nlrp5, and Bmp15) and cumulus (Btc, Ptgs2, Sema3a, Ptx3, Il6, Nts, Alcam, and Cspg2) genes to the ovulatory signal, whereas the expression of these genes was not altered in WNT4-deficient COCs from Wnt4(tm1.1Boer/tm1.1Boer);Tg (CYP19A1-cre)1Jri mice. Despite altered gene expression, cumulus expansion appeared normal in Fzd1(-/-) COCs both in vitro and in vivo. Together, these results indicate that Fzd1 is required for normal female fertility and may act in part to regulate oocyte maturation and cumulus cell function, but it is unlikely to function as the sole ovarian WNT4 receptor.

High between-animal variability in the number of embryos produced by multiple ovulation and embryo transfer (MOET) and ovum pick-up and in vitro production (OPU–IVP) methods remains a major limit to the development of embryo biotechnologies in cattle. The measurement of anti-Müllerian hormone (AMH) endocrine concentrations in cows can help to predict their follicular and ovulatory responses to gonadotrophin treatment. The present study aimed to provide practical information for a simple prognostic method based on AMH measurement in Holstein cows. Accurate AMH concentrations could be measured with ELISA in blood or plasma. In cows undergoing repeated OPU protocols over 1 year, the AMH concentrations measured in plasma samples collected before each gonadotrophin treatment were found to be highly repeatable and were tightly correlated with follicular responses. From data obtained at both an experimental station and farm settings, it was possible to propose AMH cut-off values to identify low-responding cows. Gonadotrophin-stimulated cows producing fewer than 15 large follicles at oestrus and fewer than 10 embryos in MOET protocols could be discarded efficiently with plasma AMH concentrations below 87 and 74 pg mL–1, respectively. In conclusion, we propose a prognostic method based on a single AMH measurement to improve the results of embryo biotechnologies.

National audience; The strong between-animal variability in the number of ovulations and embryos produced after ovarian stimulation by gonadotropins is a major limit to the development of embryo biotechnologies in cattle. In reproductive medicine, anti-mullerian hormone (AMH) is now widely used as an endocrine marker of the ovarian follicular reserve. In the cow, as in the woman, AMH is secreted by the granulosa cells of growing follicles. We have shown recently that in the cow, AMH is a very good endocrine marker of the population of small antral follicles that constitute the direct target of ovarian stimulatory treatments. AMH concentration measured in plasma before treatment varies between animals and is positively correlated to the number of ovulations and transferable embryos produced after an ovarian stimulatory treatment. Interestingly, AMH concentrations can remain stable over several months for each animal. Moreover, the number of embryos produced after ovarian stimulation is highly repeatable and has a relatively good heritability. From these observations, we propose the determination of AMH concentration in the plasma of a potential donor cow as a simple predictive method to evaluate both its level of ovarian activity and its capacity to produce high or low numbers of embryos. Optimal conditions for implementing this diagnostic test in cattle remain to be defined considering the age, the breed, the physiological status and the environmental factors related to breeding conditions for each animal.; Chez les bovins, l'existence d'une forte variabilité entre animaux du nombre d'ovulations et du nombre d'embryons produits après stimulation ovarienne constitue une limite majeure au développement des biotechnologies de l'embryon. Nous avons montré récemment que chez la vache, comme chez la femme, l'hormone antimüllérienne (AMH), sécrétée par les cellules de granulosa des follicules en croissance, est un très bon marqueur endocrinien de la folliculogenèse basale et, en particulier de la population de petits follicules à antrum qui est la principale cible des traitements de stimulation ovarienne utilisant des hormones gonadotropes. Les concentrations plasmatiques d'AMH mesurées avant traitement sont variables entre animaux et positivement corrélées aux nombres d'ovulations et d'embryons transférables produits après traitement de stimulation ovarienne. En revanche, pour un même animal, les concentrations plasmatiques d'AMH apparaissent stables sur une durée de plusieurs mois. En parallèle, il existe une bonne répétabilité individuelle et une relativement bonne héritabilité du nombre d'embryons produits après stimulation ovarienne. Ces résultats permettent de proposer la mesure de la concentration plasmatique d'AMH comme une méthode prédictive simple d'évaluation du niveau d'activité ovarienne et de la capacité de production d'embryons par une vache donneuse potentielle. Les conditions optimales pour la réalisation de ce diagnostic, prenant en compte l'âge de l'animal, sa race, son état physiologique et les facteurs environnementaux liés à ses conditions d'élevage, restent encore à définir.

In cattle, the embryo production rate after superovulation varies between individuals and is difficult to predict. Recently, we proposed that anti-Müllerian hormone (AMH) plasma levels measured before treatment can help predict superovulatory responses. To establish whether blood measurement of AMH can help predict the number of embryos produced by a given cow after superovulation, data collected over 4 years from 45 dairy cows submitted to repeated embryo production were analysed in a retrospective study. A high within-animal repeatability (0.38 and 0.36) and a strong effect of the father of the donor cow (P < 0.01) were observed for the numbers of collected and transferable embryos, respectively. AMH concentration, measured in the plasma of donor cows during first lactation and several months before the start of the embryo production campaigns, was found to be highly correlated with the maximal number of collected (P < 0.0001) and transferable (P < 0.01) embryos per cow. In conclusion, the capacity of embryo production is a repeatable and probably heritable trait in the cow, and blood measurement of AMH in potential donor cows could be of value in determining a cow’s intrinsic capacity to produce transferable embryos.

International audience; The major limitation to the development of embryo production in cattle is the strong between-animal variability in ovulatory response to FSH-induced superovulation, mainly due to differences in ovarian activity at the time of treatment. This study aimed to establish whether anti-Müllerian hormone (AMH) was an endocrine marker of follicular populations in the cow, as in human, and a possible predictor of the ovarian response to superovulation. Anti-Müllerian hormone concentrations in plasma varied 10-fold between cows before treatment and were found to be highly correlated with the numbers of 3- to 7-mm antral follicles detected by ovarian ultrasonography before treatment (r=0.79, P

International audience; Development of follicular cysts is a frequent ovarian dysfunction in cattle. Functional changes that precede cyst formation are unknown, but a role for anti-Müllerian hormone (AMH) in the development of follicular cysts has been suggested in humans. This study aimed to characterize intrafollicular steroids and AMH during follicular growth in a strain of beef cows exhibiting a high incidence of occurrence of follicular cysts. Normal follicular growth and cyst development were assessed by ovarian ultrasonography scanning during the 8 days before slaughtering. Experimental regression of cysts was followed by rapid growth of follicles that reached the size of cysts within 3-5 days. These young cysts exhibited higher intrafollicular concentrations of testosterone, estradiol-17beta, and progesterone than large early dominant follicles did in normal ovaries, but they exhibited similar concentrations of AMH. Later-stage cysts were characterized by hypertrophy of theca interna cells, high intrafollicular progesterone concentration, and high steroidogenic acute regulatory protein mRNA expression in granulosa cells. Progesterone and AMH concentrations in the largest follicles (> or =10 mm) and cysts were negatively correlated (r = -0.45, P < 0.01). Smaller follicles (

Development of follicular cysts is a frequent ovarian dysfunction in cattle. Functional changes that precede cyst formation are unknown, but a role for anti-Müllerian hormone (AMH) in the development of follicular cysts has been suggested in humans. This study aimed to characterize intrafollicular steroids and AMH during follicular growth in a strain of beef cows exhibiting a high incidence of occurrence of follicular cysts. Normal follicular growth and cyst development were assessed by ovarian ultrasonography scanning during the 8 days before slaughtering. Experimental regression of cysts was followed by rapid growth of follicles that reached the size of cysts within 3-5 days. These young cysts exhibited higher intrafollicular concentrations of testosterone, estradiol-17beta, and progesterone than large early dominant follicles did in normal ovaries, but they exhibited similar concentrations of AMH. Later-stage cysts were characterized by hypertrophy of theca interna cells, high intrafollicular progesterone concentration, and high steroidogenic acute regulatory protein mRNA expression in granulosa cells. Progesterone and AMH concentrations in the largest follicles (or =10 mm) and cysts were negatively correlated (r = -0.45, P0.01). Smaller follicles (10 mm) exhibited higher intrafollicular testosterone and estradiol-17beta concentrations in ovaries with cysts compared to normal ovaries. During follicular growth, AMH concentration dropped in follicles larger than 5 mm in diameter and in a similar way in ovaries with and without cysts. In conclusion, enhanced growth and steroidogenesis in antral follicles10 mm preceded cyst formation in cow ovaries. Intrafollicular AMH was not a marker of cystic development in the cow, but low AMH concentrations in cysts were associated with luteinization.

Background Valosin containing protein (VCP) is a critical mediator of protein homeostasis and may represent a valuable therapeutic target for several forms of cancer. Overexpression of VCP occurs in many cancers, and often in a manner correlating with malignancy and poor outcome. Here, we analyzed VCP expression in canine lymphoma and assessed its potential as a therapeutic target for this disease. Methods VCP expression in canine lymphomas was evaluated by immunoblotting and immunohistochemistry. The canine lymphoma cell lines CLBL-1, 17–71 and CL-1 were treated with the VCP inhibitor Eeyarestatin 1 (EER-1) at varying concentrations and times and were assessed for viability by trypan blue exclusion, apoptosis by TUNEL and caspase activity assays, and proliferation by propidium iodide incorporation and FACS. The mechanism of EER-1 action was determined by immunoblotting and immunofluorescence analyses of Lys48 ubiquitin and markers of ER stress (DDIT3), autophagy (SQSTM1, MAP1LC3A) and DNA damage (γH2AFX). TRP53/ATM-dependent signaling pathway activity was assessed by immunoblotting for TRP53 and phospho-TRP53 and real-time RT-PCR measurement of Cdkn1a mRNA. Results VCP expression levels in canine B cell lymphomas were found to increase with grade. EER-1 treatment killed canine lymphoma cells preferentially over control peripheral blood mononuclear cells. EER-1 treatment of CLBL-1 cells was found to both induce apoptosis and cell cycle arrest in G1. Unexpectedly, EER-1 did not appear to act either by inducing ER stress or inhibiting the aggresome-autophagy pathway. Rather, a rapid and dramatic increase in γH2AFX expression was noted, indicating that EER-1 may act by promoting DNA damage accumulation. Increased TRP53 phosphorylation and Cdkn1a mRNA levels indicated an activation of the TRP53/ATM DNA damage response pathway in response to EER-1, likely contributing to the induction of apoptosis and cell cycle arrest. Conclusions These results correlate VCP expression with malignancy in canine B cell lymphoma. The selective activity of EER-1 against lymphoma cells suggests that VCP will represent a clinically useful therapeutic target for the treatment of lymphoma. We further suggest a mechanism of EER-1 action centered on the DNA repair response that may be of central importance for the design and characterization of VCP inhibitory compounds for therapeutic use. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1489-1) contains supplementary material, which is available to authorized users.