Tal Raz

Major mouse placental compartments revealed by diffusion-weighted MRI, contrast-enhanced MRI, and fluorescence imaging.

Significance Fluid motion measurements can provide valuable insight regarding the structure and function of developing placentas. This study presents to our knowledge the first MRI characterization of multicompartmental diffusion and incoherent flow in pregnant mice at different gestation stages, made possible by methods herein introduced for the single-scan acquisition of diffusion-encoded images in the challenging environment associated with in vivo embryonic studies. These methods were combined with a customized contrast agent to reveal a freely diffusive maternal blood pool, a strongly perfused fetal blood flow, and an intermediate behavior for the trophoblastic labyrinth cell layer. Structural features associated with these dynamics were corroborated with ex vivo fluorescence microscopy and are discussed within the context of the anatomical structure of developing mouse placentas. Mammalian models, and mouse studies in particular, play a central role in our understanding of placental development. Magnetic resonance imaging (MRI) could be a valuable tool to further these studies, providing both structural and functional information. As fluid dynamics throughout the placenta are driven by a variety of flow and diffusion processes, diffusion-weighted MRI could enhance our understanding of the exchange properties of maternal and fetal blood pools—and thereby of placental function. These studies, however, have so far been hindered by the small sizes, the unavoidable motions, and the challenging air/water/fat heterogeneities, associated with mouse placental environments. The present study demonstrates that emerging methods based on the spatiotemporal encoding (SPEN) of the MRI information can robustly overcome these obstacles. Using SPEN MRI in combination with albumin-based contrast agents, we analyzed the diffusion behavior of developing placentas in a cohort of mice. These studies successfully discriminated the maternal from the fetal blood flows; the two orders of magnitude differences measured in these fluids’ apparent diffusion coefficients suggest a nearly free diffusion behavior for the former and a strong flow-based component for the latter. An intermediate behavior was observed by these methods for a third compartment that, based on maternal albumin endocytosis, was associated with trophoblastic cells in the interphase labyrinth. Structural features associated with these dynamic measurements were consistent with independent intravital and ex vivo fluorescence microscopy studies and are discussed within the context of the anatomy of developing mouse placentas.

Single-Molecule Sequencing Reveals Estrogen-Regulated Clinically Relevant lncRNAs in Breast Cancer.

Estrogen receptor (ER)α-positive tumors are commonly treated with ERα antagonists or inhibitors of estrogen synthesis, but most tumors develop resistance, and we need to better understand the pathways that underlie the proliferative and tumorigenic role of this estrogen-activated transcription factor. We here present the first single-molecule sequencing of the estradiol-induced ERα transcriptome in the luminal A-type human breast cancer cell lines MCF7 and T47D. Sequencing libraries were prepared from the polyadenylated RNA fraction after 8 hours of estrogen or vehicle treatment. Single-molecule sequencing was carried out in biological and technical replicates and differentially expressed genes were defined and analyzed for enriched processes. Correlation analysis with clinical expression and survival were performed, and follow-up experiments carried out using time series, chromatin immunoprecipitation and quantitative real-time PCR. We uncovered that ERα in addition to regulating approximately 2000 protein-coding genes, also regulated up to 1000 long noncoding RNAs (lncRNAs). Most of these were up-regulated, and 178 lncRNAs were regulated in both cell lines. We demonstrate that Long Intergenic Non-protein Coding RNA 1016 (LINC01016) and LINC00160 are direct transcriptional targets of ERα, correlate with ERα expression in clinical samples, and show prognostic significance in relation to breast cancer survival. We show that silencing of LINC00160 results in reduced proliferation, demonstrating that lncRNA expression have functional consequences. Our findings suggest that ERα regulation of lncRNAs is clinically relevant and that their functions and potential use as biomarkers for endocrine response are important to explore.

Ovarian Dendritic Cells Act as a Double-Edged Pro- Ovulatory and Anti-Inflammatory Sword

Ovulation and inflammation share common attributes, including immune cell invasion into the ovary. The present study aims at deciphering the role of dendritic cells (DCs) in ovulation and corpus luteum formation. Using a CD11c-EYFP transgenic mouse model, ovarian transplantation experiments, and fluorescence-activated cell sorting analyses, we demonstrate that CD11c-positive, F4/80-negative cells, representing DCs, are recruited to the ovary under gonadotropin regulation. By conditional ablation of these cells in CD11c-DTR transgenic mice, we revealed that they are essential for expansion of the cumulus-oocyte complex, release of the ovum from the ovarian follicle, formation of a functional corpus luteum, and enhanced lymphangiogenesis. These experiments were complemented by allogeneic DC transplantation after conditional ablation of CD11c-positive cells that rescued ovulation. The pro-ovulatory effects of these cells were mediated by up-regulation of ovulation-essential genes. Interestingly, we detected a remarkable anti-inflammatory capacity of ovarian DCs, which seemingly serves to restrict the ovulatory-associated inflammation. In addition to discovering the role of DCs in ovulation, this study implies the extended capabilities of these cells, beyond their classic immunologic role, which is relevant also to other biological systems.

Folliculogenesis, embryo parameters and post‐transfer recipient pregnancy rate following equine follicle‐stimulating hormone (eFSH) treatment in cycling donor mares

BACKGROUND Induction of multiple ovulations, or superovulation, may potentially increase the efficiency of equine embryo transfer programs. Our objective was to investigate the effects of equine follicle-stimulating hormone (eFSH) treatment on the success rate of embryo transfer programs in mares. METHODS In the research facility of the University of Saskatchewan, Canada, we studied 12 donor mares and 37 recipient mares during the physiological breeding season. Donor mares were used in two consecutive oestrous cycles: the first served as the control cycle and in the second an eFSH regimen was applied (eFSH cycle). In the control cycle, mares were administered human chorionic gonadotropin (hCG) to induce ovulation when a follicle ≥35 mm in diameter was detected by transrectal ultrasonographic examination. In the second oestrous cycle, twice-daily eFSH treatment was initiated when a follicle ≥25 mm was detected and treatment ceased when a follicle ≥35 mm was present, at which time hCG was administered. All donor mares were artificially inseminated while in oestrus using fresh semen collected from a stallion of proven fertility. At 8 days post-ovulation, embryos were recovered transcervically and transferred individually to the uterus of a synchronised recipient mare. RESULTS The eFSH treatment stimulated the ovary and resulted in greater numbers of ovulations and recovered embryos; however the recovered embryos tended to have a lower morphological grade than the control embryos, and the recipient pregnancy rate per transferred embryo was lower than anticipated. CONCLUSION The numbers of recipient pregnancies and foals born that resulted from eFSH treatment were not different from the control.

Chronic Akt1 deficiency attenuates adverse remodeling and enhances angiogenesis after myocardial infarction.

Background—Akt1 is a key signaling molecule in multiple cell types, including endothelial cells. Accordingly, Akt1 was proposed as a therapeutic target for ischemic injury in the context of myocardial infarction (MI). The aim of this study was to use multimodal in vivo imaging to investigate the impact of systemic Akt1 deficiency on cardiac function and angiogenesis before and after MI. Methods and Results—In vivo cardiac MRI was performed before and at days 1, 8, 15, and 29 to 30 after MI induction for wild-type, heterozygous, and Akt1-deficient mice. Noninfarcted hearts were imaged using ex vivo stereomicroscopy and microcomputed tomography. Histological examination was performed for noninfarcted hearts and for hearts at days 8 and 29 to 30 after MI. MRI revealed mildly decreased baseline cardiac function in Akt1 null mice, whereas ex vivo stereomicroscopy and microcomputed tomography revealed substantially reduced coronary macrovasculature. After MI, Akt1–/– mice demonstrated significantly attenuated ventricular remodeling and a smaller decrease in ejection fraction. At 8 days after MI, a larger functional capillary network at the remote and border zone, accompanied by reduced scar extension, preserved cardiac function, and enhanced border zone wall thickening, was observed in Akt1–/– mice when compared with littermate controls. Conclusions—Using multimodal imaging to probe the role of Akt1 in cardiac function and remodeling after MI, this study revealed reduced adverse remodeling in Akt1-deficient mice after MI. Augmented myocardial angiogenesis coupled with a more functional myocardial capillary network may facilitate revascularization and therefore be responsible for preservation of infarcted myocardium.

In search of signaling pathways critical for ovarian graft reception: Akt1 is essential for long-term survival of ovarian grafts

OBJECTIVE To explore the role of Akt1, a principle modulator of angiogenesis, in ovarian graft reception and to investigate whether Akt1 deficiency can alter ovarian graft reception. DESIGN Experimental mouse model. SETTING Research institute. ANIMAL(S) Donors: Akt1 knockout (Akt1(-/-)) and wild types (Akt1(+/+)) mice. Recipients: CD-1 nude immune deficient female mice. INTERVENTION(S) Ovaries from Akt1(-/-) and Akt1(+/+) mice transplanted in the biceps femoris muscle of immunocompromised CD-1 mice, and ovarian graft viability, perfusion, and revascularization explored in vivo by magnetic resonance imaging (MRI). MAIN OUTCOME MEASURE(S) Vascular density and permeability of newly formed graft blood vessels quantified by dynamic contrast-enhanced MRI 7, 14, 30, and 60 days after grafting as indicators for angiogenesis and reestablishment of blood perfusion. RESULT(S) The Akt1(-/-) ovarian grafts showed a gradual decrease in angiogenic response with time after transplantation, ultimately leading to complete or near-complete graft destruction coinciding with massive follicular loss. Sixty days after transplantation, the mean blood volume fraction (fBV) and vessel permeability (PS) were statistically significantly lower in Akt1(-/-) transplants compared with Akt1(+/+). CONCLUSION(S) Akt1 is essential for ovarian graft reception. However, surprisingly the impact of Akt1 deficiency was most profound not in the early stages of angiogenesis but rather in long-term survival of the graft.

The Effect of Perfusate Volume on Amikacin Concentration in the Metacarpophalangeal Joint Following Cephalic Regional Limb Perfusion in Standing Horses

OBJECTIVE To determine the influence of 3 perfusate volumes on amikacin concentration in the metacarpophalangeal joint following cephalic regional limb perfusion (RLP) in standing horses. ANIMALS Seven healthy horses. METHODS Three perfusate volumes (100, 60, and 30 mL), containing 2 grams of amikacin, were tested during intravenous RLP at the cephalic vein, placing the tourniquet at mid antebrachium, in standing sedated horses. Synovial fluid was collected from the metacarpophalangeal joint before perfusion and at 30 and 120 minutes after perfusion. Serum samples were taken from the jugular vein at the same time points. Samples were analyzed for amikacin concentrations and a repeated measures ANOVA, followed by least squares difference pairwise comparisons to identify differences in amikacin concentration across perfusate volumes. Differences were considered significant at P<.05. RESULTS The mean amikacin concentration in synovial fluid at 30 minutes after perfusion was significantly higher following perfusate volume of 100 mL (579 μg/mL), compared to volumes of 60 mL (227 μg/mL) or 30 mL (282 μg/mL) (P<.05). When a threshold of 160 μg/mL was used, more horses reached the synovial therapeutic threshold following perfusate volume of 100 mL (100%), than horses receiving 60 mL (43%) and 30 mL (57%) at 30 minutes after injection. CONCLUSION The use of 100 mL volume for RLP at the cephalic vein in standing horses resulted in higher concentration of amikacin in the synovial fluid and is recommended for use in clinical cases.

Feeders of Free-Roaming Cats: Personal Characteristics, Feeding Practices, and Data on Cat Health and Welfare in an Urban Setting of Israel

Cat feeders serve as an important source of available food for free-roaming cats (FRCs) and can play a central role in providing data on FRC distribution, welfare, and health. Data on cat feeder personalities as well as a better understanding of their feeding practices offer relevance for decision making concerning FRC population control strategies. The current study surveyed 222 FRC feeders who responded to a municipal trap-neuter-return (TNR) campaign in an Israeli central urban setting. The aim of the study was to describe their personal characteristics, feeding practices, and the FRC populations they feed. Feeders were divided into four groups according to the number of cats they claimed to feed per day (group 1: fed up to 5 cats, group 2: fed 6–10 cats, group 3: fed 11–20 cats, and group 4: fed ≥21 cats). Most feeders were women (81%), with a median age of 58 years (range 18–81). The feeders reported an overall feeding of 3337 cats in 342 different feeding locations. Feeders of group 4 comprised 15.31% (n = 34) of all feeders but fed 56% (n = 1869) of the FRC in 37.42% (n = 128) of the feeding locations. “Heavy” feeders (groups 3 and 4) reported that they traveled significantly longer distances in order to feed the cats. Commercial dry food consisted of 90% of the food they provided, with 66% of them feeding once a day, with less food per cat per day than the other feeder groups. Interestingly, “heavy” feeders were usually singles, had on average fewer offspring, a clear preference for owning cats as pets, and lived in lower income neighborhoods. According to the feeders’ reports on the FRC populations they fed, 69.7% (2325/3337) cats were neutered and 11.8% (395/3337) were kittens. In addition, they reported that 1.6% (54/3337) of the cats were limping, 2% (67/3337) suffered from a systemic disease, 4% (135/3337) had skin lesions, and 3.9% (130/3337) were suffering from a chronic disability. Abundance of kittens and morbidity rate were significantly and negatively associated with neutering rate. These findings are in accordance with the suggestion that neutering may potentially improve cat welfare by reducing morbidity. Collaboration by the authorities with these heavy feeders, who represent a small number of FRC feeders and feed substantial FRC numbers, may be significant for the control and monitoring of FRC populations and their resources.

Evaluation of two oestrus synchronization regimens in eFSH-treated donor mares.

Reliable methods for regulating oestrus and superovulation in equine embryo transfer (ET) programs are desirable. The objective in this study was to compare two oestrus synchronization methods combined with equine follicle-stimulating hormone (eFSH) treatment in an ET program. In the progesterone and estradiol-17β (P&E) group, mares (n=12) were given progesterone and estradiol-17β, daily for 10 days, followed by prostaglandin (PG)F(2α) on the last day. In the PG group, mares (n=12) were given PGF(2α) 5 days post-ovulation. In both groups donor mares were allocated to eFSH therapy, and were subsequently bred. Embryo recovery and transfer were performed routinely. The interval to ovulation (mean ± SEM, range) was not statistically different between donor mares in the P&E group (10.2±0.3, 9-12 days) and donor mares in the PG group (8.7±0.7, 4-12 days). Among donor mares, the synchrony of ovulations was higher following the P&E regimen (P<0.05); however, there was a tendency (P<0.06) for fewer ovulations than in the PG group (1.5±0.3 vs. 2.5±0.4 ovulations, respectively). Embryo recovery (0.9±0.3 vs. 1.4±0.3 embryo/recovery) and recipient pregnancy rate per transferred embryo (4/9, 44% vs. 4/15, 27%) were similar. It was concluded that the P&E regimen was more reliable for synchronization of oestrus in eFSH-treated mares but the fewer ovulations may curtail any advantage of this regimen.

Genetic and Pharmacological Modulation of Akt1 for Improving Ovarian Graft Revascularization in a Mouse Model

ABSTRACT Ovarian tissue cryopreservation and transplantation is one of a few available treatments for fertility preservation in women diagnosed with cancer. Rapid revascularization is essential for reducing hypoxic damage after grafting and protecting the primordial follicles reserve. Using a mouse model of heterotopic ovarian graft transplantation, we have delineated the role of endothelial Akt1 expression using longitudinal magnetic resonance imaging follow-up to quantify angiogenic response. Endothelial Akt1 activation in ovarian grafts promoted angiogenesis to support the graft during posttransplantation hypoxic period. Similarly, simvastatin therapy activated Akt1 at the transplantation site and improved the revascularization and vascular support of ovarian grafts. These results serve as an important first step toward pharmacological intervention to improve revascularization of ovarian grafts and restoration of fertility in cancer survivors. The pro-angiogenic effects reported here may extend beyond improving ovarian graft reception in fertility preservation and could potentially be used for different organ or tissue transplantation.