High-fat diet during pregnancy promotes fetal skeletal muscle fatty acid oxidation and insulin resistance in an ovine model.

Maternal diet during pregnancy is associated with offspring metabolic risk trajectory in humans and animal models, but the prenatal origins of these effects are less clear. We examined the effects of a high-fat diet (HFD) during pregnancy on fetal skeletal muscle metabolism and metabolic risk parameters using an ovine model. White-faced ewes were fed a standardized diet containing 5% fat wt/wt (CON), or the same diet supplemented with 6% rumen-protected fats (11% total fat wt/wt; HFD) beginning 2 wk before mating until midgestation (GD75). Maternal HFD increased maternal weight gain, fetal body weight, and low-density lipoprotein levels in the uterine and umbilical circulation but had no significant effects on circulating glucose, triglycerides, or placental fatty acid transporters. Fatty acid (palmitoylcarnitine) oxidation capacity of permeabilized hindlimb muscle fibers was >50% higher in fetuses from HFD pregnancies, whereas pyruvate and maximal (mixed substrate) oxidation capacities were similar to CON. This corresponded to greater triacylglycerol content and protein expression of fatty acid transport and oxidation enzymes in fetal muscle but no significant effect on respiratory chain complexes or pyruvate dehydrogenase expression. However, serine-308 phosphorylation of insulin receptor substrate-1 was greater in fetal muscle from HFD pregnancies along with c-jun-NH2 terminal kinase activation, consistent with prenatal inhibition of skeletal muscle insulin signaling. These results indicate that maternal high-fat feeding shifts fetal skeletal muscle metabolism toward a greater capacity for fatty acid over glucose utilization and favors prenatal development of insulin resistance, which may predispose offspring to metabolic syndrome later in life.NEW & NOTEWORTHY Maternal diet during pregnancy is associated with offspring metabolic risk trajectory in humans and animal models, but the prenatal origins of these effects are less clear. This study examined the effects of a high-fat diet during pregnancy on metabolic risk parameters using a new sheep model. Results align with findings previously reported in nonhuman primates, demonstrating changes in fetal skeletal muscle metabolism that may predispose offspring to metabolic syndrome later in life.

Peptidylarginine deiminase enzymes and citrullinated proteins in female reproductive physiology and associated diseases†.

Citrullination, the post-translational modification of arginine residues, is catalyzed by the four catalytically active peptidylarginine deiminase (PAD or PADI) isozymes and alters charge to affect target protein structure and function. PADs were initially characterized in rodent uteri and, since then, have been described in other female tissues including ovaries, breast, and the lactotrope and gonadotrope cells of the anterior pituitary gland. In these tissues and cells, estrogen robustly stimulates PAD expression resulting in changes in levels over the course of the female reproductive cycle. The best-characterized targets for PADs are arginine residues in histone tails, which, when citrullinated, alter chromatin structure and gene expression. Methodological advances have allowed for the identification of tissue-specific citrullinomes, which reveal that PADs citrullinate a wide range of enzymes and structural proteins to alter cell function. In contrast to their important physiological roles, PADs and citrullinated proteins are also involved in several female-specific diseases including autoimmune disorders and reproductive cancers. Herein, we review current knowledge regarding PAD expression and function and highlight the role of protein citrullination in both normal female reproductive tissues and associated diseases.

Microdiscectomy for sciatica: reality check study of long-term surgical treatment effects of a Lumbosacral radicular syndrome (LSRS).

PURPOSE: It remains unclear whether the long-term results of RCTs regarding the outcome of microdiscectomy for lumbosacral radicular syndrome (LSRS) are generalizable. The purpose of this study was to determine the external validity of the outcome preseneted in RCTs after microdicectomy for LSRS in a patient cohort from a high-volume spine center. METHODS: Between 2007 and 2010, 539 patients had a single level microdiscectomy for MRI disk-related LSRS of whom 246 agreed to participate. Questionnaires included visual analogue scores (VAS) for leg pain, RDQ, OLBD, RAND-36 and Likert scores for recovery, leg and back pain. Lumbar re-operation(s) were registered. RESULTS: Mean age was 51.3, and median time of follow-up was 8.0 years. Re-operation occurred in 64 (26%) patients. Unfavorable perceived recovery was noted in 85 (35%) patients, and they had worse leg and back pain than the 161 (65%) patients with a favorable recovery: median VAS for leg pain 28/100 mm versus 2/100 mm and median VAS for back pain 9/100 mm versus 3/100 mm, respectively. In addition, the median RDQ and OLBD scores differed significantly: 9 vs 3 for RDQ and 26 vs 4 for OLBD, respectively (p < 0.001). CONCLUSION: In this cohort study, the long-term results after microdiscectomy for LSRS were less favorable than those obtained in RCTs, possibly caused by less strict patient selection than in RCTs. Our findings emphasize that patients, who do not meet the same inclusion criteria for surgery as in RCTs, should be informed about the chances of a less favorable result.

The Roles of Extracellular Vesicles and Organoid Models in Female Reproductive Physiology.

Culture model systems that can recapitulate the anatomy and physiology of reproductive organs, such as three-dimensional (3D) organoid culture systems, limit the cost and welfare concerns associated with a research animal colony and provide alternative approaches to study specific processes in humans and animals. These 3D models facilitate a greater understanding of the physiological role of individual cell types and their interactions than can be accomplished with traditional monolayer culture systems. Furthermore, 3D culture systems allow for the examination of specific cellular, molecular, or hormonal interactions, without confounding factors that occur with in vivo models, and provide a powerful approach to study physiological and pathological reproductive conditions. The goal of this paper is to review and compare organoid culture systems to other in vitro cell culture models, currently used to study female reproductive physiology, with an emphasis on the role of extracellular vesicle interactions. The critical role of extracellular vesicles for intercellular communication in physiological processes, including reproduction, has been well documented, and an overview of the roles of extracellular vesicles in organoid systems will be provided. Finally, we will propose future directions for understanding the role of extracellular vesicles in normal and pathological conditions of reproductive organs, utilizing 3D organoid culture systems.

MicroRNA-mRNA Networks in Pregnancy Complications: A Comprehensive Downstream Analysis of Potential Biomarkers.

Pregnancy complications are a major cause of fetal and maternal morbidity and mortality in humans. The majority of pregnancy complications initiate due to abnormal placental development and function. During the last decade, the role of microRNAs (miRNAs) in regulating placental and fetal development has become evident. Dysregulation of miRNAs in the placenta not only affects placental development and function, but these miRNAs can also be exported to both maternal and fetal compartments and affect maternal physiology and fetal growth and development. Due to their differential expression in the placenta and maternal circulation during pregnancy complications, miRNAs can be used as diagnostic biomarkers. However, the differential expression of a miRNA in the placenta may not always be reflected in maternal circulation, which makes it difficult to find a reliable biomarker for placental dysfunction. In this review, we provide an overview of differentially expressed miRNAs in the placenta and/or maternal circulation during preeclampsia (PE) and intrauterine growth restriction (IUGR), which can potentially serve as biomarkers for prediction or diagnosis of pregnancy complications. Using different bioinformatics tools, we also identified potential target genes of miRNAs associated with PE and IUGR, and the role of miRNA-mRNA networks in the regulation of important signaling pathways and biological processes.

LIN28-let-7 axis regulates genes in immortalized human trophoblast cells by targeting the ARID3B-complex.

Abnormal placental development is one of the main etiological factors for intrauterine growth restriction (IUGR). Here, we show that LIN28A and LIN28B are significantly lower and lethal-7 (let-7) microRNAs (miRNAs) significantly higher in term human IUGR vs. normal placentas. We hypothesize that let-7 miRNAs regulate genes with known importance for human placental development [high-mobility group AT-hook 1 (HMGA1), transcriptional regulator Myc-like (c-myc), vascular endothelial growth factor A (VEGF-A), and Wnt family member 1 (WNT1)] by targeting the AT-rich interacting domain (ARID)-3B complex. ACH-3P cells with LIN28A and LIN28B knockout (DKOs) significantly increased let-7 miRNAs, leading to significantly decreased ARID3A, ARID3B, and lysine demethylase 4C (KDM4C). Similarly, Sw.71 cells overexpressing LIN28A and LIN28B (DKIs) significantly decreased let-7 miRNAs, leading to significantly increased ARID3A, ARID3B, and KDM4C. In ACH-3P cells, ARID3A, ARID3B, and KDM4C make a triprotein complex [triprotein complex comprising ARID3A, ARID3B, and KDM4C (ARID3B-complex)] that binds the promoter regions of HMGA1, c-MYC, VEGF-A, and WNT1. ARID3B knockout in ACH-3P cells disrupted the ARID3B-complex, leading to a significant decrease in HMGA1, c-MYC, VEGF-A, and WNT1. DKOs had a significant reduction, whereas DKIs had a significant increase in HMGA1, c-MYC, VEGF-A, and WNT1, potentially due to regulation by the ARID3B-complex. This is the first study showing regulation of let-7 targets in immortalized human trophoblast cells by the ARID3B-complex.-Ali, A., Anthony, R. V., Bouma, G. J., Winger, Q. A. LIN28-let-7 axis regulates genes in immortalized human trophoblast cells by targeting the ARID3B-complex.

A multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep.

Sex is an important biological variable as many physiological as well as disease processes differ between females and males. The fundamental biological distinction between females and males starts with chromosomal sex, and the establishment of XX and XY cells and tissues. Polymerase Chain Reaction (PCR) is a simple and effective method to easily determine chromosomal or genetic sex of cells and tissues. The goal of this study was to develop a simple multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep. Primers were designed to amplify a fragment of the autosomal gene myogenin (MYOG) and sex determining region on the Y chromosome (SRY). PCR analysis was performed on a variety of genomic DNA samples isolated from fetal sheep skeletal muscle, brain, liver, and placenta, and revealed a single 259 bp band for MYOG in XX females, and a 259 bp band for MYOG and a 167 bp band for SRY in XY males. Amplicons were clearly distinguishable by gel electrophoresis, and their sequences revealed 100% identity to the known ovine MYOG and SRY sequence. The reported multiplex PCR genotyping assay provides a rapid means to distinguish XX and XY sheep tissues using low volume samples.

The Role of LIN28-let-7-ARID3B Pathway in Placental Development.

Placental disorders are a major cause of pregnancy loss in humans, and 40-60% of embryos are lost between fertilization and birth. Successful embryo implantation and placental development requires rapid proliferation, invasion, and migration of trophoblast cells. In recent years, microRNAs (miRNAs) have emerged as key regulators of molecular pathways involved in trophoblast function. A miRNA binds its target mRNA in the 3'-untranslated region (3'-UTR), causing its degradation or translational repression. Lethal-7 (let-7) miRNAs induce cell differentiation and reduce cell proliferation by targeting proliferation-associated genes. The oncoprotein LIN28 represses the biogenesis of mature let-7 miRNAs. Proliferating cells have high LIN28 and low let-7 miRNAs, whereas differentiating cells have low LIN28 and high let-7 miRNAs. In placenta, low LIN28 and high let-7 miRNAs can lead to reduced proliferation of trophoblast cells, resulting in abnormal placental development. In trophoblast cells, let-7 miRNAs reduce the expression of proliferation factors either directly by binding their mRNA in 3'-UTR or indirectly by targeting the AT-rich interaction domain (ARID)3B complex, a transcription-activating complex comprised of ARID3A, ARID3B, and histone demethylase 4C (KDM4C). In this review, we discuss regulation of trophoblast function by miRNAs, focusing on the role of LIN28-let-7-ARID3B pathway in placental development.

Trophectoderm-Specific Knockdown of LIN28 Decreases Expression of Genes Necessary for Cell Proliferation and Reduces Elongation of Sheep Conceptus.

LIN28 inhibits let-7 miRNA maturation which prevents cell differentiation and promotes proliferation. We hypothesized that the LIN28-let-7 axis regulates proliferation-associated genes in sheep trophectoderm in vivo. Day 9-hatched sheep blastocysts were incubated with lentiviral particles to deliver shRNA targeting LIN28 specifically to trophectoderm cells. At day 16, conceptus elongation was significantly reduced in LIN28A and LIN28B knockdowns. Let-7 miRNAs were significantly increased and IGF2BP1-3, HMGA1, ARID3B, and c-MYC were decreased in trophectoderm from knockdown conceptuses. Ovine trophoblast (OTR) cells derived from day 16 trophectoderm are a useful tool for in vitro experiments. Surprisingly, LIN28 was significantly reduced and let-7 miRNAs increased after only a few passages of OTR cells, suggesting these passaged cells represent a more differentiated phenotype. To create an OTR cell line more similar to day 16 trophectoderm we overexpressed LIN28A and LIN28B, which significantly decreased let-7 miRNAs and increased IGF2BP1-3, HMGA1, ARID3B, and c-MYC compared to control. This is the first study showing the role of the LIN28-let-7 axis in trophoblast proliferation and conceptus elongation in vivo. These results suggest that reduced LIN28 during early placental development can lead to reduced trophoblast proliferation and sheep conceptus elongation at a critical period for successful establishment of pregnancy.

BRCA1 regulates HMGA2 levels in the Swan71 trophoblast cell line.

During early placental development, tumor suppressors and oncogenes work synergistically to regulate cell proliferation and differentiation in a restrained manner compared with the uncontrollable growth in cancer. One example of this partnership is the regulation of the oncofetal protein HMGA2 by BRCA1. BRCA1 forms a repressor complex with ZNF350 and CtIP to bind to the promoter of HMGA2, preventing transcription. Chromatin immunoprecipitation determined BRCA1 forms this repressor complex in human trophoblast cells, suggesting a role in the placenta. Furthermore, miR-182 has been shown to target BRCA1 mRNA in ovarian cancer cells, blocking the formation of the BRCA1 repressor complex and allowing increased transcription of HMGA2. miR-182 was one of the first miRNAs described as elevated in the serum and placentas of preeclamptic women. Therefore, we hypothesized that BRCA1 is essential for normal trophoblast cell development. We used CRISPR-Cas9 genome editing and miR-182 overexpression to decrease BRCA1 protein in the Swan71 cell line. HMGA2 was significantly increased in the BRCA1 KO and miR-182 overexpressing cells compared to controls. We also determined that BRCA1 repressor complex binding to HMGA2 was significantly reduced in BRCA1 KO and miR-182 overexpressing cells compared with controls, leading us to conclude that increased HMGA2 was because of decreased binding of the BRCA1 repressor complex. Finally, we found that the caspase activity was significantly higher in BRCA1 KO and miR-182 overexpressing cells suggesting an increased amount of apoptosis. These data suggest that BRCA1 is an important regulator of the oncofetal protein HMGA2 and promotes cell survival in human placental cells.

LIN28B regulates androgen receptor in human trophoblast cells through Let-7c.

LIN28B is an RNA-binding protein necessary for maintaining pluripotency in stem cells and plays an important role in trophoblast cell differentiation. LIN28B action on target gene function often involves the Let-7 miRNA family. Previous work in cancer cells revealed that LIN28 through Let-7 miRNA regulates expression of androgen receptor (AR). Considering the similarities between cancer and trophoblast cells, we hypothesize that LIN28B also is necessary for the presence of AR in human trophoblast cells. The human first-trimester trophoblast cell line, ACH-3P was used to evaluate the regulation of AR by LIN28B, and a LIN28B knockdown cell line was constructed using lentiviral-based vectors. LIN28B knockdown in ACH-3P cells resulted in significantly decreased levels of AR and increased levels of Let-7 miRNAs. Moreover, treatment of ACH-3P cells with Let-7c mimic, but not Let-7e or Let-7f, resulted in a significant reduction in LIN28B and AR. Finally, forskolin-induced syncytialization and Let-7c treatment both resulted in increased expression of syncytiotrophoblast marker ERVW-1 and a significant decrease in AR in ACH-3P. These data reveal that LIN28B regulates AR levels in trophoblast cells likely through its inhibitory actions on let-7c, which may be necessary for trophoblast cell differentiation into the syncytiotrophoblast.

Do older patients with acute or subacute subdural hematoma benefit from surgery?

PURPOSE: According to the international guidelines, acute subdural hematomas (aSDH) with a thickness of >10 mm, or causing a midline shift of >5 mm, should be surgically evacuated. However, high mortality rates in older patients resulted in ongoing controversy whether elderly patients benefit from surgery. We identified predictors of outcome in a single-centre cohort of elderly patients undergoing surgical evacuation of aSDH or subacute subdural hematoma (saSDH). MATERIALS AND METHODS: This retrospective study included all patients aged ≥65 years undergoing surgical evacuation of aSDH/saSDH from 2000 to 2015. One-year outcome was dichotomized into favourable (Glasgow Outcome Scale (GOS) 4-5) and unfavourable (GOS 1-3). Predictors of outcome were identified by analysing patient characteristics. RESULTS: Eighty-four patients aged ≥65 years underwent craniotomy for aSDH/saSDH during the 16 year time period. Twenty-five percent regained functional independence, 11% survived severely disabled, and 64% died. Most patients died of respiratory failure following withdrawal of artificial respiration or following restriction of treatment. Age of the SDH or Glasgow Coma Scores ≤8/intubation did not predict unfavourable outcome. All patients with bilaterally absent pupillary light reflexes died, also those who still exhibited one normal-sized pupil. CONCLUSION: The low number of operated patients per year probably suggests that this cohort represents a selection of patients who were judged to have good chances of favouring from surgery. Functional independence at one-year follow-up was reached in 25% of patients, 64% died. Patients with bilaterally absent pupillary light reflexes did not benefit from surgery. The tendency to restrict treatment because of presumed poor prognosis may have acted as a self-fulfilling prophecy.

Shifting perspectives from "oncogenic" to oncofetal proteins; how these factors drive placental development.

Early human placental development strongly resembles carcinogenesis in otherwise healthy tissues. The progenitor cells of the placenta, the cytotrophoblast, rapidly proliferate to produce a sufficient number of cells to form an organ that will contribute to fetal development as early as the first trimester. The cytotrophoblast cells begin to differentiate, some towards the fused cells of the syncytiotrophoblast and some towards the highly invasive and migratory extravillous trophoblast. Invasion and migration of extravillous trophoblast cells mimics tumor metastasis. One key difference between cancer progression and placental development is the tight regulation of these oncogenes and oncogenic processes. Often, tumor suppressors and oncogenes work synergistically to regulate cell proliferation, differentiation, and invasion in a restrained manner compared to the uncontrollable growth in cancer. This review will compare and contrast the mechanisms that drive both cancer progression and placental development. Specifically, this review will focus on the molecular mechanisms that promote cell proliferation, evasion of apoptosis, cell invasion, and angiogenesis.

Endplate changes after lumbar discectomy with and without implantation of an annular closure device.

BACKGROUND: The implantation of a bone-anchored annular closure device (ACD) might be associated with the developed new endplate changes (EPC) after surgery. METHODS: A post hoc analysis has been done in patients from a prospective randomized multicenter study. All patients underwent limited lumbar discectomy with intraoperative randomization into the groups limited lumbar discectomy alone or additional ACD implantation. Low-dose lumbar computed tomography (CT) and clinical investigations were performed preoperatively and 12 months after the operation. RESULTS: A total of 554 patients were randomized. After exclusion of dropouts, the per-protocol population included 493 patients (251 in the control group and 242 in the ACD group); the follow-up rate was ≥ 90%. The number of patients showing EPC at baseline was similar in both groups. The number of patients showing EPC and the total EPC lesion area significantly increased in both groups over time, but significantly increased more in the EPC group for the superior and inferior endplate (all P < 0.0001). There was no association of pre-existing number and size of EPC with sex, age, or smoking habits. Correlation of clinical variables showed no relation with number, size, and increase of EPC area after surgery. CONCLUSIONS: Patients with primary lumbar disc herniation show EPC in the corresponding segments. There is a significant increase of lesion number and size within 12 months after discectomy. This increase is significantly more pronounced in the ACD group. Presence and growth of EPC is not correlated with low-back pain or ODI.

Circulating miRNAs as Potential Alternative Cell Signaling Associated with Maternal Recognition of Pregnancy in the Mare.

During early pregnancy, the conceptus and mare communicate to establish pregnancy. Cell-secreted vesicles (e.g., exosomes) have been reported in serum. Exosomes contain bioactive materials, such as miRNA, that can mediate cell responses. We hypothesized that a) exosomes are present in mare circulation and quantity varies with pregnancy status, b) exosomes contain miRNAs unique to pregnancy status, and c) miRNAs target pathways in endometrium based upon pregnancy status of the mare. First, serum samples were obtained from mares in a crossover design, with each mare providing samples from a pregnant and nonmated control cycle (n = 3/sample day) on Days 12, 14, 16, and 18 postovulation. Flow cytometry revealed the presence of serum microvesicles in mares in two different-sized populations (greater than or less than 100 nm), validated by transmission electron microscopy. Second, serum was collected on Days 9, 11, and 13 (n = 4/day), and endometrial biopsies were collected on Days 11 and 13 (n = 3/day) from pregnant and nonmated mares. Total RNA from serum exosomes was evaluated with quantitative RT-PCR using equine-specific miRNA sequences. A total of 12 miRNAs were found in different quantities on the specified days. Pathway analysis suggested that miRNAs targeted focal adhesion molecules (FAMs). Transcripts corresponding to FAMs were evaluated in endometrial biopsies. Protein levels and localization for PAK6 and RAF1 were further evaluated. Our data suggest that serum exosomes contain miRNA that differ based upon pregnancy status, and may affect mRNA expression related to focal adhesion pathway in the endometrium, with a potential role in maternal recognition of pregnancy.

Effects of age on follicular fluid exosomal microRNAs and granulosa cell transforming growth factor-ß signalling during follicle development in the mare.

Age-related decline in fertility is a consequence of low oocyte number and/or low oocyte competence resulting in pregnancy failure. Transforming growth factor (TGF)-ß signalling is a well-studied pathway involved in follicular development and ovulation. Recently, small non-coding RNAs, namely microRNAs (miRNAs), have been demonstrated to regulate several members of this pathway; miRNAs are secreted inside small cell-secreted vesicles called exosomes. The overall goal of the present study was to determine whether altered exosome miRNA content in follicular fluid from old mares is associated with changes in TGF-ß signalling in granulosa cells during follicle development. Follicular fluid was collected at deviation (n=6), mid-oestrus (n=6) and preovulation (n=6) for identification of exosomal miRNAs from young (3-12 years) and old (20-26 years) mares. Analysis of selected TGF-ß signalling members revealed significantly increased levels of interleukin 6 (IL6) in granulosa cells from mid-oestrus compared with preovulatory follicles, and collagen alpha-2(I) chain (COL1A2) in granulosa cells from deviation compared with preovulatory follicles in young mares. In addition, granulosa cells from old mares had significantly altered levels of DNA-binding protein inhibitor ID-2 (ID2), signal transducer and activator of transcription 1 (STAT1) and cell division cycle 25A (CDC25A). Finally, changes in exosomal miRNA predicted to target selected TGF-ß members were identified.

Effects of aging on gene expression and mitochondrial DNA in the equine oocyte and follicle cells.

We hypothesised that advanced mare age is associated with follicle and oocyte gene alterations. The aims of the study were to examine quantitative and temporal differences in mRNA for LH receptor (LHR), amphiregulin (AREG) and epiregulin (EREG) in granulosa cells, phosphodiesterase (PDE) 4D in cumulus cells and PDE3A, G-protein-coupled receptor 3 (GPR3), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15) and mitochondrial (mt) DNA in oocytes. Samples were collected from dominant follicles of Young (3-12 years) and Old (≥20 years) mares at 0, 6, 9 and 12h after administration of equine recombinant LH. LHR mRNA declined after 0h in Young mares, with no time effect in Old mares. For both ages, gene expression of AREG was elevated at 6 and 9h and EREG was expression was elevated at 9h, with higher expression in Old than Young mares. Cumulus cell PDE4D expression increased by 6h (Old) and 12h (Young). Oocyte GPR3 expression peaked at 9 and 12h in Young and Old mares, respectively. Expression of PDE3A increased at 6h, with the increase greater in oocytes from Old than Young mares at 6 and 9h. Mean GDF9 and BMP15 transcripts were higher in Young than Old, with a peak at 6h. Copy numbers of mtDNA did not vary over time in oocytes from Young mares, but a temporal decrease was observed in oocytes from Old mares. The results support an age-associated asynchrony in the expression of genes that are essential for follicular and oocyte maturation before ovulation.

Sex reversal in C57BL/6J XY mice caused by increased expression of ovarian genes and insufficient activation of the testis determining pathway.

Sex reversal can occur in XY humans with only a single functional WT1 or SF1 allele or a duplication of the chromosome region containing WNT4. In contrast, XY mice with only a single functional Wt1, Sf1, or Wnt4 allele, or mice that over-express Wnt4 from a transgene, reportedly are not sex-reversed. Because genetic background plays a critical role in testis differentiation, particularly in C57BL/6J (B6) mice, we tested the hypothesis that Wt1, Sf1, and Wnt4 are dosage sensitive in B6 XY mice. We found that reduced Wt1 or Sf1 dosage in B6 XY(B6) mice impaired testis differentiation, but no ovarian tissue developed. If, however, a Y(AKR) chromosome replaced the Y(B6) chromosome, these otherwise genetically identical B6 XY mice developed ovarian tissue. In contrast, reduced Wnt4 dosage increased the amount of testicular tissue present in Sf1+/- B6 XY(AKR), Wt1+/- B6 XY(AKR), B6 XY(POS), and B6 XY(AKR) fetuses. We propose that Wt1(B6) and Sf1(B6) are hypomorphic alleles of testis-determining pathway genes and that Wnt4(B6) is a hypermorphic allele of an ovary-determining pathway gene. The latter hypothesis is supported by the finding that expression of Wnt4 and four other genes in the ovary-determining pathway are elevated in normal B6 XX E12.5 ovaries. We propose that B6 mice are sensitive to XY sex reversal, at least in part, because they carry Wt1(B6) and/or Sf1(B6) alleles that compromise testis differentiation and a Wnt4(B6) allele that promotes ovary differentiation and thereby antagonizes testis differentiation. Addition of a "weak" Sry allele, such as the one on the Y(POS) chromosome, to the sensitized B6 background results in inappropriate development of ovarian tissue. We conclude that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, this dosage-sensitivity is genetic background-dependant, and the mouse strains described here are good models for the investigation of human dosage-sensitive XY sex reversal.

Identification of microRNAs in exosomes isolated from serum and umbilical cord blood, as well as placentomes of gestational day 90 pregnant sheep.

Despite reports that circulating levels of maternal serum exosomes increase during pregnancy and that placenta-specific microRNAs (miRNAs) have been identified in humans, little is known about exosomes and miRNAs during pregnancy in agriculture animals. In this study, we characterized the expression of 94 miRNAs in ovine placentomes at gestation day (GD) 90 by real-time PCR, and then investigated the presence of these miRNAs in exosome samples isolated from maternal jugular blood in non-pregnant ewes and at GD30 and GD90 and in umbilical blood collected at GD90. In maternal jugular exosome samples, 13 miRNAs were present in lower and 12 miRNAs were present in higher amounts at GD90 compared to non-pregnant (GD0) or GD30. Additionally, 12 miRNAs were present in higher amounts in umbilical venous exosomes compared to umbilical arterial exosomes; only miR-132 was lower in exosomes isolated from umbilical venous blood than from umbilical arterial blood. In placentome samples, miR-34c and miR135a abundance was higher in cotyledon tissue than in caruncle, while miR-183 and miR-379 amounts were higher in caruncle than cotyledon tissue. Only miR-379 was differentially expressed in all serum exosomes and placentome samples. Pathway analysis predicted that differentially expressed maternal serum exosomal miRNAs target Cellular Growth and Proliferation and Organ Development pathways, while umbilical serum exosomal and placentomes miRNAs were predicted to target cellular development and organismal/embryonic development.

Regulation of ACVR1 and ID2 by cell-secreted exosomes during follicle maturation in the mare.

BACKGROUND: Ovarian follicle growth and maturation requires extensive communication between follicular somatic cells and oocytes. Recently, intercellular cell communication was described involving cell-secreted vesicles called exosomes (50-150 nm), which contain miRNAs and protein, and have been identified in ovarian follicular fluid. The goal of this study was to identify a possible role of exosomes in follicle maturation. METHODS: Follicle contents were collected from mares at mid-estrous (~35 mm, before induction of follicular maturation) and pre-ovulatory follicles (30-34 h after induction of follicular maturation). A real time PCR screen was conducted to reveal significant differences in the presence of exosomal miRNAs isolated from mid-estrous and pre-ovulatory follicles, and according to bioinformatics analysis these exosomal miRNAs are predicted to target members belonging to the TGFB superfamily, including ACVR1 and ID2. Granulosa cells from pre-ovulatory follicles were cultured and treated with exosomes isolated from follicular fluid. Changes in mRNA and protein were measured by real time PCR and Western blot. RESULTS: ACVR1 mRNA and protein was detected in granulosa cells at mid-estrous and pre-ovulatory stages, and real time PCR analysis revealed significantly lower levels of ID2 (an ACVR1 target gene) in granulosa cells from pre-ovulatory follicles. Exposure to exosomes from follicular fluid of mid-estrous follicles decreased ID2 levels in granulosa cells. Moreover, exosomes isolated from mid-estrous and pre-ovulatory follicles contain ACVR1 and miR-27b, miR-372, and miR-382 (predicted regulators of ACVR1 and ID2) were capable of altering ID2 levels in pre-ovulatory granulosa cells. CONCLUSIONS: These data indicate that exosomes isolated from follicular fluid can regulate members of the TGFB/BMP signaling pathway in granulosa cells, and possibly play a role in regulating follicle maturation.