ABSTRACT
OBJECTIVE: To determine whether increasing the dose of ulipristal acetate (UPA)-containing emergency contraception (EC) improves pharmacodynamic outcomes in individuals with obesity. STUDY DESIGN: We enrolled healthy, regularly-cycling, confirmed ovulatory, reproductive-age individuals with body mass index (BMI) >30 kg/m2 and weight >80 kg in a randomised crossover study. We monitored participants with transvaginal ultrasound and blood sampling for progesterone, luteinising hormone (LH), and estradiol every other day until a dominant follicle measuring >15 mm was visualised. At that point, participants received either oral UPA EC 30 mg or 60 mg and returned for daily monitoring up to 7 days. After a no treatment washout cycle, participants returned for a second monitored cycle and received the other UPA dose. Our primary outcome was the proportion of subjects with no follicle rupture 5 days post-dosing (yes/no). For reference, we also enrolled a control group with BMI <25 kg/m2 and weight <80 kg who received UPA EC 30 mg during a single cycle. We also obtained blood samples for pharmacokinetic parameters for UPA and its active metabolite, N-monodemethyl-UPA (NDM-UPA) as an optional substudy. RESULTS: We enrolled a total of 52 participants with BMI >30 kg/m2 and 12 controls, with the following cycles completed: 12 controls, 49 UPA 30 mg, and 46 UPA 60 mg. The entire cohort demographics were a mean (SD) age of 29.8 (3.4) years and BMI by group: controls 22.5 (1.4) kg/m2, group 1 37.9 (6.7) kg/m2, and group 2 39.3 (5.4) kg/m2. All 12 (100%) of controls had a delay of at least 5 days for follicle rupture. Among the high BMI group, dosing groups (UPA EC 30 mg vs 60 mg) were similar in the proportion of cycles without follicle rupture over 5 days post-UPA dosing (UPA 30 mg: 47/49 (96%), UPA 60 mg: 42/46 (91%), Fisher's exact test p=0.43). However, after excluding cycles where dosing occurred too late (after LH surge), a delay of at least 5 days occurred in all participants at both doses. The 60 mg UPA dose resulted in a twofold increase in maximum observed concentration and the area under the curve of both UPA and NDM-UPA levels compared with 30 mg. CONCLUSION: A standard 30 mg dose of UPA is sufficient to delay ovulation regardless of BMI or weight. Results of our study do not support dose adjustment for body size.
ABSTRACT
Huntington's disease (HD) arises from expanded CAG repeats in exon 1 of the Huntingtin (HTT) gene. The resultant misfolded HTT protein accumulates within neuronal cells, negatively impacting their function and survival. Ultimately, HTT accumulation results in cell death, causing the development of HD. A nonhuman primate (NHP) HD model would provide important insight into disease development and the generation of novel therapies due to their genetic and physiological similarity to humans. For this purpose, we tested CRISPR/Cas9 and a single-stranded DNA (ssDNA) containing expanded CAG repeats in introducing an expanded CAG repeat into the HTT gene in rhesus macaque embryos. Analyses were conducted on arrested embryos and trophectoderm (TE) cells biopsied from blastocysts to assess the insertion of the ssDNA into the HTT gene. Genotyping results demonstrated that 15% of the embryos carried an expanded CAG repeat. The integration of an expanded CAG repeat region was successfully identified in five blastocysts, which were cryopreserved for NHP HD animal production. Some off-target events were observed in biopsies from the cryopreserved blastocysts. NHP embryos were successfully produced, which will help to establish an NHP HD model and, ultimately, may serve as a vital tool for better understanding HD's pathology and developing novel treatments.
Subject(s)
Huntingtin Protein , Macaca mulatta , Animals , Macaca mulatta/genetics , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Huntington Disease/genetics , Blastocyst/metabolism , Trinucleotide Repeat Expansion/genetics , Embryo, Mammalian/metabolism , CRISPR-Cas Systems/genetics , Female , Disease Models, AnimalABSTRACT
We generated and characterized a rhesus macaque induced pluripotent stem cell (iPSC) line using induced reprogramming of fibroblasts isolated from a rhesus macaque fetus. The fibroblasts were expanded and then reprogrammed using non-integrating Sendai virus technology. This line is available as riPSC05. The authenticity of riPSC05 was confirmed through the expression of pluripotent and self-renewal markers, in vitro-directed differentiation towards three germ layers (ectoderm, mesoderm, and endoderm), karyotyping, and STR analysis.
Subject(s)
Induced Pluripotent Stem Cells , Animals , Induced Pluripotent Stem Cells/metabolism , Cellular Reprogramming , Macaca mulatta , Cell Differentiation , Karyotyping , Fibroblasts/metabolismABSTRACT
PROBLEM: Anovulatory infertility is commonly associated with hyperandrogenemia (elevated testosterone, T), insulin resistance, obesity, and white adipose tissue (WAT) dysfunction associated with adipocyte hypertrophy. However, whether hyperandrogenemia and adipocyte hypertrophy per se induce a proinflammatory response is unknown. METHOD OF STUDY: Young adult female rhesus macaques were exposed to an obesogenic Western-style diet (WSD) in the presence of elevated circulating testosterone (T+WSD) or a low-fat control diet with no exogenous T. Immune cells residing in visceral omental white adipose tissue (OM-WAT), corpus luteum and the contralateral ovary, endometrium, lymph nodes, bone marrow, and peripheral blood mononuclear cells were characterized by flow cytometry during the luteal phase of the reproductive cycle. RESULTS: Following one year of treatment, T+WSD animals became more insulin-resistant and exhibited increased body fat and adipocyte hypertrophy compared to controls. T+WSD treatment did not induce macrophage polarization toward a proinflammatory phenotype in the tissues examined. Additionally, T+WSD treatment did not affect TNFα production by bone marrow macrophages in response to toll-like receptor agonists. While the major lymphoid subsets were not significantly affected by T+WSD treatment, we observed a significant reduction in the frequency of effector memory CD8+ T-cells (Tem) in OM-WAT, but not in other tissues. Notably, OM-WAT Tem frequencies were negatively correlated with insulin resistance as assessed by the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR). CONCLUSION: This study shows that short-term T+WSD treatment induces weight gain, insulin resistance, and adipocyte hypertrophy, but does not have a significant effect on systemic and tissue-resident proinflammatory markers, suggesting that adipocyte hypertrophy and mild hyperandrogenemia alone are not sufficient to induce a proinflammatory response.
Subject(s)
Hyperandrogenism , Insulin Resistance , Polycystic Ovary Syndrome , Humans , Animals , Female , Macaca mulatta , Insulin Resistance/physiology , Testosterone/pharmacology , Leukocytes, Mononuclear , Hyperandrogenism/complications , Adipocytes/pathology , Hypertrophy/complications , DietABSTRACT
Metformin is used by women during pregnancy to manage diabetes and crosses the placenta, yet its effects on the fetus are unclear. We show that the liver is a site of metformin action in fetal sheep and macaques, given relatively abundant OCT1 transporter expression and hepatic uptake following metformin infusion into fetal sheep. To determine the effects of metformin action, we performed studies in primary hepatocytes from fetal sheep, fetal macaques, and juvenile macaques. Metformin increases AMP-activated protein kinase (AMPK) signaling, decreases mammalian target of rapamycin (mTOR) signaling, and decreases glucose production in fetal and juvenile hepatocytes. Metformin also decreases oxygen consumption in fetal hepatocytes. Unique to fetal hepatocytes, metformin activates stress pathways (e.g., increased PGC1A gene expression, NRF-2 protein abundance, and phosphorylation of eIF2α and CREB proteins) alongside perturbations in hepatokine expression (e.g., increased growth/differentiation factor 15 [GDF15] and fibroblast growth factor 21 [FGF21] expression and decreased insulin-like growth factor 2 [IGF2] expression). Similarly, in liver tissue from sheep fetuses infused with metformin in vivo, AMPK phosphorylation, NRF-2 protein, and PGC1A expression are increased. These results demonstrate disruption of signaling and metabolism, induction of stress, and alterations in hepatokine expression in association with metformin exposure in fetal hepatocytes. ARTICLE HIGHLIGHTS: The major metformin uptake transporter OCT1 is expressed in the fetal liver, and fetal hepatic uptake of metformin is observed in vivo. Metformin activates AMPK, reduces glucose production, and decreases oxygen consumption in fetal hepatocytes, demonstrating similar effects as in juvenile hepatocytes. Unique to fetal hepatocytes, metformin activates metabolic stress pathways and alters the expression of secreted growth factors and hepatokines. Disruption of signaling and metabolism with increased stress pathways and reduced anabolic pathways by metformin in the fetal liver may underlie reduced growth in fetuses exposed to metformin.
Subject(s)
Metformin , Pregnancy , Female , Animals , Sheep , Metformin/pharmacology , AMP-Activated Protein Kinases/metabolism , Hepatocytes/metabolism , Glucose/metabolism , Fetus/metabolism , Mammals/metabolismABSTRACT
CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.
Subject(s)
CRISPR-Cas Systems , DNA Breaks, Double-Stranded , CRISPR-Cas Systems/genetics , Gene Editing , DNA End-Joining Repair , GenomeABSTRACT
Maternal overnutrition increases inflammatory and metabolic disease risk in postnatal offspring. This constitutes a major public health concern due to increasing prevalence of these diseases, yet mechanisms remain unclear. Here, using nonhuman primate models, we show that maternal Western-style diet (mWSD) exposure is associated with persistent pro-inflammatory phenotypes at the transcriptional, metabolic, and functional levels in bone marrow-derived macrophages (BMDMs) from 3-year-old juvenile offspring and in hematopoietic stem and progenitor cells (HSPCs) from fetal and juvenile bone marrow and fetal liver. mWSD exposure is also associated with increased oleic acid in fetal and juvenile bone marrow and fetal liver. Assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling of HSPCs and BMDMs from mWSD-exposed juveniles supports a model in which HSPCs transmit pro-inflammatory memory to myeloid cells beginning in utero. These findings show that maternal diet alters long-term immune cell developmental programming in HSPCs with proposed consequences for chronic diseases featuring altered immune/inflammatory activation across the lifespan.
Subject(s)
Bone Marrow , Hematopoietic Stem Cells , Humans , Animals , Female , Diet, Western/adverse effects , Primates , Immunity, InnateABSTRACT
The role of prostaglandins (PGs) in the ovulatory process is known. However, the role of the ATP binding cassette subfamily C member 4 (ABCC4), transmembrane PG carrier protein, in ovulation remains unknown. We report herein that ABCC4 expression is significantly upregulated in preovulatory human granulosa cells (GCs). We found that PGE2 efflux in cultured human GCs is mediated by ABCC4 thus regulating its extracellular concentration. The ABCC4 inhibitor probenecid demonstrated effective blocking of ovulation and affects key ovulatory genes in female mice in vivo. We postulate that the reduction in PGE2 efflux caused by the inhibition of ABCC4 activity in GCs decreases the extracellular concentration of PGE2 and its ovulatory effect. Treatment of female mice with low dose of probenecid as well as with the PTGS inhibitor indomethacin or Meloxicam synergistically blocks ovulation. These results support the hypothesis that ABCC4 has an important role in ovulation and might be a potential target for non-hormonal contraception, especially in combination with PGE2 synthesis inhibitors. These findings may fill the gap in understanding the role of ABCC4 in PGE2 signaling, enhance the understanding of ovulatory disorders, and facilitate the treatment and control of fertility.
Subject(s)
Contraceptive Agents , Dinoprostone , Humans , Female , Mice , Animals , Dinoprostone/metabolism , Contraceptive Agents/metabolism , Contraceptive Agents/pharmacology , Probenecid/metabolism , Probenecid/pharmacology , Ovarian Follicle/metabolism , Ovulation/physiology , Membrane Transport Proteins/metabolism , ATP-Binding Cassette Transporters/metabolism , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolismABSTRACT
The advent of directed gene-editing technologies now over 10 years ago ushered in a new era of precision medicine wherein specific disease-causing mutations can be corrected. In parallel with developing new gene-editing platforms, optimizing their efficiency and delivery has been remarkable. With their development, there has been interest in using gene-editing systems for correcting disease mutations in differentiated somatic cells ex vivo or in vivo or for germline gene editing in gametes or 1-cell embryos to potentially limit genetic diseases in the offspring and in future generations. This review details the development and history of the current gene-editing systems and the advantages and challenges in their use for somatic cell and germline gene editing.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Humans , Mutation , Germ Cells , Precision MedicineABSTRACT
Polycystic ovary syndrome (PCOS) is associated with irregular menstrual cycles, hyperandrogenemia, and obesity. It is currently accepted that women with PCOS are also at risk for endometriosis, but the effect of androgen and obesity on endometriosis has been underexplored. The goal of this study was to determine how testosterone (T) and an obesogenic diet impact the progression of endometriosis in a nonhuman primate (NHP) model. Female rhesus macaques were treated with T (serum levels approximately 1.35 ng/ml), Western-style diet (WSD; 36% of calories from fat compared to 16% in standard monkey chow) or the combination (T + WSD) at the time of menarche as part of a longitudinal study for ~7 years. Severity of endometriosis was determined based on American Society for Reproductive Medicine (ASRM) revised criteria, and staged 1-4. Stages 1 and 2 were associated with extent of abdominal adhesions, while stages 3 and 4 were associated with presence of chocolate cysts. The combined treatment of T + WSD resulted in earlier onset of endometriosis and more severe types associated with large chocolate cysts compared to all other treatments. There was a strong correlation between glucose clearance, homeostatic model assessment for insulin resistance (HOMA-IR), and total percentage of body fat with presence of cysts, indicating possible indirect contribution of hyperandrogenemia via metabolic dysfunction. An RNA-seq analysis of omental adipose tissue revealed significant impacts on a number of inflammatory signaling pathways. The interactions between obesity, hyperandrogenemia, and abdominal inflammation deserve additional investigation in NHP model species.
Subject(s)
Diet, Western , Endometriosis , Insulin Resistance , Polycystic Ovary Syndrome , Testosterone , Animals , Female , Humans , Body Mass Index , Endometriosis/complications , Longitudinal Studies , Macaca mulatta , Obesity/metabolism , Polycystic Ovary Syndrome/metabolism , Testosterone/pharmacology , Diet, Western/adverse effectsABSTRACT
Maternal obesity adversely impacts the in utero metabolic environment, but its effect on fetal hematopoiesis remains incompletely understood. During late development, the fetal bone marrow (FBM) becomes the major site where macrophages and B lymphocytes are produced via differentiation of hematopoietic stem and progenitor cells (HSPCs). Here, we analyzed the transcriptional landscape of FBM HSPCs at single-cell resolution in fetal macaques exposed to a maternal high-fat Western-style diet (WSD) or a low-fat control diet. We demonstrate that maternal WSD induces a proinflammatory response in FBM HSPCs and fetal macrophages. In addition, maternal WSD consumption suppresses the expression of B cell development genes and decreases the frequency of FBM B cells. Finally, maternal WSD leads to poor engraftment of fetal HSPCs in nonlethally irradiated immunodeficient NOD/SCID/IL2rγ-/- mice. Collectively, these data demonstrate for the first time that maternal WSD impairs fetal HSPC differentiation and function in a translationally relevant nonhuman primate model.
Subject(s)
Diet, Western , Stem Cells , Female , Pregnancy , Humans , Mice , Animals , Macaca mulatta , Mice, Inbred NOD , Mice, SCID , Diet, Western/adverse effectsABSTRACT
In women, excess androgen causes polycystic ovary syndrome (PCOS), a common fertility disorder with comorbid metabolic dysfunctions including diabetes, obesity, and nonalcoholic fatty liver disease. Using a PCOS mouse model, this study shows that chronic high androgen levels cause hepatic steatosis while hepatocyte-specific androgen receptor (AR)-knockout rescues this phenotype. Moreover, through RNA-sequencing and metabolomic studies, we have identified key metabolic genes and pathways affected by hyperandrogenism. Our studies reveal that a large number of metabolic genes are directly regulated by androgens through AR binding to androgen response element sequences on the promoter region of these genes. Interestingly, a number of circadian genes are also differentially regulated by androgens. In vivo and in vitro studies using a circadian reporter [Period2::Luciferase (Per2::LUC)] mouse model demonstrate that androgens can directly disrupt the hepatic timing system, which is a key regulator of liver metabolism. Consequently, studies show that androgens decrease H3K27me3, a gene silencing mark on the promoter of core clock genes, by inhibiting the expression of histone methyltransferase, Ezh2, while inducing the expression of the histone demethylase, JMJD3, which is responsible for adding and removing the H3K27me3 mark, respectively. Finally, we report that under hyperandrogenic conditions, some of the same circadian/metabolic genes that are upregulated in the mouse liver are also elevated in nonhuman primate livers. In summary, these studies not only provide an overall understanding of how hyperandrogenism associated with PCOS affects liver gene expression and metabolism but also offer insight into the underlying mechanisms leading to hepatic steatosis in PCOS.
Subject(s)
Hyperandrogenism , Non-alcoholic Fatty Liver Disease , Polycystic Ovary Syndrome , Androgens/metabolism , Androgens/pharmacology , Animals , Disease Models, Animal , Epigenesis, Genetic , Female , Histones/metabolism , Humans , Hyperandrogenism/complications , Mice , Non-alcoholic Fatty Liver Disease/complications , Non-alcoholic Fatty Liver Disease/genetics , Polycystic Ovary Syndrome/metabolismABSTRACT
OBJECTIVE: To assess whether dose escalation (ie, doubling the dose) of emergency contraception that contains levonorgestrel (LNG) improves pharmacodynamic outcomes in individuals with obesity. METHODS: We enrolled healthy, reproductive-age individuals with regular menstrual cycles, body mass index (BMI) higher than 30, and weight at least 176 lbs in a randomized pharmacodynamic study. After confirming ovulation (luteal progesterone level greater than 3 ng/mL), we monitored participants with transvaginal ultrasonography and blood sampling for progesterone, luteinizing hormone, and estradiol every other day until a dominant follicle measuring 15 mm or greater was visualized. At that point, participants received either oral emergency contraception with LNG 1.5 mg or 3 mg (double dose) and returned for daily monitoring for up to 7 days. Our primary outcome was the difference in the proportion of participants with no follicle rupture 5 days postdosing (yes or no) between groups. The study had 80% power to detect a 30% difference in the proportion of cycles with at least a 5-day delay in follicle rupture (50% decrease). RESULTS: A total of 70 enrolled and completed study procedures. The two groups had similar baseline demographics (mean age 28 years, BMI 38). We found no difference between groups in the proportion of participants without follicle rupture more than 5 days post-LNG dosing (LNG 1.5 mg: 18/35 [51.4%]; LNG 3.0 mg: 24/35 [68.6%], P=.14). Among participants with follicle rupture before 5 days, the time to rupture did not differ between groups (day at 75% probability of no rupture is day 2 for both groups). CONCLUSION: Individuals with higher BMIs and weights experience a higher risk of failure of emergency contraception with LNG and exhibit an altered pharmacokinetic profile. However, the simple strategy of doubling the dose does not appear to be an effective intervention to improve outcomes. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, 02859337.
Subject(s)
Contraception, Postcoital , Levonorgestrel , Adult , Female , Humans , Obesity/drug therapy , Ovulation , ProgesteroneABSTRACT
Mutations in the MYO7A gene lead to Usher syndrome type 1B (USH1B), a disease characterized by congenital deafness, vision loss, and balance impairment. To create a nonhuman primate (NHP) USH1B model, CRISPR/Cas9 was used to disrupt MYO7A in rhesus macaque zygotes. The targeting efficiency of Cas9 mRNA and hybridized crRNA-tracrRNA (hyb-gRNA) was compared to Cas9 nuclease (Nuc) protein and synthetic single guide (sg)RNAs. Nuc/sgRNA injection led to higher editing efficiencies relative to mRNA/hyb-gRNAs. Mutations were assessed by preimplantation genetic testing (PGT) and those with the desired mutations were transferred into surrogates. A pregnancy was established from an embryo where 92.1% of the PGT sequencing reads possessed a single G insertion that leads to a premature stop codon. Analysis of single peripheral blood leukocytes from the infant revealed that half the cells possessed the homozygous single base insertion and the remaining cells had the wild-type MYO7A sequence. The infant showed sensitive auditory thresholds beginning at 3 months. Although further optimization is needed, our studies demonstrate that it is feasible to use CRISPR technologies for creating NHP models of human diseases.
Subject(s)
Usher Syndromes , Animals , Humans , CRISPR-Cas Systems , Endonucleases/genetics , Gene Editing , Macaca mulatta/genetics , Macaca mulatta/metabolism , RNA, Messenger , Usher Syndromes/genetics , RNA, Small Untranslated/metabolismABSTRACT
Hyperandrogenemia and obesity are common in women with polycystic ovary syndrome, but it is currently unclear how each alone or in combination contribute to reproductive dysfunction and female infertility. To distinguish the individual and combined effects of hyperandrogenemia and an obesogenic diet on ovarian function, prepubertal female rhesus macaques received a standard control (C) diet, testosterone (T) implants, an obesogenic Western-style diet (WSD), or both (Tâ +â WSD). After 5 to 6 years of treatment, the females underwent metabolic assessments and controlled ovarian stimulations. Follicular fluid (FF) was collected for steroid and cytokine analysis and the oocytes fertilized in vitro. Although the Tâ +â WSD females exhibited higher insulin resistance compared to the controls, there were no significant differences in metabolic parameters between treatments. Significantly higher concentrations of CXCL-10 were detected in the FF from the T group, but no significant differences in intrafollicular steroid levels were observed. Immunostaining of cleavage-stage embryos revealed multiple nuclear abnormalities in the T, WSD, and Tâ +â WSD groups. Single-cell DNA sequencing showed that while C embryos contained primarily euploid blastomeres, most cells in the other treatment groups were aneuploid. Despite yielding a higher number of mature oocytes, Tâ +â WSD treatment resulted in significantly reduced blastocyst formation rates compared to the T group. RNA sequencing analysis of individual blastocysts showed differential expression of genes involved in critical implantation processes between the C group and other treatments. Collectively, we show that long-term WSD consumption reduces the capacity of fertilized oocytes to develop into blastocysts and that the addition of T further impacts gene expression and embryogenesis.
Subject(s)
Hyperandrogenism , Animals , Blastocyst , Diet, Western/adverse effects , Embryonic Development , Female , Humans , Hyperandrogenism/complications , Macaca mulattaABSTRACT
Genome engineering is a powerful tool for in vitro research and the creation of novel model organisms and has growing clinical applications. Randomly integrating vectors, such as lentivirus- or transposase-based methods, are simple and easy to use but carry risks arising from insertional mutagenesis. Here we present enhanced-specificity tagmentation-assisted PCR (esTag-PCR), a rapid and accurate method for mapping transgene integration and copy number. Using stably transfected HepG2 cells, we demonstrate that esTag-PCR has higher integration site detection accuracy and efficiency than alternative tagmentation-based methods. Next, we performed esTag-PCR on rhesus macaque embryos derived from zygotes injected with piggyBac transposase and transposon/transgene plasmid. Using low-input trophectoderm biopsies, we demonstrate that esTag-PCR accurately maps integration events while preserving blastocyst viability. We used these high-resolution data to evaluate the performance of piggyBac-mediated editing of rhesus macaque embryos, demonstrating that increased concentration of transposon/transgene plasmid can increase the fraction of embryos with stable integration; however, the number of integrations per embryo also increases, which may be problematic for some applications. Collectively, esTag-PCR represents an important improvement to the detection of transgene integration, provides a method to validate and screen edited embryos before implantation, and represents an important advance in the creation of transgenic animal models.
ABSTRACT
OBJECTIVE: To determine the dose-dependent effect of contemporary marijuana exposure on female menstrual cyclicity and reproductive endocrine physiology in a nonhuman primate model. DESIGN: Research animal study. SETTING: Research institute environment. ANIMALS: Adult female rhesus macaques (6-12 years of age; n = 8). INTERVENTIONS: Daily delta-9-tetrahydrocannabinol (THC) edible at medically and recreationally relevant contemporary doses. MAIN OUTCOME MEASURES: Menstrual cycle length (MCL), anti-Müllerian hormone, prolactin, basal follicle-stimulating hormone (FSH), estradiol (E2) and progesterone, luteinizing hormone (LH), and thyroid-stimulating hormone. RESULTS: The average before THC weight was 6.9 kg (standard deviation, 0.8), and at the highest THC dosing, the average weight was 7.2 kg (standard deviation, 0.8). With increasing THC dosing, MCL and FSH concentrations increased, while basal E2 concentration was stable. The average MCL concentration increased 4.0 days for each mg/7 kg/day of THC (95% CI, 1.4-6.6 days). Follicle-stimulating hormone concentration increased significantly with increasing THC dose, 0.34 ng/mL for each mg/7 kg/day of THC (95% CI, 0.14-0.57 ng/mL). No significant trends were observed between THC dosing and average basal progesterone, anti-Müllerian hormone, prolactin, LH, or thyroid-stimulating hormone concentrations. CONCLUSIONS: In rhesus macaques, a dose response toward increased MCL and basal FSH concentrations but plateau of basal E2 and LH concentrations was observed with increasing THC dosing, suggesting ovulatory dysfunction. Further studies are needed to determine the effects of a longer duration of exposure and whether the significant increase in MCL and FSH concentrations results in reduced fecundity.
Subject(s)
Dronabinol , Progesterone , Animals , Anti-Mullerian Hormone , Dronabinol/pharmacology , Female , Follicle Stimulating Hormone , Luteinizing Hormone , Macaca mulatta , Menstrual Cycle , Periodicity , Prolactin , Reproductive Health , ThyrotropinABSTRACT
OBJECTIVE: To demonstrate that functional spermatids can be derived in vitro from nonhuman primate pluripotent stem cells. DESIGN: Green fluorescent protein-labeled, rhesus macaque nonhuman primate embryonic stem cells (nhpESCs) were differentiated into advanced male germ cell lineages using a modified serum-free spermatogonial stem cell culture medium. In vitro-derived round spermatid-like cells (rSLCs) from differentiated nhpESCs were assessed for their ability to fertilize rhesus oocytes by intracytoplasmic sperm(atid) injection. SETTING: Multiple academic laboratory settings. PATIENTS: Not applicable. INTERVENTIONS: Intracytoplasmic sperm(atid) injection of in vitro-derived spermatids from nhpESCs into rhesus macaque oocytes. MAIN OUTCOME MEASURES: Differentiation into spermatogenic cell lineages was measured through multiple assessments including ribonucleic acid sequencing and immunocytochemistry for various spermatogenic markers. In vitro spermatids were assessed for their ability to fertilize oocytes by intracytoplasmic sperm(atid) injection by assessing early fertilization events such as spermatid deoxyribonucleic acid decondensation and pronucleus formation/apposition. Preimplantation embryo development from the one-cell zygote stage to the blastocyst stage was also assessed. RESULTS: Nonhuman primate embryonic stem cells can be differentiated into advanced germ cell lineages, including haploid rSLCs. These rSLCs undergo deoxyribonucleic acid decondensation and pronucleus formation/apposition when microinjected into rhesus macaque mature oocytes, which, after artificial activation and coinjection of ten-eleven translocation 3 protein, undergo embryonic divisions with approximately 12% developing successfully into expanded blastocysts. CONCLUSIONS: This work demonstrates that rSLCs, generated in vitro from primate pluripotent stem cells, mimic many of the capabilities of in vivo round spermatids and perform events essential for preimplantation development. To our knowledge, this work represents, for the first time, that functional spermatid-like cells can be derived in vitro from primate pluripotent stem cells.
Subject(s)
Sperm Injections, Intracytoplasmic , Spermatids , Animals , Blastocyst , DNA , Embryonic Development , Embryonic Stem Cells , Female , Fertilization , Humans , Macaca mulatta , Male , PregnancyABSTRACT
The maternal decidua is an immunologically complex environment that balances maintenance of immune tolerance to fetal paternal antigens with protection of the fetus against vertical transmission of maternal pathogens. To better understand host immune determinants of congenital infection at the maternal-fetal tissue interface, we performed a comparative analysis of innate and adaptive immune cell subsets in the peripheral blood and decidua of healthy rhesus macaque pregnancies across all trimesters of gestation and determined changes after Zika virus (ZIKV) infection. Using one 28-color and one 18-color polychromatic flow cytometry panel we simultaneously analyzed the frequency, phenotype, activation status and trafficking properties of αß T, γδ T, iNKT, regulatory T (Treg), NK cells, B lymphocytes, monocytes, macrophages, and dendritic cells (DC). Decidual leukocytes showed a striking enrichment of activated effector memory and tissue-resident memory CD4+ and CD8+ T lymphocytes, CD4+ Tregs, CD56+ NK cells, CD14+CD16+ monocytes, CD206+ tissue-resident macrophages, and a paucity of B lymphocytes when compared to peripheral blood. t-distributed stochastic neighbor embedding (tSNE) revealed unique populations of decidual NK, T, DC and monocyte/macrophage subsets. Principal component analysis showed distinct spatial localization of decidual and circulating leukocytes contributed by NK and CD8+ T lymphocytes, and separation of decidua based on gestational age contributed by memory CD4+ and CD8+ T lymphocytes. Decidua from 10 ZIKV-infected dams obtained 16-56 days post infection at third (n=9) or second (n=1) trimester showed a significant reduction in frequency of activated, CXCR3+, and/or Granzyme B+ memory CD4+ and CD8+ T lymphocytes and γδ T compared to normal decidua. These data suggest that ZIKV induces local immunosuppression with reduced immune recruitment and impaired cytotoxicity. Our study adds to the immune characterization of the maternal-fetal interface in a translational nonhuman primate model of congenital infection and provides novel insight in to putative mechanisms of vertical transmission.