Accumulating evidence has suggested an interaction between endometriotic cells and macrophages in the endometriotic microenvironment and the potential role of this interaction in the pathogenesis of endometriosis. However, how endometriotic cells communicate with macrophages to influence their function is poorly understood. In the present study, we found that the mRNA expression and production of CC chemokine ligand 2 (CCL2) were much higher in human endometriotic epithelial cells (11Z and 12Z) than those in human endometrial epithelial cells (HES). The inhibition of CCL2 action using neutralizing antibodies substantially suppressed macrophage migration induced by endometriotic epithelial cells. The endometriosis-associated macrophages (EAMs), which are the macrophages that are stimulated by the conditionedmedium (CM) of human endometriotic cells, highly expressed the M2 phenotype markers (MRC1 and TREM2). In addition, the CM of EAMs significantly increased cell migration in 12Z cells, but no significant change was observed in cell growth. RT-PCR and antibody array analyses revealed that EAMs highly express and produce interleukin (IL) 6 compared to macrophages stimulated by the CM of HES cells. Moreover, the EAM-CM-induced migration and MMP2/9 expression in endometriotic cells were significantly attenuated by IL6 signaling inhibition. These results suggest a reciprocal activation of macrophages and endometriotic cells via the soluble factors CCL2 and IL6, which may contribute to the development of endometriosis.

The vast majority of sperm are lost from the female reproductive tract in hours following natural mating or artificial insemination in mammals. Multiple complex processes including uterine contractions, mucus barriers, and phagocytosis of sperm by neutrophils have been reported to be involved in the sperm loss, although the contribution of each process is uncertain. If phagocytosis by neutrophils has a significant role in sperm loss, inhibition of neutrophil response to sperm could potentially reduce the dose of sperm required for artificial insemination. Through the development of a quantitative in vitro assay, we have screened 74 candidate compounds for their ability to inhibit the neutrophil–sperm interaction in cattle. Nine inhibitors (GSK2126458, wortmannin, ZSTK474, PIK294, CAL-101, GSK 1059615, GDC-0941, PIK 90 and PI103) active against phosphatidylinositol 3-kinase (PI3-kinase) were most potent, and strongly reduced neutrophil–sperm interaction with an IC50 of 10 nM or less. These inhibitors did not significantly modify sperm motility, and five of the inhibitors did not affect in vitro fertilization. Examination of neutrophil–sperm interaction by time-lapse video microscopy and cell tracking analysis revealed that GSK2126458 may prevent sperm phagocytosis through inhibition of neutrophil movement and/or attachment. Twenty-four other compounds exhibited weaker inhibition (IC50 < 115 μM), and the rest did not inhibit the neutrophil–sperm interaction. Strong PI3-kinase inhibitors identified in this study may be useful to determine the contribution of neutrophil phagocytosis in the clearance of sperm from the female reproductive tract.

Summary Sentence

Neutrophil–sperm interaction is strongly inhibited by selective PI3-kinase inhibitors at nanomolar concentrations.

The secretion of mammalian Wnt ligands within the cell is dependent on the activity of Porcupine, a gene located on the X-chromosome that encodes for a membrane-bound O-acyl transferase. Here, we report that postnatal ablation of Porcupine in the uterine luminal epithelium alone results in the decrease in endometrial gland number. Despite having uterine glands, mutant females are completely infertile. Epithelial ablation of Porcupine causes defects in timely apposition of the lumen, along with failure to respond to artificial decidual induction. Interestingly, progesterone supplementation was able to rescue the initiation of decidualization, but the decidua was not maintained and subsequently resorbed. Transcriptome analysis demonstrated that deletion of Porcupine in the epithelium resulted in the stromal dysregulation of members of the Wnt signaling pathway (Lef1, Wnt4, and Wnt16), dysregulation of receptors and ligands in the Notch signaling pathway (Notch1, Notch4, and Dll4) as well as Hoxa10. Our results demonstrate the crucial requirement of Wnt signaling in the epithelium for fertility and demonstrate that epithelial Wnts regulate stromal Wnt gene expression as well as regulating the expression of essential signaling factors and effectors required for successful embryo implantation.

Summary Sentence

Epithelial Porcupine is important for pregnancy.

The ability to accurately monitor alterations in sperm motility is paramount to understanding multiple genetic and biochemical perturbations impacting normal fertilization. Computer-aided sperm analysis (CASA) of human sperm typically reports motile percentage and kinematic parameters at the population level, and uses kinematic gating methods to identify subpopulations such as progressive or hyperactivated sperm. The goal of this study was to develop an automated method that classifies all patterns of human spermmotility during in vitro capacitation following the removal of seminal plasma. We visually classified CASA tracks of 2817 sperm from 18 individuals and used a support vector machine-based decision tree to compute four hyperplanes that separate five classes based on their kinematic parameters. We then developed a web-based program, CASAnova, which applies these equations sequentially to assign a single classification to each motile sperm. Vigorous sperm are classified as progressive, intermediate, or hyperactivated, and nonvigorous sperm as slow or weakly motile. This program correctly classifies sperm motility into one of five classes with an overall accuracy of 89.9%. Application of CASAnova to capacitating sperm populations showed a shift from predominantly linear patterns of motility at initial time points to more vigorous patterns, including hyperactivated motility, as capacitation proceeds. Both intermediate and hyperactivated motility patterns were largely eliminated when sperm were incubated in noncapacitating medium, demonstrating the sensitivity of this method. The five CASAnova classifications are distinctive and reflect kinetic parameters of washed human sperm, providing an accurate, quantitative, and high-throughput method for monitoring alterations in motility.

Summary Sentence

A CASA-based support vector machine model of human sperm motility provides rapid, accurate, and quantitative analysis of all motile sperm in a population.

Kisspeptin (KISS1; encoded by Kiss1) neurons in the arcuate nucleus (ARC) coexpress tachykinin 3 (TAC3; also known as neurokinin B) and dynorphin A (PDYN). Accordingly, they are termed KNDy neurons and considered to be crucial in generating pulsatile release of gonadotropin-releasing hormone. Accumulating evidence suggests that Kiss1 and Tac3 are negatively regulated by estrogen. However, it has not been fully determined whether and how estrogen modulates Pdyn and PDYN. Here, we examined the expression of Pdyn mRNA and PDYN by in situ hybridization and immunohistochemistry, respectively, in the ARC of female rats after ovariectomy (OVX) and OVX plus low- or high-dose beta-estradiol (E₂) replacement. We also investigated the effect of E₂ on expression of Kiss1, KISS1, Tac3, and TAC3. Furthermore, colocalization of PDYN and estrogen receptor alpha (ESR1) was determined. Subsequently, we found that low-dose E₂ treatment had no effect on Pdyn mRNA-expressing cells, but increased PDYN-immunoreactive (ir) cell numbers. In contrast, high-dose E₂ treatment resulted in prominent reductions in both Pdyn mRNA-expressing and PDYN-ir cell numbers. Changes induced by low or high doses of E₂ were similarly observed in the expression of Kiss1, KISS1, Tac3, and TAC3. The majority of PDYN-ir neurons coexpressed ESR1 in all groups. Our results indicate that E₂ regulates the expression of PDYN, as well as KISS1 and TAC3, with regulation by E₂ differing according to its levels.

Summary Sentence

Estrogen regulates dynorphin expression in the arcuate nucleus of female rats with low-dose estrogen suppressing dynorphin release and high-dose estrogen inhibiting dynorphin mRNA expression.

Normal ovarian development is crucial for female reproductive success and longevity. Interruptions to the delicate process of initial folliculogenesis may lead to ovarian dysfunction. We have previously demonstrated that an early life immune challenge in the rat, induced by administration of lipopolysaccharide (LPS) on postnatal day (PND) 3 and 5, depletes ovarian follicle reserve long term. Here, we hypothesized that this neonatal immune challenge leads to an increase in peripheral and ovarian inflammatory signaling, contributing to an acute depletion of ovarian follicles. Morphological analysis of neonatal ovaries indicated that LPS administration significantly depleted PND 5 primordial follicle populations and accelerated follicle maturation. LPS exposure upregulated circulating interleukin 6, tumor necrosis factor alpha (TNFa), and C-reactive protein on PND 5, and upregulated ovarian mRNA expression of Tnfa, mitogen-activated protein kinase 8 (Mapk8/Jnk1), and growth differentiation factor 9 (Gdf9) (P < 0.05).Mass spectrometry and cell signaling pathway analysis indicated upregulation of cellular pathways associated with acute phase signaling, and cellular survival and assembly. Apoptosis assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling indicated significantly increased positive staining in the ovaries of LPS-treated neonates. These findings suggest that increased proinflammatory signaling within the neonatal ovary may be responsible for the LPS-induced depletion of the primordial follicle pool. These findings also have implications for female reproductive health, as the ovarian reserve is a major determinate of female reproductive longevity.

Summary Sentence

Neonatal immune activation acutely impacts immune-mediated early ovarian development, depleting the primordial follicle pool and upregulating inflammatory mediators.

Recent studies using several teleost models have revealed that androgens increase the size of previtellogenic (primary and/or early secondary) ovarian follicles. To explore our hypothesis that androgens drive the development of primary follicles into early secondary follicles, and to determine the mechanisms underlying these androgenic effects, we exposed juvenile coho salmon to nearphysiological and relatively sustained levels of the nonaromatizable androgen 11-ketotestosterone (11-KT). This resulted in significant growth of primary ovarian follicles after 10 and 20 days, with follicles after 20 days displaying a morphological phenotype characteristic of early secondary follicles (presence of cortical alveoli). Utilizing the same experimental approach, we then analyzed how 11-KT rapidly altered the ovarian transcriptome after 1 and 3 days of treatment. RNA-Seq analysis revealed that 69 (day 1) and 1,022 (day 3) contiguous sequences (contigs) were differentially expressed relative to controls. The differentially expressed contigs mapped to genes including those encoding proteins involved in gonadotropin, steroid hormone, and growth factor signaling, and in cell and ovarian development, including genes with putative androgen-response elements. Biological functions and canonical pathways identified as potentially altered by 11-KT include those involved in ovarian development, tissue differentiation and remodeling, and lipid metabolism. We conclude that androgens play amajor role in stimulating primary ovarian follicle development and the transition into secondary growth.

Summary Sentence

In vivo treatment of coho salmon with the androgen, 11-ketotestosterone, dramatically alters the ovarian transcriptome during primary oocyte growth, and promotes the completion of primary ovarian follicle growth.

The mycotoxin deoxynivalenol (DON) has been shown to inhibit ovarian granulosa cell function in cattle in vitro, but it is not known whether DON or its metabolite deepoxy-DON (DOM-1) affects theca cell function. The objectives of this study were to determine the effects of DON and of DOM-1 on theca cell steroidogenesis and apoptosis, and to determine the main pathways through which they act. Bovine theca cells were cultured in a nonluteinizing serum-free culture system, and challenged with DON or DOM-1 for 4 days to measure steroidogenesis and apoptosis, for 1–8 h to measure immediate-early genes, and for 5–60 min to measure phosphorylation of intracellular signaling proteins. Addition of DON decreased progesterone secretion at doses as low as 0.5 ng/ml but had no effect on testosterone secretion. Addition of DOM-1 inhibited progesterone and testosterone secretion at 0.5 ng/ml. Treatment of cells with 1 ng/ml DOM-1 increased the proportion of apoptotic cells, whereas DON had no effect. Addition of DON or DOM-1 stimulated phosphorylation of EIF2AK2,MAPK3/1, and AKT. However, DON inhibited and DOM-1 stimulatedMAPK14 phosphorylation. DON increased the levels of mRNA encoding early-immediate genes EGR1, EGR3, and FOS, whereas DOM-1 was without effect. DOM-1 but not DON increased abundance of mRNA of the endoplasmic reticulum (ER) stress-related proteins, PRKRA and ATF4.We conclude that DOM-1 has a major impact on theca function in cattle, and possibly induces theca cell apoptosis through ER stress.

Summary Sentence

Deepoxy-deoxynivalenol acts through EIF2AK2 to increase theca cell apoptosis and decrease steroid secretion.

Intracytoplasmic sperm injection (ICSI) and embryo transfer (ET) in nonhuman primates, e.g. rhesus and cynomolgus monkeys, has been widely used in researches of reproductive and developmental biology, and the success rate has been improved significantly. However, unwanted multiple pregnancy occurs frequently during the ICSI-ET in monkeys, most of which leads to miscarriages. To improve the birth rate of pregnancies and to safeguard health of host and baby monkeys, multifetal pregnancy reduction (MPR) is necessary. In this study, a total of 10monkeys with multiple pregnancies received MPR through transabdominal ultrasound-guided potassium chloride injection into beating hearts of selective fetuses. To assess MPR efficiency, 31 monkeys with normal singleton pregnancies and 25 monkeys with twin pregnancies without MPR were used as controls. The aim of the reduction is to keep only one fetus, nomatter twin or triplet pregnancy originally. Our results show that six cases of MPR were successful and all of them retained single fetus. Moreover, about 1 month (30.2 ± 1.2 days) of gestation is a better timing for MPR than later stage (50.7 ± 1.9 days). We also found that the remaining fetuses developed normally with full-term gestation and normal birth weight. In conclusion, transabdominal ultrasound-guided potassium chloride injection is a safe and effective MPR method for monkeys with multiple pregnancies.

Summary Sentence

Transabdominal ultrasound-guided potassium chloride injection is a safe and effective multifetal pregnancy reduction method for monkeys with multiple pregnancies.

Maternal liver undergoes structural and metabolic changes during pregnancy to meet the demands of the developing fetus. In rodents, this involves increased liver weight, but the mechanism remains unclear. To address this, we analyzed the histology, gene expression, and DNA methylation of livers of nonpregnant and pregnant C57/BL6 mice. Gestational liver growth in pregnant mice was accompanied by increased hepatocyte area and lower cell density (days 14 and 18). Expression of cell proliferation markers was increased on days 14 and 18. A total of 115 genes were differentially expressed on day 14 and 123 genes on day 18 (79 on both days). Pathway analysis indicated that pregnancy involves progressive increase in cell proliferation and decreased apoptosis. This was confirmed using archived data from the FVB wild-type mouse liver transcriptome. Four differentially DNA methylated and two differentially DNA hydroxymethylated regions identified on days 14 and 18 by methylome-wide analysis, but were not associated with altered gene expression. Long interspersed nuclear element-1 hypomethylation on days 14 and 18 was accompanied by increased ten-eleven translocase-2 and decreased DNA methyltransferase 3a and 3b expression. These findings suggest that gestational liver growth involves increased mitosis and hypertrophy, and decreased apoptosis contingent on pregnancy stage. Such changes may involve repetitive sequence, but not gene specific, DNA methylation.

Summary Sentence

Gestational liver growth involves hyperplasia, hypertrophy, and apoptosis which differ in contribution with progressing pregnancy.

Controllable transgene expression systems are indispensable tools for the production of animal models of disease to investigate protein functions at defined periods. However, in nonhuman primates that share genetic, physiological, and morphological similarities with humans, genetic modification techniques have not been well established; therefore, the establishment of novel transgenic models with controllable transgene expression systems will be valuable tools to understand pathological mechanism of human disease. In the present study, we successfully generated transgenic marmosets using a tetracyclin-inducible transgene expression (tet-on) system as a neurodegenerative disease model. The mutant human ataxin 3 gene controlled by the tet-on system was introduced into marmoset embryos via lentiviral transduction, and 34 transgene-introduced embryos were transferred into the uteri of surrogate mothers. Seven live offspring (TET1–7) were obtained, of which four were transgenic. Fibroblasts from TET1 and 3 revealed that inducible transgene expression had occurred after treatment with 10 μg/mL of doxycycline, while treatment with doxycycline via drinking water resulted in 1.7- to 1.8-fold inducible transgene expression compared with before treatment. One transgenic second-generation offspring (TET3-3) was obtained from TET3, and doxycycline-inducible transgene expression in its fibroblasts showed that TET3-3 maintained a high transgene expression level that matched its parent. In conclusion, we established a novel transgenic marmoset line carrying the mutant human ataxin 3 gene controlled by the tet-on system. The development of nonhuman primate models with controllable transgene expression systems will be useful for the identification of disease biomarkers and evaluation of the efficacy and metabolic profiles of therapeutic candidates.

Summary Sentence

Transgenic marmoset generation using the tetracyclin-inducible transgene expression system provides a model for human neurodegenerative disease.