High levels of inflammatory factors including chemokines have been reported in peritoneal fluid and blood of women with endometriosis. CXCL12 mediates its action by interaction with its specific receptor, CXCR4, reported to be elevated in human endometriosis lesions and in the rat model of endometriosis. Activation of the CXCR4-CXCL12 axis increases cell proliferation, migration, and invasion of cancer cells. To obtain insights into the CXCR4 expression profile in lesions and endometrium, as well as functionality of the CXCR4-CXCL12 axis in endometriosis, we analyzed the expression of CXCR4 in tissues on a human tissue array and studied CXCL12- mediated activation of proliferation, invasion, and migration in vitro. We observed differences in levels of nuclear CXCR4 expression among lesion types, being higher in ovarian lesions. Endometriotic cell lines (12Z) showed higher levels of CXCR4, proliferative and migratory potential, and AKT phosphorylation/kinase activity compared to untreated control cells (endometrial epithelial cells). CXCL12 and endometriotic stromal cell-enriched media increased proliferation of non-endometriotic epithelial cells. CXCL12 caused a significant increase in 12Z cell invasion but had no effect on migration; AMD3100, a CXCR4-specific inhibitor, significantly increased invasion of 12Z cells but decreased their migration. However, treatment with CXCL12 plus AMD3100 significantly decreased invasion and migration of 12Z cells. In conclusion, the CXCR4-CXCL12 axis is functional in endometriosis cells, but the expression of CXCR4 varies among lesions. CXCL12 promoted proliferation, migration, and invasion of endometriotic cells, while inducing AKT phosphorylation and activity, but pharmacologically blocking this axis in the absence of the ligand induced their invasiveness.

Summary Sentence

CXCL12 treatment of endometriotic cells promoted their proliferation, migration, and invasion, while inducing AKT phosphorylation and activity, but pharmacologically blocking this axis in the absence of the ligand increased their invasion capacity.

Infertility and early embryo miscarriage is linked to inadequate endometrial decidualization. Although transcriptional reprogramming is known to drive decidualization in response to progesterone, the key signaling effectors that directlymediate this hormone response are not fully known. This knowledge gap is clinically significant because identifying the early signals that directly mediate progesterone-driven decidualization will address some of the current limitations in diagnosing and therapeutically treating patients at most risk for early pregnancy loss.We recently revealed that the promyelocytic leukemia zinc finger (PLZF) is a direct target of the progesterone receptor and is essential for decidualization of human endometrial stromal cells (hESCs). The purpose of this current work was to identify the genome-wide transcriptional program that is controlled by PLZF during hESC decidualization using an established in vitro hESC culture model, siRNA-mediated knockdownmethods, and RNA-sequencing technology followed by bioinformatic analysis and validation. We discovered that PLZF is critical in the regulation of genes that are involved in cellular processes that are essential for the archetypal morphological and functional changes that occur when hESCs transform into epithelioid decidual cells such as proliferation and cell motility. We predict that the transcriptome datasets identified in this study will not only contribute to a broader understanding of PLZF-dependent endometrial decidualization at the molecular level but may advance the development of more effective molecular diagnostics and therapeutics for the clinical management of female infertility and subfertility that is based on a dysfunctional endometrium.

Summary Sentence

PLZF drives progesterone-dependent transcriptional reprogramming of the human endometrial stromal cells to enable decidualization.

Human LCN6, a lipocalin protein, exhibits predominant expression in epididymis and location on the sperm surface. However, the biological function of LCN6 in vivo remains unknown. Herein, we found that unlike human LCN6, mouse Lcn6 gene encoded two transcript variants that were both upregulated by androgen. Subsequently, we generated a conditional knockout mouse model to disrupt Lcn6 in the adult and investigate its function. In this model, spermatogenesis was normal and Lcn6 deficiency did not affect the natural birth rate of male mice or in vitro fertilization ability of their cauda epididymal sperm. Nevertheless, sperm from the cauda epididymis of the Lcn6 null mice underwent a sustained increase of acrosome reaction frequency whether capacitated or not (P < 0.01). Consistent with premature acrosome reaction, sperm from knockout mice had significantly increased intracellular calcium content when extracellular calcium was supplied (P < 0.01). These results demonstrate an important function of LCN6 in preventing calcium overload and premature acrosome reaction of sperm and suggest a potential risk factor of LCN6 deficiency for sperm maturation.

Summary Sentence

The mouse ortholog of human LCN6, of which two transcript variants exhibit specific expression in caput epididymis and positive regulation by androgen, is involved in preventing calcium overload and premature acrosome reaction of sperm.

Ovarian folliculogenesis is always of great interest in reproductive biology. However, the molecular mechanisms that control follicle development, particularly the early phase of follicle activation or recruitment, still remain poorly understood. In an attempt to decipher the gene networks and signaling pathways involved in such transition, we conducted a transcriptomic analysis (RNA-seq) on zebrafish primary growth (PG, stage I; inactive) and previtellogenic (PV, stage II; activated) follicles. A total of 118 unique microRNAs (miRNAs) (11 downregulated and 83 upregulated during PG/PV transition) and 56711 unique messenger RNAs (mRNAs) (1839 downregulated and 7243 upregulated during PG/PV transition) were identified. Real-time quantitative polymerase chain reaction analysis confirmed differential expression of 46 miRNAs from 66 candidates (66.67%). Among which, we chose to focus on 13 miRNAs (let-7a, -7b, -7c-5p, -7d-5p, -7h, -7i; miR-21, -23a-3p, -27c-3p, -107a-3p, -125b-5p, -145-3p, and -202–5p) that exhibited significant differential expression between PG and PV follicles (P ≤ 0.045*). With this 13-miRNA expression signature alone, PG follicles can be well differentiated from PV follicles by hierarchical clustering, suggesting their functional relevance during PG-to-PV transition. By overlaying predicted target genes and the differentially expressed mRNAs revealed by the RNA-seq analysis, especially those showing reciprocal miRNA-mRNA expression patterns, we shortlisted a panel of miRNA downstream targets for luciferase reporter validation. The reporter assay confirmed the interactions of let-7i:: atg4a (P = 0.01*), miR-202–5p::c23h20orf24 (P = 0.0004***), and miR-144–5p::ybx1 (P = 0.003**), implicating these potential miRNA-mRNA gene pairs in follicle activation during folliculogenesis. Our transcriptomic data analyses suggest that miRNA-mediated post-transcriptional control may represent an important mechanism underlying follicle activation.

Summary Sentence

Our transcriptomic data analyses suggest that miRNA-mediated post-transcriptional control may represent an important mechanism underlying follicle activation.

Anti-Müllerian hormone (AMH) is both a gonadal hormone and a putative paracrine regulator of neurons, the uterus, and the placenta. A mouse line with neuronal expression of AMH (Thy1.2-AMH) was generated to examine the role of paracrine AMH in the brain. The mice had normal behavior, but unexpectantly AMH was present in the circulation of the transgenic mice. Thy1.2-AMH^Tg/0 studs sired pups with a normal frequency, when mated with wild-type dams. In stark contrast, Thy1.2-AMH^Tg/0 dams rarely gave birth, with evidence of spontaneous midgestational abortion. This leads to the hypothesis that AMH influences the capacity of dams to carry concepti to term. This hypothesis was tested by mating AMH-deficient (Amh-/-), Thy1.2-AMH^Tg/0, and wildtype dams when 49-, 80-, and 111 days old, using proven wild-type studs. The litter sizes from the first two matings and the number of fetuses present on the 10th day of gestation of the third mating were recorded. Thy1.2-AMH^Tg/0 dams carried near normal numbers of midterm fetuses, but typically produced no pups, indicating that extensive late resorption of fetuses was occurring. Amh^-/- dams exhibited a lesser reduction in litter size than the Thy1.2-AMH^Tg/0 dams, with no evidence of enhanced loss of fetuses. In conclusion, this study provides the first evidence that high AMH levels can cause a miscarriage phenotype and that the absence of AMH affects reproductive output.

Summary Sentence

Prevention of the pregnancy-related decline in circulating anti-Müllerian hormone levels leads to midterm abortion.

Previous work has identified divergent mechanisms by which cervical remodeling is achieved in preterm birth (PTB) induced by hormone withdrawal (mifepristone) or lipopolysaccharide (LPS). Our current study aims to document how collagen architecture is modified to achieve premature cervical remodeling in mice treated with LPS as a model of infection-induced inflammation. Cervices were collected on gestation day (d) 15 from mice with premature cervical ripening induced by LPS and compared to d15 and d18 controls as well as a hormone withdrawal PTB model. Second harmonic generation (SHG) and electronmicroscopy were utilized for visualization of collagenmorphology and ultrastructure. LPS-mediated premature cervical ripening is characterized by unique structural changes in collagen fiber morphology. LPS treatment increased the interfibrillar spacing of collagen fibrils. A preferential disruption of collagen fiber architecture in the subepithelial region compared to midstroma region was evidenced by increased pores lacking collagen signal in SHG images in the LPS-treated mice. Coinciding with this alteration, the infiltration of neutrophils was concentrated in the subepithelial stromal region as compared to midstromal region implicating the potential role of immune cells to extracellular matrix reorganization in inflammation-induced preterm cervical ripening. The current study demonstrates a preferential disorganization of collagen interfibrillar spacing and collagen fiber structure in LPS-mediated ripening.

Maternal obesity induces pregnancy complications and disturbs fetal development, but the specific mechanisms underlying these outcomes are unclear. Circadian rhythms are implicated in metabolic complications associated with obesity, and maternal metabolic adaptations to pregnancy. Accordingly, obesity-induced circadian dysfunction may drive adverse outcomes in obese pregnancy. This study investigated whether maternal obesity alters the rhythmic expression of clock genes and associated nuclear receptors across maternal, fetal, and placental tissues. Wistar rats were maintained on a cafeteria (CAF) diet prior to and throughout gestation to induce maternal obesity. Maternal and fetal liver and placental labyrinth zone (LZ) were collected at four-hourly time points across days 15–16 and 21–22 of gestation (term = 23 days). Gene expression was analyzed by RTqPCR. Expression of the accessory clock gene Nr1d1 was rhythmic in the maternal and fetal liver and LZ of chow-fed controls, but in each case CAF feeding reduced peak Nr1d1 expression. Obesity resulted in a phase advance (approx. 1.5 h) in the rhythms of several clock genes and Ppar-delta in maternal liver. Aside from Nr1d1, expression of clock genes was mostly arrhythmic in LZ and fetal liver, and was unaffected by the CAF diet. In conclusion, maternal obesity suppressed Nr1d1 expression across maternal, fetal, and placental compartments and phase-advanced the rhythms of maternal hepatic clock genes. Given the key role of Nr1d1 in regulating metabolic, vascular, and inflammatory processes, our data suggest that disruptions to rhythmic Nr1d1 expression in utero may contribute to programmed health complications in offspring of obese pregnancies.

Summary Sentence

Obesity suppresses Nr1d1 expression in maternal, fetal, and placental tissues and phase-advances the rhythms of maternal hepatic clock genes during rat pregnancy.

Preterm birth affects 1 out of every 10 infants in the United States, resulting in substantial neonatal morbidity and mortality. Currently, there are few predictive markers and few treatment options to prevent preterm birth. A healthy, functioning placenta is essential to positive pregnancy outcomes. Previous studies have suggested that placental pathology may play a role in preterm birth etiology. Therefore, we tested the hypothesis that preterm placentae may exhibit unique transcriptomic signatures compared to term samples reflective of their abnormal biology leading to this adverse outcome. We aggregated publicly available placental villous microarray data to generate a preterm and term sample dataset (n = 133, 55 preterm placentae and 78 normal term placentae). We identified differentially expressed genes using the linear regression for microarray (LIMMA) package and identified perturbations in known biological networks using Differential Rank Conservation (DIRAC). We identified 129 significantly differentially expressed genes between term and preterm placenta with 96 genes upregulated and 33 genes downregulated (P-value <0.05). Significant changes in gene expression in molecular networks related to Tumor Protein 53 and phosphatidylinositol signaling were identified using DIRAC. We have aggregated a uniformly normalized transcriptomic dataset and have identified novel and established genes and pathways associated with developmental regulation of the placenta and potential preterm birth pathology. These analyses provide a community resource to integrate with other high-dimensional datasets for additional insights in normal placental development and its disruption.

Summary Sentence

Distinct transcriptional profiles of spontaneous preterm birth placentae compared to term placentae identify gestational regulation or potential pathogenic mechanisms.

Eukaryotic translation initiation factor 4G (EIF4G) is an important scaffold protein in the translation initiation complex. In mice, mutation of the Eif4g3 gene causes male infertility, with arrest of meiosis at the end of meiotic prophase. This study documents features of the developmental expression and subcellular localization of EIF4G3 that might contribute to its highly specific role in meiosis and spermatogenesis. Quite unexpectedly, EIF4G3 is located in the nucleus of spermatocytes, where it is highly enriched in the XY body, the chromatin domain formed by the transcriptionally inactive sex chromosomes. Moreover, many other, but not all, translation-related proteins are also localized in the XY body. These unanticipated observations implicate roles for the XY body in controlling mRNA metabolism and/or “poising” protein translation complexes before the meiotic division phase in spermatocytes.

Summary Sentence

In spermatocytes, translation initiation factor EIF4G3 localizes almost exclusively to the XY body, suggesting a role for this chromatin domain in post-transcriptional regulation of spermatogenic gene expression.

Chromatin remodeling during spermatogenesis culminates in the exchange of nucleosomes for transition proteins and protamines as an important part of spermatid development to give rise to healthy sperm. Comparative immunofluorescence analyses of equine and murine testis histological sections were used to characterize nucleoprotein exchange in the stallion. Histone H4 hyperacetylation is considered a key event of histone removal during the nucleoprotein transition to a protamine-based sperm chromatin structure. In the stallion, but not the mouse, H4 was already highly acetylated in lysine residues K5, K8, and K12 in round spermatids almost immediately after meiotic division. Time courses of transition protein 1 (TP1), protamine 1, H2A histone family member Z (H2AFZ), and testis-specific histone H2B variant (TH2B) expression in stallion spermatogenesis were similar to the mouse where protamine 1 and TP1 were only expressed in elongating spermatids much later in spermatid development. The additional acetylation of H4 in K16 position (H4K16ac) was detected during a brief phase of spermatid elongation in both species, concomitant with the phosphorylation of the noncanonical histone variant H2AFX resulting from DNA strand break-mediated DNA relaxation. The results suggest that H4K16 acetylation, which is dependent on DNA damage signaling, may be more important for nucleosome replacement in spermiogenesis than indicated by data obtained in rodents and highlight the value of the stallion as an alternative animal model for investigating human spermatogenesis. A revised classification system of the equine spermatogenic cycle for simplified comparison with the mouse is proposed to this end.

Summary Sentence

Histone H4 acetylationmarks that are necessary for nucleosome removal appear immediately after meiosis in equine but not murine spermatids.

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer found in polyvinyl chloride products such as vinyl flooring, plastic food containers, medical devices, and children's toys. DEHP is a ubiquitous environmental contaminant and is a known endocrine disrupting chemical. Little is known about the effects of prenatal DEHP exposure on the ovary and whether effects occur in subsequent generations. Thus, we tested the hypothesis that prenatal exposure to DEHP disrupts ovarian functions in the F1, F2, and F3 generations of female mice. To test this hypothesis, pregnant CD-1 mice were orally dosed with corn oil (vehicle control) or DEHP (20 and 200 µg/kg/day and 200, 500, and 750 mg/kg/day) daily from gestation day 10.5 until birth (7–28 dams/treatment group). F1 females were mated with untreated males to obtain the F2 generation, and F2 females were mated with untreated males to produce the F3 generation. On postnatal days 1, 8, 21, and 60, ovaries were collected and used for histological evaluation of follicle numbers and sera were used to measure progesterone, testosterone, 17β-estradiol, luteinizing hormone, and follicle stimulating hormone levels. In the F1 generation, prenatal exposure to DEHP disrupted body and organ weights, decreased folliculogenesis, and increased serum 17β-estradiol levels. In the F2 generation, exposure to DEHP decreased body and organ weights, dysregulated folliculogenesis, and disrupted serum progesterone levels. In the F3 generation, DEHP exposure accelerated folliculogenesis. These data suggest that prenatal exposure to DEHP leads to adverse multigenerational and transgenerational effects on ovarian function.

Summary Sentence

Developmental exposure to di(2-ethylhexyl) phthalate adversely affects ovarian functions in multiple generations of mice.