The first lineage specification during mammalian embryo development can be visually distinguished at the blastocyst stage. Two cell lineages are observed on the embryonic-abembryonic axis of the blastocyst: the inner cell mass and the trophectoderm. The timing and mechanisms driving this process are still not fully understood. In mouse embryos, cells seem prepatterned to become certain cell lineage because the first cleavage plane has been related with further embryonic-abembryonic axis at the blastocyst stage. Nevertheless, this possibility has been very debatable. Our objective was to determine whether this would be the case in another mammalian species, the bovine. To achieve this, cells of in vitro produced bovine embryos were traced from the 2-cell stage to the blastocyst stage. Blastocysts were then classified according to the allocation of the labeled cells in the embryonic and/or abembryonic part of the blastocyst. Surprisingly, we found that there is a significant percentage of the embryos (∼60%) with labeled and nonlabeled cells randomly distributed and intermingled. Using time-lapse microscopy, we have identified the emergence of this random pattern at the third to fourth cell cycle, when cells started to intermingle. Even though no differences were found on morphokinetics among different embryos, these random blastocysts and those with labeled cells separated by the embryonic-abembryonic axis (deviant pattern) are significantly bigger; moreover deviant embryos have a significantly higher number of cells. Interestingly, we observed that daughter cells allocation at the blastocyst stage is not affected by biopsies performed at an earlier stage.

The gynecological disease endometriosis is characterized by the deposition and proliferation of endometrial cells outside the uterus and clinically is linked to low body mass index (BMI). Gene expression in the liver of these women has not been reported. We hypothesized that endometriosis may impact hepatic gene expression, promoting a low BMI. To determine the effect of endometriosis on liver gene expression, we induced endometriosis in female mice by suturing donor mouse endometrium into the peritoneal cavity and measuring the weight of these mice. Dual-energy X-ray absorptiometry (DEXA) scanning of these mice showed lower body weight and lower total body fat than controls. Microarray analysis identified 26 genes differentially regulated in the livers of mice with endometriosis. Six of 26 genes were involved in metabolism. Four of six genes were upregulated and were related to weight loss, whereas two genes were downregulated and linked to obesity. Expression levels of Cyp2r1, Fabp4, Mrc1, and Rock2 were increased, whereas Igfbp1 and Mmd2 expression levels were decreased. Lep and Pparg, key metabolic genes in the pathways of the six genes identified from the microarray, were also upregulated. This dysregulation was specific to metabolic pathways. Here we demonstrate that endometriosis causes reduced body weight and body fat and disrupts expression of liver genes. We suggest that altered metabolism mediated by the liver contributes to the clinically observed low BMI that is characteristic of women with endometriosis. These findings reveal the systemic and multiorgan nature of endometriosis.

Phospholipid metabolism and signaling influences on early pregnancy events in cattle are unknown. This study aimed to characterize global phospholipid composition of oviduct and uterus during early diestrus in a model of contrasting embryo receptivity. Beef cows were treated to ovulate a larger (LF-LCL group, associated with greater receptivity) or smaller (SF-SCL group) follicle and, consequently, to present greater or smaller plasma concentrations of estradiol during proestrus-estrus, as well as progesterone during early diestrus. Oviduct and uterus (4 days after gonadotropin-releasing hormone-induced ovulation; D4) as well as the uterus (D7) were collected, and lipid profiles were monitored by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This technique allowed the identification and tissue localization of sphingomyelins (SM), phosphatidylcholines (PC), ceramides (Cer), and phosphatidylethanolamines (PE). Multivariate statistics were used to separate samples into groups with distinctly different phospholipid profiles in the uterus at D4 and D7. Different abundance of ions corresponding to specific lipids were detected on D4 (Cer [42:1], PC [31:0], PC [32:1], PC [34:4], and PC [36:4] greater for LF-LCL group; and PC [38:7], PC [38:5], PC [38:4], PC [40:7], and PC [40:6] greater for SF-SCL group) and D7 (SM [34:2], SM [34:1], PC [32:1], and PC [35:2] greater for LF-LCL group). The MALDI-MS imaging showed the spatial distributions of major phospholipids. In conclusion, distinct phospholipid profiles were associated with animals treated to show contrasting receptivity to the embryo. Functional roles of the identified phospholipids on uterine function and preimplantation embryo development deserve further studies.

Maternal RNAs are synthesized by the oocyte during its growth; some of them are utilized for oocyte-specific processes and metabolism, others are stored and used during early development before embryonic genome activation. The appropriate expression of complex sets of genes is needed for oocyte maturation and early embryo development. In spite of the basic role of noncoding RNAs in the regulation of gene expression, few studies have analyzed their role in human oocytes. In this study, we identified the microRNAs (miRNAs) expressed in human metaphase II stage oocytes, and found that some of them are able to control pluripotency, chromatin remodeling, and early embryo development. We demonstrated that 12 miRNAs are differentially expressed in women of advanced reproductive age and, by bioinformatics analysis, we identified their mRNA targets, expressed in human oocytes and involved in the regulation of pathways altered in reproductive aging. Finally, we found the upregulation of miR-29a-3p, miR-203a-3p, and miR-494-3p, evolutionarily conserved miRNAs, also in aged mouse oocytes, and demonstrated that their overexpression is antithetically correlated with the downregulation of DNA methyltransferase 3A (Dnmt3a), DNA methyltransferase 3B (Dnmt3b), phosphatase and tensin homolog (Pten), and mitochondrial transcription factor A (Tfam). We propose that oocyte miRNAs perform an important regulatory function in human female germ cells, and their altered regulation could explain the changes occurring in oocyte aging.

Inflammation plays a key role in human term and preterm labor (PTL). Intrauterine LPS has been widely used to model inflammation-induced complications of pregnancy, including PTL. It has been shown to induce an intense myometrial inflammatory cell infiltration, but the role of LPS-induced inflammatory cell activation in labor onset and fetal demise is unclear. We investigated this using a mouse model of PTL, where an intrauterine injection of 10 μg of LPS (serotype 0111:B4) was given at E16 of CD1 mouse pregnancy. This dose induced PTL at an average of 12.7 h postinjection in association with 85% fetal demise. Flow cytometry showed that LPS induced a dramatic systemic inflammatory response provoking a rapid and marked leucocyte infiltration into the maternal lung and liver in association with increased cytokine levels. Although there was acute placental inflammatory gene expression, there was no corresponding increase in fetal brain inflammatory gene expression until after fetal demise. There was marked myometrial activation of NFκB and MAPK/AP-1 systems in association with increased chemokine and cytokine levels, both of which peaked with the onset of parturition. Myometrial macrophage and neutrophil numbers were greater in the LPS-injected mice with labor onset only; prior to labor, myometrial neutrophils and monocytes numbers were greater in PBS-injected mice, but this was not associated with an earlier onset of labor. These data suggest that intrauterine LPS induces parturition directly, independent of myometrial inflammatory cell infiltration, and that fetal demise occurs without fetal inflammation. Intrauterine LPS provokes a marked local and systemic inflammatory response but with limited inflammatory cell infiltration into the myometrium or placenta.

Spontaneous preterm labor occurs in two subsets of patients with sterile intra-amniotic inflammation, a process induced by alarmins such as high-mobility group box-1 (HMGB1). Inflammasomes are implicated in the process of spontaneous preterm labor. Therefore, we investigated whether HMGB1 initiates an inflammasome-associated inflammatory response in the chorioamniotic membranes. Incubation of the chorioamniotic membranes with HMGB1 1) induced the release of mature IL-1beta and IL-6; 2) upregulated the mRNA expression of the pro-inflammatory mediators NFKB1, IL6, TNF, IL1A, IFNG, and HMGB1 receptors RAGE and TLR2; 3) upregulated the mRNA expression of the inflammasome components NLRP3 and AIM2 as well as NOD proteins (NOD1 and NOD2); 4) increased the protein concentrations of NLRP3 and NOD2; 5) increased the concentration of caspase-1 and the quantity of its active form (p20); and 6) upregulated the mRNA expression and active form of MMP-9. In addition, HMGB1 concentrations in chorioamniotic membrane extracts from women who underwent spontaneous preterm labor were greater than in those from women who had undergone spontaneous labor at term. Collectively, these results show that HMGB1 can induce an inflammatory response in the chorioamniotic membranes, which is partially mediated by the inflammasome. These results provide insight into the mechanisms whereby HMGB1 induces preterm labor and birth in mice and explain why the concentration of this alarmin is increased in women who undergo spontaneous preterm labor.

Invasive trophoblast from Day 34 horse conceptuses survives in extrauterine sites in allogeneic recipients that are immunologically naive to donor major histocompatibility complex class I antigens. The ectopic trophoblast retains its in utero characteristics, including similar lifespan, physiologic effect of its secreted product (equine chorionic gonadotropin) upon the recipient's ovaries, and induction of host immune responses. Immunologic memory has not been considered previously in this experimental system. We hypothesized that primary exposure to ectopic trophoblast would affect the recipient's immune status such that the survival time of subsequent transplants would be altered. Secondary transplant lifespans could be shortened by destructive memory responses, as has been observed in ectopic trophoblast studies in rodents, or lengthened, as occurs when male skin grafts follow multiple syngeneic pregnancies in mice. Eight mares received two closely spaced trophoblast transplants. Both grafts for each recipient were obtained from conceptuses sired by the same stallion to provide consistency in histocompatibility antigen exposure. Donor stallions were major histocompatibility complex class I homozygotes. Cytotoxic antibody production was tracked to monitor recipients' immune responses to the transplants. Detection of serum equine chorionic gonadotropin was used as a proxy for transplant lifespan. There was no significant difference between the distributions of primary and secondary transplant lifespans, despite evidence of immunologic memory. These data demonstrate that secondary ectopic trophoblast transplants in horses do not experience earlier destruction or prolonged survival following immune priming of recipients. Mechanisms responsible for the eventual demise of the transplants remain unperturbed by secondary immune responses or chronic antigenic exposure.

Beta-defensins are innate immune molecules, often described as antimicrobial peptides because of their bactericidal activity and are now known to have diverse additional functions, including cell signaling, chemoattraction, immunoregulation, and reproduction. In humans and primates, beta-defensin 126 has been shown to regulate the ability of sperm to swim through cervical mucus and to protect sperm from attack by the female immune system during transit toward the oviduct. Bovine beta-defensin 126 (BBD126) is the ortholog of human defensin 126, and computational analysis here revealed significant conservation between BBD126 and other mammalian orthologs at the N-terminus, although extensive sequence differences were detected at the C-terminus, implying possible species-specific roles for this beta-defensin in reproduction. We had previously demonstrated preferential expression of this and related beta-defensin genes in the bovine male reproductive tract, but no studies of bovine beta-defensin proteins have been performed to date. Here, we analyzed BBD126 protein using a monoclonal antibody (a-BBD126) generated against a 14 amino acid peptide sequence from the secreted fragment of BBD126. The specificity of a-BBD126 was validated by testing against the native form of the peptide recovered from bovine caudal epididymal fluid and recombinant BBD126 generated using a prokaryotic expression system. Western blot analysis of the native and recombinant forms showed that BBD126 exists as a dimer that was highly resistant to standard methods of dissociation. Immunohistochemical staining using a-BBD126 demonstrated BBD126 protein expression by epithelial cells of the caudal epididymis and vas deferens from both mature and immature bulls. BBD126 could also be seen (by confocal microscopy) to coat caudal sperm, with staining concentrated on the tail of the sperm cells. This study is the first to demonstrate beta-defensin 126 protein expression in the bovine reproductive tract and on bull sperm. Its dissociation-resistant dimeric structure is likely to have important functional implications for the role of BBD126 in bovine reproduction.

Bovine beta-defensin 126 (BBD126) exhibits preferential expression for the cauda epididymis of males, where it is absorbed onto the tail and postacrosomal region of the sperm. The aim of this study was to examine the role of BBD126 in bull sperm function. Fresh and frozen-thawed semen were incubated in the presence of different capacitating agents as well as with phosphatidylinositol-specific phospholipase C. These treatments, which have been successful in releasing beta-defensin 126 from macaque sperm, proved to be ineffective in bull sperm. This finding suggests that the protein behaves in a different manner in the bovine. The lack of success in removing BBD126 led us to use corpus epididymis sperm, a model in which the protein is not present, to study its functional role. Corpus sperm were incubated with cauda epididymal fluid (CEF) in the absence or presence of BBD126 antibody or with recombinant BBD126 (rBBD126). Confocal microscopy revealed that rBBD126 binds to corpus sperm with the same pattern observed for BBD126 in cauda sperm, whereas an aberrant binding pattern is observed when sperm are subject to CEF incubation. Addition of CEF increased motility as well as the number of corpus sperm migrating through cervical mucus from estrus cows. However, it decreased the ability of sperm to fertilize in vitro matured oocytes. The presence of the antibody failed to abrogate these effects. Furthermore, when rBBD126 was added in the absence of other factors and proteins from the CEF, an increase in motility was also observed and no negative effects in fertility were seen. These results suggest that BBD126 plays a key role in the acquisition of sperm motility in the epididymis.

Previous work characterizing ovarian bioenergetics has defined follicular metabolism by measuring metabolic by-products in culture media. However, culture conditions perturb the native state of the follicle, and these methods do not distinguish between metabolism occurring within oocytes or granulosa cells. We applied the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) at 740-nm two-photon excitation to examine the spatial distribution of free and protein-bound nicotinamide adenine dinucleotide hydride (NADH) during primordial through preovulatory stages of follicular development in fresh ex vivo murine neonatal and gonadotropin stimulated prepubertal ovaries. We obtained subcellular resolution phasor FLIM images of primordial through primary follicles and quantified the free/bound NADH ratio (relative NADH/NAD ) separately for oocyte nucleus and oocyte cytoplasm. We found that dynamic changes in oocyte nucleus free/bound NADH paralleled the developmental maturation of primordial to primary follicles. Immunohistochemistry of NAD -dependent deacetylase SIRTUIN 1 (SIRT1) in neonatal ovary revealed that increasing SIRT1 expression in oocyte nuclei was inversely related to decreasing free/bound NADH during the primordial to primary follicle transition. We characterized oocyte metabolism at these early stages to be NADH producing (glycolysis/Krebs). We extended the results of prior studies to show that cumulus and mural granulosa cell metabolism in secondary through preovulatory follicles is mainly NADH producing (glycolysis/Krebs cycle), while oocyte metabolism is mainly NADH consuming (oxidative phosphorylation). Taken together, our data characterize dynamic changes in free/bound NADH and SIRT1 expression during early follicular development and confirm results from previous studies defining antral and preovulatory follicle metabolism in culture.

There is considerable evidence that implicates oxidative stress in the pathophysiology of human pregnancy complications. However, the role and the mechanism of maintaining an antioxidant prosurvival uterine environment during normal pregnancy is largely unresolved. Herein we report that the highly active uterine unfolded protein response plays a key role in promoting antioxidant activity in the uterine myocyte across gestation. The unfolded protein response (UPR) senses the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and activates a signaling network that consists of the transmembrane protein kinase eukaryotic translation initiation factor 2 alpha kinase 3/PKR-like-ER kinase (EIF2AK3), which acts to decrease protein translation levels, allowing for a lowered need for protein folding during periods of ER stress. However, independent of its translational regulatory capacity, EIF2AK3-dependent signals elicit the activation of the transcription factor, nuclear factor erythroid 2-like 2 (NFE2L2) in response to oxidative stress. NFE2L2 binds to antioxidant response elements in the promoters of a variety of antioxidant genes that minimize the opportunities for generation of reactive oxygen intermediates. Our analysis demonstrates that in the absence of EIF2AK3, the uterine myocyte experiences increased levels of reactive oxygen species due to decreased NFE2L2 activation. Elevated levels of intracellular reactive oxygen species were observed in the EIF2AK3 null cells, and this was associated with the onset of apoptotic cell death. These findings confirm the prosurvival and antioxidant role of UPR-mediated EIF2AK3 activation in the context of the human uterine myocyte.

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.

Calcitonin gene-related peptide (CALCB), adrenomedullin (ADM), and ADM2/intermedin play critical roles in vascular adaptation during pregnancy through calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs). This study was designed to assess the predominant RAMP that associates with CALCRL to form a functional receptor in the rat uterine artery smooth muscle (RUASM). We also determined if these receptor component associations are decreased by tumor necrosis factor (TNF) alpha and if CALCB, ADM, or ADM2 can rescue CALCRL/RAMP associations. Using proximity ligation assay in RUASM cells, this study shows that CALCRL predominantly associates with RAMP1 forming a CALCB receptor, and minimally with RAMP2 and RAMP3 that confer specificity for ADM and ADM2. However, knockdown of RAMP1 mRNA increases the interaction between CALCRL and RAMP3 without affecting the association of CALCRL and RAMP2. Furthermore, CALCB, ADM, and ADM2 have no effects on the associations of CALCRL with any of the RAMPs in RUASM cells. Interestingly, CALCB reverses the TNFalpha-induced decreases in CALCRL/RAMP1 associations. Furthermore, CALCB increases ERK1/2 phosphorylation in a time-dependent manner in RUASM, and the protective effect of CALCB on TNFalpha-induced inhibition of CALCRL/RAMP1 associations was significantly blocked in presence of ERK inhibitor (PD98059). In conclusion, this study demonstrates that CALCRL predominantly associates with RAMP1 forming a CALCB-specific receptor complex in RUASM cells, which is dissociated by TNFalpha. Rescue of TNFalpha-induced dissociation of CALCRL/RAMP1 complex by CALCB in RUASM cells suggests a potential use of CALCB in developing therapeutic strategies for pregnancy-related complications that are vulnerable to abnormal levels of TNFalpha, such as fetal growth restriction and preeclampsia.

Chronic placental hypoxia is one of the root causes of placental insufficiencies that result in pre-eclampsia and maternal hypertension. Chronic hypoxia causes disruption of trophoblast (TB) development, invasion into maternal decidua, and remodeling of maternal spiral arteries. The pregnant guinea pig shares several characteristics with humans such as hemomonochorial placenta, villous subplacenta, deep TB invasion, and remodeling of maternal arteries, and is an ideal animal model to study placental development. We hypothesized that chronic placental hypoxia of the pregnant guinea pig inhibits TB invasion and alters spiral artery remodeling. Time-mated pregnant guinea pigs were exposed to either normoxia (NMX) or three levels of hypoxia (HPX: 16%, 12%, or 10.5% O₂) from 20 day gestation until midterm (39–40 days) or term (60–65 days). At term, HPX (10.5% O₂) increased maternal arterial blood pressure (HPX 57.9 ± 2.3 vs. NMX 40.4 ± 2.3, P < 0.001), decreased fetal weight by 16.1% (P < 0.05), and increased both absolute and relative placenta weights by 10.1% and 31.8%, respectively (P < 0.05). At midterm, there was a significant increase in TB proliferation in HPX placentas as confirmed by increased PCNA and KRT7 staining and elevated ESX1 (TB marker) gene expression (P < 0.05). Additionally, quantitative image analysis revealed decreased invasion of maternal blood vessels by TB cells. In summary, this animal model of placental HPX identifies several aspects of abnormal placental development, including increased TB proliferation and decreased migration and invasion of TBs into the spiral arteries, the consequences of which are associated with maternal hypertension and fetal growth restriction.

Inflammation is thought to play a critical role in the pathogenesis of placentation disorders such as recurrent miscarriages, growth restriction, and preeclampsia. Recently, neutrophil extracellular traps (NETs) have emerged as a potential mechanism for promoting inflammation in both infectious and noninfectious disorders. To investigate a pathogenic role for NETs in placentation disorders, we studied a model of antiangiogenic factor-mediated pregnancy loss in wild-type (WT) mice and in mice deficient in peptidylarginine deiminase 4 (Padi4^−/−) that are unable to form NETs. Overexpression of soluble fms-like tyrosine kinase 1 (sFlt-1), an antiangiogenic protein that is pathogenically linked with abnormal placentation disorders during early gestation, resulted in pregnancy loss and large accumulation of neutrophils and NETs in WT placentas. Interestingly, sFlt-1 overexpression in Padi4^−/− mice resulted in dramatically lower inflammatory and thrombotic response, which was accompanied by significant reduction in pregnancy losses. Inhibition of NETosis may serve as a novel target in disorders of impaired placentation.

Saccharin sodium consumption is considered safe and beneficial, owing to its very intense sweetness without any associated calories, but supporting scientific data remain sparse and controversial. Herein, we demonstrate that dose-response relationships existed with regard to administration of saccharin or sucrose to mice for 35 days, and this association involved testis-expressed sweet-tasting molecules (taste receptor type 1 subunit 3 [T1R3]; G protein alpha-gustducin [Galpha]). Mouse body weights and testis weights in middle- and low-dose saccharin-treated groups were increased with up-expressions of molecules involved in testicular sweet taste and steroidogenic (middle saccharin: steroidogenic acute regulatory protein [StAR]; P450 cholesterol side-chain cleavage enzyme [CYP11A1]; 17-alpha-hydroxylase/C17,20-lyase [CYP17A1]; low saccharin: StAR). Moreover, a high-dose saccharin-related decline in reproductive hormone levels and injuries to testis and sperm were observed to be associated with suppression of testicular T1R3 and Galpha, as well as steroidogenic-related factors (StAR; 3-beta-hydroxysteroid dehydrogenase [3-beta-HSD]; CYP11A1; CYP17A1; 17-beta-hydroxysteroid dehydrogenase [17-beta-HSD]), and activation of cleaved caspase-3. However, abnormalities of the testis and sperm in high- and middle-dose sucrose-exposed mice were related to the increased-cleaved caspase-3, but independent of T1R3 and/or Galpha. Collectively, our results clearly suggest that saccharin-induced physiologic effects on testis are associated with testicular T1R3 and Galpha, which differed from sucrose. We hence call for a reassessment of the excessive use of sweeteners in daily life, especially artificial ones, considering their potential side effects.

Precise separation of spermatogonial stem cells (SSCs) from progenitor spermatogonia that lack stem cell activity and are committed to differentiation remains a challenge. To distinguish between these spermatogonial subtypes, we identified genes that exhibited bimodal mRNA levels at the single-cell level among undifferentiated spermatogonia from Postnatal Day 6 mouse testes, including Tspan8, Epha2, and Pvr, each of which encode cell surface proteins useful for cell selection. Transplantation studies provided definitive evidence that a TSPAN8-high subpopulation is enriched for SSCs. RNA-seq analyses identified genes differentially expressed between TSPAN8-high and -low subpopulations that clustered into multiple biological pathways potentially involved in SSC renewal or differentiation, respectively. Methyl-seq analysis identified hypomethylated domains in the promoters of these genes in both subpopulations that colocalized with peaks of histone modifications defined by ChIP-seq analysis. Taken together, these results demonstrate functional heterogeneity among mouse undifferentiated spermatogonia and point to key biological characteristics that distinguish SSCs from progenitor spermatogonia.