Testis cords, embryonic precursors of the seminiferous tubules, are fundamental for testis structure and function. Delay or disruption of testis cord formation could result in gonadal dysgenesis. Although mechanisms regulating testis cord formation during sex determination have been well-studied, the genes and signaling pathways involving in testis cord maintenance after the cords have formed are not well characterized. It is now clear that the maintenance of cord structure is an active process. In this review, we summarize the recent findings regarding the regulation of testis cord integrity by a series of Sertoli cell transcription factors, including the WT1-SOX8/SOX9-beta-CATENIN-DHH network, GPR56, STIM1, and NR0B1 (also known as DAX1). In particularly, we emphasize the underappreciated role of peritubular myoid cells in testis cord maintenance and their cooperation with Sertoli cells. The regulation of the size, shape, and number of testis cords by Sertoli cell proliferation (e.g., SMAD4, GATA4, and TGF-beta signaling), Leydig cell products (e.g., ACTIVIN A), vascular development (a lesson learned from PDGF signaling), and available gonad space (as observed in Ift144 mutant mice) is also addressed. Further efforts and new genetic models are needed to unveil the gene networks and underlying mechanisms regulating testis cord integrity and morphology after sex determination.

Embryo implantation into receptive endometrium requires synergistic endometrial-blastocyst interactions within the uterine cavity and is essential for establishing pregnancy. We demonstrate that exosomes (40–150 nm nanovesicles) released from endometrial epithelial cells are an important component of these interactions. We defined the proteome of purified endometrial epithelial-derived exosomes (Exos) influenced by menstrual cycle hormones estrogen (E; proliferative phase) and estrogen plus progesterone (EP; receptive phase) and examined their potential to modify trophoblast function. E-/EP-Exos were uniquely enriched with 254 and 126 proteins, respectively, with 35% newly identified proteins not previously reported in exosome databases. Importantly, EP-Exos protein cargo was related to fundamental changes in implantation: adhesion, migration, invasion, and extracellular matrix remodeling. These findings from hormonally treated ECC1 endometrial cancer cells were validated in human primary uterine epithelial cell-derived exosomes. Functionally, exosomes were internalized by human trophoblast cells and enhanced their adhesive capacity, a response mediated partially through active focal adhesion kinase (FAK) signaling. Thus, exosomes contribute to the endometrial-embryo interactions within the human uterine microenvironment essential for successful implantation.

Although the equine oviduct clearly affects early embryo development and the selective transport of equine embryos through the oviduct indicates a reciprocal interaction, the influence of the embryo on gene expression in the oviduct remains to be determined in the horse. The aim of this study was to examine this by means of RNA sequencing. Four days after ovulation, epithelial cells ipsilateral and contralateral to the ovulation side from five cyclic and five pregnant mares were collected from the oviduct. RNA was extracted, samples were sequenced, and data analysis was performed to determine differentially expressed genes (DEGs) (P value ≤0.05 and absolute fold change ≥2) and to provide functional interpretation. A total of 10 743 transcripts were identified and 253 genes were found to be upregulated and 108 to be downregulated in the pregnant ipsilateral oviduct when compared to the cyclic ipsilateral oviduct. Comparison of the ipsilateral and the contralateral oviduct indicated 164 DEGs in pregnant mares and 77 DEGs in cyclic mares. Enriched functional categories were detected only in the comparison of pregnant and cyclic ipsilateral oviducts and showed that the equine embryo affects the expression of immune response-related genes in the oviduct, with marked upregulation of interferon-associated genes. This research represents the foundation for further assessment of the role of specific genes in the early embryo-maternal dialogue of the horse.

The lactocrine hypothesis for maternal programming of neonatal development was proposed to describe a mechanism through which milk-borne bioactive factors, delivered from mother to nursing offspring, could affect development of tissues, including the uterus. Porcine uterine development, initiated before birth, is completed postnatally. However, age- and lactocrine-sensitive elements of the neonatal porcine uterine developmental program are undefined. Here, effects of age and nursing on the uterine transcriptome for 48 h from birth (Postnatal Day [PND] = 0) were identified using RNA sequencing (RNAseq). Uterine tissues were obtained from neonatal gilts (n = 4 per group) within 1 h of birth and before feeding (PND 0), or 48 h after nursing ad libitum (PND 2N) or feeding a commercial milk replacer (PND 2R). RNAseq analysis revealed differentially expressed genes (DEGs) associated with both age (PND 2N vs. PND 0; 3283 DEGs) and nursing on PND 2 (PND 2N vs PND 2R; 896 DEGs). Expression of selected uterine genes was validated using quantitative real-time PCR. Bioinformatic analyses revealed multiple biological processes enriched in response to both age and nursing, including cell adhesion, morphogenesis, and cell-cell signaling. Age-sensitive pathways also included estrogen receptor-alpha and hedgehog signaling cascades. Lactocrine-sensitive processes in nursed gilts included those involved in response to wounding, the plasminogen activator network and coagulation. Overall, RNAseq analysis revealed comprehensive age- and nursing-related transcriptomic differences in the neonatal porcine uterus and identified novel pathways and biological processes regulating uterine development.

Trophoblasts are targets of infection by Brucella spp. but their role in the pathophysiology of pregnancy complications of brucellosis is unknown. Here we show that Brucella abortus invades and replicates in the human trophoblastic cell line Swan-71 and that the intracellular survival of the bacterium depends on a functional virB operon. The infection elicited significant increments of interleukin 8 (IL8), monocyte chemotactic protein 1 (MCP-1), and IL6 secretion, but levels of IL1beta and tumor necrosis factor-alpha (TNF-alpha) did not vary significantly. Such proinflammatory response was not modified by the absence of the Brucella TIR domain-containing proteins BtpA and BtpB. The stimulation of Swan-71 cells with conditioned medium (CM) from B. abortus-infected human monocytes (THP-1 cells) or macrophages induced a significant increase of IL8, MCP-1 and IL6 as compared to stimulation with CM from non-infected cells. Similar results were obtained when stimulation was performed with CM from infected neutrophils. Neutralization studies showed that IL1beta and/or TNF-alpha mediated the stimulating effects of CM from infected phagocytes. Reciprocally, stimulation of monocytes and neutrophils with CM from Brucella-infected trophoblasts increased IL8 and/or IL6 secretion. These results suggest that human trophoblasts may provide a local inflammatory environment during B. abortus infections either through a direct response to the pathogen or through interactions with monocytes/macrophages or neutrophils, potentially contributing to the pregnancy complications of brucellosis.

The generation of diploid spermatozoa is essential for the continuity of tetraploid lineages. The DNA content of diploid spermatozoa from allotetraploid hybrids of red crucian carp and common carp was nearly twice as great as that of haploid spermatozoa from common carp, and the durations of rapid and slow progressive motility were longer. We performed comparative proteomic analyses to measure variations in protein composition between diploid and haploid spermatozoa. Using two-dimensional electrophoresis followed by liquid chromatography tandem mass spectrometry, 21 protein spots that changed in abundance were analyzed. As the common carp and the allotetraploid hybrids are not fully sequenced organisms, we identified proteins by Mascot searching against the National Center for Biotechnology Information non-redundant (NR) protein database for the zebrafish (Danio rerio), and verified them against predicted homologous proteins derived from transcriptomes of the testis. Twenty protein spots were identified successfully, belonging to four gene ontogeny categories: cytoskeleton, energy metabolism, the ubiquitin-proteasome system, and other functions, indicating that these might be associated with the variation in diploid spermatozoa. This categorization of variations in protein composition in diploid spermatozoa will provide new perspectives on male polyploidy. Moreover, our approach indicates that transcriptome data are useful for proteomic analyses in organisms lacking full protein sequences.

The theory of fetal origins of adult disease was first proposed in 1989, and in the decades since, a wide range of other diseases from obesity to asthma have been found to originate in early development. Because mammalian oocyte development begins in fetal life it has been suggested that environmental and lifestyle factors of the mother could directly impact the fertility of subsequent generations. Cigarette smoke is a known ovotoxicant in active smokers, yet disturbingly 13% of Australian and 12% of US women continue to smoke throughout pregnancy. The focus of our investigation was to characterize the adverse effects of smoking on ovary and oocyte quality in female offspring exposed in utero. Pregnant mice were nasally exposed to cigarette smoke for 12 wk throughout pregnancy/lactation, and ovary and oocyte quality of the F1 (maternal smoke exposed) generation was examined. Neonatal ovaries displayed abnormal somatic cell proliferation and increased apoptosis, leading to a reduction in follicle numbers. Further investigation found that altered somatic cell proliferation and reduced follicle number continued into adulthood; however, apoptosis did not. This reduction in follicles resulted in decreased oocyte numbers, with these oocytes found to have elevated levels of oxidative stress, altered metaphase II spindle, and reduced sperm-egg interaction. These ovarian and oocyte changes ultimately lead to subfertility, with maternal smoke-exposed animals having smaller litters and also taking longer to conceive. In conclusion, our results demonstrate that in utero and lactational exposure to cigarette smoke can have long-lasting effects on the fertility of the next generation of females.

Ovulation and luteinization are initiated in preovulatory follicles by the luteinizing hormone (LH) surge; however, the signaling events that mediate LH actions in these follicles remain incompletely defined. Two key transcription factors that are targets of LH surge are C/EBPalpha and C/EBPbeta, and their depletion in granulosa cells results in complete infertility. Microarray analyses of these mutant mice revealed altered expression of a number of genes, including growth arrest specific-1 (Gas1). To investigate functions of Gas1 in ovulation- and luteinization-related processes, we crossed Cyp19a1-Cre and Gas1^flox/flox mice to conditionally delete Gas1 in granulosa and cumulus cells. While expression of Gas1 is dramatically increased in granulosa and cumulus cells around 12–16 h post-human chorionic gonadotropin (hCG) stimulation in wild-type mice, this increase is abolished in Cebpa/b double mutant and in Gas1 mutant mice. GAS1 is also dynamically expressed in stromal cells of the ovary independent of C/EBPalpha/beta. Female Gas1 mutant mice are fertile, exhibit enhanced rates of ovulation, increased fertility, and higher levels of Areg and Lhcgr mRNA in granulosa cells. The morphological appearance and vascularization of corpora lutea appeared normal in these mutant females. Interestingly, levels of mRNA for a number of genes (Cyp11a1, Star, Wnt4, Prlr, Cd52, and Sema3a) associated with luteinization are decreased in corpora lutea of Gas1 mutant mice as compared with controls at 24 h post-hCG; these differences were no longer detectable by 48 h post-hCG. The C/EBP target Gas1 is induced in granulosa cells and is associated with ovulation and luteinization.

Preterm birth is the largest single cause of neonatal death and morbidity. By activating cytokine- and Toll-like receptor (TLR)-signaling pathways, infection and/or inflammation are strongly associated with preterm delivery. Interferon regulatory factor-1 (IRF1) is an important regulator of the inflammatory response. The aims of this study were to establish the effect of 1) labor on IRF1 expression in human fetal membranes and myometrium, 2) prolabor mediators on IRF1 expression and activity, and 3) IRF1 small interfering RNA on the expression of prolabor mediators. IRF1 expression was higher in fetal membranes and myometrium after spontaneous term labor and in preterm fetal membranes with infection. The proinflammatory cytokine IL1B, the bacterial product fsl-1, and viral analog polyinosinic:polycytidylic acid (poly [I:C]) significantly increased IRF1 mRNA expression and transcriptional activity in human primary myometrial cells. In addition, IL1B increased IRF1 activity in primary amnion cells. IRF1 silencing in myometrial cells decreased IL1B-, fsl-1-, and poly (I:C)-induced cytokine (IL6, TNF, IL1B) and chemokine (CXCL8, CCL2) mRNA expression and IL6, CXCL8, and CCL2 release. IL1B-, fsl-1-, and poly (I:C)-induced PTGS2 mRNA expression and IL1B-induced prostaglandin release was also decreased by IRF1 silencing. In conclusion, IRF1 upregulation in fetal membranes and myometrium after term labor indicates a proinflammatory role for IRF1 in human parturition. IRF1 is involved in TLR- and cytokine-mediated signaling in human myometrium. These data provide new insights into the mechanisms associated with inflammation- and infection-associated preterm birth. IRF1 inhibitors as therapeutics for the management of spontaneous preterm birth warrants further investigation.

It has been shown that adverse obstetrical outcomes such as pre-eclampsia and intrauterine growth retardation correlate with maternal infection. In this study, we investigated mechanisms involved in infection-associated abnormalities in cytotrophoblast function. Primary human first trimester cytotrophoblast cells were isolated and treated with lipopolysaccharide (LPS). Levels of the cytokines and chemokines were measured and cytotrophoblast invasion was investigated. In addition, first trimester decidual macrophages were isolated and treated with the conditioned medium from LPS-treated cytotrophoblast cells, and macrophage migration was assessed. Coculturing decidual macrophages with cytotrophoblast cells was conducted to investigate macrophage costimulatory molecule and receptor expression and intracellular cytokine production. We found that LPS exposure increased cytotrophoblast production of pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, and chemokines IL-8, macrophage inflammatory protein (MIP)-1alpha, and CXCL12 in a dose-dependent manner. In addition, LPS decreased cytotrophoblast invasion, and its effect was Toll-like receptor 4 (TLR4)-dependent and partly TNF-alpha-dependent. Conditioned medium from LPS-stimulated cytotrophoblast cells increased decidual macrophage migration and this effect was partly TLR4-dependent. Furthermore, coculturing decidual macrophages with LPS-exposed cytotrophoblast cells up-regulated macrophage CD80 and CD86 expression and intracellular TNF-alpha and IL-12p40 production, while down-regulating macrophage CD206 and CD209 expression and intracellular IL-10 secretion. LPS-stimulated macrophages also inhibited cytotrophoblast invasion. In conclusion, our results indicate that LPS increases the production of a subset of proinflammatory cytokines and chemokines by human first trimester cytotrophoblast cells, decreases cytotrophoblast invasion, and alters the cross talk between cytotrophoblast cells and decidual macrophages.

Variations in DNA methylation levels in the placenta are thought to influence gene expression and are associated with complications of pregnancy, like fetal growth restriction (FGR). The most important cause for FGR is placental dysfunction. Here, we examined whether changes in DNA methylation, followed by gene expression changes, are mechanistically involved in the etiology of FGR. In this retrospective case-control study, we examined the association between small-for-gestational-age (SGA) children and both DNA methylation and gene expression levels of the genes WNT2, IGF2/H19, SERPINA3, HERVWE1, and PPARG in first-trimester placental tissue. We also examined the repetitive element LINE-1. These candidate genes have been reported in the literature to be associated with SGA. We used first-trimester placental tissue from chorionic villus biopsies. A total of 35 SGA children (with a birth weight below the 10th percentile) were matched to 70 controls based on their gestational age. DNA methylation levels were analyzed by pyrosequencing and mRNA levels were analyzed by real-time PCR. None of the average DNA methylation levels, measured for each gene, showed a significant difference between SGA placental tissue compared to control tissue. However, hypermethylation of WNT2 was detected on two CpG positions in SGA. This was not associated with changes in gene expression. Apart from two CpG positions of the WNT2 gene, in early placenta samples, no evident changes in DNA methylation or expression were found. This indicates that the already reported changes in term placenta are not present in the early placenta, and therefore must arise after the first trimester.

The Ggnbp2 null mutant embryos died in utero between Embryonic Days 13.5 to 15.5 with dysmorphic placentae, characterized by excessive nonvascular cell nests consisting of proliferative trophoblastic tissue and abundant trophoblast stem cells (TSCs) in the labyrinth. Lethality of Ggnbp2 null embryos was caused by insufficient placental perfusion as a result of remarkable decreases in both fetal and maternal blood vessels in the labyrinth. These defects were accompanied by a significant elevation of c-Met expression and phosphorylation and its downstream effector Stat3 activation. Knockdown of Ggnbp2 in wild-type TSCs in vitro provoked the proliferation but delayed the differentiation with an upregulation of c-Met expression and an enhanced phosphorylation of c-Met and Stat3. In contrast, overexpression of Ggnbp2 in wild-type TSCs exhibited completely opposite effects compared to knockdown TSCs. These results suggest that loss of GGNBP2 in the placenta aberrantly overactivates c-Met-Stat3 signaling, alters TSC proliferation and differentiation, and ultimately compromises the structure of placental vascular labyrinth. Our studies for the first time demonstrate that GGNBP2 is an essential factor for pregnancy success acting through the maintenance of a balance of TSC proliferation and differentiation during placental development.

Transcription factors encoded by the Dmrt gene family regulate multiple aspects of animal reproduction. Most studies investigating the Dmrt gene family were conducted in model organisms from bilateral species, with a particular emphasis on gene function in male sex determination. It is still unclear whether the E. ancora Dmrt (EaDmrt) genes found in basal metazoans such as cnidarians share similar characteristics with orthologs in other metazoans. In this study, seven full Dmrt gene transcript sequences for a gonochoric coral, Euphyllia ancora (phylum: Cnidaria; class: Anthozoa), were obtained through transcriptome data mining, RT-PCR analysis, rapid amplification of cDNA ends, and sequencing. These EaDmrts were subjected to quantitative assays measuring temporal and tissue-specific expression. Results demonstrated a unique gene expression pattern for EaDmrtE, which is enriched in female germ cells during the spawning season. Based on the phylogenetic analyses performed across the homologous Dmrt genes in metazoans, we found that the female-specific EaDmrtE gene is not related to the DM1 gene of Acropora spp. coral nor to Dmrt1 of vertebrates, which are involved in sexual reproduction, especially in sex determination (vertebrate Dmrt1). Additionally, high levels of EaDmrtE transcripts detected in unfertilized mature eggs are retained in newly formed zygotes but decrease during embryonic development. We suggest that the newly discovered gene may play a role in oogenesis and early embryogenesis as a maternal factor in corals. Therefore, the sexual reproduction-associated Dmrt gene(s) should have arisen in cnidarians and might have evolved multiple times in metazoans.

Alteration of intrauterine growth trajectory is linked to metabolic diseases in adulthood. In mammalian and, specifically, human species, pregnancies through assisted reproductive technology (ART) are associated with changes in intrauterine growth trajectory. However, it is still unclear how ART alters intrauterine growth trajectory, especially reduced fetal growth in early to midgestation. In this study, using a mouse model, it was found that ART procedures reduce fetal and placental growth at Embryonic Day 10.5. Furthermore, ART leads to decreased methylation levels at H19, KvDMR1, and Snrpn imprinting control regions in the placentae, instead of fetuses. Furthermore, in the placenta, ART downregulated a majority of parentally expressed imprinted genes, which enhance fetal growth, whereas it upregulated a majority of maternally expressed genes which repress fetal growth. Additionally, the expression of genes that regulate placental development was also affected by ART. ART also downregulated a majority of placental nutrient transporters. Disruption of genomic imprinting and abnormal expression of developmentally and functionally relevant genes in placenta may influence the placental development and function, which affect fetal growth and reprogramming.

Spermatogenesis is controlled by hormonal secretions from the hypothalamus and pituitary gland, by factors produced locally in the testis, and by direct interaction between germ cells and Sertoli cells in seminiferous tubules. Although the mammalian testis contains high levels of d-aspartate (d-Asp), and d-Asp is known to stimulate the secretion of testosterone in cultured Leydig cells, its role in testis is unclear. We describe here biochemical, immunohistochemical, and flow cytometric studies designed to elucidate developmental changes in testicular d-Asp levels and the direct effect of d-Asp on germ cells. We found that the concentration of d-Asp in mouse testis increased with growth and that fluctuations in d-Asp levels were controlled in part by its degradative enzyme, d-aspartate oxidase expressed in Sertoli cells. In vitro sperm production studies showed that mitosis in premeiotic germ cells was strongly inhibited by the addition of d-Asp to the culture medium. Moreover, immunohistochemical analysis demonstrated that d-Asp accumulated in the differentiated spermatids, indicating either transport of d-Asp to spermatids or its de novo synthesis in these cells. Such compartmentation seems to prevent premeiotic germ cells in mouse testis from being exposed to the excess amount of d-Asp. In concert, our results indicate that in mouse testis, levels of d-Asp are regulated in a spatiotemporal manner and that d-Asp functions as a modulator of spermatogenesis.

Nicotine significantly promoted apoptosis in stages I, VII, VIII, and XI spermatogonia, stages I, VII, VIII, X, and XI spermatocytes, and stages I–V, VII, and VIII elongating spermatids. To explore the underlying molecular mechanisms, sperm mRNA next-generation sequencing of nicotine-treated mice was conducted. Out of the 86 genes related to apoptosis, Tnf (tumor necrosis factor alpha) was screened to be the most significant varied transcript, and the Onto-pathway analysis indicated that the TNF apoptotic pathway was especially activated by nicotine exposure. The TNF pathway was further studied at the gene and protein levels. The results showed that RIP1, the key component in the TNF apoptotic pathway, was up-expressed in its deubiquitinated form in nicotine-treated mice testis. TRIM27, an E3 ubiquitin ligase that activated TNF apoptotic pathway through up-regulating deubiquitinated RIP1, was also overexpressed in nicotine-treated spermatocytes; moreover, four consecutive CpG sites near the Trim27 transcription start site were less frequently methylated. Finally, in vitro experiments of Trim27 overexpression and RNA interference in GC-1 spermatogonial cells confirmed that the RIP1 deubiquitination and TRIM27 hyopmethylation were both positively correlated with spermatocyte apoptosis. In summary, our study suggests that nicotine may induce murine spermatozoal apoptosis via the TNF apoptotic pathway through up-regulation of deubiquitinated RIP1 by Trim27 promoter hypomethylation.

Spermatogenesis is coordinated by the spatial and temporal expression of many transcriptional and posttranscriptional factors. The cyclic AMP-responsive element modulator (CREM) gene encodes both activator and repressor isoforms that act as transcription factors to regulate spermiogenesis. We found that the testis-expressed paralog of CstF-64, tauCstF-64 (gene symbol Cstf2t), is involved in a polyadenylation site choice switch of Crem mRNA and leads to an overall decrease of the Crem mRNAs that are generated from internal promoters in Cstf2t^−/− mice. More surprisingly, loss of tauCstF-64 also leads to alternative splicing of Crem exon 4, which contains an important activation domain. Thus, testis-specific CREMtau2 isoform protein levels are reduced in Cstf2t^−/− mice. Consequently, expression of 15 CREM-regulated genes is decreased in testes of Cstf2t^−/− mice at 25 days postpartum. These effects might further contribute to the infertility phenotype of these animals. This demonstrates that tauCstF-64 is an important stage-specific regulator of Crem mRNA processing that modulates the spatial and temporal expression of downstream stage-specific genes necessary for the proper development of sperm in mice.