Embryo imaging has long been a critical tool for in vitro fertilization laboratories, aiding in morphological assessment of embryos, which remains the primary tool for embryo selection. With the recent emergence of clinically applicable real-time imaging systems to assess embryo morphokinetics, a renewed interest has emerged regarding noninvasive methods to assess gamete and embryo development as a means of inferring quality. Several studies exist that utilize novel imaging techniques to visualize or quantify intracellular components of gametes and embryos with the intent of correlating localization of organelles or molecular constitution with quality or outcome. However, the safety of these approaches varies due to the potential detrimental impact of light exposure or other variables. Along with complexity of equipment and cost, these drawbacks currently limit clinical application of these novel microscopes and imaging techniques. However, as evidenced by clinical incorporation of some real-time imaging devices as well as use of polarized microscopy, some of these imaging approaches may prove to be useful. This review summarizes the existing literature on novel imaging approaches utilized to examine gametes and embryos. Refinement of some of these imaging systems may permit clinical application and serve as a means to offer new, noninvasive selection tools to improve outcomes for various assisted reproductive technology procedures.

Pig oocytes and embryos are highly sensitive to cryopreservation; however, tolerance to cryopreservation increases in embryos at the expanded blastocyst stage. This increased tolerance may be attributed to a decrease in cytoplasmic lipid droplets at this stage. We previously showed that an increase in the permeability of the plasma membrane in mouse oocytes to water and cryoprotectants, caused by the artificial expression of aquaporin 3, an aquaglyceroporin, enhanced tolerance to cryopreservation. In the present study, we investigated whether membrane permeability was also involved in the tolerance of pig embryos to cryopreservation. The permeability of oocytes and morulae to water and glycerol was low, whereas that of expanded blastocysts was high. Activation energy for permeability to water, glycerol, ethylene glycol, and dimethyl sulfoxide was markedly lower for expanded blastocysts than for oocytes. This suggests that water and these cryoprotectants move through expanded blastocysts predominantly by facilitated diffusion and through oocytes predominantly by simple diffusion. Aquaporin 3 mRNA was expressed in expanded blastocysts abundantly, but less so in oocytes. On the other hand, the permeability of expanded blastocysts to propylene glycol was as low as that of oocytes, and activation energy for its permeability was similar to that of oocytes, which suggests that propylene glycol moves through oocytes and embryos predominantly by simple diffusion. These results suggest that the higher tolerance of pig expanded blastocysts to cryopreservation is also related to high membrane permeability due to the expression of water/cryoprotectant channels, in addition to the decrease in cytoplasmic lipid droplets.

In mammals, sex differentiation depends on gonad development, which is controlled by two groups of sex-determining genes that promote one gonadal sex and antagonize the opposite one. SOX9 plays a key role during testis development in all studied vertebrates, whereas it is kept inactive in the XX gonad at the critical time of sex determination, otherwise, ovary-to-testis gonadal sex reversal occurs. However, molecular mechanisms underlying repression of Sox9 at the beginning of ovarian development, as well as other important aspects of gonad organogenesis, remain largely unknown. Because there is indirect evidence that micro-RNAs (miRNA) are necessary for testicular function, the possible involvement of miRNAs in mammalian sex determination deserved further research. Using microarray technology, we have identified 22 miRNAs showing sex-specific expression in the developing gonads during the critical period of sex determination. Bioinformatics analyses led to the identification of miR-124 as the candidate gene for ovarian development. We knocked down or overexpressed miR-124 in primary gonadal cell cultures and observed that miR-124 is sufficient to induce the repression of both SOX9 translation and transcription in ovarian cells. Our results provide the first evidence of the involvement of a miRNA in the regulation of the gene controlling gonad development and sex determination. The miRNA microarray data reported here will help promote further research in this field, to unravel the role of other miRNAs in the genetic control of mammalian sex determination.

The aim of the study was to characterize endometrial mRNA transcription, immunolocalization, and protein expression of interleukin (IL) 1alpha, IL1beta, IL6, and IL1RI, IL1RII, and IL6Ralpha/beta in the course of endometrosis during the estrous cycle. Additionally, the influence of IL1alpha, IL1beta, and IL6 on prostaglandin (PG) secretion and PG synthase mRNA transcription in endometrial tissue during endometrosis was investigated. The endometrial samples were obtained at the early (n = 12), mid- (n = 12), and late (n = 12) luteal phases and at the follicular (n = 12) phase of the estrous cycle. Within each of these phases, there were four samples within each category I, II, and III of endometrium, according to the Kenney classification. In experiment 1, transcription of IL1alpha, IL1beta, IL6, and their receptor's (IL1RI, IL1RII, and IL6Ralpha/beta) mRNAs and their immunolocalization and protein expression were determined using real-time PCR and immunohistochemistry, respectively. In Experiment 2, endometrial samples (n = 5 samples within categories I, II, and III) were obtained for tissue culture in the midluteal phase of the estrous cycle. The endometrial tissues were stimulated with IL1alpha (10 ng/ml), IL1beta (10 ng/ml), IL6 (10 ng/ml), and oxytocin (positive control; 10⁻⁷ M) for 24 h. The PG concentration was determined using ELISA. In addition, transcription of PTGS-2, PGES, and PGFS mRNAs was determined using real-time PCR. ILs were found to regulate PG secretion via modulation of PG synthases in equine endometrium. The alterations in IL and the expression of their receptors, and in endometrial secretory functions, were observed during the course of endometrosis, and suggest serious changes in the endometrial microenvironment. The described disturbances may be closely related to impaired endometrial processes responsible for the subfertility or the infertility in endometrosis.

Transforming growth factor-beta (TGF-B) plays an important role in embryo implantation; however, TGF-B requires liberation from its inactive latent forms (i.e., large latent TGF-B complex [LLC] and small latent TGF-B complex [SLC]) to its biologically active (i.e., monomer or dimer) forms in order to act on its receptors (TGF-BRs), which in turn activate SMAD2/3. Activation of TGF-B1 from its latent complexes in the uterus is not yet deciphered. We investigated uterine latent TGF-B1 complex and its biologically active form during implantation, decidualization, and delayed implantation. Our study, utilizing nonreducing SDS-PAGE followed by Western blotting and immunoblotting with TGF-B1, LTBP1, and latency-associated peptide, showed the presence of LLC and SLC in the uterine extracellular matrix and plasma membranous protein fraction during stages of the implantation period. A biologically active form of TGF-B1 (∼17-kDa monomer) was highly elevated in the uterine plasma membranous compartment at the peri-implantation stage (implantation and nonimplantation sites). Administration of hydroxychloroquine (an inhibitor of pro-TGF-B processing) at the preimplantation stage was able to block the liberation of biologically active TGF-B1 from its latent complex at the postimplantation stage; as a consequence, the number of implantation sites was reduced at Day 5 (1000 h), as was the number of fetuses at Day 13. The inhibition of TGF-B1 showed reduced levels of phosphorylated SMAD3. Further, the delayed-implantation mouse model showed progesterone and estradiol coordination to release the active TGF-B1 form from its latent complex in the receptive endometrium. This study demonstrates the importance of liberation of biologically active TGF-B1 during the implantation period and its regulation by estradiol.

All mammalian uteri have luminal (LE) and glandular epithelia (GE) in their endometrium. The LE mediates uterine receptivity and blastocyst attachment for implantation, and the GE synthesize and secrete or transport bioactive substances involved in blastocyst implantation, uterine receptivity, and stromal cell decidualization. However, the mechanisms governing uterine epithelial development after birth and their function in the adult are not fully understood. Here, comprehensive microarray analysis was conducted on LE and GE isolated by laser capture microdissection from uteri on Postnatal Day 10 (PD 10) and day of pseudopregnancy (DOPP) 2.5 and 3.5. This data was integrated with analysis of uteri from gland-containing control and aglandular progesterone-induced uterine gland knockout mice from PD 10 and DOPP 3.5. Many genes were expressed in both epithelia, but there was greater expression of genes in the LE than in the GE. In the neonate, GE-expressed genes were enriched for morphogenesis, development, migration, and retinoic acid signaling. In the adult, LE-expressed genes were enriched for metabolic processes and steroid biosynthesis, whereas retinoid signaling, tight junction, extracellular matrix, and regulation of kinase activity were enriched in the GE. The transcriptome differences in the epithelia support the idea that each cell type has a distinct and complementary function in the uterus. The candidate genes and regulatory networks identified here provide a framework to discover new mechanisms regulating development of epithelia in the postnatal uterus and their functions in early pregnancy.

The oviduct serves as a site for the fertilization of the ovum and the transport of the conceptus down to the uterus for implantation. In this study, we investigated the presence of adrenomedullin (ADM) and its receptor component proteins in the pig oviduct. The effect of ADM on oviductal secretion, the specific receptor, and the mechanisms involved were also investigated. The presence of ADM and its receptor component proteins in the pig oviduct were confirmed using immunostaining. Short-circuit current (I_sc) technique was employed to study chloride ion secretion in the oviductal epithelium. ADM increased I_sc through cAMP- and calcium-activated chloride channels, and this effect could be inhibited by the CGRP receptor antagonist, hCGRP8-37. In contrast, the nitric oxide synthase inhibitor, l-NG-nitroarginine methyl ester (l-NAME), could not block the effect of ADM on I_sc. In summary, ADM may increase oviductal fluid secretion via chloride secretion independent of the nitric oxide pathway for the transport of sperm and the conceptus.

The synaptonemal complex protein 1 (Sycp1) is required for the formation of crossovers that occurs during the meiotic prophase. The tissue and cell-specific expression pattern of the Sycp1 protein have been studied in mammals and fish, but data on the corresponding transcript remain scarce. In this report, we described for the first time in zebrafish the tissue- and cell-specific expression pattern of the sycp1 gene. In ovary, the expression of the sycp1 transcript was restricted to the early primary oocytes. In testis, the sycp1 transcript was observed in primary spermatocytes in agreement with a previous report describing the localization of the Sycp1 protein in those cells. Unexpectedly, sycp1 transcript expression remained high in spermatids. To gain insight on the genomic region responsible for the sycp1 gene expression pattern, we generated four independent Dr_sycp1:eGFP transgenic zebrafish lines carrying the −1482/ 338 gene fragment fused to the enhanced green fluorescent protein reporter gene. We demonstrate that this promoter fragment contains the information required for the cell-specific expression of the endogenous sycp1 gene in males and in females. However, the GFP protein and its associated fluorescence persist in spermatozoa and maturing oocytes. The Dr_sycp1:eGFP zebrafish lines have the potential to be valuable models to trace meiosis onset in zebrafish and constitute the first transgenic lines expressing the GFP reporter protein only in the male meiotic and postmeiotic cells in fish.

The hermaphrodite Pacific oyster Crassostrea gigas displays a high energy allocation to reproduction. We studied the expression of AMP-activated protein kinase (AMPK) during gametogenesis in the gonad and characterized the mRNA sequences of the AMPK subunits: the AMPK alpha mRNA sequence was previously characterized; we identified AMPK beta, AMPK gamma, and mRNAs of putative AMPK-related targets following bioinformatics mining on existing genomic resources. We analyzed the mRNA expression of the AMPK alpha, beta, and gamma subunits in the gonads of male and female oysters through a reproductive cycle, and we quantified the mRNA expression of genes belonging to fatty acid and glucose metabolism. AMPK alpha mRNA levels were more abundant in males at the first stage of gametogenesis, when mitotic activity and the differentiation of germinal cells occur, and were always more abundant in males than in females. Some targets of fatty acid and glucose metabolism appeared to be correlated with the expression of AMPK subunits at the mRNA level. We then analyzed the sex-specific AMPK activity by measuring the phosphorylation of the catalytic AMPK alpha protein and its expression at the protein level. Both the amount of AMPK alpha protein and threonine 172 phosphorylation appeared to be almost totally inhibited in mature female gonads at stage 3, at the time when accumulation of reserves in oocytes was promoted, while it remained at a high level in mature spermatozoa. Its activation might play a sex-dependent role in the management of energy during gametogenesis in oyster.

Celiac disease (CD) is an autoimmune enteropathy triggered by gluten ingestion and characterized by circulating anti-transglutaminase type 2 (anti-TG2) autoantibodies. An epidemiological link between maternal CD and increased risk of pregnancy failure has been established; however, the mechanism underlying this association is still poorly understood. Because proper endometrial angiogenesis and decidualization are prerequisites for placental development, we investigated the effect of anti-TG2 antibodies on the process of endometrial angiogenesis. Binding of anti-TG2 antibodies to human endometrial endothelial cells (HEECs) was evaluated by ELISA. Angiogenesis was studied in vitro on HEECs and in vivo in a murine model. In particular, we investigated the effect of anti-TG2 antibodies on HEEC matrix metalloprotease-2 (MMP-2) activity by gelatin zymography, cytoskeletal organization and membrane properties by confocal microscopy, and activation of extracellular signal-regulated kinases (ERKs) and focal adhesion kinase (FAK) by Western blot analysis. Anti-TG2 antibodies bound to HEECs and decreased newly formed vessels both in vitro and in vivo. Anti-TG2 antibodies impaired angiogenesis by inhibiting the activation of MMP-2, disarranging cytoskeleton fibers, changing the physical and mechanical properties of cell membranes, and inhibiting the intracellular phosphorylation of FAK and ERK. Anti-TG2 antibodies inhibit endometrial angiogenesis affecting the TG2-dependent migration of HEECs and extracellular matrix degradation, which are necessary to form new vessels. Our results identify pathogenic mechanisms of placental damage in CD.

Human placental syncytiotrophoblasts lack expression of most types of human leukocyte antigen (HLA) class I and class II molecules; this is thought to contribute to a successful pregnancy. However, the HLA class Ib antigens HLA-G, -E, and -F and the HLA class Ia antigen HLA-C are selectively expressed on extravillous trophoblast cells, and they are thought to play a major role in controlling feto-maternal tolerance. We have hypothesized that selective expression, coupled with the preferential physical association of pairs of HLA molecules, contribute to the function of HLA at the feto-maternal interface and the maternal recognition of the fetus. We have developed a unique analytical model that allows detection and quantification of the heterotypic physical associations of HLA class I molecules expressed on the membrane of human trophoblast choriocarcinoma cells, ACH-3P and JEG-3. Automated image analysis was used to estimate the degree of overlap of HLA molecules labeled with different fluorochromes. This approach yields an accurate measurement of the degree of colocalization. In both JEG-3 and ACH-3P cells, HLA-C, -E, and -G were detected on the cell membrane, while the expression of HLA-F was restricted to the cytoplasm. Progesterone treatment alone induced a significant increase in the expression level of the HLA-G/HLA-E association, suggesting that this heterotypic association is modulated by this hormone. Our data shows that the cell-surface HLA class I molecules HLA-G, -E, and -C colocalize with each other and have the potential to form preferential heterotypic associations.

Tolerance of the maternal immune system in pregnancy is important for successful pregnancy because the semiallogeneic fetus may be subject to antifetal responses. We examined maternal tolerance to the fetus using a murine system in which a model paternally inherited antigen, ovalbumin (OVA), is expressed exclusively in the fetus and placenta. By employing T cell receptor (TCR) transgenic mice specific for major histocompatibility complex class I- or class II-restricted epitopes of OVA (OT-I and OT-II) as mothers, we investigated the fate of fetus-specific CD8 and CD4 T cells, respectively, during gestation. Both OVA-specific CD8 and CD4 T cells displayed an activated phenotype in the peripheral lymphoid tissues of OVA-bred OT-I and OT-II mice, consistent with their encounter of fetal antigen. Whereas a small percentage of OVA-specific CD4 T cells were deleted in the periphery and thymus of OVA-bred OT-II mice, with evidence of TCR downregulation in the remaining T cells, deletion and TCR downregulation were not observed in OVA-bred OT-I mice. Both CD4 and CD8 T cells upregulated inducible costimulator expression in response to the fetal antigen, but only CD4 T cells consistently upregulated the inhibitory receptors programmed cell death 1 and cytotoxic T lymphocyte antigen-4. More regulatory T cells (Tregs) were present in pregnant OVA-bred than in WT-bred OT-II mice, revealing that Tregs expanded specifically in response to the fetal antigen. These data indicate that several mechanisms tolerize fetal antigen-specific maternal CD4 T cells, whereas tolerance of fetal antigen-specific CD8 T cells is less effective. The importance of these mechanisms is underscored by the finding that fetal loss occurs in OVA-bred OT-I but not OT-II mice.

The prostate gland is unique in that it undergoes rapid regression following castration but regenerates completely once androgens are replaced. Residual ductal components play an important role in the regeneration of a fully functional prostate. In this study, to examine how androgen status affects prostate structure and components, we conducted histopathological studies of the involuted and regenerated mouse dorsolateral prostate (DLP). In the castrated mouse DLP, the number of luminal epithelial cells decreased in a time-dependent manner. On Day 14 postandrogen replacement, the number of luminal epithelial cells was completely restored to the baseline level. In contrast, the number of basal epithelial cells gradually increased in the castrated mouse prostate. The Ki67-labeling index of prostate basal epithelial cells was significantly increased after castration. The number of basal epithelial cells decreased to baseline after androgen replacement. After castration, mRNA expression levels of specific growth factors, such as Fgf2, Fgf7, Hgf, Tgfa, and Tgfb, were relatively abundant in whole mouse DLPs. In organ culture experiments, basal epithelial proliferation was recapitulated in the absence of dihydrotestosterone (DHT). The proliferation of basal epithelial cells in the absence of DHT was suppressed by treatment with an FGF receptor inhibitor (PD173074). Moreover, FGF2 treatment directly stimulated the proliferation of basal epithelial cells. Taken together, these data indicated that the FGF2-FGF receptor signal cascade in the prostate gland may be one of the pathways stimulating the proliferation of basal epithelial cells in the absence of androgens.

The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters, and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells, we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33 542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis, including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were overrepresented in epididymis-selective DHS identified epithelial transcription factors, including ELF5 and ELF3, the androgen receptor, Pax2, and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium.

Oocyte-specific histone variants have been expected to play significant roles in early embryonic development, but the exact evidence and the biological function have remained unclear. Here, we present evidence that H2af1o, an oocyte-specific H2A variant, is required for cell synchrony before midblastula transition in early zebrafish embryos. The H2A variant is oocyte specific, peaks in mature eggs, and is supplied to early embryos. We constructed a series of deletion plasmids of the zebrafish h2af1o tagged with EGFP and determined the main key function regions including nuclear localization signal of N-terminal 25 amino acids and nucleosome binding region of 110–122 amino acid sequence in the C-terminus by microinjecting them into one-cell-stage zebrafish embryos. In comparison with ubiquitous H2A.X, the H2af1o was revealed to confer a more open structure than canonical H2A in the nucleosomes. Furthermore, we conducted the h2af1o-specific morpholino knockdown analysis in early embryos of zebrafish and revealed its biological function for maintaining cell synchrony division because the H2af1o deficiency disturbed cell synchrony in early cleavages before midblastula transition. Therefore, our current findings provided the first case to understand the biological function of maternal oocyte-specific histone variants in vertebrates.

Vanin-2 (VNN2) is known to be involved in inflammation and leukocyte migration, but its regulation in follicles remains unknown. The objectives of this work were to study the regulation of VNN2 transcripts in bovine follicles prior to ovulation and to characterize the control of its expression in bovine granulosa cells. VNN2 expression was studied using total RNA extracted from granulosa cells of small follicles (2–4 mm in diameter), dominant follicles obtained on Day 5 of the estrous cycle, ovulatory follicles obtained 0–24 h after human chorionic gonadotropin (hCG), and corpora lutea on Day 5 of the cycle. The results from RT-PCR analyses showed that levels of VNN2 mRNA were high in ovulatory follicles 24 h post-hCG but low in the other tissues. In ovulatory follicles, levels of VNN2 mRNA were low at 0 h but significantly up-regulated 12–24 h post-hCG. To determine factors controlling VNN2 gene expression, established primary cultures of granulosa cells isolated from bovine dominant follicles were used. Treatment with forskolin elevated VNN2 mRNA expression as observed in vivo. Mutation studies identified the minimal region conferring basal and forskolin-stimulated VNN2 promoter activities, which were dependent on chicken ovalbumin upstream promoter-transcription factor (COUP-TF), GATA, and Ebox cis-elements. Electrophoretic mobility shift assays identified COUP-TF, GATA4, and upstream stimulating factor proteins as key factors interacting with these elements. Chromatin immunoprecipitation assays confirmed basal and forskolin-induced interactions between these proteins and the VNN2 promoter in bovine granulosa cell cultures. VNN2 promoter activity and mRNA expression were markedly stimulated by forskolin and overexpression of the catalytic subunit of PKA, but inhibited by PKA and ERK1/2 inhibitors. Collectively, the findings from this study describe for the first time the gonadotropin/forskolin-dependent up-regulation of VNN2 transcripts in granulosa cells of preovulatory follicles and provide insights into some of the molecular bases of VNN2 gene expression in follicular cells.

Menopause is the permanent cessation of menstruation that results from depletion of ovarian germ cells and follicles. Although most animals experience reproductive senescence, the mechanisms differ from that in women, who may live more than one-third of their lives after menopause and consequently face the risk of a number of menopause-associated health problems. Understanding factors that influence ovarian aging may provide strategies to delay or alleviate physiological alterations that take place in postmenopausal women. The germ cell-deficient Wv mice recapitulate follicle loss, prolong postreproductive lifespan, and model many physiological changes that take place in postmenopausal women. Here, using genetic and pharmacological approaches, we found that inhibition of cyclooxygenase-1 but not cyclooxygenase-2 in Wv mice delays germ cell depletion and preserves ovarian follicles. Cyclooxygenase-1 inhibition slows down follicle maturation at the conversion of primary to secondary follicles and prolongs postnatal ovarian follicle lifespan. The current study suggests that inhibition of cyclooxygenase-1 may be able to delay ovarian aging and modulate menopausal timing.

Synthetic glucocorticoids, like dexamethasone (dex), restrict growth of the fetus and program its adult physiology, in part by altering placental phenotype. The route and timing of dex administration determine the fetal and adult outcomes, but whether these factors affect placental phenotype remains unknown. This study compared placental morphology, amino acid transport, and gene expression in mice given dex orally or by subcutaneous injection over the periods of most rapid placental (Days [D] 11–16) or fetal (D14–19) growth (term is D21). Compared with untreated and saline-injected controls, both dex treatments reduced placental weight at D16 and 19 and fetal weight and total labyrinthine volume at D19 to a similar extent. Only oral dex treatment from D11 to D16 reduced labyrinthine fetal capillary volume on D16 and increased placental ¹⁴C-methylaminoisobutyric acid (MeAIB) clearance at D19, 3 days after treatment ended. Neither route of dex treatment altered placental expression of Slc38a, Hsd11b, or the glucocorticoid receptor, Nr3c1, at D16. In contrast, both routes of dex treatment from D14 to D19 increased placental Hsd11b2 expression and labyrinthine maternal vessel volume. Furthermore, injection per se altered placental expression of Nr3c1, Hsd11b1, and specific Slc38a isoforms in an age-related manner. Overall, MeAIB clearance was not related to Slc38a transporter expression but was correlated inversely with maternal corticosterone concentrations when dex was undetectable in maternal plasma at D19. The effects of dex on placental phenotype, therefore, depend on both the route and timing of administration and may relate to local glucocorticoid availability during and after the treatment period.

During mammalian pregnancy, the immune system defies a double challenge: to tolerate the foreign growing fetus and to fight off infections that could affect both mother and fetus. Minimal disturbances to the fine equilibrium between immune activation and tolerance would compromise fetal survival. Here, we show that regulatory B10 cells are important for pregnancy tolerance in mice. The frequency of these cells increases during normal murine pregnancies, while mice presenting spontaneous abortion do not show elevated levels of regulatory B10 cells. When B10 cells are transferred to the abortion-prone mice, dendritic cells are kept in an immature state, and regulatory T cells increase, thus avoiding immunological rejection of the fetuses. In vitro, we could identify IL-10 secreted by B10 cells as the main mediator of these salutary effects. Our data add an important piece of information to the complex immune crosstalk during pregnancy. This study opens novel lines of work to better understand how to help women who have trouble in maintaining a pregnancy.

Normal pregnancy is supported by increased levels of progesterone (P4), which is secreted from ovarian luteal cells via enzymatic steps catalyzed by P450scc (CYP11A1) and HSD3B. The development and maintenance of corpora lutea during pregnancy, however, are less well understood. Here we used Cyp11a1 transgenic mice to delineate the steps of luteal cell differentiation during pregnancy. Cyp11a1 in a bacterial artificial chromosome was injected into mouse embryos to generate transgenic mice with transgene expression that recapitulated endogenous Cyp11a1 expression. Cyp11a1 transgenic females displayed reduced pregnancy rate, impaired implantation and placentation, and decreased litter size in utero, although they produced comparable numbers of blastocysts. The differentiation of transgenic luteal cells was delayed during early pregnancy as shown by the delayed activation of genes involved in steroidogenesis and cholesterol availability. Luteal cell mitochondria were elongated, and their numbers were reduced, with morphology and numbers similar to those observed in granulosa cells. Transgenic luteal cells accumulated lipid droplets and secreted less progesterone during early pregnancy. The progesterone level returned to normal on Gestation Day 9 but was not properly withdrawn at term, leading to delayed stillbirth. P4 supplementation rescued the implantation rates but not the ovarian defects. Thus, overexpression of Cyp11a1 disrupts normal development of the corpus luteum, leading to progesterone insufficiency during early pregnancy. Misregulation of the progesterone production in Cyp11a1 transgenic mice during pregnancy resulted in aberrant implantation, anomalous placentation, and delayed parturition.

The placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2; encoded by the HSD11B2 gene) plays a key role in fetal development, but its regulation is incompletely understood. We previously demonstrated that p38 MAPK was a positive regulator of placental 11beta-HSD2. However, it remains unknown if the other two MAPKs, ERK1/2 and JNK, were also involved. In the present study, we identified ERK1/2 as an important regulator of placental 11beta-HSD2. We showed that inhibition of ERK1/2 with the pharmacological inhibitor U0126 led to a 3-fold increase in 11beta-HSD2 activity, protein, and mRNA in primary human placental trophoblast cells. In contrast, the JNK inhibitor SP600125 had no effect. Furthermore, U0126 increased the HSD11B2 promoter activity by 300%, indicating that ERK1/2 regulates placental 11beta-HSD2 expression through a transcriptional mechanism. Importantly, siRNA-mediated knockdown of ERK1/2 caused a similar increase in 11beta-HSD2 protein. In addition, given that we previously showed that cadmium reduced placental 11beta-HSD2 expression via a transcriptional mechanism, but the signal transduction pathways involved remain unclear, we also addressed this question and found that treatment of trophoblast cells with cadmium led to rapid activation of ERK1/2. Importantly, U0126 completely abrogated the inhibitory effects of cadmium on placental 11beta-HSD2. Taken together, the present study not only identifies the ERK1/2 signaling pathway as a potent negative regulator of placental 11beta-HSD2 but also demonstrates that this pathway mediates cadmium repression of placental 11beta-HSD2. Thus, our present study reveals 11beta-HSD2 as an important target through which ERK1/2 may regulate human placental function and consequently fetal development.

Proper regulation of trophoblast proliferation, differentiation, and function are critical for placenta development and function. The RNA-binding protein, LIN28A, has been well characterized as a potent regulator of differentiation in embryonic stem cells; however, little is known about the function of LIN28A in the placenta. We assessed LIN28A in vitro using mouse trophoblast stem (mTS) cells and human trophoblast cells (ACH-3P). We observed that LIN28A decreased and let-7 miRNA increased when mTS cells were induced to differentiate into mouse trophoblast giant cells (mTGCs) upon the removal of FGF4, heparin and conditioned medium. Similarly, we observed that LIN28A decreased in ACH-3P cells induced to syncytialize with forskolin treatment. To assess LIN28A in vivo we examined Embryonic Day 11.5 mouse placenta and observed abundant LIN28A in the chorioallantoic interface and labyrinth layer, with little LIN28A staining in spongiotrophoblast or differentiated mTGCs. Additionally, shRNA-mediated LIN28A knockdown in ACH-3P cells resulted in increased spontaneous syncytialization, and increased levels of syncytiotrophoblast markers hCG, LGALS13, and ERVW-1 mRNA. Additionally, targeted degradation of LIN28A mRNA increased responsiveness to forskolin-induced differentiation. In contrast, targeted degradation of Lin28a mRNA in mTS cells did not alter cell phenotype when maintained under proliferative culture conditions. Together, these data establish that LIN28A has a functional role in regulating trophoblast differentiation and function, and that loss of LIN28A in human trophoblast is sufficient to induce differentiation, but does not induce differentiation in the mouse.

The mammalian target of rapamycin (mTOR) and the eukaryotic initiation factor 2 (eIF2) signaling pathways control protein synthesis in response to nutrient availability. Moreover, mTOR is a positive regulator of placental nutrient transport and is involved in the regulation of fetal growth. We hypothesized that maternal overweight, induced by a diet with high saturated fat content, i) up-regulates placental mTOR activity and nutrient transport, resulting in fetal overgrowth; ii) inhibits phosphorylation of eIF2 at its alpha subunit (eIF2alpha); and iii) leads to placental inflammation. Albino Wistar female rats were fed a control or high-saturated-fat (HF) diet for 7 wk before mating and during pregnancy. At Gestational Day 21, the HF diet significantly increased maternal and fetal triglyceride, leptin, and insulin (but not glucose) levels and maternal and fetal weights, and placental weights trended to increase. Phosphorylated 4EBP1 (T37/46 and S65) was significantly higher, and phosphorylated rpS6 (S235/236) tended to increase, in the placentas of dams fed an HF diet, indicating an activation of mTOR Complex 1 (mTORC1). Phosphorylation of AMPK and eIF2alpha was reduced in the HF diet group compared to the control. The expression and activity of placental nutrient transporters and lipoprotein lipase (LPL), as well as the activation of inflammatory pathways, were not altered by the maternal diet. We conclude that maternal overweight induced by an HF diet stimulates mTORC1 activity and decreases eIF2alpha phosphorylation in rat placentas. We speculate that these changes may up-regulate protein synthesis and contribute to placental and fetal overgrowth.

Prenatal testosterone (T) exposure impacts postnatal cardiovascular function, leading to increases in blood pressure with associated decreased endothelium-dependent vascular relaxation in adult females. Endothelial function in males is not known. Furthermore, which of the endothelial pathways contributes to endothelial dysfunction and if there exists sex differences are not known. The objective of this study was to characterize the relative contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to the impaired endothelium-dependent vasodilation in prenatal T-exposed adult males and females. Offspring of pregnant rats treated with T propionate or its vehicle were examined. Telemetric blood pressure levels and endothelium-dependent vascular reactivity were assessed with wire myography. Levels of nitric oxide synthase (NOS3) and Kcnn3 and Kcnn4 channel expression were examined in mesenteric arteries. Mean arterial pressure was significantly higher in T males and females than in controls. Endothelium-dependent acetylcholine relaxation was significantly lower in both T males and females. EDHF-mediated relaxation was specifically blunted in T males (E_max = 48.64% ± 3.73%) compared to that in control males (E_max = 81.71% ± 3.18%); however, NO-mediated relaxation was specifically impaired in T females (E_max = 36.01% ± 4.29%) compared with that in control females (E_max = 54.56% ± 6.37%). Relaxation to sodium nitroprusside and levcromakalim were unaffected with T-treatment. NOS3 protein was decreased in T females but not in T males. Kcnn3 expression was decreased in both T males and females compared to controls. These findings suggest that prenatal T leads to an increase in blood pressure in the adult offspring, associated with blunting of endothelial cell-associated relaxation and that the effects are sex-specific: EDHF-related in males and NO-related in females.

Uterine natural killer (uNK) cells remarkably increase in number after implantation. NK cells or their precursors migrate from the blood stream and contribute to the increase. However, the contribution of uNK cells present in the virgin uterus has been unclear. To elucidate this issue, we examined uterine leukocyte subsets during pregnancy in BALB/c mice. The most dramatic change was the massive decrease in CD11b⁻ or Gr-1⁻ cells at Gestation Day (gd) 6. Uterine NK cells at gd 0 were CD11b⁻, and severely decreased at gd 6. The decrease was selective, and the proportion of other cells examined did not decrease. Uterine NK cells almost recovered at gd 12. These cells at gd 12 were more mature and/or activated in terms of expression of CD11b, CD27, CD127, or B220 than at gd 0. CXCL12 expression was observed on uterine cells at gd 0 or 6, but not at gd 12, whereas CXCR4 was detected on uNK cells at gds 0 and 12. A much higher expression of IL-15 in uterine cells or interferon-gamma expression in uNK cells was observed at gd 12 than at gd 0. IL-15 receptor alpha chain was detected on uNK cells at gd 12, but not at gd 0. Taken together, these findings were consistent with our interpretation that uNK cells present at gd 0 do not contribute to the increase of uNK cell number after implantation, and NK cells or their precursors migrate into the uterus, mature, and produce interferon-gamma to support pregnancy.

Oocytes with germinal vesicles (GVs) replaced with somatic nuclei exhibit meiotic abnormalities. Although this suggests an exclusive role for GV material in meiosis, mechanisms by which a lack of GV material causes meiotic defects are unknown. Knowledge of these mechanisms will help us to understand meiotic control, nuclear-cytoplasmic interactions, and cellular reprogramming. This study showed that although oocytes with prometaphase I chromosomes replaced with primary spermatocyte nuclei (PSN) did not, oocytes with GV replaced with PSN (PSG oocytes) did display meiotic defects. Among the defects, insufficient chromosome condensation with chromosome bridges was associated with spindle abnormalities. Abnormal spindle migration, cortical nonpolarization, and the aberrant spindle caused randomly positioning of cleavage furrows, leading to large first polar bodies (PB1) and unequal allocation of chromosomes and mitogen-activated protein kinases (MAPK) between oocyte and PB1. Spindle assembly checkpoint was activated but did not stop the incorrect division. The unequal MAPK allocation resulted in differences in pronuclear formation and PB1 degeneration; oocytes receiving more MAPK were more capable of forming pronuclear rudiments, whereas PB1 receiving more MAPK degenerated sooner than those that received less. Because none of the PSG oocytes or the enucleated GV oocytes injected with sperm heads showed cortical polarization in spite of chromosome localization close to the oolemma and because the PSG oocytes receiving more MAPK could form only pronuclear rudiments and not normal pronuclei, we suggest that the GV material plays essential roles in polarization and pronuclear formation on top of those played by chromosomes or MAPK. In conclusion, using PSG oocytes as models, this study has revealed the primary pathways by which a lack of GV material cause meiotic defects, laying a foundation for future research on the role of GV material in oocyte meiotic control.

JmjC domain-containing proteins are a class of enzymes responsible for histone demethylation. Previous studies revealed that the JmjC domain-containing protein KDM3A possesses intrinsic demethylase activity toward lysine 9 of histone H3 and plays essential roles in spermiogenesis. In contrast, the biological roles of JMJD1C, a KDM3A homolog in mice, are largely unknown. Here we present the crucial role of JMJD1C in male gametogenesis. Jmjd1c-deficient males became infertile due to the progressive reduction of germ cells after 3 mo of age. Importantly, Jmjd1c-deficient testes frequently contained abnormal tubules lacking developmentally immature germ cells. JMJD1C is most abundantly expressed in undifferentiated spermatogonia in mouse testis. The numbers of ZBTB16-positive spermatogonia and apoptotic germ cells in Jmjd1c-deficient testes decreased and increased in an age-dependent manner, respectively. Our studies demonstrated that JMJD1C contributes to the long-term maintenance of the male germ line.

During spermiogenesis, histones are replaced first by transition proteins and then by protamines, resulting in a very condensed sperm DNA structure that is absolutely critical for normal sperm function. We have demonstrated previously that, despite a 9-wk recovery period, mature sperm from rats treated for 9 wk with bleomycin, etoposide, and cis-platinum (BEP), the drugs used to treat testicular cancer, have reduced levels of protamine 1 and a concomitant upregulation of specific histones, highlighting a problem in histone eviction. Here, we demonstrate that regulators of histone removal are increased in elongating spermatids following recovery; however, Ac-H4 and gammaH2AX histones remain elevated in elongating spermatids or caudal epididymal spermatozoa 9 wk post-BEP treatment. This indicates that chromatin remodelers and effector proteins that respond to histone removal cues may be a target of BEP treatment. A decrease in the expression of SMARCE1 in elongating spermatids may explain the persistent retention of histones in cauda epididymal sperm 9 wk after the cessation of BEP treatment. Remarkably, proteins implicated in the translational control and posttranslational processing of protamine 1 are also significantly elevated 9 wk post-BEP treatment, suggesting that histone eviction may dictate the DNA availability for protamine binding. Males mated to control females 9 wk after BEP treatment have reduced litter sizes; moreover, the profile of gene expression in the developing testes of their pups is altered. Altering the proportion of histones to protamine in mature spermatozoa has an adverse impact on male fecundity, with modifications to epigenetic marks potentially threatening normal progeny development.