Stem cell therapies have opened new frontiers in medicine with the possibility of regenerating lost or damaged cells. Embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, and mesenchymal stem cells have been used to derive mature cell types for tissue regeneration and repair. However, the endometrium has emerged as an attractive, novel source of adult stem cells that are easily accessed and demonstrate remarkable differentiation capacity. In this review, we summarize our current understanding of endometrial stem cells and their therapeutic potential in regenerative medicine.

In preimplantation mouse embryos, the first lineage differentiation takes place in the 8- to 16-cell-stage embryo and results in formation of the trophectoderm (TE) and inner cell mass (ICM), which will give rise to the trophoblast of the placenta and the embryo proper, respectively. Although, it is widely accepted that positioning of a cell within the embryo influences lineage differentiation, the mechanism underlying differential lineage differentiation and how it involves cell position are largely unknown. Interestingly, novel cues from the Hippo pathway have been recently demonstrated in the preimplantation mouse embryo. Unlike the mechanisms reported from epithelium-cultured cells, the Hippo pathway was found to be responsible for translating positional information to lineage specification through a position-sensing mechanism. Disruption of Hippo pathway-component genes in early embryos results in failure of lineage specification and failure of postimplantation development. In this review, we discuss the unique role of the Hippo signaling pathway in early embryo development and its role in lineage specification. Understanding the activity and regulation of the Hippo pathway may offer new insights into other areas of developmental biology that evolve from understanding of this cell-fate specification in the early embryonic cell.

Oocytes within antral follicles are thought to have restricted access to O₂, as follicle vascularity is not adjacent and both granulosa and cumulus cells are metabolically active. Indeed, measured follicular antrum partial pressure (pO₂) is regarded as low, but accurate and direct measurement represents a technical challenge that has yet to be overcome. The oocyte itself is highly dependent on oxidative phosphorylation for survival and competence for further development following fertilization, and it has been suggested that follicular pO₂ levels are correlated with this capacity for further development. It is clear that gonadotropins are involved in regulating antrum formation, follicle vascularization, cellular differentiation, and the hypoxia-inducible factors (HIF), which are mainly regulated by dissolved O₂ concentration. A newly discovered player in this story is the intracellular production of hemoglobin by both granulosa and cumulus cells, as well as the oocyte. Furthermore, cellular hemoglobin levels are dynamic, responding to the ovulatory luteinizing hormone (LH) surge. We hypothesize that this gas transport and antioxidant molecule is involved in the prevention of hypoxic response signaling by HIFs within the preovulatory antral follicle; and the transition of granulosa cells to luteal tissue by facilitating the stabilization of HIFs, enabling induction of luteinization signaling. Another possible role is by sequestering nitric oxide (NO) during the ovulatory period, which may facilitate the resumption of meiosis in the oocyte. Testing these hypotheses will be challenging but important if the regulation of ovarian function is to be fully understood.

This study examined the effect of the presence of single or multiple embryos on the transcriptome of the bovine oviduct. In experiment 1, cyclic (nonbred, n = 6) and pregnant (artificially inseminated, n = 11) heifers were slaughtered on Day 3 after estrus, and the ampulla and isthmic regions of the oviduct ipsilateral to the corpus luteum were separately flushed. Oviductal epithelial cells from the isthmus region, in which all oocytes/embryos were located, were snap-frozen for microarray analysis. In experiment 2, heifers were divided into cyclic (nonbred, n = 6) or pregnant (multiple embryo transfer, n = 10) groups. In vitro-produced presumptive zygotes were transferred endoscopically to the ipsilateral oviduct on Day 1.5 postestrus (n = 50 zygotes/heifer). Heifers were slaughtered on Day 3, and oviductal isthmus epithelial cells were recovered for RNA sequencing. Microarray analysis in experiment 1 failed to detect any difference in the transcriptome of the oviductal isthmus induced by the presence of a single embryo. In experiment 2, following multiple embryo transfer, RNA sequencing revealed 278 differentially expressed genes, of which 123 were up-regulated and 155 were down-regulated in pregnant heifers. Most of the down-regulated genes were related to immune function. In conclusion, the presence of multiple embryos in the oviduct resulted in the detection of differentially expressed genes in the oviductal isthmus; failure to detect changes in the oviduct transcriptome in the presence of a single embryo may be due to the effect being local and undetectable under the conditions of this study.

There are still major differences between in vitro production (IVP)-derived and in vivo-derived bovine blastocysts. Therefore, intrafollicular oocyte transfer (IFOT) was used in the present study to allow early embryonic development within the physiological oviductal environment, in order to avoid subsequent harmful effects of the in vitro culture environment. Using modified ovum pickup equipment, in vitro-matured oocytes were transferred into the preovulatory follicle of synchronized heifers (follicular recipients), enabling subsequent ovulation, in vivo fertilization, and in vivo development. When 1646 in vitro-matured oocytes were transferred to 28 follicular recipients, a total of 583 embryos (35.2%) were recovered in excess after uterine flushing at Day 7. Although numbers of generated extra embryos were highly variable, preovulatory follicles with a diameter of 13–14 mm delivered significantly (P < 0.05) larger amounts of extra embryos (34.3 vs. 7.3), as well as extra morulae and blastocysts (8.3 vs. 0.8), compared with follicles with a diameter of 9–10 mm. Nevertheless, the developmental rate to the blastocyst stage was lower in IFOT compared with in vitro-derived control (Vitro) embryos at Day 7 (8.0% vs. 36.5%). Likewise, cumulative developmental rates to the morula or blastocyst stage until Day 7 were lower in IFOT-derived embryos when related to the number of transferred (8.4% vs. 51.7%) or flushed (22.8% vs. 51.7%) embryos. Of the latter, IFOT-derived embryos yielded significantly lower cleavage rates compared with the Vitro controls (63.2% vs. 88.8%), and developmental rate to the morula or blastocyst stage were lower even when related to the proportion of cleaved embryos (36.8% vs. 58.2%). In contrast, lipid content and cryotolerance did not differ between IFOT and fully IVP embryos; but IFOT-derived embryos showed significantly lower lipid content (P < 0.05) and significantly higher cryotolerance compared with IVP-derived embryos cultured in CR1aa medium supplemented with estrus cow serum (ECS), but not when cultured in SOFaa medium supplemented with fatty acid-free BSA (BSA-FFA). Finally, transfer of 19 frozen-thawed IFOT-derived blastocysts to synchronized recipients (uterine recipients) resulted in pregnancy rates comparable with those obtained after transfer of fully in vivo-derived embryos or IVP-derived embryos cultured in SOFaa BSA-FFA, whereas pregnancy rate following transfer of IVP-derived blastocysts was significantly lower when they were cultured in CR1aa ECS (42.1% vs. 13.8%). All in all, seven pregnancies presumed to be IFOT derived went to term, and microsatellite analysis confirmed that five calves were indeed derived from IFOT. To our knowledge, these are the first calves born after IFOT in cattle. Interestingly, the average birth weight of IFOT-derived calves was lower than that of IVP-derived calves, even when embryos were cultured in SOFaa BSA-FFA, indicating that the environment during early embryo development might cause fetal overgrowth. Taken together, for the first time we were able to show that IFOT is a feasible technique to generate bovine blastocysts by transferring in vitro-matured oocytes derived from slaughterhouse ovaries. These IFOT-derived blastocysts closely resemble in vivo-derived blastocysts in terms of lipid content and freeze survival. Thus, the present study laid the groundwork for newly created scientific experiments enabling novel analytical possibilities. Nevertheless, IFOT-derived embryos still reached lower pregnancy rates by trend compared with in vivo-derived embryos, also implicating an important role for the maturational environment in further developmental characteristics.

Krüppel-like Factor (KLF) 13 and the closely related KLF9 are members of the Sp/KLF family of transcription factors that have collectively emerged as essential regulators of tissue development, differentiation, proliferation, and programmed cell death. Steroid hormone-responsive tissues express multiple KLFs that are linked to progesterone receptor (PGR) and estrogen receptor (ESR) actions either as integrators or as coregulators. Endometriosis is a chronic disease characterized by progesterone resistance and dysregulated estradiol signaling; nevertheless, distinct KLF members' contributions to endometriosis remain largely undefined. We previously demonstrated promotion of ectopic lesion establishment by Klf9 null endometrium in a mouse model of endometriosis. Here we evaluated whether KLF13 loss of expression in endometrial cells may equally contribute to lesion formation. KLF13 transcript levels were lower in the eutopic endometria of women with versus women without endometriosis at menstrual midsecretory phase. In wild-type (WT) mouse recipients intraperitoneally administered WT or Klf13 null endometrial fragments, lesion incidence did not differ with donor genotype. No differences were noted for lesion volume, number, proliferation status, and apoptotic index as well. Klf13 null lesions displayed reduced total PGR and ESR1 (RNA and immunoreactive protein) and altered expression of several PGR and ESR1 target genes, relative to WT lesions. Unlike for Klf9 null lesions, changes in transcript levels for PGR-A, ESR1, and Notch/Hedgehog-associated pathway components were not observed for Klf13 null lesions. Results demonstrate lack of a causative relationship between endometrial KLF13 deficiency and lesion establishment in mice, and they support the broader participation of multiple signaling pathways, besides those mediated by steroid receptors, in the pathology of endometriosis.

Mechanisms involved in the generation of spontaneous uterine contractions are not fully understood. Kit-expressing interstitial cells of Cajal are pacemakers of contractile rhythm in other visceral organs, and recent studies describe a role for Ca² -activated Cl⁻ currents as the initiating conductance in these cells. The existence and role of similar specialized pacemaker cells in the nonpregnant uterus remains undetermined. Spontaneous contractility patterns were characterized throughout the estrous cycle in isolated, nonpregnant mouse uteri using spatiotemporal mapping and tension recordings. During proestrus, estrus, and diestrus, contraction origin predominated in the oviduct end of the uterus, suggesting the existence of a dominant pacemaker site. Propagation speed of contractions during estrus and diestrus were significantly slower than in proestrus and metestrus. Five major patterns of activity were predominantly exhibited in particular stages: quiescent (diestrus), high-frequency phasic (proestrus), low-frequency phasic (estrus), multivariant (metestrus), and complex. Kit-immunopositive cells reminiscent of pacemaking ICCs were not consistently observed within the uterus. Niflumic acid (10 μM), anthracene-9-carboxylic acid (0.1–1 mM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (10 μM) each reduced the frequency of spontaneous contractions, suggesting involvement of Cl⁻ channels in generating spontaneous uterine motor activity. It is unlikely that this conductance is generated by the Ca² -activated Cl⁻ channels, anoctamin-1 and CLCA4, as immunohistochemical labeling did not reveal protein expression within muscle or pacemaker cell networks. In summary, these results suggest that spontaneous uterine contractions may be generated by a Kit-negative pacemaker cell type or uterine myocytes, likely involving the activity of a yet-unidentified Cl⁻ channel.

Ovodefensins are a novel beta defensin-related family of antimicrobial peptides containing conserved glycine and six cysteine residues. Originally thought to be restricted to the albumen-producing region of the avian oviduct, expression was found in chicken, turkey, duck, and zebra finch in large quantities in many parts of the oviduct, but this varied between species and between gene forms in the same species. Using new search strategies, the ovodefensin family now has 35 members, including reptiles, but no representatives outside birds and reptiles have been found. Analysis of their evolution shows that ovodefensins divide into six groups based on the intra-cysteine amino acid spacing, representing a unique mechanism alongside traditional evolution of sequence. The groups have been used to base a nomenclature for the family. Antimicrobial activity for three ovodefensins from chicken and duck was confirmed against Escherichia coli and a pathogenic E. coli strain as well as a Gram-positive organism, Staphylococcus aureus, for the first time. However, activity varied greatly between peptides, with Gallus gallus OvoDA1 being the most potent, suggesting a link with the different structures. Expression of Gallus gallus OvoDA1 (gallin) in the oviduct was increased by estrogen and progesterone and in the reproductive state. Overall, the results support the hypothesis that ovodefensins evolved to protect the egg, but they are not necessarily restricted to the egg white. Therefore, divergent motif structure and sequence present an interesting area of research for antimicrobial peptide design and understanding protection of the cleidoic egg.

Vertebrate eggs are arrested at metaphase of meiosis II, a state classically known as cytostatic factor arrest. Maintenance of this arrest until the time of fertilization and then fertilization-induced exit from metaphase II are crucial for reproductive success. Another key aspect of this meiotic arrest and exit is regulation of the metaphase II spindle, which must be appropriately localized adjacent to the egg cortex during metaphase II and then progress into successful asymmetric cytokinesis to produce the second polar body. This study examined the mitogen-activated protein kinases MAPK3 and MAPK1 (also known as ERK1/2) as regulators of these two related aspects of mammalian egg biology, specifically testing whether this MAPK pathway affected myosin-II function and whether myosin-II perturbation would produce some of the same effects as MAPK pathway perturbation. Inhibition of the MEK1/2-MAPK pathway with U0126 leads to reduced levels of phosphorylated myosin-regulatory light chain (pMRLC) and causes a reduction in cortical tension, effects that are mimicked by treatment with the myosin light chain kinase (MLCK) inhibitor ML-7. These data indicate that one mechanism by which the MAPK pathway acts in eggs is by affecting myosin-II function. We further show that MAPK or MLCK inhibition induces loss of normal cortical spindle localization or parthenogenetic egg activation. This parthenogenesis is dependent on cytosolic and extracellular calcium and can be rescued by hyperloading eggs with zinc, suggesting that these effects of inhibition of MLCK or the MAPK pathway are linked with dysregulation of ion homeostasis.

Female birds have been shown to manipulate offspring sex ratio. However, mechanisms of sex ratio bias are not well understood. Reduced feed availability and change in body condition can affect the mass of eggs in birds that could lead to a skew in sex ratio. We employed feed restriction in laying chickens (Gallus gallus) to induce a decrease in body condition and egg mass using 45 chicken hens in treatment and control groups. Feed restriction led to an overall decline of egg mass. In the second period of treatment (Days 9–18) with more severe feed restriction and a steeper decline of egg mass, the sex ratio per hen (proportion of male eggs) had a significant negative association with mean egg mass per hen. Based on this association, two groups of hens were selected from feed restriction group, that is, hens producing male bias with low egg mass and hens producing female bias with high egg mass with overall sex ratios of 0.71 and 0.44 respectively. Genomewide transcriptome analysis on the germinal disks of F1 preovulatory follicles collected at the time of occurrence of meiosis-I was performed. We did not find significantly differentially expressed genes in these two groups of hens. However, gene set enrichment analysis showed that a number of cellular processes related to cell cycle progression, mitotic/meiotic apparatus, and chromosomal movement were enriched in female-biased hens or high mean egg mass as compared with male-biased hens or low mean egg mass. The differentially expressed gene sets may be involved in meiotic drive regulating sex ratio in the chicken.

Endometrial epithelial cells are the first line of defense against pathogenic bacteria infecting the uterus. Innate immune responses by these polarized epithelial cells to bacteria and tissue damage are characterized by release of the chemokine (C-X-C motif) ligand 8 (CXCL8) to attract immune cells from the circulation to the site of infection, where they are regulated by the cytokine interleukin (IL) 6. The present study tested the hypothesis that IL6 is predominantly secreted apically from polarized bovine endometrial epithelial cells in response to stimuli associated with bacterial infection and tissue damage. In postpartum animals, concentrations of IL6, but not of CXCL8, were higher in uterine mucus than in peripheral blood. In vitro, polarized endometrial epithelial cells only secreted IL6 apically when treated with bacteria, the pathogen-associated molecule lipopolysaccharide, or the damage-associated molecule IL1alpha, whereas CXCL8 accumulated apically and basolaterally. Furthermore, IL6 accumulated apically irrespective of whether lipopolysaccharide was applied to the apical or basolateral surface of epithelial cells. Secretion of IL6 from epithelial cells was dependent on the trans-Golgi network but was not affected by exogenous ovarian steroids or by coculture with stromal cells. However, a confluent epithelium was essential to protect underlying stromal cells against noxious challenges, including bacteria, lipopolysaccharide, IL1alpha, and a cytolysin. In summary, when a confluent endometrial epithelial cell barrier is faced with infection and damage, chemokines attract immune cells to the uterine lumen, but IL6 is solely secreted apically to ensure immune cells are only exposed to IL6 once they reach the lumen.

Transition nuclear proteins (TNPs), major proteins found in the chromatin of condensing spermatids, have been implicated in spermatogenesis and male fertility. In this study, DNA samples were collected from 404 Chinese Holstein bulls and sequenced to identify genetic variants in the 3′-untranslated region (UTR) of TNP1 and to investigate genetic variations in the TNP1 gene and their common haplotypes. This study was also conducted to determine whether these variations affect bovine semen quality traits and expression levels by PCR-restriction fragment length polymorphism, bioinformatics analyses, quantitative real-time PCR (qPCR), and fluorescence assay. Results showed that one new single-nucleotide polymorphism (SNP; g. 528 G>A, ss1388116558) and one reported SNP (g. 442 A>G, rs110469441) were found in the 3′-UTR of the TNP1 gene. Bioinformatics analysis results revealed that both loci were located in bta-miR-532-binding and bta-miR-204-binding regions, respectively. Association studies revealed that bulls with H1H1 (AGAG) and H1H3 (AGGG) haplotype combinations exhibited a lower deformity rate than those with other haplotype combinations (P < 0.05). The qPCR results showed that the relative mRNA expression of TNP1 in bulls with H1H1 haplotype combination was significantly higher than that in bulls with H4H4 haplotype combination (P < 0.05). MicroRNA qPCR results suggested that bta-miR-532 expression was downregulated by 5-fold in adult bull testicular tissues compared with that in fetal bull testicular tissues; by contrast, bta-miR-204 expression was downregulated by 1.6-fold. Luciferase assay results also indicated that TNP1 expression was directly targeted by bta-miR-532 and bta-miR-204 in murine Leydig tumor cell lines. These results provide the first indication of g. 442 A>G-mediated and g. 528 G>A-mediated translational suppression in which SNPs altered the binding of bta-miR-204 and bta-miR-532 to the 3′-UTR of TNP1; the mediated translational suppression could be involved in the regulation of TNP1 expression and may influence the morphological characteristics of Chinese Holstein bull sperm. We propose that SNPs on the TNP1 3′-UTR may help select semen quality trait in Chinese Holstein bulls in the dairy industry.

Resistin, a hormone secreted by adipocytes, is thought to be important in reproduction. Our previous study demonstrated resistin expression in porcine ovarian follicles and its direct effect on steroidogenesis. The aim of the current study was to evaluate the effect of gonadotropins and the local ovarian factors, such as insulin-like growth factor type 1 (IGF1) and steroids (progesterone, testosterone, and 17 beta-estradiol), on the expression and secretion of resistin, as well as its steroidogenic action. Porcine ovarian follicles were exposed to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) at 50–150 ng/ml, IGF1 (10–100 ng/ml), and steroids at 10⁻⁸ to 10⁻⁶ M for 24 h. Then, mRNA, protein expression, and medium concentration of resistin were determined using real-time PCR, Western blot analysis, and ELISA, respectively. In the subsequent experiments, ovarian follicles were exposed to resistin and/or FSH, LH, IGF1, and steroids, and ovarian steroidogenesis was analyzed. Additionally, we examined the direct effect of resistin on the protein expression of receptors for gonadotropins and investigated local factors. The results showed that gonadotropins and steroids have stimulatory effects but that IGF1 has an inhibitory effect on resistin expression and secretion. Resistin decreased gonadotropins and local hormone-induced steroid secretion and inhibited 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and cytochrome P450 aromatase protein expression. Additionally, we demonstrated that resistin increased the expression of receptors for progesterone and testosterone. These findings all show that the expression and function of resistin are regulated by gonadotropins and local factors produced by ovarian follicles.

Heat stress (HS) compromises a variety of reproductive functions in several mammalian species. Inexplicably, HS animals are frequently hyperinsulinemic despite marked hyperthermia-induced hypophagia. Our objectives were to determine the effects of HS on insulin signaling and components essential to steroid biosynthesis in the pig ovary. Female pigs (35 ± 4 kg) were exposed to constant thermoneutral (20°C; 35%–50% humidity; n = 6) or HS conditions (35°C; 20%–35% humidity; n = 6) for either 7 (n = 10) or 35 days (n = 12). After 7 days, HS increased (P < 0.05) ovarian mRNA abundance of the insulin receptor (INSR), insulin receptor substrate 1 (IRS1), protein kinase B subunit 1 (AKT1), low-density lipoprotein receptor (LDLR), luteinizing hormone receptor (LHCGR), and aromatase (CYP19a). After 35 days, HS increased INSR, IRS1, AKT1, LDLR, LHCGR, CYP19a, and steroidogenic acute regulatory protein (STAR) ovarian mRNA abundance. In addition, after 35 days, HS increased ovarian phosphorylated IRS1 (pIRS1), phosphorylated AKT (pAKT), STAR, and CYP19a protein abundance. Immunostaining analysis revealed similar localization of INSR and pAKT1 in the cytoplasmic membrane and oocyte cytoplasm, respectively, of all stage follicles, and in theca and granulosa cells. Collectively, these results demonstrate that HS alters ovarian insulin-mediated PI3K signaling pathway members, which likely impacts follicle activation and viability. In summary, environmentally induced HS is an endocrine-disrupting exposure that modifies ovarian physiology and potentially compromises production of ovarian hormones essential for fertility and pregnancy maintenance.

Maternal stress has been linked to infant birth weight outcomes, which itself may be associated with health later in life. The placenta acts as a master regulator for the fetal environment, mediating intrauterine exposures to stress through the activity of genes regulating glucocorticoids, including the 11beta-hydroxysteroid dehydrogenase (HSD11B) type 1 and 2 genes, and so we hypothesized that variation in these genes will be associated with infant birth weight. We investigated DNA methylation levels at six sites across the two genes, as well as mRNA expression for each, and the relationship to infant birth weight. Logistic regressions correcting for potential confounding factors revealed a significant association between methylation at a single CpG site within HSD11B1 and being born large for gestational age. In addition, our analysis identified correlations between methylation and gene expression, including sex-specific transcriptional regulation of HSD11B2. Our work is one of the first comprehensive views of DNA methylation and expression in the placenta for both HSD11B types 1 and 2, linking epigenetic alterations with the regulation of fetal stress and birth weight outcomes.

Prenatal exposure to elevated testosterone levels induces adult life hypertension associated with selective impairments in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation in mesenteric arteries. We tested whether the angiotensin-converting enzyme inhibitor enalapril restores EDHF function through regulating the activities of small (Kcnn3) and intermediate (Kcnn4) conductance calcium-activated potassium channels in mesenteric arteries. Pregnant Sprague-Dawley rats were injected subcutaneously with vehicle or testosterone propionate (0.5 mg/kg/day from Gestation Day 15 to 19), and their 6-mo-old adult male offspring were examined. A subset of rats in these two groups was given enalapril (40 mg/kg/day) for 2 wk through drinking water. Blood pressures were assessed through carotid arterial catheter and endothelium-dependent mesenteric arterial EDHF relaxation, using wire myography. Ace and Kcnn3 and Kcnn4 channel expression levels were also examined. Renal and vascular Ace expression and plasma angiotensin II levels were increased in testosterone offspring. Blood pressure levels were significantly higher in testosterone offspring than in controls, and treatment with enalapril significantly attenuated blood pressure in testosterone offspring. EDHF relaxation in testosterone offspring was reduced compared to that in controls, and it was significantly restored by enalapril treatment. Kcnn4 channel expression and function were similar between control and testosterone rats, but it was not affected by enalapril treatment. Relaxation mediated by Kcnn3 was impaired in testosterone offspring, and it was normalized by enalapril treatment. Furthermore, enalapril treatment restored expression levels of Kcnn3 channels. These findings suggest that enalapril has a positive influence on endothelial function with improvement in EDHF relaxation through normalization of Kcnn3 expression and activity.

The aim of this study was to identify conceptus-derived proteins, in addition to IFNT, that may facilitate pregnancy recognition in cattle. Analysis of the protein content of the uterine luminal fluid (ULF) from cyclic heifers on Day 16 by nano liquid chromatography tandem mass spectrometry identified 334 proteins. Comparison of these data with 299 proteins identified in the ULF of pregnant heifers on Day 16 identified 85 proteins only present in the ULF of pregnant heifers. Analysis of Day 16 conceptus-conditioned culture medium revealed the presence of 1005 proteins of which 30 proteins were unique to ULF from Day 16 pregnant heifers. Of these 30 proteins, 12 had mRNA expression values at least 2-fold higher in abundance (P < 0.05) in the conceptus compared to the endometrium (ARPC5L, CAPG, CKMT1, CSTB, HSPA8, HSPE1, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, TKT) as determined by RNA sequencing. In addition, genes that have a significant biological interaction with the proteins (ACO2, CKMT1, CSTB, EEF2, GDI1, GLB1, GPLD1, HNRNPA1, HNRNPA2B1, HNRNPF, HSPA8, HSPE1, IDH2, KRT75, LGALS3, MSN, NUTF2, P4HB, PRKAR2A, PSMA4, PSMB5, PSMC4, SERPINA3, TKT) were differentially expressed in the endometrium of pregnant compared to cyclic heifers during the pregnancy recognition period (Days 16–18). These results indicate that 30 proteins unique to ULF from pregnant heifers and produced by short-term in vitro cultured Day 16 conceptuses could potentially be involved in facilitating the interactions between the conceptus and the endometrium during the pregnancy recognition period.

This study assessed first the impact of endometrial Tiam1/Rac1 signals and microRNA-22 (miR-22) on embryo implantation in mice, and then the expression of the above three genes in the endometrium during the embryo implantation window in the natural menstrual cycle in women with repeated implantation failure (RIF) after in vitro fertilization treatment. Four hundred fifty-two Kun-ming female mice and 200 women (70 infertility patients with RIF, 130 women as controls) were entered into this study. Endometrial Tiam1/Rac1 signals and miR-22 expression were studied in clinical and mouse samples and serum estrogen (E₂) and progesterone (P) were analyzed in clinical subjects. A pregnant mouse model based on an endometrial miR-22 and Tiam1 mRNA expression trend of patients with RIF was constructed and then the embryo implantation numbers were analyzed, and an ovariectomized mouse model was used to assess correlations of expression of these three genes with E₂ and P. The results showed that during the embryo implantation window in the natural menstrual cycle, endometrial miR-22 was significantly higher whereas Tiam1/Rac1 signals were notably lower in patients with RIF than in controls, and the P:E₂ ratio was statistically lower in the RIF group. Tiam1/Rac1 signal down-regulation and miR-22 up-regulation contributed to the inhibition of embryo implantation in mice. We also found a suppressive effect of miR-22 up-regulation on Tiam1/Rac1 signal expression, and reciprocal regulation of E₂ and P for these three genes' expression in mice. In conclusion, miR-22 up-regulation and Tiam1/Rac1 signal down-regulation inhibited embryo implantation in mice; this mechanism may be partially due to the suppressive effect of miR-22 on Tiam1 expression, and is regulated to some extent by serum E₂ and P. Our findings provide evidence that endometrial Tiam1/Rac1 signal down-regulation along with miR-22 up-regulation during embryo implantation window in the natural menstrual cycle may be one of the reasons for the failure of embryo implantation in patients with RIF.

In most vertebrates, hermaphroditism results in infertility. However, hermaphroditism occurs in 6% of teleosts, which primarily undergo protogyny. Here, to elucidate the transient stage from gonochorism to hermaphroditism, juvenile black porgies as a model animal were fed a diet containing estradiol (E2) for 3 mo, followed by withdrawal of E2 treatment. The E2-terminated fish had ectopically located oocytes in the regenerated testes. Antimüllerian hormone (amh) was strongly expressed in the Sertoli cells with type A spermatogonia and follicle cells with vitellogenic oocytes. Amh was robustly expressed in the ectopic oocytes-bordering region of regenerated testes and in testes with nonsynchronous spermatogenesis. This Amh was released by Sertoli cells and aggregated in the area containing type A spermatogonia in the ectopic oocytes-bordering region. Our in vitro results show that exogenous recombinant Amh (rAmh) can inhibit type A spermatogonia proliferation in the testis but not oogonia proliferation in the ovary. We suggest that Amh-arrested spermatogonia A may act as a boundary to block intercellular communication (i.e., prevent peptide factors released from female tissue to alter the sexual fate of type A spermatogonia) and further inhibit female growth. These results suggest that black porgy can prevent ectopic female growth in the testis and maintain male function of the digonic gonad (testes and ovary separated by the connective tissue) through Amh action. This function of amh might shed light on why the majority of syngonic fish undergo protogyny (female-to-male sex change).

Male sex determination hinges on the development of testes in the embryo, beginning with the differentiation of Sertoli cells under the influence of the Y-linked gene SRY. Sertoli cells then orchestrate fetal testis formation including the specification of fetal Leydig cells (FLCs) that produce steroid hormones to direct virilization of the XY embryo. As the majority of XY disorders of sex development (DSDs) remain unexplained at the molecular genetic level, we reasoned that genes involved in FLC development might represent an unappreciated source of candidate XY DSD genes. To identify these genes, and to gain a more detailed understanding of the regulatory networks underpinning the specification and differentiation of the FLC population, we developed methods for isolating fetal Sertoli, Leydig, and interstitial cell-enriched subpopulations using an Sf1-eGFP transgenic mouse line. RNA sequencing followed by rigorous bioinformatic filtering identified 84 genes upregulated in FLCs, 704 genes upregulated in nonsteroidogenic interstitial cells, and 1217 genes upregulated in the Sertoli cells at 12.5 days postcoitum. The analysis revealed a trend for expression of components of neuroactive ligand interactions in FLCs and Sertoli cells and identified factors potentially involved in signaling between the Sertoli cells, FLCs, and interstitial cells. We identified 61 genes that were not known previously to be involved in specification or differentiation of FLCs. This dataset provides a platform for exploring the biology of FLCs and understanding the role of these cells in testicular development. In addition, it provides a basis for targeted studies designed to identify causes of idiopathic XY DSD.

Spermatogonial stem cells (SSCs) represent a unique population of germ cells with self-renewal potential. Although reactive oxygen species (ROS) are considered toxic to germ cells, we recently showed that moderate levels of ROS are required for SSC self-renewal and that Nox1 is involved in ROS generation. In this study, we showed that self-renewal factor treatment induces Nox3 to trigger SSC self-renewal. Nox3 was transiently expressed in cultured spermatogonia by FGF2 and GDNF stimulation, whereas Nox1 was expressed predominantly during the stable phase of proliferation. Nox3 inhibition by short hairpin RNA reduced cytokine-induced ROS generation and limited the proliferation of cultured spermatogonia. Although Nox3 overexpression revealed no apparent effect, depletion of Nox3 decreased the number of SSCs in both cultured spermatogonia and freshly isolated testis cells. Our results suggest that self-renewal of SSCs is regulated by sequential activation of different Nox genes, and underscore the complexity of ROS regulation in the self-renewal division of SSCs.