The endometrium undergoes a pregnancy-delivery-repair cycle multiple times during the reproductive lifespan in females. Decidualization is one of the critical events for the success of this essential process. We have previously reported that Notch1 is essential for artificial decidualization in mice. However, in a natural pregnancy, the deletion of Notch1 (Pgr^Cre/+Notch1^f/f, or Notch1^d/d) only affects female fertility in the first 30 days of a 6-month fertility test, but not the later stages. In the present study, we undertook a closer evaluation at the first pregnancy of these mice to attempt to understand this puzzling phenomenon. We observed a large number of pregnancy losses in Notch1^d/d mice in their first pregnancy, which led to the subfertility observed in the first 30 days of the fertility test. We then demonstrated that the initial pregnancy loss is a consequence of impaired decidualization. Furthermore, we identified a group of genes that contribute to Notch1 regulated decidualization in a natural pregnancy. Gene ontogeny analysis showed that these differentially expressed genes in the natural pregnancy are involved in cell–cell and cell–matrix interactions, different from genes that have been previously identified from the artificial decidualization model, which contribute to cell proliferation and apoptosis. In summary, we determined that Notch1 is essential for normal decidualization in the mouse uterus only in the first pregnancy but not in subsequent ones.

Summary sentence

Notch1 is essential for normal decidualization in the mouse uterus only in the first pregnancy but not in subsequent ones; the initial pregnancy loss is a consequence of impaired decidualization.

Failures in hypothalamic kisspeptin/Kiss1r signaling are associated with infertility, and in vitro studies have shown that kisspeptin can modulate angiogenesis and immune activity. Because there is no in vivo research on the functional relationship between these factors in the reproductive system, especially in domestic cats, we evaluated the expression profile of kisspeptin/Kiss1r and angiogenic and immunological mediators in the genital tract of cyclic cats and of those with pyometra. The uterus of cats in diestrus exhibited greater gene and protein expression of Kiss1, as well as Vegf, Pigf, Mif, and Il6. In contrast, Kiss1r presented greater expression in proestrus/estrus, similarly to that observed for the immunostaining of INFγ, MIF, TNFα, and IL10. These factors were positively correlated with Kiss1 and/or Kiss1r, and a positive correlation between Kiss1 and Kiss1r was also observed in the uterus of cats during the estrous cycle. Cats with pyometra showed greater immunostaining of Kiss1 and Kiss1r on the endometrial surface and reduced immunostaining of Kiss1 in deep glands, whereas there was a significant reduction in Vegf, Pigf, Mif, and Il6 mRNA, and an increase in Tnf mRNA. The findings reveal that there is a gene correlation between kisspeptin/Kiss1r and angiogenic and immune mediators in the uterus of the domestic cat, which is modulated by the estrous cycle, and that pyometra affects the expression of these mediators. This study suggests, for the first time, a functional relationship between the Kiss/Kiss1r system and angiogenic and immune mediators in the female genital tract.

Summary sentence

Kisspeptin/Kiss1R system and angiogenic and immune mediators in the uterus of domestic cats are modulated by the estrous cycle and affected by pyometra, being that these mediators have a gene correlation in the uterus of these animals.

Enhancer of zeste homolog 2 (EZH2) has been extensively investigated to participate in diverse biological processes, including carcinogenesis, the cell cycle, X-chromosome inactivation, and early embryonic development. However, the functions of this protein during mammalian oocyte meiotic maturation remain largely unexplored. Here, combined with RNA-Seq, we provided evidence that EZH2 is essential for oocyte meiotic maturation in pigs. First, EZH2 protein expression increased with oocyte progression from GV to MII stage. Second, the siRNA-mediated depletion of EZH2 led to accelerated GVBD and early occurrence of the first polar body extrusion. Third, EZH2 knockdown resulted in defective spindle assembly, abnormal SAC activity, and unstable K-MT attachment, which was concomitant with the increased rate of aneuploidy. Finally, EZH2 silencing exacerbated oxidative stress by increasing ROS levels and disrupting the distribution of active mitochondria in porcine oocytes. Furthermore, parthenogenetic embryonic development was impaired following the depletion of EZH2 at GV stage. Taken together, we concluded that EZH2 is necessary for porcine oocyte meiotic progression through regulating spindle organization, maintaining chromosomal integrity, and mitochondrial function.

Summary sentence

EZH2, an important maternal factor, is demonstrated to have a non-classical function: participating in porcine oocyte meiosis through modulating spindle organization and maintaining chromosomal integrity.

Graphical Abstract

The objective of the study was to characterize the anatomical framework and sites of action of the nerve growth factor (NGF)-mediated ovulation-inducing system of llamas. The expression patterns of NGF and its receptors in the hypothalamus of llamas (n = 5) were examined using single and double immunohistochemistry/immunofluorescence. We also compare the expression pattern of the P75 receptor in the hypothalamus of llama and a spontaneous ovulator species (sheep, n = 5). Both NGF receptors (TrkA and P75) were highly expressed in the medial septum and diagonal band of Broca, and populations of TrkA cells were observed in the periventricular and dorsal hypothalamus. Unexpectedly, we found NGF immunoreactive cell bodies with widespread distribution in the hypothalamus but not in areas endowed with NGF receptors. The organum vasculosum of the lamina terminalis (OVLT) and the median eminence displayed immunoreactivity for P75. Double immunofluorescence using vimentin, a marker of tanycytes, confirmed that tanycytes were immunoreactive to P75 in the median eminence and in the OVLT. Additionally, tanycytes were in close association with GnRH and kisspeptin in the arcuate nucleus and median eminence of llamas. The choroid plexus of llamas contained TrkA and NGF immunoreactivity but no P75 immunoreactivity. Results of the present study demonstrate sites of action of NGF in the llama hypothalamus, providing support for the hypothesis of a central effect of NGF in the ovulation-inducing mechanism in llamas.

Summary sentence

Hypothalamic pathways for NGF actions in camelids.

Zebrafish gonadal sexual differentiation is an important but poorly understood subject. Previously, we have identified a novel insulin-like growth factor (Igf) named insulin-like growth factor 3 (Igf3) in teleosts. The importance of Igf3 in oocyte maturation and ovulation has been recently demonstrated by us in zebrafish. In this study, we have further found the essential role of Igf3 in gonadal sexual differentiation of zebrafish. A differential expression pattern of igf3 between ovary and testis during sex differentiation (higher level in ovary than in testis) was found in zebrafish. An igf3 knockout zebrafish line was established using TALENs-mediated gene knockout technique. Intriguingly, all igf3 homozygous mutants were males due to the female-to-male sex reversal occurred during sex differentiation. Further analysis showed that Igf3 did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells. Oocyte development was arrested at primary growth stage, and the ovary was gradually sex-reversed to testis before 60 day post fertilization (dpf). Such sex reversal was likely due to decreased germ cell proliferation by suppressing PI3K/Akt pathway in early ovaries of igf3 mutants. Estrogen is considered as a master regulator in fish sex differentiation. Here, we found that igf3 expression could be upregulated by estrogen in early stages of ovarian follicles as evidenced in in vitro treatment assays and cyp19a1a mutant zebrafish, and E2 failed to rescue the defects of igf3 mutants in ovarian development, suggesting that Igf3 may serve as a downstream factor of estrogen signaling in sex differentiation. Taken together, we demonstrated that Igf3 is essential for ovary differentiation in zebrafish.

Summary sentence

The gonad-specific Igf3 is essential for zebrafish ovary differentiation through regulating cell proliferation and interacting with estrogen.

Insulin-like growth factors (IGFs) are known for their involvement in endocrine and paracrine regulation of ovarian function. Although IGF2 is the predominant circulating and intraovarian form of IGFs in primate species, the stage-specific follicular expression, action, and regulation of IGF2 are not well defined. Therefore, experiments were conducted to investigate the follicular IGF production in response to steroid hormone regulation and the direct IGF actions on follicular development and function in vitro. Preantral follicles were isolated from rhesus macaque ovaries and cultured to the antral stage in media supplemented with follicle-stimulating hormone and insulin. Follicles were randomly assigned to treatment groups: (a) control, (b) trilostane (a steroid synthesis inhibitor), (c) trilostane + estradiol, (d) trilostane + progesterone, and (e) trilostane + dihydrotestosterone. Media was analyzed for IGF concentrations, which were correlated to follicle growth. Follicles produced IGF2, but not IGF1, at the antral stage. Steroid depletion decreased, whereas steroid replacement increased, IGF2 production by antral follicles. Media IGF2 levels correlated positively with antral follicle diameters. Macaque preantral follicles and granulosa cells were subsequently cultured without (control) and with recombinant human IGF2 supplementation. Follicle survival, growth, and paracrine factor production, as well as granulosa cell proliferation and gonadotropin receptor gene expression, were assessed. IGF2 addition increased follicle survival rates, diameters and inhibin B production, as well as granulosa cell proliferation. These data demonstrate that IGF2 produced by antral follicles, in response to steroid hormone regulation, could act as a paracrine factor that positively impacts preantral follicle development and function in primates.

Summary sentence

Insulin-like growth factor 2, but not insulin-like growth factor 1, is produced by antral follicles in response to steroid hormone regulation in nonhuman primates, and promotes preantral follicle growth with increased paracrine factor production.

Approximately, 25% of all preterm births are due to preterm premature rupture of membranes. Mice deficient in proteoglycans biglycan (Bgn) and decorin (Dcn) display abnormal fetal membranes and increased incidence of preterm birth. We conducted RNA-Seq to profile fetal membranes and identify molecular pathways that may lead to preterm birth in double knockout (DKO) mice (Bgn^–/–; Dcn^–/–) compared to wild-type (WT) at two different gestational stages, E12 and E18 (n = 3 in each group). 3264 transcripts were differentially regulated in E18 DKO vs. WT fetal membranes, and 96 transcripts differentially regulated in E12 DKO vs. WT fetal membranes (FDR < 0.05, log ₂ FC ≥ 1). Differentially regulated transcripts in E18 DKO fetal membranes were significantly enriched for genes involved in cell cycle regulation, extracellular matrix–receptor interaction, and the complement cascade. Fifty transcripts involved in the cell cycle were altered in E18 DKO fetal membranes (40↓, 10↑, FDR < 0.05), including p21 and p57 (↑), and Tgfb2, Smad3, CycA, Cdk1, and Cdk2(↓). Thirty-one transcripts involved in the complement cascade were altered (11↓, 20↑, FDR < 0.05) in E18 DKO fetal membranes, including C1q, C2, and C3 (↑). Differentially expressed genes in the top three molecular pathways (1) showed evidence of negative or purifying selection, and (2) were significantly enriched (Z-score > 10) for transcription factor binding sites for Nr2f1 at E18. We propose that in DKO mice, cell cycle arrest results in lack of cell proliferation in fetal membranes, inability to contain the growing fetus, and preterm birth.

Summary sentence

We have conducted transcriptomic profiling of fetal membranes from mice deficient in biglycan and decorin and identified molecular pathways of interest.

Uterine spiral artery (SPA) remodeling is a crucial event during pregnancy to provide enough blood supply to maternal–fetal interface and meet the demands of the growing fetus. Along this process, the dynamic change and the fate of spiral artery vascular smooth muscle cells (SPA–VSMCs) have long been debatable. In the present study, we analyzed the cell features of SPA–VSMCs at different stages of vascular remodeling in human early pregnancy, and we demonstrated the progressively morphological change of SPA–VSMCs at un-remodeled (Un-Rem), remodeling, and fully remodeled (Fully-Rem) stages, indicating the extravillous trophoblast (EVT)-independent and EVT-dependent phases of SPA–VSMC dedifferentiation. In vitro experiments in VSMC cell line revealed the efficient roles of decidual stromal cells, decidual natural killer cells (dNK), decidual macrophages, and EVTs in inducing VSMCs dedifferentiation. Importantly, the potential transformation of VSMC toward CD56⁺ dNKs was displayed by immunofluorescence-DNA in-situ hybridization-proximity ligation and chromatin immunoprecipitation assays for H3K4dime modification in the myosin heavy chain 11 (MYH11) promoter region. The findings clearly illustrate a cascade regulation of the progressive dedifferentiation of SPA–VSMCs by multiple cell types in uterine decidual niche and provide new evidences to reveal the destination of SPA–VSMCs during vascular remodeling.

Summary sentence

We illustrate a cascade regulation of the dedifferentiation and destination of SPA–VSMCs by uterine decidual niche during vascular remodeling.

Improved understanding of the molecular mechanisms underlying ascending equine placentitis holds the potential for the development of new diagnostic tools and therapies to forestall placentitis-induced preterm labor. The current study characterized the equine placental transcriptome (chorioallantois [CA] and endometrium [EN]) during placentitis (placentitis group, n = 6) in comparison to gestationally-matched controls (control group, n = 6). Transcriptome analysis identified 2953 and 805 differentially expressed genes in CA and EN during placentitis, respectively. Upstream regulator analysis revealed the central role of toll-like receptors (TLRs) in triggering the inflammatory signaling, and consequent immune-cell chemotaxis. Placentitis was associated with the upregulation of matrix metalloproteinase (MMP1, MMP2, and MMP9) and apoptosis-related genes such as caspases (CASP3, CASP4, and CASP7) in CA. Also, placentitis was associated with downregulation of transcripts coding for proteins essential for placental steroidogenesis (SRD5A1 and AKR1C1), progestin signaling (PGRMC1 and PXR) angiogenesis (VEGFA, VEGFR2, and VEGFR3), and nutrient transport (GLUT12 and SLC1A4), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could explain placental insufficiency during placentitis. Placentitis was also associated with aberrant expression of several placenta-regulatory genes, such as PLAC8, PAPPA, LGALS1, ABCG2, GCM1, and TEPP, which could negatively affect placental functions. In conclusion, our findings revealed for the first time the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during equine placentitis, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.

Summary sentence

This is the first study to characterize the equine placenta transcriptome to identify the key regulators and pathways underlying placental inflammation, separation, and insufficiency during the equine ascending placentitis.

Graphical Abstract

During the peri-implantation period of pregnancy in sheep, there is an initial period of loose apposition of the elongating conceptuses (embryos and associated placental membranes) to the endometrial luminal epithelium (LE) that is followed by adhesion of the conceptus trophectoderm to the endometrial LE for implantation. Integrins and maternal extracellular matrix (ECM) molecules are major contributors to stable adhesion at implantation, and the β3 integrin subunit (ITGB3) is implicated in the adhesion cascade for implantation in several species including the sheep. We blocked mRNA translation for trophectoderm-expressed ITGB3 by infusing morpholino antisense oligonucleotides into the uterine lumen of pregnant ewes on Day 9 to assess effects on conceptus elongation, and on Day 16 to assess effects on early placental development in sheep. Results indicate that sheep conceptuses elongate and implant to the uterine wall in the absence of ITGB3 expression by the conceptuses; however, loss of ITGB3 in conceptuses decreased the growth of embryos to Day 24 of gestation, and decreased expression of secreted phosphoprotein 1 (SPP1) and nitric oxide synthase 3 (NOS3). Abundant SPP1 was localized around the blood vessels in the placental allantoic membrane in normal sheep pregnancies. We hypothesize that NOS3 and SPP1 positively influence the development of the vasculature within the allantois, and that decreased expression of NOS3 and SPP1, in response to knockdown of ITGB3 in conceptuses, alters development of the vasculature in the allantois required to transport nutrients from the endometrium to support growth and development of the embryo.

Summary sentence

Knockdown of the ITGB3 integrin subunit in ovine conceptuses does not affect conceptus elongation or implantation, but does decrease conceptus expression of NOS3, SPP1, and embryonic growth to Day 24 of gestation, potentially due to effects on the placental vasculature.

Bovine endometrium consists of epithelial and stromal cells that respond to conceptus interferon tau (IFNT), the maternal recognition of pregnancy (MRP) signal, by increasing expression of IFN-stimulated genes (ISGs). Endometrial epithelial and stromal-cell-specific ISGs are largely unknown but hypothesized to have essential functions during pregnancy establishment. Bovine endometrial epithelial cells were cultured in inserts above stromal fibroblast (SF) cells for 6 h in medium alone or with IFNT. The epithelial and SF transcriptomic response was analyzed separately using RNA sequencing and compared to a list of 369 DEGs recently identified in intact bovine endometrium in response to elongating bovine conceptuses and IFNT. Bovine endometrial epithelial and SF shared 223 and 70 DEGs in common with the list of 369 endometrial DEGs. Well-known ISGs identified in the epithelial and SF were ISG15, MX1, MX2, and OAS2. DEGs identified in the epithelial but not SF included a number of IRF molecules (IRF1, IRF2, IRF3, and IRF8), mitochondria SLC transporters (SLC25A19, SLC25A28, and SLC25A30), and a ghrelin receptor. Expression of ZC3HAV1, an anti-retroviral gene, increased specifically within the SF. Gene ontology analysis identified the type I IFN signaling pathway and activation of nuclear factor kappa B transcription factors as biological processes associated with the epithelial cell DEGs. This study has identified biologically relevant IFNT-stimulated genes within specific endometrial cell types. The findings provide critical information regarding the effects of conceptus IFNT on specific endometrial compartments during early developmental processes in cattle.

Summary sentence

Conceptus-induced, interferon-tau-dependent genes were identified within bovine endometrial epithelial and stromal fibroblast cells providing critical information regarding conceptus-induced modifications to specific endometrial compartments in cattle.

Among a wide diversity of sexually reproducing species, male ejaculates coagulate to form what has been termed a copulatory plug. A number of functions have been attributed to copulatory plugs, including the inhibition of female remating and the promotion of ejaculate movement. Here we demonstrate that copulatory plugs also influence the likelihood of implantation, which occurs roughly 4 days after copulation in mice. Using a bead transfer method to control for differences in ejaculate retention and fertilization rates, we show that implantation rates significantly drop among females mated to genetically engineered males incapable of forming plugs (because they lack functional transglutaminase 4, the main enzyme responsible for its formation). Surprisingly, this result does not correlate with differences in circulating progesterone levels among females, an important hormone involved in implantation. We discuss three models that connect male-derived copulatory plugs to implantation success, including the hypothesis that plugs contribute to a threshold amount of stimulation required for females to become receptive to implantation.

Summary sentence

Male-derived copulatory plugs influence implantation success among their female mates.

Graphical Abstract

When women with small ovarian reserves are subjected to assisted reproductive technologies, high doses of gonadotropins are linked to high oocyte and embryo wastage and low live birth rates. We hypothesized that excessive follicle-stimulating hormone (FSH) doses during superovulation are detrimental to ovulatory follicle function in individuals with a small ovarian reserve. To test this hypothesis, heifers with small ovarian reserves were injected twice daily for 4 days, beginning on Day 1 of the estrous cycle with 35, 70, 140, or 210 IU doses of Folltropin-V (FSH). Each heifer (n = 8) was superovulated using a Williams Latin Square Design. During each superovulation regimen, three prostaglandin F_2α injections were given at 12-h interval, starting at the seventh FSH injection to regress the newly formed corpus luteum (CL). Human chorionic gonadotropin was injected 12 h after the last (8th) FSH injection to induce ovulation. Daily ultrasonography and blood sampling were used to determine the number and size of follicles and corpora lutea, uterine thickness, and circulating concentrations of estradiol, progesterone, and anti-Müllerian hormone (AMH). The highest doses of FSH did not increase AMH, progesterone, number of ovulatory-size follicles, uterine thickness, or number of CL. However, estradiol production and ovulation rate were lower for heifers given high FSH doses compared to lower doses, indicating detrimental effects on ovulatory follicle function.

Summary sentence

High doses of Folltropin-V in cattle with a low antral follicle count and small ovarian reserve are detrimental to ovulatory follicle function.

Although reactive oxygen species (ROS) are required for spermatogonial stem cell (SSC) self-renewal, they induce DNA damage and are harmful to SSCs. However, little is known about how SSCs protect their genome during self-renewal. Here, we report that Ogg1 is essential for SSC protection against ROS. While cultured SSCs exhibited homologous recombination-based DNA double-strand break repair at levels comparable with those in pluripotent stem cells, they were significantly more resistant to hydrogen peroxide than pluripotent stem cells or mouse embryonic fibroblasts, suggesting that they exhibit high levels of base excision repair (BER) activity. Consistent with this observation, cultured SSCs showed significantly lower levels of point mutations than somatic cells, and showed strong expression of BER-related genes. Functional screening revealed that Ogg1 depletion significantly impairs survival of cultured SSCs upon hydrogen peroxide exposure. Thus, our results suggest increased expression of BER-related genes, including Ogg1, protects SSCs from ROS-induced damage.

Summary sentence

OGG1 suppresses ROS-induced damages in spermatogonial stem cells.

Humanin (HN) is a mitochondrial-derived peptide that protects many cells/tissues from damage. We previously demonstrated that HN reduces stress-induced male germ cell apoptosis in rodents. HN action in neuronal cells is mediated through its binding to a trimeric cell membrane receptor composed of glycoprotein 130 (gp130), IL-27 receptor subunit (IL-27R, also known as WSX-1/TCCR), and ciliary neurotrophic factor receptor subunit (CNTFR). The mechanisms of HN action in testis remain unclear. We demonstrated in ex-vivo seminiferous tubules culture that HN prevented heat-induced germ cell apoptosis was blocked by specific anti-IL-27R, anti-gp130, and anti-EBI-3, but not by anti-CNTFR antibodies significantly. The cytoprotective action of HN was studied by using groups of il-27r^–/– or ebi-3^–/– mice administered the following treatment: (1) vehicle; (2) a single intraperitoneal (IP) injection of HN peptide; (3) testicular hyperthermia; and (4) testicular hyperthermia plus HN. We demonstrated that HN inhibited heat-induced germ cell apoptosis in wildtype but not in il-27r^–/– or ebi-3^–/– mice. HN restored heat-suppressed STAT3 phosphorylation in wildtype but not il-27r^–/– or ebi-3^–/– mice. Dot blot analyses showed the direct interaction of HN with IL-27R or EBI-3 peptide. Immunofluorescence staining showed the co-localization of IL-27R with HN and gp130 in Leydig cells and germ cells. We conclude that the anti-apoptotic effects of HN in mouse testes are mediated through interaction with EBI-3, IL-27R, and activation of gp130, whereas the role of CNTFR needs further studies. This suggests a multicomponent tissue-specific receptor for HN in the testis and links HN action with the IL-12/IL-27 family of cytokines.

Summary sentence

The cytoprotective effects of HN in mouse testes are mediated via binding with EBI-3, IL-27R, and activation of gp130. This suggests there is a multicomponent tissue-specific HN receptor in the testis and links HN with the IL-12/IL-27 family of cytokines.

Graphical Abstract

Model of humanin (HN) interaction with EBI-3, IL-27Rα, and gp130 in male germ cells at baseline and after heat induced stress. Under nonstressed condition (left part), free EBI-3 may bind with p28 to form cytokine IL-27, while IL-27Rα and gp130 may combine with each other to form the IL-27 receptor. CNTFRα may bind gp130 and IL-27Rα to form the HN membrane receptor in neuronal cells. After heat stress in the testis, exogenous HN binds with EBI-3 and interacts with gp130 plus IL-27Rα to activate the downstream Jak/STAT3 pathway. Activated STAT3 suppresses heat-induced male germ cell apoptosis. In our study, the role of CNTFRα cannot be concluded definitely. Our data suggest a different multicomponent and tissue-specific receptor for HN in the testis and linking HN action with the IL-12/IL-27 family of cytokines.