Thyroid hormones (THs) regulate a number of metabolic processes during pregnancy. After implantation, the placenta forms and enhances embryonic growth and development. Dysregulated maternal THs signaling has been observed in malplacentation-mediated pregnancy complications such as preeclampsia, miscarriage, and intrauterine growth restriction (IUGR), but the molecular mechanisms involved in this association have not been fully characterized. In this review, we have discussed THs signaling and its roles in trophoblast proliferation, trophoblast differentiation, trophoblast invasion of the decidua, and decidual angiogenesis. We have also explored the relationship between specific pregnancy complications and placental THs transporters, deiodinases, and THs receptors. In addition, we have examined the effects of specific endocrine disruptors on placental THs signaling. The available evidence indicates that THs signaling is involved in the formation and functioning of the placenta and serves as the basis for understanding the pathogenesis and pathophysiology of dysthyroidism-associated pregnancy complications such as preeclampsia, miscarriage, and IUGR.

Summary sentence

Normal THs signaling promotes placentation; while abnormal THs signaling leads to malplacentation and its associated pregnancy complications, such as preeclampsia, miscarriage and IUGR.

During pregnancy, there is increased expression of some cytokines at the fetal–maternal interface; and the clarification of their roles in trophoblast–endometrium interactions is crucial to understanding the mechanism of placentation. This review addresses the up-to-date reported mechanisms by which the members of the transforming growth factor beta superfamily regulate trophoblast proliferation, differentiation, and invasion of the decidua, which are the main phases of placentation. The available information shows that these cytokines regulate placentation in somehow a synergistic and an antagonistic manner; and that dysregulation of their levels can lead to aberrant placentation. Nevertheless, prospective studies are needed to reconcile some conflicting reports; and identify some unknown mediators involved in the actions of these cytokines before their detailed mechanistic regulation of human placentation could be fully characterized.

Summary sentence

The TGF beta superfamily are expressed in the placenta, and regulate the process of placentation through the activation of several signaling pathways.

A number of genes relevant for sex determination have been found in species with temperature-dependent sex determination. Epigenetics play a key role in sex determination, but characterization of deoxyribonucleic acid methylation of sex-related genes on temperature-dependent sex determination remains unclear. Mauremys reevesii is a typical species with temperature-dependent sex determination. In this study, we analyzed the Cytosine Guanine (CpG) methylation status of the proximal promoters, the messenger ribonucleic acid expression patterns and the correlation between methylation and expression levels of Aromatase, Forkhead box protein L2, Doublesex and mab3-related transcription factor 1, sex-determining region on Y chromosome-box 9, and anti-Müllerian hormone, which are key genes in sex determination in other species. We also analyzed the expression level of genes that encode enzymes involved in methylation and demethylation. The expression levels of Aromatase and Forkhead box protein L2 at the female producing temperature were higher than those at the male producing temperature; the expression levels of Doublesex and mab3-related transcription factor 1, sex-determining region on Y chromosome-box 9, and anti-Müllerian hormone were higher at MPT. The expression of some genes involved in methylation and demethylation is significantly different between male producing temperature and female producing temperature.

Summary sentence

The expression of messenger ribonucleic acid of genes involved in deoxyribonucleic acid methylation and demethylation affected by temperature, together with other factors, may change the methylation level of the regulatory regions of sex-related genes, which may further lead to temperature-specific expression of sex-related genes, and eventually affect the differentiation of the gonads.

Most current knowledge of sex determination in mammals has emerged from mouse and human studies. To investigate the molecular regulation of the sex determination process in cattle, we used an RNA sequencing strategy to analyze the transcriptome landscape of male and female bovine fetal gonads collected in vivo at key developmental stages: before, during, and after SRY gene activation on fetal days D35 (bipotential gonad formation), D39 (peak SRY expression), and D43 (early gonad differentiation). Differentially expressed genes (DEGs) were identified in male vs. female germinal ridges and among group genes showing similar expression profiles during the three periods. There were 143, 96, and 658 DEG between males and female fetuses at D35, D39, and D43, respectively. On D35, genes upregulated in females were enriched in translation, nuclear export, RNA localization, and mRNA splicing events, whereas those upregulated in males were enriched in cell proliferation regulation and male sex determination terms. In time-course experiments, 767 DEGs in males and 545 DEGs in females were identified between D35 vs. D39, and 3157 DEGs in males and 2008 in females were identified between D39 vs. D43. Results highlight unique aspects of sex determination in cattle, such as the expression of several Y chromosome genes (absent in mice and humans) before SRY expression and an abrupt increase in the nuclear expression of SOX10 (instead of SOX9 expression in the Sertoli cell cytoplasm as observed in mice) during male determination and early differentiation.

Summary sentence

Gene expression analysis of male and female bovine fetal gonads collected in vivo before and after SRY expression sheds light on unique aspects of sex determination and early sex differentiation in cattle.

There is general consensus that the synchronous development of the embryo and endometrium is absolutely essential for successful implantation. Recent studies have strongly suggested that embryo-secreted factors are able to deliver into the endometrial cavity/endometrium and alter its protein profile in preparation for implantation. However, there is limited research focusing on long noncoding RNA (lncRNA) changes in the endometrium that brought about by the embryonic derived factors. It has been suggested that lncRNA has intricate interplay with microRNA (miR), small (∼19–22 nucleotides), non-protein-coding RNA, to regulate protein production in the endometrium, thus controlling adhesive capacity. Here through microarray assays, we compare the lncRNA profile of the primary human endometrial epithelial cells (HEECs) that have been precultured with blastocyst-conditioned media (BCM) from embryos that implanted versus nonimplanted. Our data indicate a substantial change of lncRNA expression in HEECs, including 9 up-regulated and 12 down-regulated lncRNAs after incubation with implanted BCM. Selective knockdown of PTENP1, the most increased lncRNA after implanted BCM treatment in the HEECs, compromised the spheroid adhesion (P < 0.001). Characterization of PTENP1 confirmed its expression in the luminal epithelium with staining appeared most intense in the midsecretory phase. Furthermore, we have recorded a substantial change of miR profile upon PTENP1 knockdown in HEECs. Overexpression of miR-590-3p, a novel predicted target of PTENP1, impaired spheroid adhesion (P < 0.001). Collectively, these data have supported a novel regulation system that lncRNAs were able to participate in the regulation of implantation through association with miRs.

Summary sentence

Human endometrial epithelial cells treated with nonimplanted blastocyst conditioned media down-regulate PTENP1 expression and impair their adhesive capacity.

Prepregnancy obesity associates with adverse reproductive outcomes that impact maternal and fetal health. While obesity-driven mechanisms underlying adverse pregnancy outcomes remain unclear, local uterine immune cells are strong but poorly studied candidates. Uterine immune cells, particularly uterine natural killer cells (uNKs), play central roles in orchestrating developmental events in pregnancy. However, the effect of obesity on uNK biology is poorly understood. Using an obesogenic high-fat/high-sugar diet (HFD) mouse model, we set out to examine the effects of maternal obesity on uNK composition and establishment of the maternal–fetal interface. HFD exposure resulted in weight gain-dependent increases in systemic inflammation and rates of fetal resorption. While HFD did not affect total uNK frequencies, HFD exposure did lead to an increase in natural cytotoxicity receptor-1 expressing uNKs as well as overall uNK activity. Importantly, HFD-associated changes in uNK coincided with impairments in uterine artery remodeling in mid but not late pregnancy. Comparison of uNK mRNA transcripts from control and HFD mice identified HFD-directed changes in genes that play roles in promoting activity/cytotoxicity and vascular biology. Together, this work provides new insight into how obesity may impact uNK processes central to the establishment of the maternal–fetal interface in early and mid pregnancy. Moreover, these findings shed light on the cellular processes affected by maternal obesity that may relate to overall pregnancy health.

Summary sentence

High-fat diet promotes uterine NK cell activation in pregnancy and associates with impaired vascular remodeling within the uterus and drives altered uterine NK gene expression.

Efferent duct ligation (EDL) induces epithelial cell degeneration followed by regeneration in the epididymal initial segment. We tested here the role of androgens in the recovery phase. EDL was performed at post-natal weeks (PNW) 3, 4, 5, 6, and 7, and apoptotic and proliferating epithelial cells were quantified 24 h, and at days 2 and 2.5 post-EDL, respectively. A progressive increase in the number of apoptotic basal cells (BCs) and principal cells (PCs) was detected from PNW3 to 6, 24 h after EDL. Two days after EDL, no increase in proliferating BCs and PCs was observed at PNW3 and 4, despite the induction of apoptosis by EDL. A progressive increase in the number of proliferating BCs was then observed from PNW5 to 6, while the number of proliferating PCs remained low. 2.5 days after EDL, the number of proliferating BCs and PCs remained low at PNW3, 4, and 5, but a marked increase in the number of proliferating PCs was observed at PNW6. Flutamide pretreatment for 3 weeks followed by EDL at PNW7 dramatically decreased the number of proliferating BCs on EDL day 2, and the number of proliferating PCs on EDL day 2.5, compared to controls. We conclude that (1) BCs are the first to show recovery after EDL, followed by PCs; (2) androgens are essential for BC and PC repair after injury in the postpubertal epididymis; and (3) the prepubertal epididymis lacks repair ability following injury.

Summary Sentence

Androgen stimulation during the peripubertal and postpubertal periods is essential for epithelial repair after injury in the mouse epididymis.

High-throughput transcriptomics and proteomics approaches have recently identified a large number of germ cell–specific genes with many that remain to be studied through functional genetics approaches. Serine proteases (PRSS) constitute nearly one-third of all proteases, and, in our bioinformatics screens, we identified many that are testis specific. In this study, we chose to focus on Prss44, Prss46, and Prss54, which we confirmed as testis specific in mouse and human. Based on the analysis of developmental expression in the mouse, expression of all four genes is restricted to the late stage of spermatogenesis concomitant with a potential functional role in spermiogenesis, spermiation, or sperm function. To best understand the male reproductive requirement and functional roles of these serine proteases, each gene was individually ablated by CRISPR/Cas9-mediated ES cell or zygote approach. Homozygous deletion mutants for each gene were obtained and analyzed for phenotypic changes. Analyses of testis weights, testis and epididymis histology, sperm morphology, and fertility revealed no significant differences in Prss44, Prss46, and Prss54 knockout mice in comparison to controls. Our results thereby demonstrate that these genes are not required for normal fertility in mice, although do not preclude the possibility that these genes may function in a redundant manner. Elucidating the individual functional requirement or lack thereof of these novel genes is necessary to build a better understanding of the factors underlying spermatogenesis and sperm maturation, which has implications in understanding the etiology of male infertility and the development of male contraceptives.

Summary sentence

The testis-specific serine proteases Prss44, Prss46, and Prss54 are dispensable for male fertility based on phenotype analyses of knockout mice produced using the CRISPR/Cas9 system.

A crucial function of the epididymis is providing a surface glycocalyx that is important for sperm maturation and capacitation. Defensins are antimicrobial peptides expressed in the epididymis. In the macaque epididymis, defensin beta 126 (DEFB126) is important for sperm motility, however, it is not known whether this is the case in humans. The objectives were to determine: (1) if DEFB126 on human ejaculated sperm was correlated with sperm motility in fertile and infertile men, (2) that recombinant DEFB126 could induce immature sperm motility in vitro. Immunofluorescence staining indicated that the proportion of DEFB126-positive sperm was significantly higher in motile sperm. Furthermore, the proportion of DEFB126-labeled sperm was positively correlated with sperm motility and normal morphology. Additional studies indicated that the proportion of DEFB126-positive spermatozoa in fertile volunteers was significantly higher than in volunteers with varicocele, and in infertile volunteers with semen deficiencies. To determine the role of DEFB126 on sperm motility, the DEFB126 gene was cloned and used to generate recombinant DEFB126 in H9C2 cells (rat embryonic heart myoblast cells). Deletion mutations were created into two regions of the protein, which have been linked to male infertility. Immotile testicular spermatozoa were incubated with cells expressing the different forms of DEFB126. Full-length DEFB126 significantly increased motility of co-cultured spermatozoa. However, no increase in sperm motility was observed with the mutated forms of DEFB126. In conclusion, these results support the notion that DEFB126 is important in human sperm maturation and the potential use of DEFB126 for in vitro sperm maturation.

Summary sentence

DEFB126 is correlated with sperm motility and fertility in human.

Superoxide dismutase 1 suppresses oxidative stress within cells by decreasing the levels of superoxide anions. A dysfunction of the ovary and/or an aberrant production of sex hormones are suspected causes for infertility in superoxide dismutase 1-knockout mice. We report on attempts to rescue the infertility in female knockout mice by providing two antioxidants, ascorbic acid and/or coenzyme Q₁₀, as supplements in the drinking water of the knockout mice after weaning and on an investigation of their reproductive ability. On the first parturition, 80% of the untreated knockout mice produced smaller litter sizes compared with wild-type mice (average 2.8 vs 7.3 pups/mouse), and supplementing with these antioxidants failed to improve these litter sizes. However, in the second parturition of the knockout mice, the parturition rate was increased from 18% to 44–75% as the result of the administration of antioxidants. While plasma levels of progesterone at 7.5 days of pregnancy were essentially the same between the wild-type and knockout mice and were not changed by the supplementation of these antioxidants, sizes of corpus luteum cells, which were smaller in the knockout mouse ovaries after the first parturition, were significantly ameliorated in the knockout mouse with the administration of the antioxidants. Moreover, the impaired vasculogenesis in uterus/placenta was also improved by ascorbic acid supplementation. We thus conclude that ascorbic acid and/or coenzyme Q₁₀ are involved in maintaining ovarian and uterus/placenta homeostasis against insults that are augmented during pregnancy and that their use might have positive effects in terms of improving female fertility.

Summary sentence

The results suggest that antioxidants support fertilizing ability against oxidative stress during parturition.

As the follicle develops, the thickening of the granulosa compartment leads to progressively deficient supply of oxygen in granulosa cells (GCs) due to the growing distances from the follicular vessels. These conditions are believed to cause hypoxia in GCs during folliculogenesis. Upon hypoxic conditions, several types of mammalian cells have been reported to undergo cell cycle arrest. However, it remains unclear whether hypoxia exerts any impact on cell cycle progression of GCs. On the other hand, although the GCs may live in a hypoxic environment, their mitotic capability appears to be unaffected in growing follicles. It thus raises the question whether there are certain intraovarian factors that might overcome the inhibitory effects of hypoxia. The present study provides the first evidence suggesting that cobalt chloride (CoCl₂)-mimicked hypoxia prevented G1-to-S cell cycle progression in porcine GCs. In addition, we demonstrated that the inhibitory effects of CoCl₂ on GCs cell cycle are mediated through hypoxia-inducible factor-1 alpha/FOXO1/Cdkn1b pathway. Moreover, we identified insulin-like growth factor-I (IGF-I) as an intrafollicular factor required for cell cycle recovery by binding to IGF-I receptor in GCs suffering CoCl₂ stimulation. Further investigations confirmed a role of IGF-I in preserving G1/S progression of CoCl₂-treated GCs via activating the cyclin E/cyclin-dependent kinase2 complex through the phoshatidylinositol-3 kinase/protein kinase B (AKT)/FOXO1/Cdkn1b axis. Although the present findings were based on a hypoxia mimicking model by using CoCl₂, our study might shed new light on the regulatory mechanism of GCs cell cycle upon hypoxic stimulation.

Summary sentence

The IGF-I/PI3K/AKT/FOXO1/Cdkn1b axis is essential for preserving G1/S progression in hypoxic porcine GCs by targeting cyclin E/CDK2.

Radiation induces ovarian damage and accelerates reproductive aging. Inbred mouse strains exhibit differential sensitivity to lethality induced by total body irradiation (TBI), with the BALB/cAnNCrl (BALB/c) strain being more sensitive than the 129S2/SvPasCrl (129) strain. However, whether TBI-induced ovarian damage follows a similar pattern of strain sensitivity is unknown. To examine this possibility, female BALB/c and 129 mice were exposed to a single dose of 1 Gy (cesium-137 γ ) TBI at 5 weeks of age, and ovarian tissue was harvested for histological and gene expression analyses 2 weeks post exposure. Sham-treated mice served as controls. 1 Gy radiation nearly eradicated the primordial follicles and dramatically decreased the primary follicles in both strains. In contrast, larger growing follicles were less affected in the 129 relative to BALB/c strain. Although this TBI paradigm did not induce detectable ovarian fibrosis in either of the strains, we did observe strain-dependent changes in osteopontin (Spp1) expression, a gene involved in wound healing, inflammation, and fibrosis. Ovaries from BALB/c mice exhibited higher baseline Spp1 expression that underwent a significant decrease in response to radiation relative to ovaries from the 129 strain. A correspondingly greater change in the ovarian matrix, as evidenced by reduced ovarian hyaluronan content, was also observed following TBI in BALB/c mice relative to 129 mice. These early changes in the ovary may predispose BALB/c mice to more pronounced late effects of TBI. Taken together, our results demonstrate that aspects of ovarian damage mirror other organ systems with respect to overall strain-dependent radiation sensitivity.

Summary sentence

The ovarian response to low-dose total body radiation is mouse strain dependent.

The association between theca cells (TCs) and granulosa cells is pivotal to steroid biosynthesis in the ovary. During the late secondary follicle stage, TCs form a layer around granulosa cells, after which their steroidogenic function falls under the control of luteinizing hormone (LH) that activates the cAMP signaling pathway via a G protein-coupled receptor. In addition to perilipin-2, a marker for lipid droplets containing esters as substrates for TCs to produce steroidogenic hormones, other essential proteins, like steroidogenic acute regulatory protein (StAR), cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 2, play a role in the cascade after luteinizing hormone–choriogonadotropic hormone receptor (LH/CG-R) occupation by LH. The aim of the present study was to assess expression levels and corresponding amounts of LH/CG-R, perilipin-2, and enzymes involved in the steroidogenic pathway of TCs based on follicle stage. Immunohistochemical analysis of each of these proteins was therefore performed on ovarian samples from nine adult women, most (n = 8) with BRCA1 and/or BRCA2 mutations undergoing prophylactic bilateral oophorectomy. Pictures were taken of the theca layer of secondary, small (<3000 µm), and large (>3000 µm) antral follicles and corpora lutea at 100× magnification. ImageJ software was used to analyze the surface area and expression intensity of each protein at each stage, known as the staining index. Overall, our data showed that LH/CG-R, perilipin-2, and StAR expression increased in the course of folliculogenesis and luteinization. Similarly, cytochrome P450 11A1, cytochrome P450c17, 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 1, and 3 beta-hydroxysteroid dehydrogenase/delta 5 —> 4-isomerase type 2 expression were substantially elevated in TCs during folliculogenesis, evidenced by their coordinated action in terms of area covered and expression intensity. This study, conducted for the first time on human ovarian tissue, contributes to localizing and quantifying expression of key steroidogenic proteins at both intracellular and tissue levels. These findings may shed new light on pathological conditions involving the human ovary, such as androgen-secreting tumors of the ovary and other disorders associated with ovarian TCs in patients with polycystic ovary syndrome.

Summary sentence

This study, conducted for the first time on human ovarian tissue, contributes to localizing and quantifying expression of key steroidogenic proteins at both intracellular and tissue levels and may shed new light on pathological conditions involving the human ovary, such as androgen-secreting tumors of the ovary and other disorders associated with theca cells in PCOS patients.

Gonadotropes represent approximately 5–15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-hαCGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches.

Summary sentence

Unique RNA signatures establish the efficacy of combining adenovirus-mediated gene transfer and flow cytometry to isolate an enriched population of gonadotrope cells from the ovine pituitary gland.

Preimplantation equine embryos synthesize and secrete fibrinogen, which is a peculiar finding as fibrinogen synthesis almost exclusively occurs in the liver. This study investigated the hypothesis that conceptus-derived fibrinogen mediates cell adhesion during fixation. On day 21 of pregnancy, five integrin subunits, including ITGA5, ITGB1, ITGAV, and ITGB1, displayed significantly higher transcript abundance than on day 16 of pregnancy. Endometrial epithelial cells adhered to fibrinogen in an integrin-dependent manner in an in vitro cell adhesion assay. Bilaminar trophoblast and allantochorion expressed fibrinogen transcript, indicating that fibrinogen expression persists past fixation. Preimplantation-phase endometrium, conceptuses, and microcotyledonary tissue expressed components of the clotting cascade regulating fibrin homeostasis, leaving open the possibility that fibrinogen is converted to fibrin. Fibrinogen is likely to have functions beyond mediating cell adhesion, such trapping growth factors and triggering signaling cascades, and has remarkable parallels to the expression of fibrinogen by some tumors. The deposition of fibrinogen within tumor stroma is characteristic of breast carcinoma, and tumor-derived fibrinogen has been implicated in the metastatic potential of circulating tumor cells. DNA methylation of the fibrinogen locus in equine conceptuses was examined in comparison to liver and endometrium, and across the full gene cluster, was significantly higher for endometrium than liver and conceptus. DNA methylation of regulatory regions did not differ between liver and conceptus, and was significantly lower than in endometrium. These results, therefore, support the hypothesis of DNA methylation being a regulator of fibrinogen expression in the conceptus.

Summary sentence

Conceptus-derived fibrinogen is a mediator of cell adhesion during equine pregnancy.

Modulation of the activation status of immune cell populations during pregnancy depends on placental villous cytotrophoblast (VCT) cells and the syncytiotrophoblast (STB). Failure in the establishment of this immunoregulatory function leads to pregnancy complications. Our laboratory has been studying Syncytin-2 (Syn-2), an endogenous retroviral protein expressed in placenta and on the surface of placental exosomes. This protein plays an important role not only in STB formation through its fusogenic properties, but also through its immunosuppressive domain (ISD). Considering that Syn-2 expression is importantly reduced in preeclamptic placentas, we were interested in addressing its possible immunoregulatory effects on T cells. Activated Jurkat T cells and peripheral blood mononuclear cells (PBMCs) were treated with monomeric or dimerized version of a control or a Syn-2 ISD peptide. Change in phosphorylation levels of ERK1/2 MAP kinases was selectively noted in Jurkat cells treated with the dimerized ISD peptide. Upon incubation with the dimerized Syn-2 ISD peptide, significant reduction in Th1 cytokine production was further demonstrated by ELISA and Human Th1/Th2 Panel Multi-Analyte Flow Assay. To determine if exosome-associated Syn-2 could also be immunosuppressive placental exosomes were incubated with activated Jurkat and PBMCs. Quantification of Th1 cytokines in the supernatants revealed severe reduction in T cell activation. Interestingly, exosomes from Syn-2-silenced VCT incubated with PBMCs were less suppressive when compared with exosome derived from VCT transfected with control small interfering RNA (siRNA). Our results suggest that Syn-2 is an important immune regulator both locally and systemically, via its association with placental exosomes.

Summary sentence

Human endogenous retroviral Syncytin-2 negatively downmodulates Th1 response via its immunosuppressive domain and acts through its association to exosomes.

Preeclampsia (PE) is a complicated obstetric complication characterized by increased blood pressure, decreased trophoblast invasion, and inflammation. The growth arrest-specific 6 (Gas6) protein is known to induce dynamic cellular responses and is elevated in PE. Gas6 binds to the AXL tyrosine kinase receptor and AXL-mediated signaling is implicated in proliferation and migration observed in several tissues. Our laboratory utilized Gas6 to induce preeclamptic-like conditions in pregnant rats. Our objective was to determine the role of Gas6/AXL signaling as a possible model of PE. Briefly, pregnant rats were divided into three groups that received daily intraperitoneal injections (from gestational day 7.5 to 17.5) of phosphate buffered saline (PBS), Gas6, or Gas6 + R428 (an AXL inhibitor administered from gestational day 13.5 to 17.5). Animals dispensed Gas6 experienced elevated blood pressure, increased proteinuria, augmented caspase-3-mediated placental apoptosis, and diminished trophoblast invasion. Gas6 also enhanced expression of several PE-related genes and a number of inflammatory mediators. Gas6 further enhanced placental oxidative stress and impaired mitochondrial respiration. Each of these PE-related characteristics was ameliorated in dams and/or their placentae when AXL inhibition by R428 occurred in tandem with Gas6 treatment. We conclude that Gas6 signaling is capable of inducing PE and that inhibition of AXL prevents disease progression in pregnant rats. These results provide insight into pathways associated with PE that could be useful in the clarification of potential therapeutic approaches.

Summary sentence

Bonding of Gas6 protein to AXL receptor induces preeclamptic symptoms in the rat.

Cell reprogramming by somatic cell nuclear transfer and in induced pluripotent stem cells is associated with epigenetic modifications that are often incompatible with embryonic development and differentiation. For instance, aberrant DNA methylation patterns of the differentially methylated region and biallelic expression of H19-/IGF2-imprinted gene locus have been associated with abnormal growth of fetuses and placenta in several mammalian species. However, cloned horses are born with normal sizes and with no apparent placental anomalies, suggesting that H19/IGF2 imprinting may be epigenetically stable after reprogramming in this species. In light of this, we aimed at characterizing the equid H19 gene to determine whether imprinting is altered in somatic cell nuclear transfer (SCNT)-derived conceptuses and induced pluripotent stem cell (iPSC) lines using the mule hybrid model. A CpG-rich region containing five CTCF binding sites was identified upstream of the equine H19 gene and analyzed by bisulfite sequencing. Coupled with parent-specific and global H19 transcript analysis, we found that the imprinted H19 remains monoallelic and that on average the methylation levels of both parental differentially methylated regions in embryonic and extra-embryonic SCNT tissues and iPSC lines remained unaltered after reprogramming. Together, these results show that, compared to other species, equid somatic cells are more resilient to epigenetic alterations to the H19-imprinted locus during SCNT and iPSC reprogramming.

Summary sentence

The reprogramming of equid somatic cells by oocyte nuclear transfer (SCNT) or genetically induced pluripotency (iPSC) does not induce epigenetic alterations to the H19 imprinting.

Spermatogonial stem cells (SSCs) undergo continuous self-renewal division in response to self-renewal factors. The present study identified ephrin type-A receptor 2 (EPHA2) on mouse SSCs and showed that supplementation of glial cell-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2), which are both SSC self-renewal factors, induced EPHA2 expression in cultured SSCs. Spermatogonial transplantation combined with magnetic-activated cell sorting or fluorescence-activated cell sorting also revealed that EPHA2 was expressed in SSCs. Additionally, ret proto-oncogene (RET) phosphorylation levels decreased following the knockdown (KD) of Epha2 expression via short hairpin ribonucleic acid (RNA). Although the present immunoprecipitation experiments did not reveal an association between RET with EPHA2, RET interacted with FGFR2. The Epha2 KD decreased the proliferation of cultured SSCs and inhibited the binding of cultured SSCs to laminin-coated plates. The Epha2 KD also significantly reduced the colonization of testis cells by spermatogonial transplantation. EPHA2 was also expressed in human GDNF family receptor alpha 1-positive spermatogonia. The present results indicate that SSCs express EPHA2 and suggest that it is a critical modifier of self-renewal signals in SSCs.

Summary sentence

Mouse spermatogonial stem cells express EPHA2.

Intraflagellar transport protein 20 (IFT20) is essential for spermatogenesis in mice. We discovered that COPS5 was a major binding partner of IFT20. COPS5 is the fifth component of the constitutive photomorphogenic-9 signalosome (COP9), which is involved in protein ubiquitination and degradation. COPS5 is highly abundant in mouse testis. Mice deficiency in COPS5 specifically in male germ cells showed dramatically reduced sperm numbers and were infertile. Testis weight was about one third compared to control adult mice, and germ cells underwent significant apoptosis at a premeiotic stage. Testicular poly (ADP-ribose) polymerase-1, a protein that helps cells to maintain viability, was dramatically decreased, and Caspase-3, a critical executioner of apoptosis, was increased in the mutant mice. Expression level of FANK1, a known COPS5 binding partner, and a key germ cell apoptosis regulator was also reduced. An acrosome marker, lectin PNA, was nearly absent in the few surviving spermatids, and expression level of sperm acrosome associated 1, another acrosomal component was significantly reduced. IFT20 expression level was significantly reduced in the Cops5 knockout mice, and it was no longer present in the acrosome, but remained in the Golgi apparatus of spermatocytes. In the conditional Ift20 mutant mice, COPS5 localization and testicular expression levels were not changed. COP9 has been shown to be involved in multiple signal pathways, particularly functioning as a co-factor for protein ubiquitination. COPS5 is believed to maintain normal spermatogenesis through multiple mechanisms, including maintaining male germ cell survival and acrosome biogenesis, possibly by modulating protein ubiquitination.

Summary sentence

COPS5 is essential for mouse spermatogenesis and particularly in maintaining male germ cell survival and acrosome biogenesis.

Ataxia–telangiectasia-mutated (ATM) protein recognizes and repairs DNA double strand breaks through activation of cell cycle checkpoints and DNA repair proteins. Atm gene mutations increase female reproductive cancer risk. Phosphoramide mustard (PM) induces ovarian DNA damage and destroys primordial follicles, and pharmacological ATM inhibition prevents PM-induced follicular depletion. Wild-type (WT) C57BL/6 or Atm^+/– mice were dosed once intraperitoneally with sesame oil (95%) or PM (25 mg/kg) in the proestrus phase of the estrous cycle and ovaries harvested 3 days thereafter. Atm^+/– mice spent ∼25% more time in diestrus phase than WT. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) on ovarian protein was performed and bioinformatically analyzed. Relative to WT, Atm^+/– mice had 64 and 243 proteins increased or decreased in abundance, respectively. In WT mice, PM increased 162 and decreased 20 proteins. In Atm^+/– mice, 173 and 37 proteins were increased and decreased, respectively, by PM. Exportin-2 (XPO2) was localized to granulosa cells of all follicle stages and was 7.2-fold greater in Atm^+/– than WT mice. Cytoplasmic FMR1-interacting protein 1 was 6.8-fold lower in Atm^+/– mice and was located in the surface epithelium with apparent translocation to the ovarian medulla post-PM exposure. PM induced γ H2AX, but fewer γ H2AX-positive foci were identified in Atm^+/– ovaries. Similarly, cleaved caspase-3 was lower in the Atm^+/– PM-treated, relative to WT mice. These findings support ATM involvement in ovarian DNA repair and suggest that ATM functions to regulate ovarian atresia.

Summary sentence

The TGF beta superfamily are expressed in the placenta, and regulate the process of placentation through the activation of several signaling pathways.