Preferentially expressed antigen in melanoma (PRAME) belongs to a group of cancer/testis antigens that are predominately expressed in the testis and a variety of tumors, and are involved in immunity and reproduction. Much of the attention on PRAME has centered on cancer biology as PRAME is a prognostic biomarker for a wide range of cancers and a potential immunotherapeutic target. Less information is available about the PRAME family's function (s) during gametogenesis and in the overall reproduction process. Here, we review the current knowledge of the PRAME gene family and its function in germline development and gametogenesis. Members of the PRAME family are leucine rich repeat proteins, localized in nucleus and cytoplasm, with multifaceted roles in germ cells. As transcriptional regulators, the PRAME family proteins are involved in germline development, particularly in the maintenance of embryonic stem cell pluripotency, development of primordial germ cells, and differentiation/proliferation of spermatogenic and oogenic cells. The PRAME family proteins are also enriched in cytoplasmic organelles, such as rough endoplasmic reticulum, Golgi vesicle, germinal granules, centrioles, and play a role in the formation of the acrosome and sperm tail during spermiogenesis. The PRAME gene family remains transcriptionally active in the germline throughout the entire life cycle and is essential for gametogenesis, with some members specific to either male or female germ cells, while others are involved in both male and female gametogenesis. A potential molecular mechanism that underlies the function of PRAME, and is shared by gametogenesis and oncogenesis is also discussed.

Summary Sentence

This review summarizes the current knowledge of the PRAME gene family and its function in germline development and gametogenesis.

Current first-line treatment of patients with high-grade serous ovarian cancer (HGSOC) involves the use of cytotoxic drugs that frequently lead to recurrent tumors exhibiting increased resistance to the drugs and poor patient survival. Strong evidence is accumulating to show that HGSOC tumors and cell lines contain a subset of cells called polyploidy giant cancer cells (PGCCs) that act as stem-like, self-renewing cells. These PGCCs appear to play a key role in tumor progression by generating drug-resistant progeny produced, in part, as a consequence of utilizing a modified form of mitosis known as endoreplication. Thus, developing drugs to target PGCCs and endoreplication may be an important approach for reducing the appearance of drug-resistant progeny. In the review, we discuss newly identified regulatory factors that impact mitosis and which may be altered or repurposed during endoreplication in PGCCs. We also review recent papers showing that a single PGCC can give rise to tumors in vivo and spheroids in culture. To illustrate some of the specific features of PGCCs and factors that may impact their function and endoreplication compared to mitosis, we have included immunofluorescent images co-localizing p53 and specific mitotic regulatory, phosphoproteins in xenografts derived from commonly used HGSOC cell lines.

Summary sentence

Cytotoxic drugs frequently used in ovarian cancer treatment impact tumor cell cycle progression and the emergence of polyploid cells where genetic and epigenetic events ultimately lead to drug-resistant diploid progeny.

Graphical Abstract

In vitro fertilization (IVF) gives rise to embryos in a number of mammalian species and is currently widely used for assisted reproduction in humans and for genetic purposes in cattle. However, the rate of polyspermy is generally higher in vitro than in vivo and IVF remains ineffective in some domestic species like pigs and horses, highlighting the importance of the female reproductive tract for gamete quality and fertilization. In this review, the way the female environment modulates sperm selective migration, survival, and acquisition of fertilizing ability in the oviduct is being considered under six aspects: (1) the utero-tubal junction that selects a sperm sub-population entering the oviduct; (2) the presence of sperm binding sites on luminal epithelial cells in the oviduct, which prolong sperm viability and plays a role in limiting polyspermic fertilization; (3) the contractions of the oviduct, which promote sperm migration toward the site of fertilization in the ampulla; (4) the regions of the oviduct, which play different roles in regulating sperm physiology and interactions with oviduct epithelial cells; (5) the time of ovulation, and (6) the steroid hormonal environment which regulates sperm release from the luminal epithelial cells and facilitates capacitation in a finely orchestrated manner.

Summary sentence

Summary Sentence After mating or insemination, the region-specific and hormonally regulated morphology and secretions of the utero-tubal junction and oviduct lead to the selection of a limited sub-population of top quality spermatozoa at the site of fertilization.

Graphical Abstract

The NOTCH signaling pathway plays an important role in regulating various biological processes, including lineage specification and apoptosis. Multiple components of the NOTCH pathway have been identified in mammalian preimplantation embryos. However, the precise role of the NOTCH pathway in early embryonic development is poorly understood, especially in large animals. Here, we show that the expression of genes encoding key transcripts of the NOTCH pathway is dynamic throughout early embryonic development. We also confirm the presence of active NOTCH1 and RBPJ. By using pharmacological and RNA interference tools, we demonstrate that the NOTCH pathway is required for the proper development of bovine early embryos. This functional consequence could be partly attributed to the major transcriptional mediator, Recombination Signal Binding Protein For Immunoglobulin Kappa J Region (RBPJ), whose deficiency also compromised the embryo quality. Indeed, both NOTCH1 and RBPJ knockdown cause a significant increase of histone H3 serine 10 phosphorylation (pH3S10, a mitosis marker) positive blastomeres, suggesting a cell cycle arrest at mitosis. Importantly, RNA sequencing analyses reveal that either NOTCH1 or RBPJ depletion triggers a reduction in H1FOO that encodes the oocyte-specific linker histone H1 variant. Interestingly, depleting H1FOO results in detrimental effects on the developmental competence of early embryos, similar with NOTCH1 inhibition. Overall, our results reveal a crucial role for NOTCH pathway in regulating bovine preimplantation development, likely by controlling cell proliferation and maintaining H1FOO expression.

Summary sentence

NOTCH signaling pathway plays an indispensable role in bovine early embryonic development, which is potentially mediated by regulation of cell proliferation and expression of H1FOO.

We hypothesized that sexually dimorphic differences exist in the expression of miRNAs in amniotic fluid (AF) and maternal blood plasma (MP) in association with the process of sex determination and gonad differentiation in cattle. Amniotic fluid and MP were collected from six pregnant heifers (three carrying a single male and three a single female embryo) following slaughter on Day 39 postinsemination, coinciding with the peak of SRY expression. Samples (six AF and six MP) were profiled using an miRNA Serum/Plasma Focus PCR Panel. Differentially expressed (DE) miRNAs were identified in AF (n = 5) and associated MP (n = 56) of male vs. female embryos (P < 0.05). Functional analysis showed that inflammatory and immune response were among the 13 biological processes enriched by miRNAs DE in MP in the male group (FDR < 0.05), suggesting that these sex-dependent DE miRNAs may be implicated in modulating the receptivity of the dam to a male embryo. Further, we compared the downstream targets of the sex-dependent DE miRNAs detected in MP with genes previously identified as DE in male vs. female genital ridges. The analyses revealed potential targets that might be important during this developmental stage such as SHROOM2, DDX3Y, SOX9, SRY, PPP1CB, JARID2, USP9X, KDM6A, and EIF2S3. Results from this study highlight novel aspects of sex determination and embryo–maternal communication in cattle such as the potential role of miRNAs in gonad development as well as in the modulation of the receptivity of the dam to a male embryo.

Summary sentence

Sex-dependent differentially expressed miRNAs identified in amniotic fluid and maternal plasma on Day 39 of pregnancy are associated with gonad development as well as the modulation of the receptivity of the dam to a male embryo.

Upon fertilization, extensive chromatin reprogramming occurs during preimplantation development. Growing evidence reveals species-dependent regulations of this process in mammals. ATP-dependent chromatin remodeling factor SMARCA5 (also known as SNF2H) is required for peri-implantation development in mice. However, the specific functional role of SMARCA5 in preimplantation development and if it is conserved among species remain unclear. Herein, comparative analysis of public RNA-seq datasets reveals that SMARCA5 is universally expressed during oocyte maturation and preimplantation development in mice, cattle, humans, and pigs with species-specific patterns. Immunostaining analysis further describes the temporal and spatial changes of SMARCA5 in both mouse and bovine models. siRNA-mediated SMARCA5 depletion reduces the developmental capability and compromises the specification and differentiation of inner cell mass in mouse preimplantation embryos. Indeed, OCT4 is not restricted into the inner cell mass and the formation of epiblast and primitive endoderm disturbed with reduced NANOG and SOX17 in SMARCA5-deficient blastocysts. RNA-seq analysis shows SMARCA5 depletion causes limited effects on the transcriptomics at the morula stage, however, dysregulates 402 genes, including genes involved in transcription regulation and cell proliferation at the blastocyst stage in mice. By comparison, SMARCA5 depletion does not affect the development through the blastocyst stage but significantly compromises the blastocyst quality in cattle. Primitive endoderm formation is greatly disrupted with reduced GATA6 in bovine blastocysts. Overall, our studies demonstrate the importance of SMARCA5 in fostering the preimplantation development in mice and cattle while there are species-specific effects.

Summary sentence

The chromatin remodeler SMARCA5 is required for preimplantation development with species-specific effects in mice and cattle.

Polycystic ovary syndrome (PCOS) is a prevalent heterogeneous endocrine disorder characterized by ovulation dysfunction, androgen excess, ovarian polycystic changes, insulin resistance, and infertility. Although underlying mechanisms for PCOS are still unknown, inflammation and mitochondrial dysfunction in granulosa cells (GCs) of PCOS patients have been reported. Here, we found that Nicotinamide Adenine Dinucleotide (NAD⁺) levels in GCs of PCOS patients was significantly decreased when compared with controls. Also, we found that higher expression of inflammation factors, increased reactive oxygen species (ROS) accumulation, lower adenosine triphosphate (ATP) generation, and decreased mitochondrial membrane potential, as well as abnormal mitochondrial dynamics in GCs of PCOS patients. In addition, the NAD⁺ levels were decreased after activation of inflammation in human granulosa-like tumor cell line (KGN) treated by Lipopolysaccharide (LPS). However, supplementation of nicotinamide riboside (NR), a NAD⁺ precursor, could largely restore the NAD⁺ content, reduce ROS levels and improve mitochondrial function demonstrated by increased mitochondrial membrane potential and ATP generation in LPS-treated KGN cells. Our data suggested that inflammation decreased NAD⁺ levels in GCs of PCOS patients, while supplementation of NR could restore NAD⁺ levels and alleviated mitochondrial dysfunction in GCs of PCOS patients.

Graphical Abstract

Embryo survival and pregnancy success is increased among animals that exhibit estrus prior to fixed time-artificial insemination, but there are no differences in conceptus survival to d16. The objective of this study was to determine effects of preovulatory estradiol on uterine transcriptomes, select trophectoderm (TE) transcripts, and uterine luminal fluid proteins. Beef cows/heifers were synchronized, artificially inseminated (d0), and grouped into either high (highE2) or low (lowE2) preovulatory estradiol. Uteri were flushed (d16); conceptuses and endometrial biopsies (n = 29) were collected. RNA sequencing was performed on endometrium. Real-time polymerase chain reaction (RT-PCR) was performed on TE (n = 21) RNA to measure relative abundance of IFNT, PTGS2, TM4SF1, C3, FGFR2, and GAPDH. Uterine fluid was analyzed using 2D Liquid Chromatography with tandem mass spectrometry-based Isobaric tags for relative and absolute quantitation (iTRAQ) method. RT-PCR data were analyzed using the MIXED procedure in SAS. There were no differences in messenger RNA (mRNA) abundances in TE, but there were 432 differentially expressed genes (253 downregulated, 179 upregulated) in highE2/conceptus versus lowE2/conceptus groups. There were also 48 differentially expressed proteins (19 upregulated, 29 downregulated); 6 of these were differentially expressed (FDR < 0.10) at the mRNA level. Similar pathways for mRNA and proteins included: calcium signaling, protein kinase A signaling, and corticotropin-releasing hormone signaling. These differences in uterine function may be preparing the conceptus for improved likelihood of survival after d16 among highE2 animals.

Summary sentence

Preovulatory estradiol did not impact conceptus survival to d16; however, it did influence uterine gene/protein expressions related to adhesion, endometrial remodeling, metabolism, and immune regulation, which may explain improved pregnancy success.

Deterioration of the endometrial environment is an essential cause of recurrent miscarriage (RM). However, current studies in terms of endometrial amino acid metabolic characterization and autophagy are still inadequate. We tried to (1) identify the alternation in metabolite profiles in the RM endometrium; (2) investigate the expression of autophagy-related proteins in RM; and (3) elucidate the association between amino acid metabolism and autophagy in RM. Our results showed that glutamine metabolites were up-regulated in the endometrium of RM women. The levels of autophagy-associated proteins, LC3B, ATG12, and Beclin-1, were significantly higher in RM. Hemostasis, autophagy and IFNα signaling were the top three differentially activated signaling pathways between women with RM and normal pregnancy. Interestingly the expression of AMPK and GCN2 was significantly up-regulated in the endometrium of women with RM, and the same expression trend was also observed in the human endometrial stromal cells cultured in glutamine deprivation medium. Furthermore, inhibition of AMPK decreased the level of GCN2, indicating a positive correlation between GCN2 and AMPK. The expression of GCN2 was consistent with the expression of ATG12 and beclin-1; however, it was opposite to that of p62. Exposure to glutamine deprivation increased the level of LC3B, GCN2, ATG12, and beclin-1. Altogether, these findings suggested significant crosstalk between amino acid metabolism and autophagy. In summary, our data suggested that aberrant crosstalk between amino acid metabolism and autophagy may contribute to the impaired endometrial microenvironment of RM. Our study may provide new insight into the diagnosis of RM due to endometrial factors.

Recent evidence indicates that niclosamide is an anti-cancer compound that is able to inhibit several signaling pathways. Although niclosamide has previously been identified by high-throughput screening platforms as a potential effective compound against several cancer types, no direct binding interactions with distinct biological molecule(s) has been established. The present study identifies key signal transduction mechanisms altered by niclosamide in ovarian cancer. Using affinity purification with a biotin-modified niclosamide derivative and mass spectrometry analysis, several RNA-binding proteins (RBPs) were identified. We chose the two RBPs, FXR1 and IGF2BP2, for further analysis. A significant correlation exists in which high-expression of FXR1 or IGF2BP2 is associated with reduced survival of ovarian cancer patients. Knockdown of FXR1 or IGF2BP2 in ovarian cancer cells resulted in significantly reduced cell viability, adhesion, and migration. Furthermore, FXR1 or IGF2BP2 deficient ovarian cancer cells exhibited reduced response to most doses of niclosamide showing greater cell viability than those with intact RBPs. These results suggest that FXR1 and IGF2BP2 are direct targets of niclosamide and could have critical activities that drive multiple oncogenic pathways in ovarian cancer.

Summary sentence

FXR1 and IGF2BP2 are direct targets of niclosamide and could have critical activities that drive multiple oncogenic pathways in ovarian cancer.

Nuclear progestin receptor (PGR) is a ligand-activated transcription factor that has been identified as a pivotal mediator of many processes associated with ovarian and uterine function, and aberrant control of PGR activity causes infertility and disease including cancer. The essential role of PGR in vertebrate ovulation is well recognized, but the mechanisms by which PGR is rapidly and transiently induced in preovulatory follicles after the ovulatory LH surge are not known in lower vertebrates. To address this issue, we utilized the small freshwater teleost medaka Oryzias latipes, which serves as a good model system for studying vertebrate ovulation. In the in vitro ovulation system using preovulatory follicles dissected from the fish ovaries, we found that inhibitors of EPAC (brefeldin A), RAP (GGTI298), PI3K (Wortmannin), AKT (AKT inhibitor IV), and CREB (KG-501) inhibited LH-induced follicle ovulation, while the PKA inhibitor H-89 had no effect on follicle ovulation. The inhibitors capable of inhibiting follicle ovulation also inhibited follicular expression of Pgr and matrix metalloproteinase-15 (Mmp15), the latter of which was previously shown to not only be a downstream effector of Pgr but also a proteolytic enzyme indispensable for follicle rupture in medaka ovulation. Further detailed analysis revealed for the first time that the cAMP/EPAC/RAP/PI3K/AKT/CREB signaling pathway mediates the LH signal to induce Pgr expression in preovulatory follicles. Our data also showed that phosphorylated Creb1 is a transcription factor essential for pgr expression and that Creb1 phosphorylated by Akt1, rather than PKA, may be preferably used to induce pgr expression.

Summary sentence

EPAC/RAP/PI3K/AKT/CREB signaling mediates LH-induced cAMP signaling to induce medaka Pgr expression in ovulating follicles.

Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disease. It has been reported that chronic low-grade inflammation might participate in its pathogenesis. C1q and TNF related 6 (C1QTNF6) is a newly identified adiponectin paralog associated with inflammation. The aim of the present study was to investigate the role of C1QTNF6 in the development of chronic inflammation in PCOS and the underlying molecular mechanism. After analyzing the expression of C1QTNF6 in the serum and granulosa cells (GCs) of PCOS patients and healthy controls, we verified the roles of C1QTNF6 in inflammation through dehydroepiandrosterone-induced PCOS mouse models and cell models of lipopolysaccharide (LPS)-induced inflammation. The results demonstrated that C1QTNF6 expression in the serum and GCs of patients with PCOS was significantly elevated compared with those of the controls, and similar results were observed in the serum and ovary of PCOS mouse models. In PCOS mice and C1QTNF6-overexpressing PCOS mice, serum levels of pro-inflammatory factors including C-reactive protein (CRP), interleukin 6 (IL6), and tumor necrosis factor-α (TNFα) were increased, while the opposite effects were observed when C1QTNF6 was down-regulated in PCOS mice. Furthermore, C1QTNF6 overexpression up-regulated the levels of TNFα, IL6, and CRP and activated the AKT/NF-κB pathway in LPS-treated KGN cells, whereas C1QTNF6 knockdown and BAY-117082 (an NF-κB inhibitor) treatment resulted in the opposite effects. Taken together, our results indicate that C1QTNF6 is involved in the pathogenesis of PCOS by affecting the inflammatory response via the AKT/NF-κB signaling pathway.

Graphical Abstract

Serotonin or 5-hydroxytryptamine (5-HT) is a biogenic amine involved in regulating several functions, including development. However, its impact on human embryo development has been poorly studied. The present work investigated the expression and distribution of the main components of the serotoninergic system in human amniotic tissue and human amniotic epithelial cells (hAEC) in vitro, as an alternative model of early human embryo development. Amniotic membranes from full-term healthy pregnancies were used. Human amnion tissue or hAEC isolated from the amnion was processed for reverse transcription-polymerase chain reaction and immunofluorescence analyses of the main components of the serotoninergic system. We found the expression of tryptophan hydroxylase type 1 (TPH1), type 2 (TPH2), serotonin transporter (SERT), monoamine oxidase-A (MAOA), as well as HTR1D and HTR7 receptors at mRNA level in amnion tissue as well in hAEC. Interestingly, we found the presence of 5-HT in the nucleus of the cells in amnion tissue, whereas it was located in the cytoplasm of isolated hAEC. We detected TPH1, TPH2, and HTR1D receptor in both the nucleus and cytoplasm. SERT, MAOA, and HTR7 receptor were only observed in the cytoplasm. The results presented herein show, for the first time, the presence of the serotoninergic system in human amnion in vivo and in vitro.

Summary sentence

The results presented show the presence of the serotoninergic system in human amnion in vivo and in vitro, which opens the potential role of 5-hydroxytryptamine and the serotoninergic system in the events that occur during early human embryonic development (given the origin of the amniotic cells) in vitro.

Graphical Abstract

In humans, intrauterine growth restriction (IUGR) and preeclampsia (PE) are associated with induction of the unfolded protein response (UPR) and increased placental endoplasmic reticulum (ER) stress. Especially in PE, oxidative stress occurs relative to the severity of maternal vascular underperfusion (MVU) of the placental bed. On the premise that understanding the mechanisms of placental dysfunction could lead to targeted therapeutic options for human IUGR and PE, we investigated the roles of the placental UPR and oxidative stress in two rodent models of these human gestational pathologies. We employed a rat IUGR model of gestational maternal protein restriction, as well as an endothelial nitric oxide synthase knockout mouse model (eNOS^–/–) of PE/IUGR. Placental expression of UPR members was analyzed via qRT-PCR (Grp78, Calnexin, Perk, Chop, Atf6, and Ern1), immunohistochemistry, and Western blotting (Calnexin, ATF6, GRP78, CHOP, phospho-eIF2α, and phospho-IRE1). Oxidative stress was determined via Western blotting (3-nitrotyrosine and 4-hydroxy-2-nonenal). Both animal models showed a significant reduction of fetal and placental weight. These effects did not induce placental UPR. In contrast to human data, results from our rodent models suggest retention of placental plasticity in the setting of ER stress under an adverse gestational environment. Oxidative stress was significantly increased only in female IUGR rat placentas, suggesting a sexually dimorphic response to maternal malnutrition. Our study advances understanding of the involvement of the placental UPR in IUGR and PE. Moreover, it emphasizes the appropriate choice of animal models researching various aspects of these pregnancy complications.

Summary sentence

Gestational protein restriction in the rat or eNOS knockout in the mouse does not induce placental ER stress.

Extracellular vesicles play a crucial role in feto-maternal communication and provide an important paracrine signaling mechanism in pregnancy. We hypothesized that fetal cells-derived exosomes and microvesicles (MVs) under oxidative stress (OS) carry unique cargo and traffic through feto-maternal interface, which cause inflammation in uterine cells associated with parturition. Exosomes and MVs, from primary amnion epithelial cell (AEC) culture media under normal or OS-induced conditions, were isolated by optimized differential centrifugation method followed by characterization for size (nanoparticle tracking analyzer), shape (transmission electron microscopy), and protein markers (western blot and immunofluorescence). Cargo and canonical pathways were identified by mass spectroscopy and ingenuity pathway analysis. Myometrial, decidual, and cervical cells were treated with 1 × 10⁷ control/OS-derived exosomes/MVs. Pro-inflammatory cytokines were measured using a Luminex assay. Statistical significance was determined by paired T-test (P < 0.05). AEC produced cup-shaped exosomes of 90–150 nm and circular MVs of 160–400 nm. CD9, heat shock protein 70, and Nanog were detected in exosomes, whereas OCT-4, human leukocyte antigen G, and calnexin were found in MVs. MVs, but not exosomes, were stained for phosphatidylserine. The protein profiles for control versus OS-derived exosomes and MVs were significantly different. Several inflammatory pathways related to OS were upregulated that were distinct between exosomes and MVs. Both OS-derived exosomes and MVs significantly increased pro-inflammatory cytokines (granulocyte-macrophage colony-stimulating factor, interleukin 6 (IL-6), and IL-8) in maternal cells compared with control (P < 0.05). Our findings suggest that fetal-derived exosomes and MVs under OS exhibited distinct characteristics and a synergistic inflammatory role in uterine cells associated with the initiation of parturition.

Summary sentence

Oxidative stress-induced fetal membrane cells produce exosomes and MVs with distinct properties and cargo and may function as paracrine signalers at the feto-maternal interface during pregnancy and parturition.

Graphical Abstract

The present study aimed to identify biomarkers to assess the quality of in vitro produced (IVP) bovine embryos in the culture media. IVP embryos on Day (D) 5 of development were transferred to individual drops, where they were maintained for the last 48 h of culture. Thereafter, the medium was collected and the embryos were transferred to the recipients. After pregnancy diagnosis, the media were grouped into the pregnant and nonpregnant groups. The metabolic profiles of the media were analyzed via electrospray ionization mass spectrometry, and the concentrations of pyruvate, lactate, and glutamate were assessed using fluorimetry. The spectrometric profile revealed that the media from embryos from the pregnant group presented a higher signal intensity compared to that of the nonpregnant group; the ions 156.13 Da [M + H]⁺, 444.33 Da [M + H]⁺, and 305.97 Da [M + H]⁺ were identified as biomarkers. Spent culture medium from expanded blastocysts (Bx) that established pregnancy had a greater concentration of pyruvate (p = 0.0174) and lesser concentration of lactate (p = 0.042) than spent culture medium from Bx that did not establish pregnancy. Moreover, pyruvate in the culture media of Bx can predict pregnancy with 90.9% sensitivity and 75% specificity. In conclusion, we identified markers in the culture media that helped in assessing the most viable IVP embryos with a greater potential to establish pregnancy.

Summary sentence

The ions 156.13 Da [M + H]⁺, 444.33 Da [M + H]⁺, and 305.97 Da [M + H]⁺, as well as pyruvate and lactate are biomarkers present in the culture media of in vitro produced bovine embryos aiding the selection of embryos with greater potential to establish pregnancy.

Graphical Abstract

Sex determination requires the commitment of bipotential gonads to either a testis or an ovarian fate. Gene deletion of the kinase Map3k4 results in gonadal sex reversal in XY mice, and transgenic re-expression of Map3k4 rescues the sex reversal phenotype. Map3k4 encodes a large, multi-functional protein possessing a kinase domain and several, additional protein–protein interaction domains. Although MAP3K4 plays a critical role in male gonadal sex determination, it is unknown if the kinase activity of MAP3K4 is required. Here, we use mice expressing full-length, kinase-inactive MAP3K4 from the endogenous Map3k4 locus to examine the requirement of MAP3K4 kinase activity in sex determination. Although homozygous kinase-inactivation of MAP3K4 (Map3k4^KI/KI) is lethal, a small fraction survive to adulthood. We show Map3k4^KI/KI adults exhibit a 4:1 female-biased sex ratio. Many adult Map3k4^KI/KI phenotypic females have a Y chromosome. XY Map3k4^KI/KI adults with sex reversal display female mating behavior, but do not give rise to offspring. Reproductive organs are overtly female, but there is a broad spectrum of ovarian phenotypes, including ovarian absence, primitive ovaries, reduced ovarian size, and ovaries having follicles in all stages of development. Further, XY Map3k4^KI/KI adults are smaller than either male or female Map3k4^WT/WT mice. Examination of the critical stage of gonadal sex determination at E11.5 shows that loss of MAP3K4 kinase activity results in the loss of Sry expression in XY Map3k4^KI/KI embryos, indicating embryonic male gonadal sex reversal. Together, these findings demonstrate the essential role for kinase activity of MAP3K4 in male gonadal sex determination.

Summary sentence

Male gonadal sex determination requires the kinase activity of the multi-domain containing protein MAP3K4.

Graphical Abstract

Retinoic acid (RA) induces spermatogonial differentiation, but the mechanism by which it operates remains largely unknown. We developed a germ cell culture assay system to study genes involved in spermatogonial differentiation triggered by RA. Stimulated by RA 8 (Stra8), a RA-inducible gene, is indispensable for meiosis initiation, and its deletion results in a complete block of spermatogenesis at the pre-leptotene/zygotene stage. To interrogate the role of Stra8 in RA mediated differentiation of spermatogonia, we derived germ cell cultures from the neonatal testis of both wild type and Stra8 knock-out mice. We provide the first evidence that Stra8 plays a crucial role in modulating the responsiveness of undifferentiated spermatogonia to RA and facilitates transition to a differentiated state. Stra8-mediated differentiation is achieved through the downregulation of a large portfolio of genes and pathways, most notably including genes involved in the spermatogonial stem cell self-renewal process. We also report here for the first time the role of transcription elongation regulator-1 like (Tcerg1l) as a downstream effector of RA-induced spermatogonial differentiation.

Summary sentence

The genes stimulated by retinoic acid 8 (Stra8) and transcription elongation regulator-1 like (Tcerg1l) are necessary in early-stage male germ cell differentiation induced by retinoic acid (RA). Stra8 is critical in modulating the responsiveness of undifferentiated spermatogonia to RA.

Energy metabolism in spermatozoa is complex and involves the metabolism of carbohydrate fatty acids and amino acids. The ATP produced in the electron transport chain in the mitochondria appears to be crucial for both sperm motility and maintaining viability, whereas glycolytic enzymes in the flagella may contribute to ATP production to sustain motility and velocity. Stallion spermatozoa seemingly use diverse metabolic strategies, and in this regard, a study of the metabolic proteome showed that Gene Ontology terms and Reactome pathways related to pyruvate metabolism and the Krebs cycle were predominant. Following this, the hypothesis that low glucose concentrations can provide sufficient support for motility and velocity, and thus glucose concentration can be significantly reduced in the medium, was tested. Aliquots of stallion semen in four different media were stored for 48 h at 18°C; a commercial extender containing 67 mM glucose was used as a control. Stallion spermatozoa stored in media with low glucose (1 mM) and high pyruvate (10 mM) (LG-HP) sustained better motility and velocities than those stored in the commercial extender formulated with very high glucose (61.7 ± 1.2% in INRA 96 vs 76.2 ± 1.0% in LG-HP media after 48 h of incubation at 18°C; P < 0.0001). Moreover, mitochondrial activity was superior in LG-HP extenders (24.1 ± 1.8% in INRA 96 vs 51.1 ± 0.7% in LG-HP of spermatozoa with active mitochondria after 48 h of storage at 18°C; P < 0.0001). Low glucose concentrations may permit more efficient sperm metabolism and redox regulation when substrates for an efficient tricarboxylic acid cycle are provided. The improvement seen using low glucose extenders is due to reductions in the levels of glyoxal and methylglyoxal, 2-oxoaldehydes formed during glycolysis; these compounds are potent electrophiles able to react with proteins, lipids, and DNA, causing sperm damage.

Summary sentence

Hiigh glucose in the extender damages spermatozoa through formation of 2-oxoaldehydes.

Graphical Abstract

In-vitro maturation (IVM) of oocytes from immature females is widely used in assisted reproductive technologies. Here we illustrate that cumulus cell (CC) expansion, once considered a key indicator of oocyte quality, is not needed for oocytes to mature to the metaphase II (MII) stage and to gain nuclear and cytoplasmic competence to produce offspring. Juvenile pig oocytes were matured in four different media: (1) Basal (–gonadotropins (GN) – FLI); (2) –GN + FLI (supplement of FGF2, LIF, and IGF1); (3) +GN – FLI; and (4) +GN + FLI. There was no difference in maturation to MII or progression to the blastocyst stage after fertilization of oocytes that had been matured in –GN + FLI medium and oocytes matured in +GN + FLI medium. Only slight CC expansion occurred in the two media lacking GN compared with the two where GN was present. The cumulus-oocytes-complexes (COC) matured in +GN + FLI exhibited the greatest expansion. We conclude that FLI has a dual role. It is directly responsible for oocyte competence, a process where GN are not required, and, when GN are present, it has a downstream role in enhancing CC expansion. Our study also shows that elevated phosphorylated MAPK may not be a necessary correlate of oocyte maturation and that the greater utilization of glucose by COC observed in +GN + FLI medium probably plays a more significant role to meet the biosynthetic needs of the CC to expand than to attain oocyte developmental competence. Gene expression analyses have not been informative in providing a mechanism to explain how FLI medium enhances oocyte competence without promoting CC expansion.

Summary sentence

Gonadotropins are not required during in-vitro maturation of immature porcine oocytes when FGF2, LIF, and IGF1 are present.

In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives—such as trichostatin A—characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1–7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2–7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8–10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.

Summary sentence

Chlamydocin analogues, a novel family of inhibitors specific for class I and IIb HDACs, significantly improved the ability of mouse SCNT-derived embryos to produce offspring.

Deoxynivalenol (DON) is one of the most prevalent Fusarium mycotoxins, which cause detrimental effects on human and animal reproductive systems by inducing oxidative stress. Increasing evidence has suggested the potential roles of melatonin in protecting granulosa cells from oxidative injury, but the underlying mechanisms remain largely elusive. Here, we demonstrated that suppression of FOXO1 and endoplasmic reticulum (ER) stress was engaged in melatonin-mediated protection against oxidative damage in human granulosa cells upon DON exposure in vitro. DON induced excess reactive oxygen species accumulation, cells viability loss, reduced estradiol-17β, and progesterone production in human granulosa cells, whereas melatonin ameliorated these phenotypes. Next, we found that the protective effect of melatonin against apoptosis was via reducing ER stress because the inhibition of ER stress displayed similar protective effects during DON treatment. Moreover, melatonin provided no additional protection when ER stress was inhibited. We further found that FOXO1 is a pivotal downstream effector of melatonin and ER stress in regulating DON-induced apoptosis in human granulosa cells. Blocking of FOXO1 reduced DON-induced cells death and FOXO1 activation could be suppressed by melatonin or ER stress inhibitor. However, melatonin failed to further restore cells viability in the presence of FOXO1 inhibitor. Collectively, our results reveal a new mechanism of melatonin in protecting against DON-induced apoptosis and dysfunction by suppressing ER stress and FOXO1 in human granulosa cells.

Summary sentence

DON exposure results in human granulosa cells apoptosis and melatonin alleviates the apoptosis by suppressing FOXO1 and ER stress.

Graphical Abstract