The laboratory mouse is the most widely used animal model for studying the genetics and biology of mammalian development and reproduction. Embryonic stem cell (ESC) gene targeting technology, and the sophisticated genomic manipulations it allowed, was unique to this organism for a long period of time; this was a major factor in the mouse's rise to pre-eminence as a model system over the past three decades or so. The recent advent of CRISPR/Cas9 technology has democratized the application of genome editing to essentially all organisms. Nevertheless, the scientific infrastructure behind the mouse still makes it the organism of choice for studying molecular mechanisms of mammalian development, and for modeling human development and disease.

Embryo implantation is an intricate process which requires competent embryo and receptive endometrium. The failure of endometrium to achieve receptivity is a recognized cause of infertility. However, due to multiplicity of events involved, the molecular mechanisms governing endometrial receptivity are still not fully understood. Traditional one-by-one approaches, including western blotting and histochemistry, are insufficient to examine the extensive changes of endometrial proteome. Although genomics and transcriptomics studies have identified several significant genes, the underlying mechanism remains to be uncovered owing to post-transcriptional and posttranslational modifications. Proteomic technologies are high throughput in protein identification, and they are now intensively used to identify diagnostic and prognostic markers in the field of reproductive medicine. There is a series of studies analyzing endometrial proteomic profile, which has provided a mechanistic insight into implantation failure. These published studies mainly focused on the difference between pre-receptive and receptive stages of endometrium, as well as on the alternation of endometrial proteomics in women with reproductive failure. Here, we review recent data from proteomic analyses regarding endometrium around the time of embryo implantation and propose possible future research directions.

Summary Sentence

Based on the previous findings summarized, human endometrial proteome plays an important role around the time of embryo implantation and dysfunction of endometirla proteome is related with female reproductive failure.

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Monitoring the health of a pregnancy is of utmost importance to both the fetus and the mother. The diagnosis of pregnancy complications typically occurs after the manifestation of symptoms, and limited preventative measures or effective treatments are available. Traditionally, pregnancy health is evaluated by analyzing maternal serum hormone levels, genetic testing, ultrasonographic imaging, and monitoring maternal symptoms. However, researchers have reported a difference in extracellular vesicle (EV) quantity and cargo between healthy and at-risk pregnancies. Thus, placental EVs (PEVs) may help to understand normal and aberrant placental development, monitor pregnancy health in terms of developing placental pathologies, and assess the impact of environmental influences, such as infection, on pregnancy. The diagnostic potential of PEVs could allow for earlier detection of pregnancy complications via noninvasive sampling and frequent monitoring. Understanding how PEVs serve as a means of communication with maternal cells and recognizing their potential utility as a readout of placental health have sparked a growing interest in basic and translational research. However, to date, PEV research with animal models lags behind human studies. The strength of animal pregnancy models is that they can be used to assess placental pathologies in conjunction with isolation of PEVs from fluid samples at different time points throughout gestation. Assessing PEV cargo in animals within normal and complicated pregnancies will accelerate the translation of PEV analysis into the clinic for potential use in prognostics. We propose that appropriate animal models of human pregnancy complications must be established in the PEV field.

Summary Sentence

Experimental animal models will be essential for defining the opportunity that placental extracellular vesicles may provide for monitoring placental health and function and understanding the pathophysiology of adverse pregnancy outcomes.

There has been a significant annual increase in the number of cases of uterine leiomyomas or fibroids (UF) among women of all races and ages across the world. A fortune is usually spent by the healthcare sector for fibroid-related treatments and management. Molecular studies have established the higher mutational heterogeneity in UF as compared to normal myometrial cells. The contribution of DNA damage and defects in repair responses further increases the mutational burden on the cells. This in turn leads to genetic instability, associated with cancer risk and other adverse reproductive health outcomes. Such and many more growing bodies of literature have highlighted the genetic/molecular, biochemical and clinical aspects of UF; none the less there appear to be a lacuna bridging the bench to bed gap in addressing and preventing this disease. Presented here is an exhaustive review of not only the molecular mechanisms underlying the predisposition to the disease but also possible strategies to effectively diagnose, prevent, manage, and treat this disease.

Summary sentence

Determining and designing of DNA damage response biomarker is needed urgently to predict uterine fibroid etiology, progression, and planning of non-invasive elimination measures to lessen the impact lead by this disease on the women related healthcare system and subsequently on the economy.

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Ephrins are ligands of Eph receptors (Ephs); both of which are sorted into two classes, A and B. There are five types of ephrin-As (ephrin-A1–5) and three types of ephrin-Bs (ephrin-B1–3). Also, there are 10 types of EphAs (EphA1–10) and six types of EphBs (EphB1–6). Binding of ephrins to the Eph receptors activates signaling cascades that regulate several biological processes such as cellular proliferation, differentiation, migration, angiogenesis, and vascular remodeling. Clarification of their roles in the female reproductive system is crucial to understanding the physiology and pathology of this system. Such knowledge will also create awareness regarding the importance of these molecules in diagnostic, prognostic, and therapeutic medicine. Hence, we have discussed the involvement of these molecules in the physiological and pathological events that occur within the female reproductive system. The evidence so far suggests that the ephrins and the Eph receptors modulate folliculogenesis, ovulation, embryo transport, implantation, and placentation. Abnormal expression of some of these molecules is associated with polycystic ovarian syndrome, ovarian cancer, tubal pregnancy, endometrial cancer, uterine leiomyoma (fibroids), cervical cancer, and preeclampsia, suggesting the need to utilize these molecules in the clinical setting. To enhance a quick development of this gradually emerging field in female reproductive medicine, we have highlighted some “gaps in knowledge” that need prospective investigation.

Summary sentence

The ephrin and Eph receptors are expressed throughout the female reproductive system, regulate the functioning of this system and associate with a number of obstetric and gynecologic disorders.

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Double homeobox genes are unique to eutherian mammals. It has been proposed that the DUXC clade of the double homeobox gene family, which is present in multicopy long tandem arrays, plays an essential role in zygotic genome activation (ZGA). We generated a deletion of the tandem array encoding the DUXC gene of mouse, Double homeobox (Dux), and found it surprisingly to be homozygous viable and fertile. We characterize the embryonic development and ZGA profile of knockout (KO) embryos, finding that zygotic genome activation still occurs, with only modest alterations in 2-cell embryo gene expression, no defect in in vivo preimplantation development, but an increased likelihood of post-implantation developmental failure, leading to correspondingly smaller litter sizes in the KO strain. While all known 2-cell specific Dux target genes are still expressed in the KO, a subset is expressed at lower levels. These include numerous genes involved in methylation, blastocyst development, and trophectoderm/placental development. We propose that rather than driving ZGA, which is a process common throughout the animal kingdom, DUXC genes facilitate a process unique to eutherian mammals, namely the post-implantation development enabled by an invasive placenta.

Summary sentence

Mouse Dux is not absolutely required for viability or fertility, nor for zygotic genome activation, however the subset of Dux knockout embryos, post-implantation development fails.

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Actinomycin D (ActD) has been considered as one of the most effective and safe chemotherapeutic medications for treating a number of cancers. Although ActD has been used in the treatment of gynecological tumors and pediatric tumors for more than 50 years, the toxic effects of ActD on mammalian oocytes remain unknown. In this study, the influence of ActD on mouse and human oocyte maturation and the possible mechanisms were investigated. Notably, ActD inhibited oocyte maturation and arrested oocytes at the metaphase I (MI) stage in a dose-dependent manner. In addition, ActD arrested oocyte maturation when the oocytes were treated at different successive stages, including the germinal vesicle (GV), germinal vesicle breakdown, and MI stages. In ActD-treated oocytes, disordered chromosome condensation and irregular spindle assembly occurred, resulting in incomplete chromosome segregation and oocytes arresting at the MI phase; these results possibly occurred because ActD triggered the formation of reactive oxygen species, resulting in DNA damage and decreased ATP in mouse GV oocytes. Besides, in vivo treatment with ActD also inhibited mouse oocyte maturation. Similar effects were seen in human oocytes. Collectively, our results indicated that ActD exposure disrupted oocyte maturation by increasing DNA damage, which is a finding that might help with optimizing future methods for female fertility preservation before undergoing chemotherapy.

Summary sentence

ActD blocks oocyte maturation at MI stage by inhibiting chromosome separation and spindle assembly, perhaps through inducing nuclear DNA damage and disruption of the mitochondrial function in oocytes.

Female fertility relies on successful egg development. Besides chromosome segregation, complex structural and biochemical changes in the cytoplasmic compartment are necessary to confer the female gamete the capacity to undergo normal fertilization and sustain embryonic development. Despite the profound impact on egg quality, morphological bases of cytoplasmic maturation remain largely unknown. Here, we report our findings from the ultrastructural analysis of 69 unfertilized human oocytes from 34 young and healthy egg donors. By comparison of samples fixed at three consecutive developmental stages, we explored how ooplasmic architecture changes during meiotic maturation in vitro. The morphometric image analysis supported observation that the major reorganization of cytoplasm occurs before polar body extrusion. The organelles initially concentrated around prophase nucleus were repositioned toward the periphery and evenly distributed throughout the ooplasm. As maturation progressed, distinct secretory apparatus appeared to transform into cortical granules that clustered underneath the oocyte's surface. The most prominent feature was the gradual formation of heterologous complexes composed of variable elements of endoplasmic reticulum and multiple mitochondria with primitive morphology. Based on the generated image dataset, we proposed a morphological map of cytoplasmic maturation, which may serve as a reference for future comparative studies. In conclusion, this work improves our understanding of human oocyte morphology, cytoplasmic maturation, and intracellular factors defining human egg quality. Although this analysis involved spare oocytes completing development in vitro, it provides essential insight into the enigmatic process by which human egg progenitors prepare for fertilization.

Summary Sentence: Ultrastructural characterization and morphometric analysis of maturing human oocytes reveal complex changes in cytoplasmic architecture that are associated with the acquisition of egg's developmental competence.

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Female fertility depends greatly on the capacity of the uterus to recognize and eliminate microbial infections, a major reason of inflammation in the endometrium in many species. This study aimed to determine the in vitro effect of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the transcriptome genes expression and alternative splicing in the porcine endometrium in the mid-luteal phase of the estrous cycle during LPS-stimulated inflammation using RNA-seq technology. The endometrial slices were incubated in vitro in the presence of LPS and PPARγ agonists—PGJ₂ or pioglitazone and antagonist—T0070907. We identified 222, 3, 4, and 62 differentially expressed genes after LPS, PGJ₂, pioglitazone, or T0070907 treatment, respectively. In addition, we detected differentially alternative spliced events: after treatment with LPS-78, PGJ₂-60, pioglitazone-52, or T0070907-134. These results should become a basis for further studies explaining the mechanism of PPARγ action in the reproductive system in pigs.

Summary Sentence: PPARγ ligands regulate genes expression in the porcine endometrium during LPS-induced inflammation in vitro.

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Whey-acidic protein four-disulfide core domain (WFDC) genes display putative roles in innate immunity and fertility. In mice, a locus on chromosome 2 contains 5 and 11 Wfdc genes in its centromeric and telomeric subloci, respectively. Although Wfdc genes are highly expressed in the epididymis, their contributions to epididymal function remain elusive. Here, we investigated whether Wfdc genes are regulated in response to lipopolysaccharide (LPS)-induced epididymitis, an inflammatory condition that impairs male fertility. We induced epididymitis in mice via (i) interstitial LPS injection into epididymal initial segment and (ii) intravasal LPS injection into the vas deferens towards cauda epididymis. Interstitial and intravasal LPS induced a differential upregulation of inflammatory mediators (interleukin 1 beta, interleukin 6, tumor necrosis factor, interferon gamma, and interleukin 10) in the initial segment and cauda epididymis within 72 h post-treatment. These changes were accompanied by a time-dependent endotoxin clearance from the epididymis. In the initial segment, interstitial LPS upregulated all centromeric (Slpi, Wfdc5, Wfdc12, Wfdc15a, and Wfdc15b) and five telomeric (Wfdc2, Wfdc3, Wfdc6b, Wfdc10, and Wfdc13) Wfdc transcripts at 24 and 72 h. In the cauda epididymis, intravasal LPS upregulated Wfdc5 and Wfdc2 transcripts at 24 h, followed by a downregulation of Wfdc15b and three telomeric (Wfdc6a, Wfdc11, and Wfdc16) gene transcripts at 72 h. Pharmacological inhibition of nuclear factor kappa B activation prevented LPS-induced upregulation of centromeric and telomeric Wfdc genes depending on the epididymal region. We show that LPS-induced inflammation differentially regulated the Wfdc locus in the proximal and distal epididymis, indicating region-specific roles for the Wfdc family in innate immune responses during epididymitis.

Summary sentence

LPS-induced inflammation in the mouse epididymis differentially regulated the transcript levels of centromeric and telomeric Wfdc genes in the Wfdc locus on chromosome 2; an effect partially prevented by blocking LPS-TLR4-NFKB pathway.

Graphical Abstract

Preeclampsia (PE) is a placental disorder caused by endothelial dysfunction via trophoblast inadequate invasion activity. Adrenomedullin (ADM) and ADM2 are multifunctional peptides that can support vascular activity and placental growth. However, correlation between ADMs and trophoblast functions is currently unclear. The objective of this study was to analyze changes in expression of ADMs in placenta and HTR-8/SVneo trophoblast cells under hypoxia and their effects on invasion activity of trophoblast cells and expression of HLA-G. In placental tissues of PE, expression levels of ADM and HLA-G were significantly increased (P < 0.05) whereas expression of ADM2 was decreased compared to that in normal term placenta. Under hypoxia, expression levels of ADM, ADM2, and HLA-G and invasion ability of trophoblast cells were increased in hypoxia-inducible factor-1 (HIF-1α)- dependent manner (P < 0.05). Treatment with ADMs agonists reduced HIF-1α activity whereas enhanced invasion ability under hypoxia. However, they were not changed after cotreatment of ADMs and HIF-1α inhibitor, YC-1, although expression levels of invasion-related genes MMP2, MMP9, and Rac1 were altered (P < 0.05). ADMs also increased HLA-G expression under normoxia whereasADM2 or cotreatment of ADMs under hypoxia attenuated HLA-G expression (P < 0.05). Our findings demonstrate that altered expression of ADMs plays a critical role in placental physiology, especially in trophoblast invasion and immune-modulation under hypoxia.

Summary sentence

Altered expression of ADMs plays a critical role in placental physiology, especially in trophoblast invasion and immune-modulation under hypoxia.

Nutrient restriction (NR) has the potential to negatively impact birthweight, an indicator of neonatal survival and lifelong health. Those fetuses are termed as small for gestational age (SGA). Interestingly, there is a spectral phenotype of fetal growth rates in response to NR associated with changes in placental development, nutrient and waste transport, and lipid metabolism. A sheep model with a maternal diet, starting at Day 35, of 100% National Research Council (NRC) nutrient requirements (n = 8) or 50% NRC (n = 28) was used to assess alterations in fetuses designated NR SGA (n = 7) or NR NonSGA (n = 7) based on fetal weight at Day 135 of pregnancy. Allantoic fluid concentrations of triglycerides were greater in NR SGA fetuses than 100% NRC and NR NonSGA fetuses at Day 70 (P < 0.05). There was a negative correlation between allantoic fluid concentrations of triglycerides (R² = 0.207) and bile acids (R² = 0.179) on Day 70 and fetal weight at Day 135 for NR ewes (P < 0.05). Bile acids were more abundant in maternal and fetal blood for NR SGA compared to 100% NRC and NR NonSGA ewes (P < 0.05). Maternal blood concentrations of NEFAs increased in late pregnancy in NR NonSGA compared to NR SGA ewes (P < 0.05). Protein expression of fatty acid transporter SLC27A6 localized to placentomal maternal and fetal epithelia and decreased in Day 70 NR SGA compared to 100% NRC and NR NonSGA placentomes (P < 0.05). These results identify novel factors associated with an ability of placentae and fetuses in NR NonSGA ewes to adapt to, and overcome, nutritional hardship during pregnancy.

Summary sentence

Allantoic fluid concentrations of triglycerides and bile acids from nutrient restricted ewes at mid-gestation are negatively correlated with fetal weights near term, while placental fatty acid transporter SLC27A6 protein is reduced in small for gestational age fetuses.

Dyslipidemia is a characteristic of maternal obesity and previous studies have demonstrated abnormalities in fatty acid oxidation and storage in term placentas. However, there is little information about the effect of pre-pregnancy obesity on placental lipid metabolism during early pregnancy. The objective of this study was to determine the relationship between lipid profiles and markers of metabolism in placentas from obese and lean dams at midgestation. Mice were fed a western diet (WD) or normal diet (ND) and lysophosphatidylcholines (LPCs) and/or phosphatidylcholines (PCs) were measured in dam circulation and placenta sections using liquid chromatography–tandem mass spectrometry and mass spectrometry imaging, respectively. In WD dam, circulating LPCs containing 16:1, 18:1, 20:0, and 20:3 fatty acids were increased and 18:2 and 20:4 were decreased. In WD placenta from both sexes, LPC 18:1 and PC 36:1 and 38:3 were increased. Furthermore, there were moderate to strong correlations between LPC 18:1, PC 36:1, and PC 38:3. Treatment-, spatial-, and sex-dependent differences in LPC 20:1 and 20:3 were also detected. To identify genes that may regulate diet-dependent differences in placenta lipid profiles, the expression of genes associated with lipid metabolism and nutrient transport was measured in whole placenta and isolated labyrinth using droplet digital PCR and Nanostring nCounter assays. Several apolipoproteins were increased in WD placentas. However, no differences in nutrient transport or fatty acid metabolism were detected. Together, these data indicate that lipid storage is increased in midgestation WD placentas, which may lead to lipotoxicity, altered lipid metabolism and transport to the fetus later in gestation.

Summary sentence

At midgestation, lipid profiles are altered in the circulation of obese mouse dams and placentas with specific changes in phosphatidylcholine and lyso-phosphatidylcholine species, lipid storage, and the expression of apolipoproteins.

Graphical Abstract

Conditional knockout (cKO) mice have contributed greatly to understanding the tissue- or stage-specific functions of genes in vivo. However, the current cKO method requires considerable time and effort because of the need to generate two gene-modified mouse strains (Cre transgenic and loxP knockin) for crossing. Here, we examined whether we could analyze the germ cell-related functions of embryonic lethal genes in F0 chimeric mice by restricting the origin of germ cells to mutant embryonic stem cells (ESCs). We confirmed that the full ESC origin of spermatozoa in fertile chimeric mice was achieved by the CRISPR/Cas9 system using three guide RNAs targeting Nanos3, which induced germ cell depletion in the host blastocyst-derived tissues. Among these fertile chimeric mice, those from male ESCs with a Dnmt3b mutation, which normally causes embryo death, also produced F1 mice derived exclusively from the mutant ESCs. Thus, our new chimeric strategy readily revealed that Dnmt3b is dispensable for male germ cell development, in agreement with a previous cKO study. Our new approach enables us to analyze the germ cell functions of embryonic lethal genes in the F0 generation without using the current cKO method.

Summary Sentence

A CRISPR/Cas9 system using three guide RNAs targeting Nanos3 enabled the production of chimeric mice with spermatozoa fully derived from ESCs and confirmed that Dnmt3b is not essential for male germ cell development.

Graphical Abstract