Mediator is an evolutionarily conserved multi-subunit complex, bridging transcriptional activators and repressors to the general RNA polymerase II (Pol II) initiation machinery. Though the Mediator complex is crucial for transcription of almost all Pol II promoters in eukaryote organisms, the phenotypes of individual Mediator subunit mutants are each distinct. Here we report for the first time, the essential role of subunit Med20 in early mammalian embryo development. Although Med20 mutant mouse embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at early post-gastrulation stages. Outgrowth assays show that mutant blastocysts cannot hatch from the zona pellucida, indicating impaired blastocyst function. Assessments of cell death and cell lineage specification reveal that apoptosis, inner cell mass, trophectoderm, and primitive endoderm markers are normal in mutant blastocysts. However, the epiblast marker Nanog is ectopically expressed in the trophectoderm of Med20 mutants, indicative of defects in trophoblast specification. These results suggest that Med20 specifically, and the Mediator complex in general, are essential for the earliest steps of mammalian development and cell lineage specification.
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Wei Cui, Chelsea Marcho, Yongsheng Wang, Rinat Degani, Morgane Golan, Kimberly D. Tremblay, Jaime Rivera-Pérez and Jesse Mager
The focus of my life in science, has been to improve reproductive health for women, with an emphasis on the endometrium, the most dynamic tissue in the human body: its remarkable cyclical remodelling is essential for establishment of pregnancy. The most notable events in a woman’s endometrial cycle are menstruation and endometrial repair, regeneration of the endometrium during the proliferative phase, attainment of receptivity by the mid-secretory phase of the cycle and the embryo-maternal interactions that initiate peri-implantation events within the microenvironment of the uterine cavity. I have contributed to understanding the molecular and cellular changes underpinning these events, and how disturbance of them leads to menstrual disorders, infertility and endometrial diseases including abnormal uterine bleeding, endometriosis and endometrial cancer. My team have contributed to changes in clinical IVF practice, to a new diagnostic for endometrial receptivity in infertile women, and to enhancing endometrial repair. I have shared my world with many amazing younger scientists: it has indeed been a privileged journey.
Gabriela Hernández-Silva, Marta Durand, Fernando Larrea and Mayel Chirinos
When levonorgestrel (LNG) is given for emergency contraception during the follicular phase, it not only inhibits or delays ovulation, but also induces changes in endometrial secretions that modulate sperm functionality. In order to characterize the female reproductive tract secreted molecules that may affect human spermatozoa, we analyzed changes in the protein content of uterine flushings obtained from women during the periovulatory phase of a control and a LNG-treated menstrual cycle. Lectin affinity analysis and 2D gel electrophoresis of uterine samples showed changes in protein glycosylation patterns and the presence of 31 differentially expressed proteins (8 upregulated and 23 downregulated). Mass spectrometry and Western blot analyses of the differential expressed proteins showed lactotransferrin (LTF) as one of the upregulated molecules by LNG. In this study, LTF exhibited significant dose-related effects on sperm functionality, particularly a decrease of calcium ionophore-induced acrosome reaction and protein tyrosine phosphorylation. Overall, the results indicated that LNG promoted changes in the proteome of uterine secretions that might compromise human sperm capacitation. These data further support the participation of other mechanisms of action of LNG as emergency contraceptive, in addition to those on ovulation.
Roland Abi Nahed, Guillaume Martinez, Jean Pascal Hograindleur, Emilie Le Blévec, Sabine Camugli, Richard Le Boucher, Pierre F Ray, Jessica Escoffier, Eric Schmitt and Christophe Arnoult
For artificial insemination (AI) to be successful, it is essential that sperm delivery be perfectly timed relative to ovulation, as sperm lifespan is limited due to oxidative metabolism induced by capacitation. Extending the window of sperm capacitation could therefore increase sperm lifespan, prolong sperm-fertilizing competence and increase AI efficiency. Hyperpolarization of sperm is a crucial step in capacitation and is induced by activation of the potassium calcium-activated channel subfamily U member 1 (KCNU1, also named Slo3 or KSper). Given the essential role played by KCNU1 in capacitation, this study assessed the impact of its pharmacological inhibition on sperm lifespan. We showed that treatment of murine sperm with sub-micromolar concentrations of clofilium, a specific inhibitor of KCNU1, slowed down capacitation, decreased the rate of acrosome reaction and extended the fertilizing competence of capacitated sperm for 12 h. Clofilium also extended fertilizing competence and motility of bovine-capacitated sperm, and increased the rate of fertilization with sperm capacitated for 24 h by 100%, and the rate of blastocyst formation by 150%. Finally, toxicity experiments showed clofilium to have no impact on sperm DNA and no embryotoxicity at the concentration used to extend sperm lifespan. Our results demonstrate that clofilium prolongs fertilizing competence of aging capacitated sperm in vitro in both rodent and bovine species. To our knowledge, this is the first time the duration of sperm-fertilizing competence is shown to be extended by potassium channels blockers.
Andrée-Anne Saindon and Pierre Leclerc
Sperm adhesion molecule 1 (SPAM1) is a sperm protein possessing a hyaluronidase domain in its N-terminus and a zona pellucida-binding domain in its C-terminus. Our previous studies showed that bovine spermatozoa potentially have 2 SPAM1 isoforms that present different C-terminal domains, different origins (testis and epididymis) and different locations in spermatozoa. In this study, two approaches were taken to characterize the different SPAM1 isoforms. First, 3′-RACE experiments were done to determine the sequence of the 3′ regions of the potential transcripts. Second, by in silico analyses, we aimed to determine whether our antibody that recognizes the N-terminal domain of SPAM1 detects two SPAM1 isoforms or two highly similar, although different, proteins. We found that the 3′ regions of SPAM1 transcripts from bovine testis and caput epididymis were identical. Nevertheless, two transcript variants that differ by 90 nucleotides, encoded by an entire exon, are expressed in both tissues. Only the protein encoded by the longest SPAM1 transcript variant was confirmed in ejaculated bull spermatozoa by mass spectrometry. In silico analyses revealed a highly similar protein to SPAM1, PH-20, that could potentially be recognized by our N-terminal antibody. The presence of PH-20 transcripts was confirmed in bovine testis and the protein is present in ejaculated spermatozoa. Our N-terminal antibody possibly recognizes both SPAM1 and the highly homologous protein PH-20 instead of two SPAM1 isoforms. Identifying the proteins implicated in the fertilization process is crucial in order to elucidate their roles and to better understand the complex process of fertilization.
Dong-Kyung Lee, Kwang-Hwan Choi, Jae Yeon Hwang, Jong-Nam Oh, Seung-Hun Kim and Chang_Kyu Lee
Lipid droplets (LD) provide a source of energy, and their importance during embryogenesis has been increasingly recognized. In particular, pig embryos have larger amounts of intercellular lipid bilayers than other mammalian species, suggesting that porcine embryos are more dependent on lipid metabolic pathways. The objective of the present study was to detect the effect of stearoyl-coenzyme A desaturase 1 (SCD1) on LD formation and to associate these effects with the mRNA abundance of LD formation-related genes (SREBP, ARF1, COPG2, PLD1, and ERK2) in in vitro-produced porcine embryos. To determine the effect of SCD1 on LD formation and related genes, we examined the effects of SCD1 inhibition using CAY10566 (an SCD1 inhibitor, 50 μM) on parthenogenetic embryos. SCD1 inhibition downregulated the mRNA levels of LD formation-related genes and embryo development. Our results revealed that SCD1 functions in the regulation of LD formation via phospholipid formation and embryo development. In addition, we treated parthenogenetic embryos with oleic acid (100 μM), which led to a significant increase in the blastocyst formation rate, LD size and number compared to controls. Remarkably, the adverse effects of the SCD1 inhibitor could be counteracted by oleic acid. These data suggest that porcine embryos can use exogenous oleic acid as a metabolic energy source.
Svetlana Farberov, Raghavendra Basavaraja and Rina Meidan
The multimodular matricellular protein thrombospondin-1 (THBS1) was among the first identified endogenous antiangiogenic molecules. Recent studies have shown THBS1-mediated suppression of angiogenesis and other critical activities for corpus luteum (CL) regression. THBS1 is specifically induced by prostaglandin F2alpha in mature CL undergoing regression, whereas luteinizing signals such as luteinizing hormone and insulin reduced its expression. THBS1 interacts both synergistically and antagonistically with other essential luteal factors, such as fibroblast growth factor 2, transforming growth factor beta1, and serpin family E member 1, to promote vascular instability, apoptosis, and matrix remodeling during luteal regression. Expression of THBS1 is also downregulated by pregnancy recognition signals to maintain the CL during early pregnancy. This dynamic pattern of luteal expression, the extensive interactivity with other luteal factors, and strong antiangiogenic and proapoptotic activities indicate that THBS1 is a major determinant of CL fate.
Siennah R Miller and Nathan J Cherrington
The blood–testis barrier protects developing germ cells by limiting the entry of xenobiotics into the adluminal compartment. There is strong evidence that the male genital tract can serve as a sanctuary site, an area of the body where tumors or viruses are able to survive treatments because most drugs are unable to reach therapeutic concentrations. Recent work has classified the expression and localization of endogenous transporters in the male genital tract as well as the discovery of a transepithelial transport pathway as the molecular mechanism by which nucleoside analogs may be able to circumvent the blood–testis barrier. Designing drug therapies that utilize transepithelial transport pathways may improve drug disposition to this sanctuary site. Strategies that improve disposition into the male genital tract could reduce the rate of testicular relapse, decrease viral load in semen, and improve therapeutic strategies for male fertility.
Sathish Kumar, Geoffrey H Gordon, David H Abbott and Jay S Mishra
Adequate maternal vascular adaptations and blood supply to the uterus and placenta are crucial for optimal oxygen and nutrient transport to growing fetuses of eutherian mammals, including humans. Multiple factors contribute to hemodynamics and structuring of placental vasculature essential for term pregnancy with minimal complications. In women, failure to achieve or sustain favorable pregnancy progression is, not surprisingly, associated with high incidence of antenatal complications, including preeclampsia, a hypertensive disorder of pregnancy. While the pathogenesis of preeclampsia in women remains unknown, a role for androgens is emerging. The relationship between androgens and maternal cardiovascular and placental function deserves particular consideration because testosterone levels in the circulation of preeclamptic women are elevated approximately two- to three-fold and are positively correlated with vascular dysfunction. Preeclampsia is also associated with elevated placental androgen receptor (AR) gene expression. Studies in animal models mimicking the pattern and level of increase of adult female testosterone levels to those found in preeclamptic pregnancies, replicate key features of preeclampsia, including gestational hypertension, endothelial dysfunction, exaggerated vasoconstriction to angiotensin II, reduced spiral artery remodeling, placental hypoxia, decreased nutrient transport and fetal growth restriction. Taken together, these data strongly implicate AR-mediated testosterone action as an important pathway contributing to clinical manifestation of preeclampsia. This review critically addresses this hypothesis, taking into consideration both clinical and preclinical data.
Sheba M J MohanKumar, Priya Balasubramanian, Madhan Subramanian and P S MohanKumar
Estradiol (E2) is a female hormone that is produced largely by the ovaries, but also by the adrenal glands, fat and liver. It is present in the circulation of both males and females. Many studies in the literature have described how E2 is beneficial to the body in terms of preventing bone loss, affording protection in ischemia reperfusion injury, relieving symptoms of menopause, maintaining vaginal health and helping with ovarian failure or hypogonadism. Beneficial effects on the brain have been reported to include protection against memory loss, neuronal degeneration, changes in cognition, mood and behavior. However, the effects of E2 exposure on the neuroendocrine system have not been understood completely. This is because differences in doses, preparation and duration of exposure have produced variable results ranging from beneficial, to no change, or to detrimental. Studies in our lab over the last few years have shown that chronic exposures to low levels of E2 in young rats can produce specific effects on the neuroendocrine system. We have observed that these exposures can induce reproductive senescence, hypertension, anxiety-like behavior and cause degenerative changes in specific neuronal populations leading to hyperprolactinemia. The purpose of the review is to present evidence from the literature for these effects and to discuss the underlying molecular mechanisms.