Molecular regulators of human blastocyst development and hatching: Their significance in implantation and pregnancy outcome.

In humans, blastocyst hatching and implantation events are two sequential, critically linked and rate-limiting events for a prospective pregnancy. These events are regulated by embryo-endometrium derived molecular factors which include hormones, growth factors, cytokines, immune-modulators, cell adhesion molecules and proteases. Due to poor viability of blastocysts, they fail to hatch and implant, leading to a low 'Live Birth Rates', majorly contributing to infertility. Here, embryo-derived biomarkers analysis plays a key role to assess potential biological viability of blastocysts which are capable of implantation and prospective pregnancy. Thus far, embryo-derived biomarkers examined are mostly immune-modulators which are thought to be associated with blastocyst development-implantation and progression of pregnancy, leading to live births. There is an urgent need to develop a quantitative and a reliable non-invasive approach aiding embryo selection for elective single embryo transfer and to minimize recurrent pregnancy loss and multiple pregnancies. In this article, we provide a comprehensive review on our current knowledge and understanding of potential embryo-derived molecular regulators, that is, biomarkers, of development of human blastocysts, their hatching and implantation. We discuss their potential implications in the assessment of blastocyst implantation potential and pregnancy outcome in terms of live births in humans.

Soluble human leukocyte antigen-G is a potential embryo viability biomarker and a positive predictor of live-births in humans.

PROBLEM: Human infertility affects 15-20% of reproductive-age couples and it is mitigated by assisted reproductive technology (ART) approaches. Poor biological viability of embryos contributes to implantation failure and live birth rate (LBR). This study is aimed to examine whether or not embryo-secreted soluble human leukocyte antigen-G (sHLA-G) is (i) associated with developing embryos and (ii) able to predict successful pregnancy outcome. METHOD OF STUDY: A retrospective, multicentric study using 539 human embryo spent medium samples (E-SMs), analysed for sHLA-G levels by ELISA. Correlation analysis was performed on sHLA-G levels with developing embryonic stages, their quality scores and pregnancy outcome in terms of LBR. RESULTS: Of 539 E-SMs analysed, 445 had detectable sHLA-G (83%) with levels varying within and across clinics and, between stages of embryonic development. Levels of sHLA-G (ng/mL) were significantly (P < .05) different in E-SMs of cleavage-stage embryos versus blastocysts. There was an insignificant correlation between the sHLA-G levels and morphology scores of embryos. But, sHLA-G levels showed a positive correlation with grades of blastocysts and importantly, its levels were significantly (P < .05) higher in live-birth vis-a-vis no-birth cases. Also, levels were higher in live-births out of blastocysts-ETs versus cleavage-stage-embryo transfers. Altered levels were observed with embryos, which resulted in miscarriages. Overall, a significant (P < .0001) association of sHLA-G with live births was observed. CONCLUSION: Embryo-derived sHLA-G can be a valuable embryo viability, independent, biomarker, which can predict live-birth outcome and it could be useful as an adjunct to existing criteria for elective single embryo transfer.

Adenosine deaminase modulates metabolic remodeling and orchestrates joint destruction in rheumatoid arthritis.

Rheumatoid Arthritis (RA) is a chronic autoimmune disease associated with inflammation and joint remodeling. Adenosine deaminase (ADA), a risk factor in RA, degrades adenosine, an anti-inflammatory molecule, resulting in an inflammatory bias. We present an integrative analysis of clinical data, cytokines, serum metabolomics in RA patients and mechanistic studies on ADA-mediated effects on in vitro cell culture models. ADA activity differentiated patients into low and high ADA sets. The levels of the cytokines TNFα, IFNγ, IL-10, TGFß and sRANKL were elevated in RA and more pronounced in high ADA sets. Serum metabolomic analysis shows altered metabolic pathways in RA which were distinct between low and high ADA sets. Comparative analysis with previous studies shows similar pathways are modulated by DMARDs and biologics. Random forest analysis distinguished RA from control by methyl-histidine and hydroxyisocaproic acid, while hexose-phosphate and fructose-6-phosphate distinguished high ADA from low ADA. The deregulated metabolic pathways of High ADA datasets significantly overlapped with high ADA expressing PBMCs GEO transcriptomics dataset. ADA induced the death of chondrocytes, synoviocyte proliferation, both inflammation in macrophages and their differentiation into osteoclasts and impaired differentiation of mesenchymal stem cells to osteoblasts and mineralization. PBMCs expressing elevated ADA had increased expression of cytokines and P2 receptors compared to synovial macrophages which has low expression of ADA. Our data demonstrates increased cytokine levels and distinct metabolic signatures of RA based on the ADA activity, suggests an important role for ADA in the pathophysiology of RA joints and as a potential marker and therapeutic target in RA patients.

Expression and function of interleukin-1ß is required for hamster blastocyst hatching: Involvement of hatching-associated cathepsin proteases.

In mammals, the phenomenon of blastocyst hatching is an essential prerequisite for successful implantation. Blastocyst hatching is regulated by various molecules. Of them, cytokines, expressed by preimplantation embryos, are thought to be functionally important in blastocyst development and hatching, but their mechanistic roles are not clearly understood. Here, we examined the involvement of two cytokines, namely, interleukin-1ß (IL-1ß) and its natural antagonist, IL-1ra, in blastocyst hatching in the golden hamster. Blastocysts expressed both cytokines and their receptor, IL-1rt1. Supplementation of IL-1ß to cultured eight-cell embryos improved blastocyst hatching (84.1% ± 4.2% vs. 66.6% ± 6.8%; treated vs. control). This improvement was diminished by IL-1ra treatment (23.6% ± 12.9% vs. 76.4% ± 12.9%; treated vs. control). Interestingly, IL-1ß-treated embryos showed increased messenger RNA expression of zonalytic proteases, that is, cathepsin-L and -B by 1.9 ± 0.5- and 3.5 ± 0.1-folds, respectively. This was accompanied by their increased enzyme activities; cathepsin-L by 2.8 ± 0.7 fold and -B by 2.3 ± 0.7-fold. Strikingly, proteases and IL-1ß were intensely colocalized to trophectodermal projections of hatching blastocysts. This is the first report to show the involvement of embryonic IL-1ß in regulating hatching-associated proteases required for blastocyst hatching.

Expression of IL-1ß and implantation serine proteases is required for mouse blastocyst hatching.

Mammalian blastocyst hatching is a critically indispensable process for successful implantation. One of the major challenges in IVF clinics is to achieve superior embryonic development with intrinsically potent hatching-competent blastocyst. However, the molecular regulation of hatching phenomenon is poorly understood. In this study, we examined the expression and function of one of the cytokines, IL-1ß during blastocyst hatching in the mouse. In particular, the expression of IL-1ß (Interleukin-1ß), IL-1ra (Interleukin-1 receptor antagonist) and their functional receptor IL-1rt1 (Interleukin 1 receptor type-1) in morulae, zona intact- and hatched-blastocysts was studied. Supplementation of IL-1ß to cultured embryos accelerated blastocyst development with improved hatching (treated: 89.6 ± 3.6% vs untreated: 65.4 ± 4.1%). When embryos were treated with IL-1ra, blastocyst hatching was decreased (treated: 28.8 ± 3.1% vs untreated: 67.5 ± 3.8%). Moreover, IL-1ß and IL-1ra influenced the expression of hatching enzymes viz., implantation serine proteases (ISP1 and ISP2). While IL-1ß increased the embryonic mRNA expression of ISPs (Isp1: 2-4; Isp2: 9- to 11-fold), IL-1ra decreased expression. The protein localization studies revealed increased nuclear presence predominantly of ISP 2 in IL-1ß-treated blastocysts. This is the first report to show the functional significance of embryonic IL-1ß in regulating hatching-associated proteases, particularly ISP2. These findings have implications in our understanding of molecular regulation of blastocyst hatching and implantation failure in other species including humans.

In situ differentiation of human-induced pluripotent stem cells into functional cardiomyocytes on a coaxial PCL-gelatin nanofibrous scaffold.

Human-induced pluripotent stem cells (hiPSCs) derived cardiomyocytes (hiPSC-CMs) have been explored for cardiac regeneration and repair as well as for the development of in vitro 3D cardiac tissue models. Existing protocols for cardiac differentiation of hiPSCs utilize a 2D culture system. However, the efficiency of hiPSC differentiation to cardiomyocytes in 3D culture systems has not been extensively explored. In the present study, we investigated the efficiency of cardiac differentiation of hiPSCs to functional cardiomyocytes on 3D nanofibrous scaffolds. Coaxial polycaprolactone (PCL)-gelatin fibrous scaffolds were fabricated by electrospinning and characterized using scanning electron microscopy (SEM) and fourier transform infrared (FTIR) spectroscopy. hiPSCs were cultured and differentiated into functional cardiomyocytes on the nanofibrous scaffold and compared with 2D cultures. To assess the relative efficiencies of both the systems, SEM, immunofluorescence staining and gene expression analyses were performed. Contractions of differentiated cardiomyocytes were observed in 2D cultures after 2 weeks and in 3D cultures after 4 weeks. SEM analysis showed no significant differences in the morphology of cells differentiated on 2D versus 3D cultures. However, gene expression data showed significantly increased expression of cardiac progenitor genes (ISL-1, SIRPA) in 3D cultures and cardiomyocytes markers (TNNT, MHC6) in 2D cultures. In contrast, immunofluorescence staining showed no substantial differences in the expression of NKX-2.5 and α-sarcomeric actinin. Furthermore, uniform migration and distribution of the in situ differentiated cardiomyocytes was observed in the 3D fibrous scaffold. Overall, our study demonstrates that coaxial PCL-gelatin nanofibrous scaffolds can be used as a 3D culture platform for efficient differentiation of hiPSCs to functional cardiomyocytes.

Systems analysis of avascular necrosis of femoral head using integrative data analysis and literature mining delineates pathways associated with disease.

Avascular necrosis of femoral head (AVNFH) is a debilitating disease, which affects the middle aged population. Though the disease is managed using bisphosphonate, it eventually leads to total hip replacement due to collapse of femoral head. Studies regarding the association of single nucleotide polymorphisms with AVNFH, transcriptomics, proteomics, metabolomics, biophysical, ultrastructural and histopathology have been carried out. Functional validation of SNPs was carried out using literature. An integrated systems analysis using the available datasets might help to gain further insights into the disease process. We have carried out an analysis of transcriptomic data from GEO-database, SNPs associated with AVNFH, proteomic and metabolomic data collected from literature. Based on deficiency of vitamins in AVNFH, an enzyme-cofactor network was generated. The datasets are analyzed using ClueGO and the genes are binned into pathways. Metabolomic datasets are analyzed using MetaboAnalyst. Centrality analysis using CytoNCA on the data sets showed cystathionine beta synthase and methylmalonyl-CoA-mutase to be common to 3 out of 4 datasets. Further, the genes common to at least two data sets were analyzed using DisGeNET, which showed their involvement with various diseases, most of which were risk factors associated with AVNFH. Our analysis shows elevated homocysteine, hypoxia, coagulation, Osteoclast differentiation and endochondral ossification as the major pathways associated with disease which correlated with histopathology, IHC, MRI, Micro-Raman spectroscopy etc. The analysis shows AVNFH to be a multi-systemic disease and provides molecular signatures that are characteristic to the disease process.

Research on early mammalian development in India.

Historically, research in India on early mammalian development had only begun, rather modestly, in the last century, unlike the USA and UK. In India, initial studies were on gonadal and reproductive tissue development and function and they were limited to anatomical and histological characterization. This was followed by research on fertility regulation and contraception. Since the 1960s, a major initiative took place regarding endocrine biochemistry and the use of antifertility agents in inhibiting gonadal function and early development. Post-independence, the Indian government´s funding support enabled universities and institutions to embark on various research disciplines in biology but with no particular emphasis on developmental biology per se. Subsequently, India made significant progress in the area of mammalian reproduction and development, but not specifically in the core aspects of developmental biology. Reasons for this could be due to the nation's compulsion to invest and embark on socio-economic and infrastructure development and on research involving family planning methods for reversible-affordable contraceptives to curtail population growth. With regard to the latter, biologists were involved in hormone-based contraception research. During this pursuit, insights were achieved into basic aspects of the development of gonads, gametes and embryos. Notwithstanding this, in the post-1980s through to the present time, Indian scientists have contributed to (i) the understanding of the cellular and molecular regulation of early development, (ii) developing genetically modified mouse models, (iii) using assisted reproductive technologies, generating mammalian progeny, including humans and (iv) deriving pluripotent stem cell lines for developmental studies. This article provides a perspective on the past and current status of early mammalian development research in India.

Successful Derivation of an Induced Pluripotent Stem Cell Line from a Genetically Nonpermissive Enhanced Green Fluorescent Protein-Transgenic FVB/N Mouse Strain.

The embryonic stem cell line derivation from nonpermissive mouse strains is a challenging and highly inefficient process. The cellular reprogramming strategy provides an alternative route for generating pluripotent stem cell (PSC) lines from such strains. In this study, we successfully derived an enhanced green fluorescent protein (EGFP)-transgenic "N9" induced pluripotent stem cell (iPS cell, iPSC) line from the FVB/N strain-derived mouse embryonic fibroblasts (MEFs). The exposure of MEFs to human OCT4, SOX2, KLF4, and c-MYC (OSKM) transgenes via lentiviral transduction resulted in complete reprogramming. The N9 iPS cell line demonstrated all the criteria of a typical mouse PSC line, including normal colony morphology and karyotype (40,XY), high replication and propagation efficiencies, expression of the pluripotency-associated genes, spontaneous differentiation to three germ lineage-derived cell types, and robust potential of chimeric blastocyst formation. Taken together, using human OSKM genes for transduction, we report, for the first time, the successful derivation of an EGFP-expressing iPS cell line from a genetically nonpermissive transgenic FVB/N mouse. This cell line could provide opportunities for designing protocols for efficient derivation of PSC lines from other nonpermissive strains and developing mouse models of human diseases.

Mouse embryonic stem cell-derived cardiomyocytes cease to beat following exposure to monochromatic light: association with increased ROS and loss of calcium transients.

We earlier established the mouse embryonic stem (ES) cell "GS-2" line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420-495), green (~510-575), and red (~600-700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N-acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1) mechanistic insights into light impact on cardiomyocyte contraction, 2) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3) transient cessation of hearts during coronary artery bypass grafting, and 4) functional preservation of hearts for transplantation.

Enabling Hamster Embryo Culture System: Development of Preimplantation Embryos.

Development of preimplantation embryos, from fertilization to hatched-blastocyst stage, has been a challenging task, regardless of the mammalian species being studied. While the mouse model has been versatile for studying in vitro development of early embryos, other rodent species are important to gain insights into comparative early embryogenesis. The golden hamster (Mesocricetus auratus) offers unique advantages to study cellular and molecular regulation of gamete maturation, fertilization and preimplantation development, including the phenomenon of blastocyst hatching. Achieving in vitro fertilization and first cleavage division is relatively easy; however, subsequent development past the two-/four-cell stage had been difficult in hamsters. Pioneering research, carried out over three decades has markedly enabled successful in vitro development of one-cell embryos to blastocysts. This article provides a comprehensive perspective (historical and current) on the embryo culture systems and details an optimized culture protocols to achieve normal and viable development of preimplantation embryos in the golden hamster.

Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium.

P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES+ neural progenitors (~ 46%), TUBB3+ immature neurons (~ 6%), MAP2+ mature neurons (~ 2%), and GFAP+ astrocytes (~ 50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds.

Assessment of ovarian follicular dynamics and folliculogenesis associated endocrine profiles following gonadotropin stimulation in the bonnet monkey.

We evaluated ovarian follicular dynamics in bonnet monkeys by employing trans-abdominal ultrasonography. Following the administration of human follicle stimulating hormone (hFSH) and/or human menopausal gonadotropin (hMG), multiple follicular development was assessed and their numbers, size and growth profiles were monitored. The ultrasonograms showed that the follicular antrum appeared distinctly anechoic with well-defined hyperechoic borders. Depending on the type, quantity (12.5-25IU), and duration (6-9days) of hormones administered, the number of developing follicles was 2-12 per ovary with their lowest diameter being 2mm. With continued hormone administration, their numbers and diameters increased; which were more pronounced in animals administered with hFSH than with hMG, with follicles of 6-8mm. Interestingly, human chorionic gonadotropin (hCG) injection (2000-3000IU), when follicles acquiring >6-8mm sizes, induced the maximum expansion of antral follicles with sizes reaching up to 14mm. On days 3-5 post-hCG, the ultrasonograms showed loosely demarcated multiple hypoechoic structures and well-demarcated hyperechoic structures with anechoic/hypoechoic cores corresponding to unruptured luteinized follicles and corpora lutea, respectively. On day 4 post-hCG, there was a substantial reduction in the number of antral follicles. In stimulated animals, follicular growth, ovulation, and formation of luteal structures were accompanied by corresponding physiological changes in the serum estradiol and progesterone profiles. These findings, for the first time, showed that ultrasonographic imaging approach is useful for precise monitoring of temporal changes in follicular developmental dynamics and to time the hCG induced ovulation in the bonnet monkey.

Assessment of season-dependent body condition scores in relation to faecal glucocorticoid metabolites in free-ranging Asian elephants.

We studied seasonal and annual changes in visual body condition scores (BCSs), and assessed how these scores were related to levels of faecal glucocorticoid metabolites (fGCMs) in free-ranging Asian elephants (Elephas maximus) in the seasonally dry tropical forests of the Mysore and Nilgiri Elephant Reserves in southern India. We assessed the animals' BCS visually on a scale of 1 to 5; where 1 represents a very thin and 5 represents a very fat elephant. To understand the influence of seasonality on BCS, we sampled the population during dry (n = 398) and wet seasons (n = 255) of 2013 and 2015 while, for annual changes in BCS, we sampled nine free-ranging adult females from different family groups that had been repeatedly sighted over seven years. To evaluate the influence of body condition on fGCM, 307 faecal samples were collected from 261 different elephants and were analysed. As a parameter of adrenocortical activity, and thus stress, fGCM was measured (µg/g) in the ethanol-extracted samples using a group-specific 11-oxoaetiocholanolone EIA (antibody raised against 11-oxoaetiocholanolone-17-CMO:BSA and biotinylated-11-oxoaetiocholanolone as a label). Effect of age and season on BCS in relation to fGCM was also studied. A seasonal shift in BCS was observed as expected, i.e. individuals with low BCS were more frequent during the dry season when compared with the wet season. Concentrations of fGCM were highest in individuals with lowest BCS (BCS 1) and then significantly declined till BCS 3. fGCM levels were almost comparable for BCS 3, 4 and 5. This pattern was more conspicuous in female than in male elephants. Season-dependent BCS, hence, reflect the stress status as measured by fGCM, especially in female Asian elephants. This could be used as an important non-invasive approach to monitor the physiological health of free-ranging elephant populations.

Efficient neural differentiation of mouse pluripotent stem cells in a serum-free medium and development of a novel strategy for enrichment of neural cells.

Pluripotent stem cells (PSCs) offer an excellent model to study neural development and function. Although various protocols have been developed to direct the differentiation of PSCs into desired neural cell types, many of them suffer from limitations including low efficiency, long duration of culture, and the use of expensive, labile, and undefined growth supplements. In this study, we achieved efficient differentiation of mouse PSCs to neural lineage, in the absence of exogenous molecules, by employing a serum-free culture medium containing knockout serum replacement (KSR). Embryoid bodies (EBs) cultured in this medium predominantly produced neural cells which included neural progenitors (15-18%), immature neurons (8-24%), mature neurons (10-26%), astrocytes (27-61%), and oligodendrocytes (∼1%). Different neuronal subtypes including glutamatergic, GABAergic, cholinergic, serotonergic, and dopaminergic neurons were generated. Importantly, neurons generated in the KSR medium were electrically active. Further, the EB scooping strategy, involving the removal of the EB core region from the peripheral EB outgrowth, resulted in the enrichment of PSC-derived neural cells. Taken together, this study provides the evidence that the KSR medium is ideal for the rapid and efficient generation of neural cells, including functional neurons, from PSCs without the requirement of any other additional molecule.

Ascorbic acid-mediated enhanced cardiomyocyte differentiation of mouse ES-cells involves interplay of DNA methylation and multiple-signals.

Embryonic stem cells (ES-cells) provide a good model system to study lineage-specific differentiation. Though, the differentiation of ES-cells to cardiomyocytes is documented, a clear understanding of the molecular mechanism of differentiation and improved functional-differentiation efficiency are yet to be achieved. In this regard, ascorbic acid (Aa) is shown to be one of the effective cardiac inducers in ES-cells. But, its mechanism is poorly understood. We therefore, investigated the mechanism of Aa-mediated cardiomyocyte differentiation of ES-cells. Here, we describe the potential involvement of epigenetic (DNA methylation) as well as integrin- and Erk- signaling systems during cardiomyocyte differentiation. Transgenic GS-2 ES-cells and wild-type D3 ES-cells were differentiated to cardiomyocytes, in the presence or absence of Aa and with or without inhibitors of Erk-, collagen- and integrin- pathways. At specific time points, differentiated states of ES-cells were scored by gene expression analyses and the proportion of functional cTnI+ cardiomyocytes. DNA methylation changes of Isl-1, BMP-2, GATA-4 and α-MHC in cardiogenic cells, following stimulation with Aa, were analyzed by using methylation specific PCR (MSP). We observed that Aa, when applied in initial phase of ES-cell differentiation, consistently enhanced cardiac differentiation (99%) over that observed during spontaneous differentiation (70%). This was associated with enhanced expressions of cardiogenesis-associated genes. A two-fold increase in cTnI+ cells was observed, with appropriate myofibril arrangement. The observed effect of Aa was due to enhanced collagen and integrin signaling, coupled with a high p-ERK1/2 expression, downstream. Besides, the involvement of DNA methylation in regulating the expression of cardiac genes i.e., Isl-1 and α-MHC was also observed. Overall, this study, for the first time, demonstrates that Aa-mediated cardiac enhancement is brought about, mechanistically, through the interplay of epigenetic changes in DNA methylation of cardiac genes (Isl-1 and α-MHC) and integrin signaling system.

Cytokines and Blastocyst Hatching.

Blastocyst implantation into the uterine endometrium establishes early pregnancy. This event is regulated by blastocyst- and/or endometrium-derived molecular factors which include hormones, growth factors, cell adhesion molecules, cytokines and proteases. Their coordinated expression and function are critical for a viable pregnancy. A rate-limiting event that immediately precedes implantation is the hatching of blastocyst. Ironically, blastocyst hatching is tacitly linked to peri-implantation events, although it is a distinct developmental phenomenon. The exact molecular network regulating hatching is still unclear. A number of implantation-associated molecular factors are expressed in the pre-implanting blastocyst. Among others, cytokines, expressed by peri-implantation blastocysts, are thought to be important for hatching, making blastocysts implantation competent. Pro-inflammatory (IL-6, LIF, GM-CSF) and anti-inflammatory (IL-11, CSF-1) cytokines improve hatching rates; they modulate proteases (MMPs, tPAs, cathepsins and ISP1). However, functional involvement of cytokines and their specific mediation of hatching-associated proteases are unclear. There is a need to understand mechanistic roles of cytokines and proteases in blastocyst hatching. This review will assess the available knowledge on blastocyst-derived pro-inflammatory and anti-inflammatory cytokines and their role in potentially regulating blastocyst hatching. They have implications in our understanding of early embryonic loss and infertility in mammals, including humans.

Endocrine correlates of musth in free-ranging Asian elephants (Elephas maximus) determined by non-invasive faecal steroid hormone metabolite measurements.

The occurrence of musth, a period of elevated levels of androgens and heightened sexual activity, has been well documented for the male Asian elephant (Elephas maximus). However, the relationship between androgen-dependent musth and adrenocortical function in this species is unclear. The current study is the first assessment of testicular and adrenocortical function in free-ranging male Asian elephants by measuring levels of testosterone (androgen) and cortisol (glucocorticoid--a physiological indicator of stress) metabolites in faeces. During musth, males expectedly showed significant elevation in faecal testosterone metabolite levels. Interestingly, glucocorticoid metabolite concentrations remained unchanged between musth and non-musth periods. This observation is contrary to that observed with wild and captive African elephant bulls and captive Asian bull elephants. Our results show that musth may not necessarily represent a stressful condition in free-ranging male Asian elephants.

Expression and function of cyclooxygenase-2 is necessary for hamster blastocyst hatching.

Blastocyst hatching is critical for successful implantation leading to pregnancy. Its failure causes infertility. The phenomenon of blastocyst hatching in humans is poorly understood and the available information on this stems from studies of rodents such as mice and hamsters. We and others showed that hamster blastocyst hatching is characterized by firstly blastocyst deflation followed by a dissolution of the zona pellucida (zona) and accompanied by trophectodermal projections (TEPs). We also showed that embryo-derived cathepsins (Cat) proteases, specifically Cat-L, -B and -P act as zonalysins and are responsible for hatching. In this study, we show the expression and function of one of the potential regulators of embryogenesis, cyclooxygenase (COX)-2 during blastocyst development and hatching. The expression of COX-2 mRNA and protein was observed in 8-cell through hatched blastocyst stages and it was also localized to blastocyst's TEPs. Specific COX-2 inhibitors, NS-398 and CAY-10404, inhibited blastocyst hatching; percentages achieved were only 28.4 ± 5.3 and 32.3 ± 5.4%, respectively, compared with >90% with untreated embryos. Interestingly, inhibitor-treated blastocysts failed to deflate, normally observed during hatching. Supplementation of prostaglandins (PGs)-E2 or -I2 to cultured embryos reversed the inhibitors' effect on hatching and also the deflation behavior. Importantly, the levels of mRNA and protein of Cat-L, -B and -P showed a significant reduction in the inhibitor-treated embryos compared with untreated embryos, although its mechanism remains to be examined. These data provide the first evidence that COX-2 is critical for blastocyst hatching in the golden hamster.

Aza-induced cardiomyocyte differentiation of P19 EC-cells by epigenetic co-regulation and ERK signaling.

Stem cells in cell based therapy for cardiac injury is being potentially considered. However, genetic regulatory networks involved in cardiac differentiation are not clearly understood. Among stem cell differentiation models, mouse P19 embryonic carcinoma (EC) cells, are employed for studying (epi)genetic regulation of cardiomyocyte differentiation. Here, we comprehensively assessed cardiogenic differentiation potential of 5-azacytidine (Aza) on P19 EC-cells, associated gene expression profiles and the changes in DNA methylation, histone acetylation and activated-ERK signaling status during differentiation. Initial exposure of Aza to cultured EC-cells leads to an efficient (55%) differentiation to cardiomyocyte-rich embryoid bodies with a threefold (16.8%) increase in the cTnI+ cardiomyocytes. Expression levels of cardiac-specific gene markers i.e., Isl-1, BMP-2, GATA-4, and α-MHC were up-regulated following Aza induction, accompanied by differential changes in their methylation status particularly that of BMP-2 and α-MHC. Additionally, increases in the levels of acetylated-H3 and pERK were observed during Aza-induced cardiac differentiation. These studies demonstrate that Aza is a potent cardiac inducer when treated during the initial phase of differentiation of mouse P19 EC-cells and its effect is brought about epigenetically and co-ordinatedly by hypo-methylation and histone acetylation-mediated hyper-expression of cardiogenesis-associated genes and involving activation of ERK signaling.