Rajesh Kumar Jha

Profiling of E-cadherin, β-catenin and Ca2+ in embryo–uterine interactions at implantation

Establishment of early pregnancy is promoted by a complex network of signalling molecules that mediate cell‐to‐cell and cell‐to‐extracellular matrix communications between the receptive endometrium and the invasive trophectoderm. In this study, we have attempted to evaluate the expression profiles of cadherin and catenin during embryo implantation in the mouse. Western blotting studies along with immunocytochemical analysis revealed that E‐cadherin is expressed rather ubiquitously in the uterine epithelial cells, distinct enrichment is observed on the apical membrane in the endometrium of peri‐implantation uterus specifically at the implantation sites and not at the inter‐implanation sites. β‐Catenin also is upregulated and is specifically restricted to apical membrane of epithelial cells of implantation sites. Progesterone induced expression of E‐cadherin and 17β‐estradiol regulated the expression of catenin in implantation‐delayed uteri. Interestingly, estradiol imparted negative modulation on cadherin expression when co‐administered with progesterone. On the contrary, trophoblast exhibits a striking down regulation of cadherin, catenin and Ca2+ at peri implanting stage. These observations suggest that the trophoblasts exhibited an invasive phenotype while the endometrial epithelium displayed an adhesive phenotype during the window of implantation. Thus, embryo implantation presents an instance where two interacting surfaces showed mutually complementing interaction phenotypes.

Evaluation of poly(ADP-ribose) polymerase cleavage (cPARP) in ejaculated human sperm fractions after induction of apoptosis

OBJECTIVE To examine the presence of cleaved poly(ADP-ribose) polymerase(s) (cPARP) in ejaculated spermatozoa and determine cPARP levels following exposure to chemical or oxidative stress. DESIGN Prospective pilot study. SETTING Tertiary care academic hospital. PATIENT(S) Eight healthy men. INTERVENTION(S) Semen specimens were collected, prepared with double-density-gradient centrifugation, and divided into control, hydrogen peroxide (H(2)O(2)), H(2)O(2) + 3-aminobenzamide (3-ABA), staurosporine (STS), and STS + 3-ABA treated groups. MAIN OUTCOME MEASURE(S) Cleaved PARP and apoptosis markers by flow cytometry. RESULT(S) Cleaved PARP was detected in both neat and mature fractions. The cPARP levels were similar in both mature and immature spermatozoa. In combined mature and immature fractions, a higher percentage of late apoptotic sperm was seen in STS + 3-ABA versus STS. Higher levels of late apoptotic spermatozoa were seen in immature versus mature fractions within STS and STS + 3-ABA groups. Lower levels of cPARP were seen in immature versus mature fractions in H(2)O(2) and H(2)O(2) + 3-ABA treated groups. Cleaved PARP was related to activated caspase-3. CONCLUSION(S) Cleaved PARP is present in ejaculated human spermatozoa. Poly(ADP-ribose) polymerase inhibitors may play a different role in chemical versus oxidative stress-induced sperm damage.

Determination of poly (ADP-ribose) polymerase (PARP) homologues in human ejaculated sperm and its correlation with sperm maturation

OBJECTIVES To investigate the presence of Poly (ADP-ribose) polymerase (PARP) and evaluate its function in ejaculated spermatozoa. DESIGN Experimental study. SETTING Reproductive Research Center. PATIENT(S) Semen specimens from 18 healthy donors and 12 infertile males. INTERVENTION(S) Ejaculated spermatozoa were subjected to sperm fractionation with a double-layer density gradient, protein extraction, detection, immunoblotting, and mass spectrometry. Semen samples were exposed to PARP inducer staurosporine, or hydrogen peroxide with or without PARP inhibitor 3-aminobenzimide. Annexin V assay was examined for apoptosis by flow cytometry. RESULT(S) We detected approximately 75 kDa, approximately 63 kDa, and approximately 60 kDa PARP homologues on the immunoblot of mature and immature sperm fraction isolated from human ejaculate, which were identified as PARP-1 ( approximately 75 kDa), PARP-9 ( approximately 63 kDa), and PARP-2 ( approximately 60 kDa), respectively. Western blot analysis showed a positive correlation between the amount of PARP protein and sperm maturity. PARP proteins (75 kDa, 63 kDa, and 60 kDa) were evaluated after inducing apoptosis by hydrogen peroxide and staurosporine exposure. CONCLUSION(S) PARP-2 may have a role in prevention of oxygen species/oxidative stress and chemical (staurosporine)-induced sperm cell apoptosis. The presence of the PARP homologue, that is, PARP-1 ( approximately 75 kDa), suggests its role in preventing damage of mature sperm. Additional studies are needed to delineate the role of PARP-9 in sperm physiology. The results from our study indicate an active role for PARPs in sperm cell physiology in preventing apoptosis.

Transforming Growth Factor-Beta 1 (TGF-B1) Liberation from Its Latent Complex During Embryo Implantation and Its Regulation by Estradiol in Mouse

ABSTRACT Transforming growth factor-beta (TGF-B) plays an important role in embryo implantation; however, TGF-B requires liberation from its inactive latent forms (i.e., large latent TGF-B complex [LLC] and small latent TGF-B complex [SLC]) to its biologically active (i.e., monomer or dimer) forms in order to act on its receptors (TGF-BRs), which in turn activate SMAD2/3. Activation of TGF-B1 from its latent complexes in the uterus is not yet deciphered. We investigated uterine latent TGF-B1 complex and its biologically active form during implantation, decidualization, and delayed implantation. Our study, utilizing nonreducing SDS-PAGE followed by Western blotting and immunoblotting with TGF-B1, LTBP1, and latency-associated peptide, showed the presence of LLC and SLC in the uterine extracellular matrix and plasma membranous protein fraction during stages of the implantation period. A biologically active form of TGF-B1 (∼17-kDa monomer) was highly elevated in the uterine plasma membranous compartment at the peri-implantation stage (implantation and nonimplantation sites). Administration of hydroxychloroquine (an inhibitor of pro-TGF-B processing) at the preimplantation stage was able to block the liberation of biologically active TGF-B1 from its latent complex at the postimplantation stage; as a consequence, the number of implantation sites was reduced at Day 5 (1000 h), as was the number of fetuses at Day 13. The inhibition of TGF-B1 showed reduced levels of phosphorylated SMAD3. Further, the delayed-implantation mouse model showed progesterone and estradiol coordination to release the active TGF-B1 form from its latent complex in the receptive endometrium. This study demonstrates the importance of liberation of biologically active TGF-B1 during the implantation period and its regulation by estradiol.

STAT3 and MCL-1 associate to cause a mesenchymal epithelial transition

ABSTRACT Embryo implantation is effected by a myriad of signaling cascades acting on the embryo–endometrium axis. Here we show, by using MALDI TOF analysis, far-western analysis and colocalization and co-transfection studies, that STAT3 and MCL-1 are interacting partners during embryo implantation. We show in vitro that the interaction between the two endogenous proteins is strongly regulated by estrogen and progesterone. Implantation, pregnancy and embryogenesis are distinct from any other process in the body, with extensive, but controlled, proliferation, cell migration, apoptosis, cell invasion and differentiation. Cellular plasticity is vital during the early stages of development for morphogenesis and organ homeostasis, effecting the epithelial to mesenchymal transition (EMT) and, the reverse process, mesenchymal to epithelial transition (MET). STAT3 functionally associates with MCL-1 in the mammalian breast cancer cell line MCF7 that overexpresses STAT3 and MCL-1, which leads to an increased rate of apoptosis and decreased cellular invasion, disrupting the EMT. Association of MCL-1 with STAT3 modulates the normal, anti-apoptotic, activity of MCL-1, resulting in pro-apoptotic effects. Studying the impact of the association of STAT3 with MCL-1 on MET could lead to an enhanced understanding of pregnancy and infertility, and also metastatic tumors.

Integrin Beta 8 (ITGB8) Regulates Embryo Implantation Potentially via Controlling the Activity of TGF-B1 in Mice

ABSTRACT Integrins (ITGs) are mediators of cell-cell and cell-matrix interactions, which are also associated with embryo implantation processes by controlling the interaction of blastocyst with endometrium. During early pregnancy, ITGbeta8 (ITGB8) has been shown to interact with latent transforming growth factor (TGF) beta 1 (TGFB1) at the fetomaternal interface. However, the precise role of ITGB8 in the uterus and its association with embryo implantation has not been elucidated. Therefore, we attempted to ascertain the role of ITGB8 during the window of embryo implantation process by inhibiting its function or protein expression. Uterine plasma membrane-anchored ITGB8 was augmented at peri-implantation and postimplantation stages. A similar pattern of mRNA expression was also found during the embryo implantation period. An immunolocalization study revealed the presence of ITGB8 on luminal epithelial cells along with mild expression on the stromal cells throughout the implantation period studied; however, an intense fluorescence was noted only during the peri- and postimplantation stages. Bioneutralization and mRNA silencing of the uterine Itgb8 at preimplantation stage reduced the rate/frequency of embryo implantation and subsequent pregnancy, suggesting its indispensable role during the embryo implantation period. ITGB8 can also regulate the liberation of active TGFB1 from its latent complex, which, in turn, acts on SMAD2/3 phosphorylation (activation) in the uterus during embryo implantation. This indicates involvement of ITGB8 in the embryo implantation process through regulation of activation of TGFB1.

Association of sperm morphology and the sperm deformity index (SDI) with poly (ADP-RIBOSE) polymerase (PARP) cleavage inhibition

Apoptosis was induced in immature and mature sperm in the presence or absence of poly (ADP-ribose) polymerase (PARP) inhibitor. The association of cleaved (cPARP) with sperm morphology was examined using sperm deformity index (SDI) score. The SDI scores are associated with PARP cleavage as an early marker of apoptosis.

Integrin beta8 (ITGB8) activates VAV-RAC1 signaling via FAK in the acquisition of endometrial epithelial cell receptivity for blastocyst implantation

Integrin beta8 (ITGB8) is involved in the endometrial receptivity. The blastocyst first interacts with the luminal endometrial epithelial cells during its implantation; therefore, we have investigated the signaling of ITGB8 via FAK and VAV-RAC1 in the endometrial epithelial cells. Integrin beta8 was found elevated in epithelial cells at late-pre-receptive (day4, 1600 h) and receptive (day5, 0500 h) stages of endometrial receptivity period in the mouse. Integrins downstream molecule FAK has demonstrated an increased expression and phosphorylation (Y397) in the endometrium as well as in the isolated endometrial epithelial cells during receptive and post-receptive stages. Integrin beta8 can functionally interact with FAK, VAV and RAC1 as the levels of phosphorylated-FAK, and VAV along with the RAC-GTP form was reduced after ITGB8 knockdown in the endometrial epithelial cells and uterus. Further, VAV and RAC1 were seen poorly active in the absence of FAK activity, suggesting a crosstalk of ITGB8 and FAK for VAV and RAC1 activation in the endometrial epithelial cells. Silencing of ITGB8 expression and inhibition of FAK activity in the Ishikawa cells rendered poor attachment of JAr spheroids. In conclusion, ITGB8 activates VAV-RAC1 signaling axis via FAK to facilitate the endometrial epithelial cell receptivity for the attachment of blastocyst.

Endoglin (CD105) coordinates the process of endometrial receptivity for embryo implantation

Endoglin is a TGF-β receptor that is expressed in uterine endothelial and stromal cells in addition to trophoblast expression. However, the functional importance of endoglin in the embryo implantation process is not clear. We observed endoglin expression in the endometrium throughout the stages of its receptivity; however, its expression was enhanced during the receptive stage. Endoglin expression was predominant in epithelial cells of the lumen and glands, but showed a milder expression in stromal cells. Endoglin expression was initially observed in the primary decidual zone and later extended to the secondary decidua zone. Knockdown of endoglin via siRNA reduced the implantation sites along with the blastocyst numbers. Mouse blastocyst with endoglin-silenced endometrial epithelial cells (human and mouse origin) showed poor trophoblast outgrowth, which suggests an essential role for endoglin during endometrial receptivity. In conclusion, our findings reveal the association of endoglin with endometrial receptivity, which is important for embryo attachment.

CD44 targeting hyaluronic acid coated lapatinib nanocrystals foster the efficacy against triple-negative breast cancer

Lapatinib (LPT) is an orally administered drug for the treatment of metastatic breast cancer. For expanding its therapeutic horizon, we have prepared its nanocrystals (LPT-NCs) that were subsequently coated with hyaluronic acid (HA) to produce LPT-HA-NCs. The detailed in-vitro and in-vivo investigation of LPT-HA-NCs showed the superior anticancer activity due to active targeting to CD44 receptors than the counterparts LPT-NCs and free LPT. In the triple negative 4T1 cells induced breast tumor bearing female Balb/C mice; LPT-HA-NCs treatment caused significant retardation of tumor growth and overall increase in animal survival probability because of their higher tumor localization, increased residence time. Our findings clearly suggest that HA coated LPT-NCs formulation enhances the activity of LPT against triple negative breast cancer. It exhibited magnificent therapeutic outcome at low dose thus presenting a strategy to reduce dose administrations and minimize dose related toxicity.