Geetanjali Sachdeva

Progesterone receptors: various forms and functions in reproductive tissues.

The unequivocal role of progesterone in a variety of events like ovulation, mammary gland development, establishment and maintenance of pregnancy etc are well established. Also the data are accumulating on its role in male reproductive events. In vertebrates and humans, the biological activity of progesterone is mediated by two progesterone receptor proteins PR-A and PR-B, that arise from the same gene and are the members of nuclear receptor superfamily of transcriptional factors. Several studies have demonstrated that the blockage of progesterone receptor using antiprogestins impairs folliculogenesis, ovulation, implantation and pregnancy. Progesterone receptor (PR), have also been detected in human spermatozoa. However, unlike the conventional PR, sperm PR was localized on the membrane and showed distinct characteristics in terms of its size. There are data to demonstrate the inhibition of progesterone driven functions such as hyperactive motility, acrosome reaction on neutralization of sperm membrane PR with specific antibodies against PR. Further significant decrease in the % of PR positive spermatozoa was observed in infertile cases as compared to the fertile men. This indicated that PR can serve as the marker to define the fertilizing potential of the spermatozoa. Recently we have also shown that the PR is expressed in human testis. This reinforced that this PR protein is an inherent testicular protein and not a secretion of accessory reproductive organs. This review compiles the major observations on the forms of the progesterone receptor in various reproductive tissues.

Protein profiling of human endometrial tissues in the midsecretory and proliferative phases of the menstrual cycle

OBJECTIVE To identify the proteins displaying differential expression in midsecretory phase endometrium as compared with proliferative phase endometrium. DESIGN Prospective study with two groups of women in the midsecretory or proliferative phase. SETTING Clinical research outpatient department. PATIENT(S) Healthy, regularly cycling women of proven fertility. INTERVENTION(S) Collection of endometrial biopsy samples. MAIN OUTCOME MEASURE(S) Image analysis software was used to compare two-dimensional protein maps of midsecretory phase endometrial tissues (MSE) with maps of proliferative phase endometrial tissues (PROE) and midsecretory phase uterine fluids (MSU). Matrix-assisted laser desorption/ionization time of flight in tandem (MALDI-TOF-TOF) analysis was carried out to identify eight proteins that were differentially expressed between the two phases and also to identify the spots that shared similar coordinates in the two-dimensional maps of MSE and MSU. RESULT(S) Densitometry analysis and subsequent MALDI-TOF-TOF analysis revealed up-regulation of calreticulin, the beta chain of fibrinogen, adenylate kinase isoenzyme 5, and transferrin in the PROE and of annexin V, alpha1-antitrypsin, creatine kinase, and peroxidoxin 6 in MSE compared with the other phase. Superimposition of the two-dimensional maps of MSE on those of MSU revealed the presence of heat-shock protein 27, transferrin, and alpha1-antitrypsin precursor in both endometrial tissues and uterine secretions. CONCLUSION(S) Differentially expressed proteins identified in the present study could be of relevance in endowing the endometrium with receptivity.

Progesterone Receptor as an Indicator of Sperm Function

Abstract Expression of progesterone receptor (PR) localization on spermatozoa was determined in men with normal and abnormal spermiograms. Studies were also carried out to evaluate the potential of PR as a marker of sperm function. Progesterone receptor expression on spermatozoa from men with normozoospermia (n = 8), oligozoospermia (n = 7), asthenozoospermia (n = 8), oligoasthenozoospermia (n = 7), and teratozoospermia (n = 11) was analyzed using an immunocytochemical method with monoclonal antibodies against PR, and flow cytometry using a cell-impermeable fluorescein-tagged progesterone coupled to BSA complex (P-FITC-BSA). Both methods revealed significantly fewer (P < 0.05) PR-positive spermatozoa in men with oligozoospermia, asthenozoospermia, oligoasthenozoospermia, and teratozoospermia compared with men with normozoospermia, thereby suggesting that down-regulation of PR expression in spermatozoa may be one of the causes of male infertility. Spermatozoa from men with normozoospermia (n = 12), oligozoospermia (n = 12), asthenozoospermia (n = 12), oligoasthenozoospermia (n = 9), and teratozoospermia (n = 10) were exposed to low osmotic conditions in the hypoosmotic swelling (HOS) test and then analyzed for PR expression using P-FITC-BSA complex. A significantly higher percentage (P < 0.05) of spermatozoa with physiologically active plasma membrane (HOS+) lacked PR expression (HOS+PR−) in all categories of men with infertility, thereby suggesting that compared to the HOS test, PR expression is a better indicator of sperm function. Furthermore, PR expression in spermatozoa showed a strong (P < 0.05) positive correlation with their ability to undergo an in vitro acrosome reaction. This was observed in all study groups (i.e., normozoospermia, r = 0.8545; oligozoospermia, r = 0.8711; asthenozoospermia, r = 0.7645; oligoasthenozoospermia, r = 0.9003; and teratozoospermia, r = 0.8676). This suggests a potential role for PR in the events leading to the acrosome reaction in sperm.

Detection of Progesterone Receptor Transcript in Human Spermatozoa

Abstract The present study, to our knowledge, is the first to demonstrate presence of progesterone receptor (PR) transcript in human spermatozoa. The study shows the presence of low copy number PR mRNA in mature human spermatozoa. The PR transcript in spermatozoa was detected by reverse transcriptase-polymerase chain reaction using primers specific for the hormone binding domain and the DNA binding domain of the conventional uterine PR. Further, the cDNA sequence of the partial PR transcript from spermatozoa was found to be identical to the region spanning nucleotides 2694 to 3230 of the conventional PR full-length cDNA sequence. This study also indirectly suggests that the PR protein indeed is an intrinsic sperm protein and is not acquired through proteinaceous secretions of accessory reproductive organs.

Expression Profiles of Endometrial Leukemia Inhibitory Factor, Transforming Growth Factor β2 (TGFβ2), and TGFβ2 Receptor in Infertile Bonnet Monkeys1

Abstract The expression profiles of leukemia inhibitory factor (LIF), transforming growth factor β2 (TGFβ2), and transforming growth factor β2 receptor (TGFβ2R) were analyzed during the peri-implantation period in regularly menstruating, fertile bonnet monkeys and in animals in which endometrial nonreceptivity was induced by administering an antiprogestin, onapristone. Based on our previous experiences, a dose of 2.5 or 5 mg of onapristone was administered s.c. every third day during the menstrual cycle, because these dosages impair endometrial development without upsetting the normal gonadal endocrine profiles. Endometrial biopsy specimens were collected during the proliferative phase (estradiol levels about 200 pg/ml, n = 5) and peri-implantation period (Day 8 after midcycle peak in estradiol levels, n = 5) from normal ovulatory animals and during the peri-implantation period from onapristone-treated animals (n = 10). The biopsy specimens were processed to determine the expression patterns of LIF, TGFβ2, and TGFβ2R by immunohistochemical and reverse transcription-polymerase chain reaction (RT-PCR) methods. Levels of both protein and mRNA for LIF, TGFβ2, and TGFβ2R (analyzed by immunohistochemistry and RT-PCR, respectively) were greater in the endometrial samples collected during the peri-implantation period compared to samples collected during the proliferative phase in control animals. Treatment with either of the two doses (2.5 or 5 mg) of onapristone caused a significant (P < 0.05) down-regulation in the expression of LIF in the peri-implantation endometria. The endometrial expressions of TGFβ2 and TGFβ2R mRNAs were reduced significantly in animals treated with 5 mg of onapristone, but not in those treated with the lower dose. However, immunoreactive TGFβ2 and TGFβ2R proteins were significantly (P < 0.05) down-regulated in the endometrial samples from both the 2.5- and 5-mg-treated groups. The alterations observed in the expression patterns of LIF, TGFβ2, and TGFβ2R were specific, because the expression levels of epidermal growth factor receptor remained unaffected in the endometria from the treated groups. The present study demonstrates derangement in the expression profiles of LIF, TGFβ2, and TGFβ2R during the peri-implantation period in infertile bonnet monkeys. It may be hypothesized that TGFβ2 function is one of the early steps in the regulation of the progesterone-driven cascade of events leading to endometrial receptivity, and that any aberration in this step may adversely affect the subsequent molecular events (i.e., expression of LIF). These data also suggest that potential aberrations in the functional network of locally produced cytokines and growth factors even may occur in an endometrium exposed to the optimal peripheral hormonal levels.

Endometrial Receptivity: A Revisit to Functional Genomics Studies on Human Endometrium and Creation of HGEx-ERdb

Background Endometrium acquires structural and functional competence for embryo implantation only during the receptive phase of menstrual cycle in fertile women. Sizeable data are available to indicate that this ability is acquired by modulation in the expression of several genes/gene products. However, there exists little consensus on the identity, number of expressed/not-detected genes and their pattern of expression (up or down regulation). Methods Literature search was carried out to retrieve the data on endometrial expression of genes/proteins in various conditions. Data were compiled to generate a comprehensive database, Human Gene Expression Endometrial Receptivity database (HGEx-ERdb). The database was used to identify the Receptivity Associated Genes (RAGs) which display the similar pattern of expression across different investigations. Transcript levels of select RAGs encoding cell adhesion proteins were compared between two human endometrial epithelial cell lines; RL95-2 and HEC-1-A by quantitative real time polymerase chain reaction (q-RT-PCR). Further select RAGs were investigated for their expression in pre-receptive (n = 4) and receptive phase (n = 4) human endometrial tissues by immunohistochemical studies. JAr spheroid attachment assays were carried out to assess the functional significance of two RAGs. Results HGEx-ERdb (http://resource.ibab.ac.in/HGEx-ERdb/) helped identification of 179 RAGs, of which 151 genes were consistently expressed and upregulated and 28 consistently not-detected and downregulated in receptive phase as compared to pre-receptive phase. q-RT-PCR confirmed significantly higher (p<0.005) expression of Thrombospondin1 (THBS1), CD36 and Mucin 16 transcripts, in RL95-2 as compared to HEC-1-A. Further, the pretreatment with antibodies against CD36 and COMP led to a reduction in the percentage of JAr spheroids attached to RL95-2. Immunohistochemical studies demonstrated significantly higher (p<0.05) expression of endometrial THBS1, Cartilage Oligomeric Matrix Protein (COMP) and CD36 in the receptive phase as compared to pre-receptive phase human endometrial tissues. Conclusion HGEx-ERdb is a catalogue of 19,285 genes, reported for their expression in human endometrium. Further 179 genes were identified as the RAGs. Expression analysis of some RAGs validated the utility of approach employed in creation of HGEx-ERdb. Studies aimed towards defining the specific functions of RAGs and their potential networks may yield relevant information about the major ‘nodes’ which regulate endometrial receptivity.

Proteomics in reproductive biology: beacon for unraveling the molecular complexities.

Proteomics, an interface of rapidly evolving advances in physics and biology, is rapidly developing and expanding its potential applications to molecular and cellular biology. Application of proteomics tools has contributed towards identification of relevant protein biomarkers that can potentially change the strategies for early diagnosis and treatment of several diseases. The emergence of powerful mass spectrometry-based proteomics technique has added a new dimension to the field of medical research in liver, heart diseases and certain forms of cancer. Most proteomics tools are also being used to study physiological and pathological events related to reproductive biology. There have been attempts to generate the proteomes of testes, sperm, seminal fluid, epididymis, oocyte, and endometrium from reproductive disease patients. Here, we have reviewed proteomics based investigations in humans over the last decade, which focus on delineating the mechanism underlying various reproductive events such as spermatogenesis, oogenesis, endometriosis, polycystic ovary syndrome, embryo development. The challenge is to harness new technologies like 2-DE, DIGE, MALDI-MS, SELDI-MS, MUDPIT, LC-MS etc., to a greater extent to develop widely applicable clinical tools in understanding molecular aspects of reproduction both in health and disease.

Long circulating PEGylated PLGA nanoparticles of cytarabine for targeting leukemia

The present investigation was aimed at developing PEGylated PLGA nanoparticles of cytarabine. PLGA Nanoparticles were prepared by modified nanoprecipitation method, optimized for mean particle size (152 ± 6 nm) and entrapment efficiency (41.1 ± 0.8%) by a 32 factorial design. The PEGylated PLGA nanoparticles of cytarabine had a zeta potential of −7.5 ± 1.3 mV and sustained the release of cytarabine for 48 h by Fickian diffusion. The IC50 values for L1210 cells were 6.5, 5.3, and 2.2 µM for cytarabine, cytarabine loaded PLGA nanoparticles and cytarabine loaded PLGA-mPEG nanoparticles respectively. Confocal microscopy and flow cytometry showed that the nanoparticles were internalized by the L1210 cells and not simply bound to their surface. Biodistribution studies showed that the PEGylated nanoparticles of cytarabine were present in significantly higher concentrations in blood circulation as well as in brain and bones and avoided RES uptake as compared to the free drug.

Endometrial contraception: modulation of molecular determinants of uterine receptivity

Modulation of endometrial receptivity is a promising approach for fertility regulation since it allows a contraceptive to act specifically at the endometrium. This was corroborated by our previous observations that treatment with low doses of a pure progesterone antagonist (PA, antiprogestin), onapristone (ZK 98299), in bonnet monkeys inhibited fertility by selectively retarding endometrial development, without affecting the hypophyseal-hypothalamic function. In the present study, further investigations, undertaken to analyze the molecular repertoire of a nonreceptive primate endometrium, determined expression of: steroid hormone receptors, i.e. progesterone receptor (PR) and estrogen receptor (ER); cytokines, i.e. leukemia inhibitory factor (LIF): transforming growth factor beta (TGFbeta) and its receptor (TGFbetaR); and cell adhesion molecules, i.e. integrins (alpha(v)beta(3), alpha(1)beta(1)). These studies were conducted during the different phases of the normal menstrual cycle and following treatment with different doses of onapristone (2.5 mg, 5 mg, or 10 mg every third day for one cycle) in bonnet monkeys. The molecules were analysed collectively to explore the possibility of a correlation between expression of these markers and endometrial receptivity and to investigate whether there exists a regulatory link between expression of these molecules under in vivo conditions. Three types of expression patterns of endometrial factors were observed during the peri-implantation period following onapristone treatment: 1) LIF, alpha(v)beta(3), and alpha(1)beta(1) showed significant (P < 0.02) down regulation in glandular epithelium of endometria in animals treated with all three doses of onapristone as compared to the control group. This was indicative of their critical role in the progesterone-driven cascade leading to implantation. 2) PR, TGFbeta, and TGFbetaR remained unaffected in the endometria from 2.5 mg treated animals and showed down regulation in animals treated with 5 and 10 mg onapristone as compared to the control group, thereby suggesting that the expression of these markers may not truely reflect endometrial receptivity per se. However, their facilitatory role in preparing the endometrium for implantation can not be ruled out since continued perturbation in the expression of these molecules may affect endometrial growth, remodelling, and differentiation, which in turn may render the endometrium nonreceptive; 3) ER remained unaltered in endometria of animals rendered infertile with 2.5, 5, and 10 mg onapristone. This observation indirectly suggests that onapristone-induced endometrial changes are mediated via some specific mechanisms. The present study clearly demonstrates that endometrial non-receptivity induced at low doses of onapristone is associated with changes in the expression pattern of specific molecular markers. However, no direct correlation was observed between in vivo expression of TGFbeta, LIF, and integrins, thereby lending support to the concept that there exists redundancy or multiple pathways which regulate implantation events.

Spot-ting differences between the ectopic and eutopic endometrium of endometriosis patients

OBJECTIVE To examine proteins aberrantly expressed in the ectopic endometrium compared with eutopic endometrium from the same patient. DESIGN Experimental study. SETTING Research institute and an obstetrics and gynecology clinic (National Institute for Research in Reproductive Health and Sanjeevani Diagnostic Center and Maternity Home, India). PATIENT(S) Twenty participants with (n=11) and without (n=9) endometriosis. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Protein identification by two-dimensional (2D) electrophoresis and mass spectrometry as well as validation of the identified proteins by studying protein expression via Western blot and protein localization via immunohistochemical analysis. RESULT(S) Computer-assisted image analysis detected the presence of 53 protein spots in ectopic 2D gels that were conspicuous by their absence in the 2D maps of eutopic and control endometrium, i.e., these spots were detected only in ectopic gels. Eleven spots were identified by mass spectrometry. The expression of four of these proteins-haptoglobin, Rho-GDIα, SM-22α, and Rab37-have been validated by immunohistochemical and Western blotting analysis. CONCLUSION(S) This study assumes significance, as there are no reports on the comparison of the global protein profiles of paired eutopic and ectopic endometrium. Furthermore, the study demonstrates a definitive difference in the protein repertoire of the ectopic endometrium compared with its uterine counterpart in the same patient. Such studies are relevant in deciphering the complex biology of the endometriotic lesion.