http://hdl.handle.net/2005/11 2013-06-15T23:09:44Z http://hdl.handle.net/2005/1930 Title: Allosteric Regulation Of Proteins In The Cyclic GMP Signal Transduction Pathway Authors: Biswas, Kabir Hassan 2013-02-18T18:30:00Z http://hdl.handle.net/2005/1903 Title: Cyclic AMP In Mycobacteria Adenylyl Cyclases And Cyclic AMP Receptor Proteins Authors: Sharma, Ritu Abstract: The discovery of cyclic AMP (cAMP), nearly 50 years ago by Sutherland radically altered the appreciation of metabolic regulation. Since then the presence of cAMP and its tremendous physiological impact has been demonstrated in many prokaryotic systems. In fact, virulence mechanisms of several pathogens known today exploit cAMP dependent pathways. Interestingly the genome of Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis, encodes as many as 16 adenylyl cyclases (enzymes that convert ATP to 3’, 5’-cAMP) and 10 cyclic-nucleotide binding proteins. Recent reports show that bacterial-derived cAMP manipulates host signaling for bacterial survival, suggesting an important role for cAMP in the pathogenesis of M. tuberculosis. A large number of non-pathogenic species of mycobacteria also share and conserve several of these cAMP metabolism genes, suggesting that cAMP is not only important for pathogenesis but also may play a critical physiological role in the genus. The work carried out in this thesis aims at a better understanding of the role of cAMP by studying the adenylyl cyclases and cyclic AMP receptor proteins (CRPs) from Mycobacterium smegmatis, a non-pathogenic member of the genus. Intracellular cAMP levels in a cell are precisely maintained by modulating the activities of the adenylyl cyclases (cAMP synthesising enzymes), the phosphodiesterases (cAMP hydrolysing enzymes) and the secretion machinery, if any. To assess the role of cAMP in mycobacteria, cAMP levels were measured in M. smegmatis during growth and under various stress conditions. The results show that cAMP levels peak at log phase of growth and decline thereafter. Under acidic conditions or on perturbing the cell-wall, cellular cAMP levels are altered, which indicate a possible role of cAMP in stress adaptation. Earlier work in our laboratory has led to the identification of multiple adenylyl cyclases in the mycobacterial genomes. These cyclases are similar in sequence to the mammalian enzymes and several of them have been shown to be active in vitro displaying a diverse range of biochemical properties. The M. smegmatis genome encodes 10 adenylyl cyclase-like genes. In order to understand the role of cAMP in M. smegmatis, individual cyclases were analysed for their biochemical properties and physiological functions. The work presented in this thesis is concerned with the functional characterization of MSMEG_3578 and MSMEG_3780, two of the several adenylyl cyclases from M. smegmatis. MSMEG_3578 encodes for a protein that comprises two transmembrane domains, an extracellular receptor-like domain, a membrane anchoring HAMP domain and an intracellular cyclase domain. The cyclase domain is closely related to mammalian cyclases but lacks the canonical residues that are critical for the catalysis of class III cyclases. Interestingly, the stop codon of this gene overlaps with the start codon of the downstream gene, MSMEG_3579 (a putative cyclic nucleotide gated mechanosensitive ion channel), suggesting a functional link between the two genes. The conservation of this gene pair across the mycobacterial genus indicates the importance of this putative receptor-effector pair in the physiology of mycobacteria. Additionally, microarray analysis by various groups have shown that this gene pair in Mycobacterium tuberculosis is differentially regulated in conditions that mimic stress the bacteria may experience during infection. In order to ascertain the physiological role of MSMEG_3578, a knock-out M. smegmatis strain was generated and tested for growth and cAMP defects. The knock-out strain showed growth and cAMP profiles similar to the wild-type strain. Over-expression of MSMEG_3578 in M. smegmatis resulted in a significant rise in cAMP levels. Interestingly, over-expression of the MSMEG_3578 adenylyl cyclase in E. coli did not lead to an elevation in cAMP levels indicating that other mycobacterial proteins may be required for the activity of MSMEG_3578 in vivo. In agreement with this, the purified adenylyl cyclase domain of MSMEG_3578 was found to be biochemically inactive in vitro. Additionally, the over-expressing strain has altered colony morphology as compared to the wild type strain. Perturbation of cAMP levels by over-expression of other cyclases also leads to a similar colony morphology phenotype, indicating the phenotype to be controlled by cAMP in general rather than by a specific cyclase in the cell. MSMEG_3780 is a highly conserved, biochemically active adenylyl cyclase, speculated to play a house-keeping function in M. smegmatis. Its orthologs from M. tuberculosis (Rv1647) and M. leprae (ML1399) have been biochemically characterized earlier in our laboratory. To unravel the role of this gene in vivo, the ∆MSMEG_3780 strain was tested for growth and cAMP defects under various conditions. The deletion strain did not show any difference in growth rate or morphology when compared to the wild-type strain. However it showed a reduction in intracellular cAMP levels at the log-phase of growth. Reintroduction of the MSMEG_3780 gene in the deletion strain restored cAMP to wild-type levels, thus indicating a crucial role for this adenylyl cyclase in the maintenance of intracellular cAMP levels during logarithmic growth. In order to investigate the regulation of the MSMEG_3780 gene, its promoter activity was tested under various stress-conditions. Acid-stress specifically resulted in the repression of the MSMEG_3780 promoter activity, a condition which also leads to a decrease in cAMP levels in the cells. Further evidence for the susceptibility of the MSMEG_3780 gene to acid-stress was obtained when the ∆MSMEG_3780 strain failed to reduce intracellular cAMP content upon sustained acid-stress conditions. Since Rv1647 shares similar biochemical properties with MSMEG_3780 and can also heterodimerize with the MSMEG_3780 protein in vitro, it was interesting to test whether the M. tuberculosis ortholog could functionally complement MSMEG_3780. To assess this, a complement strain was generated that contained the Rv1647 gene under the control of MSMEG_3780 promoter, integrated into the genome of ∆MSMEG_3780 strain. Rv1647 protein was able to restore the cAMP phenotype seen on acid stress as well as the cAMP levels in the mutant strain at the log phase of growth. This study indicated the role of cAMP and MSMEG_3780 in acid adaptation and also suggested a non-redundancy of adenylyl cyclases in mycobacteria, where different individual cyclases may have unique functions in the cells. Since Rv1647 could complement the cAMP defective phenotype in ∆MSMEG_3780, this suggests functional parallels between the proteins from the two species. Bacterial adaptation to environmental stress is brought about by a rapid change in its gene expression profile. Cyclic AMP plays an important role by binding to and activating a transcriptional factor, cAMP receptor protein or CRP. We have identified two CRPs from M. smegmatis, viz., MSMEG_0539 and MSMEG_6189 that possess high similarity at the amino acid level (78% overall sequence identity). The CRP ortholog from M. tuberculosis, Rv3676 has been characterized structurally, biochemically and functionally earlier. Western blot and RT-PCR analyses showed that CRPs in M. smegmatis were present during all phases of growth. Both the CRPs were cloned, expressed and shown to bind cAMP. Since the DNA binding domains of Rv3676 and the two M. smegmatis CRPs are nearly identical, the CRPs from M. smegmatis were predicted to bind similar target sequences. Interestingly, a CRP site was identified in the promoter of the MSMEG_3780 gene, suggesting a possible feed-forward or feed-back loop, where the enzymatic product of the adenylyl cyclase now governs its own gene expression. We performed Electrophoretic Mobility Gel Shift Assays (EMSAs) with M. smegmatis lysates to show that CRP binds to the MSMEG_3780 promoter at the CRP site. Subsequent Chromatin Immunoprecipitation (ChIP) assays confirmed that CRP binding to the MSMEG_3780 promoter occurred in vivo. To investigate the role of CRP in the regulation of the MSMEG_3780 gene, luciferase reporter assays with the wild-type and CRP site mutant promoters were carried out. Results suggest that CRP regulates the MSMEG_3780 gene under osmotic stress. However, CRP did not play any role in basal expression of MSMEG_3780 during growth. To assess which CRP of the two is functionally linked to the MSMEG_3780 promoter, we carried out a footprint assay with purified CRPs. It was intriguing to note that both the CRPs were in fact able to bind the promoter albeit under different conditions. Whereas MSMEG_6189 bound the promoter both in the presence and absence of cAMP, MSMEG_0539 bound the promoter only in the presence of cAMP. MSMEG_6189 thus deviates from the accepted CRP paradigm that seeks an absolute requirement of cAMP for specific DNA binding. The present study identifies cAMP as an important stress signal in M. smegmatis. Using MSMEG_3780 as a model gene, the role of cAMP in mycobacteria was studied. The two divergent CRPs that were characterized may function and dictate cAMP-mediated and perhaps independent functions in cells. Taken together, our results provide compelling evidence for the important role of cAMP in the general physiology and stress adaptation in M. smegmatis. 2013-01-27T18:30:00Z http://hdl.handle.net/2005/1318 Title: Characterization Of Down Regulated Genes In Astrocytoma Authors: Bhanja, Poulomi Abstract: Gliomas are the most common primary brain tumors and include astrocytomas, oligodendrogliomas and oligoastrocytomas. Astrocytomas have a high frequency of occurrence as compared to the other gliomas and several studies including ours have focused on understanding the etiology, biology and genetics of this disease. Based on the degree of malignancy, astrocytomas have been graded from I to IV. Grade I or pilocytic astrocytomas are benign tumors and have limited infiltration. On the contrary, Grade II-IV astrocytomas also referred to as diffusely infiltrating astrocytomas (DA, Grade II), anaplastic astrocytomas (AA, Grade III) and glioblastoma multiforme (GBM, Grade IV), have the tendency of diffusely infiltrating the normal brain parenchyma. GBM is characterized by uncontrolled proliferation and resistance to apoptosis, rampant invasion, recalcitrance to most established therapies etc which makes them the most aggressive of all gliomas with a median survival of about 12 months. This makes it imperative to initiate further studies to understand the molecular basis of this disease. Gene expression profiling studies have been central to this effort. In recent years, several Microarray studies have provided crucial insights into the biological role of novel genes not previously associated with astrocytomas. In a previous Microarray study, several differentially regulated genes in astrocytoma were identified in our laboratory. In addition to many up regulated genes, several down regulated genes were also identified in this study. Down regulated genes are interesting to study because of their relevance as possible tumor suppressor genes. Hence, we decided to characterize the regulation and functional significance of few down regulated genes. The specific objectives of the study are as follows 1)To validate novel down‐regulated genes in astrocytomas identified by a previous Microarray study. 2)To understand the mechanism of down-regulation of a few selected gene. 3)Functional characterization of DIRAS2, a novel astrocytoma down‐regulated gene with respect to its possible role in astrocytoma progression. Towards these objectives, we identified 21 genes as differentially down-regulated across all grades of astrocytoma based on a previous Microarray study from our lab and data from literature. Real time qRT-PCR analysis performed on these 21 genes confirmed their down-regulation in all grades of astrocytoma as compared to normal brain tissues. From these 21 genes, we short-listed 10 of the most consistently down-regulated genes for further analysis. These genes were DIRAS2, IGFBP9, MAL2, MBP, OLFM1, PACSIN1, RAB26, SYT1, SYT5 and VSNL1. We also confirmed the expression of two of the genes, OLFM1 and RAB26 at the protein level by performing immunohistochemical analysis on an independent set of 38 tissues that included 10 normal tissues and 28 tissues from different grades of astrocytoma. OLFM1 was found to be down-regulated in a grade specific manner. RAB26 expression was found to be strikingly high in all the low grade astrocytomas in comparison to high grade astrocytomas which made it an interesting gene to study functionally. On functional characterization, we found that RAB26 over‐expressing LN229 cells showed significantly reduced invasion compared to the vector transfected cells suggesting RAB26 could have a tumor-suppressing role in astrocytomas. In order to investigate whether transcriptional modulation could play a role in the down-regulation of these 10 genes, we searched for transcription factor binding sites in approximately 2kb 5’ flanking region of each gene. Intriguingly one or more PAX6 binding sites were present in all their promoters. In light of the fact that PAX6 has been proposed as a tumor‐suppressor in astrocytomas, we predicted that some of these genes could be targets of PAX6 transactivation and could possibly mediate some of the tumor‐suppressive actions of PAX6. PAX6 has been proposed as a down stream target of Notch signaling in the context of eye development. Similar to this observation, upon activation of Notch signaling with a virus expressing human intracellular domain of Notch (Ad-NIC-1), PAX6 expression was found to be induced in glioma cell lines suggesting PAX6 to be a novel NOTCH target in astrocytomas. In addition, Ad-NIC-1 infection could also induce the expression of OLFM1, RAB26, MAL2 and MBP in U343 cells. We could also demonstrate that Ad-NIC-1 co-operates with PAX6 in the regulation of these four genes in cell lines expressing endogenous PAX6, namely U343 and U251. Intriguingly, in a cell-line lacking PAX6 expression (LN229), Ad-NIC-1 could not induce OLFM1, RAB26 and MBP, although we could see induction of MAL2. Interestingly, PAX6 overexpression in LN229 cells in the absence of Ad-NIC-1 could induce OLFM1, RAB26 and MAL2. In contrast, infection of Ad-NIC-1 on the PAX6 over-expressing cells seemed to have an antagonistic effect on the expression of OLFM1, RAB26 and MBP, suggesting that Ad-NIC-1 antagonizes PAX6 actions in these cells. Ad-NIC-1 infection resulted in increased apoptosis in a PAX6 independent manner in U343 cells, which as previously mentioned has high levels of PAX6 endogenous expression. Conversely, Ad-NIC-1 could not induce apoptosis in LN229 cells, which has negligible expression of PAX6. We could also demonstrate that apoptosis induced in U343 cells could be in a p53 dependent manner. Activation of AMPK pathway and inhibition of the mTOR pathway as a consequence of p53 induction could also explain the Ad-NIC-1 mediated apoptosis that was seen in these cells. Thus, we have proposed that Notch signaling could possibly have a tumor-suppressing role in the presence of PAX6. We also suggest that down-regulation of OLFM1, RAB26, MAL2 and MBP via the NOTCH-PAX6 axis could be a possible molecular mechanism for the down-regulation of these genes. With respect to the third objective, we sought to characterize DIRAS2 with respect to its function in astrocytomas. DIRAS2 was identified as a down‐regulated gene in all grades of astrocytoma by our Microarray study. We were also able to validate the down‐regulation of DIRAS2 in all grades of astrocytomas. DIRAS2 also bears significant homology to RIG1 (also known as DIRAS1), which has been proposed as a tumor suppressor gene in astrocytomas. In the light of these data, we predicted that DIRAS2 could be a tumor suppressor gene in astrocytomas. Overexpression of DIRAS2 in two glioma cell lines U87 and C6 did not reveal any appreciable change in proliferation. Strikingly when the DIRAS2 over-expressing clones were grown in the absence of serum, there was marked increase in proliferation with respect to vector transfected clones along with a distinct change in morphology. Decorin expression in the DIRAS2 over-expressing clones was found to be up regulated and could be responsible for the altered morphology as well as enhanced viability in absence of serum. Interestingly along with Decorin expression, we also observed an increase in phosphor-SMAD2 levels indicative of activated TGF‐β signaling in the DIRAS2 over-expressing clones in the absence of serum. In the soft agar and migration/invasion assays, the results across the two cell lines, U87 and C6 were contrasting. DIRAS2 over-expressing clones of U87 cells formed visibly larger and increased number of colonies as compared to vector transfected clones and there was about a three fold increase in invasion with respect to that seen in vector transfected clones in the matrigel invasion assay. On the other hand, DIRAS2 over-expressing C6 clones formed colonies of smaller size compared to vector transfected clones and a marked decrease in migration was observed in the DIRAS2 over-expressing clones of C6. The discrepancies in the results in these two cell lines could be attributed to the presence of other regulators of DIRAS2 function unique to each of the two cell lines. Although in the present study, the results with respect to its predicted function as a tumor-suppressor gene has not been conclusive, the role of DIRAS2 in tumorigenesis may depend on the cellular context in which the protein is expressed. Overall in this study, we have identified a novel down regulated gene signature in astrocytomas consisting of OLFM1, RAB26, MAL2 and MBP. Furthermore, we have proposed that inhibition of NOTCH and PAX6 signaling pathways could be responsible for the down-regulated expression of OLFM1, RAB26, MAL2 and MBP in astrocytomas. Collectively, these results suggest that astrocytomas with activated Notch1 and/or Pax6 signaling could have good prognosis due to the tumor suppressive actions of OLFM1, RAB26, MAL2 and MBP 2011-07-26T18:30:00Z http://hdl.handle.net/2005/1347 Title: Studies On Intracrine Regulators Of Ovarian Function : Examination Of Progesterone Action On Structure And Function Of Corpus Luteum In The Monkey Authors: Suresh, P S Abstract: The control of reproductive cycles in higher primates is largely dependent on negative and positive feedback mechanisms by both steroidal and non-steroidal substances of the ovaries which regulate the function of hypothalamo-pituitary system. To gain insights into the role of INH A, the non steroidal ovarian hormone in the feedback control of pituitary FSH secretion, studies were conducted to examine the interrelationships of hormones throughout the menstrual cycle of the bonnet macaque. The findings of chapter II provide a detailed description of endocrine hormone profile during the menstrual cycle of the bonnet macaques with special attention to the feedback role of INH A on pituitary FSH secretion. To characterize the endocrine profile of different hormones, both ovarian (E2, P4, INH A) and pituitary (FSH, LH) hormones were measured daily for more than 40 days. To further examine the site of secretion of INH A and its relationship with pituitary FSH dynamics, surgical lutectomy and pharmacological induction of luteolysis employing the third generation GnRH R antagonist, Cetrorelix (CET) studies were carried out in the subsequent experiments. The results obtained from these studies suggest that INH A and P4 secreted from the CL during luteal phase regulate pituitary FSH secretion. The selective rise in FSH observed during the late menstrual cycle and during menstruation (referred to as luteo-follicular transition), as has been reported previously in higher primates, considered necessary for initiation of follicular growth and recruitment of follicles for ensuing menstrual cycle was characterized in the monkey. Surgical lutectomy and induction of luteolysis by CET experiments suggested that increased GnRH secretion is essential for this selective rise in FSH following withdrawal of inhibition by P4 and INH A. In clinical cases of reproductive ageing, the shortened follicular phase in human females has been identified to be the result of occurrence of early onset of FSH rise during the luteal-follicular transition period. The cause(s) of declining fertility with age in women who still have regular menstrual cycles are not clear, but issues of relationship between dysregulation of selective FSH rise in the late luteal phase and associated infertility could be examined using bonnet monkey as a model system. INH A is secreted in significant quantities by CL in higher primates and the feto placental unit suggesting its importance during fertility and pregnancy. Apart from the negative feedback regulation of pituitary FSH secretion, the complete repertoire of actions of this hormone during pregnancy is yet to be fully understood. The data presented in this thesis is the first comprehensive report showing the endocrine hormone profile of gonadotropins and ovarian hormones including INH A throughout the menstrual cycle of the bonnet macaque. The characterization of INH A profile in bonnet monkey will be of significant value for studies examining the role of INH A in higher primates. Dimeric inhibin has been suggested to be important for regulation of fertility and reproductive functions. Also, inhibin-α (one of the subunits of the dimeric protein) knock out mice model has provided convincing evidence that it acts as a tumour suppressor. A great deal of new information has been generated in recent years regarding the potential clinical usefulness of monitoring inhibin levels in blood and biological fluids in gynaecological diseases, pathological pregnancies and other disorders. Emerging clinical roles of inhibin have made INH A an important candidate molecule to study its molecular regulation. The results presented in chapter II suggested that LH regulates luteal INH A secretion (induction of luteolysis by CET administration experiment). As a first step towards understanding molecular regulation of inhibin-α expression in the macaque CL, in silico promoter analysis of macaque inhibin-α was performed and it revealed several transcriptional factor binding sites that were conserved across species. In rats FSH up regulates while preovulatory LH surge suppresses inhibin-α mRNA expression in the ovary and this suppression has been suggested to be necessary for occurrence of secondary FSH surge during metestrus. To address differential regulation of inhibin-α by LH and FSH in rat ovary during the periovulatory period, studies employing immature rats were carried out and the results are presented in chapter III. The results suggest that immature rat ovaries respond to exogenous gonadotropins in terms of LH signaling (cAMP production), luteinization (P4 production) and as well induction of ICER expression required for repression of inhibin-α subunit expression. PDE4 inhibitor (rolipram) treatment enhanced the ovarian cAMP concentrations suggesting that PDE4 play a major role in controlling intraovarian cAMP concentrations in rat ovaries. However increased cAMP concentrations did not appear to up regulate the ICER expression at the time point examined in this study. In higher primates time course of second FSH surge and continued synthesis and secretion of INH A in the CL are different from non primate species. In the monkey, the second FSH rise occurs during the late luteal phase and experiments have been carried out to examine the regulation of inhibin-α subunit expression by ICER. Expressions of ICER (mRNA/protein) and INH A were examined during different stages of CL and the results indicated no clear inverse relationship between the ICER and inhibin-α mRNAs. With no conclusive role for the ICER in regulating luteal inhibin-α observed in the study, the role of transcriptional activators in the regulation of inhibin-α like GATA4, SF-1, β-catenin were further examined. Since luteal INH A secretion was dependent on pituitary LH as determined earlier in chapter II, expressions of transcriptional activators were examined in CL of different stages and also during induced luteolysis and the results are described in chapter IV. In conclusion, our results indicate cross talk between WNT, cAMP and P38 MAP kinase signaling pathways in the regulation of luteal INH A secretion. The pituitary gonadotropin, LH, is the primary luteotropin in primate species acting to maintain the structure and function of the CL during the menstrual cycle. However whether the actions of LH are direct or mediated by local factors such as P4 remain unknown. Moreover, P4 secretion which is dominant during luteal phase has any role in regulating CL structure and function is not clearly defined. To address these and issues concerning P4 actions, initially, experiments were performed in the rat model to study the importance of P4 in the regulation of ovarian functions. An antiprogestin, RU486, was employed as a tool to uncover the PR regulated pathways during ovulation in rats and the findings are presented in the chapter V. The results indicated that blockade of PR action by RU486 during gonadotropin-induced superovulation resulted in inhibition of follicular rupture and ovulation in immature rats. Further to understand the downstream effectors of PR action, and to identify the candidate target genes of PR activation, semi-quantitative RT-PCR and western blot analyses were performed. The results obtained indicated that betacellulin, a member of EGF family and MMP-9 a proteolytic enzyme, were markedly repressed in response to RU486 treatment in rat ovaries. Also, the down stream pathway of EGF signaling leading to activation of ERK was markedly repressed in RU486 treated ovaries. It was next examined what role the P4/PR system has in the regulation of CL structure and function. Surprisingly, PR expression is absent in CL of rats, while it is present in higher primates. Experiments were carried out to examine intracrine actions of P4 in the regulation of CL structure and function in monkeys. The recently reported model system of induced luteolysis yet capable of responsive to trophic support from the laboratory provided an ideal opportunity to examine direct effects of P4 on structure and function of CL in the monkey. A series of pilot experiments were carried out in monkeys experiencing summer amenorrhea, to determine dose and mode of administration of exogeneous P4 to simulate mid luteal phase circulating P4 concentrations in monkeys subjected to induced luteolysis. Based on the results of pilot experiments, implantation of Alzet pumps containing 97.5mg of P4 was selected for maintaining mid luteal phase P4 concentrations. The microarray data of induced luteolysis previously deposited by the laboratory in NGBI’s gene expression omnibus were mined for identification and validation of differentially expressed genes of PR and its target genes following LH depletion and LH replacement experiments. Expressions of PR, PR cofactors and expressions of PR downstream target genes through out the luteal phase and in CL from day1 of menses were also examined. Analysis of expressions of genes revealed that of the 45 genes identified to be regulated by LH treatment, 4 genes were found to be responsive to P4, and 14 were identified to be responsive to both P4 and LH. Morphology of CL tissue sections revealed that P4 treatment appeared to have reversed the induced-luteolysis changes. In another experiment, implantation of P4 during late luteal phase (i.e., the period of declining P4 concentrations) for 24h caused changes in expressions of genes associated with tissue remodeling and morphology of luteal cells. Taken together, the results suggest that induced luteolysis plus P4 replacement model is suitable for assessing the effects of P4 on CL function. The results also suggest that CL could serve as target tissue for examining the genomic and non genomic actions of P4. In summary, studies carried out in the present thesis provides a comprehensive endocrine hormone profile throughout the menstrual cycle of the bonnet monkey with special emphasis on time course of INH A and FSH secretion which is very useful for future investigations. Studies have been carried out in rats and monkeys with different experimental model systems to address molecular mechanisms underlying inhibin-α regulation in the ovary in general and CL in particular. Experimental findings in monkeys could help elucidate the underlying molecular nature of CL functionality and extrapolate to understand luteal insufficiency and infertility producing conditions in humans. Also different model systems have been validated to examine the actions of P4/PR system in rats and monkeys and more importantly to address the direct effects of P4 upon monkey CL structure and function were established. Future investigations based on findings of these studies should help clarify relative roles for LH and P4 during maintenance of CL function and luteolysis. 2011-08-08T18:30:00Z