M R Sairam

McGill University, Montréal, Quebec, Canada

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Publications (155)618.21 Total impact

  • H Zhang · X Chen · M R Sairam
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    ABSTRACT: Visceral adiposity represents a high risk factor for type 2 diabetes, metabolic syndrome, and cardiovascular disease as well as various cancers. While studying sex hormone imbalance-induced early obesity and late onset of insulin resistance in FSH receptor knock out female mice, we identified a novel mesenteric estrogen-dependent adipose gene (MEDA-4) selectively up-regulated in a depot-specific manner in mesenteric adipose tissue. Meda-4 cloned from both mouse and human adipose tissue codes for a 34-kDa cytosolic protein with 91% homology. Mouse Meda-4 mRNA is expressed highest in visceral adipose tissue and localizes predominantly in the adipocyte fraction. Human MEDA-4 is also more abundant in omental fat than sc depot in obese patients. In 3T3-L1 cells endogenous Meda-4 expression increases early during differentiation, and its overexpression promotes differentiation of preadipocytes into adipocytes and enhances glucose uptake. Conversely, short hairpin RNA-mediated knockdown of Meda-4 reduces both adipogenic and glucose uptake potential. In promoting adipogenesis, Meda-4 up-regulates transcription factor peroxisome proliferator-activated receptor-γ2. Meda-4 promotes lipid accumulation in adipocytes, regulating adipocyte fatty acid-binding protein 2, CD36, lipoprotein lipase, hormone-sensitive lipase, acyl-Coenzyme A oxidase-1, perilipin-1, and fatty acid synthase expression. 17β-Estradiol reduced Meda-4 expression in mesenteric adipose tissue of ovariectomized mice and in 3T3-L1 adipocytes. Thus our study identifies Meda-4 as a novel adipogenic gene, capable of promoting differentiation of preadipocytes into adipocytes, increasing lipid content and glucose uptake in adipocytes. Therefore it might play an important role in adipose tissue expansion in normal and aberrant hormonal conditions and pathophysiological states.
    Endocrinology 04/2012; 153(6):2665-76. DOI:10.1210/en.2011-2008 · 4.50 Impact Factor
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    H Zhang · X Chen · M R Sairam
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    ABSTRACT: We sought to characterise novel genes dysregulated by sex hormonal imbalances that induce obesity and metabolic disorder in a setting of oestrogen deficiency and androgen dominance in follicle-stimulating hormone receptor (For [also known as Fshr]) knockout female mice. Transcriptome analysis of mesenteric adipose tissue (MAT) of mutants revealed novel genes. One novel gene named Meda-7 was selected for study. Meda-7 was cloned from mouse and human adipose tissue; its expression, hormonal regulation and function were characterised. Mouse Meda-7 is richly expressed in deep visceral adipose tissue and encodes a 22 kDa secreted protein with 71% homology to human mesenteric oestrogen-dependent adipose gene- 7 (MEDA-7) protein. Both have six conserved cysteines like many cytokines. In obese patients, MEDA-7 is more abundant in omental than subcutaneous fat. Meda-7 is downregulated in For-knockout female MAT at 5 months (obese state) followed by steep upregulation at 9 months (prediabetic condition) when mutants progress towards the metabolic syndrome. Meda-7 is expressed predominantly in the stromal-vascular cell fraction. In this fraction,M1-proinflammatorymacrophages are rich in Meda-7. Meda-7 dysregulation in 5-month-old For-knockout MAT is restored by oestrogen, but treatment has no effect in older mutants. Overabundance of MEDA-7 in HEK-293 cells enhances cell proliferation via p42/44 mitogen-activated protein kinases. Secreted MEDA-7 attenuates insulin-stimulated glucose uptake in 3T3-L1 adipocytes, while downregulating glucose transporter-4 and upregulating both monocyte chemotactic protein-1 and suppressor of cytokine signalling-3. Downstream activity of the insulin signalling mediator, phospho-AKT, is also downregulated. MEDA-7 is a hormone-regulated adipokine/proinflammatory cytokine that is implicated in causing chronic inflammation, affecting cellular expansion and blunting insulin response in adipocytes.
    Diabetologia 06/2011; 54(9):2368-80. DOI:10.1007/s00125-011-2212-7 · 6.67 Impact Factor
  • X Chen · H Zhang · J P Aravindakshan · W H Gotlieb · M R Sairam
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    ABSTRACT: In studying the age dependence and chronology of ovarian tumors in follicle stimulating hormone receptor knockout mice, we identified a novel ovarian tumor associated gene-12 (OTAG-12), which is progressively downregulated and maps to Chr. 8B3.3. OTAG-12 protein overexpression in mouse ovarian and mammary tumor cells suggested powerful anti-proliferative effects. In human epithelial ovarian cancers (OCs) and OC cell lines, OTAG-12 mRNA expression is downregulated in comparison with normal ovaries. Cloning and identification revealed that human OTAG-12 mapping to gene-rich Chr. 19p13.12 is expressed in three spliced forms: hOTAG-12a, hOTAG-12b and hOTAG-12c, of which b is predominant in the normal ovary. Functionally active hOTAG-12b is a simple protein with no disulfide bonds and a nuclear localization signal is present in all variants. Transfection of OTAG-12 variants in OC and tumorigenic HEK293 cells confirmed nuclear localization. hOTAG-12b overexpression in OC and HEK293 cells effectively suppressed cell growth, anchorage-dependent and independent colony formation followed by apoptosis, whereas hOTAG-12a and hOTAG-12c had no such effects. Deletion mutants identified the critical importance of carboxyl terminus for hOTAG-12b function. Doxycycline-inducible growth inhibition of HEK293 cells by hOTAG-12a was associated with effects on G2 cell cycle arrest and apoptosis induction. hOTAG-12b expression rendered tumorigenic cells more sensitive to four apoptotic stimuli including etoposide-a topoisomerase-II inhibitor. Doxycycline-induced hOTAG-12b expression blocked xenograft tumor growth in nude mice, whereas hOTAG-12a was ineffective. Although p53-pathway-dependent apoptotic agents could upregulate endogenous hOTAG-12b and p53 in UCI-101/107 OC cells, hOTAG-12b could also induce apoptosis in p53-null and platinum-resistant SKOV3 OC cells and Doxycycline-induced hOTAG-12b did not alter p53. Further study showed that hOTAG-12b increases mRNAs of pro-apoptotic genes such as BAD, GADD45α and CIEDB, while inhibiting anti-apoptotic NAIP and Akt1 expression, suggesting that hOTAG-12b-induced apoptosis might be p53-independent. These results indicate that hOTAG-12b is a putative ovarian tumor suppressor gene warranting further studies.
    Oncogene 02/2011; 30(25):2874-87. DOI:10.1038/onc.2011.11 · 8.46 Impact Factor
  • J Libman · M. San Gabriel · M R Sairam · A Zini
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    ABSTRACT: The aetiology of sperm DNA damage is likely multi-factorial with abnormal compaction of nuclear DNA, abortive apoptosis and oxidative stress implicated as potential causes of DNA damage. The objective of this study was to evaluate DNA damage in spermatozoa from wild-type (WT) and FSH receptor knock-out (FORKO) mice, compare the relative susceptibility of spermatozoa from these animals to oxidative DNA damage, and examine the protective effect of the antioxidant catalase on sperm DNA damage. Epididymal spermatozoa from FORKO mice (n = 5) and WT controls (n = 5) were extracted and incubated with or without catalase. Sperm DNA damage was assessed immediately after epididymal extraction (time 0 control) and following 2-h incubation at 37 °C. DNA damage was measured by the sperm chromatin structure assay and the results expressed as the %DNA fragmentation index or %DFI. Freshly retrieved epididymal spermatozoa from WT mice had a significantly lower mean (±SD) %DFI than that of FORKO mice (2.7 ± 1.8 vs. 6.4 ± 2.9%, p < 0.05). Prolonged (2-h) incubation of FORKO mice spermatozoa resulted in a significant increase in %DFI compared with the time 0 control (17.9 ± 9.2% vs. 6.4 ± 2.9%, respectively, p < 0.05) and the addition of catalase protected these spermatozoa from DNA damage (9.8 ± 4.1 vs. 17.9 ± 9.2%, respectively, p < 0.05). However, incubation of WT mice spermatozoa did not increase %DFI significantly (5.8 ± 5.0 vs. 2.7 ± 1.8, respectively, p > 0.05) and the addition of catalase (vs. no catalase) did not result in a significant reduction in %DFI (5.8 ± 5.0 vs. 7.7 ± 6.5%, respectively, p > 0.05). These data indicate that catalase may protect sperm nuclear DNA from oxidative stress in vitro. The data also demonstrate the differential susceptibility of WT and FORKO mice spermatozoa to oxidative stress.
    International Journal of Andrology 12/2010; 33(6):818-22. DOI:10.1111/j.1365-2605.2009.01032.x · 3.70 Impact Factor
  • J Aravindakshan · X L Chen · M R Sairam
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    ABSTRACT: Among gynecologic malignancies ovarian cancer is the deadliest and most difficult to detect at early stages. As ovarian tumors have long latency and are relatively more frequent in postmenopausal women, revealing chronological changes in model systems might help in the discovery of novel molecular targets and diagnostic biomarkers for disease detection and management. Follitropin receptor knockout (FORKO) mice with early and sustained sex steroid hormone disharmony develop various age-dependent ovarian abnormalities including increased incidence ovarian tumors in complete absence of ovulation. These mutants show various tumor cell types including those related to ovarian surface epithelium around 12-15 months of age. To explore why the FORKO mice develop ovarian tumors later in life, we assessed global gene expression changes during the pre-tumor period (at 8 months). Age-matched wild-type and FORKO mice were compared to gain a comprehensive view of genes that are misregulated, even before overt tumors appear in mutants. Applying a conservative 2-fold change to detect changes, our study identified 476 genes (338 upregulated and 138 downregulated) to be altered between 8-month-old FORKO and wild-type ovaries. Using Ingenuity Pathway Analysis (IPA), we found highly significant alterations in five functional networks in pre-tumor stage FORKO ovaries. Notably, the top network to change in 8-month-old FORKO ovaries was associated with functions implicated in immune system development and function. We selected 9 immune related genes that are reportedly altered in Epithelial Ovarian Cancer (EOC) in women and confirmed their expression and chronology of changes in FORKO ovaries before and after tumor development. Our data indicate that immune surveillance mechanisms are compromised with in a 4-month window of tumorigenic alterations. In addition, expression of previously unrecognized genes misregulated in the dysfunctional FORKO ovaries suggests mechanisms not yet appreciated to date. We propose that a better understanding of genes that change before overt tumors develop could provide useful insights into ovarian carcinogenesis and open the door to additional new targets for treating ovarian cancers.
    Molecular and Cellular Endocrinology 11/2010; 329(1-2):37-46. DOI:10.1016/j.mce.2010.05.015 · 4.41 Impact Factor
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    ABSTRACT: A homozygous missense mutation, C566T, in the follicle stimulation hormone receptor (FSHR) gene has been linked to premature ovarian failure. The disease leads to infertility in a normal karyotype female with an elevated follicle stimulating hormone (FSH) and decreased serum estrogen level. Female mice carrying mutated FSHR gene, called follitropin receptor knockout (FORKO), display similar phenotype and are sterile because of a folliculogenesis block at a primary stage. We investigated the effects of bilateral intraovarian injection of an adenovirus expressing a normal copy of human FSHR on the reproductive system of 6-10 weeks female FORKO mice. Ad-LacZ was injected directly into each ovary of the control group. Animals were sacrificed at 2, 4, 8 and 12 weeks post-injection and tissues collected for evaluation. Treated mice showed estrogenic changes in daily vaginal smear whereas control animals remained fixated in the diestrus stage. Histological evaluation showed on average 26±4 follicles/ovary in treated group with 8±2 follicles at the antral stage compared with only 5±2 with zero follicles at antral stage in Ad-LacZ control mice. There was no significant change in serum level of progesterone, however, estrogen level increased 2-3-fold (P < 0.02) and FSH decreased by up to 50% (P < 0.04) in treated animals. FSHR mRNA was detected in the ovaries of the treated group. In conclusion, intra-ovarian injection of an adenovirus expressing human FSHR gene is able to restore FSH responsiveness and reinitiate ovarian folliculogenesis as well as resume estrogen production in female FORKO mice. Ad-LacZ injections indicate the absence of systemic viral dissemination or germ line transmission of adenovirus DNA to offspring. © The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected] /* */
    Molecular Human Reproduction 04/2010; 16(4):241-50. DOI:10.1093/molehr/gaq003 · 3.75 Impact Factor
  • M. R. SAIRAM
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    ABSTRACT: The subunits of ovine lutropin prepared by acid dissociation and salt precipitation were characterized by end group analysis, tryptic peptide mapping, SDS gel electrophoresis and biological activity. No evidence of internal peptide cleavage was found in the α subunit. The subunits possessed low activity. The α and β subunits recombined effectively to generate a complex that had full receptor binding activity and in vitro biological activity. The recombinants of subunits prepared by countercurrent distribution showed only 50% activity in both assays. The salt precipitation method α subunit could be completely reduced and reoxidized in the absence of denaturants. The reoxidized α subunit combines with the native β subunit generating full activity. However, this recombined hormone tends to lose activity with time, suggesting that the reoxidation may not fully restore the native structure of the reduced α subunit. The native lutropin α subunit effectively combined with follitropin β subunit generating complete follitropin activity.
    International journal of peptide and protein research 08/2009; 14(2):153-160. DOI:10.1111/j.1399-3011.1979.tb01738.x
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    ABSTRACT: On the basis of the observation that in the hormonotoxin oLH-gelonin conjugation of the toxin occurs via the α-subunit of lutropin, an attempt was made to develop a general method for generation of similar hybrid proteins involving other glycoprotein hormones. In this approach sites suitable for conjugation would be first introduced into the α-subunit, hybridized with any native hormone specific β-subunit of choice (LH-β, FSH-β, TSH-β) following which the toxic component gelonin would be added on in the form of gelonin-S-S- α—β complex. Thus, thiolated lutropin α-subunit recombined well with free lutropin β-subunit, yielding a hybrid which was active in terms of receptor binding, immunoreactivity, and steroidogenic properties. However, subsequent conjugation with thiolated gelonin, a ribosome inactivating protein, resulted in dissociation of β-subunit from its non-covalent union with the thiolated α-subunit. It is concluded that the addition of positively charged gelonin at particular sites on the α-subunit led to the destabilization of the lutropin quaternary structure.
    European Journal of Allergy and Clinical Immunology 01/2009; 35(1):46 - 51. DOI:10.1111/j.1399-3011.1990.tb00719.x · 1.30 Impact Factor
    International journal of peptide and protein research 01/2009; 35(1):46-51.
  • J Aravindakshan · X L Chen · M R Sairam
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    ABSTRACT: Most ovarian tumors in women occur upon aging. Follitropin receptor knockout (FORKO) mice are sterile and have age-dependent abnormalities including increased ovarian tumor incidence. To explore why atrophic ovaries of FORKO mice become tumorigenic later in life, we compared gene expression profiles by microarray at different ages. Here we show an unexpected ovarian expression of GDNF and its bimodal regulation. GDNF was down-regulated at a young age but up-regulated in aging FORKO mice prior to tumor appearance. Immunohistochemistry localized GDNF in the oocyte as well as somatic granulosa and stromal cells. GDNF protein also showed an age-dependent increase in the ovary, being lower in young mutants and increasing by 6 months. We found evidence for GDNF up-regulation in GC tumors and a potential role for androgen. The peripheral expression pattern and functions of this powerful neurotropic factor suggest mediation of processes involved in pathology of ovarian compartments.
    Biochemical and Biophysical Research Communications 01/2007; 351(2):507-13. DOI:10.1016/j.bbrc.2006.10.069 · 2.30 Impact Factor
  • M. R. Sairam · Y. Yang · R. Tiwari-Pandey
    Fertility and Sterility 09/2005; 84. DOI:10.1016/j.fertnstert.2005.07.025 · 4.59 Impact Factor
  • X. L. Chen · J. Aravindakshan · Y. Yang · M. R. Sairam
    Fertility and Sterility 09/2005; 84. DOI:10.1016/j.fertnstert.2005.07.146 · 4.59 Impact Factor
  • N Danilovich · D Maysinger · M.R. Sairam
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    ABSTRACT: Increased life expectancy leads to increased age-associated health issues in both sexes. For menopausal women the most important of these appear to result from the severe estrogen deficiency caused by ovarian dysfunction. The consequences among others include hot flashes, osteoporosis, obesity, impaired memory, higher incidence of Alzheimer's disease and cardiovascular disease. Ovarian function and steroidogenesis are influenced by pituitary gonadotropins, including follicle-stimulating hormone (FSH), whose actions are mediated through ovarian receptors. This article highlights our recent data pertinent to aging as derived from a novel genetically modified animal model [the FORKO mouse (FOllitropin Receptor KnockOut) lacking the FSH receptor. FORKO female mice experience a chronic depletion of estrogen (E2) from early development, and have phenotypes similar to aging women, with ovarian failure, obesity, skeletal changes, and ovarian tumors. A variety of findings support the conclusion that E2 deficiency in FORKO mice is responsible for their neural impairments associated with glial cell hypertrophy, region-specific brain cells loss, and abnormal behavior. Findings from mice with FSH receptor haploinsufficiency mice ('menopausal mice') are also shedding light on the molecular basis of menopausal conditions that include degeneration of the hippocampus. Many phenotypes noted in the null condition also occur in +/- females but in an age related manner. Thus, the FORKO mouse becomes an excellent model to investigate mechanisms underlying age-related changes especially when these events are accelerated, as in menopausal women. Opportunities abound to assess the potential benefits/adverse effects of hormone replacement regimen on various targets.
    Experimental Gerontology 11/2004; 39(11-12):1669-78. DOI:10.1016/j.exger.2004.05.009 · 3.49 Impact Factor
  • M. R. Sairam · A. Balla · Y. Yang · N. Danilovich · M. Wang
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    ABSTRACT: Introduction: Androgens important for normal ovarian function and produced by the thecal and stromal compartments are used by the granulosa cells for production of estrogens. Androgen excess has adverse effects on reproduction and causes related metabolic disturbances. Methods: We examined ovarian hormones, LH levels, follicular dynamics, markers and metabolic parameters at different ages in genetically altered female mice with complete or partial disruption of the FSH-receptor gene. Results: Sterile null females (FORKO mice) produce excess androgen as early as 3 weeks with sustained high levels in adult life. Estradiol is undetectable and progesterone is also reduced. Circulating androgen excess is associated with extensive hypertrophy of the theca and stromal cells with early elevated levels of LH in circulation. Follicular dynamics are drastically altered, ranging from accelerated recruitment to eventual slow down and early loss of oocytes. These phenomena are also reproducible in haploinsufficient females that show early reproductive senescence (similar to middle-aged women). Study of developmental markers such as antimullerian hormone, transcription factor GATA-4, estrogen receptor (ER) β and androgen receptor has provided a working model to explore molecular interactions. In addition to ovarian dysfunction early androgen excess (and estrogen deficiency) led to visceral obesity, abnormal lipid profiles, glucose intolerance, hyperinsulinemia and presumptive insulin resistance in the mutants.
    International Congress Series 09/2004; 1271:296-299. DOI:10.1016/j.ics.2004.05.087
  • V Sriraman · M R Sairam · A Jagannadha Rao
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    ABSTRACT: Spermatogenesis is a complex process, and previous studies have clearly established the role for testosterone in its maintenance. However, the role of FSH remains controversial, although several lines of evidence suggest its importance in initiation of spermatogenesis. In the present study, the relative roles of FSH and LH have been evaluated using specific antisera capable of neutralizing endogenous hormones in adult male rats following ethanedimethylsulphonate (EDS) treatment. Restoration of spermatogenesis in EDS-treated rats was monitored following FSH or LH deprivation by histological analysis and flow cytometry. Deprivation of FSH resulted in a reduction of seminiferous tubule diameter and spermatogonial number, which was much more drastic than that observed following LH deprivation. More importantly, FSH deprivation was associated with a significant reduction in the number of pachytene spermatocytes. These results provide evidence for a definite role for FSH in regulation of spermatogenesis, in addition to confirming the role of LH in spermatogenesis via testosterone.
    Reproductive biomedicine online 03/2004; 8(2):167-74. DOI:10.1016/S1472-6483(10)60512-3 · 3.02 Impact Factor
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    V Sriraman · M R Sairam · A J Rao
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    ABSTRACT: The relative role of LH and FSH in regulation of differentiation of Leydig cells was assessed using an ethane 1,2-dimethylsulfonate (EDS)-treated rat model in which endogenous LH or FSH was neutralized from day 3 to day 22 following EDS treatment. Serum testosterone and the in vitro response of the purified Leydig cells to human chorionic gonadotropin (hCG) was monitored. In addition RNA was isolated from the Leydig cells to monitor the steady-state mRNA levels by RT-PCR for 17alpha-hydroxylase, side chain cleavage enzyme, steroidogenic acute regulatory protein (StAR), LH receptor, estrogen receptor (ER-alpha) and cyclophilin (internal control). Serum testosterone was undetected and the isolated Leydig cells secreted negligible amount of testosterone on stimulation with hCG in the group of rats that were treated with LH antiserum following EDS treatment. RT-PCR analysis revealed the absence of message for cholesterol side chain cleavage enzyme and 17alpha-hydroxylase although ER-alpha and LH receptor mRNA could be detected, indicating the presence of undifferentiated precursor Leydig cells. In contrast, the effects following deprival of endogenous FSH were not as drastic as seen following LH neutralization. Deprival of endogenous FSH in EDS-treated rats led to a significant decrease in serum testosterone and in vitro response to hCG by the Leydig cells. Also, there was a significant decrease in the steady-state mRNA levels of 17alpha-hydroxylase, cholesterol side chain cleavage enzyme, LH receptor and StAR as assessed by a semiquantitative RT-PCR. These results establish that while LH is obligatory for the functional differentiation of Leydig cells, repopulation of precursor Leydig cells is independent of LH, and also unequivocally establish an important role for FSH in regulation of Leydig cell function.
    Journal of Endocrinology 02/2003; 176(1):151-61. · 3.72 Impact Factor
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    J Tam · N Danilovich · K Nilsson · M R Sairam · D Maysinger
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    ABSTRACT: The follitropin receptor knockout (FORKO) mouse undergoes ovarian failure, thereby providing an animal model to investigate the consequences of the depletion of circulating estrogen in females. The estrogen deficiency causes marked defects in the female reproductive system, obesity, and skeletal abnormalities. In light of estrogen's known pleiotropic effects in the nervous system, our study examined the effects of genetically induced estrogen-testosterone imbalance on this system in female FORKO mice. Circulating concentrations of 17-beta-estradiol (E2) in FORKO mice are significantly decreased (FORKO -/-: 1.13+/-0.34 pg/ml; wild-type +/+: 17.6+/-3.5 pg/ml, P<0.0001, n=32-41); in contrast, testosterone levels are increased (-/-: 37.7+/-2.3 pg/ml; wild-type +/+: 3.9+/-1.7 pg/ml, P<0.005, n=25-33). The focus was on the activities of key enzymes in the central cholinergic and peripheral nervous systems, on dorsal root ganglia (DRGs) capacity for neurite outgrowth, and on the phosphorylation state of structural neurofilament (NF) proteins. Choline acetyltransferase activity was decreased in several central cholinergic structures (striatum 50+/-3%, hippocampus 24+/-2%, cortex 12+/-3%) and in DRGs (11+/-6%). Moreover, we observed aberrations in the enzymatic activities of mitogen-activated protein kinases (extracellular-regulated kinase and c-Jun N-terminal kinase) in the hippocampus, DRGs, and sciatic nerves. Hippocampal and sensory ganglia samples from FORKO mice contained hyper-phosphorylated NFs. Finally, explanted ganglia of FORKO mice displayed decreased neurite outgrowth (20-50%) under non-treated conditions and when treated with E2 (10 nM). Our results demonstrate that genetic depletion of circulating estrogen leads to biochemical and morphological changes in central and peripheral neurons, and underlie the importance of estrogen in the normal development and functioning of the nervous system. In particular, the findings suggest that an early and persisting absence of the steroid leads to neurodegenerative changes and identify several key enzymes that may contribute to the process. This model provides a system to explore the consequences of circulating estrogen deprivation and other hormonal imbalances in the nervous system.
    Neuroscience 10/2002; 114(2):493-506. DOI:10.1016/S0306-4522(02)00278-6 · 3.36 Impact Factor
  • W Xing · M R Sairam
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    ABSTRACT: Expression and activation of follicle-stimulating hormone receptor (FSHR) in the granulosa and Sertoli cells are required for normal development of the ovarian follicles and germ cells. However, little is known regarding the mechanisms by which FSHR expression is regulated. We fused an ovine FSHR promoter to a luciferase gene to understand the promoter regulation in two gonadal cell lines. Deletion studies revealed that the strongest promoter was at -200 to +163 relative to the transcription start site. One of cis-elements protected from DNase I digestion was mapped to between +32 and +54 of the 174-base pair (bp) minimal promoter. Electrophoretic mobility shift assay with a 26-bp probe (+32 to +57) and nuclear extracts from Sertoli (15P1) and granulosa (JC-410) cell lines demonstrated a sequence-specific DNA-protein complex. Southwestern analysis detected a 43-kDa protein bound to the 26-bp probe. Gel supershift with upstream stimulatory factor 1 and 2 (USF-1/2) antibodies revealed that the DNA-protein complex contained these two transcription factors. Mutation within the E-box of the promoter abolished the sequence-specific binding and the minimal promoter activity but also greatly reduced the transcription of the proximal promoters by 49%-70%. These data suggest that the USF-1/2 binding to the promoter is required for the expression of the ovine FSHR in the gonadal cells.
    Biology of Reproduction 03/2001; 64(2):579-89. · 3.32 Impact Factor
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    P. Suresh Babu · N Danilovich · M R Sairam
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    ABSTRACT: The acquisition of FSH receptor(s) during follicular growth and their coupling to signaling pathways are key events in follicular development and dominance. However, little is known about the precise nature of the FSH receptor(s) involved in the growth-promoting phases of hormone action. To investigate the hormonal regulation of a newly discovered, alternatively spliced, growth factor type 1 receptor (designated FSH-R3) for the hormone, we examined expression in the adult mouse and the effect of PMSG treatment in the immature mouse ovary. Using RT-PCR and primers based on the established sheep ovarian transcript, a part of the FSH-R3 message was amplified only in wild-type (+/+), but not in the FSH-R knockout (-/-), mouse ovary. Semiquantitative RT-PCR using 3'-end primers specific for FSH-R1 (G(s)-coupled) and FSH-R3 indicated expression levels of the latter to be higher when follicular growth was induced by PMSG. Using FSH-R3-specific peptide IgG, FSH-R3 protein was detected by Western blotting in extracts of adult mouse ovary and was localized in granulosa cell membrane of mature follicles. In the immature mouse, levels of FSH-R3 protein that increased after PMSG administration in a time-dependent manner were also localized only on granulosa cell membranes of large follicles. The results reveal for the first time the expression of a different growth-promoting receptor for FSH in the developing and cycling mouse ovary. These observations introduce a new paradigm in the control of ovarian function.
    Endocrinology 02/2001; 142(1):381-9. DOI:10.1210/en.142.1.381 · 4.50 Impact Factor
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    ABSTRACT: Immunization of adult male rabbits with a synthetic luteinizing hormone-receptor peptide (LH-RP; representing amino-acids 21-41 of the extracellular domain of the rat LH receptor) resulted in production of high-titer antibodies capable of interacting with particulate and cell-based LH receptors. The antibody produced was able to inhibit binding of 125I-labeled human chorionic gonadotropin (hCG) to a particulate sheep luteal LH receptor preparation by 40%-50%. Maximal inhibitory activity was correlated with high antibody titer. Immunocytometry revealed that the antibody could directly bind to cells having LH receptors, such as rat granulosa and Leydig cells. The antibodies recognized a 77-kilodalton membrane protein in Western blots of mouse testicular extracts. Interaction of endogenous Leydig cell LH receptor with the LH-RP antibody resulted in both hormone agonist and antagonistic activities. The hormone-mimicking activity (increase in serum testosterone over control) was confined only to the early phase of immunization when the antibody titer was low. Blockade of LH receptor during the later part of immunization resulted in a significant reduction in serum testosterone over controls and inhibition of spermatogenesis. DNA flow cytometry showed that a specific and significant inhibition of meiosis (transformation of primary spermatocytes to round and elongated spermatids P < .01) and spermiogenesis (transformation of round spermatids to elongated spermatids P < .0001) occurred following blockade of LH function.
    Journal of Andrology 01/2001; 22(6):992-8. DOI:10.1002/j.1939-4640.2001.tb03440.x · 2.47 Impact Factor

Publication Stats

3k Citations
618.21 Total Impact Points


  • 1992–2012
    • McGill University
      • • Division of Experimental Medicine
      • • Department of Animal Science
      Montréal, Quebec, Canada
  • 1981–2010
    • Montreal Heart Institute
      Montréal, Quebec, Canada
  • 1979–2010
    • Instituto de Investigación Clínica de Occidente
      Zapopan, Jalisco, Mexico
  • 1976–2009
    • Université de Montréal
      • • Faculty of Veterinary Medicine
      • • Department of Medicine
      Montréal, Quebec, Canada
  • 1997
    • Institut de Génétique et de Biologie Moléculaire et Cellulaire
      Strasburg, Alsace, France
  • 1990
    • Georgia Health Sciences University
      Augusta, Georgia, United States
  • 1986
    • University of North Carolina at Chapel Hill
      North Carolina, United States
  • 1971–1986
    • University of California, San Francisco
      • Division of Hospital Medicine
      San Francisco, California, United States