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Mapping genetic heterogeneity of viable and non-viable sperm
exposure continues to be a major public health problem in the United States and Russia, thus these results add to our understanding of reproductive health risks from childhood lead exposure. Supported by: NIH grants R01ES0014370, P30ES000002, EPA grant R82943701 and Russian Science Foundation grant 14-45-0065 (for OS and RH). O-22 Monday, October 17, 2016 12:00 PM ALLEVIATING EFFECTS OF VARIOUS CONCENTRATIONS OF ETHANOLIC EXTRACT OF LYCOPODIUM CLAVATUM ON AGING INDUCED TESTICULAR PATHOLOGY IN WISTAR ALBINO RATS. G. Lakshmanana P. Seppan.b aDepartment of Anatomy, Research Scholar, Chennai, India; bDepartment of Anatomy, Assosciate Proffessor, Chennai, India. OBJECTIVE: Delayed paternity in the modern society and decreased sexual activity provides reason to research on the effects of ageing on male reproductive system and their possible treatment modalities. The crude ethanolic extract of Lycopodium clavatum commonly known as ‘‘club moss’’ has been reported to have good antioxidant properties. Traditionally lycopodium is used to treat sexual complaints of old aged men and is known to increase their ‘potency’ and considered as the ‘balm of old man’. However there is no scientific validation to confirm this effect. DESIGN: To analyze the efficacy of ethanolic extract of Lycopodium clavatum on aging induced alteration in testicular function and to find its effective therapeutic concentration. MATERIALS AND METHODS: Aged male wistar albino rats (24 months) were randomly divided into four groups (n¼12), first group received distilled water, and the other three groups received ethanolic extract of Lycopodium clavatum at dosage of 250, 500, 1000 mg / kg. b.w daily for 60 days by gavage. At the end of experimental period, serum testosterone was estimated. Sperm parameters including count, viability, motility, membrane permeability and nuclear condensation were done. Biochemical analyses of testicular antioxidants were done. Testicular mRNA expression of antioxidants and apoptotic genes were estimated by qPCR. The results were statistically analysed. RESULTS: Aging induced pathological changes were noted in the aged untreated animals. Beneficial effects were significant in Lycopodium clavatum treated groups when compared to untreated aged rats. Depleted testosterone level in untreated aged rat was improved in all the treated rats however, significant improvement was seen with 500mg dosage. Sperm parameters were significantly improved in aged rats with 500 mg dosage when compared to the other two treated aged groups i.e. 250 & 1000mg. The gene expression studies emphasized the maximum beneficial effects under 500mg dosage. CONCLUSIONS: This observation confirms that Lycopodium clavatum has therapeutic effect under aging induced reproductive disorder and the effect seem to be dosage dependent. Thus results signifies that finding effective dose for given animal is a key factor for maximum therapeutic outcome. O-23 Monday, October 17, 2016 12:15 PM MAPPING GENETIC HETEROGENEITY OF VIABLE AND NON-VIABLE SPERM. L. Nagirnaja,a M. J. Noordam,b D. Conrad.a a Genetics, Washington University School of Medicine, St. Louis, MO; bClinical Genetics, Maastricht University, Maastricht, Netherlands. OBJECTIVE: This study aimed to describe the genetic heterogeneity of viable and non-viable sperm selected from the total ejaculate of a healthy man. DESIGN: Whole-exome sequencing was performed for bulk DNA of live and dead sperm in comparison to blood. Additionally, three DNA pools of 200 live and 200 dead sperm from the same healthy male donor were amplified with multiple annealing and looping-based amplification cycles (MALBAC) whole-genome amplification prior to identical sequencing procedures. MATERIALS AND METHODS: Sperm selection was performed by FACS (LIVE/DEAD Sperm Viability Kit staining). Exome sequencing libraries were prepared with Nextera Rapid Capture Exome kit and run on HiSeq2000. Six pools of 200 sperm were subjected to MALBAC amplification prior to exome sequencing, reads were processed with BWA, PicardTools and samtools. De novo mutations were called with MuTect treating blood as a reference. Mutations from MALBAC pre-amplified samples were considered if confirmed by at least two pools of sperm DNA. Significance was tested with Fisher’s exact test or Mann-Whitney U-test (P<0.05 significant). RESULTS: Comparative exome sequencing of sperm DNAs identified increased number of mutations in bulk dead versus live sperm (42 vs 38,
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respectively). Due to the increased resolution in detecting rare mutations, sequencing of small pools of sperm revealed higher rates of de novo mutations with almost double counts in dead sperm (313 vs 172) and yielding in germline mutation rates of 4.6x10-9 per bp of exome (0.29/sperm) in live and 6.5x10-9 (0.51/sperm) in dead cells. Germline mutations had propagated to an average of 11% sperm cells (mean variant allele fraction, VAF¼0.11) in bulk and 17% (VAF¼0.17) in small sperm pools. Functional characterization of the identified de novo mutations with integrated CADD annotation predicted an increased deleterious impact of the rare variants identified in small pools of sperm compared to bulk (P¼1.5x10-7) and a similar increase in the ratio of non-synonymous to synonymous amino acid changes (Ka/Ks 3.4 vs 1.9, P¼0,05). In small pools of dead sperm, the upper 5th percentile of mutations with the largest CADD score (n ¼ 15) included variants in four genes highly relevant for sperm function (ADCY10, TSGA10, SPEF1 and BBS1). CONCLUSIONS: Characterizing the genetic differences between viable and non-viable sperm is an important step towards developing robust assays for mutation detection from bulk ejaculate. The results highlight the contribution of rare mutations that potentially define the viability outcome of sperm. Furthermore, the increased rate of de novo mutations in dead sperm has relevant implications when selecting sperm in vitro for assisted reproduction procedures Supported by: This work was supported by U.S. National Institutes of Health (grants R01HD078641 and R01MH101810 to D.F.C.).
O-24 Monday, October 17, 2016 12:30 PM MOLECULAR MECHANISMS BEHIND GHRELIN-MEDIATED PREVENTION OF POST-SURGICAL ADHESIONS. E. Bianchi,a K. Boekelheide,b M. Sigman,c S. J. Hall,d K. Hwang.b aDivision of Urology, Brown University, Providence, RI; bBrown University, Providence, RI; cSurgery (Urology), Brown University and Lifespan, Providence, RI; dPathology, Saunderstown, RI. OBJECTIVE: Postoperative adhesions are a leading cause of infertility, chronic pelvic pain, and intestinal obstruction. A new reproducible mouse model of induction of adhesion has been developed to demonstrate the capability of ghrelin to reduce post-surgical adhesions in a growth hormone secretagogue receptor (GHSR-1a)-dependent manner1. The present study was designed to assess the molecular mechanisms and the GHSR-1a signaling pathway activated by ghrelin in preventing post-surgical adhesions. DESIGN: In Vivo and In Vitro experiments were performed. Post-surgical adhesions were created in C57BL/6 wild type mice. Ghrelin or saline intraperitoneal injections were given from two days before surgery to 1, 4 and 20 days after surgery. An In Vitro model to test the ghrelin-activated GHSR-1a signaling pathway has been optimized, characterized by lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages in the presence or absence of ghrelin. MATERIALS AND METHODS: Post-surgical adhesion-induced mice were sacrificed at 1, 4 and 20 days after surgery and adhesions were objectively scored. Peritoneal ischemic buttons were harvested to determine gene and protein expression levels of pro-fibrogenic factors by RT-PCR and Western blot. RAW 264.7 cells were LPS-stimulated in the presence or absence of ghrelin and protein and gene levels of tumor necrosis factor (TNF) were assessed by immunofluorescence and RT-PCR. RESULTS: The anti-fibrotic effect of ghrelin seems to be associated with blockage of transforming growth factor beta (TGF-b) signaling via downregulation of Tgfb3 and Tgfbr2 and up-regulation of inhibitory proteins, such as Smad6 and Smad7. p38 mitogen-activated protein kinases (p38MAPK) signaling is a compensatory pathway that amplifies the response to TGF-b via a non-canonical pathway. Protein levels of phospho-p38 MAPK were attenuated in ghrelin-treated ischemic buttons. LPS-stimulated murine macrophages pre-treated with ghrelin (100nM) showed reduction in TNF mRNA and protein levels. CONCLUSIONS: These findings suggest that ghrelin inhibits the TGF-b/ Smads and p-38 MAPK signaling pathways activated during the inflammatory response at the onset of injury before the granulation-remodeling phase occurs. Future studies will be focused on ghrelin-activated GHSR-1a signaling pathway. Gene expression analysis will be performed using Mouse Gene 2.0 ST microarrays to detect the altered mRNA transcripts when compared to control using linear models of microarray analysis (q<0.05). Reference: 1. Bianchi E, Boekelheide K, Sigman M, Lamb DJ, Hall SJ, Hwang K. Ghrelin ameliorates adhesions in a postsurgical mouse model. J Surg Res 2016; 201: 226-34. Supported by: Lifespan Hospital.
Vol. 106, No. 3, Supplement, September 2016
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respectively). Due to the increased resolution in detecting rare mutations, sequencing of small pools of sperm revealed higher rates of de novo mutations with almost double counts in dead sperm (313 vs 172) and yielding in germline mutation rates of 4.6x10-9 per bp of exome (0.29/sperm) in live and 6.5x10-9 (0.51/sperm) in dead cells. Germline mutations had propagated to an average of 11% sperm cells (mean variant allele fraction, VAF¼0.11) in bulk and 17% (VAF¼0.17) in small sperm pools. Functional characterization of the identified de novo mutations with integrated CADD annotation predicted an increased deleterious impact of the rare variants identified in small pools of sperm compared to bulk (P¼1.5x10-7) and a similar increase in the ratio of non-synonymous to synonymous amino acid changes (Ka/Ks 3.4 vs 1.9, P¼0,05). In small pools of dead sperm, the upper 5th percentile of mutations with the largest CADD score (n ¼ 15) included variants in four genes highly relevant for sperm function (ADCY10, TSGA10, SPEF1 and BBS1). CONCLUSIONS: Characterizing the genetic differences between viable and non-viable sperm is an important step towards developing robust assays for mutation detection from bulk ejaculate. The results highlight the contribution of rare mutations that potentially define the viability outcome of sperm. Furthermore, the increased rate of de novo mutations in dead sperm has relevant implications when selecting sperm in vitro for assisted reproduction procedures Supported by: This work was supported by U.S. National Institutes of Health (grants R01HD078641 and R01MH101810 to D.F.C.).
O-24 Monday, October 17, 2016 12:30 PM MOLECULAR MECHANISMS BEHIND GHRELIN-MEDIATED PREVENTION OF POST-SURGICAL ADHESIONS. E. Bianchi,a K. Boekelheide,b M. Sigman,c S. J. Hall,d K. Hwang.b aDivision of Urology, Brown University, Providence, RI; bBrown University, Providence, RI; cSurgery (Urology), Brown University and Lifespan, Providence, RI; dPathology, Saunderstown, RI. OBJECTIVE: Postoperative adhesions are a leading cause of infertility, chronic pelvic pain, and intestinal obstruction. A new reproducible mouse model of induction of adhesion has been developed to demonstrate the capability of ghrelin to reduce post-surgical adhesions in a growth hormone secretagogue receptor (GHSR-1a)-dependent manner1. The present study was designed to assess the molecular mechanisms and the GHSR-1a signaling pathway activated by ghrelin in preventing post-surgical adhesions. DESIGN: In Vivo and In Vitro experiments were performed. Post-surgical adhesions were created in C57BL/6 wild type mice. Ghrelin or saline intraperitoneal injections were given from two days before surgery to 1, 4 and 20 days after surgery. An In Vitro model to test the ghrelin-activated GHSR-1a signaling pathway has been optimized, characterized by lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages in the presence or absence of ghrelin. MATERIALS AND METHODS: Post-surgical adhesion-induced mice were sacrificed at 1, 4 and 20 days after surgery and adhesions were objectively scored. Peritoneal ischemic buttons were harvested to determine gene and protein expression levels of pro-fibrogenic factors by RT-PCR and Western blot. RAW 264.7 cells were LPS-stimulated in the presence or absence of ghrelin and protein and gene levels of tumor necrosis factor (TNF) were assessed by immunofluorescence and RT-PCR. RESULTS: The anti-fibrotic effect of ghrelin seems to be associated with blockage of transforming growth factor beta (TGF-b) signaling via downregulation of Tgfb3 and Tgfbr2 and up-regulation of inhibitory proteins, such as Smad6 and Smad7. p38 mitogen-activated protein kinases (p38MAPK) signaling is a compensatory pathway that amplifies the response to TGF-b via a non-canonical pathway. Protein levels of phospho-p38 MAPK were attenuated in ghrelin-treated ischemic buttons. LPS-stimulated murine macrophages pre-treated with ghrelin (100nM) showed reduction in TNF mRNA and protein levels. CONCLUSIONS: These findings suggest that ghrelin inhibits the TGF-b/ Smads and p-38 MAPK signaling pathways activated during the inflammatory response at the onset of injury before the granulation-remodeling phase occurs. Future studies will be focused on ghrelin-activated GHSR-1a signaling pathway. Gene expression analysis will be performed using Mouse Gene 2.0 ST microarrays to detect the altered mRNA transcripts when compared to control using linear models of microarray analysis (q<0.05). Reference: 1. Bianchi E, Boekelheide K, Sigman M, Lamb DJ, Hall SJ, Hwang K. Ghrelin ameliorates adhesions in a postsurgical mouse model. J Surg Res 2016; 201: 226-34. Supported by: Lifespan Hospital.
Vol. 106, No. 3, Supplement, September 2016