The Father s Early Contribution to the Birth of the Child: the Role of Paternal RNAs DNA Cell Biol. 1995;14:155-61
SPERMATOGENESIS Mol. Human Reprod. (1997) 3:15-19 www.advancedfertility.com/abnormal-ivf-egg-pictures.htm
TEMPORAL CONSTRAINTS FOR PATERNAL EFFECTS ZGA 2 hrs 30 hrs 38-42 wks
EXPERIMENTAL STRATEGY RNA extracted Pool - 9 ejaculates Individual - 1 ejaculate Synthesis of spermatozoal and testes crna probes Human adult testes RNAs pooled from 19 different samples cdna synthesis and quality control Sperm penetration assay Human Sperm + Zona Free hamster eggs PRM2 The Lancet. 2002 360:773-777 Analysis of gene filter arrays L S O Z 30 Z 3h Hu Ha T Nature 2004 429:154
EVALUATING SPERM RNAS 1999-2015 12 2002 20,000 3,000 2007 2010 2012 (0.008 GB) (0.024 GB) (95 GB) (1000 GB)
DEFAULT STATE: SURVEILLANCE TO ENSURE OPTIMAL CONTRIBUTION
SPERMATOZOAL RNAs 18S 28S rrna (~89%) sncrna (~1%) Other Intergenic EE Intronic EE Exonic EE ERVL-MaLR lncrna Intronic RE Transcript Coding EE, expressed element; RE, retained element Nucleic Acids Res.( 2013) 41:4104-17
Increasing Integrity 1000 Abundant Sperm Transcripts INTEGRITY OF CODING TRANSCRIPTS Quintiles 1 2 Normalized Local Sequencing Coverage High 3 Average (Uniform) 4 Low 5' 3' Transcript Coding Region 5 Nucleic Acids Res. (2013) 41:4104-17
RELATIVE LEVELS OF ACSBG2 10 INDIVIDUALS Hum Reprod Update. 2013 Jul 14
DISTRIBUTION OF SPERMATOZOAL sncrnas Promoter L13 UTR L15 UTR TSS 34c 375 184 152 mirna ALU pirna Other mirna pirna Other ALU L1 5 UTR L1 3 UTR Promoter TSS
POTENTIAL SPERM RNA FUNCTIONS Confrontation and consolidation Translation of intact paternal mrnas AAAAA A Translational regulation by paternal mirna AAAAAA Legend AAAAA Paternal Maternal mrna mirna primicrorna pirna RNA fragments Ribosome Protein Dicer Embryonic genome DNA Transcriptional regulation by paternal RNA fragments Transcriptional regulation by paternal mirnas Promoter Activation of paternal primirnas by maternal DICER Hum Reprod Update. 2013 Jul 14
TRANSGENERATIONAL EPIGENETIC INHERITANCE http://jaxmice.jax.org/strain/000049.html RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse. Rassoulzadegan et. al., (2006) Nature. 441:469-74 RNA induction and inheritance of epigenetic cardiac hypertrophy in the mouse. Wagner, et. al., 2008) Dev Cell. (14:962-9. The mir-124-sox9 paramutation: RNA-mediated epigenetic control of embryonic and adult growth. Grandjean et. al., (2009) Development. 136:3647-55. Microinjection of mir-221 and mir-222 induced paramutated white-spotted phenotype that was heritable Microinjection of mir-1 targets Cdk9 cardiac hypertrophy mir-124 targets Sox9 giant phenotype and twin pregnancies
RESPONDING TO THE ENVIRONMENT Do you want to make a permanent change? Will times get better? Solution: Transmit a responsive state without altering the primary structure of DNA Consequences of environmental change: Metabolic disorders Type-2 diabetes Cardiovascular disease Infertility parallels obesity bell weather for metabolic syndrome?
STRESS & TRANSGENERATIONAL INHERITANCE Paternal stress exposure alters sperm microrna content and reprograms offspring HPA stress axis regulation. Rodgers, et. al., (2013) Neurosci. 33: 9003 9012. Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice. Gapp., et.al., (2014) Nature Neruoscience 17:667-9. Parental olfactory experience influences behavior and neural structure in subsequent generations. Dias and Ressler. (2014) Nat Neurosci. 7:89-96.
Cardiovascular and diabetes mortality determined by nutrition during parents' and grandparents' slow growth period. Kaati, et.al., ( 2002) Eur J Hum Genet. 11:682-8. Epidemiological evidence from three Swedish cohorts born in 1890, 1905 and 1920 followed to 1995. Food access extrapolated from historical data. Question: Can overeating during a child's slow growth period (SGP), i.e., before prepubertal peak, influence descendants' risk of death from cardiovascular disease and diabetes?
Cardiovascular and diabetes mortality determined by nutrition during parents' and grandparents' slow growth period. Kaati, et.al., (2002) Eur J Hum Genet. 11:682-8. Results: Limited access to food during the father's SGP, limits the child s cardiovascular disease risk A paternal grandfather surrounded by a bounty of food during SGP increases the grandchild s risk of diabetes Conclusion: A nutrition-linked mechanism through the male line seems to have influenced the risk for cardiovascular and diabetes mellitus mortality
TRANSGENERATIONAL DIET Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Carone, et al., Cell. (2010) 143:1084-96. Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy. Lambrot, et. al., NATURE (2013) COMMUNICATIONS 4:2889 Paternal obesity initiates metabolic disturbances in two generations of mice with incomplete penetrance to the F2 generation and alters the transcriptional profile of testis and sperm microrna content. Fullston, et al., FASEB J. (2013) 27:4226-43.
Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals. Carone, et al., (2010) Cell. 143:1084-96. Low protein diet High fat diet Control diet altered lipid/cholesterol biosynthesis gene expression and products decreased islet cells and reduced glucose tolerance (daughters) Genome-wide methylation essentially unaltered Reduction of H3K27me3 at few promoters
Paternal obesity initiates metabolic disturbances in two generations of mice with incomplete penetrance to the F2 generation and alters the transcriptional profile of testis and sperm microrna content: Fullston, et. al. (2013) FASEB J. 27:4226-43. Experimental Design: Male Control Diet (CD) vs High Fat (HF) F 0 all offspring raised on CD Observations: F 0 Paternal obesity adiposity 21% F1 Adiposity & insulin resistance F2 67% adiposity daughters and 24% adiposity sons Conclusions: F 1 males program obesity and insulin resistance in F 2 females F 1 females program obesity, impaired metabolic response in F 2 males Mechanism: mirna profile maintained to F 2 candidates e.g., mir-205
TO BE RESOLVED How is the information relayed to the male gamete? Is it a temporary or permanent change? Does it cross the blood-testis barrier to be delivered to sperm? Modulated response: - RNA production - stability - acquisition?
GENETIC FLOW OF INFORMATION? Soma-to-Germline Transmission of RNA in Mice Xenografted with Human Tumour Cells: Possible Transport by Exosomes. Cossetti et al. (2014) PLoS One. 9:e101629. Xenograft of human A-375 EGFP - Blood exosomes & mature sperm cells (+) http://ezbiosystems.com/view2.asp?d_id=214
Quantitative and stoichiometric analysis of the microrna content of exosomes Chevilleta et al., (2014) PNAS 111:4888 14893 - Sources of exosomes: plasma, seminal fluid, dendritic, mast and ovarian cells - If mirnas are distributed homogenously across all exosomes each exosome would contain 0.01 copies of the most abundant mirna - consolidation of mirnas into exosomes Sperm 50 fg RNA/cell + (0.3 fg sncrna/cell) Somatic 40,000 fg RNA/cell
Wayne State University Ob/Gyn & Molecular Medicine & Genetics Alumni: J. Nelson S. M. Wykes J. Kramer C. Ostermeier R. Martins A. Linnemann K. Drennan A. Platts C. Lalancette E. Anton S. Mao S. Selvaraju G. Johnson, M. Jodar, M. Estill, R. Goodrich, E. Sendler Current Collaborators: Georgia Regents Univ.: M.P. Diamond Harvard Univ.: R. Hauser CReATe: S. Moskovtsev, C. Librach CMMG: H. Heng, R. P-Regis, F. Luca Univ. Leeds: D. Miller Univ. Hawaii: W. S. Ward Contributors: Univ. Utah: D. Carrell, B. Emery Baylor College of Medicine: D. Lamb LANL: N. Doggett GBSF: J. Bode Oakland University: G. Singh Environmental Protection Agency: D. Dix