Advances in Bull Fertility and Semen Evaluation From Sperm Phenotype to Genome and Back Peter Sutovsky, PhD, Dr.h.c. Division of Animal Sciences, and Departments of Obstetrics, Gynecology and Women’s Health, University of Missouri, Columbia, MO 62511 [email protected] Thank You Brazil 2015 TRUMAN Sutovsky Lab Intro • Established 2001 • Male fertility in livestock and humans • Assisted Reproductive Technologies/Therapy (ART) • Improvement of Artificial Insemination (AI) Ubiquitination Targets Proteins for Degradation by The 26S Proteasome UPS in Gametogenesis, Fertilization & Development – USDA Projects 1998-2019 EPIDIDYMAL SPERM MAJOR ZYGOTIC MITOCHONDRIAL PRONUCLEAR MATURATION – GENOME INHERITANCE: Sperm DEVELOPMENT & HISTONE Defective sperm ACTIVATION mitophagy after MODIFICATION ubiquitination fertilization: FERTILIZATION: Sperm-zona penetration & anti- polyspermy defense SPERMATID DIFFERENTIATION, OVARY & OOCYTE - Oocyte SPERM CAPACITATION ACROSOMAL maturation, cortical granule Sperm remodeling in BIOGENESIS biogenesis, meiotic spindle preparation for function fertilization Guinea pig Ostrich H. sapiens..? Zebra finch Opossum Drosophila Large Animal Models For every physiological problem, there will be some animal of choice, or a few such animals, on which it can be most conveniently studied. (The Krogh Principle) Goals • Better understand reproductive process in mammals • Improve reproductive health, well being and increase the efficiency of assisted reproductive therapy in humans • Increase the efficiency od reproductive performance in food animals (AI, IVF, ET, sexing, cryopreservation, reprogenomics) Market Need/Opportunity Annual semen sales: $1.5 billion (dairy), $250 million (beef), $600 million (swine) Trend & Hotspots: • Genomics • Sexed semen • Higher AI fertility • Maximized AI dose production from genetically valuable sires Problems & Solutions • Suboptimal conception rates, early pregnancy loss • Lack of objective semen analysis methods • Mitigation of sperm damage from cryopreservation, sexing • Fertility biomarker discovery & validation • Improvement of AI dose • Maximize extension of semen from sires with valuable genomes outline 1. Brief overview of defective sperm phenotypes and their causes 2. Conventional and objective methods for semen analysis 3. Biomarkers of sperm quality/fertility 4. Biomarkers of sperm capacitation – Zinc signature 5. Genomics of sperm phenotype Teratospermia • Generally poor morphology, motility, viability • Various causes – age, toxicants, heat stress, nutrition Causes of Reduced Sperm Quality • Age • Health (infections) • Nutrition (malnutrition, trace element imbalance, toxicants) • Environment (heat stress, pollution) • Genetics • Damage from collection and processing (cryopreservation/cryocapacitation, sexing) Heat Stress • May be partially, completely reversible • Cause variety of sperm defects • Can be studied by scrotal insulation Infectious Bacterial Disease • Leucocytospermia • Increase in amorphous cellular debris • Semen quality may recover after antibiotic treatment • Case in point: ubiquitin and PAFR (WBC marker) levels recovered in yearling bulls treated with antibiotics Sutovsky et al., J. Androl. 2006 Feed Toxicants • Gossypol (cottonseed feed, Chenoweth 2000) • Decapitation, head defects, midpiece lesions, cytoplasmic droplet (CD) retention Corkscrew Nuclear crest Proximal CD Distal reflex Distal CD Genetic Causes • Single nucleotide polymorphisms (SNP)/point mutations • Differences in copy number of multi-copy genes • Epigenetic aberrations • Early example: Dag defect • Often recessive, manifested only in homozygous configuration Heritable Sperm Defects • Globozoospermia (round headed sperm syndrome) • Pyriform head • Knobbed acrosome • Fibrous sheath dysplasia (stump tail) • Multi-nuclear, multi- flagellar defect • Asthenozoospermia (flagellar defects) 2. Semen Analysis First (Human) Sperm Observations Van Leeuvenhook & Hamm 1677-79 Conventional Semen Analysis 1909 A careful Microscopic examination is a valuable aid in determining the nature of chronical diseases of generative organs Spermatorrhea – seminal weakness* *may be a result of marital excess University of Missouri 1917 Frederick B. Mumford 1868-1946 Conventional Semen Analysis 2019 • Front line semen assessment (volume, density, color, swirl) • Sperm Count • Motility • Appearance/Morphology • Contaminants (Leukocytes, spermatids, epithelial cells, residual bodies, cellular debris). • Not always correlated with field AI fertility Good or Bad? Conventional Detection of “Good “Bad Sperm” Marker Semen Analysis Fertility” Protein PAFr Ubiquitin SOLUTION: Biomarker-based Sperm Quality/ Fertility Assays • Biomarker-based, objective, automated • Correlate with conventional semen parameters and field AI fertility • Inexpensive, simple, and predictive of sires’ future fertility 3. Biomarker Identification by Comparing Normal vs. Defective Sperm Fraction Proteomes 1-13 Infertile Defective Normal 14-21 Fertile Surface-Enhanced Laser Desorption/Ionization (SELDI) P62 UBA Negative Biomarkers of Male Fertility • Vital stains (mitopotential, acrosome, live/dead) • Ubiquitin (UBB) • 15-lipoxygenase (ALOX15) • Lectin ligands (LCA, PNA) • PAWP (WBP2NL) • Aggresome (AGG) Sperm Flow Cytometry Flow Cell Photo Laser Detectors Side Scatter Forward Scatter Flow Cytometry – Histogram & Scatter Diagram Normal Histogram Abnormal Histogram Image-based FC: Cytometer & Microscope in One Box Normal Sperm (R3) Defective Sperm (R2) Amnis Corp., Seattle WA [Buckman et al., 2009, Systems Biol. Reprod. Med., 55(5-6):244-251] Ubiquitin (UBB) • Protein recycling peptide tags defective proteins, binds covalently to the surface of defective spermatozoa • • Ubiquitinated proteins of testicular origin may be carried over by defective spermatozoa Apocrine Secretion of Ubiquitin in the Epididymis From Hermo and Jacks, 2002 UBC-GFP Rat (Baska et al., 2008) Defective Sperm Ubiquitination Sutovsky et al., 2001, J. Cell Sci. 114:1665-75. Possible Roles of Defective Sperm Coating • Clustering of defective spermatozoa • Prevention of autoimmune infertility • Degradation of defective spermatozoa Ubiquitin Correlates With PARAMETER r Breeding Soundness Parameters Scrotal circumference -0.79 in Yearling Bulls Color (1-4) -0.59 Semen Volume -0.42 BSE identifies young bulls satisfactory for Swirl (0-5) -0.64 breeding based on a physical examination, Strength of Motility (SMR; 0-5) -0.71 reproductive organ examination including % Progressive Motility -0.72 of scrotal circumference and semen analysis % Normal Sperm -0.73 % Head Abnormalities +0.65 USDA, Forth Keogh, MT (n=207 Yearlings) % Total Abnormalities +0.74 TABLE: Highest correlations* between sperm UBI and % Live Sperm -0.71 BSE parameters obtained in 5 flow cytometry UBI trials Hemocytometer dilution -0.37 testing a total of 207 bulls Concentration -0.56 Pearson’s r-coefficient (0.01-0.99) Contaminants +0.29 Ubiquitin Correlates with AI Fertility Indexes %M2 (good MedM3 (Bad Sperm Med-All MedM1 MedM2 Sperm) %M1 ) %M3 Services, ATA 02-07 0.070911 0.241528 0.085717 -0.15057 -0.23675 0.402741 -0.13591 Services, ERCR 02-07 0.297884 -0.00446 0.279037 0.1108 -0.28891 0.171768 0.24182 Services, RBE II 01-07 0.267008 -0.16777 0.221209 -0.04858 -0.26394 0.082796 0.314891 DEV, ATA 02-07 -0.13288 0.244449 -0.10763 -0.50733 -0.07998 0.322993 -0.26919 DEV, ERCR 02-07 0.180263 0.40738 0.142119 -0.26655 -0.3231 0.351187 0.076113 DEV, RBE II 01-07 -0.09158 0.318927 -0.1193 -0.64229 -0.09412 0.222514 -0.09654 RBE II Index 01-07 0.07999 0.306967 0.03692 -0.58223 -0.32076 0.427711 0.011327 Services, CFI 02-07 0.267276 0.08448 0.245291 0.03589 -0.31763 0.26772 0.167393 DEV, CFI 02-07 0.008237 0.504845 -0.02311 -0.44093 -0.25192 0.425151 -0.12445 CFI Index, 02-07 0.11139 0.526457 0.079442 -0.47993 -0.34474 0.481186 -0.04369 • N=15 sires/85 collections Aggresomes (AGG) • Proteinaceous inclusion bodies that form when the ubiquitin-proteasome machinery is overloaded (cellular stresses, ROS overproduction) • Degraded by autophagosome, bind SQSTM1 • Sperm mitochondrial sheath has aggresome-like properties • Detected by ProteoStat Aggresome Detection Kit (Enzo), a 488 nm excitable molecular rotor dye that binds to denatured proteins Does Size Matter? HYPOTHESIS: Length of sperm tail mitochondrial sheath (MS) correlates with conventional semen and sperm parameters, and with fertility in bulls used for artificial insemination (AI) service. Fit Plot for Pregnancy and Fit Plot for Fertility Index and Mitochondrial Sheath Length Mitochondrial Sheath Length Fertility Index Fertility Pregnancy Rate (%) Rate Pregnancy Expected Progeny Difference (EPD) Correlations Mitochondrial Sheath (MS) Length Reproduction Correlations Avg MS Length:Yearling Weight -0.61 Nelore 9.74±0.31 µm Avg MS Length:Bull Fertility Index 0.38 Avg MS Length:Weaning Weight -0.59 Angus 9.35±0.28 µm Avg MS Length:% Live Sperm 0.41 Avg MS Length:Residual Average -0.50 Daily Gain P<0.0001 Avg MS Length:Motility 0.04 Avg MS Length:Calving Ease -0.61 Std Dev MS Length:% Dead and 0.44 Modified Sperm Acrosome Avg MS Length:Maternal Milk EPD -0.63 Grace Wiley, Eriklis Nogueira, Camile Sanches, Karl Kerns, Peter Sutovsky Avg MS Length:Fat Thickness EPD -0.58 Future is in Multiplexing Kennedy et al., 2014, Mol Reprod. Dev. 81:436-449 Ubiquitinated Spermatozoa Lack PAWP Protein Postacrosomal Sheath WW-Domain- Binding Protein (WBP2NL/PAWP) • Resides in the post-acrosomal sheath of the sperm head perinuclear theca (PT) • Promotes oocyte activation and pronuclear