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Sperm Decondensation and Male Pronuclear Formation in Bovine Intracytoplasmic Sperm Injection

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Sperm Decondensation and Male Pronuclear Formation in Bovine Intracytoplasmic Sperm Injection Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School June 2020 Sperm Decondensation and Male Pronuclear Formation in Bovine Intracytoplasmic Sperm Injection Lauren Gatenby Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Animal Sciences Commons Recommended Citation Gatenby, Lauren, "Sperm Decondensation and Male Pronuclear Formation in Bovine Intracytoplasmic Sperm Injection" (2020). LSU Master's Theses. 5179. https://digitalcommons.lsu.edu/gradschool_theses/5179 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. SPERM DECONDENSATION AND MALE PRONUCLEAR FORMATION IN BOVINE INTRACYTOPLASMIC SPERM INJECTION A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for degree of Master of Science in The School of Animal Sciences by Lauren Nicole Gatenby B.S., Louisiana State University, 2017 August 2020 ACKNOWLEDGEMENTS First and foremost, I would like to thank and express my deepest gratitude to my major professor, Dr. Kenneth Bondioli. Without his support, patience, encouragement, extensive knowledge, and mentoring abilities this would not have been possible. His contributions to both my life and education are innumerable. It has truly been a privilege to learn under his guidance and I will always be grateful. I would also like to extend a special thanks to the members of my graduate committee, Dr. Zongliang (Carl) Jiang and Dr. Christopher Green, for all of their advice and support. Their input has been greatly appreciated and has helped set a high standard of education. I would also like to thank my fellow graduate students, whose encouragement, advice, and friendship will never be forgotten. They are the only people who truly appreciate the effort and desire required to succeed in Dr. Bondiloi’s program. With special thanks to Emilio Gutierrez, Fabian Diaz, Emily Dzekunskas, and Hao Ming, whose friendship and expertise throughout this experience and completion of this thesis will always be remembered and appreciated. Finally, my gratitude to my mother and to my family is greater than words can express. My mother has been an outstanding role model and example of a strong successful woman. Without her hard work and support throughout my life, I would not be where I am today. My family, especially my uncle and godfather, Mitchell Veronie, have always provided me with unconditional love and support, which has helped me through every adverse situation. I am truly grateful to have all of these people in my life who support me while perusing my education. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS . ii LIST OF TABLES . iv LIST OF FIGURES . v LIST OF ABBREVIATIONS . .. vi ABSTRACT . .. vii CHAPTERS I. INTRODUCTION . 1 II. LITERATURE REVIEW . 3 2.1. Intracytoplasmic Sperm Injection . 3 2.2. Bovine ICSI Pitfalls and Limitations . 7 2.3. Sperm Treatments for Bovine ICSI . 14 III. SPERM DECONDENSATION AND MALE PRONUCLEAR FORMATION FOLLOWING BOVINE INTRACYTOPLASMIC SPERM INJECTION . 21 3.1. Introduction . 21 3.2. Materials and Methods . 24 3.3. Results . 30 3.4. Discussion . 39 IV. SUMMARY AND CONCLUSIONS . 44 APPENDIX. MEDIA FORMULATIONS AND STOCK SOLUTIONS . 48 LITERATURE CITED . 55 VITA . 64 iii LIST OF TABLES Table 1. Sperm undergoing decondensation following DTT treatment. 31 Table 2. Effect of DTT treatment of sperm on fertilization rates of injected bovine oocytes. 37 Table 3. Effect of progesterone treatment of sperm on fertilization rates of injected bovine oocytes. 38 iv LIST OF FIGURES Figure 1. Morphological changes seen in spermatozoa following a 5mM treatment of DTT for 1 hour. 32 Figure 2. Changes in decondensation of DTT treated bovine spermatozoa over time. 33 Figure 3. Flow cytometry output for progesterone treated and nontreated sperm. 34 Figure 4. FITC-PNA binding to the exposed acrosome 15 minutes after progesterone treatment under DIC microscopy. 35 Figure 5. Abnormal and normal staining patterns in fertilized zygotes using progesterone treated sperm. 39 v LIST OF ABBREVIATIONS 1PN 1 pronucleus 2PN 2 pronuclei BSA Bovine serum albumin DAG 1,2-diacylglycerol DMAP 6- dimethylaminopurine DTT Dithiothreitol ER Endoplasmic reticulum FITC-PNA Fluorescein isothiocyanate-conjugated peanut agglutinin HEP Heparin ICSI Intracytoplasmic sperm injection IP3 Inositol-triphosphate IVF In vitro fertilization LPC Lysophosphatidylcholine MPF Maturation promoting facto P4 Progesterone PBS Phosphate Buffered Saline PIP2 Phosphatidyl 4,5-bisphosphate PLCz Phospholipase C zeta PVP Polyvinylpyrrolidone SOF Synthetic Oviductal Fluid vi ABSTRACT This study assessed the effects both DTT and progesterone have on bovine spermatozoa to induce decondensation or the acrosome reaction in order to facilitate male pronuclear formation after ICSI. Sperm prepared by swim-up in a 5 mM concentration of DTT displayed time dependent morphological changes resulting in decondensation over a 6-hour period. An estimated 90% of treated sperm displayed early changes in morphology after the second hour of incubation. Sperm displaying partial decondensation of the nucleus was estimated as 42% by the 3rd hour and increased to 62% by the 5th hour of incubation. Fully decondensed sperm or sperm with completely dispersed DNA increased from 20% at hour 5 to 50% at hour 7. No such changes were seen in sperm not treated with DTT (p < 0.01). Progesterone was used to induce an acrosome reaction in sperm which were first capacitated with heparin (10 µg/mL) prior to treatment with progesterone (10 µM). The acrosome reacting population was measured using a conjugated FITC-PNA stain and observations using fluorescent microscopy and cytometric flow analysis. Sperm undergoing an acrosome reaction at hour 1 was 80.2% and increased to 89.3% after 2 hours. Sperm that completed the acrosome reaction increased from 49.8% at hour 1 and 62.5% at hour 2. Only 14.2% completed acrosome reactions in sperm not treated with progesterone (p < 0.05). Using these observations with DTT and progesterone treated sperm, ICSI was performed with both treatments to assess if rates of male pronuclei formation and successful fertilization could be improved. Injection of sperm treated with DTT resulted in 28.3% of embryos with 2 pronuclei (2PN) after activation and 16 hours of culture, compared to 6% in control injections (p < 0.001). ICSI with progesterone treated sperm also resulted vii in a higher number of 2PN embryos, with 38.1% compared to 10.1% in control injections (p < 0.001). These results show that both DTT and progesterone have effects on sperm which allow for higher rates of male pronuclear formation after ICSI by utilizing either the physical approach of DTT or the sperm’s physiological response to progesterone. viii CHAPTER I. INTRODUCTION Intracytoplasmic sperm injection (ICSI) has been a valuable tool in many species for its ability to not only overcome male factor infertility problems, but also to eliminate the risk of polyspermy commonly seen with in vitro fertilization (IVF), and can assist in implementing other technologies (Catt et al., 1995). For species with limited gamete availability ICSI has the potential of bypassing the need to optimize IVF conditions. However, bovine ICSI lacks the success rates that has been observed in other mammals (Águila et al., 2017). For example, in humans (Tesarik, 1998) and mice (Kimura et al., 1995) the injection of a spermatozoon directly into the cytoplasm of the oocyte is sufficient for activation and to initiate decondensation and formation of the sperm’s nucleus into the male pronucleus to join with the female genome to complete a diploid embryo. This is not the case for bovine ICSI, where injection of a spermatozoon is not sufficient for activation or sperm decondensation (Ock et al., 2003). While the exact reason has yet to be elucidated, it is suspected to be a combination of both oocyte and sperm factors. It has been shown that in vitro matured bovine oocytes may be deficient in glutathione, an essential antioxidant which plays a major role in sperm decondensation, giving the oocytes a reduced capacity to form a male pronucleus (Curnow et al., 2010). This is coupled with bovine sperm’s resistance to decondensation, as there is a greater stability of the sperm nucleus due to a high density of protamine disulfide bonds and tightly packed nuclear structure compared to other species (Hutchison et al., 2017). Despite these challenges, ICSI has the potential to be a valuable tool in cattle reproduction. ICSI provides some advantages over IVF in 1 using genetic material more conservatively, as well as being able to improve male factor infertility. However, a more valuable application is perhaps in implementing genetic editing, CRISPR/Cas9, more efficiently and studying aspects of fertilization. Improving on this technology in cattle may also provide insight into improving ICSI for other species, including humans, and shed knowledge on fundamental events during fertilization. 2 CHAPTER II. LITERATURE REVIEW 2.1. Intracytoplasmic Sperm Injection The process of successfully combining mammalian oocytes and sperm in vitro to create a viable embryo first began with rabbits in the 1950’s, which initiated a decades long cascade in reproductive studies
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