Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Colleagues working in similar research fields, post docs and graduate students, technicians, undergraduate students, embryologists, and producers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided??Training: Graduate students and post-docs were shown and mastered several techniques including collection of mouse and bovine oocytes, handling of mouse and bovine sperm, percoll gradients, performing in vitro fertilization and sperm ICSI injections as well as regular microinjections. Students and post-docs were also exposed to a variety of biochemical and molecular biology techniques. Professional development: Graduate and undergraduate students received training and learned a variety of laboratory techniques in addition to enhancing their skills in developing and testing hypotheses, experimental design and data analysis. They were taught all of the procedures and assays necessary to perform these experiments and gained access to state of art imaging equipment. Further professional development included the opportunity to present their results at the Fertilization Gordon Conferences and local seminars. Our undergraduate students will present findings obtained in the laboratory at a Symposium for undergraduate students that takes place on our campus. Both graduate and undergraduate students present their results at our weekly lab meetings. PI and graduate students participated on weekly and monthly seminar series, which included external speakers and student presentations. PIs met individually with students at least once a week to go over results and planning next set of experiments. How have the results been disseminated to communities of interest?Manuscripts were published in peer-reviewed journals. Graduate students and post-doc presented data at the Gordon Conferences (international meeting) and SSR. Graduate students and PI participated in different on Campus events. PI is director of Frontiers in Reproduction, a six-week long course in reproductive biology that meets annually in Woods Hole, MA. He presents two lectures in the course. What do you plan to do during the next reporting period to accomplish the goals?Future studies: We will conduct studies to determine how both treatments affect energy production, how the consequent decrease in ATP levels induces significant changes in a subset of sperm molecules, and how these changes impact pre- and post-fertilization events. We also hypothesize that upon rescue, i.e. re-addition of energy, other subset of molecules are up-regulated. We will use NMR and mass spectrometry to identify these changes in metabolites produced upon different steps of the A23187 and the SER treatments. We will examine how SER and/or SER+A23187-treated sperm affects the distribution of PLCzeta 1. We will subsequently examine how these treatments impact the initiation of Ca2+ oscillations and embryo development. New studies will be started to examine Ca2+ influx channels in bovine oocytes that are needed to support the Ca2+ oscillations associated with fertilization. These channels are presently unknown. These studies will require an electrophysiological rig that is presently available in our core facility and that we have already used to record mouse eggs. We will base our studies on initial findings being presently conducted in mouse oocytes and eggs.
Impacts
What was accomplished under these goals?
Impact: Fertilization is a complex process that requires gametes to undergo appropriate maturation. The quality of the gametes not only impacts fertilization rates, which under in vivo conditions cannot be appreciated by the producer, but also pregnancy rates and development to term, which can be monitored by producers and directly impact their profits. In general, less fertilization means fewer offspring, reduced available replacement animals and on occasions the need to purchase replacements animals. Likewise, in a dairy farm, it might mean cows with greater average of open days during the lactation. Further, reduced conceptions rates are a well-documented and growing problem of the dairy industry worldwide. Thus, the focus of the Massachusetts project is to understand the basic molecular mechanisms that need to be optimized to prepare gametes for successful fertilization and embryo development. The notion is that improving the fertilization competence of gametes would increase the developmental competence of the resulting embryos, which will improve conception rates. The overall goal of this proposal is to use bovine gametes to perform intracytoplasmic sperm injection (ICSI) to identify the molecular steps that are compromised/altered both in in vitro matured oocytes and in frozen sperm and that undermine the success of this procedure in this species. To achieve this goal, the following three objectives will be performed: (1) examine the Ca2+ responses of bovine oocytes following injection of bull sperm and compare them to those of fertilized by conventional in vitro fertilization procedures, (2) determine whether the defects of the success of ICSI lies on the initiation of an inadequate Ca2+ activation signal or the inability of the oocyte to reprogram the sperm nucleus; (3) pre-treat sperm with novel treatments that modify the energy levels of sperm to determine if it increases the success of this technique. The combined results from these studies would make possible the development of protocols to assess oocyte quality, as the ability to initiate oscillations is directly related to oocyte quality and developmental competence. We intend to ultimately use this approach to evaluate oocyte quality on oocytes matured under different in vitro conditions to improve these conditions. Further, this approach would also allow the evaluation of oocytes from high producing animals, which would determine if indeed the fertilization competence is already compromised ahead of fertilization. This knowledge could be utilized to develop procedures that ameliorate the loss of oocyte developmental competence caused by metabolic stress in high producing animals. In the past academic year, we explored how exposing sperm prior to fertilization to changes in the internal concentrations of Ca2+ and/or the metabolic/energy levels might affect fertilization rates both after IVF and ICSI. The procedures we are using in these studies involve "Sperm Energy Recovery after starvation" (SER) and/or the calcium ionophore A23187. For SER treatment sperm are incubated in media depleted of all nutrients (starvation) until they became motionless, and thereafter individual nutrients can be added back (recovery/rescue). For A23187, sperm are exposed to the ionophore for a short period of time, after which it is washed away. In mice, these procedures have greatly improved both the rates of fertilization in strains of mice that are subfertile, and significantly after SER the rates of post-implantation embryo development and the number of offspring per litter are improved. Our preliminary results suggest that these treatments may have similar favorable effects in bull sperm. What was accomplished? Objective 2: Objective A: Identify intracellular signaling pathways and gene expression regulatory mechanisms within the ovary, embryo, or female reproductive tract that promote oocyte growth and maturation, fertilization, early embryonic development, and establishment and maintenance of pregnancy Major findings, Key Outcomes: · Treatment of bull sperm with ionophore A23187 in standard TALP medium rapidly immobilized bull sperm. Important, when A23187 was washed away 10 minutes later, bull sperm rapidly regained general motility, and these sperm displayed parameters consistent with hyperactive motility such as decrease linearity, increased VCL (curvilinear velocity) and ALH (lateral head displacement). · When ionophore A23187-treated bull sperm were functionally tested for in vitro fertilization rates, ionophore treated sperm performed better than control sperm, but not better than heparin-treated sperm. However, the combined treatment of ionophore A23187+ heparin, produced significantly better results than either of the treatments alone, especially when the outcomes were evaluated as total number of blastocysts and total number of hatched blastocysts. · Sperm Energy Recovery after starvation" (SER) treated bull sperm, just as mouse sperm treated by the same procedure, responded to the restoration of energy with better motility patterns, and increased rates of pre-implantation of embryo development. · SER-treated bull sperm also resulted in better fertilization rates following ICSI, greater percentages of sperm induced oscillations, the rates of blastocysts development changed dramatically. · SER-treated sperm seem to undergo differential re-localization of sperm PLCzeta, the sperm molecule responsible for the initiation of oscillations at fertilization in all mammals. In conclusion, the treatment of bull sperm with procedures that change either the intracellular concentration of Ca2+ or availability of energy substrates appears to enhance many functional aspects of bull sperm that are needed for effective fertilization. Enhanced parameters include progressive motility, ability to fertilize in vitro as well post sperm injection, and the distribution of PLCzeta1. This proposal seeks to understand the molecular mechanisms underpinning these changes with the long-term goal to improve ART in the bovine and other species of agricultural importance.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Navarrete FA, Aguila L, Martin-Hidalgo D, Tourzani DA, Luque GM, Ardestani G, Garcia-Vazquez FA, Levin LR, Buck J, Darszon A, Buffone MG, Mager J, Fissore RA, Salicioni AM, Gervasi MG, Visconti PE. Transient Sperm Starvation Improves the Outcome of Assisted Reproductive Technologies. Front Cell Dev Biol. 2019 Nov 5;7:262
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Progress 11/24/17 to 09/30/18
Outputs
Target Audience:Target Audiences:Colleagues working in similar research fields, post docs and graduate students, technicians, undergraduate students, embryologists, and producers. Efforts:presentations at professional meetings, peer-reviewed journal articles and reviews,formal classroom instruction, experiential learning opportunities, experiment station meetings. Changes/Problems:No Major problems were encountered. However, obtaining bovine ocoytes continues to be a problem and they are very expensive. Nevertheless, we are still able to obtain them and work with them. All the techniques are available. We have not performed electrophysiology in bovine oocytes, but we expect to attempt this technique this summer. What opportunities for training and professional development has the project provided?Training:Graduate students and post-docs were shown several techniques including collection of mouse and bovine oocytes, ICSI injections and regular microinjections and a variety of biochemical and molecular biology techniques. Professional development: Graduate and undergraduate students received training and learned a variety of laboratory techniques in addition to enhancing their skills in developing and testing hypotheses, experimental design and data analysis.They were taught all of the procedures and assays necessary to perform these experiments and gained access to state of art imaging equipment. Further professional development included the opportunity to present their results at the Society for the Study of Reproduction held in New Orleans in the summer of 2018, and the Fertilization Gordon Conferences that will be held at Holderness, NH, in July of 2019. Our undergraduate students will present findings obtained in the laboratory at a Symposium for undergraduate students that will take place on our campus as well as lab meetings.PI and graduate students participated in weekly and monthly seminar series, which included external speakers and student presentations. Lab meetings were held on a weekly basis and the PI met individually with students at least once a week to go over results and planning next experiments. How have the results been disseminated to communities of interest?Manuscripts were published in peer-reviewed journals. Graduate students and post-doc presented data at the Gordon Conferences (international meeting). Graduate students and PI participated in different on Campus events. PI is director of Frontiers in Reproduction, a six-week long course in reproductive biology that meets annually in Woods Hole, MA. He presents two lectures in the course. What do you plan to do during the next reporting period to accomplish the goals?Future studies: In the first year of this proposal we showed that treatments that alter the energy status of sperm during capacitation enhance the Ca2+ responses following ICSI. This is an important result, as one of the limitations of ICSI in the bovine is to initiate persistent oscillations. Nevertheless, we did not examine the molecular mechanism that might be enhancing the initiation of the responses. Therefore, here we hypothesize that PLCzeta release is facilitated by this treatment. We will therefore examine using immunofluorescence and super-resolution if the location and/or distribution of PLCzeta is altered by this treatment. In a second goal, we will use different activation procedures to induce bovine egg activation. For this procedure, we will inject a constitutive active form of CaMKII that have shown is able to active mouse eggs with high efficiency. Initial studies will determine the concentration and rates of egg activation, and future studies will examine pre-implantation embryo development. New studies will be started to examine the Ca2+influx channels in bovine oocytes, which presently are unknown. These studies will require an electrophysiological rig that is presently available in our core facility and that we have already used to record mouse eggs. We don't expect difficulties developing this approach.
Impacts
What was accomplished under these goals?
Objective 2:Objective A:Identify intracellular signaling pathways and gene expression regulatory mechanisms within the ovary, embryo, or female reproductive tract that promote oocyte growth and maturation, fertilization, early embryonic development, and establishment and maintenance of pregnancy Major findings, Key Outcomes: Using X-ray Fluorescence Microscopy (XFM), a technique that enables quantitation of the total metal ion pool, which consists of labile (loosely bound) metals as well as metals tightly bound to proteins, membranes, we found that similar to findings in mouse eggs, bovine MII eggs contained significantly more Zn relative to Fe and Cu. ·Examination of the distribution of labile Zn2+in bovine eggs using the cell-permeable Zn2+-responsive fluorophore, FluorZin-3-AM, we find that Zn2+ located to discrete punctuate structures at the cell periphery. Given that this fluorescence dissipates upon treatment with the heavy metal chelator TPEN, which has affinity for Zn2+, the data suggest that labile Zn2+is sequestered in cortical structures. ·To determine the association between Ca2+ increases and Zn2+ exocytosis, Zn2+ sparks, we induced an intracellular Ca2+increase with Ionomycin and monitored simultaneously intracellular Ca2+ changes and extracellular Zn2+. In all cases, an intracellular Ca2+ increase caused an increase in extracellular Zn2+release. ·Additional examination of extracellular Zn2+releasewithout monitoring Ca2+ showed that the amplitude of the extracellular Zn2+releasecould be quantified, which showed that significant differences could be observed among eggs such that approximately only 50% of the eggs showed high to median rises whereas the other half showed low or no Zn2+ release. These results suggest that there are differences in egg quality following in vitro maturation, and that quantification of the levels of Zn2+release could be used to optimize in vitro maturation conditions. The assumption is that fewer Zn2+accumulation in vesicles would suggest lower oocyte quality. Some possible reasons underlying this could be that lower Zn2+means :1- fewer transporters capable of carrying Zn2+into the cell, or that the ability to sequester Zn2+ into vesicles is reduced, etc. If more extensive studies show that this is indeed the case, the follow up studies will focus on addressing these questions. ·Unlike the mouse system, in vitro fertilization in the bovine system demands well over 60 min prior to the initiation of oscillations. Given this limitation, we induced fertilization-like oscillations following injection ofbPLC-zeta cRNA, which encodes for the sperm's enzyme that triggers the Ca2+responses at fertilization.We found that in the majority of eggs that initiated oscillations following injection of PLCzeta cRNA, from 1 to 3 Zn2+sparks were observed, and in a minority of eggs, Zn2+release was not observed. ·In conclusion, our studies show that Zn2+release occurs following fertilization in the bovine as it happens in other mammalian species. The ability to detect this release, may offer some opportunities to assess oocyte quality. For example: oThis Zn2+release can be quantified allowing quantification of each cell's content. oSince the dye to detect Zn2+release is extracellular, the possible cellular damage caused by exposure to UV should be minimized. Therefore, examined eggs could be used for in vitro development. oDifferences in Zn2+ release were observed among oocytes following Ionomycin exposure, and it would be of interest to determine if the levels of Zn2+ release affect the oocytes' ability to support pre-implantation embryo development. In addition, this method could be used to evaluate and improve in vitro maturation conditions in the bovine.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Que EL, Duncan FE, Lee HC, Hornick JE, Vogt S Fissore RA, O'Halloran TV, Woodruff TK. 2019. Bovine eggs release zinc in response to parthenogenetic and sperm-induced egg activation. Theriogenology. 127:127-41.
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