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Colorado State University
1.
Gonzalez-Castro, Raul A.
Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection.
Degree: PhD, Biomedical Sciences, 2018, Colorado State University
URL: http://hdl.handle.net/10217/191446 
► When performing intracytoplasmic sperm injection (ICSI), many in vivo mechanisms for sperm selection are bypassed; however, sperm must still be capable of activating the oocyte…
(more)
▼ When performing intracytoplasmic sperm injection (ICSI), many in vivo mechanisms for sperm selection are bypassed; however, sperm must still be capable of activating the oocyte for successful fertilization. Limited information is available for horses on the effect of sperm preparation method and sperm characteristics that affect ICSI outcome. The overall objectives of this dissertation were to: 1) study the association between sperm sorting methods, sperm population characteristics, and equine ICSI outcome, and 2) characterize sperm oocyte activating factors in stallion sperm, such as phospholipase C zeta (PLCz) and postacrosomal WW binding protein (
PAWP). In Experiment 1, a microfluidic device was used to sort frozen-thawed sperm from stallions (n=19), which resulted in a sperm subpopulation with improved motility, morphology, viability and DNA integrity (P<0.05) compared to the original sample. Then, microfluidic sorting was compared with the swim-up procedure and density gradient centrifugation. Swim-up was the least effective method to separate equine sperm. Microfluidic sorting and density gradient centrifugation sorted a sperm subpopulation with similar parameters, improving motility, viability and DNA integrity. After ICSI (n=45), no differences (P>0.3) were observed for cleavage and embryo development among sorting methods. In Experiment 2, sperm population parameters from which individual sperm were selected for injection were analyzed immediately after ICSI and correlated with the outcome. Sperm morphology, viability, membrane integrity measurement of hypoosmotic swelling and DNA integrity were evaluated in frozen-thawed sperm (n=114) used for ICSI in a program. Among sperm parameters, viability correlated positively with normal morphology and membrane integrity (P<0.05). Normal sperm morphology and DNA integrity were not predictive of ICSI outcome. Viability was predictive of cleavage and blastocyst formation, and membrane integrity was predictive of early pregnancy (P<0.05). In Experiment 3, PLCz and
PAWP were identified, localized and quantified in stallion sperm, and the relationship with other sperm parameters was investigated. PLCz was identified as a 71 kDa protein and located in the acrosomal and postacrosomal region, midpiece and principal piece of the tail.
PAWP was identified by two bands of ~28 and ~32 kDa, located in the postacrosomal region, midpiece and principal piece of the tail. The expression of PLCz and
PAWP correlated positively (P=0.04) when analyzed for sperm of 14 stallions. Flow cytometric assessment was feasible for PLCz, but not for
PAWP. Expression and percentages of positive labeled sperm for PLCz varied among stallions (n=21). Expression of PLCz was higher in live than dead sperm (P<0.005), and DNA fragmentation correlated negatively with PLCz expression (P<0.04). In conclusion, microfluidic sorting and density gradient centrifugation resulted in a subpopulation of sperm with high quality parameters for ICSI. The probability of sperm-injected oocytes to develop into an embryo…
Advisors/Committee Members: Carnevale, Elaine (advisor), Grahman, James (advisor), Seidel, George (committee member), Gerrit, Bouma (committee member), Ann, Hess (committee member).
Subjects/Keywords: microfluidics; PLC zeta; stallion; PAWP; ICSI; sperm
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APA (6th Edition):
Gonzalez-Castro, R. A. (2018). Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection. (Doctoral Dissertation). Colorado State University. Retrieved from http://hdl.handle.net/10217/191446
Chicago Manual of Style (16th Edition):
Gonzalez-Castro, Raul A. “Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection.” 2018. Doctoral Dissertation, Colorado State University. Accessed April 16, 2021.
http://hdl.handle.net/10217/191446.
MLA Handbook (7th Edition):
Gonzalez-Castro, Raul A. “Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection.” 2018. Web. 16 Apr 2021.
Vancouver:
Gonzalez-Castro RA. Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection. [Internet] [Doctoral dissertation]. Colorado State University; 2018. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/10217/191446.
Council of Science Editors:
Gonzalez-Castro RA. Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection. [Doctoral Dissertation]. Colorado State University; 2018. Available from: http://hdl.handle.net/10217/191446
Queens University
2.
Shi, Mengqi.
Identification of YAP as sperm-PAWP’s predominant binding partner in MII-arrested oocytes and the relevance of this WWI domain modular interaction to oocyte activation
.
Degree: Biomedical and Molecular Sciences, 2016, Queens University
URL: http://hdl.handle.net/1974/14382
► PAWP, a candidate sperm-borne oocyte activating factor, induces oocyte activation and acts upstream of the calcium signalling pathway, however, PAWP’s downstream signalling pathway in oocyte…
(more)
▼ PAWP, a candidate sperm-borne oocyte activating factor, induces oocyte activation and acts upstream of the calcium signalling pathway, however, PAWP’s downstream signalling pathway in oocyte cytoplasm remains to be uncovered. Data from our lab suggested that the interacting partner of PAWP, at least in the frog (Xenopus laevis) model may be YAP, a highly expressed protein in amphibian and mammalian oocytes. Therefore, the objectives of this study were to confirm that PAWP’s predominant binding partner in Xenopus laevis oocyte is YAP; to determine if mammalian oocyte activation is also dependent on PAWP-YAP interaction; and to verify that the PAWP-YAP interaction during oocyte activation is dependent on the WWI domain module. By immunohistochemistry, YAP was localized predominantly in the cytosol of metaphase II-arrested Xenopus laevis oocytes, where presumably the PAWP-YAP interaction occurs. Utilizing Far Western blotting, YAP was identified as the predominant binding partner of PAWP, in metaphase II-arrested frog (Xenopus laevis), swine (Sus scrofa) and mouse (mus musculus) oocytes. The specificity of this interaction was then tested on Far Western blotting of mouse ovarian and oocyte cytosolic extracts, by competition with both wild-type and point-mutated recombinant WWI domains derived from YAP. The removal of GST from the wild-type WWI-GST fusion protein was a requirement for effective blockage of WWI module interaction between PAWP and YAP. As expected, the mutated WWI domain was ineffective in inhibiting the PAWP-YAP interaction. To conclude, this study identified YAP as the predominant binding partner of PAWP in both amphibian and mammalian oocytes, and showed this interaction is dependent on the WWI modular interaction. The results allow us to test the functional relevance of this WWI modular interaction during oocyte activation in vivo, in the future.
Subjects/Keywords: Oocyte Activation
;
Sperm-borne Oocyte Activating Factor
;
PAWP
;
Fertilization
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APA (6th Edition):
Shi, M. (2016). Identification of YAP as sperm-PAWP’s predominant binding partner in MII-arrested oocytes and the relevance of this WWI domain modular interaction to oocyte activation
. (Thesis). Queens University. Retrieved from http://hdl.handle.net/1974/14382
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Shi, Mengqi. “Identification of YAP as sperm-PAWP’s predominant binding partner in MII-arrested oocytes and the relevance of this WWI domain modular interaction to oocyte activation
.” 2016. Thesis, Queens University. Accessed April 16, 2021.
http://hdl.handle.net/1974/14382.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Shi, Mengqi. “Identification of YAP as sperm-PAWP’s predominant binding partner in MII-arrested oocytes and the relevance of this WWI domain modular interaction to oocyte activation
.” 2016. Web. 16 Apr 2021.
Vancouver:
Shi M. Identification of YAP as sperm-PAWP’s predominant binding partner in MII-arrested oocytes and the relevance of this WWI domain modular interaction to oocyte activation
. [Internet] [Thesis]. Queens University; 2016. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/1974/14382.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Shi M. Identification of YAP as sperm-PAWP’s predominant binding partner in MII-arrested oocytes and the relevance of this WWI domain modular interaction to oocyte activation
. [Thesis]. Queens University; 2016. Available from: http://hdl.handle.net/1974/14382
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Queens University
3.
Qin, Zheng.
Contribution of a Sperm Protein, PAWP, to the Signal Transduct Pathway During Vertebrate Fertilization
.
Degree: Anatomy and Cell Biology, 2008, Queens University
URL: http://hdl.handle.net/1974/992
► PAWP, postacrosomal sheath WW domain binding protein, is a novel sperm protein identified as a candidate sperm borne, oocyte-activating factor (SOAF). PAWP induces both early…
(more)
▼ PAWP, postacrosomal sheath WW domain binding protein, is a novel sperm protein identified as a candidate sperm borne, oocyte-activating factor (SOAF). PAWP induces both early and later egg activation events including meiotic resumption, pronuclear formation and egg cleavage. Based on the fact that calcium increase is universally accepted as the sole requirement for egg activation, we hypothesized that PAWP is an upstream regulator of the calcium signaling pathway during fertilization. Intracellular calcium increase was detected by two-photon laser scanning fluorescence microscopy following microinjection of recombinant PAWP into Xenopus oocytes, bolstering our hypothesis and suggesting the involvement of a novel PAWP-mediated signaling pathway during fertilization. The N-terminal of PAWP shares a high homology to WW domain binding protein while the C-terminal half contains a functional PPXY motif, which allows it to interact with group I WW domain proteins. These structural considerations together with published data indicating that PPXY synthetic peptide derived from PAWP inhibits ICSI-induced fertilization led to the hypothesis that PAWP triggers egg activation by binding to a group I WW domain protein in the oocyte. By far-Western analysis of oocyte cytoplasmic fraction, PAWP was found to bind to a 52 kDa protein. The competitive inhibition studies with PPXY synthetic peptide, WW domain constructs, and their point mutants demonstrated that the interaction between PAWP and its binding partner is specifically via the PPXY-WW domain module. The 52 kDa protein band crossreacted with antibodies against group I WW domain protein YAP in Western blot assay, indicating that this 52 kDa PAWP binding partner is either YAP or a YAP-related protein. In addition, the far-Western competitive inhibition studies with recombinant GST fusion protein YAP and another WW domain-containing protein, TAZ, demonstrated that the binding of PAWP to its binding partner was significantly reduced by TAZ, providing evidence that TAZ could be the 52 kDa protein candidate. Mass spectrometry was employed to identify this PAWP binding partner candidate. However, due to the low abundance of the candidate protein and the complexity of the sample, several strategies are still needed to enrich this protein. This study correlates PAWP induced meiotic resumption and calcium efflux at fertilization and uncovers a 52 kDa candidate WW domain protein in the oocyte cytoplasm that most likely interacts with PAWP to trigger egg activation.
Subjects/Keywords: PAWP
;
Signal Transduct Pathway
;
Fertilization
Record Details
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Qin, Z. (2008). Contribution of a Sperm Protein, PAWP, to the Signal Transduct Pathway During Vertebrate Fertilization
. (Thesis). Queens University. Retrieved from http://hdl.handle.net/1974/992
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Qin, Zheng. “Contribution of a Sperm Protein, PAWP, to the Signal Transduct Pathway During Vertebrate Fertilization
.” 2008. Thesis, Queens University. Accessed April 16, 2021.
http://hdl.handle.net/1974/992.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Qin, Zheng. “Contribution of a Sperm Protein, PAWP, to the Signal Transduct Pathway During Vertebrate Fertilization
.” 2008. Web. 16 Apr 2021.
Vancouver:
Qin Z. Contribution of a Sperm Protein, PAWP, to the Signal Transduct Pathway During Vertebrate Fertilization
. [Internet] [Thesis]. Queens University; 2008. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/1974/992.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Qin Z. Contribution of a Sperm Protein, PAWP, to the Signal Transduct Pathway During Vertebrate Fertilization
. [Thesis]. Queens University; 2008. Available from: http://hdl.handle.net/1974/992
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Queens University
4.
Aarabi, Mahmoud.
A Functional, Comparative and Clinical Analysis of Sperm-Borne Oocyte Activating Factor, PAWP
.
Degree: Anatomy and Cell Biology, 2013, Queens University
URL: http://hdl.handle.net/1974/8354
► Successful fertilization depends upon the activation of metaphase II arrested oocytes by sperm-borne oocyte activating factor (SOAF). Failure of oocyte activation is considered as the…
(more)
▼ Successful fertilization depends upon the activation of metaphase II arrested oocytes by sperm-borne oocyte activating factor (SOAF). Failure of oocyte activation is considered as the cause of treatment failure in a proportion of infertile couples. SOAF induces the release of intracellular calcium in oocyte which leads to meiotic resumption and pronuclear formation. Calcium release is either in the form of single calcium transient in echinoderm and amphibian oocytes or several calcium oscillations in ascidian and mammalian oocytes. Although the SOAF attributes are established, it is not clear which sperm protein(s) play such role. Sperm postacrosomal WW binding protein (PAWP) satisfies a developmental criteria set for a candidate SOAF. This study shows that recombinant human PAWP protein or its transcript acts upstream of calcium release and fully activates the amphibian and mammalian oocytes. Interference trials provided evidence for the first time that PAWP mediates sperm-induced intracellular calcium release through a PPXY/WWI domain module in Xenopus, mouse and human oocytes. Clinical applications of PAWP were further investigated by prospective study on the sperm samples from patients undergoing intracytoplasmic sperm injection (ICSI). PAWP expression level, analyzed by flow cytometry, was correlated to ICSI success rate and embryonic development. This study also explored the developmental expression of the other SOAF candidate, PLCζ in male reproductive system and its function during fertilization. Our findings showed for the first time that PLCζ most likely binds to the sperm head surface during epididymal passage and is expressed in epididymis. We demonstrated that PLCζ is also compartmentalized early in spermiogenesis and thus could play an important role during spermiogenesis. Detailed analysis of in vitro fertilization revealed that PLCζ disappears from sperm head during acrosome reaction and is not detectable during sperm incorporation into the oocyte cytoplasm. In conclusion, this dissertation provides evidence for the essential non-redundant role of sperm PAWP in amphibian and mammalian fertilization; recommends PAWP as a biomarker for prediction of ICSI outcomes in infertile couples; and proposes that sperm PLCζ may have functions other than inducing oocyte activation during fertilization.
Subjects/Keywords: PLCz
;
Fertilization
;
Sperm
;
YAP
;
WWI Domain
;
Oocyte Activation
;
Intracytoplasmic Sperm Injection
;
Assisted Reproductive Technologies
;
Infertility
;
PAWP
Record Details
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Record Details
Similar Records
Cite
« Share
❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Aarabi, M. (2013). A Functional, Comparative and Clinical Analysis of Sperm-Borne Oocyte Activating Factor, PAWP
. (Thesis). Queens University. Retrieved from http://hdl.handle.net/1974/8354
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Aarabi, Mahmoud. “A Functional, Comparative and Clinical Analysis of Sperm-Borne Oocyte Activating Factor, PAWP
.” 2013. Thesis, Queens University. Accessed April 16, 2021.
http://hdl.handle.net/1974/8354.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Aarabi, Mahmoud. “A Functional, Comparative and Clinical Analysis of Sperm-Borne Oocyte Activating Factor, PAWP
.” 2013. Web. 16 Apr 2021.
Vancouver:
Aarabi M. A Functional, Comparative and Clinical Analysis of Sperm-Borne Oocyte Activating Factor, PAWP
. [Internet] [Thesis]. Queens University; 2013. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/1974/8354.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Aarabi M. A Functional, Comparative and Clinical Analysis of Sperm-Borne Oocyte Activating Factor, PAWP
. [Thesis]. Queens University; 2013. Available from: http://hdl.handle.net/1974/8354
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
. 
Open Access Theses and Dissertations