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The non-human primate as a model for mitochondrial inheritance
system is the result of disturbed membrane lipid distribution and related apoptotic signaling.
Conclusion: CD9 protein and mRNA are present at different stages of human oocytes. The role of CD9 in human sperm-egg fusion and fertilization failure requires further research.
P-446 The non-human primate as a model for mitochondrial inheritance. Carol A. Brenner, Ethan Jacoby. Univ of New Orleans, New Orleans, LA. Objective: Non-human primates are our closest relatives and their embryo morphology and preimplantation development closely resembles that of humans. It is for that reason why non-human primates such as rhesus monkeys may be used for animal models for mitochondrial inheritance. It has long been thought that transmission of mitochondria between generations is strictly maternal. However the development of more invasive techniques of assisted reproduction has increased that concern that the mechanisms of uni-parental mitochondrial maternal inheritance may be altered and as a result could compromise the integrity of the offspring as well as the adult. For example, there is now evidence for the persistence of human paternal mitochondria in “abnormal” human oocytes and embryos. The aim of this initial study is to establish the non-human primate as a model of mitochondrial inheritance. Design: Retrospective clinical study. Materials and Methods: Samples of rhesus bloods have been collected from mother, father and offspring from babies generated from normal IVF cycles. Samples have also been collected from discarded embryos prior to embryo transfer. Blood DNA was prepared using the Gentra Puragene DNA isolation kit while DNA from single oocytes, embryos and blastomeres were also isolated using a simple PCR lysis procedure. The DNA sequence for the D-loop from the HV1/HV2 regions was amplified using primate consensus primers. Samples from over 100 oocytes are in the process of being DNA sequenced. Results: So far data from a limited number of rhesus samples has established that mitochondrial are maternally inherited in normal IVF. Similarly, there is considerable sequence variation in the mitochondrial D-loop to identify maternal rhesus embryos as well as offspring. Conclusions: Non-human primates are an excellent model to study mitochondrial inheritance. By using such models we will be able to manipulate assisted reproductive technologies such as ICSI, germinal vesicle transplantation, embryo cell nuclear transfer (SCNT) as well as somatic cell nuclear transfer(SCNT) and evaluate whether the embryos exhibit any abnormal mitochondrial reprogramming or elimination of the paternal mitochondrial. It is possible that a failure or delay of sperm mitochondrion degradation by the oocyte could provide a window of opportunity for the survival of paternal mitochondria which may result in an abnormal heteroplasmic propagation of both maternal and paternal mtDNA in the primate embryo. P-447 CD9 is expressed on human oocytes. Serdar Coskun, Atif Elnour, Ali Hellani, Tawfig Gaafar. King Faisal Specialist Hosp and Research Ctr, Riyadh, Saudi Arabia; Sulaiman Al-Habib Medical Ctr, Riyadh, Saudi Arabia. Objective: CD9 is an integral membrane protein with four transmembrane domains and expressed in variety of cells including mouse oocytes. CD9 deficient mice have severely reduced female fertility because of sperm-egg fusion failure. Intracytoplasmic sperm injection (ICSI) of oocytes lacking CD9 showed normal fertilization which suggest an important role for CD9 in natural fertilization process. The objective of this study was to investigate the expression of CD9 in human oocytes. Design: Oocytes that were not used in ICSI after removing cumulus cells were utilized to detect CD9 protein and mRNA. Materials and Methods: Germinal vesicle (GV) and Metaphase (M) I stage human oocytes were obtained from women undergoing ICSI. Some of MI oocytes were cultured overnight to obtain MII stage oocytes. CD9 protein at the different stages of oocytes was detected by immunofluorescence staining, and mRNA from individual oocytes by fluorescent reverse transcription-polymerase chain reaction (RT-PCR). Results: A total of 70 oocytes were utilized in immunofluorescent staining (11 negative control, 25 GV, 23 MI and 21 MII). All the oocytes were positively stained for CD9 protein while negative controls did not exhibit any staining. Among 12 oocytes used in RT-PCR, 10 of them showed CD9 mRNA expression.
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Abstracts
P-448 The effect of bilateral salpengectomy and tubal ligation on ovarian function. Munire Erman Akar, Tonguc Gunduz, Omur Taskin, Bilal Trak. Medical Sch of Akdeniz Univ, Antalya, Turkey. Objective: To evaluate the effects of bilateral salpingectomy and tubal ligation on ovarian function, in a rat model. Study design: Forty-eight female Whistar albino rats weighing 200-250g are divided equally into three groups. All rats underwent laparotomy, while no specific intervention was made to the first group. Bilateral tubal ligation by Pomeroy’s technique, bilateral salpingectomy was done to the second and third groups, respectively.Serum cholesterol,triglyceride,HDL, LDL, E2, FSH, and LH levels were measured before and four months after the operation. All rats were then individually caged and fed on demand for 4 months. Afterwards, the rats were sacrificed and underwent bilateral oophorectomy. Materials and Methods: A pathologist blinded to the groups made histological examination by counting number of healthy tertiary follicles and corpora lutea in each ovary. The results of the groups were statistically compared by one-way ANOVA using post-hoc Bonferroni correction. Results: Rats in group 1 had significantly higher number of healthy tertiary follicles than every other group. Rats in group 1 also had significantly more corpora lutea than those in group 2 and group 3. There was no significant difference in serum cholesterol,triglyceride,LDL and HDL levels between group 1,group 2 and group 3.Rats in group 1 had significantly lower LH and FSH levels when compared with group 2 and group 3. Conclusion: Salpingectomy and tubal ligation may affect ovarian function, which in turn may reflect to ovarian histology and serum hormon levels in rats.
P-450 Platelet-activating factor-receptor antibody impairs human sperm-ova interaction. Scott M. Slayden, Andrew A. Toledo, Dorothy Mitchell-Leef, Carlene W. Elsner, Hilton I. Kort, William E. Roudebush. Reproductive Biology Assoc, Atlanta, GA. Objective: Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3phosphocholine; PAF], a unique and potent phospholipid, is present in human sperm and its content is related to sperm motility, fertilization and pregnancy potential. Sperm with poor motility have altered mRNA PAFreceptor sequences as well as depressed PAF-receptor mRNA and protein expression levels. Sperm may be unable to produce sufficient PAF quantities and, or be able to bind with the PAF ligand, and, or be incapable of processing the signal. Additionally, anti-PAF antibodies inhibit embryo development and phospholipid antibodies interfere with pregnancy. Therefore the study objective was to determine the effect of antibodies to the PAF-receptor on gamete interaction. Design: Human sperm subjected to the sperm penetration assay (SPA) in the presence of PAF-receptor antibody. Materials and Methods: Normal, motile sperm were isolated from semen by silica particle suspension-washing (Promotor, CERES Fertility, San Diego, CA, USA) and incubated in test yolk buffer (Refrigeration Medium, Irvine Scientific, Santa Ana, CA, USA) for 48-72 hours at 4°C to induce capacitation. Zona-free hamster ova (EmbryoTech Laboratories, Wilmington, MA, USA) and washed capacitated human sperm were co-incubated (37°C; 3.5 hrs) in the presence or absence (control) of a PAF-receptor monoclonal antibody [1: 10] in modified HTF (CERES Fertility). Following incubation, ova were washed free of bound non-penetrated sperm cells and evaluated for penetrations under phase contrast light microscopy. Data were analyzed by Student’s t-test. Results: A total of 26 patient specimens were subjected to the SPA in the presence of the PAF-receptor monoclonal antibody. There was a significant (P⬍0.001) reduction in the mean (⫹SEM) number of penetrations per patient in the PAF-receptor antibody group (4.668⫹0.835) than the control group (10.742⫹1.207). Conclusion: The data suggests that endogenous PAF found in human
Vol. 80, Suppl. 3, September 2003
Conclusion: CD9 protein and mRNA are present at different stages of human oocytes. The role of CD9 in human sperm-egg fusion and fertilization failure requires further research.
P-446 The non-human primate as a model for mitochondrial inheritance. Carol A. Brenner, Ethan Jacoby. Univ of New Orleans, New Orleans, LA. Objective: Non-human primates are our closest relatives and their embryo morphology and preimplantation development closely resembles that of humans. It is for that reason why non-human primates such as rhesus monkeys may be used for animal models for mitochondrial inheritance. It has long been thought that transmission of mitochondria between generations is strictly maternal. However the development of more invasive techniques of assisted reproduction has increased that concern that the mechanisms of uni-parental mitochondrial maternal inheritance may be altered and as a result could compromise the integrity of the offspring as well as the adult. For example, there is now evidence for the persistence of human paternal mitochondria in “abnormal” human oocytes and embryos. The aim of this initial study is to establish the non-human primate as a model of mitochondrial inheritance. Design: Retrospective clinical study. Materials and Methods: Samples of rhesus bloods have been collected from mother, father and offspring from babies generated from normal IVF cycles. Samples have also been collected from discarded embryos prior to embryo transfer. Blood DNA was prepared using the Gentra Puragene DNA isolation kit while DNA from single oocytes, embryos and blastomeres were also isolated using a simple PCR lysis procedure. The DNA sequence for the D-loop from the HV1/HV2 regions was amplified using primate consensus primers. Samples from over 100 oocytes are in the process of being DNA sequenced. Results: So far data from a limited number of rhesus samples has established that mitochondrial are maternally inherited in normal IVF. Similarly, there is considerable sequence variation in the mitochondrial D-loop to identify maternal rhesus embryos as well as offspring. Conclusions: Non-human primates are an excellent model to study mitochondrial inheritance. By using such models we will be able to manipulate assisted reproductive technologies such as ICSI, germinal vesicle transplantation, embryo cell nuclear transfer (SCNT) as well as somatic cell nuclear transfer(SCNT) and evaluate whether the embryos exhibit any abnormal mitochondrial reprogramming or elimination of the paternal mitochondrial. It is possible that a failure or delay of sperm mitochondrion degradation by the oocyte could provide a window of opportunity for the survival of paternal mitochondria which may result in an abnormal heteroplasmic propagation of both maternal and paternal mtDNA in the primate embryo. P-447 CD9 is expressed on human oocytes. Serdar Coskun, Atif Elnour, Ali Hellani, Tawfig Gaafar. King Faisal Specialist Hosp and Research Ctr, Riyadh, Saudi Arabia; Sulaiman Al-Habib Medical Ctr, Riyadh, Saudi Arabia. Objective: CD9 is an integral membrane protein with four transmembrane domains and expressed in variety of cells including mouse oocytes. CD9 deficient mice have severely reduced female fertility because of sperm-egg fusion failure. Intracytoplasmic sperm injection (ICSI) of oocytes lacking CD9 showed normal fertilization which suggest an important role for CD9 in natural fertilization process. The objective of this study was to investigate the expression of CD9 in human oocytes. Design: Oocytes that were not used in ICSI after removing cumulus cells were utilized to detect CD9 protein and mRNA. Materials and Methods: Germinal vesicle (GV) and Metaphase (M) I stage human oocytes were obtained from women undergoing ICSI. Some of MI oocytes were cultured overnight to obtain MII stage oocytes. CD9 protein at the different stages of oocytes was detected by immunofluorescence staining, and mRNA from individual oocytes by fluorescent reverse transcription-polymerase chain reaction (RT-PCR). Results: A total of 70 oocytes were utilized in immunofluorescent staining (11 negative control, 25 GV, 23 MI and 21 MII). All the oocytes were positively stained for CD9 protein while negative controls did not exhibit any staining. Among 12 oocytes used in RT-PCR, 10 of them showed CD9 mRNA expression.
S268
Abstracts
P-448 The effect of bilateral salpengectomy and tubal ligation on ovarian function. Munire Erman Akar, Tonguc Gunduz, Omur Taskin, Bilal Trak. Medical Sch of Akdeniz Univ, Antalya, Turkey. Objective: To evaluate the effects of bilateral salpingectomy and tubal ligation on ovarian function, in a rat model. Study design: Forty-eight female Whistar albino rats weighing 200-250g are divided equally into three groups. All rats underwent laparotomy, while no specific intervention was made to the first group. Bilateral tubal ligation by Pomeroy’s technique, bilateral salpingectomy was done to the second and third groups, respectively.Serum cholesterol,triglyceride,HDL, LDL, E2, FSH, and LH levels were measured before and four months after the operation. All rats were then individually caged and fed on demand for 4 months. Afterwards, the rats were sacrificed and underwent bilateral oophorectomy. Materials and Methods: A pathologist blinded to the groups made histological examination by counting number of healthy tertiary follicles and corpora lutea in each ovary. The results of the groups were statistically compared by one-way ANOVA using post-hoc Bonferroni correction. Results: Rats in group 1 had significantly higher number of healthy tertiary follicles than every other group. Rats in group 1 also had significantly more corpora lutea than those in group 2 and group 3. There was no significant difference in serum cholesterol,triglyceride,LDL and HDL levels between group 1,group 2 and group 3.Rats in group 1 had significantly lower LH and FSH levels when compared with group 2 and group 3. Conclusion: Salpingectomy and tubal ligation may affect ovarian function, which in turn may reflect to ovarian histology and serum hormon levels in rats.
P-450 Platelet-activating factor-receptor antibody impairs human sperm-ova interaction. Scott M. Slayden, Andrew A. Toledo, Dorothy Mitchell-Leef, Carlene W. Elsner, Hilton I. Kort, William E. Roudebush. Reproductive Biology Assoc, Atlanta, GA. Objective: Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3phosphocholine; PAF], a unique and potent phospholipid, is present in human sperm and its content is related to sperm motility, fertilization and pregnancy potential. Sperm with poor motility have altered mRNA PAFreceptor sequences as well as depressed PAF-receptor mRNA and protein expression levels. Sperm may be unable to produce sufficient PAF quantities and, or be able to bind with the PAF ligand, and, or be incapable of processing the signal. Additionally, anti-PAF antibodies inhibit embryo development and phospholipid antibodies interfere with pregnancy. Therefore the study objective was to determine the effect of antibodies to the PAF-receptor on gamete interaction. Design: Human sperm subjected to the sperm penetration assay (SPA) in the presence of PAF-receptor antibody. Materials and Methods: Normal, motile sperm were isolated from semen by silica particle suspension-washing (Promotor, CERES Fertility, San Diego, CA, USA) and incubated in test yolk buffer (Refrigeration Medium, Irvine Scientific, Santa Ana, CA, USA) for 48-72 hours at 4°C to induce capacitation. Zona-free hamster ova (EmbryoTech Laboratories, Wilmington, MA, USA) and washed capacitated human sperm were co-incubated (37°C; 3.5 hrs) in the presence or absence (control) of a PAF-receptor monoclonal antibody [1: 10] in modified HTF (CERES Fertility). Following incubation, ova were washed free of bound non-penetrated sperm cells and evaluated for penetrations under phase contrast light microscopy. Data were analyzed by Student’s t-test. Results: A total of 26 patient specimens were subjected to the SPA in the presence of the PAF-receptor monoclonal antibody. There was a significant (P⬍0.001) reduction in the mean (⫹SEM) number of penetrations per patient in the PAF-receptor antibody group (4.668⫹0.835) than the control group (10.742⫹1.207). Conclusion: The data suggests that endogenous PAF found in human
Vol. 80, Suppl. 3, September 2003