Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro

Reproduction ◽  
2000 ◽  
pp. 127-135 ◽  
Author(s):  
W Bone ◽  
NG Jones ◽  
G Kamp ◽  
CH Yeung ◽  
TG Cooper
Keyword(s):  
Zona Pellucida ◽  
Glucose Utilization ◽  
Cumulus Cells ◽  
Glycolytic Enzyme ◽  
Male Rats ◽  
Fertilization In Vitro ◽  
Swimming Pattern ◽  
Principal Piece ◽  
Free Media

The effects of the male antifertility agent ornidazole on glycolysis as a prerequisite for fertilization were investigated in rats. Antifertility doses of ornidazole inhibited glycolysis within mature spermatozoa as determined from the lack of glucose utilization, reduced acidosis under anaerobic conditions and reduced glycolytic enzyme activity. As a consequence, cauda epididymidal spermatozoa from ornidazole-fed rats were unable to fertilize rat oocytes in vitro, with or without cumulus cells, which was not due to transfer of an inhibitor in epididymal fluid with the spermatozoa. Under IVF conditions, binding to the zona pellucida was reduced in spermatozoa from ornidazole-fed males and the spermatozoa did not undergo a change in swimming pattern, which was observed in controls. The block to fertilization could be explained by the disruption of glycolysis-dependent events, since reduced binding to the zona pellucida and a lack of kinematic changes were demonstrated by control spermatozoa in glucose-free media in the presence of respiratory substrates. The importance of glycolysis for binding to, and penetration of, the zona pellucida, and hyperactivation in rats is discussed in relation to the glycolytic production of ATP in the principal piece in which local deprivation of energy may explain the reduced force of spermatozoa from ornidazole-fed males.

319 PROTEIN GENE PRODUCT 9.5 REGULATES SPERM PENETRATION DURING PORCINE FERTILIZATION IN VITRO

2007 ◽  
Vol 19 (1) ◽  
pp. 275
Author(s):  
Y. J. Yi ◽  
G. Manandhar ◽  
M. Sutovsky ◽  
C. S. Park ◽  
P. Sutovsky
Keyword(s):  
Gene Product ◽  
Zona Pellucida ◽  
Protein Gene ◽  
Cumulus Cells ◽  
Fertilization In Vitro ◽  
Protein Gene Product 9.5 ◽  
Dapi Staining ◽  
Protein Gene Product ◽  
Oviductal Fluid

The protein gene product 9.5 (PGP9.5) belongs to a family of ubiquitin C-terminal hydrolases (UCHs), which regenerate monoubiquitin from ubiquitin–protein complexes or polyubiquitin chains by cleaving the amide linkage next to the C-terminal glycine of ubiquitin. Identified in the acrosome of boar spermatozoa, we hypothesized that PGP9.5 might regulate sperm–zona pellucida interactions during porcine IVF. The cumulus–oocyte complexes isolated from slaughterhouse ovaries were cultured in TCM-199 media for 44 h at 38.5�C, 5% CO2 in air. After completion of in vitro maturation (IVM), cumulus cells were removed by 0.1% hyaluronidase, and metaphase II (MII) oocytes were used for IVF. In Experiment 1, oocytes were co-incubated with different sperm concentrations (1 � 106, 5 � 105, and 1 � 105 sperm mL-1) in TBM medium with or without anti-PGP9.5 antibody (1 : 50 dilution). In Experiment 2, oocytes were inseminated with 1 � 106 sperm mL-1 in TBM medium containing different concentrations of extracted oviductal fluids (0, 0.1, 0.5, 1, 2, and 3 �g mL-1) for 6 h. After IVF, oocytes were transferred into NCSU23 medium containing 0.4% BSA for further culture. The fertilization rates were evaluated by DAPI staining at 13 to 19 h. Data were analyzed by ANOVA and Duncan's multiple range test using the SAS program. Polyspermy was increased by the addition of anti-PGP9.5 antibody to the IVF medium (56.5–60.2% at polyspermy). This PGP9.5-antibody-induced polyspermy increase was sustained even with decreasing sperm concentrations. The polyspermy rates were reduced by the addition of isolated porcine oviductal fluid to IVF medium (50.4, 44.8, 28.0, 31.1, 1.6, and 0.0% at oviductal fluid concentrations of 0, 0.1, 0.5, 1, 2, and 3 µg mL−1, respectively). Biochemical analysis by Western blotting detected the appropriate 24-kDa PGP9.5 band in porcine oviductal fluid used for these experiments. Enzymatic UCH activity comparable to activity of recombinant UCH-L3 was detected in sperm extract, whole spermatozoa, and isolated oviductal fluid by fluorometric assay using fluorogenic UCH-substrate ubiquitin-AMC. This UCH activity was not reduced by the general protease inhibitor phenyl methyl sylfonyl fluoride, but it was reduced in a statistically significant manner (P < 0.05) by the specific UCH-inhibitor ubiquitin aldehyde. In conclusion, the polyspermy increased with different concentrations of sperm in the anti-PGP9.5 antibody, and PGP9.5 was detected in oviductal fluids, suggesting that PGP9.5 is involved in the sperm–zona pellucida interaction during porcine fertilization. This work was supported by the US Department of Agriculture (USDA-NRI grant #2002-35203-12237 to P.S.), the F21C Program of The University of Missouri–Columbia (P.S.), and the ERC Program of the Korea Science and Engineering Foundation (KOSEF grant no. R11-2002-100-00000-0 to Y.J. Yi and C.S. Park).

scholarly journals Changes in the properties and composition of zona pellucida of pigs during fertilization in vitro

Reproduction ◽  
1992 ◽  
Vol 95 (2) ◽  
pp. 431-440 ◽  
Author(s):  
Y. Hatanaka ◽  
T. Nagai ◽  
T. Tobita ◽  
M. Nakano
Keyword(s):  
Zona Pellucida ◽  
Fertilization In Vitro

IN VITRO STUDIES ON CARBOHYDRATE METABOLISM IN THE VITAMIN-B6-DEPRIVED RAT

1955 ◽  
Vol 33 (1) ◽  
pp. 562-567
Author(s):  
John R. Beaton
Keyword(s):  
Carbohydrate Metabolism ◽  
Vitamin B6 ◽  
Glucose Utilization ◽  
Female Rats ◽  
Male Rats ◽  
Vitamin B ◽  
Significant Difference ◽  
Aldolase Activity ◽  
Food Consumed

Following earlier studies on carbohydrate metabolism in the vitamin-B6-deprived rat, in vitro investigations have been carried out. In all cases, comparisons were made between tissues from vitamin-B6-deprived and pair-fed control animals so that differences in the amount of food consumed would not affect the interpretation of experimental results. No significant difference was found in glucose utilization by muscle nor in liver cytochrome oxidase activity. Liver aldolase activity was significantly decreased and the activity of plasma alkaline phosphatase was significantly increased in the vitamin-B6-deprived rats. In vitamin-B6-deprived female rats, but not male rats, liver catalase activity was significantly increased. These results are discussed in the light of earlier observations indicating disturbances in carbohydrate metabolism in the vitamin-B6-deprived rat.

203 PRETREATMENT OF BOVINE SPERM OR OOCYTE WITH ANTIBODY TO LIPOCALIN-TYPE PROSTAGLANDIN D SYNTHASE INHIBITS FERTILIZATION

2008 ◽  
Vol 20 (1) ◽  
pp. 180
Author(s):  
R. F. Gonçalves ◽  
V. H. Barnabe ◽  
G. J. Killian
Keyword(s):  
Polyclonal Antibody ◽  
Gradient Centrifugation ◽  
Cumulus Cells ◽  
Fertilization In Vitro ◽  
Prostaglandin D Synthase ◽  
Rabbit Polyclonal Antibody ◽  
Development In Vitro ◽  
Triton X ◽  
Bovine Oocytes

Lipocalin-type prostaglandin D synthase (L-PGDS) has been identified in cow uterine tube fluid (UTF), and as fertility-associated protein in the Holstein bull seminal plasma, but its function is unclear. A previous study demonstrated that L-PGDS is associated with the bovine zona pellucida, and that antibody incubated with UTF decreased embryo development in vitro. This study was conducted to determine whether IVF of bovine oocytes would be affected by pretreating either the sperm or oocytes, or both, with L-PGDS antibody. In vitro-matured bovine oocytes were incubated for 1 h in IVF TALP medium supplemented with penicillamine, hypotaurine, epinephrine, and heparin containing (a) no antibody, or (b) a rabbit polyclonal antibody against recombinant bovine L-PGDS (α L-PGDS; 1:2000). Frozen–thawed spermatozoa were washed by a 45:90% layered Percoll gradient centrifugation and incubated for 1 h in IVF TALP with (a) no antibody, or (b) α L-PGDS. For this study we had 4 different treatments: (1) no antibody (control), (2) α L-PGDS at fertilization time, (3) α L-PGDS-treated oocytes, or (4) α L-PGDS-treated sperm. Oocytes were inseminated with 10 � 104 washed spermatozoa in 4-well culture dishes. After 18 h (39�C, 5% CO2 in air), oocytes were vortexed to remove cumulus cells and accessory spermatozoa, and fixed in 3.7% paraformaldehyde and 10% Triton X–100 for 1 h. Oocytes were washed and transferred to a solution with PBS, 0.3% BSA, and 1% Triton for 1 h, stained with Hoechst 33342, and observed in the presence of 2 pronuclei in the cytoplasm. There were 4 replicates of 200 to 250 oocytes for fertilization assays. Data were analyzed by SAS. Addition of α L-PGDS with sperm, oocytes, or both significantly decreased fertilization (P < 0.05) compared with the control: (1) 89.2 � 2.0%; (2) 19.4 � 2.0%; (3) 27.2 � 3.1%; or (4) 14.1 � 3.4%. These studies demonstrated that a rabbit polyclonal antibody against recombinant bovine L-PGDS reacts with both oocytes and spermatozoa, resulting in inhibition of fertilization in vitro, and has a possible role in bovine fertilization. This study was supported by FAPESP #2007/00363-5 and #2006/06008-0, Brazil.

Processing of human IVF/IVM oocytes for single cell RNA sequencing

2020 ◽  
Author(s):  
Kristina W. Olsen ◽  
Jennifer R. Gruhn ◽  
Eva R. Hoffmann ◽  
Marie Louise Grøndahl
Keyword(s):  
Single Cell ◽  
Fertility Preservation ◽  
Rna Sequencing ◽  
Zona Pellucida ◽  
Cumulus Cells ◽  
Antral Follicles ◽  
Step Procedure ◽  
Single Cell Rna Sequencing ◽  
Human Ivf

Abstract The protocol describes the step-by-step procedure of isolating and preparing human oocytes 1) oocytes donated in connection with IVF/ICSI treatment and 2) in vitro matured (IVM) oocytes from small antral follicles donated in connection to fertility preservation. It details how cumulus cells and the zona pellucida are enzymatically removed to ensure that only the oocyte is analyzed. We have successfully used this protocol in connection with SMART-Seq2 (Takara).This oocyte preparation has recently been used in Sankar et al. 2020 (Sankar et al. 2020).

scholarly journals 25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 162
Author(s):  
S. Akagi ◽  
B. Tsuneishi ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Zona Pellucida ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Functional Peptide ◽  
Vitro Development

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378). Table 1. Development, cell number, and ICM ratio of NT aggregates

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