Effects of oocyte collection time and injection position on pronucleus formation and blastocyst development in round spermatid injection in mouse

Zygote ◽  
2005 ◽  
Vol 13 (3) ◽  
pp. 249-253 ◽  
Author(s):  
Man-Xi Jiang ◽  
Yue-Liang Zheng ◽  
Shu-Zhen Liu ◽  
Cai-Xia Yang ◽  
Li-Sheng Zhang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Round Spermatid ◽  
Blastocyst Development ◽  
Oocyte Collection ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Round Spermatid Injection ◽  
Viable Embryo ◽  
Collection Time

The injection of spermatozoa into mouse, human and rabbit oocytes at specific times and positions can result in different rates of viable embryo development. However, it is not clear how the timing and position of round spermatid injection (ROSI) affect pronucleus (PN) formation and blastocyst development of mice. First, we determined the changes in relative position of the first polar body and the spindle, carried out ROSI from 11.5 to 13 h post-hCG administration, then activated by Sr2+, and finally compared the development of ROSI zygotes, including the formation of pronuclei and development of blastocyst. Between 11.5 and 13 h post-hCG administration, the rate of 2PN formation by ROSI at 3 o'clock was the highest among all treated oocytes. Moreover, the blastocyst rate of zygotes with two pronuclei (2PN) was up to 27.41%. These results suggest that the time and position of ROSI can significantly influence the formation of 2PN, that the rates of 2PN formation are closely correlated with blastocyst formation and that the formation of 2PN is necessary for later embryo development.

scholarly journals Dielectrophoretic behavior of in vitro-derived bovine metaphase II oocytes and zygotes and its relation to in vitro embryonic developmental competence and mRNA expression pattern

Reproduction ◽  
2007 ◽  
Vol 133 (5) ◽  
pp. 931-946 ◽  
Author(s):  
Salilew-Wondim Dessie ◽  
Franca Rings ◽  
Michael Hölker ◽  
Markus Gilles ◽  
Danyel Jennen ◽  
...  
Keyword(s):  
Polar Body ◽  
Mrna Level ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Non Invasive ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Selection Of ◽  
Metaphase Ii Oocytes

Selecting developmentally competent oocytes and zygotes based on their morphology is more often influenced by personal judgments and lacks universal standards. Therefore, this experiment was conducted to investigate the rate of development and mRNA level of dielectrophoretically separated oocytes and zygotes to validate dielectrophoresis (DEP) as non-invasive option for selection of oocytes and zygotes. In the first experiment, metaphase II oocytes with (PB+) and without (PB−) first polar body and zygotes were subjected to DEP at 4 MHz and 450 μm electrode distance and classified into fast, very fast, slow, and very slow depending on the time elapsed to reach one of the electrodes in the electric field. Parthenogenetic activation was employed to monitor the embryonic development of dielectrophoretically classified oocytes. The result revealed that at 6 and 7 days of post-activation, the blastocyst rate of very slow dielectrophoretic PB+and PB−oocytes was significantly (P< 0.05) lower than other groups. Similarly, in zygotes, the blastocyst rate at 7 days post-insemination was higher (P< 0.05) in the very fast dielectrophoretic categories when compared with the slow and very slow categories. In the second experiment, mRNA level was analyzed in the very fast and very slow dielectrophoretic PB+oocytes and zygotes respectively using the bovine cDNA microarray. The result showed that 36 and 42 transcripts were differentially regulated between the very fast and very slow dielectrophoretic categories PB+oocytes and zygotes respectively. In conclusion, dielectrophoretically separated oocytes and zygotes showed difference in the rate of blastocyst development accompanied by difference in transcriptional abundances.

379 EFFECT OF SUPPLEMENTARY INOSITOL 1,4,5-TRIPHOSPHATE INJECTION ON FERTILIZATION AND EMBRYO DEVELOPMENT AFTER BOVINE ICSI

2007 ◽  
Vol 19 (1) ◽  
pp. 305
Author(s):  
T. K. Suh ◽  
G.E. Seidel, Jr
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Formation Rate ◽  
Cortical Granule ◽  
Blastocyst Development ◽  
Exact Test ◽  
Injection Process ◽  
First Polar Body ◽  
Membrane Bound

During fertilization in mammals, sperm membrane-bound phospholipase C zeta induces breakdown of ooplasmic membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP2), which leads to the production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The IP3 induces intracellular Ca2+ oscillations ([Ca2+]i), which trigger inactivation of maturation-promoting factor (MPF) and mitogen-activated kinase (MAPK), resulting in decondensation of the sperm head, resumption of meiosis, extrusion of a second polar body, cortical granule exocytosis, formation of pronuclei (PN), and entry into the first cell cycle. In bovine ICSI, injection of a single spermatozoon into an oocyte does not consistently induce [Ca2+]i oscillations, as was observed in IVF, and this may, at least in part, explain the low rates of fertilization and embryo development. Although IP3 has been used as a powerful activator for nuclear transferred zygotes or parthenogenetic oocytes, few studies have evaluated the effect of IP3 injection on normal fertilization and embryo development after ICSI. The objective of this study was to determine the effect of injecting IP3 during bovine ICSI on fertilization and embryo development in vitro. Chemically defined media (CDM) were used throughout (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585–596). The injection volume of IP3, which was dissolved in calcium- and magnesium-free PBS, was approximately 0.01 nL, and the total dose for injection along with a spermatozoon was about 250 �M, which was determined by the 2PN formation rate in preliminary experiments. Semen from 3 bulls was used to produce embryos in 5 replicates. Oocytes obtained from slaughterhouse ovaries were matured in vitro in CDM-M for 22–23 h under 5% CO2 in air at 38.5�C, and oocytes with a first polar body were used for ICSI. Motile sperm from frozen–thawed semen were used for sperm injection, with or without IP3 in a 50-�L drop of GMOPS medium, with a piezo-driven pipet of 7–8 �m inner diameter. After ICSI, presumptive zygotes were cultured in CDM-1 for 3 days, and then in CDM-2 for 5 days at 39�C under 5% CO2/5% O2/90% N2. Cleavage and blastocyst development were evaluated at the end of each culture period. Data were subjected to Fisher&apos;s exact test. Cleavage in control and IP3 groups was 36.4 and 50.0%, respectively (P &lt; 0.05). Respective blastocyst rates per oocyte were 5.5 and 13.0% (P &gt; 0.05). This study showed that injection of IP3 during the sperm injection process improved cleavage of bovine oocytes after ICSI.

Effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection rabbit embryos

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Yue-Liang Zheng ◽  
Man-Xi Jiang ◽  
Yan-Ling Zhang ◽  
Qing-Yuan Sun ◽  
Da-Yuan Chen
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
First Polar Body ◽  
Injection Methods ◽  
Blastocyst Rate ◽  
Repeated Aspiration ◽  
Rabbit Embryos

This study assessed the effects of oocyte age, cumulus cells and injection methods on in vitro development of intracytoplasmic sperm injection (ICSI) rabbit embryos. Oocytes were recovered from female rabbits superovulated with PMSG and hCG, and epididymal sperm were collected from a fertile male rabbit. The oocyte was positioned with the first polar body at 12 o'clock position, and a microinjection needle containing a sperm was inserted into the oocyte at 3 o'clock. Oolemma breakage was achieved by aspirating ooplasm, and the aspirated ooplasm and sperm were re-injected into the oocyte. The injected oocytes were cultured in M199 medium containing 10% fetal calf serum at 38 °C with 5% CO2 in air. The results showed that oocytes injected at 1 h post-collection produced a higher (p<0.05) fertilization rate than those injected at 4 or 7 h post-collection. Blastocyst rate in the 1 h group was higher (p<0.05) than in the 7 h group. Denuded oocytes (group A) and oocytes with cumulus cells (group B) were injected, respectively. Rates of fertilization and development of ICSI embryos were not significantly different (p<0.05) between the two groups. Four ICSI methods were applied in this experiment. In methods 1 and 2, the needle tip was pushed across half the diameter of the oocyte, and oolemma breakage was achieved by either a single aspiration (method 1) or repeated aspiration and expulsion (method 2) of ooplasm. In methods 3 and 4, the needle tip was pushed to the oocyte periphery opposite the puncture site, and oolemma breakage was achieved by either a single aspiration (method 3) or repeated aspiration and expulsion (method 4) of ooplasm. Fertilization rate in method 2 was significantly higher (p<0.05) than in methods 1 and 3. Blastocyst rates were not significantly different (p<0.05) among methods 1, 3 and 4, but method 2 produced a higher (p<0.05) blastocyst rate than method 3.

111 SLOW FREEZING AND VITRIFICATION OF IN VITRO-MATURED DOMESTIC CAT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Slow Freezing ◽  
Developmental Potential ◽  
Freezing Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Chi Square Analysis

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

98 BLASTOCYST DEVELOPMENT RATE OF CLONED CAT EMBRYOS USING SERIAL CLONING

2007 ◽  
Vol 19 (1) ◽  
pp. 166
Author(s):  
X. J. Yin ◽  
H. S. Lee ◽  
E. G. Choi ◽  
X. F. Yu ◽  
B. H. Choi ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Second Generation ◽  
Assisted Reproductive Technologies ◽  
Polar Body ◽  
Donor Cell ◽  
Fibroblast Cell ◽  
Cumulus Cells ◽  
Reproductive Technologies ◽  
Blastocyst Development ◽  
First Polar Body

Domestic cats are a useful research model to develop assisted reproductive technologies for the conservation of endangered felids. Previously, we produced cloned offspring derived from somatic cell nuclear transfer of ear skin fibroblasts obtained from a deaf, odd-eyed, male Turkish Angora. The aim of this study was to assess the cloning efficiency of the fibroblasts derived from a cloned cat. Fibroblast cell lines were established from 6-mm skin biopsies taken from a deaf, odd-eyed, male Turkish Angora and his clone. The protocol for nuclear transfer was described previously (Yin et al. 2005 Reproduction 129, 245–249). Briefly, cumulus cells were removed from the ova by gently pipetting them into TCM-199 supplemented with 0.1% hyaluronidase. The denuded oocytes were then cultured in TCM-199 supplemented with 0.2 �g mL-1 demecolcine for 1 h and placed into TCM-199 containing 5 �g mL-1 cytochalasin B and 0.2 �g mL-1 demecolcine. The first polar body and protruded chromatin plate were removed with a beveled micropipette. Micromanipulation was used to place a single donor cell nucleus into the perivitelline space of enucleated ova. The ovum-cell couplets were fused and pulse activated. The activated couplets were cultured in 500 �L of CRI medium supplemented with 0.3% BSA for 2 days. The cleaved embryos were cultured in CRII medium supplemented with 10% FBS for 5 days. The cleavage and blastocyst development rates were 38.5% and 3.5% for second generation cloned embryos. A total of 310 second generation cloned embryos were transplanted to 9 surrogates, and 2 pregnancies at 30 days were determined by ultrasonography. One pregnancy was aborted at 40 days of gestation; the second pregnancy continued. These results indicate that the serial cloning of a cat can be generated efficiently up until pregnancy. This work was supported by KOSEF (grant #M10525010001-05N2501-00110).

44 PRODUCTION OF LIVE PIGLETS AFTER CRYOPRESERVATION OF IMMATURE PORCINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 136
Author(s):  
T. Somfai ◽  
K. Kikuchi ◽  
K. Yoshioka ◽  
F. Tanihara ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Polar Body ◽  
Petri Dish ◽  
Developmental Competence ◽  
Basic Medium ◽  
High Survival ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Vitrification Solution

Development to term of vitrified porcine follicular oocytes is reported in the present study. Immature cumulus-oocyte complexes (COC) were collected from slaughtered prepubertal gilts and were vitrified according to our method published recently (Somfai et al. 2013 J. Reprod. Dev., in press). Briefly, after pretreatment with 7.5 μg mL–1 of cytochalasin B (CB) for 30 min in modified NCSU-37 (a basic medium, BM) at 38.5°C, groups of 88 to 121 COC were equilibrated in a mixture of 2% ethylene glycol (EG), 2% propylene glycol (PG), and 7.5 μg mL–1 CB for 13 to 15 min. Then, COC were washed in vitrification solution (17.5% EG, 17.5% PG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BM) and then dropped with 2 μL of vitrification solution onto the surface of aluminum foil floating on liquid nitrogen (LN2). Microdroplets (each containing 10–25 COC) were transferred into cryotubes. After storage in LN2 for 2 to 4 weeks, the oocytes were warmed by dropping the microdroplets directly into 2.5 mL of warming solution (0.4 M trehalose in BM) kept in a 35-mm Petri dish on a 42°C hotplate for less than 1 min. Then, the warming dish was placed on a 38°C hotplate and COC were consecutively transferred for 1-min periods into BM containing 0.2, 0.1, or 0.05 M trehalose at 38°C. The COC were matured in vitro for 44 h using porcine oocyte medium (POM) supplemented with 10% follicular fluid (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Then, oocytes were denuded, and their live/dead status and nuclear maturation were determined by their morphology and the presence of the first polar body, respectively. To assess their developmental competence, vitrified and non-vitrified (control) oocytes were in vitro fertilized (IVF; Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) and then in vitro cultured in porcine zygote medium-5 (PZM-5; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Blastocyst rates were recorded on Days 5, 6, and 7 of culture (Day 0 = the day of IVF). The experiment was replicated 4 times. Data were analysed with 1-way ANOVA and the Tukey test. The results revealed that 86.4% (364/424) of oocytes survived after vitrification, which was significantly lower (P < 0.05) than that of controls [100% (326/326)]. Live oocytes in vitrified and control groups did not differ statistically in terms of nuclear maturation (63.9 v. 65.3%). Blastocyst rates of surviving vitrified oocytes were significantly lower compared with controls on Days 5 (2.4 v. 12.7%), 6 (4.8 v. 17.6%), and 7 (5.6 v. 18.4%). To test their ability to develop to term, 16 and 27 blastocysts on Day 5 developing from vitrified COC were transferred into 2 recipients. Both recipients became pregnant and farrowed a total of 10 live piglets (4 and 6 piglets, respectively). These data demonstrate that large groups of immature porcine oocytes could be cryopreserved by this method showing high survival and maturation rates. Furthermore, despite a low rate of blastocyst development, transfer of Day-5 blastocysts generated from vitrified oocytes resulted in piglet production for the first time in the world. Partially supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program.

53 EFFECT OF THE NUCLEAR REPROGRAMMING TIME ON IN VITRO DEVELOPMENT OF EQUINE AGGREGATED CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 174
Author(s):  
R. Olivera ◽  
C. Alvarez ◽  
I. Stumpo ◽  
G. Vichera
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Nuclear Reprogramming ◽  
Chi Square ◽  
In Vitro Development ◽  
First Polar Body ◽  
Group 3 ◽  
Group 2 ◽  
Vitro Development

The time allowed for nuclear reprogramming is considered an essential factor for the efficiency of cloning and has not been evaluated in equine aggregated cloned embryos. The aim of our work was to assess the effect of different timing of activation stimulus after fusion of adult equine fibroblast cells to enucleated equine oocytes on embryo development and embryo quality. We processed a total of 1874 equine ovaries, recovering 3948 oocytes, of which 1914 (48.5%) had extruded the first polar body after 24 h of maturation. Oocyte collection, maturation, and the NT procedure were performed as described by Lagutina et al. (2007 Theriogenology 67, 90–98). Reconstructed oocytes (RO) were activated at 3 different times after cell fusion: (1) 1 h, (2) 1.5 h, and (3) 2 h. Activation was performed using 8.7 µM ionomycin for 4 min, followed by a 4-h culture in a combination of 1 mM DMAP and 5 mg mL–1 of cycloheximide. The RO were cultured in the well of the well system, aggregating 3 RO per well. The RO were cultured in DMEM-F12 with 5% fetal bovine serum (FBS) and antibiotics. Cleavage (48 h after activation), blastocyst, and expanded blastocyst rates (8–9 days) were assessed. In vitro development was compared using the chi-square test (P < 0.05). A total of 1608 RO were cultured. Cleavage was significantly lower in group 3 with respect to the other 2 groups [(1): 396/450, 88%; (2): 540/639, 84.5%; (3): 365/519, 70.3%]. There were no significant differences in blastocyst rates within the 3 groups considering the number of total RO [(1): 19/450, 4.2%; (2): 23/639, 3.6%; (3): 15/519, 2.9%] or aggregated RO per well [(1): 12.7%; (2): 10.8%; (3): 8.7%]. However, the rate of blastocyst expansion was higher (P < 0.05) in group 2 than in group 3 [(1): 17/19, 89.5%; (2): 23/23, 100%; (3): 11/15, 73.3%]. In conclusion, the timing of nuclear reprogramming did not affect blastocyst rates but affected cleavage rates and blastocyst quality. This indicates that 1 h before activation stimulus is enough for embryo development of equine aggregated cloned embryos.

242 USE OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN IN VITRO PREMATURATION OF BOVINE OOCYTES SUBJECTED TO PARTHENOGENETIC ACTIVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 200
Author(s):  
T. H. C. De Bem ◽  
R. Rochetti ◽  
P. R. L. Pires ◽  
F. F. Bressan ◽  
P. R. Adona ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Polar Body ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Parthenogenetic Activation ◽  
Bovine Oocytes ◽  
Hatching Rates

Prematuration provides an additional time for oocyte capacitation and maturation in an attempt to improve in vitro embryo production (IVP) rates and allows media supplementation during this period for IVP. The aim of this study was to use brain-derived neurotropic factor (BDNF) in prematuration to improve maturation of bovine oocytes subjected to parthenogenetic activation and cultured with different media. Oocytes were subjected to prematuration in TCM-199 medium supplemented with 10 µm butyrolactone I, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin for 24 h in the absence of BDNF (control) or in the presence of 10 ng mL–1 BDNF (BD). Oocytes were then in vitro-matured (IVM) in TCM-199 medium supplemented with 10% FCS, 0.5 µg mL–1 FSH, 5.0 µg mL–1 LH, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin at 38.5�C under 5% CO2 in air. After 19 h oocytes were denuded using hyaluronidase and vortexing for 3 min for the 1st polar body (1PB) selection. Those which extruded the 1PB were maintained in IVM until 26 h, when parthenogenetic activation was performed (5 min in 5 µm ionomycin, followed by 3 h in 2 mm 6-DMAP). Activated oocytes were then transferred to in vitro culture (IVC) for embryo development evaluation. Embryos from both groups were cultured in SOF medium with 2.5% FCS, 0.05 g mL–1 BSA, 0.2 mm pyruvate, and 10 mg mL–1 gentamicin. Cleavage rates on the second day of in vitro culture (D2), embryo production at Days 7 and 8 (D7 and D8), and hatching rate at Day 8 were evaluated. Data regarding 1PB extrusion, cleavage, blastocyst development on D7 and D8, and blastocyst D8 hatching rates of three replicates were analyzed by chi-square test at 5% significance using the BIOESTATS 4.0 software. Control and BD, respectively, did not show differences (P > 0.05) regarding 1PB extrusion (n = 164, 63.81%, and n = 175, 66.79%) or cleavage (n = 117, 71.34%, and n = 138, 78.86%). However, for control and BD, respectively, blastocyst development on D7 (n = 63, 38.41%, and n = 89, 50.86%), D8 (n = 63, 38.41%, and n = 91, 52.00%), and hatching on D8 (n = 22, 34.92%, and n = 39, 43.82%) were all significantly higher for BD when compared with control (P < 0.05). In conclusion, BDNF during prematuration improved in vitro embryo development by increasing blastocyst and hatching rates of parthenogenetic embryos.

Round spermatids stained with MitoTracker can be used to produce offspring more simply

Zygote ◽  
2005 ◽  
Vol 13 (1) ◽  
pp. 55-61 ◽  
Author(s):  
Takafusa Hikichi ◽  
Satoshi Kishigami ◽  
Nguyen Van Thuan ◽  
Hiroshi Ohta ◽  
Eiji Mizutani ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Round Spermatid ◽  
Testicular Cells ◽  
Infertility Treatments ◽  
Developmental Capacity ◽  
20 Nm ◽  
Harmful Effects ◽  
Round Spermatid Injection ◽  
Selection Of

Although both intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) are used in infertility treatments, the rate of offspring achieved with ROSI is low compared with that achieved with ICSI. The difficulty in correctly selecting round spermatids from testicular cells is one of the causes of this phenomenon. We easily selected live round spermatids from testicular cells stained with 20 nM MitoTracker, which visualizes mitochondria without killing the cell. Using this method, we divided round spermatids into three groups based on the polarization of their mitochondria, and performed ROSI. The rate of successful offspring achieved with MitoTracker-stained ROSI was the same in all groups. This indicates that changes in the polarization of mitochondria in round spermatids are not directly related to the developmental capacity of subsequently fertilized embryos. Because this staining has no harmful effects on embryo development, the selection of spermatids by MitoTracker under a fluorescence microscope should be useful in research into and the treatment of infertility.

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