Adult ; Animals ; Female ; Frameshift Mutation/genetics* ; Genes, sry ; Gonadal Dysgenesis, 46,XY/genetics* ; Humans ; Menarche ; Ovary/pathology* ; Oviducts/pathology* ; Sex-Determining Region Y Protein/genetics*

OBJECTIVE: This study was conducted to investigate the efficacy of laser-assisted hatching (LAH) and various vitrification times for embryonic development and blastocyst cell numbers. METHODS: First, 2-cell and 8-cell embryos were collected by flushing out the oviducts. In the control groups, they were vitrified for 8 or 10 minutes without LAH. The LAH groups underwent quarter laser zona thinning-assisted hatching before vitrification (4, 6, and 8 minutes or 4, 7, and 10 minutes, respectively). After incubation, double-immunofluorescence staining was performed. RESULTS: The hatched blastocyst rate 72 hours after the 2-cell embryos were thawed was significantly higher in the 2LAH-ES8 group (33.3%) than in the other groups (p < 0.05). In the control-8 group (22.1±4.6), the cell number of the inner cell mass was higher than in the LAH groups (p < 0.05). The number of trophectoderm cells was higher in the 2LAH-ES6 group (92.8±8.9) than in the others (p < 0.05). The hatched blastocyst rate 48 hours after the 8-cell embryos were thawed was higher in the 8LAH-ES4 group (45.5%) than in the other groups, but not significantly. The inner cell mass cell number was highest in the 8LAH-ES7 group (19.5±5.1, p < 0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group (73.2±12.1) than in the other groups, but without statistical significance. CONCLUSION: When LAH was performed, 2-cell embryos with large blastomeres had a lower hatched blastocyst rate when the exposure to vitrification solution was shorter. Conversely, 8-cell embryos with small blastomere had a higher hatched blastocyst rate when the exposure to vitrification solution was shorter.
Animals ; Blastocyst ; Blastomeres ; Cell Count ; Embryonic Development* ; Embryonic Structures* ; Female ; Flushing ; Herpes Zoster ; Mice* ; Oviducts ; Pregnancy ; Vitrification*

OBJECTIVE: The goal of this study was to ascertain optimal assisted hatching (AH) method in frozen embryo transfer. We compared the effect of depending on whether mechanical or laser-AH was performed before or after the vitrification of embryo development rate and blastocyst cell numbers. METHODS: In order to induce superovulation, pregnant mare's serum gonadotropin followed by human chorionic gonadotropin were injected into 4- to 5-week-old female mice. 2-cell embryos were then collected by flushing out the oviducts. The Expanded blastocysts were recovered after the collected embryos were incubated for 48 hours, and were then subjected to artificial shrinkage (AS) and cross-mechanical AH (cMAH) or quarter-laser zona thinning-AH (qLZT-AH) were carried out using the expanded blastocysts before or after vitrification. After 48 hours of incubation, followed by vitrification and thawing (V-T), and blastocysts were fluorescence stained and observed. RESULTS: The rate of formation of hatched blastocysts after 24 and 72 hours of incubation was significantly higher in the AS/qLZT-AH/V-T group than in the other groups (p<0.05). The cell number of the inner cell mass was higher in AS/V-T/non-AH and AS/V-T/cMAH groups than those of others (p<0.05). In the control group, the number of trophectoderm and the total cell number were higher than in the AS-AH group (p<0.05). CONCLUSION: The above results suggest that AS and AH in vitrification of expanded blastocysts lead to the more efficient formation of hatched blastocysts in mice.
Animals ; Blastocyst* ; Cell Count* ; Chorionic Gonadotropin ; Embryo Transfer ; Embryonic Development ; Embryonic Structures ; Female ; Fluorescence ; Flushing ; Gonadotropins ; Herpes Zoster ; Humans ; Mice* ; Oviducts ; Pregnancy ; Superovulation ; Vitrification*

Male pseudohermaphroditism is not commonly reported in veterinary medicine. Here, a 3-year-old Maltese/poodle mixed dog presented with malformed external genitalia and episodic hematuria. Inspection and palpation of the external genitals showed a malformed penis, shortened prepuce, external urethral orifice, and cryptorchidism. There was no urethral meatus at the tip of the penis. The urethral opening was situated between the prepuce and the penis. The anterior half of the prepuce was absent, and the penis was free and exposed to both trauma and licking. Plain radiographic examination showed absence of an os penis in the penis. A double-contrast cystograph showed the suspected uterus as well as the cystic calculi. A hypoechoic space was seen at the dorsal portion of the urinary bladder. The space was suspected to be the uterus. A sagital ultrasonograph showed cystic calculi in the urinary bladder. During surgery to remove cystic calculi, hypoplastic testes as well as the uterus were observed. Histological examination of the testes showed the seminiferous tubules and interstitial cells. The sertoli cells and spermatogonia were adjacent to the basement membrane. No evidence of spermatogenesis was found. Striated squamous epithelial cells and smooth muscle cells were found in the uterus. This dog had vestigial oviducts as well as a uterus with male-appearing external genitals.
46, XY Disorders of Sex Development* ; Animals ; Basement Membrane ; Calculi ; Child, Preschool ; Cryptorchidism ; Disorders of Sex Development ; Dogs* ; Epithelial Cells ; Genitalia ; Hematuria ; Humans ; Male ; Myocytes, Smooth Muscle ; Oviducts ; Palpation ; Penis ; Seminiferous Tubules ; Sertoli Cells ; Spermatogenesis ; Spermatogonia ; Testis ; Urinary Bladder ; Uterus ; Veterinary Medicine

OBJECTIVE: In search of an ideal method of assisted hatching (AH), we compared the effects of conventional micropipette-AH and laser-AH on the blastocyst formation rate (BFR) and blastocyst cell numbers. METHODS: Four- to five-week-old ICR female mice were paired with male mice after superovulation using Pregnant mare's serum gonadotropin (PMSG) and hCG. The two-cell embryos were flushed from the oviducts of female mice. The retrieved two-cell embryos underwent one of five AH procedures: single mechanical assisted hatching (sMAH); cross mechanical assisted hatching (cMAH); single laser assisted hatching (sLAH); quarter laser assisted hatching (qLAH); and quarter laser zona thinning assisted hatching (qLZT-AH). After 72 hours incubation, double immunofluorescence staining was performed. RESULTS: Following a 72 hours incubation, a higher hatching BFR was observed in the control, sMAH, cMAH, and sLAH groups, compared to those in the qLAH and qLZT-AH groups (p<0.05). The hatched BFR was significantly higher in the qLAH and qLZT-AH groups than in the others (p<0.05 for each group). The inner cell mass (ICM) was higher in the control and sMAH group (p<0.05). The trophectoderm cell number was higher in the cMAH and qLAH groups (p<0.05). CONCLUSION: Our results showed that the hatched BFR was higher in groups exposed the the qLAH and qLZT-AH methods compared to groups exposed to other AH methods. In the qLAH group, although the total cell number was significantly higher than in controls, the ICM ratio was significantly lower in than controls.
Animals ; Blastocyst ; Cell Count ; Embryonic Development* ; Embryonic Structures ; Female ; Fluorescent Antibody Technique ; Gonadotropins ; Herpes Zoster ; Humans ; Male ; Mice* ; Oviducts ; Pregnancy ; Superovulation

BACKGROUND AND OBJECTIVES: Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. METHODS AND RESULTS: Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA-4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. CONCLUSIONS: Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.
Animals ; Blastocyst ; Buffaloes ; Collagen ; Collagen Type I ; Drug Combinations ; Embryonic Stem Cells ; Extracellular Matrix ; Feeder Cells ; Female ; Fibroblasts ; Fibronectins ; Granulosa Cells ; Humans ; Laminin ; Mental Competency ; Models, Animal ; Oviducts ; Proteoglycans ; Stage-Specific Embryonic Antigens ; Stem Cell Research

We constructed transgenic chicken bioreactor vector, driven by chicken ovalbumin promoter, lentiviral vector and cytomegalovirus (CMV) promoter control vector encoding green fluorescent protein (GFP) and luciferase (Luc) as reporter genes. The three vectors were used to transfect or infect chicken primary oviduct epithelial cells, embryo fibroblasts cells, mouse 3T3-L1 preadipocytes cells and bovine mammary epithelial cells. High efficient and specific expression vector for transgenic chicken bioreactor was determined by detecting fluorescence and luciferase activity. Reporter gene analysis showed that chicken ovalbumin promoter expression vector was not cell type-specific in these four different cells. Additionally, luciferase reporter analysis illustrated that the chicken ovalbumin promoter activity was over 100 times lower than that of the CMV promoter in four different cells. Both of these two reporter genes were expressed in those four different cells infected by lentiviral expression vectors. Similarly, the GFP reached the similar expression level in cells infected by lentivirus and cells transfected with CMV promoter plasmid vectors when the multiplicity of infection was 20. In conclusion, the transgenic chicken bioreactor vector under the control of chicken ovalbumin promoter was not highly efficient and cell type-specific. However, the efficient expression and extensiveness oflentiviral vector could be used for studying chicken oviduct bioreactor.
3T3-L1 Cells ; Animals ; Animals, Genetically Modified ; Cattle ; Chickens ; genetics ; Cytomegalovirus ; genetics ; metabolism ; Epithelial Cells ; cytology ; Female ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Lentivirus ; genetics ; metabolism ; Luciferases ; genetics ; Mice ; Ovalbumin ; genetics ; Oviducts ; cytology ; metabolism ; Promoter Regions, Genetic ; genetics ; Transfection

We studied the influence of the first intron and 3'-regulatory region of ovalbumin gene (ov) on oviduct-specific transgene expression. The 3'-regulatory region in the oviduct-specific expression vector containing human tissue kallikrein (hK1) cDNA was replaced with bovine growth hormone (BGH) poly A, and the first intron was deleted by restriction enzyme digestion, resulting in five new vectors pOV2K, pOV3K, pOV4K, pOV5K and pOV6K. After mixing with polyethylenimine, we injected same copies of the five vectors via wing vein route into laying hens and compared their expression levels by quantitative assay for enzymatic activities in the egg whites. Among the five vectors tested, the pOV2K containing both the 5'- and 3'-regulatory regions expressed highest level of rhK1 activity, followed by pOV3K with the 3'-regulatory region replaced with BGH poly A, and then by the first intron-shortened vectors pOV4K, pOV5K and pOV6K. These data suggest that both the first intron and 3'-regulatory region of ov gene have enhancing effect on transgene expression in oviduct cells, which should be included in oviduct-specific expression vectors.
Animals ; Animals, Genetically Modified ; Cattle ; Chickens ; Cloning, Molecular ; Female ; Gene Transfer Techniques ; Growth Hormone ; genetics ; Humans ; Introns ; genetics ; Ovalbumin ; genetics ; Oviducts ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Regulatory Sequences, Nucleic Acid ; genetics ; Tissue Kallikreins ; genetics ; Transgenes ; genetics

Isolated torsion of the fallopian tube is very uncommon during pregnancy. It usually confuses with acute appendicitis and torsion of the ovary. The diagnosis is usually established during operation, and a salpingectomy is almost always necessary. However, the early diagnosis and surgical treatment of the disease are mandatory to preserve oviduct. Because of safety and feasibility, laparoscopic surgery has recently become major treatment method.
Animals ; Appendicitis ; Diagnosis ; Early Diagnosis ; Fallopian Tubes* ; Female ; Laparoscopy ; Ovary ; Oviducts ; Pregnancy* ; Salpingectomy

OBJECTIVE: This study is to analyze the direct effects of hyperprolactinemia, cause of anovulation and infertility, on ovarian function. METHODS: The prepubertal female Sprague-Dawley (SD) rats were obtained and ovulation was induced with PMSG and hCG s.c.. The rats were divided into four groups, which received the following treatments IP : saline 0.2 ml, 150 ug PRL, 300 ug PRL, 300 ug PRL plus 300 ug naloxone. The animals were killed and the oviducts were evaluated for the presence of ova. The ovary were then removed and evaluated under light microscopy. For changes of follicular t-PA and PGE2 concentration after PRL, immature female SD rats were stimulated as described above. At four hours after the hCG injection the rats were killed and the ovaried were removed. Each isolated ovaries were incubated in culture plate containing incubation medium or 300 ng PRL to be tested. And PRL plus gonadotropin in incubation medium was tested because of change of PGE2 concentration. After incubation period, t-PA and PGE2 were measured by EIA. Differences between groups were assessed by two-way ANOVA of variance followed Mann-Whitney U test or Kruskal-Wallis test for multiple comparisons. p<0.05 was considered statistically significant. RESULTS: As result, prolactin transiently suppresses ovulation, especially with its increased concentration not by altering the ovarian morphology. But ovulation inhibition was reversed by naloxone injection. The level of t-PA in control and prolactin-treated group increased steadily in response to human chorionic gonadotropin administration, yet lower in prolactin-treated group. But PGE2 concentration was increased in gonadotropin mixed groups but not affected in prolactin-treated group despite a significant blockade of ovulation. CONCLUSION: Thus, further studies on the effect of high level prolactin on ovulatory function would significantly contribute toward the patient with hyperprolactinemia for managing infertility and maintaining appropriate female reproductive function.
Animals ; Anovulation ; Chorionic Gonadotropin ; Dinoprostone* ; Female ; Gonadotropins ; Humans ; Hyperprolactinemia ; Infertility ; Microscopy ; Naloxone ; Ovary ; Oviducts ; Ovulation ; Ovulation Inhibition ; Ovum ; Prolactin* ; Rats* ; Rats, Sprague-Dawley