bluuebunny/crossref_metadata_2025 huggingface dataset:
183 MITOGEN-ACTIVATED PROTEIN KINASES ARE ACTIVATED BY ADENOSINE TRIPHOSPHATE IN RAT OVARIAN SURFACE EPITHELIAL CELLS
Authors: Hwang K.-A. , Choi K.-C.
Published: August 2011

Extracellular ATP has been suggested to play a role in cellular proliferation and intracellular calcium concentrations (Ca2+) in ovarian cells. To investigate the role of ATP in rat ovarian folliculogenesis or steroidogenesis, we examined the expression of the P2U purinoceptor (P2U-R) and the effect of ATP on growth stimulation in rat ovarian surface epithelial (OSE) cells. Rat OSE cells were isolated and cultured in DMEM media with 10% fetal bovine serum. Our results indicated that P2U-R mRNA was expressed and that ATP exerted a growth-stimulatory effect in rat OSE cells. To investigate the mechanism of the growth-stimulatory effect, we examined the activation of mitogen-activated protein kinases (MAPK) by ATP. Treatment with ATP resulted in MAPK activation in these cells, whereas the stimulatory effect of ATP in cellular proliferation and MAPK activation was completely abolished in the presence of PD98059 [a MAPK/ERK kinase (MEK) inhibitor] and staurosporin [a protein kinase C (PKC) inhibitor], suggesting that the growth-stimulatory effect of ATP may be mediated via PKC-dependent MAPK activation in rat OSE cells. In a time-dependent study, ATP significantly increased MAPK activity at 5 to 20 min, and the activated MAPK in these cells declined to the control level after 20 min. Similarly, treatment with ATP significantly induced MAPK activation after 5 min and sustained it for 60 min in these OSE cells. In addition, treatment with ATP resulted in substantial phosphorylation of Elk-1, confirming that ATP action is mediated by activation of MAPK. In conclusion, we demonstrated that P2U-R was expressed and that ATP induced growth stimulation in rat OSE cells. Furthermore, treatment with ATP resulted in activation of the MAPK cascade and phosphorylation of Elk-1 in these cells. Taken together, these results suggest that the MAPK cascade may be involved in growth stimulation in response to ATP in rat OSE cells.

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; No. 2010-0003093).
187 DIFFERENTIAL EXPRESSIONS OF GROWTH AND DIFFERENTIATION FACTOR 9 AND BONE MORPHOGENETIC PROTEIN 15 GENES IN OVARIES OF THE CALF AND COW
Authors: Hosoe M. , Ushizawa K. , Hayashi K.-G. , Takahashi T.
Published: August 2011

It has been reported that prepubertal calf oocytes are less developmentally competent than those obtained from cows. The bone morphogenetic protein (BMP) family of proteins regulate folliculogenesis and the ovulation rate in mammals. Of the members of the BMP family, growth and differentiation factor 9 (GDF9) and BMP15 are oocyte-derived proteins that play critical roles in granulosa cell proliferation and differentiation. In the present study, we characterised the gene expression of bovine GDF9 and BMP15 in calf and adult cow ovaries. The ovaries obtained from 4 calves at 9 to 11 months old and 4 cows at 4 to 6 years old. For quantitative real-time RT-PCR (qPCR), cumulus–oocyte complexes (COC) and mural granulosa cells were collected by aspiration of follicles 2 to 5 mm in diameter from an ovary from each animal. Ovaries from the other side were used for in situ hybridization. The COC and mural granulosa cells were separated and cultured for 22 h according to the protocol for oocyte maturation. Total RNA was isolated from denuded oocytes, cumulus cells, and mural granulosa cells. Bovine glyceraldehyde-3-phosphate dehydrogenase was used to normalise qPCR efficiency. For in situ hybridization, the collected ovaries were immediately fixed with 4% formaldehyde-PBS and embedded in a paraffin block. In situ hybridization was carried out with digoxigenin-labelled RNA probes. We confirmed there was no contamination of oocytes in the collected cumulus and mural granulosa cells by determining the mRNA expression of germ cell–oocyte markers (ZAR1 and VASA). Two, 16, 7, and 10 COC were collected from the ovary on one side of each calf, and 14, 22, 29, and 33 COC were collected from an ovary of each adult cow. Two COC from the calves could not be used for qPCR analysis. Both GDF9 and BMP15 mRNA were detected in oocytes and cumulus cells at the end of maturation culture, whereas only GDF9 mRNA was detected in mural granulosa cells. Quantitative PCR detection revealed that BMP15 and GDF9 mRNA expression of the cumulus cells from adult ovaries was significantly greater than that from calf ovaries. The expression of GDF9 mRNA was significantly greater in calf oocytes than in oocytes from cows. However, BMP15 mRNA expression in the oocytes of calf and adult ovaries was not significantly different. In mural granulosa cells, the intensities of GDF9 mRNA expression were not significantly different between calves and cows. These qPCR results were also ascertained by in situ hybridization. In conclusion, we clarified that the characteristics of bovine GDF9 and BMP15 mRNA expression in oocytes and cumulus cells were different between calves and cows. Our results indicate the possibility that the calf oocytes were less developmentally competent because of excess GDF9 on the oocytes, deficiencies of GDF9 and BMP15 on the cumulus cells, or both.
188 CALBINDIN-D28k IS INVOLVED IN THE APOPTOTIC PATHWAY AND PLAYS A ROLE AS AN ANTIAPOPTOTIC GENE IN HYDROGEN PEROXIDE-INDUCED CELL DEATH OF HUMAN ENDOMETRIAL ISHIKAWA CELLS
Authors: Hyun S.-H. , Jung E.-M. , Jeung E.-B.
Published: August 2011

Calbindin-D28k (CaBP-28k), a calcium-binding protein, buffers intracellular Ca2+ and eventually has antiapoptosis properties in neuron, osteoblast, and male germ cells. Although endometrial cancer is the most common invasive gynecological malignancy, CaBP-28k expression in apoptosis signalling is poorly understood. In this study, we investigated whether CaBP-28k expression is regulated by H2O2-induced apoptosis signalling in human endometrial Ishikawa cells. Ishikawa cells were treated with H2O2 in a dose-dependent manner (0, 0.25, 0.5, 1.0, 1.5, 2 mM) and a time-dependent manner (0, 15, 30, 60, 90, 120 min). The protein expressions of Bax, p53, and caspase 3 were determined by Western blot analysis. Treatment of Ishikawa cells with H2O2 induced an increase in Bax and p53 expression at the translational level at 1 mM for 1 h. Interestingly, overexpression of CaBP-28k caused a decrease in Bax, p53, and caspase 3 on H2O2-induced apoptosis in the Ishikawa cells. These results suggest that expression of CaBP-28k blocked up-regulation of apoptotic gene expression. In addition, knockdown of CaBP-28k expression using a small inhibitory RNA resulted in an elevation of H2O2-induced cell death and a increase in Bax, p53, and caspase 3 in H2O2-induced apoptosis, whereas cell survival was increased in overexpressing CaBP-28k cells, providing additional evidence that the induction of CaBP-28k expression may be associated with survival signalling during H2O2-mediated oxidative cell death. Taken together, these results imply that CaBP-28k expression is involved in the apoptotic pathway and may play a role as an antiapoptotic gene in the human endometrial Ishikawa cells.
189 THE CALCIUM EXCHANGERS NCKX3 AND NCX1 ARE DISTINCTLY EXPRESSED AND REGULATED BY STEROIDS IN THE HUMAN ENDOMETRIUM DURING THE MENSTRUAL CYCLE
Authors: Jeung E.-B.
Published: August 2011

Plasma membrane sodium–calcium exchangers are an important component of intracellular calcium homeostasis and electrical conduction. The potassium-dependent or sodium–calcium exchangers NCKX3 (gene SLC24A3) and NCX1 (gene SLC8A1) play a critical role in the transport of intracellular calcium across the cell membrane in exchange for extracellular sodium ions. The transcripts SLC24A3 and SLC8A1 are most abundant in the brain and smooth muscle, but many other tissues, particularly the uterus, aorta, and intestine, also express this gene at lower levels. However, the expression and physiological roles of NCKX3 and NCX1 are largely unknown in the human endometrium during the menstrual cycle. Thus, we examined the endometrial expression of NCKX3 and NCX1 at the transcriptional and translational levels at different phases of the menstrual cycle. Our findings revealed that NCKX1 and NCKX3 were differentially expressed during the menstrual cycle. The endometrial expression of NCKX3 mRNA and protein was enhanced up to 1.5- to 2.5-fold at the early proliferative phase, midproliferative phase, and early secretory phase compared with other phases, whereas a significant alteration in NCX1 expression during the human menstrual cycle was not observed. Subsequent immunohistochemical analysis revealed a large number of uterine NCKX3 and NCX1 proteins in the cytoplasm of luminal and glandular epithelial cells throughout the menstrual cycle. Taken together, these results suggest that human endometrial NCKX3 is abundantly expressed in the endometrium and that NCKX3 may be involved in reproductive function during the menstrual cycle in the human endometrium.
190 EXPRESSION OF APOPTOTIC GENES IS MEDIATED BY CALCIUM-BINDING PROTEINS, CALBINDINS, IN THE UTERUS OF CALBINDIN-D9k AND CALBINDIN-D28k KNOCKOUT MICE
Authors: Jung E.-M. , Jeung E.-B.
Published: August 2011

Calcium (Ca2+) is known to be an important regulator in apoptotic signalling. The uterine calbindins, calbindin-D9k (CaBP-9k) and calbindin-28k (CaBP-28k), are known to be involved in the regulation of myometrial activities by regulating intracellular Ca2+. In addition, the uterine calbindins are expressed in the mouse endometrium and are highly regulated during implantation and development. The aim of the present study was to evaluate the regulation of apoptosis in the uterus of immature CaBP-9k, CaBP-28k, and CaBP-9k/28k knockout (KO) mouse models. Our findings indicate that Bax protein levels were enhanced in the uterus of CaBP-28k and CaBP-9k/28k KO mice compared with wild-type mice, and no difference was observed in Bcl-2 protein expression. In addition, the levels of caspase 3, 6, and 7 protein were induced in both CaBP-28k and CaBP-9k/28k KO mice compared with wild-type mice. These results suggest that the absence of calbindins may induce an increase in apoptotic signalling. In addition, we investigated the expression of the endoplasmic reticulum stress genes by Western blot analysis in calbindin KO mice. These results showed that CHOP and BiP proteins were increased in CaBP-28k and CaBP-9k/28k KO mice compared with wild-type mice, and no difference was observed in the expression of PDI and IRE1α proteins. It is of interest that the expression of CaBP-28k may block up-regulation of apoptosis-related genes. In summary, this study implies that CaBP-28k may decrease apoptosis-related gene expression in the uterine tissue of immature mice.
194 EXPRESSION OF STANNIOCALCIN FAMILY GENES DURING PREIMPLANTATION STAGE BOVINE EMBRYO DEVELOPMENT
Authors: Mamo S. , Al Naib A. , O'Hara L. , Fair T. , Lonergan P.
Published: August 2011

Stanniocalcins (STC) are a small family of secreted homodimeric glycoprotein hormones consisting of STC1 and STC2. A previous study in Drosophila (Tolias and Stroumbakis 1998 Dev. Genes Evol. 208, 274–282) indicated that maternally derived STC is required during embryogenesis. However, little information is available for mammalian embryos. The aim of this study was to examine the expression of STC and assess their roles during the preimplantation stage of bovine embryo development. Immature cumulus–oocyte complexes were aspirated from follicles of bovine ovaries collected at a local abattoir and matured in vitro for 24 h at 39°C under an atmosphere of 5% CO2 in air with maximum humidity in TCM-199 supplemented with 10% (vol/vol) fetal calf serum and 10 ng mL–1 of epidermal growth factor. Matured cumulus–oocyte complexes were inseminated with fertile bull semen (Day 0). Embryos were cultured in vitro, and subsequently, 4 pools of 10 embryos each at the zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst stages were collected from 4 different replicate cultures and stored at –80°C until analysis. Total RNA was isolated using an RNeasy Micro Kit and a random primer was used during cDNA synthesis. The expression of STC1, STC2, and reference genes (YWHAZ, PPIA, SDHA) was examined. Quantitative real-time PCR was used to compare transcript abundance, and data were normalized to the geometric averages of the reference genes. The expression levels were analysed using the relative standard curve method, and means were compared using Student’s t-test. Despite being members of the same family and having large sequence similarity, the expression of each gene was unique and stage dependent during embryo development. Expression of STC1 was detected in all the stages examined. Expression was transiently reduced at the 2-cell stage, with no significant change until the 8-cell stage but with a slight increase at the 16-cell stage. In contrast, STC2 was barely detectable before the 8-cell stage. Expression at the 8- and 16-cell stages was significantly (P < 0.0001) higher compared with all other stages, with a peak at the 16-cell stage. This significantly higher expression pattern of STC2 during the critical stages of maternal to zygotic control of development may suggest an important role during this critical period of embryo development.

Supported by Science Foundation Ireland (07/SRC/B1156).
195 SPERMATOZOAL PROTEIN MARKERS FOR ANGUS BULL FERTILITY
Authors: Wang X. , Kaya A. , Memili E.
Published: August 2011

Fertility is one of the most economically important traits controlling animal reproduction. Despite its significant economic impact, there are no reliable markers to predict semen quality. The objective of this study was to identify spermatozoal proteins associated with bull fertility, the ability of the sperm to fertilize the oocyte and support embryonic development. To accomplish our objectives, we isolated total spermatozoal proteins from 4 Angus bulls with different fertility phenotypes. Next, differentially expressed proteins were determined using two-dimensional differential in-gel electrophoresis (2D-DIGE), followed by sequencing of the most differentially expressed proteins. Immunoblotting experiments were conducted to confirm expression of key proteins detected by 2D-DIGE. Our results from 2D-DIGE experiments showed approximately 2000 detectable spermatozoal proteins. Of these comprehensive lists of proteins, we identified 80 of the proteins with the highest differential expression in spermatozoa from high- and low-fertility bulls. Diverse sets of differentially expressed proteins known to play roles in sperm motility, metabolism, and cell morphology were identified. These proteins included outer dense fibre of sperm tails 2 and manganous superoxide, heat shock protein, tubulins, α-enolase, and acrosomal vesicle protein-1. Expression profiles of outer dense fibre of sperm tails 2 and manganous superoxide dismutase were confirmed using immunoblotting. The findings are significant because they help us understand the fundamental biology of the male gamete and early development. In addition, the identified proteins can be used as molecular markers to predict bull fertility, an economically important trait.

Funded in part by the Mississippi Agricultural and Forestry Experiment Station, American Angus Association, and Alta Genetics, Inc.
197 MOLECULAR CLONING AND EXPRESSION OF THE CASHMERE GOAT IZUMO GENE
Authors: Na R. H. , Liang L. , Fu L.
Published: August 2011

During the fertilization process, complex events are involved in the fusion between the reacted spermatozoa and the mature oocyte. Fusion implies that many proteins are present on the cell membrane of the gametes. Recently, a new protein, Izumo, has been shown to play a role in the sperm–egg fusion. Izumo was identified through the generation of a monoclonal antibody that inhibits this fusion. This protein belongs to the immunoglobulin superfamily type I transmembrane protein. Izumo can be detected only after acrosome reaction at the spermatozoal surface. The cashmere goat Izumo gene was identified and cloned by 3′ and 5′ rapid amplification of cDNA ends-PCR. The expression of cashmere goat Izumo was examined by RT-PCR and Northern blotting. The full-length cDNA of cashmere goat Izumo contains 1536 bp and an open reading frame of 1035 bp, encoding a polypeptide of 344 amino acids with a calculated molecular mass of 38.76 kDa and a theoretical isoelectric point of 8.18. This predicted amino acid sequence showed 89.82% amino acid identity with the bovine Izumo. The deduced amino acid sequence contained 3 conserved domains of the signal peptide, immunoglobulin-like domain, and transmembrane region. Reverse transcription-PCR and Northern blotting analysis showed that cashmere goat Izumo transcripts were highly expressed in the testis, caput, corpus, cauda epididymis. Cashmere goat Izumo may play a role in the biological process of fertilization.

This work was supported by the National Natural Science Foundation (No. 30560103 and No.30740043), China, and the China Postdoctoral Science Foundation.
202 KNOCK-DOWN AND DEVELOPMENTAL EFFICIENCY OF Arhgap15 GENE KNOCK-DOWN EMBRYOS
Authors: Yang B.-C. , Lee H.-C. , Hwang S. , Jeon I.-S. , Lee D.-K. , An J.-H. , Noh E.-H. , Han Y.-J. , Kim E.-Y. , Hwang K.-C. , Park S.-B.
Published: August 2011

The technique of RNA interference (RNAi) knock-down is a powerful tool for the analysis of gene function in mammalian cells and eggs. Rho GTPase-activating protein 15 (Arhgap15) is closely related to anaemia and immunity, especially as caused by trypanosome. This study was performed to investigate the effect of Arhgap15 gene knock-down on the developmental competence of bovine embryos in vitro. Bovine fibroblast cells were treated with 50 and 100 nM concentrations of Arhgap15 RNAi, respectively. After 24 h of transfection, the control group showed no change in Arhgap15 mRNA level, whereas mRNA expression in the RNAi-treated donor cells was obviously decreased in a dose-dependent manner. These RNAi-treated cells were then transferred into enucleated bovine oocytes to analyse the consequence of RNAi-mediated Arhgap15 gene knock-down. In the control group, cleavage and blastocyst development rates were 75% (102/136) and 14.7% (20/136), whereas those in the knock-down embryos were 81.6% (120/147) and 24.5% (36/147), respectively. The occurrence of cell death by apoptosis was examined in Day 7 blastocysts. Apoptotic cells numbered 12 ± 3.2 in control embryos and 8.9 ± 4.8 in knock-down embryos. Therefore, it can be concluded that RNAi-mediated Arhgap15 gene knock-down in somatic cells did not affect the developmental competence of bovine cloned embryos.

This work was supported by grant 120080401034062 from the BioGreen 21 Program, Rural Development Administration, Republic of Korea.
203 PLASMA MEMBRANE Ca2+-PUMPING ATPase 1 IS ABUNDANTLY EXPRESSED AND DISTINCTLY REGULATED BY ESTROGEN IN HUMAN ENDOMETRIUM DURING THE MENSTRUAL CYCLE
Authors: Yang H. , Jeung E.-B.
Published: August 2011

Plasma membrane Ca2+-pumping ATPases (PMCA) play a critical role in maintaining cellular Ca2+ homeostasis. The PMCA mRNA are encoded on 4 genes, designated PMCA1 to PMCA4. In a previous study, we found that both PMCA1 and PMCA4 are expressed at similar levels in astrocytes and in neurons. Although PMCA1b is expressed in the uterus of rats during the oestrous cycle, the expression of PMCA1 and its potential roles has not been elucidated during the menstrual cycle in the human endometrium. Thus, in the current study, the expression pattern of PMCA1 was examined to predict its roles in the human endometrium during the menstrual cycle. Human uterine tissues (total n = 40) were separated into 3 groups according to menstrual cycle phase: menstrual phase, proliferative phase (early, mid, late), and secretory phase (early, mid, late). Using real-time PCR and Western blot analysis, uterine expression of PMCA1 mRNA and protein increased to 1.5-fold in the early-, mid- and late-proliferative phases in the endometrium of the human uterus, compared with other menstrual phases. In addition, uterine PMCA1 was abundantly localised in the cytoplasm of the luminal and glandular epithelial cells in the menstrual phases, indicating that this protein may participate in the uterine Ca balance of the human endometrium during the menstrual cycle. Taken together, these results suggest that a high level of uterine PMCA1 expression may be involved in reproductive functions during the menstrual cycle of humans.
213 THE EFFECTS OF SERICIN SUPPLEMENTATION IN IN VITRO CULTURE MEDIUM ON THE DEVELOPMENT AND CRYOSURVIVAL OF BOVINE IN VITRO-MATURED - IN VITRO-FERTILIZED EMBRYOS
Authors: Inaba Y. , Hosoe M. , Teramoto H. , Geshi M.
Published: August 2011

The objective of this study was to investigate the feasibility of the silk protein sericin as an alternative protein supplement for bovine embryo cultures. The effects of sericin supplementation in in vitro culture (IVC) medium on the development and cryosurvival of bovine IVM-IVF embryos were investigated. Cumulus–oocyte complexes were collected from 2- to 8-mm follicles of ovaries obtained from a local abattoir. They were matured for 20 to 22 h in TCM-199 supplemented with 5% fetal bovine serum (FBS), 0.002 AU mL–1 of FSH, and 1 μg mL–1 of oestradiol-17β at 38.5°C under an atmosphere of 5% CO2 in air. After IVF (Day 0), presumptive zygotes were cultured in SOF medium (IVC medium) containing amino acids and supplemented with either 5% FBS (FBS group: n = 400) or sericin at 3 different concentrations (wt/vol; 0.05%: n = 493; 0.1%: n = 419; or 0.15%: n = 520; sericin groups) at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2 for 5 days. They were then transferred into each IVC medium supplemented with 0.1 mM β-mercaptoethanol and cultured for an additional 4 days (9 days in total). Cleavage rates were recorded on Day 2 of IVC. The excellent expanded blastocysts harvested on Days 7 and 8 were used for freezing (FBS group: n = 51, 0.05% sericin group: n = 56, 0.1% sericin group: n = 44, 0.15% sericin group: n = 44). They were frozen in m-PBS supplemented with 1.5 M ethylene glycol, 0.1 M sucrose, 20% calf serum, and 4 mg mL–1 of BSA. After thawing, they were cultured in TCM-199 supplemented with 20% FBS and 0.1 mM β-mercaptoethanol under the same atmosphere used for IVC for 72 h. Rates of the embryos that reexpanded and developed to the hatching and hatched blastocyst stages were determined at, respectively, 24, 48, and 72 h after thawing. Rates of cleavage and blastocyst formation were expressed as mean ± SD and were analysed by ANOVA. The post-thaw survival rates of frozen embryos were analysed by Fisher’s exact test and chi-square test. Four replications were performed. Cleavage and blastocyst formation rates did not differ among the groups (FBS: 61.6 ± 15.1 and 22.1 ± 3.5%; 0.05% sericin: 71.6 ± 12.0 and 19.4 ± 6.7%; 0.1% sericin: 70.3 ± 4.7 and 18.4 ± 5.6%; 0.15% sericin: 66.9 ± 10.3 and 17.9 ± 6.9%, respectively). There were no significant differences in post-thaw survival rates after 24 h among the FBS (88.2%), 0.05% sericin (92.9%), 0.1% sericin (84.1%), and 0.15% sericin (93.2%) groups. However, post-thaw survival rates after 72 h in the 0.05% sericin (83.9%) and FBS (82.4%) groups were significantly higher than those in the 0.1% sericin (56.8%) and 0.15% sericin (61.4%) groups (P < 0.05). These results indicate the feasibility of sericin as an alternative protein supplement for bovine embryo culture. Additionally, in this study, 0.05% sericin was shown to be the best concentration for survival of the resultant embryos after freezing and thawing.
214 EFFECT OF LONG-DISTANCE TRANSPORTATION OF OVARIES ON THE DEVELOPMENT OF IN VITRO-MATURED - IN VITRO-FERTILIZED - IN VITRO-CULTURED BOVINE EMBRYOS
Authors: Kanae M. , Hisaichi K. , Jaswant S. , Osamu D.
Published: August 2011

Keeping ovaries for a long time before oocyte recovery affects the maturation rate and blastocyst rate (BL rate), but a change in the temperature of the ovary at the time of transportation does not affect the BL rate (Ribeiro et al. 2008 Anim. Reprod. Sci. 108, 171–179). The objective of this study was to investigate the effect of long-distance transportation of ovaries on the in vitro development of bovine oocytes into blastocysts. Ovaries in the transportation group (Group T) were collected from a slaughterhouse, placed inside a Thermos flask, and transported to the laboratory within 17 to 21 h. The Thermos flask was covered with a freezer pack in a foam polystyrene box (Nakatate et al. 2006 Reprod. Fertil. Dev. 18, 193). The temperature of the Thermos flask was measured with a temperature recorder. Cumulus–oocyte complexes (COC) were collected by aspirating 2- to 6-mm follicles from the ovaries. The COC were matured in TCM-199 supplemented with 5% calf serum and 0.02 AU mL–1 of FSH. Groups of 20 COC were incubated in 100-μL drops of IVM media at 38.5°C under an atmosphere of 5% CO2 in air for 20 h. After 18 h of gamete co-culture (3 × 106 sperm mL–1), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2. Embryonic development was evaluated at 48 h after IVF (total cleavage rates, CL rate) and on Days 7 to 9 (BL rate). The ovaries in the control group (Group C) were collected from a slaughterhouse different from that of the Group T ovaries and transported to the laboratory in saline (23°C) in a Thermos flask within 3 h. These ovaries were then used for the collection of COC in the same way as for Group T. Data were analysed by chi-square test. A total of 25 experiments were performed using the ovaries from Groups C and T (n = 3945 v. n = 7218). The CL rate and the BL rate for Group T were significantly lower than those for Group C (41.8 v. 62.0% and 16.1 v. 29.7%; P < 0.01). In Group T, the duration of ovary transportation did not affect the CL rate (within 19 h, between 19 and 20 h, and more than 20 h), but the BL rate was significantly decreased with an increase in transport time (18.7 v. 16.1 v. 14.8%). The temperature change in the Thermos flask during transportation ranged from 12.0 to 25.1°C; the temperature was maintained virtually constant or decreased. The CL rates of the groups that underwent a temperature (t) change (t ≤ 5.0 and 5.0 < t ≤ 10.0) were not significantly different, but the BL rate in the group with 5.0 < t ≤ 10.0 was significantly lower than that of the other group (16.7 v. 13.5%; P < 0.01). These results suggest that the long-distance transportation of ovaries affects in vitro embryo development. Moreover, a decrease in temperature during transportation may have an adverse effect on blastocyst formation.
219 IN VITRO FERTILIZATION RATE OF MATURED PIG OOCYTES BY FROZEN - THAWED KOLBROEK PIG SPERM CELLS
Authors: Mapeka M. H. , Lehloenya K. C. , Mphaphathi M. L. , Nedambale T. L.
Published: August 2011

No studies have investigated the IVF rate of South African indigenous Kolbroek sperm cells following cryopreservation. The objective of this study was to test if frozen–thawed Kolbroek pig sperm cells could penetrate pig oocytes matured in vitro. Pig ovaries were collected from a local abattoir and cumulus–oocytes complexes were obtained by aspiration and were then in vitro matured in TCM-199 supplemented with 10% pig follicular fluid, 10% fetal bovine serum, and 1 μg mL–1 of FSH and LH. Following 44 h of incubation, 200 matured pig oocytes were randomly assigned to 2 treatments with frozen–thawed and fresh (control) Kolbroek pig sperm cells. For IVF, Kolbroek sperm cells were in vitro capacitated using Brackett and Oliphant’s sperm wash medium. Matured pig oocytes and sperm cells were co-incubated for 24 h in Brackett and Oliphant’s IVF medium. Following fertilization, presumptive zygotes were in vitro cultured at 39°C in 5% CO2, 5% O2, and 90% N2. Rate of fertilization was identified by the number of cleaved zygotes. Data were analysed by ANOVA. The total motility of Kolboek pig sperm cells used for IVF was 40% for frozen–thawed sperm cells and 80% for fresh sperm cells. The results showed that Kolbroek pig sperm cells were able to penetrate pig oocytes in vitro. However, no significant (P < 0.05) difference was observed in the percentage of cleavage of pig oocytes fertilized with either frozen–thawed (13.25%) or fresh (13.0%) Kolbroek pig sperm cells. The percentage of embryos that developed to the morulae stage was 2% in frozen–thawed sperm cells and was 0% in fresh Kolbroek sperm cells. Furthermore, oocytes fertilized with Kolboek sperm cells did not develop to the blastocyst stage in either treatment. In conclusion, this study demonstrated that frozen–thawed Kolbroek sperm cells are able to fertilize matured pig oocytes in vitro.

This study was funded by the Department of Agriculture Forestry and Fishery, ARC, DST-PDP (RT19000), and National Research Foundation (NRF, Grant No. RT21 and 24000).
222 COMPARISON OF COMMERCIAL IN VITRO EMBRYO PRODUCTION OF BRAHMAN DONORS UNDER BRAZILIAN v. PANAMANIAN MANAGEMENT
Authors: Chen J. R. S. , Nasser L. F. , Penteado L. , Mendizabal M. , Basso A. C. , Pontes J. H. F. , Bionaz M. , Wheeler M. B.
Published: August 2011

Brazil is a leading country in the world in commercial use of in vitro-produced bovine embryos, with approximately 200 000 transfers per year (IETS; Thibier, 2009). This model of large-scale commercial in vitro bovine embryo production is now available for Panamanian producers. Because of the tropical environment in Panama, the most popular breed is the Brahman, a Zebu type of cattle that has been shown to have more follicles emerging per follicular wave than Bos taurus type of cattle and, consequently, that produce more oocytes per session of follicular aspiration. This characteristic, added to the embryo production results, permits such a biotechnology to be implemented on a commercial scale and incorporated into the reproduction management of a herd. A comparison of oocyte number and quality, cleavage, and embryo production was made using the same in vitro production system (InVitro Brazil, Mogi Mirim, São Paulo, Brazil) for Brahman donors, both in Brazil and in Panama. Data were compared using a z-test analysis (Table 1). The percentage of cleaved zygotes was greater (P < 0.001) with the Panamanian Brahman donors as compared with the Brazilian Brahman donors (73 v. 69%, respectively). However, the percentage of blastocysts/cleaved zygotes was greater (P < 0.01), indicating a higher blastocyst production rate from the Brazilian donors. No other differences were observed. Thus, in vitro embryo production with Brahman donors could be used as a tool to improve and spread superior genetics within a Panamanian herd and could also serve as a model for other Central American and Caribbean countries under similar management systems.

Table 1.Panama and Brazil in vitro Brahman embryo production1

This work was supported by BORN Animal Biotechnology, Panama City, Panama.
223 CHANGE IN THE DISTRIBUTION OF RNA IN BOAR SPERM DURING CULTURE IN A CAPACITATION MEDIUM CONTAINING CAFFEINE
Authors: Okudaira Y. , Funahashi H.
Published: August 2011

Mammalian sperm are known to contain various types of RNA. Recently, translation of RNA to proteins occurring during capacitation was reported to be important for sperm function and fertilization. The objective of this study was to examine the change in the distribution of RNA in boar sperm during culture in a capacitation medium containing caffeine by using fluorescence specifically binding to RNA. The sperm-rich fraction from Berkshire boars (n = 4) was diluted (cells mL–1) with modified Modena solution containing 20% seminal fluid, cooled to 15°C for 4 h, and kept at the same temperature until use. Stored, diluted semen was washed by centrifugation (1500 rpm for 35 min at room temperature) in a Percoll gradient (45/90%). A sperm suspension (concentration 1 × 107 cells mL–1) in modified TCM-199 containing 0.4% BSA and 5 mM caffeine was then prepared and cultured in an atmosphere of 5% CO2 in air at 39°C for 1 or 4 h. Before and after culture, sperm were stained by using SYTO RNA Select Green Fluorescence Cell Stain (Molecular Probes, Eugene, OR) according to the manufacturer’s protocol, and then the mounted specimens were observed and the intensity of fluorescence images was measured under a fluorescence microscope (BIOREVO, Keyence, Osaka, Japan). Viability of sperm was also determined following SYBR-Green–propidium iodide staining under a fluorescence microscope. Statistical analyses were carried out by ANOVA and with a Bonferroni-Dunn post-hoc test (P < 0.05). Although the viability of sperm decreased before (96.7%) and 1 h after the start of culture (79.8%), it did not decline until 4 h after the start of culture (80.9%). Before culture, fluorescence indicating the presence of RNA was observed at the head, especially the postacrosomal region and the midpiece region of the sperm. The intensity of fluorescence changed during culture. The fluorescence intensity of RNA at the sperm head region was higher (P < 0.01) at 1 h of culture (36.11 × 104) than before culture (30.50 × 104) and at 4 h of culture (28.60 × 104). The intensity of RNA at the midpiece region was higher (P < 0.01) at 1 h (11.42 × 104) and was lower (P < 0.01) at 4 h of culture (4.93 × 104) than before culture (8.45 × 104). From these results, we concluded that the distribution and content of RNA changes drastically during culture in a capacitation medium containing caffeine. Additional study of the kinetics of sperm RNA during capacitation is ongoing to further understand the post-transcriptional regulation.
225 EFFECTS OF PEDIGREE, MEAT QUALITY, AND MEAT QUANTITY OF SLAUGHTERHOUSE DONOR COWS ON OOCYTE RECOVERY, EMBRYO DEVELOPMENT, AND PREGNANCY AFTER EMBRYO TRANSFER
Authors: Park Y. S. , Park H. D.
Published: August 2011

In conventional in vitro embryo production, oocytes are obtained from various ovaries, and donor characteristics, such as pedigree type, meat quality, or meat quantity, are not considered. The aim of this study was to examine the effects of pedigree type and meat quality and quantity of slaughterhouse donor cows on oocyte recovery, embryo development, and pregnancy rates. The ovaries of individual Korean native cows were obtained from a slaughterhouse. The ear tag numbers were obtained 24 h after slaughter, and the donor data were compared according to the pedigree type (no registration to third bloodline registration), meat quantity according to the percentage of carcass weight (more than 69%, 66 to 69%, and below 66%; Grades A, B, and C, respectively), and meat quality according to the marbling grade (marbling grade 8 to 9, 6 to 7, 4 to 5, 2 to 3, and 1; Grades 1++, 1+, 1, 2, and 3, respectively). There were 390 donors in total, and each group had 30 donors. Recipients (Holstein heifers; n = 222) were synchronized with a progesterone device, and a single blastocyst was transferred nonsurgically on Day 7 from the onset of oestrus. Data on embryo development were compared by Duncan’s multiple range tests, and pregnancy rate was analysed by chi-square test. Values of P < 0.05 were considered to indicate a significant difference. No significant difference was detected in the average number of oocytes recovered (17.5 ± 2.9 to 22.9 ± 1.2%) or blastocyst development (16.4 ± 2.6 to 19.5 ± 3.4%) from cows of different pedigrees. A higher number of oocytes were recovered from Grade A (22.9 ± 1.0) than from Grade B (19.8 ± 0.7; P < 0.05) cows. However, the meat quantity grade had no significant effect on blastocyst development (14.3 ± 1.2 to 15.0 ± 0.8%). A greater number of oocytes were recovered from the Grade 1++ (25.0 ± 1.6), 1+ (27.7 ± 2.4), and 2 (23.6 ± 1.5) groups than from the Grade 3 group (19.7 ± 1.0; P < 0.05). The developmental rate to the blastocyst stage was significantly higher in the Grades 2 and 3 groups (22.1 ± 2.2 and 19.4 ± 1.7) than in the Grade 1++, 1+, and 1 groups (9.0 ± 1.2 to 13.8 ± 2.0). Pregnancy rate was not affected by pedigree type, meat quantity, or meat quality (41.7 to 58.6%). We concluded that 1) pedigree type had no effect on oocyte recovery and embryo development; 2) meat quantity affected oocyte recovery but did not affect embryo development; 3) high meat quality (Grades 1++ and 1+) resulted in greater oocyte recovery but lower embryo development; and 4) pregnancy rate remained unaffected by pedigree type, meat quantity, and meat quality.

This research was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
231 SEMINAL QUALITY IN WHOOPING CRANE (GRUS AMERICANA) IS AFFECTED BY STAGE OF BREEDING SEASON BUT NOT BY AGE OF INDIVIDUAL
Authors: Brown M. E. , Crosier A. , Lynch W. , Converse S. J. , Chandler J. , Olsen G. , French J. , Wildt D. E. , Songsasen N.
Published: August 2011

The whooping crane is one of the most critically endangered species in North America. The species underwent a severe genetic bottleneck with only 16 individuals remaining in the wild as of 1942. Captive breeding began in 1966 and continues to produce chicks for release in order to establish new wild populations. However, captive birds experience poor reproduction with approximately 40% of eggs being infertile. Males have been known to reach sexual maturity at 5 years of age and continue to reproduce almost as long as the duration of their adult life (i.e. 40 years). Understanding factors affecting seminal quality may assist in identifying and correcting causes of suboptimal reproduction. Our objectives were to determine the influence of age and reproductive seasonality on seminal quality. We hypothesised that seminal quality variations among whooping cranes and ejaculates within a given individual over time were due to bird age and stage of breeding season. In 2010, twenty-nine whooping cranes of 5 age groups housed at Patuxent Wildlife Research Center (Laurel, MD, USA) were studied: ≤5 years (n = 3); 6–10 years (n = 7); 11–15 years (n = 7); 16–20 years (n = 4); >20 years old (n = 8). Semen was collected using a manual manipulation technique at 3 stages of the breeding season: early (March, n = 29) mid (April, n = 24), and late (May, n = 14). Samples were evaluated for seminal volume and sperm concentration, motility, and morphology, with data evaluated by analysis of variance. Bird age had no influence on seminal quality, whereas stage of breeding season affected seminal volume and the proportion of sperm with normal morphology (95% confidence interval). Specifically, samples collected during Mid breeding season had the highest volume (mean ± SEM; early: 42.0 ± 8.0 μL; mid: 66.0 ± 15.2 μL; late: 39.7 ± 17.8 μL), but lowest proportions of structurally normal sperm (early: 78.4 ± 3.7%: mid: 61.5 ± 3.2%; late: 69.7 ± 3.4%). There was a significant difference (P = 0.06) in sperm concentration among stages of the breeding season (early: 66.3 ± 18.8 × 106 sperm mL–1; mid: 179.2 ± 46.2 × 106 sperm mL–1; late: 91.4 ± 47.8 × 106 sperm mL–1). Sperm motility was unaffected by season (early: 36.4 ± 3.5%; mid: 45.9 ± 4.1%; late: 48.0 ± 4.9%). In summary, there is a peak in seminal quality that corresponds with higher volume and more sperm during the mid stage of the season, although with higher instances of structural abnormalities. Despite the small founder base for this species, males in this population produce sperm with no variation in seminal quality across a wide variation in age.
234 MORPHOLOGICAL CHARACTERIZATION OF TESTES FROM INSULIN RECEPTOR SUBSTRATE (IRS) 2-DEFICIENT MICE
Authors: Griffeth R. J. , Burks D. J.
Published: August 2011

In mammals, reproduction is controlled by the hypothalamic-pituitary-gonadal axis but it is also affected by changes in energy homeostasis and metabolism. Insulin exhibits pleiotropic effects that are tissue and development dependent. In humans, sperm from males with diabetes exhibit severe structural defects and lower motility and concentration. Insulin receptor substrate 2 (IRS2) is a key mediator in the insulin/insulin-like growth factor-1 signalling pathway. Deletion of IRS2 in mice leads to diabetes and causes female infertility and impaired male fertility. The objective of this study was to determine if the absence of IRS2 in male mice causes a testicular phenotype. Mice were categorized into 3 groups based on genotyping and blood glucose concentrations: wildtype (WT), knockout normal (KOn), or knockout diabetic (KOd); n = 6 mice per group. Testes weighed significantly less in KOn (107.5 ± 10.4 mg) and KOd (112.9 ± 11.1 mg) mice, which corresponds to a reduction in testes size of 46 and 43%, respectively, compared with WT (197.8 ± 29.9 mg; P < 0.001). To determine the difference between the size of the testes in KO and WT mice, the diameter of the seminiferous tubules and the length of the seminiferous epithelium were measured. In both KO groups, both parameters were significantly reduced (P < 0.001). The diameter of the seminiferous tubules of KOn (123.0 ± 12.6 μm) and KOd (137.1 ± 13.1 μm) was reduced by 42 and 35%, respectively, compared with WT (210.5 ± 16.5 μm). The length of the seminiferous epithelium of the KOn (44.0 ± 5.5 μm) and KOd (41.5 ± 6.9 μm) was reduced by 43 and 45%, respectively, compared with WT (76.6 ± 7.25 μm). Because the number of Sertoli cells determines the size of the testes and the number of germ cells that can be supported during spermatogenesis, Sertoli cells were counted. The number of Sertoli cells per cross section of seminiferous tubule was significantly lower in KO v. WT mice (KOn 13.7 ± 2.5, KOd 15 ± 2.8, WT 24.1 ± 2.5; P < 0.001), corresponding to a 43% reduction in KOn and a 38% reduction in KOd. To determine how the differences in morphology affect spermatogenesis, epididymal sperm were counted. As expected, there were fewer sperm in KO mice than WT mice (KOn 3.80 × 106, KOd 8.58 × 106, WT 1.34 × 107). After in vitro capacitation, sperm from all groups were motile; however, sperm from WT mice displayed a more characteristic motility pattern. In summary, there was a reduction in all morphological criteria studied in the testes of KO mice compared with WT. Although IRS2-deficient mice are initially able to breed, when they become severely diabetic, they are infertile. It appears that reductions in testicular morphology and declining numbers of sperm are likely causes of the reduced fertility. The data suggest that insulin signalling in the testes is important for normal testicular development and function and that metabolic abnormalities such as diabetes lead to reduced fertility.
236 PROTEIN TYROSINE PHOSPHORYLATION IN BOAR SPERM DURING CO-CULTURE WITH OVIDUCTAL EPITHELIAL CELLS
Authors: Luño V. , López-Úbeda R. , Gadea J. , Matás C.
Published: August 2011

Essentials steps of the capacitation are coordinated in the caudal isthmus of the oviduct, where sperm are stored in intimate contact with the epithelium. A crucial event involved in capacitation is the protein tyrosine phosphorylation. The aim of this study was to characterise the different protein tyrosine phosphorylation patterns in boar sperm after the co-culture with oviducal epithelial cells (POEC). Epithelial cells were stripped from the oviducts of cycling sows and cultivated for 7 days on Petri dishes. Sperm (with and without treatment by discontinuous gradient of isotonic Percoll) were added and co-incubated for 1 h. Six different samples were analysed by indirect immunofluorescence to determine the localization of proteins phosphorylated in tyrosine residues (Tardif et al. 2001 Biol. Reprod. 65, 784–792): ejaculated control, ejaculated bound and unbound POEC, sperm washed through Percoll, and sperm Percoll-washed bound and unbound POEC. Four patterns were determined according to their surface distribution: nonphosphorylated spermatozoa, head and/or tail phosphorylated, equatorial segment phosphorylated, equatorial segment, and head and/or tail phosphorylated. Sperm without any treatment showed lower tyrosine phosphorylation levels than sperm washed through Percoll (66.90% v. 1.90%; P < 0.01). The most common tyrosine phosphorylation distribution in sperm adhesion to POEC was located in equatorial segment (57.78%; P < 0.01). A significant difference regarding head phosphorylation (P < 0.01) was found between sperm bound to oviductal epithelium and unbound sperm (0.00% v. 4.75%). Binding to POEC occurred mainly in sperm with nonphosphorylated heads and tails, whereas higher proportions of equatorial segment and head and/or flagellum phosphorylated cells were found in unbound population (50.25% v. 83.75%; P < 0.01). The data of this study showed differences in the sperm-POEC binding in relation to protein tyrosine phosphorylation patterns, indicating a selective function in the sperm-oviductal epithelial cells interaction.

Supported by Fundación Séneca 08752/PI/08.
240 FUNCTIONAL TRAITS OF CAT SPERM DURING DISTINCT MATURATION STATUS
Authors: Perez E. G. A. , Nichi M. , Baptista Sobrinho C. A. , Góes P. A. A. , Dalmazzo A. , Gurgel J. R. , Rocha C. C. , Silva R. O. C. , Barnabe R. C. , Barnabe V. H.
Published: August 2011

Sperm recovery from the caudae epididymides can be advantageous for preserving semen of endangered animal species. In this context, the domestic cat is a suitable model for the study of sperm physiology in endangered feline species and the research on epididymal sperm preservation combined with the use of reproductive biotechnologies including intracytoplasmic sperm injection (ICSI). The aim of the present study was to examine the sperm collected from the cauda and caput of the cat epididymis using functional tests. Testicles and epididymides from 5 adult tomcats were collected by orchiectomy and maintained at 4°C for 4 h, until semen collection. Semen samples were collected from the epididymal tail and head by careful dissection. Samples were then analysed for motility by computer assisted sperm analysis (CASA; only for the caudal sperm). The 3-3′ diaminobenzidine stain was used as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity, the simple stain (fast green/Bengal rose) as an index of acrosome integrity, and the measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Statistical analysis was performed using the SAS System for Windows (SAS Institute Inc., Cary, NC, USA; least significant differences test and Spearman correlation; P < 0.05). No motility was observed in samples collected from the epididymal head, whereas samples from the tail showed 50.0 ± 4.2% motile spermatozoa. Surprisingly, more spermatozoa with high mitochondrial activity were found in the epididymal head than in samples from the tail (74.0 ± 3.5 v. 50.0 ± 4.3%, respectively). Similarly, samples collected from the head showed a higher susceptibility against the attack of ROS (31.9 ± 5.5 v. 16.3 ± 7.1 ng of TBARS/106 sperm, respectively). Furthermore, epididymal head sperm showed a lower percentage of sperm with intact membrane and a higher percentage of sperm with intact acrosome (44.9 ± 3.3 and 78.4 ± 1.8 v. 66.4 ± 4.2 and 56.7 ± 4.4%, respectively). Our results demonstrate that, during maturation, feline sperm are subjected to high oxidative stress, as shown by the lipid peroxidation assay, which would lead to structural damage to biomolecules, DNA, lipids, carbohydrates and proteins, as well as other cellular components, such as mitochondria, and acrosomal impairment. Similar results were found in humans, in which higher levels of oxidative stress occurred in the post-testicular environment. The plasma membrane seems to be more resistant to damages. This may be due to the described rearrangement in the lipid profile occurring during maturation, but studies to test this hypothesis are still underway.