bluuebunny/crossref_metadata_2025 huggingface dataset:
227 PHOSPHOINOSITIDE-3-KINASE SIGNALING AND THE CONTROL OF SPERM MOTILITY IN THE PORCINE OVIDUCT
Authors: Satake N. , Watson P. F. , Holt W. V.
Published: January 2008

Prior to ovulation, interactions of spermatozoa with the oviductal epithelium lead to the formation of a sperm reservoir; here we investigate the hypothesis that the physiological state of the stored spermatozoa is modulated by the epithelial cells. In the pig, where modulation of sperm motility is sensitive to environmental components, the high bicarbonate content of the oviductal fluid would be expected to stimulate motility to its maximal extent; however, as this is incompatible with reservoir formation, we postulate that a signaling pathway counteracts bicarbonate stimulation and depresses motility (Satake et al. 2006 J. Exp. Biol. 209, 1560–1572). We chose to investigate a signaling mechanism which may suppress sperm motility through phosphoinositide-3-kinase (PI3K). Boar spermatozoa were exposed to solubilized proteins derived from the oviduct epithelial apical plasma membrane (sAPM). We exploited the motility activation effects of bicarbonate on Percoll-washed boar spermatozoa to see whether it was countered by sAPM and reversed by PI3K inhibitors. Washed spermatozoa (n = 9 boars) were incubated (10 min at 38�C) in Tyrode's medium in the presence of sAPM (25 and 50 µg mL–1) or PI3K inhibitors (LY294002 or wortmannin) prior to activation with 15 mm bicarbonate/CO2, and then incubated for a further 35 min. Sperm motility was monitored at 5-min intervals using a Hobson Sperm Tracker (Hobson Tracker, Ltd., Sheffield, UK). Samples were also analyzed with a flow cytometer for mitochondrial activity using JC-1. PATN analysis (Belbin 1993 PATN Pattern Analysis Package, Div. Wildlife & Ecol., CSIRO, Canberra, Australia) was used to distinguish four motility-based sperm subgroups within the data. The proportions of fast and progressive spermatozoa showed significant and rapid (<5 min) bicarbonate-induced increases when there was no sAPM treatment, or when PI3K inhibitors were present. However, there was no significant bicarbonate-induced increase in the presence of sAPM alone, or when spermatozoa were exposed to a mixture of wortmannin and sAPM. PI3K inhibition by LY294002 reversed the sAPM-induced motility suppression (P < 0.001). Mitochondrial membrane potential was also suppressed by sAPM and restored by the presence of LY294002 (P < 0.01). These results support the hypothesis that the sperm–oviduct interaction is modulated by an intracellular signaling pathway involving PI3K and appropriate receptors.
228 RECIPIENT PREPARATION FOR SPERMATOGONIAL STEM CELL TRANSPLANTATION: ALTERATION IN TESTICULAR CELL COMPONENTS FOLLOWING TRANSIENTLY INDUCED ISCHEMIA
Authors: Schuenemann G. M. , Mendis-Handagama S. M. L. C. , Prado T. M. , Adair H. S. , Schrick F. N.
Published: January 2008

Depletion of endogenous spermatogonial stem cells using busulfan (Brinster et al. 2003 Biol. Reprod. 69, 412–420) or irradiation (Izadyar et al. 2003 Reproduction 126, 765–774) have been used in preparation of recipient animals prior to transplantation; however, both techniques are not without compromises (severe bone marrow depression or specialized radiotherapy equipment required). Induced testicular ischemia in rams altered spermatic epithelium with germ cell-depleted seminiferous tubules (Markey et al. 1994 Reprod. Fertil. 101, 643–650). The objective was to evaluate testicular transiently induced ischemia (using elastrator bands) in Jersey calves on testicular morphology and development. Treatments (at 27 ± 5 days of age) consisted of control (0, n = 4), banding for periods of 2 h (2, n = 4), 4 h (4, n = 4), and 8 h (8, n = 4). After castration (age: 60 ± 5 days), the right testis of each animal was used for calculation of cell components per testis according to the point counting method. Data were analyzed using the MIXED procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Body weight (59.8 ± 6.2 kg) and scrotal circumference (SC) at banding (9.1 ± 0.2 cm) did not differ between treatments. Fresh testis weight (TW), scrotal temperature immediately before banding removal (ST), and daily scrotal circumference growth (SC) were decreased (4 and 8 h) in ischemic testes compared to controls (Table 1: P < 0.05). In addition, Sertoli and Leydig cells were severely reduced in the 8-h ischemic treatment (Table 1: P < 0.05). Transiently induced ischemia significantly decreased the number of germ cells in the 8-h group, compared to the 0-, 2-, and 4-h groups (Table 1: P < 0.05). These results suggest that transiently induced ischemia significantly decreases the number of germ, Sertoli, and Leydig cells in the testis. Therefore, these present findings could be applicable for preparation of recipient animals through depletion of endogenous germ cells within the seminiferous tubules. This procedure may provide a suitable environment for transplanted donor germ cell colonization in prepubertal recipient bulls.

Table 1. Parameters evaluated under transient-induced ischemia treatments
229 EFFECTS OF NONYLPHENOL ON MOTILITY, ACROSOMAL INTEGRITY, AND MITOCHONDRAL MEMBRANE POTENTIAL OF EPIDIDYMAL RAT SPERM
Authors: Uguz C. , Varisli O. , Agca C. , Agca Y.
Published: January 2008

Nonylphenol (NP), a derivative of an estrogenic environmental endocrine disrupter called alkylphenol polyethoxylates (APEs), has been shown to have toxic, estrogenic, and carcinogenic effects on both aquatic and terrestrial organisms. NP is widely used in common cosmetic products and detergents. The primary objective these studies was to determine the effects of NP (dissolved in either ethanol or dimthyl sulfoxide (Me2SO)) on rat sperm viability characteristics such as motility, acrosomal integrity, and mitochondrial membrane potential. The epididymal rat sperm were suspended (50 � 106 spermatozoa mL–1) in sodium bicarbonate-buffered Tyrode's lactate solution containing 3 mg mL–1 bovine serum albumin (BSA) after removal of the cauda epididymis of sexually mature Sprague Dawley male rats (n = 5). Aliquots of 0.5 mL sperm suspension were added into 5-mL test tubes containing 1, 10, 100, 250, 500, and 1000 µg NP mL–1. As negative controls, no NP–no solvent, no NP+ethanol, and no NP+Me2SO were used. All sperm treatments were incubated for 1, 2, 3, and 4 h at 37�C with 5% CO2 in air. Computer-aided sperm analysis system (Hamilton Thorne, Beverly, MA, USA) was used to determine sperm motility characteristics. An epifluorescent microscopy method was used to determine sperm acrosomal status after staining with 20 µg mL–1 Alexa Fluor-488-PNA (Molecular Probes, Eugene, OR, USA) and a flow cytometric method was used to determine mitochondrial membrane potential after staining with 2 µg mL–1 JC-1 (Molecular Probes). The data were analyzed by ANOVA by using SAS (SAS Institute, Inc., Cary, NC, USA). The results showed that, as compared to negative controls (no NP–no solvent, no NP+ethanol), exposure of rat sperm to 250, 500, or 1000 µg NP mL–1 was highly detrimental to their motility (P < 0.05). Total loss of sperm motility was observed after exposure to 500 µg NP mL–1. However, the time course did not seem to have a significant effect on sperm motility (P > 0.05). Nonylphenol induced dose-dependent acrosomal reaction in rat sperm (P < 0.05). The effects of NP on the mitochondrial membrane potential (MMP) of rat sperm were also significant (P < 0.05). The percentage of rat sperm with high MMP declined drastically after exposure to 100 and 250 µg NP mL–1 as compared to negative controls (P < 0.05). However, duration of exposure to NP did not have an effect on MMP of rat sperm (P > 0.05). In conclusion, these data obtained from epididymal rat sperm provide important information about the mechanism of detrimental effects of NP on mammalian sperm, which must be seriously considered in the case of short- or long-term exposure to NP in humans as well as livestock species.
236 PARTHENOGENETIC ACTIVATION OF IN VITRO-MATURED OVINE OOCYTES WITH STRONTIUM
Authors: Zhu J. , Campbell K. H. S.
Published: January 2008

The objective of the present experiments was to examine whether strontium could activate in vitro-matured ovine oocytes. Oocytes were collected and matured as previously described (Lee and Campbell 2006 Biol. Reprod. 74, 691–698). Briefly, selected cumulus–oocyte complexes were cultured in modified TCM-199 medium supplemented with 20% sheep serum and hormones for 22–23 h, at 39°C, 5% CO2 in air. Matured oocytes were randomly divided into four groups and treated as follows: (1) cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (2) electrically activated in Ca2+-containing medium, then cultured in 10 mm strontium + 5 μg mL–1 cytochalasin B in Ca2+-free CZB medium for 4–5 h; (3) electrically activated in Ca2+-containing medium and then cultured in SOF medium containing 5 μg mL–1 cytochalasin B for 4–5 h; and (4) electrically activated in Ca2+-free medium and then transferred into SOF medium + 5 μg mL–1 cytochalasin B for 4–5 h. This experiment was repeated three times. Activation rates based on the number of pronuclear formations/the number of oocytes cultured were 96.7% (147/152), 95.9% (116/121), 75.9% (101/133), and 43.0% (56/107) in Groups 1–4, respectively. After 7 days of culture in SOF medium, 26.8%, 33.3%, 19.6%, and 0% of oocytes in Groups 1, 2, 3, and 4 developed to the blastocyst stage, respectively. Significant differences in blastocyst rate were observed across these groups except between groups 1 and 2 (P < 0.01). However, there were no significant differences in mean number of nuclei/blastocyst across Groups 1, 2, and 3 (P > 0.05). Our results demonstrated that in vitro-matured ovine oocytes can be effectively activated with strontium alone, resulting in an activation rate of 96.7% and a blastocyst rate of 26.8% (blastocysts/oocytes). Also, a combination of strontium and electrical pulses could benefit sheep oocyte activation and embryo development to the blastocyst stage (95.9% and 33.3%, respectively). We conclude that strontium is an effective activator for sheep oocyte activation and it could be used for sheep nuclear transfer.

Table 1. Parthenogenetic development of oocytes activated by SrCl2+ and electrical pulses
237 EXPRESSION OF THE GENES HSP 70.1, ZAR-1, AND MATER IN BOVINE OOCYTES SUBMITTED TO PREMATURATION AND/OR IN VITRO MATURATION
Authors: Adona P. R. , Biase F. H. , Braga F. C. , De Bem T. H. C. , Rochetti R. , Leal C. L. V.
Published: January 2008

The present study aimed to assess the transcripts for the proteins that embryos require, histone 2a (H2a-FZ), heat shock 70 kDa protein 1A (HSP 70.1), zygote arrest 1 (ZAR-1), and maternal antigen (MATER), in bovine oocytes submitted to prematuration (PM) culture and/or in vitro maturation (IVM). Follicles (2–6 mm diameter) were aspirated from slaughterhouse-derived ovaries. Oocytes were selected and randomly distributed among treatments. For PM, oocytes were cultured 24 h in TCM-199 medium supplemented with 10 µm butyrolactone I, 0.2 mm pyruvate, and 10 µg mL–1 gentamicin (BGV group). Part of the prematured oocytes were washed and transferred to IVM culture (BMII group). For IVM, oocytes were cultured in TCM-199 supplemented with 10% FCS, 5.0 µg mL–1 LH, 0.5 µg mL–1 FSH, 0.2 mm pyruvate, and 10 µg mL–1 gentamicin for 22 h. As controls one group of oocytes was collected immediately after aspiration (GV group) and another group was matured in vitro (MII group) without undergoing prematuration. All cultures were carried out in 100-µL droplets of culture medium under mineral oil at 38.5�C and 5% CO2 in air. Oocytes from each treatment were denuded and frozen at 80�C (3 pools of 200 oocytes) in PBS with 0.1% polyvinyl alcohol (PVA) and 1 UI µL–1 RNase inhibitor. RNA was extracted using the RNeasy Protect Kit (Qiagen, S�o Paulo, Brazil) according to the manufacturer's instructions. The extracted RNA was used for reverse transcription by the enzyme Improm-II Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturer's instructions. cDNA was produced in a thermocycler for 60 min at 42�C, followed by warming to 70�C for 15 min and cooling to 4�C for freezing at –20�C. Relative quantification of the transcripts for the genes H2a-FZ, HSP 70.1, ZAR-1, and MATER was performed by real-time PCR using the SYBR GREEN Kit (Applied Biosystems do Brasil, Sao Paulo, Brazil) according to manufacturer's instructions. Data were analyzed by the REST� software (Relative Expression Software Tool; Pfaffl et al. 2002 Nucl. Acids Res. 30, e36) 2005 BETA V1.9.9; a level of significance of 5% was considered to show differences among transcripts using H2a-FZ to normalize data. No differences were observed (P < 0.05) for the transcripts HSP 70.1, ZAR-1, and MATER, respectively, when comparing GV (1.0, 1.0, and 1.0), MII (0.37 � 0.1, 0.37 � 0.05, and 0.43 � 0.1), and GV with BGV (0.43 � 0.15, 0.54 � 0.2, and 0.33 � 0.25). However, a difference was detected (P < 0.05) between BGV (1.0, 1.0, and 1.0) and BMII (0.17 � 0.1, 0.13 � 0.05, and 0.3 � 0.2), but not between (P > 0.05) MII (1.0, 1.0, and 1.0) and BMII (0.52 � 0.25, 0.5 � 0.1, and 0.6 � 0.25) for transcripts HSP 70.1, ZAR-1, and MATER, respectively. A reduction in transcripts in group BMII may influence oocyte competence, reducing embryo development and/or quality.
238 EFFECTS OF LEPTIN SUPPLEMENTATION ON NUCLEAR AND CYTOPLASMIC IN VITRO MATURATION OF RABBIT OOCYTES
Authors: Arias-Alvarez M. , Garcia-Garcia R. M. , Revuelta L. , Rebollar P. G. , Lorenzo P. L.
Published: January 2008

Reproductive function is affected substantially by nutritional status. Leptin is a peptide secreted mainly by adipocytes that reflects the amount of body fat and acts as a modulator of oocyte quality. The aim of this study was to analyze, for the first time in the rabbit, the influence of leptin on meiotic and cytoplasmic maturation (cortical granule (CG) migration) of rabbit oocytes in vitro (IVM). Cumulus–oocyte complexes (COCs) were collected from 25 young New Zealand white female rabbits (<3 parturitions) in 3 replicates. COCs were aspirated from ovarian follicles >1 mm in size and were matured in TCM-199 medium, containing sodium pyruvate, sodium bicarbonate, BSA, and 10 ng mL–1 epidermal growth factor (EGF), and supplemented with 0, 10, or 100 ng mL–1 leptin. A total of 163 COCs were treated progressively with hyaluronidase (2 mm), 0.5% pronase, 4% paraformaldehyde, 0.02% Triton X-100, and 7.5% BSA after the maturation period. Oocytes were incubated with 100 mg mL–1 fluorescein isothiocyanate (FITC)-conjugated Lens culinaris agglutinin (LCA) for CG staining and with 4′,6-diamino-2-phenylindole (DAPI) for nuclear staining, and observed under a confocal laser-scanning microscope. In addition, 17 ovulated oocytes recovered from oviducts at 20 h post- GnRH were used as in vivo-maturated controls for CG distribution. Most of the ovulated oocytes at metaphase II (MII, 100%) presented CGs located in the cortex beneath the plasma membrane (61.1 ± 11.8%). Addition of 10 ng mL–1 leptin to IVM medium significantly increased the rate of oocytes reaching MII, compared to the 0 and 100 ng mL–1 leptin concentrations (P < 0.05). The percentage of oocytes showing CG migration to the cortex was significantly increased in the 10 ng mL–1 leptin treatment group (Table 1) compared to that in the 100 ng mL–1 leptin group (P < 0.05) and tended to be higher than that in the 0 ng mL–1 leptin group (P < 0.08). The rest of the oocytes showed homogeneous CG distribution, as they were not cytoplasmic maturated. Moreover, both in vivo- and in vitro-matured oocytes had a GC-free domain overlying the MII spindle. In conclusion, addition of leptin to IVM medium at physiological dose (10 ng mL–1) improves both meiotic and cytoplasmic maturation of rabbit oocytes, whereas an excessive leptin concentration does not exert a beneficial effect. These findings suggest a physiological role for leptin in the relationship between nutritional status and regulation of oocyte maturation.

Table 1. Nuclear maturation and CG distribution of oocytes after IVM

This research was supported by AGL05-196 and UCM-CM research program (920249). MAA received a grant from CM and FSE. RMGG was supported by the Juan de la Cierva-MEC Program.
243 NUCLEOSIDES REDUCE THE POTENTIAL OF THE MITOCHONDRIAL INNER MEMBRANE AND THE DEVELOPMENTAL COMPETENCE OF MOUSE OOCYTES DURING IN VITRO MATURATION
Authors: Fujii W. , Funahashi H.
Published: January 2008

Although nucleosides such as adenosine exist in follicular fluid and decrease during oocyte maturation (Eppig et al. 1985 Biol. Reprod. 33, 1041–1049), the role of nucleosides is still unclear. The present study was undertaken to determine the effect of nucleoside on nuclear and cytoplasmic maturation of mouse oocytes. Oocyte–cumulus complexes (OCCs)were collected from the large antral follicles of C57BL/6J female mice (3–5 weeks old) 4 h after a combination injection of equine chorionic gonadotropin (eCG) and hCG with a 48-h interval, and cultured in maturation medium (αMEM containing 3 mg mL–1 BSA, 0.23 mm Na pyruvate and antibiotics) with or without ribo- and deoxyribo-nucleosides (10–11 µg mL–1) for 12 h. Statistical analyses in this study were carried out by ANOVA and Bonferroni/Dunn's post hoc test. Regardless of the presence of nucleosides, a majority of oocytes developed to the metaphase-II stage in vitro, and the incidence was not different with in vivo-matured oocytes. However, the mitochondrial membrane potential (MMP) of oocytes was significantly lower when the oocytes were matured in the presence of nucleosides, as compared with nucleoside-free controls. In the scanning experiment of MMP level during in vitro maturation (IVM), the MMP of oocytes maturing in vivo increased between 8 and 12 h after hCG injection, whereas no rises of MMP was observed in oocytes matured in vitro in the presence of nucleosides. To examine the affecting period of nucleosides, OCCs were cultured for the first 4 h or the latter 8 h of IVM in the presence of nucleosides. MMP of the oocytes was significantly lower only when the OCCs were exposed to nucleosides for the latter 8 h of IVM. To determine if adenosine in the nucleosides (10 µg mL–1) affects the MMP of oocytes, OCCs were exposed to adenosine or a mixture of guanosine, cytidine, and uridine during the latter 8 h of IVM. The MMP of oocytes exposed to adenosine was lower than that of in vivo-matured oocytes and of oocytes exposed to a mixture of guanosine, cytidine, and uridine. To determine the effect of nucleosides on the developmental competence, oocytes exposed to adenosine during the latter 8 h of IVM were cultured in kSOMaa containing 1 mg mL–1 BSA after activation in the presence of 10 mm SrCl2 and 5 µg mL–1 cytochalasin B for 6 h. Pronuclear formation and early development of those were compared with artificially activated oocytes matured in vivo or in the absence of nucleosides. The incidences of pronuclear formation and cleavage of oocytes matured in the presence of adenosine following activation were extremely decreased, as compared with control oocytes matured in vivo or in the absence of adenosine. These observations indicate that nucleosides, at least adenosine, inhibit an increase of MMP of oocytes during the latter half of meiotic maturation, and detrimentally affect the developmental competence of murine oocytes.
246 FUNCTIONAL ANALYSIS OF Sebox DURING OOCYTE MATURATION AND EARLY EMBRYOGENESIS BY RNAi
Authors: Kim K. H. , Kim E. Y. , Lee H. S. , Lee K. A.
Published: January 2008

Previously, we found that the skin-embryo-brain-oocyte homeobox (Sebox) gene was highly expressed in germinal vesicle (GV)-stage oocytes. The function of Sebox is unknown as yet; thus the objective of this study was to determine the role(s) of Sebox during oocyte maturation and early embryogenesis using RNA interference (RNAi). Cumulus-free GV oocytes were collected from 4-week-old ICR mice 48 h after pregnant mare serum gonadotropin (PMSG) injection. The expression pattern of Sebox mRNA was evaluated in various tissues, including gonads, oocytes, and embryos at different developmental stages. To determine the role of Sebox in oocyte maturation and preimplantation embryonic development, Sebox dsRNA was microinjected into the cytoplasm of GV oocytes and pronuclear (PN)-stage embryos, respectively. After Sebox RNAi, phenotype and maturation rates were recorded prior to measuring changes in Sebox mRNA and protein concentration. RNA content was determined by RT-PCR and protein content was determined by oocyte dot blot. Anti-Sebox antibody was produced against a 14 amino acid synthetic peptide corresponding to residues 67–80. Additionally, chromosomal status of the oocytes was confirmed by orcein staining, while spindle shape was confirmed by immunofluorescence staining. Sebox mRNA was ubiquitous in adult mice tissues, with relatively high expression in the brain and ovary. Expression of Sebox was detected in oocytes, granulosa cells, and theca cells. Sebox mRNA was highly expressed from GV up to 2-cell-stage embryos, but dramatically decreased afterward in later developmental stages. Sebox dsRNA microinjection into GV oocytes resulted in a markedly decreased Sebox mRNA (80%) and protein (70%) expression. However, Sebox RNAi resulted in rates in MII oocytes (82%) similar to that of control (87%) and the buffer injection (80%) group during in vitro maturation. Sebox RNAi did not affect the spindle and chromosomal organizations of the MII oocytes. But microinjection of Sebox dsRNA into PN embryos inhibited preimplantational embryo development and blocked at the 2-cell stage (79%). Results suggest that the Sebox gene is not related to the regulation of oocyte nuclear maturation in mice. However, we concluded that Sebox is a new entry of maternally expressed homeobox gene that is involved in the regulation of early embryogenesis, especially at the 2-cell block.
This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-311-E00067).
251 DEVELOPMENT OF A TECHNIQUE FOR STAINING CHROMOSOME AND SPINDLE OF MI AND MII BABOON OOCYTES
Authors: Ridha-Albarzanchi M. , Alanssari S. , Taiyeb A. , Dunkin K. , Beifuss K. , Chowdhary B. , Eddy C. , Liu L. , Bernstein L. , Kraemer D.
Published: January 2008

The objective of this study was to develop a technique for staining baboon oocytes in order to detect chromosome and spindle disorganizations in abnormal oocytes. Baboon oocytes were retrieved by laparoscopy at Southwest National Primate Center/San Antonio following ovulation induction. The metaphase-I (MI) and metaphase-II (MII) oocytes were placed in one-mL sterile plastic tubes containing 10% formaldehyde solution. The tubes were sent by regular mail from San Antonio to the Reproductive Sciences Laboratory at Texas A&M University/College Station. The duration of oocyte fixation was from two to three weeks. The technique of baboon oocyte staining was based on the modification of the mouse oocyte staining protocol. A total of nine oocytes were collected for staining. The age of the baboon was six years. The oocytes were washed four times each, 20–30 min at room temperature, with PBS containing 10 µL Triton, 0.2 g dry milk, 2 g bovine serum albumin, and 0.75 g of glycine. The oocytes were kept over night in a refrigerator. The oocytes were stained with primary antibody solution (Monoclonal Anti Beta-Tubulin, Sigma Co., St. Louis, MO, USA) for 60 min at 37�C. After staining, five washes were performed for 20–30 min each at room temperature (RT). The oocytes were stained with secondary antibody solution (Alexa fluor 488, Invitrogen Co., Carlsbad, CA, USA) for 60 min at 37�C. Three washes were carried out at the end of the second staining, 20–30 min each at RT. Final staining was performed with Vectashieldreg propidium iodide (PI; Vector Laboratories, Burlingame, CA, USA) on a barrier slide which was then covered with a cover slide. Each slide contained 2–3 stained oocytes. The barrier slides were kept in a refrigerator for at least 48–72 h before imaging the oocytes. Chromosomes were stained with PI (red) and micro-tubular spindles were stained with anti Beta-tubulin antibodies + Alexa 488 (green). Cumulus cells appeared as bright red dots. The chromosomes of the immature oocytes (MI) did not yet appear completely aligned at the metaphase plate. The mature oocytes (MII) had extruded the polar body and it was clearly visible. The chromosomes looked elegantly organized at the metaphase plate. One chromosome was found to be disorganized (not aligned at the methaphase plate) in some oocytes which may be due to the side effect of superovulation treatment, but the spindles looked organized. In conclusion, the technique is found to be suitable for study of chromosome and spindle disorganizations which may cause recurrent abortions, birth defects, and infertility in nonhuman and human primates.
257 THE PRESENCE OFAMMONIUM IN CHEMICALLY DEFINED MATURATION MEDIUM INHIBITS PORCINE OOCYTE NUCLEAR MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT IN VITRO
Authors: Yuan Y. , Krisher R. L.
Published: January 2008

Exogenous supplementation with amino acids is important to the success of oocyte IVM and embryonic development in vitro. However, free ammonium ions will be generated by amino acid degradation or as a by-product of amino acid metabolism. Ammonium in embryo culture medium has detrimental effects on embryo development and viability. Nevertheless, the effects of ammonium in maturation medium on oocyte nuclear maturation and resulting developmental potential are still unknown. The objective of this study was to examine the effect of ammonium in maturation medium on porcine oocyte nuclear maturation and subsequent embryonic development after parthenogenetic activation (PA). Cumulus–oocyte complexes were matured in a chemically defined medium, Purdue Porcine Medium (PPM; experimental treatments), or TCM199 with 10% porcine follicle fluid (TCM + pFF; positive control) for 44 h, in 7% CO2 in air at 38.7°C. For experimental treatments, ammonium chloride was added to PPM: 0 mm, 0.02 mm. 0.2 mm, 2 mm, and 20 mm. To examine nuclear maturation, oocytes were fixed and stained after IVM (20–40/treatment/replicate; 6 replicates). Each oocyte was scored from 1 to 7 based on meiotic stage. Oocytes were activated by exposure to a DC electrical pulse of 120 V mm–1, 80 μs. The PA embryos were cultured in porcine zygote medium (PZM) containing 2 mm 6-DMAP for 6 h, then in PZM supplemented with 4 mg mL–1 BSA for 6 days, at which time cleavage, blastocyst development, and blastocyst cell number were determined (40–60/treatment/replicate; 4 replicates). Data were analyzed by ANOVA with Bonferroni multiple comparison test; percentage data were arcsin transformed (significance, P < 0.05). There was no significant difference in maturation between TCM + pFF (6.17 ± 0.11), 0 mm (5.76 ± 0.14), 0.02 mm (5.70 ± 0.13), or 0.2 mm (5.61 ± 0.14), but maturation was significantly decreased in 2 mm (4.57 ± 0.15) and 20 mm (3.76 ± 0.14) ammonium. Embryonic development is shown in Table 1. Blastocyst development (of total activated oocytes) was significantly reduced in 0.2 mm, 2 mm, and 20 mm groups compared to the positive control. In conclusion, the presence of 0.2 mm ammonium in maturation medium compromised oocyte developmental competence, although nuclear maturation was not affected until ammonium concentrations reached 2 mm. These results demonstrate the deleterious effects of ammonium on both nuclear and cytoplasmic maturation in vitro, and point to the importance of minimization of ammonium build-up during this time period.

Table 1. Development of parthenogenetically activated oocytes after exposure to ammonium during IVM
259 ADDITION OF CHOLESTEROL TO FLOW-SORTED BULL SPERMATOZOA TO ENHANCE CRYOSURVIVAL DOES NOT AFFECT THE ACROSOME REACTION
Authors: Crichton E. G. , Graham J. K. , Schenk J. L.
Published: January 2008

Treating bull sperm with cholesterol-loaded cyclodextrins (CLCs) improves post-thaw motility and viability (Purdy and Graham 2004 Biol. Reprod. 71, 522–527). Unpublished research in our laboratory demonstrated similar improvements for cryosurvival of CLC-treated sex-sorted bull sperm. Our objective was to determine the effects of CLCs on the dilauroylphosphatidylcholine (PC12)-induced acrosome reaction of flow-sorted and non-sorted (control), frozen/thawed bull sperm. Sperm concentrations (3 ejaculates, 4 bulls) were adjusted to 200 × 106 sperm mL–1 with TALP without Ca++. Sperm for sorting were stained (63 μm Hoechst 33342, 34.5°C, 45 min), and then incubated an additional 15 min with 2.5 mg CLC in TALP (+CLC) or an equivalent volume of TALP without CLCs (–CLC), adjusted to 100 × 106 sperm mL–1 with 4% egg yolk TALP containing 0.002% food dye, filtered, and ‘bulk’ sorted (Schenk et al. 1999 Theriogenology 52, 1375–1391) into 2.0 mL aliquots of 20% egg yolk Tris buffer. Controls were extended in 20% egg yolk Tris A to result in a final concentration of 21 × 106 sperm mL–1. All samples were cooled to 5°C over 90 min, and Tris buffer with (controls) or without (flow-sorted) egg yolk (12% glycerol) was added (v/v). Sorted samples were centrifuged (850g, 20 min), and pellets were suspended in 20% egg yolk/6% glycerol Tris, resulting in 21 × 106 sperm mL–1. Sperm were loaded into 0.25-mL straws and frozen over LN2 vapor. Straws were subsequently thawed (30 s, 37°C), and 25 million sperm/treatment were centrifuged through 60% Percoll® to remove egg yolk. Pellets were adjusted to 20 × 106 sperm mL–1 with TALP containing 2.0 mm Ca++. Aliquots (30 μL) were stained with propidium iodide (PI) and fluorescein isothiocyanate–peanut agglutinin (FITC-PNA); treated with 0, 25, or 35 μL PC12; and brought to a constant volume (300 μL) with TALP. TALP (30 μL) containing 42 mm Ca++ was added, resulting in final concentrations of 0, 68, and 95 μm PC12 and 6 mm Ca++. Following incubation (38°C, 35–40 min) 50 000 sperm were analyzed on an SX MoFlo® (Dako, Fort Collins, CO, USA) to determine the percentage of live acrosome-reacted sperm/live sperm(PI –ve, FITC +ve). There were no differences between CLC-treated and non-treated sorted or non-sorted sperm (Table 1) suggesting that, as previously shown for non-sorted sperm (Purdy and Graham 2004), treatment of flow-sorted sperm with CLCs does not interfere with the progression of the acrosome reaction.

Table 1. Effect of treatment with CLCs on % live acrosome-reacted (mean ± SEM) non-sorted and flow-sorted cryopreserved bull spermatozoa
261 LABELING SEX-SPECIFIC DNA SEQUENCES IN MAMMALIAN SPERM
Authors: Didion B. A. , Bleher R.
Published: January 2008

While flow cytometric separation of X- andY-chromosome- bearing sperm has advanced to the point of acceptance in the commercial production of sex-preselected cattle, it is important to continue researching this area to improve efficiencies. For example, the difference in DNA sequence between the X- andY-chromosomes has merit as a foundation for an alternative sperm sexing approach that could enable the complete separation and use of an entire ejaculate. We used synthetic DNA mimics conjugated to a fluorescent dye for in situ detection of Y-chromosomes in metaphase preparations of porcine somatic cells and spermatozoa. Peptide nucleic acids (PNA) are synthetic compounds with higher affinity and stability than conventional DNA probes and are used as specific hybridization probes to complementary DNA. The application of PNA probes was demonstrated previously in telomere analysis studies, and we confirmed their efficacy using a CY3-(CCCTAA)3 PNA to probe bull and boar sperm telomeric sequences. Using male porcine somatic cells and theY-chromosome as a template, we arranged for the synthesis of a CY3-conjugated PNA to bind 13-15 base pairs of unique, Y-chromosome sequence. By testing different labeling conditions, we found that brief incubation of metaphase chromosomes with the PNA produced a localized signal on theY-chromosome. No signals were present when chromosomes of porcine female somatic cells were incubated with the PNA probes. Because chromosomes occupy non-random territories in all cell nuclei including those in sperm, we expected to find centrally located signals in 50% of fixed boar sperm when these were treated with the same PNA as used for the somatic cells. We found the signals present in 161 of 302 (53.3%) sperm to consist of a single, centrally located, round fluorescent dot in the sperm head. Further research is required to establish the uptake of PNA in live sperm toward evaluation of this approach for sperm sexing.
262 THE EFFECT OF TRIS AND Botu-Bov® FOR BOVINE SEXED SPERM CRYOPRESERVATION
Authors: Freitas C. P. , Dell'Aqua-Junior J. A. , Papa F. O. , Alvarenga M. A. , Crespilho A. M. , Landim-Alvarenga F. C.
Published: January 2008

Sexing of sperm by flow cytometry has been applied worldwide. However, the sorting as well as the cryopreservation process causes damage to the sperm cells. Therefore, the sexed sperm need enhanced protection during the freezing process. Thus, the aim of the present study was to compare the effect of two different extenders for sperm cryopreservation. Two ejaculates from 10 bulls (Bos taurus and Bos indicus) of reproductive age and used in commercial AI programs were used. The ejaculates were collected with an artificial vagina and the semen was analyzed and prepared for separation by flow cytometry. After sorting, the samples were split into two groups for freezing in TRIS-egg yolk or Botu-Bov� (Biotech Botucatu, Ltda., Sao Paulo, Brazil). Sperm were evaluated by computer-aided semen analysis (CASA) and fluorescent probes to determine plasma and acrosomal membrane integrity. The fertility of the spermwas also tested in an IVF program. Statistical analysis of sperm parameters was achieved using theTukey test with a significant level of P ≤ 0.05. Embryo production data after IVF were analyzed by chi-square test. There was a significant improvement in progressive motility with the use of Botu-Bov, compared with TRIS (67.4% v. 56.7%, respectively). There was also a statistical difference for curvilinear velocity (VCL) and lateral head displacement (ALH), with TRIS showing higher values than Botu-Bov (VCL = 206.6 v. 157.3 µm s–1, and ALH = 8.0 v. 5.9 µm, respectively). According to the literature, an ALH higher than 7 µm associated with a high VCL characterizes a vigorous and disordered movement indicating hyperactivity. The sperm frozen with the Botu-Bov had a higher straight movement (STR; 85.1%) and linearity (57.4%) than the TRIS group (79.8% and 45.6%, respectively). These features indicate a straighter and more uniform movement when Botu-Bov was used. The morphological analyses using fluorescent probes showed higher proportions of intact plasma and acrosomal membranes for Botu-Bov than for TRIS (50.1% v. 39.4% and 85.3% v. 71.2% for Botu-Bov and TRIS, respectively). Blastocyst formation after IVF was 21% (68/327; blastocysts/matured oocytes) for the TRIS group. This result was statistically different from the 30% (75/252) of blastocysts obtained with Botu-Bov and from the 34% (43/128) obtained in the control group. These results suggest that Botu-Bov provides better conditions for the maintenance of the viability of frozen sexed semen than does TRIS, probably due to the high concentration of essential and nonessential amino acids present in the Botu-Bov extender.
This work was supported by Sexing Technologies – Brazil and HG Lagoa da Serra.
264 SUITABILITY OF BOVINE SEXING PRIMERS FOR SEX DETERMINATION OF CAPRINE CELLS
Authors: Herr C. , Valentin C. , Greeson K.
Published: January 2008

Recent studies on PCR-based sex determination of caprine cells use amplification of a single-copy Y-chromosome-specific sequence. Our study investigated the suitability of widely used bovine sexing primers for their usefulness in sexing caprine cells. The common use of these sexing primers is to amplify BRY.4, a Y-specific sequence found in bovine cells (Reed et al. 1995 U.S. Pat# 5459038). The major advantage in amplifying these sequences is that they are multiple repeated DNA sequences. This enhances the sensitivity for use on as few as one cell. Early in their use, a problem was discovered amplifying BRY.4 for bovine sexing; one or more copies of a homologous repeat sequence were present in female cells. Copies in female cells differed by the presence of a Hinf1 restriction site. By digesting the DNA with Hinf1 either prior or subsequent to PCR amplification, it was possible to eliminate the troublesome sequence. Our study investigated whether a site homologous to theY-specific sequence was also present in female caprine cells and, if so, it too contained the Hinf1 restriction site, making possible the elimination of the troublesome sequence. The components of the PCR reaction mix were: buffer (50 mm Tris, 1% dextran T-500, 50 mm KCl, 2.5 mm MgCl2, and 0.035% 2- mercaptoethanol), deoxyribonucleotides (5 µm) (Boehringer Mannheim, Basel, Switzerland), primers (5´-GAACTTTCAAGCAGCTGAGGC-3´ and 5´-GATTGTTGATCCCACAGAAGG-3´) (2.5 µm) (custom synthesis, IDT Inc, Coralville, IA, USA), and Taq polymerase (2.7 U) (AmpliTaq DNA Polymerase, Stoeffel Fragment, Perkin Elmer, Branchburg, NJ, USA). Lymphocytes from one male and three female goat species (Saanen, Nubian/Alpine mix, and Albino) were used as the source of DNA. Approximately 100 cells/2 µL were used to ensure the amplification of the sequence homologous to theY-repeat. The DNA replication was carried out using a Corbett Rapid Thermocycler (Model FTS-IS, Corbett Research, Montlake, Australia) in 20-µL volumes, and all assays were run with positive and negative DNA controls. After replication, some samples were digested with Hinf1 (20 U) (New England Biolabs, Ipswich, MA, USA) for 2 h at 38�C. A 3% agarose gel was run in an agarose gel electrophoresis apparatus (Model 52000, IBI-Shelton Scientific, Peosta, IA, USA) for 30 min at 180 volts. All caprine species samples were run in duplicate. The DNA from three female undigested samples banded at the same position as the male samples (130 bp). The DNA from all three female digested samples banded at two positions (50 bp and 80 bp), both lower than the male samples. The male DNA remained unaltered after the Hinf1 digest, which suggests that the Hinf1 site is not present in the amplified DNA from male cells. The assay generated strong sexing bands with as few as 6 male lymphocytes, the lowest number of cells tested. These primers seem suitable if used in conjunction with Hinf1 digestion.
268 TIMED INSEMINATION OF HEIFERS WITH SEXED SPERM
Authors: Schenk J. L. , Seidel G. E.
Published: January 2008

The objective of this field trial was to compare pregnancy rates in Angus heifers time-inseminated with sexed sperm 55–56 h or 67–68 h after CIDR removal and administration of 25 mg prostaglandin F2α (PGF2α). Timed insemination eliminates labor costs associated with visual detection of estrus. Anecdotally, use of sexed sperm in commercial timed insemination environments has resulted in reduced pregnancy rates compared to rates for visually observed estrus. However, the optimal fixed time of insemination for sexed sperm has not been studied previously. Sperm were stained at 200 � 106 sperm mL–1 with 49 µm Hoechst 33342 for 45 min at 34.5�C and then diluted to 100 � 106 sperm mL–1 for sorting. Sperm were sexed by flow cytometry/cell sorting at 40 psi using a quasi-cw (pulsed; Vanguard 350-HDM, Spectra-Physics, Mountain View, CA, USA) laser operating at 125 mW, resulting in >91% X-chromosome-bearing sperm. Sorted sperm(2 � 106 total) were packaged into 0.25-mL straws at 10 � 106 sperm mL–1 and cryopreserved. Straws contained >43% progressively motile sperm immediately after thawing for 30 s in a 37�C water bath, as determined by 'track' motility. Angus heifers (n = 67) were synchronized for estrus with IM administration of 100 µg gonadotropin-releasing hormone (GnRH) and a CIDR in place for 7 days, followed by 25 mg PGF2α IM. Heifers were inseminated in the uterine body either 55–56 h (the timing currently recommended for unsexed semen) or 67–68 h after CIDR removal, accompanied by 100 µg GnRH IM, balanced across two inseminators with sperm from two Angus bulls. Estrus was visually detected three times daily but not used as the basis for insemination. Two months post-insemination, pregnancywas determined using ultrasound. Datawere subjected to chi-square analysis. Pregnancy rates for heifers inseminated 55–56 h and 67–68 h after CIDR removal were 34% (n = 11/32) and 46% (n = 16/35), respectively (P > 0.1). Estrus was visually detected for 85% (n = 57/67) of the heifers. Four heifers not detected in estrus became pregnant (40%). Respective pregnancy rates resulting from inseminations 0–12 h and 18–24 h after observed onset of estrus were 25% (7/28) and 55% (16/29), respectively (P < 0.05). Based on the limited scope of this preliminary field trial, insemination of heifers with sexed sperm at 67–68 h post-CIDR removal may be preferable to insemination at 55–56 h. It appears to be best to wait 18 to 24 h post-onset of observed estrus to inseminate heifers with sexed sperm when not using fixed-time insemination procedures. A larger field trial is warranted to more accurately identify the optimal fixed time for insemination using sexed sperm.
27 EFFECT OF ACTIVATION METHODS AND BIOPSY SAMPLING ON NUCLEAR TRANSFER BLASTOCYST PRODUCTION AND CLONING EFFICIENCY IN A HORSE
Authors: Choi Y. H. , Hartman D. L. , Fissore R. A. , Bedford S. J. , Hinrichs K.
Published: January 2008

In the horse, rates of blastocyst production after nuclear transfer are low. This study was conducted to examine the effect of activation via injection of different volumes of sperm extract or via injection of murine mRNA for PLC-ζ, a sperm-specific protein which induces Ca2+ oscillations in all species thus far studied, on blastocyst development after nuclear transfer. Two biopsy samples from the same horse were also examined for cloning efficiency. Donor fibroblasts cultured from a skin biopsy taken from a 19-year-old mare were treated with 15 µm R-roscovitine for 18 to 24 h before direct injection into enucleated ooplasts. Ionomycin treatment, 5 µm for 4 min, was used in all activation treatments, as the combination of ionomycin with sperm extract provided the highest blastocyst development and foaling rates in our previous report (Hinrichs et al. 2007 Reproduction 134, 319–325). All treatments were followed by incubation in 2 mm 6-dimethylaminopurine for 4 h. Differences in blastocyst development between treatments were analyzed using Fisher's exact test. In Experiment 1, reconstructed oocytes were injected with sperm extract for 0.1, 0.2, 0.4, 0.8, or 1.6 s at a fixed pipette diameter and injection pressure, and then treated with ionomycin. The blastocyst rate (9.8%) for 0.1 s was significantly higher than that for 0.2 s (0%) or 0.8 s (1.4%). In Experiment 2, murine PLC-ζ mRNA (0.25 µg µL–1) was injected into reconstructed oocytes 20 to 30 min before or after ionomycin treatment and compared with a control treatment (injection of 2 to 4 pL sperm extract 20 to 50 min after ionomycin exposure). There were no differences in blastocyst development among treatments (0, 4.5, and 5.6%, respectively). Transfer of 10 blastocysts produced in Experiments 1 and 2 resulted in 5 pregnancies: however, all were lost before 70 days of gestation. In Experiment 3, a second skin biopsy was obtained from the same mare and cells from this tissue sample were used for nuclear transfer concurrently with cells from the first sample, using the control method above. Blastocyst production was higher using cells from the second biopsy sample (4/23 v. 0/23; P = 0.05). Transfer of these four blastocysts yielded four pregnancies, two of which continued to term and produced viable foals. These results indicate that blastocyst development after injection of sperm extract is dependent upon the volume injected, that injection of murine PLC-ζ mRNA does not improve blastocyst formation under the given conditions, and that the efficiency of cloning may vary with biopsy sample even from the same animal.
275 IN VITRO DIFFERENTIATION OF PORCINE MESENCHYMAL STEM CELLS INTO NEURON-LIKE CELLS
Authors: Mohana Kumar B. , Song H. J. , Kang E. J. , Kim M. K. , Ock S. A. , Balasubramanian S. , Rho G. J.
Published: January 2008

The unique self-renewal ability for multilineage differentiation makes mesenchymal stem cells (MSCs) a promising cell source for regenerative medicine. However, before adapting in clinical trials, there is a need for a systematic evaluation of the in vitro differentiation process and the functional identities of cells generated using animal model systems. We have previously reported that MSCs derived from porcine umbilical cord blood (pUCB), post-natal bone marrow, and young adult bone marrow with typical phenotypic properties differentiate in vitro into various cells types of mesodermal lineage (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90; Mohana Kumar et al. 2007 Mol. Cells, in press). In the present study, we examined whether porcine MSCs (pMSCs) obtained from different origins, such as UCB, post-natal (immediately after birth) bone marrow, and young adult (6 months old) bone marrow are capable of neuronal differentiation in vitro. Subconfluent cultures of pMSCs (2–5 passages) were induced to become neuroectodermal cells following the protocol described earlier (Woodbury et al. 2000 J. Neurosci. Res. 61, 364–370), with minor modifications. After induction, 75 80% of cells exhibited substantial morphological changes including appearance of a more spherical shape, with multipolar and extending processes arranged into a network-like structure. Besides acquiring a neuronal phenotype, pMSCs derived from different origins displayed the expression pattern of characteristic neural-specific markers including early, intermediate, and late neural cell types. Differentiated cells expressed markers such as nestin, microtubule-associated protein-2 (MAP-2), neuronal nuclei (NeuN), βIII-tubulin, neurofilament-M (NF-M), glial fibrillary acidic protein (GFAP), galactocerebroside (GalC), and myelin basic protein (MBP) by immunofluorescence analysis. Expression pattern was further confirmed by analyzing selected neuronal transcripts by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Neurally differentiated pMSCs from different origins showed significantly (P < 0.05) higher relative abundance of nestin, βIII-tubulin, NF-M, and GFAP, compared to cells in control. In conclusion, the morphological observations and expression of neuronal-specific markers support the fact that pMSCs from different origins possess the ability to differentiate in vitro into neuron-like cells upon exposure to appropriate stimuli. Furthermore, these findings provide additional support for the emerging concept that the establishment of cell lines from pMSCs can have a pivotal role in standardizing cell replacement strategies.