The expression patterns of α-smooth muscle actin (SMA) and vimentin in the bovine testis indicate the maturation status of seminiferous tubules. The present study demonstrates the effects of impaired spermatogenesis resulting from increased testicular temperature after scrotal insulation and pathological lesions in subfertile bull testes on the changes in immunohistochemical expression of α-SMA and vimentin. Scrotal insulation induced degenerative changes of seminiferous tubules, and subfertile bull testes demonstrated characteristic mixed atrophied lesions; dysplastic lesions were seen in one bull. The increased intensity of peritubular α-SMA in the dysplastic area was distinct and indicated shrinkage of the seminiferous tubules. The mixed atrophied lesions revealed unaltered expression of peritubular α-SMA. However, considerable distortion was observed in the expression of α-SMA in severely degenerated tubules after insulation, which may indicate the heat sensitivity of peritubular α-SMA or its relation with spermatogenic activity under sudden heat stress. The vimentin expression pattern in the degenerated tubules of post-insulated testes was unaltered. However, the Sertoli cell-only tubules of mixed atrophied subfertile bull testes were characterized by an increase in vimentin of strong intensity resembling that in the transforming pattern, which may indicate the reversion of Sertoli cell maturity in such cases.

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This study aimed to clarify the mechanism of the spacing of murine embryos along the metrial and anti-metrial (MA) axis of the uterus using our newly developed experimental model. The model mice were produced by keeping mice in the supine position from the pre-implantation to implantation period. The starting points and periods of restraint of the mice in the supine position were set variously during the peri-implantation. Then, the position of the embryo was evaluated morphologically. In only one group (set in the instrument from the second day of pregnancy, Day 2, to Day 5), strong disruption of embryo spacing along the MA axis was observed. On the other hand, there was little abnormality in embryo positioning in the groups that were treated from Day 3 to Day 5 or from Day 3 to Day 6. These results suggested that determination of the position of the embryo in the MA axis is not related to duration of the experiment (2 days or 3 days), but is related to the starting time-point of the experiment, at Day 2 or Day 3. In conclusion, the period between Days 2 and 3 is critical for determination of the position of the embryo along the MA axis.

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The 4-nitrophenol (PNP) in diesel exhaust particles (DEP) has been identified as a vasodilator and is a known degradation product of the insecticide parathion. In this study, the protective effect of quercetin, a potent oxygen free radical scavenger and metal chelator, against the oxidative damage of PNP on cultured testicular cells was studied in male embryonic chickens. Testicular cells from Day 18 embryos were cultured in serum-free McCoy's 5A medium and challenged with quercetin (1.0 μg/ml) alone or in combinations with PNP (10^-7-10^-5 M) for 48 h. The oxidative damage was estimated by measuring cell viability, content of malondialdehyde (MDA), activity of superoxide dismutase (SOD) and glutathione peroxidation (GSH-Px) activity. The results showed that exposure to PNP (10^-5 M) induced condensed nuclei, vacuolated cytoplasm and a decrease in testicular cell viability and spermatogonial cell number. Exposure to PNP induced lipid peroxidation by elevation of the content of MDA. Exposure to PNP also decreased GSH-Px activity and SOD activity. However, simultaneous supplementation with quercetin restored these parameters to the same levels as the control. Consequently, PNP induced oxidative stress in spermatogonial cells, and dietary quercetin may attenuate the reproductive toxicity of PNP to restore the intracellular antioxidant system in the testicular cells of embryonic chickens.

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This study was conducted to study the kinetics of initial cell divisions in relation with the cleavage patterns in viable (with the ability to develop to the blastocyst stage) and non-viable bovine embryos and parthenotes. The kinetics of in vitro development and cleavage patterns were observed by time lapse cinematography. The length of the first and second but not third cell cycle differed significantly between the viable and non-viable embryos after IVF or parthenogenesis. Viable embryos had significantly shorter first and second cell cycles than non-viable ones. The presence of fragments, protrusions and unequally-sized blastomeres was associated with an extended one-cell stage and reduced ability to develop to the blastocyst stage; however, the lengths of the second and third cell cycles were not altered. Oocytes showing direct division from one cell to 3 or 4 blastomeres showed similar developmental ability and embryonic cell numbers to those showing normal division, although, with a high frequency of chromosomal abnormalities. Our results suggest that the differences in the first cell cycles between viable and non-viable embryos were not sperm-related, whereas direct cleavage of 1-cell embryos to 3 or more blastomeres and protrusion formation are related to sperm-driven factors. The length of the first and second cell cycles and the cleavage pattern should be examined simultaneously to predict developmental competence of embryos at early cleavage stages.

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Anestrus is one of the most important reproductive disorders in dairy buffaloes. The clinical features of anestrus in buffaloes, however, have not been well described. The objectives of this study were to describe the causes of anestrus in buffaloes and their reproductive performance after treatment under field conditions in southern Nepal. Of 135 anestrus buffalo cows, 61.4% had true anestrus with ovarian dysfunction and 33.3% had silent ovulation. In 111 buffalo heifers, 76.6% were in true anestrus and 18.9% had silent ovulation. The duration of anestrus after calving was longer than 6 months in 83% of buffalo cows and 61.5% of the buffalo cows had durations longer than 10 months. The interval between the last breeding and diagnosis of anestrus was more than 5 months in 67.4% of cows and heifers. Treatment of anestrus with prostaglandin F₂α in cows and heifers with a corpus luteum resulted in higher pregnancy rates within one (P<0.01) and two months (P<0.05) after treatment as compared with treatment with a vitamin/mineral mixture. Buffalo cows and heifers with inactive ovaries bearing a dominant follicle were also successfully treated with gonadotropin releasing hormone, resulting in higher pregnancy rate within one month after treatment (P<0.05). In conclusion, the predominant cause of anestrus in dairy buffaloes in this region was true anestrus with inactive ovaries, and the duration of anestrus after calving as well as breeding was extremely long. Routine reproductive examination and adequate hormone treatment may improve the reproductive performance of these buffaloes.

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The feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells (PGCs) by rapid cooling (i.e., vitrification) of dechorionated whole embryos at the 14- to 20-somite stage was investigated. Initially, we examined the glass-forming properties and embryo toxicities of six cryoprotectants: methanol (MeOH), ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG) and 1,3-butylene glycol (1,3-BG). According to the results of glass-forming and embryo toxicity tests, pretreatment solution (PS) containing 2 or 3 M cryoprotectant and vitrification solution (VS) containing 5 M cryoprotectant and 0.5 M sucrose were prepared using each cryoprotectant. Dechorionated embryos, the PGCs of which were visualized by injection of green fluorescence protein-nos1 3'UTR mRNA, were cooled rapidly by plunging into liquid nitrogen after serial exposure to PS and VS. All embryos cooled with MeOH, PG and 1,3-BG showed ice formation during cooling, and few embryos had live PGCs after warming. Most embryos cooled with GC did not show ice formation; however, few embryos had live PGCs. All embryos cooled with EG and most embryos cooled with DMSO had live PGCs when the embryos did not show ice formation during cooling. Based on the number of live PGCs in fresh embryos, the maximum survival rates of PGCs recovered from embryos cooled with EG and DMSO were estimated to be about 40 and 20%, respectively. The present study indicates that rapid cooling of dechorionated whole embryos, especially using EG-based solutions, could be utilized as a simple and promising tool for cryopreservation of PGCs.

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Progesterone (P4) is synthesized in the luteal cells of many species. The objective of the present study was to determine the expression pattern of P4 receptor (PR) mRNA and the distribution of PR protein in the bovine corpus luteum (CL) during the estrous cycle. The gene expression of PR in the bovine CL throughout the estrous cycle was determined by real-time PCR analysis, and the PR protein expression was evaluated by immunohistochemistry. Messenger RNA of PR was clearly expressed in the CL throughout the estrous cycle. The level of PR mRNA in the CL was highest at the early stage of the estrous cycle and was higher at the mid and late stages than at the regressed stage (P<0.01). In regard to the distribution of PR, the protein was expressed in both small and large luteal cells and in vascular endothelial cells throughout the estrous cycle. These results suggest that P4 has a role in regulating luteal and endothelial cell function in the bovine CL, especially at the early luteal stage as an autocrine/paracrine regulator.

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Vascular endothelial growth factor (VEGF) is a well known angiogenic factor that has been suggested to play some physiological roles in reproductive organs. To clarify whether VEGF is involved in regulating bovine endometrial function locally, in experiment 1, we determined the expression of VEGF, VEGF receptor (VEGFR) 1 and VEGFR2 throughout the estrous cycle in endometrial tissues. Endometrial tissue was collected at estrus (Day 0), the early I (Days 2-3), early II (Days 5-6), mid (Days 8-12) and late luteal stages (Days 15-17) and the follicular stage (Days 19-21). RT-PCR and Western blotting analysis revealed that VEGF mRNA expression at estrus was higher than at the early I, early II and late luteal stages (P<0.05), whereas VEGF protein content was greatest at the early I luteal stage and decreased thereafter. VEGFR1 mRNA expression was lower at estrus and at the early I and early II luteal stages than at the other stages, whereas VEGFR1 protein expression did not change significantly throughout the estrous cycle (P<0.05). VEGFR2 mRNA expression was higher at the mid and late luteal stages than at the early I and early II luteal stages, and VEGFR2 protein was higher at the mid and late luteal stages than at estrus (P<0.05). In experiment 2, to determine the effect of VEGF on prostaglandin (PG) F2α and PGE2 production by endometrial cells, cultured endometrial epithelial and stromal cells were exposed to VEGF (0, 5, 50, 100 and 200 ng/ml) for 24 h. VEGF (200 ng/ml) stimulated PGF2α production by stromal cells (P<0.05), but not PGE2 production. VEGF did not affect PG production by endometrial epithelial cells. The overall results suggest that VEGF and its receptors are regulated throughout the estrous cycle and that VEGF participates in the local regulation of bovine endometrial function by a selective modulation of PGF2α production in stromal cells in an auto- and/or paracrine manner.

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The objective of the present study was to investigate the potential mechanisms regulating cellular FLICE-like inhibitory protein (cFLIP), an anti-apoptotic factor, in the bovine corpus luteum (CL). Expression of cFLIP mRNA was highest at the developing stage and then decreased significantly during the mid, late and regressed stages (P<0.05). Western blot analysis revealed that expression of the long isoform of cFLIP (cFLIP_L) protein was high during the early and developing luteal stages, remained steady during the mid and late luteal stages and then decreased significantly (P<0.05) by the regressed stage. However, the expression levels of the short isoform of cFLIP (cFLIP_S) remained low during the early, developing and mid luteal stages. Immunostaining of cFLIP was strongest in the cytoplasm of luteal and non-luteal cells, including endothelial and immune cells, remained high during the early, developing and mid luteal stages and then decreased significantly (P<0.05) in the late and regressed luteal stages. Immunostaining of cFLIP was observed only in macrophage-like cells in the regressing CL. However, cultured mid luteal cells had a higher percentage of cFLIP-positive cells and a lower percentage of TUNEL-positive cells than luteal cells treated with tumor necrosis factor α (TNF)/interferon γ (IFNG; P<0.01). These results indicate downregulation of cFLIP during structural luteal regression, suggesting that cFLIP plays a survival role in the bovine CL.

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The Japanese black bear (Ursus thibetanus japonicus) is a typical seasonal breeder that has a mating season in early summer. Spermatogenesis and testicular steroidogenesis are known to develop and regress annually; however, its molecular mechanism has not yet been investigated. In the present study, we clarified the mRNA sequence of 5 steroidogenic enzymes (P450scc, 3βHSD, P450c17, 17βHSD3 and P450arom) using RT-PCR and RACE methods and the localization of these gene expressions in the bear testis using an in situ hybridization technique. The amino acid sequence deduced from each mRNA sequence had high homology with the corresponding sequences of other species and possessed a motif typical of the P450 family or short chain alcohol dehydrogenase family. Expression of P450scc, 3βHSD and P450c17 mRNA in interstitial tissue indicated that conversion from cholesterol to androstenedione occurs in Leydig cells. On the other hand, the mRNA of 17βHSD3, which plays a central role in synthesizing testosterone, was detected not only in the interstitium but also inside the seminiferous tubules, along the basement membrane. P450arom mRNAs were distributed in the seminiferous tubules. These results suggest the possibility of testosterone and estradiol-17β synthesis inside the seminiferous tubules in the bear testis. We expect that the results of this study will be useful for further investigation of the molecular mechanism of steroidogenic seasonality in the bear testis.

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The current study was performed to follow up the circulating hormonal changes and to correlate the findings with the physiological activity of the corpus luteum (CL) and placenta during pregnancy in goats. Blood samples were collected weekly from five goats during pregnancy for measuring steroid and protein hormones. A gradual increase was observed in immunoreactive (ir-) inhibin, with maximal levels at the 17th week. The plasma concentrations of estradiol and prolactin (PRL) showed nearly similar patterns during pregnancy, where they declined to basal levels during the first 4 weeks post-breeding and then increased significantly, with the maximal concentration during late pregnancy. The plasma FSH and LH concentrations were maintained at basal levels throughout the gestation period. The plasma progesterone concentration abruptly increased in the first week post-breeding and remained at high values throughout the pregnancy period. Immunohistochemical localization of inhibin alpha, beta_A, beta_B and steroidogenic enzymes cytochrome P450 aromatase, 3alpha-hydroxysteroid dehydrogenase (3betaHSD), cytochrome 17alpha-hydroxylase P450 and cholesterol side-chain cleavage cytochrome P450 in the cyclic and pregnant goat CL revealed positive immunoreactivity without affinity differences between the luteal and pregnancy stages. The placental syncytiotrophoblasts also showed positive staining, except for inhibin beta_A and 3betaHSD. The giant binucleate cells of the placenta showed positive immunoreactions to PRL. These results suggest that the high concentrations of ir-inhibin, estradiol and PRL during late pregnancy are of placental origin and that the placenta may have a vital role in the maintenance of pregnancy, regulation of mammary growth and preparation for kidding and lactation in goats.

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Detection of returning estrus in dairy cows after AI and re-insemination without delay are important in shortening the calving to conception interval. The objectives of this study were to show the effectiveness of CIDR insertion 12 to 19 days after AI on returning estrus and shortening the calving to conception interval in dairy cows. Seventy-nine dairy cows from two commercial dairy farms were synchronized for first postpartum estrus using a CIDR-Heatsynch protocol, and 76 cows (96.2%) showed estrus signs within 2 days after EB injection and were inseminated. The cows were then divided randomly into two groups. Thirty-seven cows were treated with a CIDR from 12 to 19 days after AI (CIDR group), while the other 39 cows were not treated and served as a control group. Milk samples were collected twice weekly from one week before the commencement of the CIDR-Heatsynch protocol until 7 to 9 days after removal of device. Detection rates of returning estrus 20 to 25 days after AI (within 6 days after removal of the device) were 30.4% in the CIDR group and 47.6% in the control group. According to the progesterone profiles, almost half of the non-pregnant cows that did not show estrus 20 to 25 days after AI had high progesterone concentrations from days 20 to 25, 59.1% in the CIDR group and 50.0% in control group. The calving interval was not significantly different between the CIDR (162 ± 50 days) and control groups (151 ± 40 days). In conclusion, CIDR insertion 12 to 19 days after AI did not increase the detection rate of returning estrus. As a consequence, there was no effect of the CIDR treatment on the calving to conception interval.

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The present study compared the developmental ability of miniature pig embryos cloned with fetal fibroblasts (FFs), bone marrow-derived mesenchymal stem cells (MSCs) and differentiated (osteocytes, adipocytes and chondrocytes) MSCs. MSCs were isolated from an approximately 1-month-old female miniature pig (T-type, PWG Micro-pig^®, PWG Genetics Korea). MSCs were differentiated into osteocytes, adipocytes and chondrocytes under controlled conditions and characterized by cell surface antigen profile using specific markers. These differentiated or undifferentiated MSCs, as well as FFs of miniature pig, were transferred into enucleated oocytes of domestic pigs. Data from 10 replicates involving 1567 cloned embryos were assessed in terms of developmental rates. The in vitro development rate to the blastocyst stage of embryos cloned with undifferentiated MSCs was significantly (P<0.05) higher than that of embryos cloned with differentiated MSCs and FFs. Surgical transfer of 523 two-cell stage embryos cloned with undifferentiated MSCs into five synchronized domestic pig recipients resulted in 5 cloned miniature pig offspring (1 stillborn and 4 viable) from 2 pregnant recipients. The results imply that MSCs might be multipotent and that they can be used to produce viable cloned miniature pigs that cannot be easily reproduced with differentiated somatic cells.

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The present study determined vascular changes in the bovine corpus luteum (CL) at Day 16 (early maternal recognition period) and Day 40 in early pregnancy and compared them to the CL from Day 12 and Day 16 of the estrous cycle. The CLs were analyzed in the central and peripheral regions, where site-depending features of vessels and angiogenic factors are evident. The same protein level of the endothelial cell marker von Willebrand factor was retained in the CL from Day 16 of the estrous cycle to Day 40 of early pregnancy. The protein level of pericytes and smooth muscle cells was determined using smooth muscle α-actin; the level decreased at Day 40 of early pregnancy in both regions of the CL. No significant change in the expressions of vascular endothelial growth factors VEGF₁₆₄ and VEGF₁₂₀ mRNA occurred from Day 16 of the estrous cycle until Day 40 of early pregnancy. Angiopoietin (ANGPT)-2 / ANGPT-1 mRNA ratio (an index of instability of vasculature) increased in the periphery at Day 16 of the estrous cycle and then decreased until Day 40 of early pregnancy. The results suggest that there is no difference in vascular structure between non-pregnant and pregnant luteal tissue during the early maternal recognition period (Day 16). Also, luteal rescue by early pregnancy may be not associated with further blood vessel formation but rather may be related to the decrease of blood vessels per unit of area and blood vessel stabilization in the bovine CL.

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The aim of this study is to provide evidence of the existence of the adenylyl cyclase 10 (ADCY10) ortholog proteins in boar spermatozoa. Experiments with RT-PCR techniques, nucleotide sequence analyses and Northern blot analyses revealed that boar testes exclusively express approximately 5.1-kbp RNA, the nucleotide sequence of which is highly similar to that of human ADCY10. Database analyses with CDART suggested that pig ADCY10 ortholog proteins conserve two catalytic domains of adenylyl cyclase. Western blot techniques and indirect immunofluorescence with a specific antiserum to pig recombinant ADCY10 ortholog proteins showed that 48-kDa and 70-kDa truncated forms of pig ADCY10 ortholog proteins are localized in the equatorial segments and connecting pieces of boar ejaculated spermatozoa. Finally, cell imaging techniques with fluo-3/AM indicated that incubation with sodium bicarbonate (an ADCY10 activator) can initiate the calcium influx in the boar sperm heads that is controlled via the cyclic AMP signaling cascades. These results are consistent with the suggestion that functional ADCY10 ortholog proteins exist in the heads of boar spermatozoa. This is the first direct evidence of the existence of ADCY10 proteins in the heads of mammalian spermatozoa.

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The efficiency of a porcine embryo vitrification method that uses water-soluble films of pullulan, a naturally-occurring polysaccharide polymer, was compared with two other types of vitrification methods using different devices and solutions for vitrification and warming. Blastocysts collected in vivo and vitrified by the conventional straw (ST), Cryotop^® (MVC) or pullulan film vitrification (PFV) methods were stored in liquid nitrogen for a certain period of time, after which the cryoprotective agents were removed by stepwise dilution. Fresh embryos were used as controls for the non-vitrification group. The vitrified-warmed embryos were incubated in TCM199 with 0.1 mM β-mercaptoethanol and 20% fetal bovine serum for 24 h at 38.5 C in humidified air with 5% CO₂ to evaluate their viability. The survival rate of embryos in the ST group (48.3%) was significantly lower than that of those in the MVC (70.7%), PFV (79.0%) and non-vitrification (94.4%) groups. The oxygen consumption rate after vitrification was significantly lower than that before vitrification in the ST group, but was not significantly different in the MVC and PFV groups. Both the oxygen consumption rates of embryos after warming and the live cell numbers in the ST group were lower than those in the MVC group, while they did not differ significantly between the PFV and MVC groups. There was a correlation between the oxygen consumption rate and the number of live cells in vitrified embryos after warming. Our results demonstrated that in vivo-derived porcine embryos could be vitrified using pullulan films.

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The effects of mechanical vibration during in vitro maturation and/or in vitro culture after artificial activation of pig oocytes on maturation and development were examined. In addition, the optimal conditions were applied to in vitro production of blastocysts derived from miniature pig somatic cell nuclear transfer (SCNT) embryos. Mechanical vibration during in vitro maturation did not affect the rates (60.5 ± 1.9-69.5 ± 2.2%) of oocytes reaching the metaphase-II stage. However, the blastocyst formation rates after activation of oocytes matured with mechanical vibration for 5 sec at intervals of 30-60 min or for 10 sec at intervals of 60 min were significantly (P<0.05) higher than those of oocytes matured without mechanical vibration (25.7 ± 2.0-28.1 ± 2.7% vs. 12.3 ± 1.4% and 25.8 ± 1.8% vs. 15.7 ± 1.9%, respectively). In contrast, mechanical vibration during in vitro culture after activation did not affect the blastocyst formation (11.6 ± 5.2-16.5 ± 3.0%) of oocytes. Mechanical vibration for 5 sec at intervals of 60 min during in vitro maturation of oocytes did not affect fusion (66.8 ± 3.5-72.1 ± 3.1%) with miniature pig somatic cells after enucleation. However, the blastocyst formation rate of SCNT embryos was improved (P<0.05) by mechanically vibrating recipient oocytes for 5 sec at intervals of 60 min during in vitro maturation, regardless of the presence or absence of the same treatment during in vitro culture (17.6 ± 2.5% vs. 9.4 ± 0.9% and 13.0 ± 0.3% vs. 7.4 ± 0.9%, respectively). The results indicated that mechanical vibration enhances the cytoplasmic maturation of in vitro-matured pig oocytes, resulting in improvement of their parthenogenetic development. In addition, it was shown that in vitro maturation of oocytes with mechanical vibration can be applied to efficient production of blastocysts derived from miniature pig SCNT embryos.

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Reversine, a 2-(4-morpholinoanilino)-6-cyclohexylaminopurine analog, can induce dedifferentiation of myogenic lineage-committed cells into multipotent mesenchymal progenitor cells, from which osteoblasts and adipocytes redifferentiate under lineage-specific inducing conditions. Although the molecular mechanism of how reversine causes dedifferentiation of a differentiated cell has not been fully elucidated, we speculated that it would be involved in reprogramming. In the present study, we examined whether reversine can enhance the development of somatic cell nuclear transfer (SCNT) embryos by improving the reprogramming state of the somatic cell nuclei. As donor cells, we used miniature pig fetal fibroblasts transfected with a plasmid construct containing a mouse Oct-3/4 promoter and enhanced green fluorescent protein (EGFP) cDNA. When the nuclei of these transfected cells are reprogrammed to an undifferentiated state in the SCNT embryos, EGFP expression is expected to commence under the control of the Oct-3/4 promoter. After SCNT, the resulting embryos were treated with 5 μM reversine for different durations (0, 6, 12, 18 and 24 h) or at different concentrations (0, 1, 5 and 10 μM) of reversine for 12 h and then cultured in vitro. When embryos were treated with 5 μM reversine for 12 h, the blastocyst formation rate was significantly (P<0.01) higher than that of embryos without reversine treatment. However, the strength and pattern of EGFP expression in the embryos were not affected by the same treatment. A normal-looking fetus was obtained 21 days after transfer of embryos treated with 5 μM reversine for 12 h into recipients. The present findings indicate that treatment with reversine is beneficial for enhancement of the in vitro development of miniature pig SCNT embryos, although the underlying mechanism is still unclear.

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