Embryonic stem cells (ES cells) were first established in the mouse, and they represent a population of pluripotent, undifferentiated cells derived from early embryos that is capable of proliferating without any limitation in an undifferentiated state. These cells retain the ability to differentiate in vitro or in vivo into derivates of all three germ layers, and when injected into blastocysts, they can participate in the formation of all tissues, including gonads (germ-line chimeras). It is possible to transfect them with a gene of interest, and the resulting transgenic cell lines can also be used for production of chimeras. Unfortunately, mammalian germ-line chimeras that can carry an inserted gene into their progeny have only been produced in the mouse. Logically, before application of stem cell therapies into a human medicine, it is necessary to verify the efficiency and safety of these methods with an acceptable animal model. The pig is currently used as a very convenient animal for pre-clinical applications, and therefore establishment of porcine ES cell lines is highly needed; unfortunately, no convincing ES cell lines have been produced in this species (and other domestic animals) to date. In this article, we discuss the recent advances in this field, especially oriented on possible reasons and obstacles why derivation of porcine ES cell lines is still unsuccessful.

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A high cyclic adenosine monophosphate (cAMP) level in fully-grown immature oocytes prevents meiotic resumption. In Xenopus, inhibitory cAMP is synthesized within oocytes depending on a stimulatory α-subunit of G-protein (Gsα). In the present study, we examined whether ooplasmic Gsα is involved in meiotic arrest of porcine oocytes. First, we studied the presence of Gsα molecules in porcine oocytes by immunoblotting, and this suggested the presence of reported isoforms (45 and 48 kDa) not only in cumulus cells but also in porcine oocytes. Then we injected an anti-Gsα antibody into porcine immature oocytes and found that inhibition of ooplasmic Gsα functions significantly promoted germinal vesicle breakdown of the oocytes, whose spontaneous meiotic resumption was prevented by 3-isobutyl-l-methylxanthine (IBMX) treatment. Although cyclin B synthesis and M-phase promoting factor (MPF) activation were largely prevented until 30 h of culture in IBMX-treated oocytes, injection of anti-Gsα antibody into these oocytes partially recovered cyclin B synthesis and activated MPF activity at 30 h. These results suggest that meiotic resumption of porcine oocytes is prevented by ooplasmic Gsα, which may stimulate cAMP synthesis within porcine oocytes, and that synthesized cAMP prevents meiotic resumption of oocytes through the signaling pathways involved in MPF activation.

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Hypoxia is a consistent challenge for aquatic animals. It is a pressing environmental problem; hypoxia can cause cranial edema and ovarium dysfunction in fish. Although several studies have reported the effect of hypoxic insult to the visual system, the hypoxic effect on perinatal animals and in particular their offspring has yet to be elucidated. In this study, activated caspase-3 activity was investigated using immunohistochemistry in order to examine the perinatal hypoxic damage in offspring fish. Offspring were divided into groups based on different time points of sacrifice. This allowed assessment of ocular development for different age groups. The results indicated that perinatal hypoxia induced ocular developmental defects in the offspring. The defects took the form of trabecular cell death and fibre degeneration, corneal thinning and lens fibre derangement. A concomitant change in intraocular pressure was recorded by tonometer in the experimental animals compared with the controls. Further investigation should be initiated to develop strategies to prevent developmental disability due to perinatal hypoxia and to increase survivability of the offspring.

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Freeze-dried mouse spermatozoa can be used for normal embryonic development after injection into oocytes, thus indicating that freeze-drying is a useful method for the storage and transportation of genetic materials from animals. We recently reported that storage of freeze-dried mouse spermatozoa requires maintenance at temperatures lower than -80 C for long-term preservation and a pressure of 0.37 mbar at primary drying and that these conditions significantly improve the developmental rate to the blastocyst stage. In this study, we examined the influence of transportation and preservation conditions on freeze-dried spermatozoa. Freeze-dried spermatozoa stored for 2 or 2.5 years at 4 or -80 C were transported round trip overland between Shizuoka and Hokkaido prefectures in Japan or by air between Japan and Belgium. The freeze-drying conditions consisted of primary drying at pressures of 0.04, 0.37 and 1.03 mbar and secondary drying at a pressure of 0.001 mbar. Embryos (2-cell stage) from freeze-dried spermatozoa dried at 0.04 mbar and stored at 4 C for 2 years with and without overland transportation did not develop to term. The development rates of embryos from spermatozoa stored at -80 C for up to 2 years and transported overland, by air and without transportation were 8, 1 and 28%, respectively. The development rates of embryos from spermatozoa without transportation were significantly higher than with transportation (P<0.05). These data indicate that freeze-dried spermatozoa stored at -80 C with and without transportation can retain their ability to generate viable offspring after storage for up to 2 years. However, there are limitations to be considered in the transportation of freeze-dried spermatozoa at ambient temperature.

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Mos and the mitogen-activated protein kinase (MAPK) cascade have been established as crucial regulators of second meiotic metaphase arrest, the so-called CSF arrest, in mammalian oocytes. They are also thought to play a role in regulating mitotic metaphase arrest of early mammalian embryos. In the present study, we examined whether mitotic arrest is induced in early mouse embryos by activation of extracellular signal-regulated kinases (ERKs), which are major MAPKs in mouse eggs, and their substrate, p90Ribosomal S6 kinase (RSK), as reported in Xenopus embryos. Wild-type Mos (wt-Mos), degradation-resistant Mos mutant (P2G-Mos) or constitutive active mutant of MAPK/ERK kinase, MEK (SDSE-MEK), was expressed in early mouse embryos by injecting the respective expression vectors into the pronucleus of fertilized eggs, and the developmental rates were then examined up to 72 h after insemination. Expression of P2G-Mos and SDSE-MEK succeeded in activating ERKs and RSK in developing mouse embryos, while wt-Mos failed to activate them in spite of expression of mos mRNA, indicating that the wt-Mos protein is unstable in early mouse embryos. Although the activated levels of ERKs and RSK in the vector-injected embryos were comparable to those of meiotically arrested mouse oocytes, their developmental rates were identical to those of the control embryos. These results suggest that activation of MAPK and RSK does not induce mitotic arrest in early mouse embryos. The present study indicates that there are large physiological differences between early mouse embryos and mouse oocytes and that CSF arrest of mouse eggs in mitosis should be discussed separately from that in meiosis.

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Since very few oocytes grow completely in vivo, in vitro growth (IVG) of ovarian oocytes may provide a new source of functional oocytes. The long-term effects of in vitro maturation (IVM) of oocytes and in vitro culture of fertilized eggs have been reported; however, the effects of IVG of oocytes are unknown. Here in, we report the long-term effects of IVG of oocytes. Ovaries from 1-day-old mice containing non-growing oocytes were cultured for 10 days; the isolated follicles were then cultured for 11 days. Secondary follicles from 10-day-old mice were also cultured for 11 days. The nuclei of oocytes collected from the IVG and Graafiais follicles of adult mice were transferred to enucleated oocytes grown in vivo, respectively. Developmental competence was examined following IVM of the reconstituted oocytes. Chronologically, oocytes of 1-day-old, 10-day-old and adult mice were cultured for 22, 12 and 1 day(s). The result showed that the reconstituted eggs developed into pups at high rates after nuclear transfer and in vitro fertilization (IVF) in all the experimental groups (29-45%). However, the pups from reconstituted eggs containing the nuclei of 22-day cultured oocytes were heavier than the control pups (P<0.05). We concluded that long-term culture of oocytes did not affect their nuclear ability to develop to term; however, fetal growth was affected by the culture duration or culture conditions during the initial phase of follicular growth.

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Diesel exhaust particles (DEPs) contain polycyclic aromatic hydrocarbons (PAH) that bind to aryl hydrocarbon receptors (AhRs) and decrease sperm production. Since it is not clear if AhR mediates DEP toxicity, we investigated the effect of DEPs in four strains of mice that have different AhR responsiveness. We treated BALB/c, C57BL/6, ICR and DBA/2 mice with DEP suspensions and compared their toxicity in each strain. In both the vehicle- and DEP-treated groups, ethoxyresorufin-O-deethylase (EROD) activity, as an indirect index of AhR activity, was increased in the order of BALB/c > C57BL/6 > ICR > DBA/2. Only BALB/c and C57BL/6 mice had significantly lower daily sperm production (DSP) than vehicle-treated mice. All strains exhibited increased sperm abnormalities. In particular, the C57BL/6, ICR and DBA/2 mice exhibited significantly increased abnormalities. A significant correlation was found between EROD activity and DSP or incidence of morphologically abnormal sperm. These data suggest that DEP toxicity may affect the male reproductive system in an AhR-dependent manner.

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Assessment of the developmental ability of oocytes following freezing and thawing is an important step for optimizing oocyte cryopreservation techniques. However, the in vitro fertilization of frozen-thawed mouse oocytes is often inefficient because of incomplete capacitation of spermatozoa in the absence of surrounding cumulus cells. This study was undertaken to determine whether the oocyte cryopreservation efficiency of different strains of mice could be assessed from the development of oocytes following parthenogenetic activation and intracytoplasmic sperm injection (ICSI). Oocytes were collected from hybrid (C57BL/6 × DBA/2) F1 or inbred (C57BL/6J, C3H/HeN, DBA/2J and BALB/cA) strains and were vitrified in a solution containing ethylene glycol, DMSO, Ficoll and sucrose. In the first series of experiments, oocytes were activated parthenogenetically by Sr²⁺ treatment after warming. The oocytes from the inbred strains, but not those of the F1 hybrid, were diploidized by cytochalasin treatment to obtain a sufficient number of blastocysts. In all strains tested, parthenogenetic embryos derived from vitrified oocytes developed into blastocysts at rates between 23 and 68%. In the second series of experiments, vitrified oocytes from each strain were injected with homologous spermatozoa after warming. Normal offspring were obtained from all strains at rates between 5 and 26% per embryo transferred. Thus, the feasibility of oocyte cryopreservation protocols can be assessed easily by in vitro development of parthenogenetic embryos or by in vivo development of ICSI embryos. Moreover, the oocytes of these four major inbred strains of mice can be cryopreserved safely for production of offspring.

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Without using sperm, artificial oocyte activation is essential for current assisted reproductive technologies, particularly somatic cell nuclear transfer and round spermatid injection. Strontium has been widely used as an activator of oocytes especially in the mouse, by which efficient oocyte activation requires Ca²⁺-free medium. In this study, we examined whether Sr²⁺ can efficiently activate oocytes in Ca²⁺-containing culture media when calcium is chelated. Ethylene glycol-bis (β-aminoethyl ether) -N, N, N', N'-tetraacetic acid (EGTA) was added to three standard culture media (CZB, M16 and KSOM) for mouse embryos because it preferentially binds Ca²⁺ rather than Sr²⁺. We found that treatment with 5 mM Sr²⁺ and 2 mM EGTA left fewer than 1% of oocytes at the MII stage, which is comparable to that of Ca²⁺-free medium. As a result, addition of 2 mM EGTA along with 5 mM Sr²⁺ in either CZB, M16 or KSOM made more than 80% of available activated oocytes, which was comparable to or better than 72% in a Ca²⁺-free Sr²⁺ medium, since EGTA-Sr²⁺ activation led to significantly less oocyte degeneration than Ca²⁺-free Sr²⁺ activation. Furthermore, we demonstrated that this activation method can support the birth of cloned embryos. Thus, addition of EGTA to typical Ca²⁺-containing culture media can easily produce activation media that does not interfere with embryonic development.

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Intracytoplasmic sperm injection (ICSI)-mediated gene transfer has recently been shown to be an effective technique for producing transgenic pigs; however, the types of sperm pretreatment having the most beneficial effects on post-ICSI embryogenesis or transgenic efficiency have not been clarified. In the present study, we performed ICSI-mediated gene transfer using pig sperm subjected to various pretreatments and determined the developmental potential of sperm-injected oocytes and introduction efficiency of exogenous DNA. Embryos were then transferred to recipient pigs to confirm gene transfer efficiency during the fetal period. When ICSI was performed using unfrozen sperm heads with tails removed by piezo-pulse, the rates of blastocyst formation (14.2%, 17/120) and transgene (EGFP) expression (11.8%, 2/17) were both low. When unfrozen sperm heads were used that were removed by sonication, EGFP expression efficiency (11/21, 52.4%) improved significantly (P<0.05). Pretreatment of unfrozen sperm with a surfactant or acrosomal reaction did not further improve the rates of blastocyst formation and EGFP expression. However, use of the heads of sperm frozen-thawed with or without a cryoprotective agent resulted in rates of blastocyst formation and EGFP expression that tended to be generally high (23.0%, 14/61-33.8%, 26/77 and 42.9%, 6/14-66.7%, 10/15). A total of 219 in vitro matured oocytes were fertilized by ICSI-mediated gene transfer using the heads of frozen-thawed sperm and then transferred into two recipient pigs. Seven fetuses were obtained, and EGFP expression and integration of the transgene (10-30 copies) were confirmed in two of the seven fetuses. Use of unfrozen sperm thus confers no advantages on ICSI-mediated gene transfer, and although further investigations are needed, frozen-thawed sperm heads appear to be useful in ICSI-mediated gene transfer.

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The aim of this study was to determine the changes in the mRNA expression of Toll-like receptors (TLRs) in hen ovarian follicles during follicular growth and in response to lipopolysaccharide (LPS). White follicles and the fifth largest to largest follicles (WF and F₅-F₁, respectively) were collected from laying hens. To examine the effects of LPS, the laying hens were treated intravenously with LPS (1 mg/kg BW) 0, 3, 6, 12 and 24 h before examination. Expressions of TLRs and IL-1β in the theca and granulosa layers were examined by semi-quantitative RT-PCR. Immunocytochemistry was performed to identify immunoreactive TLR-4. The theca layer expressed TLR-2, TLR-4, TLR-5 and TLR-7, whereas the granulosa layer expressed only TLR-4 and TLR-5. The expression of TLR-4 and TLR-5 in the theca layer increased significantly during follicular growth. In the granulosa layer, the expression of TLR-5 increased, but that of TLR-4 was unchanged. Expression of TLR-4 increased significantly during the period of 6 to 12 h of LPS treatment in the theca layer and during the period of 12 to 24 h in the granulosa layer of F₃. Immunoreaction products for TLR-4 were observed in theca interna and granulosa layers of WF and F₅-F₁, with the greater amount observed in the theca interna. LPS treatment significantly increased expression of IL-1β in the theca layer after 3 h and in the granulosa layer during the period of 12 to 24 h. These results suggest that TLRs are expressed in ovarian follicles and that TLR-4 and TLR-5 expression increase with the growth of follicles. Enhanced expression of TLR-4 and IL-1β by LPS in the theca and granulosa layers suggests possible roles of TLR in recognition of microorganisms.

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The aim of present experiment was to evaluate the plasma concentrations of estrone sulfate (E₁S) and progesterone (P₄) during late gestation in recipient cows transferred embryos produced by nuclear transfer (NT) and in vitro fertilization (IVF). Blood samples were collected from recipients transferred embryos produced by NT (n=9) and IVF (n=13) at 160, 220, 240, 260 and 270 d of gestation and then at 5 d intervals until parturition. Plasma samples were analyzed for E₁S and P₄ by ELISA. One NT and three IVF cows aborted between days 220 and 260 of gestation. Two NT and one IVF cow had prolonged gestation (over 290 d). One IVF cow had an overweight fetus (50 kg) after abortion (257 d). The patterns of changes in the concentrations of E₁S during late gestation in the NT and IVF cows were almost identical. The NT and IVF cows that aborted had prolonged gestation and much higher E₁S concentrations than the average. One NT cow aborted after 220 d of gestation and had a sudden high increase in its E₁S concentration from 160 d to 220 d of gestation. The NT and IVF cows that had prolonged gestation also had significantly higher (P<0.05) P₄ concentrations than the average. These results raise the possibility that the E₁S and P₄ profiles can be used to monitor some late gestational problems, such as higher birth weight, abortion and prolonged gestation.

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Aberrant gene expression in somatic cell nuclear-transferred (NT) embryos due to abnormal epigenetic modifications of the donor nucleus likely accounts for much of the observed diminished viability and developmental abnormalities. We compared the expression of 13 developmentally important genes in individual 8-cell and blastocyst stage NT embryos produced from adults female cumulus cells and adult male skin fibroblast cells with low and high incidences of neonatal abnormalities [1, 2]. In vitro-fertilized (IVF) embryos were used as control embryos. Among the genes tested, the relative abundance of Glut-1, IGF-1R, E-cad, and Cx43 transcripts varied significantly between the two types of NT embryos at the 8-cell stage. The relative abundance of manganese super oxide dismutase (MnSOD) and Stat3 transcripts was significantly higher in IVF embryos compared with both types of NT embryos. At the blastocyst stage, there was a significant difference in the relative expression of only one gene, Bcl-2, between the two types of NT embryos. Although the level of Glut-1 expression did not vary between the two types of NT blastocysts, its expression in both types of NT blastocysts was significantly lower than that in IVF blastocysts. The MnSOD expression level tended to be higher in NT blastocysts. The gene expression profile for any single gene, however, was highly variable among individual embryos and was independent of embryo morphology. The present study demonstrated that the expression profiles of the 13 genes examined in Day 9 NT blastocysts produced from two different types of donor cells with different incidences of neonatal abnormalities are largely indistinguishable.

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The concentrations of various components of follicular fluid were compared among three groups of follicles (small, <5 mm; medium: 5-10 mm; large, >10 mm) with a control that consisted of the components of umbilical serum using seven pregnant Antarctic minke whales. Follicular oocytes recovered from the follicles were also used for measurement of oocyte diameter after removing the cumulus cells. The mean diameter of the ooplasm in the oocytes from the large follicles (143.2 μm) was significantly greater than those from the small (127.1 μm) and medium (131.7 μm) follicles, although there were no significant differences in diameter of the whole oocyte and thickness of the zona pellucida among the three follicular sizes. The osmolarity of the follicular fluid from the small follicles (363.3 mOsmol) was significantly lower than that of the medium follicles (388.9 mOsmol) and tended to be lower than that of large (381.9 mOsmol) follicles, respectively, both of which were similar to that of the umbilical serum (379.5 mOsmol). There was no significant difference in the concentrations of all components of the follicular fluid between the medium and large follicles. As compared with the values of the umbilical serum, the total-protein, glucose, albumin and chlorine concentrations of the follicular fluid from the medium and large follicles were significantly higher, and the total cholesterol and calcium concentrations were significantly lower. The concentrations of lactic acid (85.3-136.0 mg/dl) of the follicular fluid from the three groups of follicles were significantly lower than that of the umbilical serum (360.0 mg/dl). Among the follicles, the follicular fluid from the small follicles (136.0 mg/dl) contained a significantly higher concentration of lactic acid than that from the large follicles (85.3 mg/dl). The progesterone concentrations were not significantly different among the fluid from the three group of follicles and the umbilical serum: however, the estradiol 17-β concentrations of the follicular fluid increased with the size of the follicle (14.3 and 34.6 ng/ml for small and large follicles, respectively). These results offer new information concerning whale reproductive physiology, especially for improvement of in vitro oocyte maturation and related technologies in whales.

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Mammalian ovaries are endowed with a huge number of small oocytes in primordial follicles (primordial oocytes). The mechanism regulating initiation of oocyte growth and follicular development is not well understood. Several growth factors and cytokines are known to be involved in oocyte growth and follicular development. Herein, the involvement of KIT, a receptor tyrosine kinase, and its ligand, KIT ligand (KL), in the initiation of porcine oocyte growth was examined. At first, KIT expression was examined immunohistochemically in primordial oocytes from neonatal (10-20 days) and prepubertal (about 6 months) pigs. Similar expression of KIT was detected in all oocytes from both the neonatal and prepubertal pigs. Next, to examine the growth of primordial oocytes, ovarian tissues containing primordial oocytes were xenotransplanted into immunodeficient SCID mice. Primordial oocytes from the neonatal pigs grew with follicular development as described previously, whereas those from the prepubertal pigs did not initiate growth in the xenografts after 2 months. To stimulate the growth of primordial oocytes from the prepubertal pigs, they were cultured in a medium supplemented with KL (50 and 100 ng/ml) for 1 or 3 days before xenografting. After 2 months, however, the oocytes did not grow, and the primordial follicles did not develop, although a higher number of primordial oocytes survived in the KL-treated tissues. These results suggest that KIT-KL might not be associated with the growth initiation of porcine primordial oocytes, although they do enhance the survival of the oocytes.

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The progesterone (P₄) profiles and macroscopic vulvar changes of female Malayan tapirs were investigated in order to understand their fundamental reproductive physiology and to search for visual indicators of estrus. Blood was collected once or twice a week from seven female Malayan tapirs kept at four zoos. Serum or plasma P₄ concentrations were determined by radioimmunoassay. The P₄ concentrations changed cyclically throughout the years, and a total of 56 cycles was confirmed in the seven females. The length of the estrous cycle based on the P₄ profiles was 43.6 ± 2.0 days; however, this mean includes great variation in length, from 21 to 84 days. Mucous discharge from the vulva and vulvar swelling were seen when the P₄ concentrations were low before the beginning of a rise in most cases. In conclusion, captive female Malayan tapirs have variations of approximately 1 to 3 months in estrous cycle length, and visual changes in the vulva are helpful in estimating estrus in female Malayan tapirs.

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Tumor necrosis factor (TNF) α can induce both cell death and proliferation by binding to either TNF receptor (TNFR) 1 or 2. In the granulosa cells of porcine ovaries, TNFα is considered to act as an anti-apoptotic/survival factor during follicular atresia. As a first step toward elucidating the function of TNFR2 in regulating follicular development/growth and atresia in porcine ovaries, we isolated the porcine (Sus scrofa) cDNA encoding TNFR2, which was identified from a cDNA library prepared from the follicular granulosa cells of pig ovaries. Porcine TNFR2 (1,125 bp, 375 amino acid residues), which contains specific amino acid region of transmembrane, indicated high identities with human and murine TNFR2 (78 and 69% at mRNA level, respectively; 73 and 61% at protein level, respectively), suggesting that the function of porcine TNFR2 is similar to that of human and murine homologues. Understanding the expression patterns of porcine TNFR2 mRNA in various organs, which we confirmed by reverse transcription polymerase chain reaction analysis, would help to elucidate the physiological role of TNFR2 in the regulation of apoptosis in porcine organs.

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The objective of this study was to investigate the effect of dosage and number of days of follicle stimulating hormone (FSH) treatment on superovulatory response in controlled internal drug release (CIDR)-treated Korean native cows. Forty cows underwent two superovulatory treatments with a crossover design. Cows, at random stages of the estrous cycle, received a CIDR together with injections of 1 mg estradiol benzoate and 50 mg progesterone, and gonadotropin treatment began 4 days later. The cows were divided into 2 groups based on the dosage and number of days of treatment with porcine FSH; a total of 28 mg FSH was given in twice daily intramascular injections in decreasing doses over 4 days (5, 5, 4, 4, 3, 3, 2 and 2 mg; T1 group, n=20) or a total of 24 mg FSH was given in twice daily decreasing doses over 3 days (5, 5, 4, 4, 3 and 3 mg; T2 group, n=20). This was followed by the alternate treatment in the subsequent superovulation. The cows were treated identically in all other respects. PGF_2α (25 mg and 15 mg) was given with the 5^th and 6^th injections of FSH, CIDR were withdrawn at the 6^th FSH injection and the cows received 200 μg GnRH 36 h after CIDR withdrawal. The cows were artificially inseminated twice, at 48 and 60 h after CIDR withdrawal, using commercial semen from four Korean native bulls, and embryos were recovered 6 or 7 days after the 2^nd insemination. The numbers of corpora lutea (CL; 7.9 ± 1.0 vs. 8.3 ± 1.1) and large follicles (1.2 ± 0.2 vs. 1.3 ± 0.3) present at the time embryo recovery, as detected by ultrasonography, did not differ between the T1 and T2 groups (P>0.05). Similarly, the numbers of total ova/embryos (6.2 ± 0.9 vs. 6.4 ± 1.1), transferable embryos (3.4 ± 0.8 vs. 3.2 ± 0.7), degenerate embryos (0.8 ± 0.2 vs. 1.0 ± 0.3) and unfertilized ova (2.1 ± 0.5 vs. 2.2 ± 0.5) did not differ between the groups (P>0.05). These data indicate that a reduced dose (24 vs. 28 mg) and number of treatments (6 vs. 8) of FSH for superovulation of CIDR-treated Korean native cows does not affect the embryo yield.

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Inhibition of specific gene expression using RNA interference (RNAi) is a valuable tool for functional analysis of a target gene. However, there is little information available concerning RNAi for analysis of gene function in relation to the reproductive physiology of follicular cells in ruminants. Thus, the aim of this study was to evaluate the interfering effect of small interference RNA (siRNA) on expression of cyclooxygenase-2 (Cox-2) mRNA and prostagrandin F_2α (PGF _2α) production in bovine cumulus-granulosa (CG) cells. Bovine CG cells were collected from aspirated follicles and cultured. After reaching confluency, two experiments were conducted. In experiment 1, to investigate the effective concentration of siRNA, 0, 100, 250 and 500 pM of Cox-2 siRNA was introduced into the CG cells, respectively. After 24 h, the amount of Cox-2 mRNA expression was measured by RT-PCR and real-time PCR. In experiment 2, to investigate the time required for effective interference of siRNA and Cox-2 activity, 250 pM siRNA was introduced for 0, 3, 6, 12 and 24 h. After culture, the amount of Cox-2 mRNA expression was measured and the culture medium was collected to determine the PGF_2α concentration by enzyme immunoassay. The Cox-2 mRNA expression was not affected by introduction of 100 pM siRNA into CG cells for 24 h, but 250 and 500 pM Cox-2 siRNA significantly reduced the Cox-2 mRNA expression. Moreover, the significant suppressive effect of 250 pM siRNA was observed 6 h after introduction, and the reduction of mRNA expression by RNAi became more obvious over 12 h. On the other hand, the PGF_2α concentration in the culture medium was not significantly different 12 h after siRNA introduction; however, the PGF_2α concentration 24 h after siRNA introduction was significantly decreased compared with the control at the same time point. These results suggest that gene silencing of Cox-2 with siRNA is capable of analyzing the function and expression of specific genes in bovine CG cells.

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This study evaluated the pregnancy rates following either a controlled internal drug release (CIDR)-based timed artificial insemination (TAI) or an embryo transfer (TET) protocol compared with that following a single PGF_2α injection and AI after estrus (AIE) in lactating repeat breeder dairy cows. Fifty-three lactating dairy cows diagnosed as repeat breeders were used in this study and were randomly assigned to the following three treatments. (1) Cows, at random stages of the estrous cycle, received a CIDR device and 2 mg estradiol benzoate (EB; Day 0), a 25 mg PGF_{2 α} injection at the time of CIDR removal on Day 7 and a 1 mg EB injection on Day 8. The cows then received TAI 30 h (Day 9) after the second EB injection using dairy semen (TAI group, n=13). (2) Cows, at random stages of the estrous cycle, received the same hormonal treatments as in the TAI group. The cows then received TET on Day 16 using frozen-thawed blastocysts or morula embryos collected from Korean native cattle donors (TET group, n=13). (3) Cows, at the luteal phase, received a 25 mg injection of PGF_2α and AIE using dairy semen (control group, n=27). The ovaries of the cows in the TET group were examined by transrectal ultrasonography to determine ovulation of the preovulatory follicles, and blood samples were collected for serum progesterone (P4) analysis. The pregnancy rate was significantly higher in the TET group (53.8%) than in the control (18.5%) or TAI (7.7%) groups (P<0.05). The ultrasonographic observations demonstrated that all the cows in the TET group ovulated the preovulatory follicles and concomitantly formed new corpora lutea. Accordingly, the mean serum P4 concentration remained constant between Day 0 and Day 7 of the luteal phase, decreased dramatically on Day 8 (P<0.01) and subsequently increased by Day 16 (P<0.01). These data suggest that the CIDR-based TET protocol can be used to effectively increase the pregnancy rate in lactating repeat breeder dairy cows.

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Cell-to-cell interaction via cell contact-dependent pathway is essentially important for maintenance and regulation of corpus luteum (CL) integrity and its physiological actions. The objective of the present study was to evaluate the mRNA expression of the cell adhesion molecules (CAMs) that are constituent factors of gap junctions [connexin (Cx) 43] and adherence junctions (VE-, E-, N-cadherin) in two types of endothelial cells from the mid CL and in CL tissue during the estrous cycle and PGF_2α-induced luteolysis in the cow. Specific mRNA expression for Cx43 and N-cadherin was detected in cytokeratin-positive (CK+) and cytokeratin-negative (CK-) luteal endothelial cells (EC) and fully luteinized granulosa cells (LGC). E-cadherin mRNA was expressed in CK+EC and LGC, but not in CK-EC. VE-cadherin mRNA was expressed in both CK+ and CK-EC. During the estrous cycle, Cx43 mRNA expression was significantly lower in the regressing CL. VE-cadherin expression also tended to increase in the mid CL and increased significantly in the regressing CL. E-cadherin mRNA expression was higher in the early and late CL than in the mid- and regressing CL. N-cadherin mRNA expression gradually increased from the early to late CL followed by a decrease in the regressing CL. During PGF_2α-induced luteolysis, Cx43 mRNA expression appeared to increase, and VE-cadherin and E-cadherin mRNA significantly increased at 24 h. N-cadherin mRNA expression decreased 2 and 4 h after PGF_2α administration. Collectively, expression of the mRNAs for CAMs was different in the two types of luteal endothelial cells and fully luteinized granulosa cells and changed independently in the CL during the estrous cycle and PGF_2α-induced luteolysis in the cow. The results suggest that CAMs play physiological roles in cell-to-cell communication to regulate both gap and adherence junctions during CL development and regression in the cow.

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The `male effect' is a well-known phenomenon in female sheep and goats, whereby pheromone-induced activation of reproductive function occurs. In a previous study, we showed that the genes for elongation of long-chain fatty acids family member 5 (ELOVL5) and stearoyl-CoA desaturase 1 (SCD1) increased their expression significantly, concomitant with induction of pheromone synthesis. Therefore, these genes were considered to be prime candidate genes for pheromone synthesis. In the present study, we performed in situ hybridization to investigate where these two genes are expressed in goat skin. Strong positive signals were detected for both genes in the head skin of the male goat, which is the main site of pheromone production, and were mainly in the basal layer of the sebaceous gland cells, with the remaining cells showing negligible signals. None of the cells in the rump skin of the male goat or the head skin of the orchidectomized goat, neither of which produce pheromone, exhibited strong positive signals. The present study demonstrates that expression of these two candidate genes for pheromone synthesis is primarily localized in the sebaceous glands of the pheromone-producing skin region.

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To investigate the effect of hypothyroidism on gonadal and adrenal functions in male Japanese quail (Coturnix japonica), hypothyroidism was induced in male adult Japanese quail by daily administration of 2-Mercapto-1-methylimidazole (methimazole) in their drinking water. Four weeks after methimazole treatment, the Japanese quail were scarificed, and the plasma concentrations of free triiodothyronine (FT3), free thyroxine (FT4), total T3 (TT3), total T4 (TT4), corticosterone, testosterone, LH and immunoreactive (ir) inhibins were measured by radioimmunoassy, the testes and adrenal glands were removed and weighed and the thyroid glands and testes were fixed in 4% paraformaldehyde for histological observation. The results showed that the hypothyroidism induced by methimazole caused a significant decrease in body and testes weight; the plasma levels of FT3, FT4 and TT4 significantly decreased, and the hypothyroid quail possessed a greater number of small follicles and more follicular epithelial cells in the thyroid gland. In addition, hypothyroidism resulted in a significant decrease in the plasma concentrations of corticosterone, LH, testosterone and ir-inhibin. Furthermore, no spermatogenesis was found in the seminiferous tubules of the methimazole treatment groups. These results clearly demonstrate that hypothyroidism caused both gonadal and adrenal disturbances in the adult male Japanese quail.

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Legend to Table 1 should have appeared.

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