Prolactin (PRL) is known to be one of the hormone involved in the induction of maternal behavior. Recent observation that PRL receptor (PRL-R)-knock-out (KO) mice show a deficiency in pup contact-induced maternal behavior indicates that PRL-R plays an essential role in the induction of the maternal behavior. However, PRL-KO mice exhibit no abnormality in the maternal behavior. This discrepancy in the maternal behavior between PRL-R- and PRL-KO mice suggests that actions of maternal PRL or placental lactogens (PLs) to the fetus and/or the pups are critical for the maternal behavior at adulthoods. In rat brain, the expression of mRNA for the long form PRL-R increases during maternal behavior in males and females, suggesting that PRL actions on the brain are important for maternal behavior. Expressions of rat PRL-R gene are known to be regulated in a tissue-specific manner by the promoters for multiple first exons. In addition to the three known first exons, E1₁, E1₂, and E1₃, we have recently identified two novel first exons, E1₄ and E1₅. E1₄ is expressed only in the brain with higher levels at pregnant and lactating periods compared to dioestrous period. The regulatory mechanisms for the expression of PRL-R gene in the brain associated with maternal behavior and molecular events responsive to the action of PRL on the brain to induce maternal behavior are discussed.

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We found that two inbred strains of Hatano rats bred selectively for shuttlebox avoidance responses, had different characteristics of responses to stress and adrenal function. The characteristics of male puberty, including preputial separation, sperm number, and plasma hormone levels during development, were investigated and compared between the two strains. Body weight in high-avoidance (HAA) rats was consistently heavier than that in low-avoidance (LAA) rats. Although the absolute testicular weight was heavier in HAA rats than in LAA rats during the juvenile period, after the peripubertal period the inverse finding was observed. Preputial separation occurred about 7 days earlier in HAA rats than in LAA rats. The testicular and epididymal sperm head number in HAA rats was greater than that in LAA rats during the peripubertal period, but there were no differences after sexual maturation. A steep increment of plasma gonadotropin levels was observed in HAA rats, and the levels were higher in HAA rats than in LAA rats during the peripubertal period. Plasma testosterone levels began to rise at a younger age in HAA rats than in LAA rats. It was concluded that HAA males sexually mature earlier than LAA males, and that these apparent strain differences in puberty are primarily dependent on gonadotropin levels during sexual maturation.

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Porcine nuclear transfer embryos were reconstructed by intracytoplasmic nuclear injection of cumulus cell nuclei into recipient oocytes matured in vitro by two types of method (NCSU23-based and TCM199-based methods). Remodelling of the transferred nuclei and in vitro development of the reconstructed embryos were investigated. Intracytoplasmic nuclear injection did not induce activation of the recipient oocytes regardless of the type of maturation method (NCSU23-based: 61/61, 100%, TCM199-based: 49/50, 98%). When the reconstructed embryos were examined 1 h after nuclear injection many of them (NCSU23-based: 22/29, 76%; TCM199-based: 19/23, 83%) possessed condensed chromosomes or a chromosome array resembling the maternal metaphase plate. The proportion of the reconstructed embryos possessing condensed chromosomes arranged in a scattered fashion showed a tendency to increase, when they were examined 2-5 h after nuclear injection. Reconstructed embryos were electrically activated either 2-2.5 h or 3.5-4 h after nuclear injection, followed by fixation/staining to examine formation of pronucleus-like nuclei (pseudo-nuclei). Regardless of the nuclear injection/activation interval tested, 68% (36/53) of the reconstructed embryos developed pseudo-pronuclei ranging in number between 1 and 4. When the reconstructed embryos were cultured for 7 days to assess their developmental competence, 5 (5/98) to 11% (12/111) developed to blastocysts regardless of the type of the recipient oocyte and nuclear injection/activation intervals. These results show that intracytoplasmic injection of porcine cumulus cell nuclei achieves a high rate of nuclear remodelling and that the reconstructed embryos are capable of developing into blastocysts.

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This study aimed to develop an antiserum specific to follicle stimulating hormone (FSH), for a wide range of species, free from cross-reactivity against luteinizing hormone. An antiserum against recombinant porcine FSH (rec-pFSH) expressed in insect cells was raised in a rabbit and its specificity was characterized by time-resolved fluoroimmunoassay (TR-FIA) and immunohistochemistry. The titer of the anti-rec-pFSH antiserum was about 1:5,000 for use in TR-FIA. The inhibition curve of TR-FIA with rec-pFSH paralleled that of pituitary-derived FSH, indicating that the antigenicities of the two FSH preparations do not differ significantly. The anti-rec-pFSH antiserum showed low reactivities with FSHs prepared from sheep and rat, suggesting it was species- and hormone-specific, contrary to our intention. The TR-FIA showed that this antiserum did not react with pituitary extracts from mammals other than the pig. However, immunohistochemistry demonstrated that this antiserum reacted with the specific cells not only of the porcine anterior pituitary but also of the cow, goat, and horse. The antiserum also immunostained pituitary cells of the cow, goat and horse. Therefore, this anti-rec-pFSH antiserum will be of limited use in studies involving competitive immunoreactions due to its species-specific reactivity, but it should be useful for non-competitive techniques, such as immunohistochemistry.

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With the eventual application of the embryo culture system (ECS) first developed for chickens to endangered avian species in mind, we examined some of the basic features of the ECS in the chick. We demonstrate that embryos cultured in System II for 72 h show a delay in development as well as a wider distribution of the developmental stages attained (range Stages 12-20; mode Stages 17-18) than the control intact embryos (range 15-21; mode 19), and that the developmental stage of the embryos at the time of transfer to System III significantly affected subsequent viability and hatchability. When the Stage 18 embryos were transferred to System III, they showed the best rates of viability and hatchability in the present study. The implications of the results as applied to endangered avian species are discussed.

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Primordial germ cells circulating in the embryonic vascular system (cPGCs) were isolated from 2.5-day-old chick embryos and cultured individually in suspension for 5 days in embryonic stem-cell medium (ESM) adjusted to pH 8.0 supplemented with fetal bovine serum. Most cells died within 5 days, during which time only a few cells (16-23%) underwent mitosis once, and only a very few cells (less than 3%) twice. The use of conditioned medium (CM) derived from embryonic fibroblasts enhanced the viability and proliferation of the cPGCs, resulting in survival of up to one-third of the cells for 5 days. A fraction of the cells (3-5%) even divided 3 times. The in vitro cultured cPGCs maintain the capacity to migrate into the gonadal area. The chick cPGCs cultured in vitro for up to 5 days in the CM produced germ-line chimeras upon injection into the bloodstream of 2-day-old quail embryos.

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To assess the effects of different transgene forms on integration efficiency, plasmids containing human growth hormone gene under the control of the metallothionein promoter (pMThGH) in circular, linearized forms and lyposome trapped linearized plasmid were introduced into fertilized loach eggs. Prior to microinjection, plasmids were identified for the presence of human growth hormone (hGH) gene by digestion with restriction enzymes (EcoRI, BamHI, BglII) and by polymerase chain reaction (PCR). The linearized form of pMThGH was generated by digesting circular plasmids with BamHI. Liposome trapped linearized plasmid was prepared in the presence of dioleyl phosphatidyl choline, cholesterol and pMThGH. The presence of hGH genes in the genomic DNA of loach developed from microinjected eggs was identified by a PCR method using two hGH-specific primers. PCR products of the positive individuals showed one band corresponding to 479 bp, while those of negative and control individuals showed no corresponding band. Integration rates of hGH genes into fish chromosomes were 6%, 12% and 11% for circular, linearized plasmids and lyposome trapped linearized plasmid, respectively. In accordance with above order, the average body weight of transgenic loach at 6 months was increased by 1.63, 1.73 and 1.68 times over controls, respectively. The results indicate that plasmids in linearized form and lyposome trapped linearized plasmid were more efficiently integrated into loach chromosomes than circular ones, but there was little significant difference in body weight among the 3 groups of transgenic fish.

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The aim of this study was to determine the influence of the homogeneity of genomic components and ploidy on the in vitro development of porcine parthenogenetic oocytes to the blastocyst stage. In vitro matured oocytes were subjected to a single pulse of electro-stimulation (El-St; 100 μsec, 1,500 V/cm) for activation. First, the activated oocytes were cultured for 6, 16, 18, 20 and 22 h after El-St, and examined for the timing of the first cleavage of parthenogenetic haploids. Next, the effects of the timing and duration of cytochalasin B (CB) treatment on inhibition of the first cleavage of haploids were examined in order to produce homogenous parthenogenetic diploids. Then the developmental ability to the blastocyst stage was compared among activated oocytes with genomic components of haploid (without CB treatment), homogeneous diploid (nn-diploid, CB treatment for 6 h from 20 h after El-St), and heterogeneous diploid (nn'-diploid, CB treatment for 4 h immediately after El-St). Most haploids were at the prophase to telophase of the first cleavage between 16 and 22 h after El-St. When the haploids were treated with 5.0 μg/ml CB for 6 h from 20 h after El-St, their first cleavage was efficiently suppressed, and most of them (84%) became diploids. The frequency of parthenogenetic development to the blastocyst stage was significantly lower in haploids (5%) than in nn-diploids (48%, P<0.01) or nn'-diploids (57%, P<0.01). These results shows that ploidy of activated oocytes, but not the homology of genomic components, affects the development of porcine parthenogenetic oocytes to the blastocyst stage.

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To reveal the specific regulatory molecules that control granulosa cell apoptosis during follicular atresia, we immunohistochemically examined the localization of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors in porcine ovaries. A marked reduction in the expression of decoy receptor-1 (DcR1), which has high affinity for TRAIL, was demonstrated in granulosa cells of atretic follicles, but no marked differences were seen in expression of TRAIL or other TRAIL-receptors (death receptor-4 or death receptor-5) in granulosa cells between healthy and atretic follicles. No positive staining for DcR2 was seen. We presum that TRAIL and its receptors are involved in induction of apoptosis in granulosa cells during atresia, and that DcR1 plays an inhibitory role in granulosa cell apoptosis.

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Previously, we histochemically demonstrated the expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its receptors, death receptor-4 (DR4), death receptor-5 (DR5) and decoy receptor-1 (DcR1) in granulosa cells of porcine follicles. However, TRAIL can induce both cell death and cell proliferation. In the present study, reverse transcription polymerase chain reaction and in situ hybridization analyses revealed increased mRNA expression of TNF receptor-associated death domain protein (TRADD), which transmits the death signal from DR4 and/or DR5 to intracellular apoptosis-signal transduction components, in granulosa cells was demonstrated only in atretic follicles but not in healthy follicles. These findings indicate that TRADD is involved in induction of apoptosis in granulosa cells, and that the TRAIL-receptor system induces apoptosis in granulosa cells during atresia in porcine ovaries.

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It is well accepted that glutamate stimulates in vivo and in vitro gonadotropin-releasing hormone (GnRH) and/or luteinizing hormone (LH) release. The present study aimed to determine whether the arcuate nucleus (ARC) has a role in exerting the modulating effect of estrogen on GnRH response to glutamate. The effects of estrogen on glutamate-induced in vitro GnRH release from either the ARC-median eminence (ME) or ME fragment were examined in female rats. Both high and low doses of estradiol-17 β treatment in ovariectomized OVX animals enhanced glutamate-induced GnRH release from the ARC-ME fragment but not from the ME fragment. This suggests that the ARC mediates the facilitating effect of estrogen on glutamate-induced GnRH release from the ARC-ME. The facilitating effect of estrogen was also observed on GnRH release from the ARC-ME treated with selective excitatory amino acid agonists, i.e., N-methyl-d-aspartate (NMDA), kainate, and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. These results suggest that the ARC plays an important role in facilitating estrogen actions on GnRH response to glutamate via both NMDA and non-NMDA receptors. The estrogen-induced enhancement of GnRH secretory response to glutamate in the ARC-ME region could partly contribute to the induction of preovulatory GnRH/LH surge.

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The present study investigated the in vitro viability of cryopreserved-thawed ovine embryos by three methods, slow freezing (ethylene glycol (EG)-3 step method), freezing for direct transfer (Direct method) and vitrification (Experiment 1), and the in vivo viability by two transfer methods, direct ET (d-ET) and stepwise ET (s-ET), for the Direct method and vitrified embryos, respectively (Experiment 2). In vivo produced embryos (morula and blastocyst) were recovered from 79 superovulated and artificially inseminated ewes, and were cryopreserved by three methods: EG-3 step method, Direct method and vitrification. In Experiment 1, all thawed embryos were removed from the respective cryoprotectants and cultured for 144 h. There were no significant differences among in the mean survival rates, normal hatching rates (by 72 h) and hatching rates (by 144 h) of the three methods. The viability of frozen-thawed embryos by the EG-3 step method tended to be higher than that of the other methods, but some of the viable embryos frozen by the EG-3 step method had delayed hatching or were degenerated (28.6% vs. 16.7% for Direct method and 6.7% for vitrification). In Experiment 2, the frozen-thawed embryos were transferred by s-ET and d-ET. The pregnancy and lambing rates of both transfer methods for embryos frozen-thawed by the Direct method were 25.0%, but the subsequent viability of embryos in d-ET was higher than that of s-ET (the survival rates of transferred embryos, 18.8% vs. 10.0%, and the efficiency of embryonic utility, 18.8% vs. 3.7%). Sixty-nine percent (20/29) of vitrified-warmed embryos with normal morphology were transferred to 8 ewes by s-ET resulting in a lambing rate of 62.5%. The present results indicate that direct transfer of frozen-thawed ovine embryos is practical, and vitrification is a useful cryopreservation method with less influence on embryonic developmental ability.

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The unavailability of homologous antibodies to avian thyrotropin (TSH) has been a major hindrance in the study of avian reproduction and metabolism. Here we report the production and characterization of an anti-peptide antibody specific to chicken TSH β subunit. According to the cDNA sequence of chicken TSH β subunit, a synthetic peptide corresponding to the amino acid residues 35-50 (Tb3550; Asp-Ser-Asn-Gly-Lys-Lys-Leu-Leu-Leu-Lys-Ser-Ala-Leu-Ser-Gln-Asn) was prepared to produce polyclonal antibody in rabbits. This peptide sequence is highly specific to avian TSH β and was designed for the antibody to show no cross-reaction to pituitary and other chicken proteins with known amino acid sequences. Anti-Tb3550 recognized a 18.2-kDa band on immunoblot of the cephalic lobe homogenate, but not of the caudal lobe homogenate, of chicken anterior pituitary gland. Positive immunohistochemical staining with anti-Tb3550 was restricted to the cephalic lobe, whereas the cells positive to anti-chicken luteinizing hormone were scattered throughout the anterior pituitary. Anti-Tb3550 did not bind either to purified chicken luteinizing hormone or to follicle stimulating hormone preparation. These results indicate that this antibody specifically recognizes chicken TSH β subunit. This antibody should be useful for immunochemical study of avian TSH and for tracing TSH activity during the purification of the hormone.

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