Environmental pollutants are known to exert endocrine-disrupting effects on several hormonal axes of animals, including reproduction and development. Many of these xenobiotics modulate the estrogen-receptor signaling pathway(s) in agonistic or antagonistic ways. Some of the compounds are themselves estrogenic (so-called xenoestrogens, environmental estrogens, or ecoestrogens), and are classified as synthetic estrogens, phyto- or fungal estrogens, alkylphenol ethoxylates; certain non-coplanar polyclorinated biphenyls (PCBs), etc. Other molecules are anti-androgenic, e.g., p, p'-dichloro-diphenyl-dichloroethylene (DDE); while still others disrupt estrogen function in a manner that is dose, species and tissue specific, e.g., certain co-planar PCBs and dioxin-like molecules (e.g., tetrachlorodibenzo-p-dioxin [TCDD]). Exposure to some compounds has been correlated with skewing of sex ratios in aquatic species, and feminization and demasculinization of male animals; declines in human sperm counts; and overall diminution in fertility of birds, fish, and mammals. This review will cover these various xenobiotics and evaluate them for their estrogen-modulating effects; and then further concentrate on TCDD specifically. Dioxins are produced as by-products of herbicide overuse, from paper bleaching, plastics manufacture, and waste incineration. TCDD has been correlated with altered fecundity and endometriosis in monkeys, and with certain cancers in experimental animals and humans. TCDD is also correlated with reproductive deficits in many laboratory species. In summary, we believe that some of the reproductive deficits from endocrine-disrupting xenobiotics may be attributable to the modulation of the estrogen-signaling pathway, in positive and negative manners, depending on dose, species, and tissue specificity.

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The advent of ultrasonography over a decade ago has contributed tremendously in the field of bovine reproduction. Ultrasound imaging has shown that cattle exhibit 2 or 3 waves of follicular development during an estrous cycle. Ultrasonography allows monitoring of individual follicles as they grow and/or regress over time and patterns of follicular development can thus be determined with relative precision. Other important applications of ultrasonography in bovine reproduction includes; pregnancy diagnosis, fetal sex determination, folliculocenteses, diagnosis of abnormalities of the reproductive organs, monitoring of treatment of ovarian cysts, monitoring of postpartum genital resumption, ultrasound-guided centesis and male genital ultrasonography. Recent applications of ultrasonography in bovine reproduction includes doppler ultrasonography for genital blood flow and mammary ultrasonography. The use of ultrasonography is indispensable in most studies in which morphology (structure) and function are correlated. In this paper, the literature pertaining to the use of ultrasonography in bovine reproduction is reviewed. The applications and limitations of ultrasonography are highlighted. The non-invasive nature of ultrasonography makes it an excellent clinical and research tool in bovine reproduction.

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Recent reports have shown that sperm cells incubated with foreign DNA in vitro are able to transfer the DNA into eggs at fertilization. The present study examined if an injection of DNA into the testis in vivo could generate transgenic animals via sperm ejaculated. We prepared 3 gene constructs; human growth hormone (hGH), hGH receptor (hGHR) and mouse leptin (mOB) genes fused to the promoter regions of the mouse genes for whey acidic protein (WAP), metallothionein-I (MT) and mouse mammary tumor virus (MMTV), respectively. Each gene construct mixed with cationic liposome was injected into bilateral testes of male rats or mice, and the males mated with females 3 or 4 days later. A female rat mated with a male treated with MT/hGHR gene gave birth to 17 pups, 3 of which were found to carry the transgene. The expression of hGHR mRNA was demonstrated in the liver, kidney, muscle and brain after treating with zinc in drinking water. At present, the transmission of the exogenous gene to the descendants was confirmed up to the F₄ generation. Three female rats mated with 3 different males injected with MMTV/mOB fusion gene produced 10, 14 and 12 pups, respectively. The genome of 2 out of these 36 pups harbored MMTV-mOB gene, though expression of mOB mRNA was not detected. Two female mice were mated with 2 male mice injected with WAP/hGH gene and produced 15 and 16 pups, 3 of which incorporated the gene. The expression of hGH mRNA in the mammary gland in these mice was confirmed. These results indicate that exogenous DNA injected into the testis as a liposome-complex can be transferred into eggs via sperm and expressed in the postpartum progeny.

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We have recently shown that DNAs injected into the testis of either rats or mice can be transferred into eggs via sperm at fertilization and expressed and transmitted in the descendants (testis-mediated gene transfer). In the present study, we investigated the association of epididymal and ejaculated sperm with DNA injected into the testis of rats. In the first experiment, a chimeric gene of mouse metallothionein-I promoter (MT)/human growth hormone receptor (hGHR) labeled with FITC was prepared. This gene construct was mixed with cationic liposome and injected into rat testis, and 4 days later the cauda epididymis was dissected out and prepared for histological examination. A confocal microscopic observation showed that not only the head, but also the tail, of almost all spermatozoa in the epididymis was labeled with FITC. In the next experiment, 4 days after the injection of MT/hGHR gene encapsulated by liposome into the testis, the male was allowed to mate with females and spermatozoa were recovered from the uterus in the next morning. Spermatozoa were incubated in the presence or absence of deoxyribonuclease (DNase) I and then genomic DNA was extracted. The MT/hGHR gene was detected only in the spermatozoa incubated without DNase by means of PCR. These results support the notion that, in the case of our testis-mediated gene transfer, exogenous DNA injected with liposomes is not integrated into genome of the sperm and the integration occurs after fertilization.

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The placenta produces prolactin (PRL)- or growth hormone-like hormones known as placental lactogens (PLs) which play roles in the maternal endocrine system and fetal growth development. We previously cloned a cDNA encoding a novel rat PL, PL-I mosaic (PL-Im), which is specifically expressed at mid-pregnancy. In this study, the biological activity of PL-Im was evaluated by producing the recombinant protein (recPL-Im) by means of a baculovirus/insect cell expression system. RecPL-Im demonstrated binding activity for PRL receptor in a radioreceptor assay (RRA) system with rat liver membrane fraction and ¹²⁵I-ovine PRL (oPRL). In addition, the activity of recPL-Im is comparable to oPRL in the Nb2 cell proliferation assay. Interestingly, recPL-Im stimulated progesterone secretion in a concentration-dependent manner in a primary culture of luteal cells from pregnant rat, whereas oPRL had no effect on luteal cells. The luteotropic function of recPL-Im was accompanied by suppression of the secretion of 20α-dihydroprogesterone (20α-OHP) in the culture. PL-Im therefore has ability to bind PRL-receptor and express PRL-like activity. In addition, recPL-Im has luteotropic hormone (LTH) activity and its activity is qualitatively different from that of PRL.

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Objectives of this study were to examine effects of GnRH-PGF2α-GnRH treatment and fixed-time AI (OVSYNCH) on the conception rate in Holstein-Friesian dairy herds with poor estrus detection efficiency in Eastern region of Hokkaido. In the first experiment ten cows, which were found to have normal ovarian cycle, were first injected intramuscularly with 100 μg fertirelin acetate (an analog of GnRH), followed by an intramuscular injection of 25 mg dinoprost (PGF2α-THAM) at an interval of 7 days. A second dose of fertirelin acetate was administered 48 h after the dinoprost injection. All cows were artificially inseminated (AI) 24 h after the second fertirelin injection. Milk progesterone profile and ultrasonography indicated that ovulation was synchronized in all 10 cows, and 70% cows conceived. In the second experiment a total of 336 cows with postpartum anestrus were divided into two groups. A group of 185 cows were subjected for OVSYNCH, while the other group of 139 anestrus cows with functional corpus luteum were given 25 mg dinoprost and inseminated artificially 3 days after the dinoprost treatment and 12 anestrus cows with inactive ovaries were injected with 100 μg fertirelin acetate and inseminated artificially at heat and inserved as a control. Of 185 cows treated with OVSYNCH, 153 cows were inseminated artificially and 82 cows (53.6%) conceived. A conception rate in control cows treated with dinoprost and bred 3 days later was 20.9%. The OVSYNCH has been proved to be a useful tool for improving reproductive performance in dairy herds with a low estrus detection rate.

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To show the effect of intravaginal progesterone-releasing device (CIDR) on turnover of DF and follicular growth after CIDR insertion on day 16 of estrous cycle and to compare the follicular growth during CIDR treatment between heifers with two or three follicular waves, four heifers were given CIDR for 7 days at late luteal phase on day 16 of estrous cycle. The presence of CIDR in the vagina containing progesterone prevented for the animals to return to estrus at the normal time and significantly increased cycle length, as compared to the preceding cycle (26.3 ±0.5 vs 20.8 ±1.5 days, p<0.05). Ovarian follicular dynamics were characterized by waves of follicular development occurring at different times during the estrous cycle. In the stage 1, four heifers had two (n=2) or three (n=2) follicular waves per estrous cycle. However, during the cycle of CIDR treatment, all heifers had three follicular waves per cycle. The CIDR treatment caused an extension of dominant phase of the ovulatory DF selected in the third follicular wave in heifers with 3 follicular waves per cycle before CIDR treatment. In heifers with two follicular waves per cycle before CIDR treatment, after CIDR treatment the DF underwent atresia without delay and followed emergence of the new follicular wave, in which no extension of dominant phase of the DF or ovulatory follicle was shown. The CIDR treatment did not affect the follicular growth during the subsequent cycle. In conclusion, when CIDR treatment commenced at late luteal phase, CIDR may cause an extension of an interval from emergence to ovulation of ovulatory DF in heifer with three follicular waves. And also, the CIDR treatment does not delay atresia process of DF in preceding cycle and is developed the new follicular waves in heifers with two follicular waves per cycle before CIDR treatment

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The present study was carried out to establish aggregation procedures of bovine inner cell mass (ICM) cells and tetraploid embryos to produce cloned cattle in a different way from nuclear transfer. Bovine two-cell embryos produced by in vitro fertilization were exposed to the electric pulse to fuse 2 blastomeres resulting in tetraploid embryos. Nine (18.8%) of 48 embryos fused by electrofusion developed to blastocyst stage. Three (16.7%) aggregates consisting of two putative tetraploid embryos and one clump of ICM cells developed to blastocyst. And the aggregates which were aggregated with a clump of bovine mammary cells and a clump of ICM cells which were stained with Hoechst also developed to blastocysts. These blastocysts were illuminated by UV, overall or some parts of all ICM of the aggregates treated were observed to luminesce. This raises the possibility that the clumps of the cells can form some parts of ICM of the aggregates. These results indicate that the aggregates of a clump of ICM cells or mammary cells and tetraploid embryos developed to blastocyst stage and that some parts of ICMs of the aggregates were originated from the clump of cells.

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Two experiments were conducted to enhance the corpus luteum (CL) function for increasing lambing rate of ewes treated with GnRH or hCG after artificial insemination (AI) during the non-breeding season. Ewes (Experiment 1: n=102, Experiment 2: n=37) were pretreated with a controlled internal drug release dispenser (CIDR) for 12 days and 500 IU equine chorionic gonadotropin (eCG) 1 day before CIDR removal. In Experiment 1, the ewes were treated on Day 12 (Day 0=CIDR removal): 1) Group I; GnRH (100 μg), 2) Group II; hCG (500 IU), 3) Group III; 0.6% saline (2 ml) for control. Pregnancy and lambing rates and prolificacy were not significantly different among the groups. But, there was significant (P<0.01) difference in the lambing rate between parous (27%) and non-parous (72%) in Group I. On Day 17, plasma progesterone (P₄) levels of Group I were significantly (P<0.01) lower than those of Group III, but on the contrary, the plasma P₄ levels of Group II were significantly (P<0.01) higher than those of Group III. In Experiment 2, the ewes were treated: 1) Group I; hCG (500 IU) on Day 6, 2) Group II; hCG (500 IU) on Day 9, 3) Group III; no treatment for control. Pregnancy and lambing rates and prolificacy were not significantly different among the groups, but on Days 12 and 15, the plasma P₄ levels of Groups I and II were significantly (P<0.01) higher than those of Group III. The present results indicate that a single hCG treatment on Days 6, 9 and 12 after CIDR removal stimulates CL and increased P₄ concentration, but the increased P₄ levels did not reflect on the fertility of treated ewes.

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The vomeronasal sensory epithelium of goats native to Korea was examined morphologically by light and electron microscopy. The vomeronasal organ is a tubular structure situated bilaterally at the base of the nasal septum. The sensory epithelium lines the medial wall of the vomeronasal lumen and consists of receptor, supporting, and basal cells. The receptor and supporting cells have prominent microvilli on the luminal surface. In the basal region, four types of basal cells can be distinguished morphologically, two of which are attached to the basal lamina. The morphological characteristics of Korean goat vomeronasal sensory epithelium are discussed in comparison with those of other mammalian vomeronasal epithelia.

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A transgene in which polyoma enhancer was fused between the 5' upstream region of PGK-RU5/lacZ and that of PGK/neo was microinjected into pronuclei of mouse zygotes. These embryos were cultured in the presence (G-418 treatment) or absence (control treatment) of 150 μg/ml G-418 and those surviving were subsequently examined for the pattern of lacZ expression. The rate of development to the blastocyst stage after culture for 3.5 days was significantly reduced by the G-418 treatment (32.8%, 59/180 vs 17.6%, 40/227, P<0.05). The overall proportion of embryos expressing lacZ gene did not change significantly with treatment (72.8%, 131/180 vs 66.1%, 150/227). However, 54.2% (32/59) and 85.0% (34/40) of the blastocyst stage embryos obtained from the control and G-418 treatment groups showed signs of lacZ activity, respectively (P<0.05). These direct comparisons between the developmental potential and lacZ expression of embryos revealed that the transgene employed in this study can retain both neo and lacZ functions. Blastocysts were classified by blue deposits in the cytoplasm according to pattern and intensity. Almost all lacZ positive blastocysts were mosaic being composed of unstained and stained regions, and weakly or spotty stained and strongly stained regions within whole embryos. In both groups, all of the blastocysts in which the intensity of the staining cytoplasm was scored as weak or spotty, were categorized as having staining in less than 1/3 the area of whole embryo (56.2%, 18/32 vs 41.2%, 14/34). In the remaining blastocysts which had intense staining, the number of blastocysts scored as having staining in more than 1/3 the total area tended to increase on G-418 treatment (25.0%, 8/32 vs 38.2%, 13/34), but there was no clear relationship between the pattern of X-gal staining and the treatment. These results suggest that mouse embryos without exogenous DNA after microinjection can be eliminated during the preimplantation period.

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A serum-free culture system was used to allow determination of the fatty acid composition of bovine embryos during in vitro development. The proportion of embryos developing to the blastocyst stage in serum-free medium (TCM199 supplemented with bovine serum albumin, insulin, apotransferrin and transforming growth factor-α; BITTα) was as high as in serum-supplemented medium (TCM199+5% calf serum). Bovine blastocysts grown in serum-supplemented medium contained abundant lipid droplets in the cytoplasm. The fatty acid composition of bovine immature oocytes, embryos at 2-cell and blastocyst stages cultured in serum-free or serum-supplemented medium was determined by capillary column gas chromatography. Additionally, the fatty acid composition of calf serum alone was determined. Myristic acid (59.6%) was the most abundant fatty acid in immature oocytes followed by docosahexaenoic acid (12.3%). The fatty acid profiles of two-cell embryos derived in the serum-free medium were similar to those of immature oocytes. In contrast, two-cell embryos derived in the serum-supplemented medium showed high levels of palmitic (27.6%) and stearic (27.2%) acids and a low level of myristic acid (21.9%). For blastocysts grown in the serum-free medium, myristic acid (35.7%) was high and palmitic (17.0%) and stearic (12.5%) acids were moderately high. On the other hand, blastocysts developed in the serum-supplemented medium retained high levels of palmitic (30.5%) and stearic (24.2%) acid compositions and had considerably increased levels of palmitoleic (16.3%) and oleic (12.1%) acids. High levels of oleic (17.3%), palmitic (16.5%) and stearic (12.3%) acids were detected in calf serum, which were similar to the profile of the 2-cell and blastocyst stage embryos cultured with serum. This study shows that bovine embryos grown in serum-supplemented medium showed different morphology and fatty acid composition when compared to those cultured in serum-free medium.

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A 369-bp cassette DNA fragment (LACFE) was constructed for simultaneous detection of exogenous gene integration and sexing of preimplantation bovine embryos. The LACFE consists of a part of the bovine lactoferrin gene, including three Dpn I sites and a pair of Y chromosome-specific primer sequences for polymerase chain reaction (PCR) of both ends. Bovine pronucleus embryos were microinjected with the LACFE modified by DNA adenine methylase. After 7 to 9 days culture in vitro, the blastocysts were bisected and the demi-embryos were analyzed by the PCR method following the nuclease treatment with Dpn I and Bal 31 nucleases (digestion-PCR) or without the nucleases (unmodified PCR), respectively. Results of sexing between a pair of demi-embryos agreed each other in 95 of 102 blastocysts. Ten of 102 demi-embryos were positive for the exogenous gene by unmodified PCR. On the other hand, 3 of 102 demi-embryos were positive by digestion-PCR, and included in the 10 demi-embryos. The digestion-PCR may exclude false-positive embryos, and prove to be an efficient selection of preimplantation bovine transgenic embryos. These results indicate that LACFE is useful for the simultaneous detection of the integration of exogenous genes and the sexing of preimplantation bovine embryos using only a single pair of primers. As the LACFE can be introduced into a construct of desirable transgene for microinjection, the LACFE is widely applicable for detection and sexing of transgenic embryos under the same analytical conditions.

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