Mammalian oogenesis occurs concomitantly with folliculogenesis in a coordinated manner in the ovaries. In vitro growth (IVG) culture systems of the oocytes have been developed as a new technology for utilizing incompetent oocytes in the ovary as a source of mature oocytes as well as for studying oogenesis, folliculogenesis, and oocyte-somatic cell interactions. The results of IVG experiments have suggested that direct association of oocytes and surrounding granulosa cells supports oocyte viability and growth through the gap junctions, which are efficient conduits for low molecular weight substances. It has been revealed that granulosa cells metabolize some molecules which are in turn transported into the oocytes. IVG systems have also provided evidence that FSH promotes the development of follicles at secondary or later stages by its stimulation of proliferation and differentiation of granulosa cells, and perhaps by its anti-apoptotic effects. In addition, interactions between granulosa cell-derived KIT ligands and oocyte KIT receptors have been suggested as initiating oocyte growth and follicular development. Furthermore, recent findings suggest there are growth factors derived from oocytes such as GDF-9 and BMP-15. With such factors, oocytes participate in follicular development by regulating the differentiation of surrounding somatic cells. These bidirectional communications between oocytes and somatic cells are important for oocyte growth and follicular development. IVG systems should provide further information regarding oogenesis and folliculogenesis in the ovary.

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In the course of profiling alterations of gene expression in the male reproductive system induced by anti-androgenic agents, 28 genes expressed in the testis or pituitary of adult rats were examined shortly after subacute administration of the well-known anti-androgen, flutamide (FM). FM (25 mg/kg/day) was orally administered to male rats for six days. On day 8 (D8) after the first dose of FM, intratesticular testosterone (T) levels had dramatically increased, but daily sperm production on D36 was significantly decreased. The mRNA levels of testicular and pituitary genes on D8 were measured by semiquantitative RT-PCR. Among the six testicular steroidogenic enzyme genes, the mRNAs of the P450 side chain cleavage, P450 17 α/C_17-20 lyase, and 3β-hydroxysteroid dehydrogenase type I (3βHSD) genes significantly increased, whereas 17β-hydroxysteroid dehydrogenase type III slightly decreased. Among the three steroid receptors examined, androgen receptor (AR) and glucocorticoid receptor (GR) mRNAs were significantly down-regulated (29% and 35%, respectively) in the testis, but there was no change in estrogen receptor α. There were no clear changes in expression of the gonadotropin receptors and Sertoli cell specific genes, but a slight increase was observed in expression of the lactose dehydrogenase-c mRNA, a germ cell specific gene. Among the three immediate early genes, c-myc mRNA was increased approximately 1.4-fold. In the pituitary, on the other hand, mRNAs for LHβ and FSHβ subunits and gonadotropin releasing hormone receptor had increased significantly. These results show that subacute FM administration first affected hypothalamus/pituitary hormone gene expression, then altered gonadotropin secretion, and subsequently induced over-expression of testicular steroidogenic enzyme genes. However, the significant up-regulation of 3βHSD and down-regulation of AR mRNAs, despite the higher level of intratesticular T, might be explained by an antagonistic action of hydroxyflutamide retained in the testis. The profiles of alterations in gene expression observed will provide important information for the screening of adult male animals for anti-androgenic chemicals.

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The objective of this study was to determine plasma concentrations of luteinizing hormone (LH), progesterone (P4) and estradiol-17β (E2) in Mangalica gilts (M), a Hungarian native breed, and compare them with Landrace gilts (L) during the peri- and post-ovulatory periods. The estrous cycle of gilts was synchronised by Regumate^® feeding, and ovulation was induced with a gonadotropin-releasing hormone (GnRH) agonist. Blood sampling was carried out via indwelling jugular catheters three times a day and in 2-h intervals during a 16-h period after the GnRH application. The concentrations of LH, E2 and P4 were determined by immunoassays. Gilts of both breeds showed a typical gonadotropin and gonadal hormone secretion pattern. Preovulatory E2 peaks were observed on day 2 (M) and day 4 (L) after the last Regumate^® feeding. Highest E2 concentration was different between M and L breeds (46.5 ± 5.7 vs. 26.0 ± 6.8 pg/ml, P < 0.05). Maximum LH levels measured up to 6 h after GnRH were not different between M and L breeds (11.5 ± 4.1 vs. 6.6 ± 2.3 ng/ml). Both LH amounts during surge (41.1 ± 15.9 vs. 27.5 ± 6.1 ng/ml) and total over LH release (73.4 ± 22.2 vs. 50.0 ± 8.7 ng/ml) did not differ significantly between M and L breeds. P4 concentrations started to rise on day 6 after Regumate^® feeding and increased significantly from 0.6 ± 0.3 and 0.7 ± 0.4 ng/ml to maximal 14.0 ± 2.4 and 11.3 ± 2.1 ng/ml in M and L breeds, respectively. Mean P4 secretion was higher in M on days 10-15 (12.9 ± 2.6 vs. 9.3 ± 2.2 ng/ml; P<0.05). At the same time the number of corpora lutea was lower in M compared to L (10.3 ±1.5 vs. 17.8 ± 5.0, P<0.05). In our experiment, there was no evidence that differences in the secretion of analysed hormones during the peri- and post-ovulatory periods are a possible cause of usually lower fecundity in Mangalica gils.

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The effect of interferon-τ on in vitro development of bovine embryos was investigated. After in vitro fertilization, embryos developed to the morula stage were cultured for 3 days in TCM-199 or CR1 medium containing BSA or FCS supplemented with or without recombinant IFN-τ produced by a baculovirus expression system. Addition of baculovirus-expressed IFN-τ (100 ng/ml) significantly promoted development to the blastocyst stage in both culture media. Addition of E. coli expressed IFN-τ (2 μg/ml) also significantly promoted the embryonic development. Supplementation of BSA or FCS did not affect the growth-promoting effect of IFN-τ. To determine whether the growth-promoting effect of IFN-τ is related to the interferon type I receptors that bind to type I interferon such as IFN-α, embryos were cultured with IFN-α. Although IFN-α significantly promoted the development, a much higher concentration (25 μg/ml) was required than IFN-τ. A reverse transcription polymerase chain reaction analysis revealed the expression of mRNA encoded type-I IFN receptor subunit from morula to blastocyst stage embryos. The overall results suggest a novel function for IFNs in promoting embryonic development and the effect may be related to type-I IFN receptor expressed in the early stages of preimplantation embryos.

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Sex determination of livestock is performed to achieve the objectives of livestock breeding programmes. Techniques for sex determination have evolved from karyotyping to detecting Y-specific antigens and recently to the polymerase chain reaction (PCR), which appears to be the most sensitive, accurate, rapid and reliable method to date. In this study, a PCR-based sex determination method for potential application in goat breeding programmes was developed. Primers were designed to amplify a portion of the X amelogenin gene (Aml-X) on the X chromosome to give a 300 bp product and Sry gene on the Y chromosome to give a 116 bp product. PCR optimization was performed using DNA template extracted from a whole blood sample of Jermasia goats (German Fawn × Katjang) of both sexes. It was possible to identify the sex chromosomes by amplifying both male- and female-specific genes simultaneously in a duplex reaction with males yielding two bands and females yielding one band. The Aml-X primer set, which served as an internal control primer, did not interfere with amplification of the Y-specific sequence even when a low amount of DNA (1 ng) was used. The duplex reaction subjected to a blind test showed 100% (14/14) concordance, proving its accuracy and reliability. The primer sets used were found to be highly specific and were suitable for gender selection of goats.

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To reveal the molecular mechanism of selective follicular atresia in porcine ovaries, we investigated the changes in the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor (DR4) proteins and TRAIL mRNA in granulosa cells during follicular atresia. Immunohistochemical, Western immunoblotting and reverse transcription-polymerase chain reaction analyses (RT-PCR) revealed that significant increases in TRAIL protein and mRNA levels but not DR4 protein were changed during atresia. The RT-PCR product was confirmed to be porcine TRAIL by the cDNA sequence determination. An in vitro apoptosis inducing assay using cultured granulosa cells prepared from healthy follicles showed that TRAIL could activate caspase-3 and induce apoptotic cell death in the cells. The present findings confirm that TRAIL induces apoptosis in granulosa cells during atresia in porcine ovaries.

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Protein tyrosine phosphatase (PTP) expression was examined in preimplantation mouse embryos. We previously reported that SHP-2, LAR, PTPT9, SHP-1, and mRPTPB were expressed in preimplantation mouse embryos. Here, we examined changes in the expression levels of these PTPs during preimplantation development. cDNA was obtained by reverse transcription of embryo mRNA, amplified with 10 PCR cycles, and then subjected to real-time fluorescence-monitored PCR. Experiments with an mRNA dilution series revealed that the data obtained matched the quantities of mRNA used. The measurements obtained with real-time fluorescence-monitored PCR showed that the expression of each PTP mRNA changed dynamically, and that each had a different expression pattern. This suggests that PTPs are involved in the regulation of growth and differentiation during preimplantation development.

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Bisphenol A (BPA), a candidate endocrine disruptor (ED), is considered to bind to estrogen receptors and to regulate expressions of estrogen responsive genes. It has also shown evidence of affecting the reproductive, immunological and nervous systems of mammalian embryos. However, the effects of BPA on placentae, a central organ of feto-maternal interlocution, are still unclear. To reveal the mechanisms of BPA effects on placentae in mammals, we compared the mRNA expression of 20 nuclear receptors between placentae of vehicle controls and those of orally BPA exposed pregnant mice by a DNA microarray technique. In murine placentae, mRNAs of 11 nuclear receptors were not detected. However, greater than 1.5 fold changes in mRNA expression of nine nuclear receptors between vehicle control and BPA treated mice were noted. Moreover, remarkable changes in mRNA expression of six non-nuclear receptor proteins were induced by BPA exposure. There were various differences in the effects of BPA on the expression of these mRNAs between the placentae with male embryos and those with female embryos. Such embryo-sex dependent differences are interesting and important pointers to understanding of the endocrine disrupting effect of BPA. The present data indicate that BPA affects the expression of nuclear receptor mRNAs in placentae and may disrupt the physiological functions of placentae.

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The Immunoassay (p.101) should be as follows:

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