Masahide Nishibori

Luteinizing Hormone Receptor Formation in Cumulus Cells Surrounding Porcine Oocytes and Its Role During Meiotic Maturation of Porcine Oocytes

Abstract We investigated the formation of LH receptor (LHR) in cumulus cells surrounding porcine oocytes and the role of LHR in meiotic maturation of oocytes. At least three splice variants of LHR mRNA were detected in cumulus cells, in addition to the full-length form. Low levels of three types of products were seen in cumulus cells from cumulus oocytes complexes (COCs), whereas the full-length form was significantly increased by 12-h cultivation with FSH. The addition of FSH also significantly increased the binding level of biotinylated hCG to COCs. The formation of LHR in FSH-stimulated cumulus cells was not affected by additional 0.5 mM phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and the oocytes were synchronized to the germinal vesicle (GV) II stage by exposure to 0.5 mM IBMX and FSH for 20 h. The binding of LH to its receptor induced a further increase in cAMP level and progesterone production and acceleration of meiotic progression to the metaphase I stage. The oocytes cultured with LH for 24 h following cultivation with FSH and IBMX were used for in vitro fertilization. At 6 days after in vitro fertilization, blastocyst rate in oocytes matured under these conditions was significantly higher than that of oocytes cultured in the absence of LH. Treatment of oocytes with FSH and 0.5 mM IBMX to express LH receptor in cumulus cells while holding oocytes at the GV II stage is a very beneficial way to produce in vitro-matured oocytes, which have high developmental competence.

Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro.

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.

Karyotypic evolution in the Galliformes: an examination of the process of karyotypic evolution by comparison of the molecular cytogenetic findings with the molecular phylogeny

To define the process of karyotypic evolution in the Galliformes on a molecular basis, we conducted genome-wide comparative chromosome painting for eight species, i.e. silver pheasant (Lophura nycthemera), Lady Amherst’s pheasant (Chrysolophus amherstiae), ring-necked pheasant (Phasianus colchicus), turkey (Meleagris gallopavo), Western capercaillie (Tetrao urogallus), Chinese bamboo-partridge (Bambusicola thoracica) and common peafowl (Pavo cristatus) of the Phasianidae, and plain chachalaca (Ortalis vetula) of the Cracidae, with chicken DNA probes of chromosomes 1–9 and Z. Including our previous data from five other species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and blue-breasted quail (Coturnix chinensis) of the Phasianidae, guinea fowl (Numida meleagris) of the Numididae and California quail (Callipepla californica) of the Odontophoridae, we represented the evolutionary changes of karyotypes in the 13 species of the Galliformes. In addition, we compared the cytogenetic data with the molecular phylogeny of the 13 species constructed with the nucleotide sequences of the mitochondrial cytochrome b gene, and discussed the process of karyotypic evolution in the Galliformes. Comparative chromosome painting confirmed the previous data on chromosome rearrangements obtained by G-banding analysis, and identified several novel chromosome rearrangements. The process of the evolutionary changes of macrochromosomes in the 13 species was in good accordance with the molecular phylogeny, and the ancestral karyotype of the Galliformes is represented.

Contribution of mutation, recombination, and gene conversion to chicken MHC-B haplotype diversity.

The Mhc is a highly conserved gene region especially interesting to geneticists because of the rapid evolution of gene families found within it. High levels of Mhc genetic diversity often exist within populations. The chicken Mhc is the focus of considerable interest because of the strong, reproducible infectious disease associations found with particular Mhc-B haplotypes. Sequence data for Mhc-B haplotypes have been lacking thereby hampering efforts to systematically resolve which genes within the Mhc-B region contribute to well-defined Mhc-B-associated disease responses. To better understand the genetic factors that generate and maintain genomic diversity in the Mhc-B region, we determined the complete genomic sequence for 14 Mhc-B haplotypes across a region of 59 kb that encompasses 14 gene loci ranging from BG1 to BF2. We compared the sequences using alignment, phylogenetic, and genome profiling methods. We identified gene structural changes, synonymous and non-synonymous polymorphisms, insertions and deletions, and allelic gene rearrangements or exchanges that contribute to haplotype diversity. Mhc-B haplotype diversity appears to be generated by a number of mutational events. We found evidence that some Mhc-B haplotypes are derived by whole- and partial-allelic gene conversion and homologous reciprocal recombination, in addition to nucleotide mutations. These data provide a framework for further analyses of disease associations found among these 14 haplotypes and additional haplotypes segregating and evolving in wild and domesticated populations of chickens.

Poultry production profile and expected future projection in Bangladesh

The aim of this paper is to review the current status of poultry production and its future prospects in Bangladesh, covering both rural and commercial poultry production. As an important sub-sector of livestock production, the poultry industry in Bangladesh plays a crucial role in economic growth and simultaneously creates numerous employment opportunities. The poultry industry, as a fundamental part of animal production, is committed to supplying the nation with a cheap source of good quality nutritious animal protein in terms of meat and eggs. Two main systems of poultry production are common in Bangladesh nowadays: commercial poultry production – where birds are kept in total confinement, and traditional scavenging or semi-scavenging poultry production. Approximately 20% of the protein consumed in Bangladesh originates from poultry. With the exception the dip in production due to the recent Avian Influenza outbreak, the growth of this industry in terms of standards of commercialization, is very rapid. A gap still exists between the requirement and supply of poultry meat and eggs within the recent frame-work of the informal marketing system that is currently used. Among poultry species, the chicken population is dominant over others, at almost 90%, followed by ducks (8%) and a small number of quail, pigeons and geese. Free range ‘backyard’ and scavenging poultry, that are traditionally reared by rural women and children, still play an important role in generating family income, in addition to improving the familys diet with eggs and meat. Productive and reproductive performance of indigenous birds is relatively very low (35–40 eggs and 1–1.5 kg meat per bird per year), but genetic improvements by selective breeding, along with adequate nutrition and proper management, looks promising and quite possible. Commercial poultry production in Bangladesh, is conducted on an industrial scale and is growing tremendously in spite of recent difficulties but is expected to make a significant contribution to the economic development of the country. A national poultry policy is expected to be approved by the government shortly, which, when implemented, will improve the organization of production and marketing, allowing increased stability and security of output throughout the year. In addition, efforts should be taken to ensure safety standards of poultry meat and eggs for human consumption. Experts from the government, research institutes, universities, NGOs and other relevant sectors need to work in a collaborative manner in order to allow sustainable production and fight challenges jointly when they appear from time to time. Corresponding attention to research and development will allow the poultry sector to flourish in Bangladesh. As government funding is limited, industrialists need to come forward either to establish their respective research facilities or to provide funds to universities and research institutes in order to undertake research works of national and international importance.

Indigenous naked neck chicken: a valuable genetic resource for Bangladesh.

The present review work aims at determining the potential usefulness of indigenous naked neck (INN) chicken (D. Nana) for poultry production in a hot-humid climate. INN chicken has good heat dissipation mechanism and well adaptive to harsh tropical environment and nutrition, and is highly resistant to disease and superior to indigenous full-feathered and exotic egg-type or exotic naked neck counterparts in terms of growth rate, egg production, egg quality and meat yield traits. It can produce double the standard number of eggs under improved nutrition and management conditions. Crossbreds of INN with exotic chicken can perform even better than that of exotic chicken in respect of productive and reproductive traits. Consumers prefer the meat and eggs of INN chickens for reasons of pigmentation, leanness, taste, firmness, and they are also used in special dishes. INN chicken prices are typically higher compared with those of products from exotic stocks. There are a very few published papers on the molecular aspects of INN chickens, although this is essential to determine genetic distance or relationship within or between INN chicken and indigenous full-feathered (IFF) varieties (D. nana) for future breeding plans. Therefore INN strains may be a promising and worthy genetic resource for the development of a breed or strain through selective or crossbreeding program suited to Bangladesh and in other countries where similar environments and socio-economic conditions exist. Thus, the present review provides genetic and performance information on INN chickens which may be useful for further improvement of tropical breeds.

Changes in the expression of gallinacins, antimicrobial peptides, in ovarian follicles during follicular growth and in response to lipopolysaccharide in laying hens (Gallus domesticus)

The aim of this study was to identify the types of gallinacin genes (GALs) expressed in ovarian follicles and to determine the changes in their expression during follicular growth and in response to lipopolysaccharide (LPS). Follicles at different stages of growth were collected from laying hens (n = 5) and LPS-injected hens (n = 3). The expression of GALs in the theca and granulosa layers was examined by semi-quantitative RT-PCR. The expression of GAL-1, -2, -7, -8, -10, and -12 in the theca layer and GAL-1, - 8, -10, and -12 in the granulosa layer was identified in white and yellow follicles. The expression of these genes was not changed in the theca and granulosa layers during follicular growth except for a decrease in that of GAL-1 in theca. The expression of GAL-1, -7, and -12 in the theca layer of the third largest follicles was increased in response to LPS at a dose of 1 mg/kg body weight and this increase was induced within 3 h and maintained until 12h postinjection. Granulosa layers did not respond to LPS until 12h injection. These results show that six and four types of GALs are expressed in the theca and granulosa layers of healthy follicles respectively, and their levels do not change with follicular growth except for GAL-1 in theca. Elevated levels of GAL-1, -7, and -12 expression in theca in response to LPS suggest that the theca cells expressing these GALs function to eliminate LPS-containing bacteria.

LH reduces proliferative activity of cumulus cells and accelerates GVBD of porcine oocytes

It has been reported that LH receptor (LHR) mRNA is not detected in cumulus cells of porcine cumulus-oocyte complexes (COCs) just after collection from small antral follicles. The present study showed that the formation of LHR in cumulus cells was up-regulated by the cultivation with 20 ng/ml FSH. When the newly synthesized receptors were stimulated by 1.0 microg/ml LH, significantly higher levels of cAMP and progesterone production in cumulus cells were observed as compared with those of COCs cultured with FSH. A loss of proliferative activity of cumulus cells was induced by the additional LH to FSH-containing medium; however, the inhibitory effect was overcome by progesterone receptor antagonist RU486. Furthermore, the addition of LH also accelerated ongoing GVBD in cumulus cells-enclosed oocytes. These results revealed that during in vitro meiotic maturation of porcine COCs, progesterone secreted by FSH- and LH-stimulated cumulus cells reduced proliferative activity of cumulus cells; the changes of cumulus cells might be involved in inducing meiotic resumption of porcine oocytes.

Identification of quantitative trait loci affecting shank length, body weight and carcass weight from the Japanese cockfighting chicken breed, Oh-Shamo (Japanese Large Game)

We performed a quantitative trait locus (QTL) analysis to map QTLs controlling shank length, body weight, and carcass weight in a resource family of 245 F2 birds developed from a cross of the large-sized, native, Japanese cockfighting breed, Oh-Shamo (Japanese Large Game), and the White Leghorn breed of chickens. Interval mapping revealed three significant QTLs for shank length on chromosomes 1, 4 and 24 at the experiment-wise 5% level, and a suggestive shank length QTL on chromosome 27 at the experiment-wise 10% level. For body weight two QTLs, one significant and the other suggestive, were identified on chromosomes 4 and 24, respectively. As expected, QTLs for carcass weight, which was highly correlated with body weight (r = 0.95), were detected at the same chromosomal locations as the detected body weight QTLs. Interestingly, the chromosomal locations containing these body weight and carcass weight QTLs coincided with those of two of the four shank length QTLs detected. No QTL with an epistatic interaction effect was discovered for any trait. The total contribution of all detected QTLs to genetic variance was 98.4%, 27.0% and 25.9% for shank length, body weight and carcass weight, respectively, indicating that most shank length QTLs have been identified but many body weight and carcass weight QTLs have been overlooked by the present analysis because of a low coverage rate of the 88 microsatellite markers used here (approximately 46% of the whole genome).

The ubiquitin ligase gene (WWP1) is responsible for the chicken muscular dystrophy

Chicken muscular dystrophy with abnormal muscle (AM) has been studied for more than 50 years, but the gene responsible for it remains unclear. Our previous studies narrowed down the AM candidate region to approximately 1 Mbp of chicken chromosome 2q containing seven genes. In this study, we performed sequence comparison and gene expression analysis to elucidate the responsible gene. One missense mutation was detected in AM candidate genes, while no remarkable alteration of expression patterns was observed. The mutation was identified in WWP1, detected only in dystrophic chickens within several tetrapods. These results suggested WWP1 is responsible for chicken muscular dystrophy.