The development of reproductive system in prepubertal gilts was affected not only by gonadotropins but also by other cytokines and growth factors. Recent studies have revealed that Neurotrophins (NTs) which are well known for their essential roles in neural system also expressed in mammalian reproductive organs and involved in the regulation of mammalian reproduction. Dietary energy level is an important factor affecting domestic animal reproductive performance including central effects on Hypothalamic Pituitary Gonadal (HPG) axis and local effects on gonads. In the present study, the expression of Neurotrophin-4 (NT-4) which is a member of NTs in reproductive tissues of prepubertal gilts including ovary, oviduct and uterus was studied by immunohistochemistry. The effects of dietary energy on NT-4 mRNA expression were also investigated by quantitative PCR. The results show that NT-4 was localized in all classes of follicles in ovaries of prepubertal gilts including primordial follicles, primary follicles and secondary follicles, NT-4 was localized in mucosal epithelial cells in oviducts and glandular epithelium and endometrial epithelium in uteri. Furthermore, different dietary energy levels altered mRNA expression level of NT-4 in ovaries, uteri and oviducts of prepubertal gilts. NT-4 mRNA expression level in ovaries and uteri was higher in high dietary energy group than that in mid and low-energy groups. In oviduct, the expression pattern of NT-4 mRNA among three different dietary energy groups is similar as that occurred in in ovaries and uteri but with more significant differences. These results suggest that NT-4 may be one of the signals that links metabolic status and neuroendocrine control of reproduction in pig.

The factor in the germline alpha (FIGLA) is a basic helix-loop-helix transcription factor that is essential for folliculogenesis and regulates expression of zona pellucida genes in mouse and human. But bovine homologue has not as yet been confirmed experimentally. Here, researchers present the first cDNA cloning and transcript expression analysis of the bovine FIGLA gene. Using RT-PCR and quantitative real-time PCR, researchers revealed that expression within adult cattle tissues is limited to the ovary. The researchers found that Germinal Vesicle (GV) oocytes, Metaphase II (MII) oocytes, 4 and 8 cell embryos, morula and blastocysts were all shown to express mRNA for FIGLA and expression were different in bovine oocytes and IVF embryos at different stages (p<0.05) with the highest expression in Germinal Vesicle (GV) oocytes and lower expression in 8 cells and blastocysts embryos. There was no difference in mRNA levels for FIGLA gene among bovine IVF, PA (Parthenogenetic Activated) and NT (Nuclear Transfer) blastocysts (p>0.05). In situ hybridization the FIGLA RNA was only localized in ovarian follicle. The persistence of FIGLA in adult cowes suggests that it may regulate additional pathways that are essential for bovine ovarian and embryonic development.

The expression and localization of neurotrophin 4 (NT4) and its receptor, tyrosine kinase B (TRKB), in the bovine oviduct, and their interaction with gonadotrophins in bovine oviduct epithelial cells (BOECs), were examined. Transcripts for NT4 and TRKB were detected by reverse transcription polymerase chain reaction (RT-PCR) in bovine oviducts in the follicular and luteal phases, and their proteins were immunolocalized in BOECs. Based on real time PCR, NT4 mRNA did not differ significantly between the two phases of the cycle, although TRKB mRNA expression was higher (P < 0.05) in the luteal phase than that in follicular phase. The BOECs were treated with various concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in vitro; for NT4, mRNA and protein were higher (P < 0.05) than those in the control (based on real time PCR and enzyme-linked immunosorbent assay (ELISA) assays). The effects of NT4 and the TRKB inhibitor (K252a) on the expression of LH receptor (LHR) and FSH receptor (FSHR) in the oviduct epithelial cells were also studied using a monolayer culture model. Expression levels of LHR and FSHR mRNA in BOECs treated with various concentrations of NT4 were higher (P < 0.05) than those in the control. However, these expressions were blocked by treatment with K252α. We concluded that neurotrophin 4 may have a role in regulating the function of bovine oviducts by interacting with gonadotrophins.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) performs essential roles in the regulation of follicular development and the level at which it is expressed may reflect the steroidogenic capacity of follicles and their physiological status. The aim of this study was to investigate the expression of 3 beta-HSD protein and mRNA in normal follicles categorized by size into small (2 to 4 mm), medium (5 to 7 mm), large (8 to 10 mm) and cystic follicles (> 21 mm). The expression levels of 3 beta-HSD protein and mRNA were evaluated by western blotting and quantitative real-time PCR, respectively; whereas, the immunolocalization of 3 beta-HSD was examined in normal and cystic follicles. The results indicated that the expression of 3 beta-HSD protein and mRNA increased as follicles grew. High levels of 3 beta-HSD protein and mRNA (P < 0.01) were found in cystic follicles. Immunoreactions of 3 beta-HSD were localized in the theca cells in the normal and cystic follicles, with reactions indicating 3 beta-HSD also to be present in the granulosa cells of the cystic follicles, but to be less evident in or absent from the granulosa cells of normal follicles. Our results revealed that the expression and localization of 3 beta-HSD differed in the cystic follicles and normal follicles and suggest that 3 beta-HSD in cystic follicles plays an essential role in the formation and persistence of cysts. Our findings provide important additional insights into the pathogenesis of follicular cysts in sows.

3β-Hydroxysteroid dehydrogenase (3β-HSD) performs essential roles in the regulation of follicular development and the level at which it is expressed may reflect the steroidogenic capacity of follicles and their physiological status. The aim of this study was to investigate the expression of 3β-HSD protein and mRNA in normal follicles categorized by size into small (2 to 4 mm), medium (5 to 7 mm), large (8 to 10 mm) and cystic follicles (> 21 mm). The expression levels of 3β-HSD protein and mRNA were evaluated by western blotting and quantitative real-time PCR, respectively; whereas, the immunolocalization of 3β-HSD was examined in normal and cystic follicles. The results indicated that the expression of 3β-HSD protein and mRNA increased as follicles grew. High levels of 3β-HSD protein and mRNA (P < 0.01) were found in cystic follicles. Immunoreactions of 3β-HSD were localized in the theca cells in the normal and cystic follicles, with reactions indicating 3β-HSD also to be present in the granulosa cells of the cystic follicles, but to be less evident in or absent from the granulosa cells of normal follicles. Our results revealed that the expression and localization of 3β-HSD differed in the cystic follicles and normal follicles and suggest that 3β-HSD in cystic follicles plays an essential role in the formation and persistence of cysts. Our findings provide important additional insights into the pathogenesis of follicular cysts in sows. Key words : 3β-Hydroxysteroid dehydrogenase (3β-HSD), expression, follicular cysts, sows.

1. The goose major histocompatibility complex (MHC) class IIB cDNA (Ancy-MHCII) was cloned by homology cloning and rapid amplification of cDNA ends by polymerase chain reaction (RACE-PCR), and the genomic structure and tissue expression were investigated. 2. Three different 5′-RACE sequences (Ancy-MHC II5′-1, Ancy-MHC II5′-2, Ancy-MHC II5′-3), one 3′-RACE sequence (Ancy-MHC II-3′) and two different full length Ancy-MHC IIB cDNA sequences (Ancy-CD01, Ancy-CD02), which came from different alleles at one locus or different loci, were determined. 3. The genomic organisation is composed of 6 exons and 5 introns, with a longer intron region than that of the chicken. The alleles encode 259 and 260 amino acids in the mature protein. 4. The number of non-synonymous substitutions (dN) in the peptide-binding region of exon 2 from 8 alleles was higher than that of the synonymous substitutions (dS). 5. Tissue-specific expression of Ancy-MHC II mRNA was detected in an adult goose using RT-PCR. These results showed that Ancy-MHC II mRNA was expressed in the lung, spleen, liver, intestine, heart, kidney, pancreas, brain, skin and muscle. This is consistent with the expression of MHC class IIB in various tissues from the chicken. 6. Sequences from goose, snipe and duck clustered together when compared with known MHC class IIB sequences from the other species, significantly differing from mammals and aquatic species, indicating a pattern consistent with accepted evolutionary pathways.

Neurotrophins (NTs) belong to a family of soluble homodimeric polypeptide growth factors and are widely recognized for their essential roles in central and peripheral nervous systems. One such neurotrophin, Brain-Derived Neurotrophic Factor (BDNF) was originally described in the nervous system but has now been shown to be expressed in reproductive system. In this study, the researchers examined the presence and different expression levels of BDNF mRNA in porcine reproductive organs during different stages of estrous cycle and in pig oocytes in Germinal Vesicle (GV) and in vitro matured Metaphase II (MII) stage. In oviduct and uterus, BDNF mRNA expression was higher than that in ovary (p<0.05). The expression level in luteal phase ovary is higher than that in follicular phase ovary but the difference between them was not significant (p>0.05), a similar but more significant change occurred in oviduct (p<0.05). However, the expression levels in uterus were on an opposite trend i.e., a higher level of mRNA for BDNF was found in follicular phase uterus instead of in luteal phase uterus. BDNF mRNA was also detected in GV oocytes and in vitro matured MII oocytes with significantly higher amounts in GV oocytes than in MII oocytes (p<0.01). These results suggest a possible role for BDNF in the regulation and modulation of pig reproductive function and oocyte maturation.

The neurotrophins, required for the survival and differentiation of the nervous system, are known to be important for the development of the reproductive tissues.However, the signals initiating the growth of follicles, gamete development, and transport and the development of zygote in the reproductive system of cows remain ambiguous.The purpose of the present study was to identify the transcripts and proteins of Neurotrophin 4 (NT4) and its receptor tyrosine kinase B (TrkB) in bovine reproductive tissues.The transcripts and immunoreactivity of NT4 and TrkB proteins were detected by reverse transcription polymerase chain reaction and western blot analysis.Using immunohistochemistry, the specific immunoreactivity of NT4 and TrkB were detected in the oocytes of primordial follicles and in the growing primary follicles.The NT4 and TrkB immunoreactivity was predominantly observed in granulosa cells, cumulus granulosa cells, cumulus oocyte complexes, theca cells of mature follicles, as well as in the oviduct epithelial cells, uterine gland cell, and epithelium cells of the uterus during the follicular and luteal phases in cows.Expressions of NT4 and TrkB mRNAs were not significantly different among the ovary, oviduct, and uterus of the follicular phase.For the luteal phase, the expression of NT4 mRNA in the ovary was significantly higher than that in the oviduct and uterus, and the expression of TrkB mRNA in the oviduct was significantly higher than that in the ovary and uterus, as determined by fluorescence quantitative reverse transcription polymerase chain reaction.The expression of NT4 mRNA was significantly higher than that of TrkB mRNA in the ovary and uterus, whereas NT4 mRNA expression was lower than that of TrkB mRNA in the oviduct during the luteal phase.The present study hypothesizes that NT4 participates in the regulation of both gonads and extra-gonadal reproductive tissues in cows.(

在DMEM培养液中分别添加不同质量浓度的脑源性神经生长因子(Brain-derived neurotrophic factor,BD NF)和神经生长因子(Nerve growth factor,NGF),观察其对牛颗粒细胞体外生长的影响.结果显示:在DMEM中添加20μg/L的BDNF,可促进牛颗粒细胞的体外生长;在DMEM中添加5μg/L的NGF,可促进牛颗粒细胞的体外生长.结果表明,在DMEM培养液中添加一定质量浓度的NGF和BDNF可促进牛颗粒细胞的体外生长.

Accumulating of proline in plant subjected to adverse environment is one of the most important adaptations,which can reduce damage on plant.Proline metabolism,and proline functions in plants encountered with adverse environments were summarized in this paper.The consentaneous conclusion indicated that the expression of P5CS gene was up-induced,and the increasing expression of proline synthetase gene could help for increasing the proline accumulation in plants,which could improve plant tolerance to adverse circumstance.The study was to determine effective methods of sample preparation of two-dimensional electrophoresis of follicular fluid in sows,to improve its resolution and reproducibility.The sample were depleted the high abundant proteins by ProteoExtract Albumin/IgG Remove Kit,and then concentrated by Acetone precipitation,TCA-acetone precipitation method and ultrafiltration method to compare the effect on electrophoresis.Sample prepared by TCA-acetone precipitation,obtained more and clearer protein spots,and high resolution and better reproducibility of 2-DE maps than that of others.It proved that TCA-acetone precipitation is suitable for preparing the samples of follicular fluid in sows,which could provide a foundamental guarantee for further analysis of 2-DE maps and finding biomarkers.

In order to understand the variations of genomic organization of the major histocompatibility complex (MHC) and provide data for the studies on disease resistance of avian species, the MHC class II polymorphism in Chinese Z-goose was investigated for the first time in the present study. Eight alleles, which probably came from different loci, were found in six different geese with only one obvious band in the restriction fragment length polymorphism data. The numbers of nonsynonymous substitutions (dN) in peptide binding region of exon 2 were higher than that of synonymous substitutions (dS), and no stop codons or frameshift mutations were found in this region, indicating that balance selection was in operation, and the sequences are not likely to be pseudogenes. In addition, we successfully obtained five different long MHC class II fragments (about 1,162 bp) in six geese and found that the length of intron 1 was longer than that in chicken and some other birds, but intron 2 seemed to be intermediate in length. The phylogenetic tree appeared to branch in an order consistent with accepted evolutionary pathway.

In order to clarify the function of protein in follicular fluids from sows during follicular development, we detected the changes of total protein content in different diameter follicles. The experiments were separated into four groups of small follicles(3~5 mm diameter), middle follicles(5~8 mm diameter), large follicles(8～10 mm diameter), and cystic follicles(diameter 21mm). Follicular fluids were collected from different diameter of the follicles with syringe. The protein samples in follicular fluids were extracted by the method of TCA/acetone pecipitation and quantificated by ProtaQuant Assay Kit. The mean concentrations of protein for the fluids were 4 440 μg/mL for small follicles (3~5 mm diameter), 976 μg/mL for middle follicles (5~8 mm diameter), 686 μg/mL for large follicles (8～10 mm diameter), and 537.2 μg/mL for cystic follicles (diameter 21mm). The results indicated that total protein concentrations in follicular fluids of 3~5 mm were significantly higher than those of other groups (P 0.01);there were no differences among other groups (P 0.05);the concentrations of cystic fluids were lowest. Variability in the concentrations of protein within follilces suggested that total protein content was decreased as follicle diameter increased. The change of protein concentrations may be related to the follicle maturation. Lower protein concentration in cystic fluids would give new theoretical foundation to the pathology and etiology of ovarian follicular cysts.

To explore the effects of long-term treatment of different dietary energy levels on ovarian expression of mRNAs for LHR and FSHR,the present study was performed in nine growth-matched littermate crossbred (Land-race×Large White X Duroc) prepubertal gilts. At approximately 30 kg of body weight and 50 day of age,gilts were housed with individual feeding stalls and placed on a normal level of feeding for 90 days (dl-90) with free ac-cess to water and food throughout the whole research. From d91 ,littermates were divided and randomly assigned to one of the following three treatment lines for 3 weeks till d112:Group H,Group M, and Group L, fed the high energy level diet (n = 3, digestible energy 14.87 MJ/kg), moderate energy level diet (n = 3, digestible energy 12.39 MJ/ kg), and low energy level diet (n = 3, digestible energy 9.98 MJ/kg), respectively. When gilts were slaughtered on d112 after 3 weeks energy treatment, both ovaries of every gilts were collected,snap frozen in liquid nitrogen and re-tained at -80℃ for use to determine and analysis the relative amount of ovarian LHR and FSHR mRNAs using semi-quantitative RT-PCR. Results showed that the effect of dietary energy treatment was notable: the ovarian ex-pression of mRNAs for LHR and FSHR was significantly higher (P<0.05) in gilts treated with high energy diet compared to gilts treated with moderate and low energy diets, while gilts treated with low energy diet had a signifi-cantly lower (P<0.05) ovarian LHR and FSHR expression compared with gilts treated with moderate and high en-ergy diets. These results revealed that ovarian expression of I.HR and FSHR in prepubertal gilts increased as the lev-el of dietary energy intake elevated,i, e. , high energy diet can markedly enhance ovarian expression of mRNAs for LHR and FSHR,whereas energy deficit markedly suppress the expression.

AbstractNine prepubertal gilts (JunMu No. 1) were randomly allocated into three groups (n=3) and fed with a high-energy diet (Group H), a low-energy diet (Group L), or a moderate-energy diet (Group M) for 14 days. Free access to water was provided throughout the research period. Ovaries and uteri were collected after the energy treatments, and processed for determination of the absolute quantities of insulin-like growth factor receptor (IGF-1R) and epidermal growth factor receptor (EGFR) mRNA, using real-time polymerase chain reaction (PCR). The expression of IGF-1R and EGFR mRNA in ovaries and uteri was significantly ranked as: Group H>Group M>Group L (P<0.05). This result suggests that high energy intake markedly enhanced the ovarian and uterine expression of IGF-1R and EGFR in prepubertal gilts, whereas insufficient energy intake markedly inhibited such expression. IGF-1R and EGFR may be involved in mediating the effects of energy intake on the development of the reproductive system in prepubertal gilts.

In livestock production,there is a high incidence of intrauterine growth retardation(IUGR),which may increas the neonatal mortality and impact the postnatal growth and health.This paper reviews the main factors causing IUGR.Understanding the causes of IUGR is of great significance for reducing the incidence and improving livestock production level.

The FQ-PCR technique was used to study the expression of FSHR mRNA in adult boar testes and epididymis.The results showed that FSHR mRNA was expressed in testis and epididymis.The level of FSHR mRNA in testis((1.66±0.14)×107 copies/ml) was significantly higher than that in epididymis((3.54±0.25)×105 copies/ml)(P0.01).