Journal of Reproduction and Development

Publications in this journal

• Article: Evaluation of Zona Pellucida Function for Sperm Penetration during In Vitro Fertilization in Pigs.

  Fuminori Tanihara, Michiko Nakai, Hiroyuki Kaneko, Junko Noguchi, Takeshige Otoi, Kazuhiro Kikuchi
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  ABSTRACT: In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP- oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP- oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP- oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP- oocytes at 1-10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP- oocytes. Finally, we performed IVF using ZP- oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.
  Journal of Reproduction and Development 05/2013;
• Article: Comparison of Ethylene Glycol and Propylene Glycol for the Vitrification of Immature Porcine Oocytes.

  Tamás Somfai, Michiko Nakai, Fuminori Tanihara, Junko Noguchi, Hiroyuki Kaneko, Naomi Kashiwazaki, István Egerszegi, Takashi Nagai, Kazuhiro Kikuchi
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  ABSTRACT: Our aim was to optimize a cryoprotectant treatment for vitrification of immature porcine cumulus-oocyte complexes (COCs). Immature COCs were vitrified either in 35% ethylene glycol (EG), 35% propylene glycol (PG) or a combination of 17.5% EG and 17.5% PG. After warming, the COCs were in vitro matured (IVM), and surviving oocytes were in vitro fertilized (IVF) and cultured. The mean survival rate of vitrified oocytes in 35% PG (73.9%) was higher (P<0.05) than that in 35% EG (27.8%). Oocyte maturation rates did not differ among vitrified and non-vitrified control groups. Blastocyst formation in the vitrified EG group (10.8%) was higher (P<0.05) than that in the vitrified PG group (2.0%) but was lower than that in the control group (25.0%). Treatment of oocytes with 35% of each cryoprotectant without vitrification revealed a higher toxicity of PG on subsequent blastocyst development compared with EG. The combination of EG and PG resulted in 42.6% survival after vitrification. The maturation and fertilization rates of the surviving oocytes were similar in the vitrified, control and toxicity control (TC; treated with EG+PG combination without cooling) groups. Blastocyst development in the vitrified group was lower (P<0.05) than that in the control and TC groups, which in turn had similar development rates (10.7%, 18.1% and 23.3%, respectively). In conclusion, 35% PG enabled a higher oocyte survival rate after vitrification compared with 35% EG. However, PG was greatly toxic to oocytes. The combination of 17.5% EG and 17.5% PG yielded reasonable survival rates without toxic effects on embryo development.
  Journal of Reproduction and Development 05/2013;
• Article: Characterization of Alpha-fetoprotein Levels in Three Dolphin Species: Development of Sensitive Immunoassays for Analysis of the Pregnancy-associated Variations.

  Yuka Morita, Naoshi Hiramatsu, Toshiaki Fujita, Haruna Amano, Etsuko Katsumata, Kazutoshi Arai, Toshihide Iwasaki, Takashi Todo, Akihiko Hara
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  ABSTRACT: A single radial immunodiffusion (SRID) assay and a chemiluminescent immunoassay (CLIA) were initially developed for alpha-fetoprotein (AFP) of the striped dolphin. Utilizing these developed assays, we investigated pregnancy-associated changes in the levels of AFP in the sera of fetuses and pregnant females of three dolphin species; samples were either collected from captive individuals or obtained as fishery by-products. The concentrations of AFP in the fetal serum ranged from 419.0 to 2,026.3 µg/ml in the striped dolphin, 12.6 to 1,218.7 µg/ml (for an AFP equivalent; eqAFP) in the common bottlenose dolphin and 770.6 to 3,129.1 µg eqAFP/ml in the Risso's dolphin. AFP levels decreased with increased fetal size in fetuses over 20 cm in length. The concentrations of AFP in sera of pregnant females ranged from 7.18 to 8,068.7 ng/ml in the striped dolphin, 6.6 to 1,241.1 ng eqAFP/ml in the common bottlenose dolphin and 3.4 to 2,868.7 ng eqAFP/ml in the Risso's dolphin. The levels in most pregnant females were equal to or lower than those found in males and nonpregnant individuals, although a few pregnant females exhibited extremely high levels (in the range of hundreds to thousands of nanograms per milliliter). Such high levels of AFP were not observed during pseudopregnancy. To our knowledge, this is the first report on basal profiles for serum AFP levels in small odontocetes. The profiles indicated that AFP may play a significant role during embryonic development, although maternal levels do not appear to be a diagnostic biomarker for monitoring pregnancy.
  Journal of Reproduction and Development 05/2013;
• Article: Effects of Downregulating Oct-4 Transcript by RNA Interference on Early Development of Porcine Embryos.

  Nobuyuki Sakurai, Takashi Fujii, Tsutomu Hashizume, Ken Sawai
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  ABSTRACT: The objective of this study was to investigate the role of the POU family transcription factor, Oct-4, in the early development of porcine embryos. We attempted Oct-4 downregulation of porcine early embryos by RNA interference, and evaluated Oct-4 suppression of developmental competencies and gene transcripts in porcine embryos. Injection of specific siRNA resulted in a distinct decrease in Oct-4 mRNA and protein expression in porcine embryos until at least the morula stage. Although the porcine embryos injected with Oct-4 siRNA were able to develop to the morula stage, these embryos failed to form blastocysts. Gene transcripts of caudal-like transcription factor (Cdx2) and fibroblast growth factor 4 (Fgf4), which were involved in segregation of the trophectderm and functionalization of the inner cell mass, were unchanged by Oct-4 siRNA injection. Our results indicated that Oct-4 is an important factor for porcine embryos and, in particular, for the regulation of porcine blastocyst formation.
  Journal of Reproduction and Development 04/2013;
• Article: Timing of Puberty and Its Relationship with Body Growth and Season in Male Raccoons (Procyon lotor) in Hokkaido.

  Minami W Okuyama, Michito Shimozuru, Go Abe, Mariko Nakai, Mariko Sashika, Ken-Ichiro Shimada, Nobuhiro Takahashi, Daisuke Fukui, Ryohei Nakamura, Toshio Tsubota
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  ABSTRACT: The raccoon (Procyon lotor), indigenous to North America, has naturalized in Japan as an invasive alien species, having been introduced into the country in the 1970s. In Hokkaido, the northernmost island of Japan, feral raccoons have been increasing in number and spreading throughout the island. The age at the onset of puberty for raccoons is important for estimating individual lifetime reproductive success and population growth. The present study investigated the timing of and potential factors affecting the onset of puberty in male raccoons in Hokkaido. External characteristics and histology of testes were studied in 151 male feral raccoons and in 1 captive juvenile. For the majority of feral yearling raccoons, prepubertal development began in May, and spermatozoa production began in October prior to their second mating season. However, some larger juveniles attained puberty during the juvenile period. The captive juvenile, which was fed throughout the winter, attained puberty only 11 months after birth. These results suggest that if male raccoons can achieve enough body growth before the first mating season, puberty can be attained early. In both juveniles and yearlings, spermatozoa production was only observed after autumn. This timing coincided with the recrudescence of seasonally active spermatogenesis in adult males. Therefore, attaining puberty in male raccoons appears to require both adequate body nutrient development and several environmental factors that control seasonal testicular changes.
  Journal of Reproduction and Development 04/2013;
• Article: Selection of In Vitro-Matured Porcine Oocytes Based on Localization Patterns of Lipid Droplets to Evaluate Developmental Competence.

  Kou Hiraga, Yumi Hoshino, Kentaro Tanemura, Eimei Sato
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  ABSTRACT: Localization patterns of lipid droplets in the cytoplasm of porcine oocytes were evaluated as a novel marker for in vitro maturation (IVM) of oocytes with high developmental competence. Porcine oocytes were cultured in TCM-199, which is a complete synthetic medium, for 44 h at 38.5 C. Localization patterns were divided into 2 classes: lipid droplets localized uniformly in the whole cytoplasm (class I) and those that were centrally located (class II). After IVM in TCM-199, 60% of matured oocytes exhibited the class II pattern. To investigate the relation between the distribution of lipid droplets and the developmental rate of the oocyte, the developmental rates of class I and class II oocytes were compared after in vitro fertilization (IVF). Class II oocytes showed a significantly higher rate of blastocyst development than class I oocytes. These results suggest that porcine oocytes with high developmental competence can be selected based on the localization patterns of lipid droplets.
  Journal of Reproduction and Development 04/2013;
• Article: Expression of Glucocorticoid Receptor α and Its Regulation in the Bovine Endometrium: Possible Role in Cyclic Prostaglandin F2α Production.

  Mariko Kuse, Hwa-Yong Lee, Tomas J Acosta, Takuo Hojo, Kiyoshi Okuda
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  ABSTRACT: Cortisol (Cr), the most important glucocorticoid (GC), is well known to suppress uterine prostaglandin F2α (PGF) production. However, the details of the regulatory mechanisms controlling the cyclic changes in endometrial PGF production remain unclear. Here we investigated the expression of the GC receptor (GC-Rα), the actions of cortisol throughout the estrous cycle and the regulatory mechanism of GC-Rα in the bovine endometrium. The levels of GC-Rα protein were greater at the mid-luteal stage (Days 8-12) than at the other stages. Cr more strongly suppressed PGF production at the mid-luteal stage than at the follicular stage. GC-Rα expression was increased by progesterone (P4) but decreased by estradiol-17β (E2) in cultured endometrial stromal cells. The overall results suggest that ovarian steroid hormones control the cyclic changes in endometrial PGF production by regulating GC-Rα expression in bovine endometrial stromal cells.
  Journal of Reproduction and Development 04/2013;
• Article: Seasonal Changes in Immunoreactivity of Inhibin/Activin Subunits in the Epididymis of Wild Ground Squirrels (Citellus dauricus Brandt).

  Mengyuan Zhang, Xia Sheng, Rongbo Sun, Qinglin Li, Haolin Zhang, Jiao Zhou, Meiyu Xu, Qiang Weng, Gen Watanabe, Kazuyoshi Taya
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  ABSTRACT: The inhibin/activin subunits (α, βA and βB) have been found in epididymal tissue of many mammals, but there have been no data available for wild seasonal breeders so far. The aim of this study was to investigate the immunoreactivities of inhibin/activin α, βA and βB subunits in the epididymis of wild ground squirrels during the breeding and nonbreeding seasons. Immunohistochemistry and Western blotting were performed to detect the epididymal immunolocalizations and immunoreactivities of the three subunits. Strong immunostaining of α subunit was present in the interstitial part of the caput epididymis and epithelial parts of the corpus epididymis and cauda epididymis during the breeding season, whereas no α subunit was found in the nonbreeding season. βA and βB subunits were expressed in all cell types of the epithelium throughout the whole seasonal cycle, and immunostaining in the breeding season was likely stronger compared with that of the nonbreeding season. These results suggested that the epididymis might be a potential source of inhibin and activin in the wild male ground squirrel, and the secretion of epididymal inhibin and activin showed distinct seasonal changes. Furthermore, inhibin and activin might function as paracrine and/or autocrine factors that have an effect on the epididymis.
  Journal of Reproduction and Development 03/2013;
• Article: Downregulation of Lymphatic Vessel Formation Factors in PGF2α-induced Luteolysis in the Cow.

  Akane Nitta, Koumei Shirasuna, Sayo Nibuno, Heinrich Bollwein, Takashi Shimizu, Akio Miyamoto
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  ABSTRACT: Prostaglandin F2α (PGF2α) induces luteolysis in cows and causes infiltration of immune cells, which resembles inflammatory immune response. Since the general immune response is mediated by the lymphatic system, we hypothesized that luteolysis is associated with generation of an immune response that involves lymphatic vessels in the bovine corpus luteum (CL). The CL was obtained from Holstein cows at the mid-luteal phase (days 10-12, ovulation = day 0) by ovariectomy at various time points after PGF2α injection. Lymphatic endothelial cell (LyEC) marker, endothelial hyaluronan receptor 1 (LYVE1), levels decreased significantly 12 h after PGF2α injection. Podoplanin, another LyEC marker, decreased from 15 min after PGF2α injection. PGF2α also diminished mRNA expression of lymphangiogenic factors, such as vascular endothelial growth factor (VEGF) C, VEGFD and VEGF receptor 3 (VEGFR3). During PGF2α-induced luteolysis, the levels of mRNA expression of tumor necrosis factor α (TNFα; the major pro-inflammatory cytokine) and chemokine (C-X-C motif) ligand 1 (neutrophil chemokine) were increased. On the other hand, chemokine (C-C motif) ligand 21, which regulates outflow of immune cells from tissues via the lymphatic vessels during an immune response, was decreased. This study demonstrated that the lymphatic network in the CL is disrupted during luteolysis and suggests that during luteolysis, immune cells can induce a local immune response in the CL without using the lymphatic vessels.
  Journal of Reproduction and Development 03/2013;
• Article: Season- and Age-related Reproductive Changes Based on Fecal Androgen Concentrations in Male Koalas, Phascolarctos cinereus.

  Satoshi Kusuda, Hisashi Hashikawa, Masato Takeda, Hideki Ito, Atsushi Goto, Jun Oguchi, Osamu Doi
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  ABSTRACT: The purposes of the present study were to clarify age- and season- related androgen patterns, and to compare the reproductive physiology between Japanese captive koala populations and Australian populations. To measure fecal androgens, feces were collected from male koalas (4.2 to 13.8 years of age) kept in Japanese zoos. Fecal androgens were extracted with methanol from the lyophilized samples and determined by enzyme immunoassay using 4-androstene-3,17-dione antibody. Fecal androgen concentration in male koalas increased after sexual maturation and remained relatively high until old age. In the survey with the Japanese zoo studbook of koalas, copulation (conception) month showed a pyramid shape with a peak in March to June (60.7%) in koalas born and reared in Japanese zoos and from July to April with the highest concentration in September to January (69.7%) in Australian institutes. Japanese zoo koala populations have a characteristic physiological cycle adapted to Japan's seasonal changes. The suitable month of year for copulation or conception in Japan is diametrically opposed to that in Australia. Mean fecal androgen concentrations by month in the males born and reared in Japan indicated annual changes with the highest concentration in May and the lowest value in November. Fecal androgen analysis may be a noninvasive alternative tool to monitor circulating testosterone and may be helpful in understanding reproductive activity and physiology in male koalas.
  Journal of Reproduction and Development 03/2013;
• Article: Progesterone Profiles in the Caudal Vena Cava and Jugular Vein in Response to Pulsatile Luteinizing Hormone Stimulation Induced by GnRH Treatment During the Mid-luteal Phase in Lactating Dairy Cows.

  Natsumi Endo, Kiyosuke Nagai, Tomomi Tanaka, Hideo Kamomae
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  ABSTRACT: The aim of this study was to examine whether increased frequency of luteinizing hormone (LH) pulses influences luteal progesterone (P4) secretion by measuring progesterone concentrations at the secreted (caudal vena cava) and circulating levels (jugular vein) in lactating dairy cows. Cows received six intravenous administrations of 2.5 μg of GnRH (gonadorelin acetate, n=4) or 2 ml saline (n=3) at 1-h intervals on 12.4 ± 0.4 (mean ± SE) days after ovulation. Blood samples were collected from the caudal vena cava and jugular vein every 12 min for 12 h (6 h before and after treatment). During the 6 h after treatment, frequency of LH pulses (5.3 ± 0.3 and 3.0 ± 0.0 pulses/6 h) and mean LH concentration (0.50 ± 0.06 and 0.38 ± 0.05 ng/ml) were greater (P<0.05) in GnRH-treated cows than in saline-treated cows. Mean P4 concentration and amplitude of P4 pulses in the caudal vena cava during the 6 h after treatment were greater (P<0.05) in GnRH-treated cows than in saline-treated cows, but the frequency of P4 pulses was not different between the groups. Mean P4 concentration in the jugular vein during the 6 h after treatment was also higher (P<0.05) in GnRH-treated cows than in saline-treated cows (7.0 ± 1.3 and 5.4 ± 0.9 ng/ml). These results indicate that the increased frequency of LH pulses stimulates progesterone secretion from the functional corpus luteum and brings about higher P4 concentrations in the circulating blood in lactating dairy cows.
  Journal of Reproduction and Development 03/2013;
• Article: Vitreous Cryopreservation of Human Preantral Follicles Encapsulated in Alginate Beads with Mini Mesh Cups.

  Jiang Bian, Tao Li, Chenhui Ding, Weijie Xin, Bo Zhu, Canquan Zhou
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  ABSTRACT: To completely avoid ice crystal formation and thus get a higher survival rate, vitrification methods have been commonly used for cryopreservation of oocytes and embryos. However,currently used vitrification methods for oocytes and embryos are not suitable for the cryopreservation of preantral follicles (PFs). In the present study, stainless steel mesh was fabricated into mini mesh cups to vitrify isolated PFs. Moreover, isolated follicles were encapsulated and then subjected to vitreous cryopreservation to facilitate in vitro culture/maturation of follicles after warming. The results showed that the percentages of viable follicles did not differ significantly between the vitrification group and fresh group soon after warming (81.25% vs. 85.29%, P>0.05) and after a 7-day culture period (77.78% vs. 83.33%, P>0.05). No difference in mean follicular diameter was observed between cryopreserved and fresh follicles when cultured in vitro. Transmission electron microscopic analysis revealed that vitreous cryopreservation could maintain the ultrastructure of follicles in alginate beads. In conclusion, the present vitrification method could efficiently cryopreserve isolated human ovarian follicles encapsulated by calcium alginate, which could be put into immediate use (in vitro culture/ maturation) after warming. However, more follicles and some detailed biochemical analyses are required to further investigate the effects of vitrification on the long-term growth of human encapsulated PFs.
  Journal of Reproduction and Development 03/2013;
• Article: Nicotinamide: a Class III HDACi Delays In Vitro Aging of Mouse Oocytes.

  Ah Reum Lee, Satoshi Kishigami, Tomoko Amano, Kazuya Matsumoto, Teruhiko Wakayama, Yoshihiko Hosoi
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  ABSTRACT: Postovulatory mammalian oocyte developmental potential decreases with aging in vivo and in vitro. Aging oocytes typically show cellular fragmentation and chromosome scattering with an abnormally shaped spindle over time. Previously, it was shown that histone acetylation in the mouse oocyte increased during aging and that treatment with trichostatin A (TSA), an inhibitor for class I and II histone deacetylases (HDACs), enhanced the acetylation, that is, aging. In this study, we examined the effect of nicotinamide (NAM), an inhibitor for class III HDACs, on in vitro aging of mouse oocytes as well as TSA. We found that treatment with NAM significantly inhibited cellular fragmentation, spindle elongation and astral microtubules up to 48 h of culture. Although presence of TSA partially inhibited cellular fragmentation and spindle elongation up to 36 h of culture, treatment with TSA induced chromosome scattering at 24 h of culture and more severe cellular fragmentation at 48 h of culture. Further, we found that α-tubulin, a nonhistone protein, increased acetylation during aging, suggesting that not only histone but nonhistone protein acetylation may also increase with oocyte aging. Thus, these data indicate that protein acetylation is abnormally regulated in aging oocytes, which are associated with a variety of aging phenotypes, and that class I/II and class III HDACs may play distinct roles in aging oocytes.
  Journal of Reproduction and Development 03/2013;
• Article: Functional Analysis of Nocturnin, a Circadian Deadenylase, at Maternal-to-zygotic Transition in Mice.

  Satoshi Nishikawa, Yuki Hatanaka, Mikiko Tokoro, Seung-Wook Shin, Natsumi Shimizu, Takuji Nishihara, Rie Kato, Atsushi Takemoto, Tomoko Amano, Masayuki Anzai, Satoshi Kishigami, Yoshihiko Hosoi, Kazuya Matsumoto
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  ABSTRACT: Degradation of maternally stored mRNAs after fertilization is an essential process for mammalian embryogenesis. Maternal mRNA degradation depending on deadenylases in mammalian early embryos has been mostly speculated, rather than directly demonstrated. Previously, we found that gene expression of nocturnin, which functions as a circadian clock-controlled deadenylase in mammalian cells, was clearly changed during the maternal-to-zygotic transition (MZT). Here, we investigated the possible role of nocturnin during mouse MZT. First, we examined the expression profile and localization of nocturnin in mouse oocytes and early embryos. The abundance of Nocturnin mRNA level was significantly decreased from the MII to 4-cell stages and slightly increased from the 8-cell to blastocyst stages, whereas the Nocturnin protein level was almost stable in all examined cells including GV and MII oocytes and early embryos. Nocturnin was localized in both the cytoplasm and the nucleus of all examined cells. We then examined the effect of loss or gain of Nocturnin function on early embryonic development. Knockdown of Nocturnin by injection of Nocturnin antisense expression vector into 1-cell embryos resulted in the delay of early embryonic development to the early blastocyst stage. Moreover, Nocturnin-overexpressed embryos by injection of Nocturnin expression vector impaired their development from the 1-cell to 2-cell or 4-cell stages. These results suggest that precise expression of nocturnin is critical to proper development of early mouse embryos. Functional analysis of nocturnin may contribute to the understanding of the possible role of the deadenylase at mouse MZT.
  Journal of Reproduction and Development 03/2013;
• Article: Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos.

  Benson H Morrill, Lindsay Cox, Anika Ward, Sierra Heywood, Randall S Prather, S Clay Isom
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  ABSTRACT: The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx platform. The average depth of sequencing coverage was 14,611 for IVV and 17,068 for PA. Quantitative analysis of the methylation profiles of both input samples for each genomic locus showed distinct differences in methylation profiles between IVV and PA samples for six of the target loci, and subtle differences in four loci. It was concluded that high throughput sequencing technologies can be effectively applied to provide a powerful, cost-effective approach to targeted DNA methylation analysis of embryonic and other reproductive tissues.
  Journal of Reproduction and Development 02/2013;
• Article: Early Regression of Spermatogenesis in Boars of an Inbred Duroc Strain Caused by Incident Orchitis/Epididymo-orchitis.

  Junko Noguchi, Michiko Nakai, Kazuhiro Kikuchi, Hiroyuki Kaneko, Noriaki Imaeda
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  ABSTRACT: In the process of establishment of an inbred Duroc pig strain, males with size asymmetry of the testes were frequently observed. To clarify the possible causes of this asymmetry, we examined the testes and epididymides of 67 males of the F4-F7 generations at 35-100 weeks of age. Testicular weights showed a wide variation (120-610 g). When the weights of the testes were compared bilaterally, 35 of the 67 males showed more than a 10% difference. Histological examination of testes from this asymmetry group revealed a range of seminiferous tubule disruption including disappearance of all germ cells, but not Sertoli cells, in the epithelium. Focal lesions associated with the degenerated tubules were observed. Trends of incident fibrosis or hyalinization of these lesions were seen in aged males of the asymmetry group. Besides this abnormality of spermatogenesis, infiltration of mononuclear inflammatory cells around the tubule was frequently observed in the asymmetry group (32.9%, compared with 1.6% in males showing testis symmetry). In severe cases, the inflammatory cells were concentrated in the intertubular region instead of Leydig cells. Cellular infiltration was also observed around the epididymal duct and blood vessels, but its incidence did not differ between the symmetry and asymmetry groups. Testicular testosterone levels were significantly increased in the asymmetry group, but those of E(2) and inhibin did not differ between the two groups. These histopathological features indicate that disruption of spermatogenesis after orchitis/epididymo-orchitis could induce testicular atrophy. Genetic predispositions for this trait may cause prevalent retrograde infections, resulting in orchitis/epididymo-orchitis.
  Journal of Reproduction and Development 02/2013;
• Article: In Vitro Fertilization and Development of Porcine Oocytes Matured in Follicular Fluid.

  Budiyanto Agung, Takeshige Otoi, Dai-Ichiro Fuchimoto, Shoichiro Senbon, Akira Onishi, Takashi Nagai
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  ABSTRACT: This study was conducted to assess the fertilization and development of porcine oocytes matured in a solo follicular fluid (pFF) using different in vitro culture systems and insemination periods. Cumulus-oocyte complexes (COCs), follicular cells (FCs), and pFF were collected from the follicles of ovaries. The pFF was used as a maturation medium (MpFF) after supplementation with follicle stimulating hormone (FSH) and antibiotics. The COCs were matured in a 15 ml test tube containing 3.5 ml of MpFF with FCs (5.2 × 10(6) cells/ml; rotating culture system) or 2 ml of MpFF without FCs in a 35-mm petri dish (static culture system) for 44 to 48 h. After maturation culture, oocytes were co-incubated with frozen-thawed spermatozoa for 5 h and then cultured for 7 days. The total mean rates of sperm penetration, normal fertilization, male pronucleus (MPN) formation, cleavage, and development to the blastocyst stage of oocytes after insemination were significantly higher (P<0.01) in the rotating culture system than in the static culture system. In conclusion, compared with the static culture system, the rotating culture system is adequate for the production of developmentally competent porcine oocytes when MpFF is used as a maturation medium.
  Journal of Reproduction and Development 02/2013;
• Article: Expression Pattern Implicates a Potential Role for LRF in the Process of Implantation in Uteri and Development of Preimplantation Embryos in Mice.

  Yanzhou Yang, Yaping Jin, Amanda C Martyn, Pengfei Lin, Yujie Song, Fenglei Chen, Linyong Hu, Chenchen Cui, Xiao Li, Qian Li, Ray Lu, Aihua Wang
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  ABSTRACT: Luman/CREB3 recruitment factor (LRF or CREBRF) was identified as a regulator of Luman (or CREB3) that is involved in the unfolded protein response during endoplasmic reticulum stress. Luman is implicated in a multitude of functions ranging from viral infection and immunity to cancer. The biological function of LRF, however, is unknown. In this paper, we report that uteri of pregnant mice and embryos displayed enhanced LRF expression at all stages, and the expressed LRF was found to be localized specifically at implantation sites. On the other hand, uteri of mice induced for delayed implantation or pseudopregnant mice showed low levels of LRF expression, suggesting that LRF mediates uterine receptivity during implantation. Further, expression of LRF was found to be modulated by steroid hormones such as progesterone and estradiol. This study thereby identifies a potential role for LRF in the process of implantation in uteri and development of preimplantation embryos in mice.
  Journal of Reproduction and Development 02/2013;
• Article: Dynorphin-Kappa Opioid Receptor Signaling Partly Mediates Estrogen Negative Feedback Effect on LH Pulses in Female Rats.

  Mst Parvin Mostari, Nahoko Ieda, Chikaya Deura, Shiori Minabe, Shunji Yamada, Yoshihisa Uenoyama, Kei-Ichiro Maeda, Hiroko Tsukamura
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  ABSTRACT: Accumulating evidence suggests that the arcuate nucleus (ARC) kisspeptin/neurokinin B (NKB)/dynorphin (KNDy) neurons play a role in estrogen negative feedback action on pulsatile gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release. The present study aimed to determine if dynorphin (Dyn) is involved in estrogen negative feedback on pulsatile GnRH/LH release. The effect of the injection of nor-binaltorphimine (nor-BNI), a kappa-opioid receptor (KOR) antagonist, into the third cerebroventricle (3V) on LH pulses was determined in ovariectomized (OVX) adult female rats with/without replacement of negative feedback levels of estradiol (low E(2)). The mean LH concentrations and baseline levels of LH secretion in nor-BNI-injected, low E(2)-treated rats were significantly higher compared with vehicle-treated controls. On the other hand, the nor-BNI treatment failed to affect any LH pulse parameters in OVX rats without low E(2) treatment. These results suggest that Dyn is involved in the estrogen negative feedback regulation of pulsatile GnRH/LH release. The low E(2) treatment had no significant effect on the numbers of ARC Pdyn (Dyn gene)-,Kiss1- and Tac2 (NKB gene)-expressing cells. The treatment also did not affect mRNA levels of Pdyn and Oprk1 (KOR gene)in the ARC-median eminence region, but significantly increased the ARC kisspeptin immunoreactivity. These findings suggest that the negative feedback level of estrogen suppresses kisspeptin release from the ARC KNDy neurons through an unknown mechanism without affecting the Dyn and KOR expressions in the ARC. Taken together, the present result suggests that Dyn-KOR signaling is a part of estrogen negative feedback action on GnRH/LH pulses by reducing the kisspeptin release in female rats.
  Journal of Reproduction and Development 02/2013;
• Article: Induction of the Expressions of Antioxidant Enzymes by Luteinizing Hormone in the Bovine Corpus Luteum.

  Syota Kawaguchi, Ryosuke Sakumoto, Kiyoshi Okuda
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  ABSTRACT: Luteoprotective mechanisms of luteinizing hormone (LH) involved in the maintenance of bovine corpus luteum (CL) function have not been completely clarified. Since antioxidant enzymes are well documented as antiapoptotic factors in the CL of many mammals, we hypothesized that the luteoprotective action of LH is mediated by stimulating the local production and action of antioxidant enzymes. To test the above hypothesis, in the present study, we examined the mechanisms involved in the luteoprotective actions of LH. Cultured bovine luteal cells obtained from the CL at the mid-luteal stage (days 8-12 of the estrous cycle) were treated with LH (10 ng/ml), onapristone (OP; an specific progesterone receptor antagonist, 100 μM) and diethyldithiocarbamate [DETC; an inhibitor of superoxide dismutase (SOD), 100 μM] for 24 h. LH in combination with or without OP significantly increased the mRNA and protein expressions of manganese SOD (Mn-SOD) and catalase (CATA) and SOD activity. While LH alone significantly increased the mRNA and protein expressions of SOD containing copper and zinc (Cu,Zn-SOD), OP in combination with or without LH significantly decreased the mRNA and protein expressions of Cu,Zn-SOD. In addition, Cu,Zn-SOD, Mn-SOD and CATA mRNA expressions were higher at the mid luteal phase than the other luteal phases. LH in combination with DETC significantly decreased LH-increased cell viability. The overall results suggest that LH increases cell viability by LH-increased antioxidant enzymes, resulting in maintenance of CL function during the luteal phase in cattle.
  Journal of Reproduction and Development 02/2013;