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; a 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.
Luteinizing hormone (LH) regulates several ovarian functions. However, the luteoprotective mechanisms of LH involved in the maintenance of bovine corpus luteum (CL) function are not well understood. Since prostaglandin F2α (PGF), PGE2 and progesterone (P4) are well documented as antiapoptotic factors in the bovine CL, we hypothesized that LH protects the CL by stimulating the local production and action of PGF, PGE2 and P4. Cultured bovine luteal cells obtained at the mid-luteal stage (days 8–12 of the estrous cycle) were treated with LH (10 ng/ml), onapristone (OP: a specific P4 receptor antagonist, 100 μM) and indomethacin [INDO; a cyclooxygenase (COX) inhibitor, 100 μM] for 24 h. LH with and without OP significantly increased the mRNA and protein expressions of COX-2, PGF synthase and carbonyl reductase (P<0.05) but not the mRNA and protein expressions of COX-1 and PGE synthase in bovine luteal cells. In addition, these treatments significantly increased PGF and P4 production (P<0.05) but not PGE2 production. Luteal cell viability was significantly increased by LH alone (P<0.05), but LH-increased cell viability was reduced by LH in combination with INDO as well as OP (P<0.05). The overall results suggest that LH prevents luteal cell death by stimulating luteal PGF and P4 production and supports CL function during the luteal phase in cattle.
In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of Xist during the preimplantation period in both sexes. We found that this aberrant Xist expression could be impeded by deletion of Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of Xist RNA signals remained within single embryos. After implantation, the dysregulated Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.
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.
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.
Germ cell differentiation in reverse-sexed reproductive organs and interspecies germ line chimeras provides insight into the mechanism of germ cell development and represents a useful tool for conservation of endangered birds. We investigated the migration and survival capacity of male chicken primordial germ cells (PGCs) in female chicken embryos and in quail and Korean ring-necked pheasant embryos of both sexes. Interestingly, the PGCs were successfully reintroduced in all cases. Furthermore, the cells survived in the recipient gonads until hatching regardless of sex and species of the recipient. In the case of male recipient chickens, PGC-derived offspring were produced. However, the reverse-sexed female chickens, quails and pheasants of both sexes did not generate any male donor PGC-derived progeny. These results suggest that migration and survival circuits in chicken PGCs are conserved in both sexes and between avian species during embryonic development.
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.
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 E2). The mean LH concentrations and baseline levels of LH secretion in nor-BNI-injected, low E2-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 E2 treatment. These results suggest that Dyn is involved in the estrogen negative feedback regulation of pulsatile GnRH/LH release. The low E2 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.
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 E2 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.
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.
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.
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.
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.
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.
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.
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