Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. JMY is a critical nucleation-promoting factor (NPF); however, its role in the development of mammalian embryos is poorly understood. In the current study, we investigated the functional roles of the NPF JMY in porcine embryos. Porcine embryos expressed JMY mRNA and protein, and JMY protein moved from the cytoplasm to the nucleus at later embryonic developmental stages. Knockdown of JMY by RNA interference markedly decreased the rate of blastocyst development, validating its role in the development of porcine embryos. Furthermore, injection of JMY dsRNA also impaired actin and Arp2 expression, and co-injection of actin and Arp2 mRNA partially rescued blastocyst development. Taken together, our results show that the NPF JMY is involved in the development of porcine embryos by regulating the NPF-Arp2-actin pathway.
Krüppel-like protein Gli-similar 1 (GLIS1) is known as a direct reprogramming factor for the generation of induced pluripotent stem cells. The objective of this study was to investigate the role of GLIS1 in the preimplantation development of bovine embryos. GLIS1 transcripts in in vitro-matured oocytes and 1-cell to 4-cell stage embryos were detected, but they were either absent or at trace levels at the 8-cell to blastocyst stages. We attempted GLIS1 downregulation of bovine early embryos by RNA interference and evaluated developmental competency and gene transcripts, which are involved in zygotic gene activation (ZGA) in GLIS1-downregulated embryos. Injection of specific siRNA resulted in a distinct decrease in GLIS1 transcript in bovine embryos at the 4-cell stage. Although the bovine embryos injected with GLIS1-siRNA could develop to the 16-cell stage, these embryos had difficulty in developing beyond the 32-cell stage. Gene transcripts of PDHA1 and HSPA8, which are transcribed after ZGA, showed lower level in GLIS1 downregulated embryos. It is possible that GLIS1-downregulated embryos fail to initiate ZGA. Our results indicated that GLIS1 is an important factor for the preimplantation development of bovine embryos.
During mammalian spermatogenesis, spermatogenic cells undergo mitotic division and are subsequently divided into haploid spermatids by meiotic division, but the dynamics of sex chromosomes during spermatogenesis are unclear in vivo. To gain insight into the distribution of sex chromosomes in the testis, we examined the localization of sex chromosomes before and after meiosis in mouse testis sections. Here, we developed a method of fluorescence in situ hybridization (FISH) using specific probes for the X and Y chromosomes to obtain their positional information in histological testis sections. FISH analysis revealed the sex chromosomal position during spermatogenesis in each stage of seminiferous epithelia and in each spermatogenic cell. In the spermatogonia and leptotene spermatocytes, sex chromosomes were distantly positioned in the cell. In the zygotene and pachytene spermatocytes at prophase I, X and Y chromosomes had a random distribution. After meiosis, the X and Y spermatids were random in every seminiferous epithelium. We also detected aneuploidy of sex chromosomes in spermatogenic cells using our developed FISH analysis. Our results provide further insight into the distribution of sex chromosomes during spermatogenesis, which could help to elucidate a specific difference between X and Y spermatids and sex chromosome-specific behavior.
Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival.
Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.
An experimental ischemia (EI)-induced mouse model was used to analyze pathological and biochemical alterations in testes. Initial morphological changes were observed in Sertoli cells of EI testes at the light microscopic level. Examination of the ultrastructure using transmission electron microscopy confirmed that Sertoli cells were partially detached from the basement membrane of the seminiferous epithelium and that the cell membranes of adjacent Sertoli cells were not joined. The functional integrity of the blood-testis barrier (BTB) was assessed using the lanthanum tracer technique. Lanthanum had penetrated into the spaces between adjacent Sertoli cells in the adluminal compartment up to the lumen of the seminiferous epithelium in EI testes. Proteome analysis showed that the expression of heat shock protein (HSP) 70 was significantly upregulated in EI testes. Western blot analysis confirmed that the expression of HSP70 increased in a time-dependent manner after the EI procedure. HSP70 immunostaining was observed in spermatocytes and in round and elongated spermatids in EI testes. Our results suggest that a change in the junctions between adjacent Sertoli cells on the basal compartment is involved in the BTB disruption in EI testes. Therefore, male infertility caused by the BTB disruption could be associated with heat stress induced by ischemia.
Once deposited in the female tract, sperm face a series of challenges that must be overcome to ensure the presence of an adequate normal sperm population close to the site of fertilization. Our aim was to evaluate the influence of the uterine milieu on boar sperm morphology. In experiment 1, sperm morphology was evaluated in the backflow (60 min after insemination) and within the uterotubal junction (UTJ) (collected ~24 h after insemination) following intrauterine sperm deposition (n = 6) and compared with the morphology of the sperm in the insemination dose. In experiment 2, the influence of the uterine fluid (UF) on sperm morphological modifications was evaluated. For this purpose, ejaculated (n = 4) and epididymal (n = 4) sperm were in vitro incubated with or without UF for 2 and 24 h. In both experiments, sperm were classified as normal, having a cytoplasmic droplet (proximal or distal) or having tail defects. The results of experiment 1 pointed to an increase in morphologically abnormal sperm collected in the backflow (27.70%) and a reduction of the same in the UTJ (2.12%) compared with the insemination dose (17.75%) (P < 0.05). In experiment 2, incubation of ejaculated sperm with UF did not provoke any morphological modifications; however, when epididymal sperm were incubated with UF, a pronounced increase in the percentage of normal sperm was evident after 24 h compared with the initial dose (from 25.77% to 53.58%, P < 0.05), mainly due to distal cytoplasmatic droplet shedding (53.22 vs. 20.20%). In conclusion, almost all the sperm that colonize the UTJ had a normal morphology, with part of the abnormal sperm having been discarded in the backflow and part selected/modified on their way to the oviduct. UF seems to influence cytoplasmic distal droplet removal, as demonstrated previously in seminal plasma.
The structure and functions of placentas were examined in 3 species of rorqual whales, common minke (Balaenoptera acutorostrata), Bryde’s (B. brydei) and sei (B. borealis) whales, with the aim of confirming the structural characteristics of the chorion, including the presence of the areolar part, and clarifying steroidogenic activities and fetomaternal interactions in the placentas of these whales. Placentas were collected from the second phase of the Japanese Whale Research Program under Special Permit in the North Pacific (JARPN II). Histological and ultrastructural examinations revealed that these whale placentas were epitheliochorial placentas with the interdigitation of chorionic villi lined by monolayer uninucleate cells (trophoblast cells) and endometrial crypts as well as folded placentation by fold-like chorionic villi. Moreover, well-developed pouch-like areolae were observed in the placentas, and active absorption was suggested in the chorionic epithelial cells of the areolar part (areolar trophoblast cells). Berlin blue staining showed the presence of ferric ions (Fe3+) in the uterine glandular epithelial cells and within the stroma of chorionic villi in the areolar part. An immunohistochemical examination revealed tartrate-resistant acid phosphatase (TRAP; known as uteroferrin in uteri) in the cytoplasm of glandular cells and areolar trophoblast cells. This result suggested that, in cetaceans, uteroferrin is used to supply iron to the fetus. Furthermore, immunoreactivity for P450scc and P450arom was detected in trophoblast cells, but not in areolar trophoblast cells, suggesting that trophoblast cells synthesize estrogen in whale placentas. Therefore, we herein immunohistochemically revealed the localization of aromatase and uteroferrin in cetacean placentas during pregnancy for the first time.
In this study, the effect of heat shock on frozen-thawed blastocysts was evaluated using in vitro-produced (IVP) bovine embryos. In experiment 1, the effects of 6 h of heat shock at 41.0 C on fresh blastocysts were evaluated. HSPA1A expression as a reflection of stress was increased by heat shock (P < 0.05), but the expressions of the quality markers IFNT and POU5F1 were not affected. In experiment 2, frozen-thawed blastocysts were incubated at 38.5 C for 6 h (cryo-con) or exposed to heat shock at 41.0 C for 6 h (cryo-HS). Then, blastocysts were cultured at 38.5 C until 48 h after thawing (both conditions). Cryo-HS blastocysts exhibited a decreased recovery rate: HSPA1A expression was dramatically increased compared with that in fresh or cryo-con blastocysts at 6 h, and IFNT expression was decreased compared with that in cryo-con blastocysts at 6 h (both P < 0.05). Cryo-con blastocysts at 6 h also exhibited higher HSPA1A expression than fresh blastocysts (P < 0.05). At 48 h after thawing, the number of hatched blastocysts and blastocyst diameter were lower in cryo-HS blastocysts (P < 0.05). Cryo-con blastocysts showed lower POU5F1 levels at 48 h than fresh, cryo-con or cryo-HS blastocysts at 6 h (P < 0.05), but their POU5F1 levels were not different from those of cryo-HS blastocysts at 48 h. These results indicated that application of heat shock to frozen-thawed blastocysts was highly damaging. The increase in damage by the interaction of freezing-thawing and heat shock might be one reason for the low conception rate in frozen-thawed embryo transfer in summer.
This study was designed to compare the efficiency of the Cryotop method and that of two methods that employ a micro volume air cooling (MVAC) device by analyzing the survival and development of bovine oocytes and blastocysts vitrified using each method. In experiment I, in vitro-matured (IVM) oocytes were vitrified using an MVAC device without direct contact with liquid nitrogen (LN2; MVAC group) or directly plunged into LN2 (MVAC in LN2 group). A third group of IVM oocytes was vitrified using a Cryotop device (Cryotop group). After warming, vitrified oocytes were fertilized in vitro. There were no significant differences in cleavage and blastocyst formation rates among the three vitrified groups, with the rates ranging from 53.1% to 56.6% and 20.0% to 25.5%, respectively; however, the rates were significantly lower (P < 0.05) than those of the fresh control group (89.3% and 43.3%, respectively) and the solution control group (87.3% and 42.0%, respectively). In experiment II, in vitro-produced (IVP) expanded blastocysts were vitrified using the MVAC, MVAC in LN2 and Cryotop methods, warmed and cultured for survival analysis and then compared with the solution control group. The rate of development of vitrified-warmed expanded blastocysts to the hatched blastocyst stage after 24 h of culture was lower in the MVAC in LN2 group than in the solution control group; however, after 48–72 h of culture, the rates did not significantly differ between the groups. These results indicate that the MVAC method without direct LN2 contact is as effective as the standard Cryotop method for vitrification of bovine IVM oocytes and IVP expanded blastocysts.
The corpus luteum (CL) is a temporary endocrine gland producing a large amount of progesterone, which is essential for the establishment and maintenance of pregnancy. Galectin-1 is a β-galactose-binding protein that can modify functions of membrane glycoproteins and is expressed in the CL of mice and women. However, the physiological role of galectin-1 in the CL is unclear. In the present study, we investigated the expression and localization of galectin-1 in the bovine CL and the effect of galectin-1 on cultured luteal steroidogenic cells (LSCs) with special reference to its binding to the glycans on vascular endothelial growth factor receptor-2 (VEGFR-2). Galectin-1 protein was highly expressed at the mid and late luteal stages in the membrane fraction of bovine CL tissue and was localized to the surface of LSCs in a carbohydrate-dependent manner. Galectin-1 increased the viability in cultured LSCs. However, the viability of LSCs was decreased by addition of β-lactose, a competitive carbohydrate inhibitor of galectin-1 binding activity. VEGFR-2 protein, like galectin-1, is also highly expressed in the mid CL, and it was modified by multi-antennary glycans, which can be recognized by galectin-1. An overlay assay using biotinylated galectin-1 revealed that galectin-1 directly binds to asparagine-linked glycans (N-glycans) on VEGFR-2. Enhancement of LSC viability by galectin-1 was suppressed by a selective inhibitor of VEGFR-2. The overall findings suggest that galectin-1 plays a role as a survival factor in the bovine CL, possibly by binding to N-glycans on VEGFR-2.
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are new tools for producing gene knockout (KO) animals. The current study reports produced genetically modified pigs, in which two endogenous genes were knocked out. Porcine fibroblast cell lines were derived from homozygous α1,3-galactosyltransferase (GalT) KO pigs. These cells were subjected to an additional KO for the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene. A pair of ZFN-encoding mRNAs targeting exon 8 of the CMAH gene was used to generate the heterozygous CMAH KO cells, from which cloned pigs were produced by somatic cell nuclear transfer (SCNT). One of the cloned pigs obtained was re-cloned after additional KO of the remaining CMAH allele using the same ZFN-encoding mRNAs to generate GalT/CMAH-double homozygous KO pigs. On the other hand, the use of TALEN-encoding mRNAs targeting exon 7 of the CMAH gene resulted in efficient generation of homozygous CMAH KO cells. These cells were used for SCNT to produce cloned pigs homozygous for a double GalT/CMAH KO. These results demonstrate that the combination of TALEN-encoding mRNA, in vitro selection of the nuclear donor cells and SCNT provides a robust method for generating KO pigs.
Two studies were conducted with the overarching goal of determining the extent to which lipolytic changes relate to germinal vesicle breakdown (GVBD) in bovine oocytes matured under thermoneutral or hyperthermic conditions. To this end, cumulus-oocyte complexes underwent in vitro maturation for 0, 2, 4, 6 or 24 h at 38.5 (first study) or 38.5 and 41.0 C (second study; heat stress applied up through first 12 h only, then shifted to 38.5 C). Independent of maturation temperature, triglyceride and phospholipid content decreased markedly by 2 h of in vitro maturation (hIVM; P < 0.0005). Content was lowest at 24 hIVM with no detectable impact of heat stress when exposure occurred during first 12 hIVM. Germinal vesicle breakdown occurred earlier in oocytes experiencing heat stress with effects observed as soon as 4 hIVM (P < 0.0001). Germinal vesicle breakdown was associated with lipolytic changes (R2 = 0.2123 and P = 0.0030 for triglyceride content; R2 = 0.2243 and P = 0.0026 for phospholipid content). ATP content at 24 hIVM was higher in oocytes experiencing heat stress (P = 0.0082). In summary, GVBD occurs sooner in heat-stressed oocytes. Although marked decreases in triglyceride and phospholipid content were noted as early as 2 hIVM and preceded GVBD, lipolytic changes such as these are not likely serving as an initial driver of GVBD in heat-stressed oocytes because changes occurred similarly in oocytes matured at thermoneutral conditions.
This study compared the responses shown by lactating dairy cows to four different P4-based protocols for AI at estrus. Cows with no estrous signs 96 h after progesterone intravaginal device (PRID) removal were subjected to fixed-time AI (FTAI), and their data were also included in the study. In Experiment I, follicular/luteal and endometrial dynamics were assessed every 12 h from the beginning of treatment until AI. The estrous response was examined in Experiment II, and fertility was assessed in both experiments. The protocols consisted of a PRID fitted for five days, along with the administration of different combinations of gonadotropin releasing hormone (GnRH), equine chorionic gonadotropin and a single or double dose (24 h apart) of prostaglandin F2α. In Experiment I (40 cows), animals receiving GnRH at the start of treatment showed a significantly higher ovulation rate during the PRID insertion period while estrus was delayed. In Experiment II (351 cows), according to the odds ratios, cows showing luteal activity at the time of treatment were less likely to show estrus than cows with no signs of luteal activity. Treatment affected the estrous response and the interval from PRID removal to estrus but did not affect conception rates 28–34 days post AI. Primiparous cows displayed a better estrous response than multiparous cows. Our findings reveal acceptable results of 5-day P4-based protocols for AI at estrus in high-producing dairy cows. Time from treatment to estrus emerged as a good guide for FTAI after a 5-day P4-based synchronization protocol.
Diploid germ cells are thought to have pluripotency potential. We recently described a method to derive pluripotent stem cells (PSCs) from cultured spermatogonial stem cells (SSCs) by depleting Trp53 and Dmrt1, both of which are known suppressors of teratomas. In this study, we used this technique to analyze the effect of this protocol in deriving PSCs from the male germline at different developmental stages. We collected primordial germ cells (PGCs), gonocytes and spermatogonia, and the cells were transduced with lentiviruses expressing short hairpin RNA against Dmrt1 and/or Trp53. We found that PGCs are highly susceptible to reprogramming induction and that only Trp53 depletion was sufficient to induce pluripotency. In contrast, gonocytes and spermatogonia were resistant to reprogramming by double knockdown of Dmrt1 and Trp53. PSCs derived from PGCs contributed to chimeras produced by blastocyst injection, but some of the embryos showed placenta-only phenotypes suggestive of epigenetic abnormalities of PGC-derived PSCs. These results show that PGCs and gonocytes/spermatogonia have distinct reprogramming potential and also suggest that fresh and cultured SSCs do not necessarily have the same properties.
The objectives of the present study were to develop a programmable piggyback syringe pump for bovine superovulation and to evaluate the effects of a four-times-a-day injection regimen using the pump. Non-lactating Holstein cows were treated with a total of 30 armour units of porcine FSH by injection four times a day with the pump (study, n = 9) or injection twice a day manually (control, n = 9) for four consecutive days from D10 of the estrous cycle. The pump-driven program successfully induced superovulation in all cows tested. The numbers of small (3– < 5 mm in diameter) and large (≥ 10 mm in diameter) follicles were greater in the study group on D11-13 and D14, respectively. There were fewer unovulated follicles detected on D21 (7 days after estrus) in the study group than in the control group (1.2 ± 0.4 and 3.2 ± 0.6, respectively).