Intrafollicular spontaneous parthenogenetic development of dromedary camel oocytes.

Dromedary camel oocytes are unique in their capability for intrafollicular and in vitro spontaneous parthenogenetic activation (SPA) and development. This study was designed for (a) observing the incidence of SPA and development of dromedary camel oocytes retrieved from ovaries; (b) assessing intrafollicular development of dromedary camel oocytes using histological examination; (c) evaluating the abilities of dromedary camel oocytes to mature, SPA, and develop in vitro; and (d) identifying the transcript abundance of Cdx2 messenger RNA (mRNA) expression in different stages of SPA and developed camel embryos. The results revealed that 2.33% of oocytes retrieved from dromedary camel ovaries were SPA and developed to blastocyst stage. Serial sections of dromedary camel ovaries also demonstrated the presence of 1.4 SPA and parthenotes per ovary, which included from two-cell to the blastocysts with demarcated trophectoderm and inner cell mass layers. A total of 2.6% in vitro matured dromedary camel oocytes developed into morulae. The SPA and developed dromedary embryos expressed transcript abundance for Cdx2 mRNA with the highest (p < .05) at the blastocyst. The present work determines for the first time the intrafollicular oocytes from the dromedary camel display SPA, and the parthenotes can develop into blastocysts and expressing Cdx2 mRNA.

Roles of interferon-stimulated gene 15 protein in bovine embryo development.

Interferon (IFN)-stimulated gene 15 (ISG15) is one of several proteins induced by conceptus-derived Type I or II IFNs in the uterus, and is implicated as an important factor in determining uterine receptivity to embryos in ruminants. But little is known about the role the ISG15 gene or gene product plays during embryo development. In the present study, both the expression profile and function of ISG15 were investigated in early bovine embryos in vitro. ISG15 mRNA was detectable in Day 0, 2, 6 and 8 bovine embryos, but IFN-τ (IFNT) mRNA only appeared from Day 6. This means that embryonic expression of ISG15 on Days 0 and 2 was not induced by embryonic IFNT. However, ISG15 mRNA expression paralleled the expression of IFNT mRNA in Day 6 and 8 embryos. ISG15-lentivirus interference plasmid (ISG15i) was injected into 2-cell embryos to knockdown ISG15 expression. This resulted in decreases in the proportion of hatching blastocysts, the diameter of blastocysts and cell number per diameter of blastocysts compared with control embryos. In addition, ISG15i inhibited IFNT, Ets2 (E26 oncogene homolog 2) mRNA and connexion 43 protein expression in Day 8 blastocysts, whereas exogenous IFNT treatment (100ngmL-1, from Day 4 to Day 8) improved ISG15 mRNA and connexion 43 protein expression. In conclusion, it appears that ISG15 is involved in early bovine embryo development and that it regulates IFNT expression in the blastocyst.

Inhibition of NF-κB promotes autophagy via JNK signaling pathway in porcine granulosa cells.

The transcription factor nuclear factor-κB (NF-κB) plays an important role in diverse processes, including cell proliferation and differentiation, apoptosis and inflammation. However, the role of NF-κB in porcine follicle development is not clearly elucidated. In this study, we demonstrated that follicle stimulating hormone (FSH) increased the level of inhibitor of NF-κB (IκB) protein and promoted the cytoplasmic localization of p65, indicating that FSH inhibits the activation of NF-κB in porcine granulosa cells. Moreover, inhibition of NF-κB by FSH or another specific inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC), could activate JNK signaling and enhance autophagic activity in porcine granulosa cells. Knockdown of RelA (p65) Subunit of NF-κB by RNA interference abrogated the activation of JNK signaling pathway and the increase of autophagic protein expression by FSH. Meanwhile, the functional significance of FSH or PDTC-mediated autophagy were further investigated. Our results demonstrated that the increased autophagy promoted progesterone secretion in porcine granulosa cells. Blockage of autophagy by chloroquine obviated the FSH or PDTC-induced progesterone production. Taken together, these results indicate that inhibition of NF-κB increased autophagy via JNK signaling, and promote steroidogenesis in porcine granulosa cells. Our results provide new insights into the regulation and function of autophagy in mammalian follicle development.

Recombinant bovine interferon-τ enhances in vitro development of bovine embryos by upregulating expression of connexin 43 and E-cadherin.

Interferon-τ (IFNT), produced in ruminants by embryonic trophoblastic cells before implantation, is involved in the maternal recognition of pregnancy. It is a pleiotropic molecule that alters the synthesis of endometrial proteins and inhibits the proliferation of some cells. The present study investigated the effects of recombinant bovine IFNT on the development of early-stage bovine embryos and the molecular mechanism underlying this effect. This study demonstrated that expression of mRNA encoding type I IFN receptor subunits was detectable from d 4 to 8 in in vitro fertilized (IVF) bovine embryos. A considerable number of IVF (n = 1,941) and parthenogenetic activated (n = 1,552) bovine embryos demonstrated that supplementing the culture medium with IFNT (100 ng/mL) produced a greater percentage of blastocysts, and the total cell number within the resulting blastocysts was higher. In addition, IFNT upregulated the expression levels of both mRNA and protein for connexin 43 (GJA1) and E-cadherin (CDH1) and expression levels for granulocyte-macrophage colony-stimulating factor and insulin-like growth factor 2 mRNA but not for their proteins in d 8 embryos. However, IFNT inhibited mRNA expression for leukemia inhibitory factor (LIF), LIF receptor α, and the sodium/potassium-transporting ATPase subunit ß-1. We concluded that IFNT promoted the development of bovine embryos by upregulating the expression of GJA1 and CDH1. Thus, supplementing embryo cultures or transfer medium with IFNT may stimulate embryo development and improve embryo transfer efficiency.

Follicle-stimulating hormone regulates pro-apoptotic protein Bcl-2-interacting mediator of cell death-extra long (BimEL)-induced porcine granulosa cell apoptosis.

The pro-apoptotic protein Bim (B-cell lymphoma-2 (Bcl-2)-interacting modulator of cell death) has recently been identified and shown to promote cell death in response to several stimuli. In this report, we investigated the role of Bim in porcine follicular atresia. Initially, Bim cDNA was cloned and characterized from porcine ovarian tissue. Porcine Bim had three alternative splicing variants (Bim-extra long, Bim-long, and Bim-short), all containing the consensus Bcl-2 homology 3 domain. We then found the Bim-extra long (Bim(EL)) protein, the most abundant isoform of Bim, was strongly expressed and co-localized with apoptotic (TUNEL-positive) granulosa cells from porcine atretic follicles. Furthermore, overexpression of Bim(EL) triggered apoptosis in granulosa cells. In primary granulosa cell cultures under basal conditions, we observed that Bim(EL) expression was dampened by treatment with follicle-stimulating hormone (FSH). The role of the PI3K/Akt pathway in the regulation of repression was clarified by the use of the PI3K inhibitor, LY294002, and by transfection with Akt siRNA. Forkhead Box Protein O3a (FoxO3a), a well defined transcriptional activator of Bim, was phosphorylated at Ser-253 and inactivated after FSH stimulation. Also, FSH abolished FoxO3a nuclear accumulation in response to LY294002. Finally, chromatin immunoprecipitation assays demonstrated that FoxO3a directly bound and activated the bim promoter. Taken together, we conclude that Bim(EL) induces porcine granulosa cell apoptosis during follicular atresia, and its expression is regulated by FSH via the PI3K/Akt/FoxO3a pathway.

Oocyte-secreted growth differentiation factor 9 inhibits BCL-2-interacting mediator of cell death-extra long expression in porcine cumulus cell.

Oocyte-secreted factors (OSFs) maintain the low incidence of cumulus cell apoptosis. In this report, we described that the presence of oocytes suppressed the expression of proapoptotic protein BCL-2-interacting mediator of cell death-extra long (BIMEL) in porcine cumulus cells. Atretic (terminal deoxynucleotidyl transferase dUTP nick end labeling-positive) cumulus cells strongly expressed BIMEL protein. The healthy cumulus- oocyte complex exhibited a low BIMEL expression in cumulus cell while the removal of oocyte led to an about 2.5-fold (P < 0.5) increased expression in oocytectomized complex (OOX). Coculturing OOXs with denuded oocytes decreased BIMEL expression to the normal level. The similar expression pattern could also be achieved in OOXs treated with exogenous recombinant mouse growth differentiation factor 9 (GDF9), a well-characterized OSF. This inhibitory action of GDF9 was prevented by the addition of a phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Luciferase assay further demonstrated that BIM gene expression was forkhead box O3a (FOXO3a)-dependent because mutation of FOXO3a-binding site on the BIM promoter inhibited luciferase activities. Moreover, the activity of BIM promoter encompassing the FOXO3a-binding site could be regulated by GDF9. Additionally, we found that GDF9 elevated the levels of phosphorylated AKT and FOXO3a, and this process was independent of the SMAD signal pathway. Taken together, we concluded that OSFs, particularly GDF9, maintained the low level of BIMEL expression in cumulus cell through activation of the PI3K/FOXO3a pathway.

The disturbances of endoplasmic reticulum calcium homeostasis caused by increased intracellular reactive oxygen species contributes to fragmentation in aged porcine oocytes.

Accumulating evidence indicates that cellular and molecular abnormalities occur during oocyte aging, including fragmentation, increases in intracellular reactive oxygen species (ROS), and abnormal Ca(2+) oscillations. The objective of the present study was to characterize the relationships between intracellular ROS, Ca(2+) homeostasis of endoplasmic reticulum (ER), and fragmentation in aged porcine MII oocytes. Prolonged culture (36 h) of porcine oocytes resulted in elevated intracellular ROS level, impaired ER Ca(2+) homeostasis (i.e., Ca(2+) storage, Ca(2+) rising patterns after electroactivation, and the cluster distribution of ER), and increased fragmentation rates. However, when the porcine oocytes were treated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester), an intracellular Ca(2+) chelator, the fragmentation was significantly inhibited during in vitro aging. In order to pursue the underlying mechanisms, H2O2 and cycloheximide (CHX) were used to artificially increase or inhibit, respectively, the intracellular ROS levels in aged porcine oocytes during in vitro culture. The results demonstrated that incubation of porcine MII oocytes with H2O2 damaged the ER clusters and the Ca(2+) regulation of ER, leading to a high proportion of fragmented oocytes. In contrast, CHX, an intracellular inhibitor of ROS generation, prevented both increase of ROS level and damage of the ER Ca(2+) homeostasis in porcine oocytes during aging, resulting in low fragmentation rate. We conclude that the increased intracellular ROS damaged the ER clusters and ER Ca(2+) homeostasis, resulting in a disorder in ooplasmic free Ca(2+), which caused the fragmentations seen in porcine MII oocytes during aging.

Differential expression pattern of ISG15 in different tissue explants and cells induced by various interferons.

Interferon stimulated gene 15 (ISG15), an ubiquitin cross-reactive protein, can conjugate to target proteins. Unlike ubiquitination, protein modification by ISG15 does not target protein for degradation, but enhances the cellular response to interferon (IFN), which plays a key role in antiviral responses. In this study, Western blot and/or immunocytochemistry were performed to explore the ISG15 expression patterns in explants of bovine endometrium, mammary gland and kidney, as well as Madin-Darby bovine kidney (MDBK), endometrial and mammary cells stimulated by IFN-α, -ß, and -τ. Western blot indicated that there are differential minimum antiviral units among recombinant bovine interferon-α (rbIFN-α, 10(2) IU/mL), rbIFN-ß (10(3) IU/mL) and rbIFN-τ (10(4) IU/mL) in regard to stimulating saturation expression of free and ISG15-conjugated proteins by MDBK cells and endometrial and mammary explants. These results were further confirmed through immunocytochemical analysis of MDBK, endometrial and mammary cells. For the first time it has been shown that the expression pattern of ISG15-conjugated proteins occurs in a tissue-specific manner. Furthermore, the present findings provide the first evidence of 10- to 100-fold differences in minimum antiviral units of rbIFN-α, rbIFN-ß, and rbIFN-τ in regard to stimulating saturation expression of ISG15.

Effects of melatonin on in vitro development of mouse two-cell embryos cultured in HTF medium.

Melatonin is capable of improving the developmental capacity of ovine, porcine and bovine embryos in vitro. However, whether melatonin possesses similar benefits to the in vitro mouse embryonic development has yet to be determined. In this study, we assessed the effects of various concentrations of melatonin (10-13 to 10-3 M) on the in-vitro development of mouse embryos cultured in HTF medium for 96 hr; embryos cultured without melatonin were used as control. The in vitro development of mouse two-cell embryos significantly benefited from treatment with melatonin in a concentration-dependent manner. The effects of melatonin on the rates of blastocyst formation, hatching/hatched blastocysts and cell number per blastocyst were bi-phasic; all significantly increased by melatonin at 10-13 to 10-5 M and decreased by melatonin at 10-3 M. Maximal benefit of melatonin on in vitro mouse 2-cell embryo development was achieved at a concentration of 10-9 M. In comparison to control, 10-9 M melatonin increased blastocyst formation rate from 48.08 +/- 5.25% to 82.08 +/- 2.34% (p < 0.05), hatched blastocyst rate from 25.65 +/- 11.79% to 66.47 +/- 4.94% (p < 0.05), and cell number per blastocyst 62.71 +/- 5.97 to 77.91 +/- 10.63 (p < 0.05). Thus, our datas demonstrated firstly that melatonin has beneficial effects on the in vitro development of 2-cell mouse embryos cultured in HTF medium.

Melatonin exists in porcine follicular fluid and improves in vitro maturation and parthenogenetic development of porcine oocytes.

This study focused on the effect of melatonin on in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Melatonin was measured in porcine follicular fluid of follicles of different sizes in the same ovary. Melatonin exists in follicular fluid, and the concentration is approximately 10(-11) m. Its concentration decreased as the diameter of follicle increased, which suggests an effect of melatonin on oocyte maturation. Therefore, immature oocytes were cultured in vitro in maturation medium supplemented with melatonin (10(-11), 10(-9), 10(-7), 10(-5) and 10(-3) m) or without melatonin. The oocytes at maturation stage were collected and activated. The parthenogenetic embryos were cultured and observed in medium supplemented with or without melatonin. Fresh immature oocytes without melatonin treatment were used as control. When only maturation medium was supplemented with 10(-9) m melatonin, the cleavage rate, blastocyst rate and the cell number of blastocyst (70 +/- 4.5%, 28 +/- 2.4% and 50 +/- 6.5%) were significantly higher (P < 0.05) than that of controls; when only culture medium was supplemented with melatonin, the highest cleavage rate, blastocyst rate and the cell number of blastocyst was observed at 10(-7) m melatonin, which were significantly higher than that of controls (P < 0.05). The best results (cleavage rates 79 +/- 8.4%, blastocyst rates 35 +/- 6.7%) were obtained when both the maturation and culture medium were supplemented with 10(-9) m melatonin respectively (P < 0.05). In conclusion, exogenous melatonin at the proper concentration may improve the in vitro maturation of porcine oocytes and their parthenogenetic embryonic development. Further research is needed to identify the effect of melatonin on in vitro and in vivo oocyte maturation and embryo development in porcine.

Abnormal changes in mitochondria, lipid droplets, ATP and glutathione content, and Ca(2+) release after electro-activation contribute to poor developmental competence of porcine oocyte during in vitro ageing.

The present study aims to investigate major changes in porcine oocytes during ageing in vitro. After the oocytes were cultured for 44, 56, 68 and 80 h, changes to porcine oocytes in ultrastructure, mitochondrial distribution, glutathione (GSH) and ATP content, Ca(2+) release patterns and developmental competence after electro-activation were observed. Mitochondria were evenly distributed in oocytes at 44 h, aggregated in clusters or in peripheral cytoplasm at 68 h and dimly dispersed throughout ooplasm at 80 h. Mitochondrial shape during ageing was also observed by transmission electron microscopy (TEM) at the same time intervals. Most mitochondria were spherical at 44 h, and became elongated when the culture time was extended to 68 h and 80 h. Moreover, mitochondrial clustering became increasingly loose from 56 h. Lipid droplets in oocytes appeared prominent and electron-dense at 44 h, but electron density was lost at 56 h. Lipid droplets were solidified as of 68 h. There was an age-dependent decrease in ATP content per oocyte. Glutathione content per oocyte decreased significantly and remained lower after 56 h. Amplitudes of [Ca(2+)] rise decreased dramatically following 56 h, and the time required for [Ca(2+)] to plateau became shorter after electro-activation with prolonged culture time. Cleavage and blastocyst rates of aged oocytes progressively decreased, while the fragmentation rate gradually increased after electro-activation. It is concluded that abnormal changes in mitochondria, lipid droplets, Ca(2+) release after electro-activation, and ATP and GSH content in oocytes during ageing may result in poor developmental competence of parthenotes.

Vitrification of farmed blue fox oocytes in ethylene glycol and DMSO-based solutions using open-pulled straw (OPS).

Farmed blue fox was used as a model to develop cryopreservation protocol for nondomestic canine species. We report here the developmental potential of farmed blue fox oocytes after vitrification with a two-step OPS method. Oocytes were collected and pre-cultured for 0, 24, 48, 72 hours respectively before cryopreservation. Vitrification of oocytes was achieved by a 30 sec treatment in 10% ethylene glycol (EG) or 10% EG + 10% dimethyl sulfoxide (DMSO) at 25 degree C followed by a 25 sec equilibration in EFS30 (30% (v/v) EG +21% (w/v) Ficoll +0.35M sucrose) or EDFS30 (15% (v/v) EG +15% (v/v) DMSO +21% (w/v) Ficoll +0.35M sucrose), before plunging into liquid nitrogen. The survival of oocytes after vitrification was assessed morphologically immediately after warming, and cultured for in vitro maturation. For comparison, control oocytes were cultured for in vitro maturation for 96 hours. The best result was obtained when oocytes were pre-cultured for 72 hours, first exposed to 10 percent EG + 10% DMSO and vitrified in EDFS30. The survival percentage of oocytes under these conditions was not significantly different (P > 0.05) from that of the control.

Surgical embryo transfer resulted in birth of live offspring in farmed blue fox.

Finland blue fox (Alopex lagopus) has great reputation in pelt industry around the world for its large size and top-ranking fur quality; however, both the herd size and the average survival rate of purebred offspring are rather low in production systems in China. Surgical transfer of blue fox embryos was investigated as a means to increase the population fox and also as a possible means to conserve endangered canine species. The animals were chosen on the basis of synchrony in natural oestrus. During the reproductive season of blue fox, 59 embryos were flushed from 6 farmed donors 9-11 days after the first insemination, and 53 embryos were transferred surgically into the uteri of the 6 paired recipients with natural synchronized oestrous. Two of the recipients littered 46-49 days after embryo transfer; one gave birth to 7 pups and the other 1 pup. This report describes the first successful embryo transfer in the farmed blue fox in China.

Evaluation of three hormonal protocols for anovulatory lactating cows under regulations restricting the use of estrogenic compounds.

When European Union regulations restricted the use of estrogenic compounds in food-producing animals, refined hormonal protocols were no longer applicable for anovulatory cows. However, Ovsynch and its adaptations are routinely and uniformly applied to all cows regardless of ovarian function. To evaluate their efficacy on anovulatory cows, 143, 147 and 144 anovulatory cows received Ovsynch, Presynch and G6G protocols, respectively. In comparison, 150 cyclic cows were bred without using a synchronized protocol. Results showed that cows in the Presynch group had luteolysis responding to the last prostaglandin F2α (PGF2α ) injection greater than the Ovsynch group. The serous progesterone levels at the first gonadotropin-releasing hormone of Ovsych and the last PGF2α injection was greater in the G6G group than the other two hormonal treatment groups. Concentrations of Ca2+ and total protein in cervical mucus in all three hormone-treated groups before artificial insemination (AI) were significantly different from the controls. The G6G group obtained a greater pregnancy rate compared with Ovsynch and Presynch, but significantly less than the controls. For open cows in the Ovsynch group, estrus rate within 24 days after the first AI was significantly less than the controls. In conclusion, the G6G treatment resulted to better reproductive performance in anovulatory cows.

Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection.

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.

Cited2 protein level in cumulus cells is a biomarker for human embryo quality and pregnancy outcome in one in vitro fertilization cycle.

OBJECTIVE: To determine whether the levels of CBP/p300 interacting transactivator with ED-rich tail 2 (Cited2) protein in cumulus cells (CCs) derived from patients undergoing IVF related to infertility factors, embryo quality, and clinical outcomes in one IVF cycle. DESIGN: Retrospective analysis of human CCs. SETTING: Public hospital and university. PATIENT(S): A total of 103 (conventional) IVF patients and 32 intracytoplasmic sperm injection patients. INTERVENTION(S): All CCs from each patient's oocytes were considered as one sample. The patients were divided into two groups according to whether the Cited2/ß-actin levels in their CCs were above or below the mean level detected for all patients. MAIN OUTCOME MEASURE(S): Embryo quality and clinical outcomes of IVF patients. RESULT(S): The oocytes derived from the group of patients whose CCs showed lower Cited2 levels displayed higher fertilization, transferable embryo, and implantation rates. Moreover, the patients in this group were more likely to have a successful pregnancy outcome. Among different infertility factors, a total of 78.6% of patients with polycystic ovary syndrome had a higher Cited2 level in CCs. Additionally, patients with a lower basal FSH level belonged to the higher Cited2 levels group. The expression of two genes (phosphoenolpyruvate carboxykinase 1 [PCK1] and progesterone receptor [PR]) and the glucose content in CCs were also markedly increased in CCs derived from patients with higher Cited2 levels. CONCLUSION(S): The present findings imply that Cited2 level in CCs is associated with polycystic ovary syndrome, embryo quality, and pregnancy outcome of IVF patients.

A weekly postpartum PGF2α protocol enhances uterine health in dairy cows.

Postpartum uterine health in dairy cows is crucial for the maintenance of good reproductive performance. In order to improve uterine health and reduce puerperal intrauterine infection, 608 Holstein cows received a weekly PGF2α protocol (3 i.m. injections of PGF2α at 7, 14 and 21 d postpartum). For comparison, 593 cows in the control group received injections of sterile saline at the same time. Uterine score at 14 d postpartum, the prevalence of endometritis at 21-27 d postpartum, and subsequent reproduction performance was evaluated. Cows in the treated group exhibited higher tonicity (P<0.05) of the uterus, with less prevalence of endometritis (10.4%, 63/608 vs. 34.6%, 205/593; P<0.001), and required shorter time to the first AI postpartum (67.5±3.4 d vs. 84.4±3.7 d, P<0.05) and to pregnancy (114.5±5.4 d vs. 131.4±5.8 d, P<0.05). In conclusion, the present study demonstrated that uterine health in Holstein cows can be promoted while puerperal infection can be suppressed by this weekly postpartum PGF2α protocol.

Lysosomes are involved in induction of steroidogenic acute regulatory protein (StAR) gene expression and progesterone synthesis through low-density lipoprotein in cultured bovine granulosa cells.

Progesterone is an important steroid hormone in the regulation of the bovine estrous cycle. The steroidogenic acute regulatory protein (StAR) is an indispensable component for transporting cholesterol to the inner mitochondrial membrane, which is one of the rate-limiting steps for progesterone synthesis. Low-density lipoprotein (LDL) supplies cholesterol precursors for progesterone formation, and the lysosomal degradation pathway of LDL is essential for progesterone biosynthesis in granulosa cells after ovulation. However, it is currently unknown how LDL and lysosomes coordinate the expression of the StAR gene and progesterone production in bovine granulosa cells. Here, we investigated the role of lysosomes in LDL-treated bovine granulosa cells. Our results reported that LDL induced expression of StAR messenger RNA and protein as well as expression of cholesterol side-chain cleavage cytochrome P-450 (CYP11A1) messenger RNA and progesterone production in cultured bovine granulosa cells. The number of lysosomes in the granulosa cells was also significantly increased by LDL; whereas the lysosomal inhibitor, chloroquine, strikingly abolished these LDL-induced effects. Our results indicate that LDL promotes StAR expression, synthesis of progesterone, and formation of lysosomes in bovine granulosa cells, and lysosomes participate in the process by releasing free cholesterol from hydrolyzed LDL.

Age-associated potency decline in bovine oocytes is delayed by blocking extracellular Ca(2+) influx.

Oocyte aging due to delayed fertilization is associated with declining quality and developmental potential. Intracellular calcium (Ca(2+)) concentration ([Ca(2+)]i) regulates oocyte growth, maturation, and fertilization and has also been implicated in aging. Using bovine oocytes, we tested the hypothesis that oocyte aging could be delayed by reducing [Ca(2+)]ivia blocking the influx of extracellular Ca(2+) or chelating ooplasmic free Ca(2+). After IVM, cumulus-oocyte complexes or denuded oocytes were cultured in medium supplemented with 1-octanol, phorbol 12-myristate 13-acetate, or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM) to manipulate [Ca(2+)]i. Addition of 1-mM 1-octanol increased blastocyst development rates in the cumulus-oocyte complexes aged for 6 hours by IVF and for 6, 12, and 24 hours by parthenoactivation, and this effect was independent of the presence of cumulus cells. The intracellular levels of ATP, Glutathione, and Glutathione disulfide were not affected by 1-octanol, but [Ca(2+)]i was significantly decreased. When oocytes were cultured in Ca(2+)-free medium for 12 hours, the blastocyst development rate was greater and the beneficial effects of 1-octanol on oocyte aging were abolished. However, when the medium was supplemented with phorbol 12-myristate 13-acetate, [Ca(2+)]i increased and the blastocyst development rate decreased. Moreover, BAPTA-AM reduced [Ca(2+)]i and increased blastocyst development rates after IVF or parthenoactivation. We conclude that the age-associated developmental potency decline was delayed by blocking the influx of extracellular Ca(2+) or reducing ooplasmic free Ca(2+). 1-Octanol, BAPTA-AM, or Ca(2+)-free medium could be used to lengthen the fertilization windows of aged bovine oocytes.

A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro.

No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation.