The supply of human oocytes is very limited. This restricts not only certain assisted reproduction procedures in IVF clinics where recipients wait for oocytes from donors, but also development of some promising approaches, like therapeutic nuclear transfer with subsequent derivation of patient compatible embryonic stem cells. Moreover, in some patients, collected oocytes exhibit certain specific defects, and logically, we can expect that after fertilization, the embryos arising from these defective oocytes may not develop or that their development might eventually be compromised. For this reason, an increased effort to determine how to repair oocytes is evident in the literature. In general, abnormalities (defects) can be detected in different oocyte components, the zona pellucida, cytoplasm, nucleus (chromosomes) and nucleolus. Whereas defects of a nuclear component are impossible (nuclear DNA) or very hard to repair (nucleolus), zona pellucida abnormalities and cytoplasm defects (for example, if containing mutated mitochondrial DNA, mtDNA) can be repaired in some cases with the help of micromanipulation schemes. In the present article, we will briefly outline the current methodological approaches that can be used to repair the oocyte or one-cell stage embryo.
The maturation and developmental competence of the oocyte is acquired during folliculogenesis. It is still unclear whether follicle size is associated with the levels of transcript and protein encoding molecules contributing to the fertilization ability of the porcine oocyte. Follicles were dissected from porcine ovaries after slaughter and classified as small (< 3 mm), medium (3-5 mm) or large (>5 mm), aspirated cumulus-oocyte complexes were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH) using a brilliant cresyl blue (BCB) test, real-time quantitative PCR reaction methods, western-blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoproteins pZP1, pZP2, pZP3, pZP3 alpha and integrins beta 1 and beta 2, as well as the levels of pZP3 and integrin beta 2 proteins. We observed significantly higher levels of pZP1, pZP3 and integrin beta1 and beta2 transcripts in oocytes collected from medium follicles as compared with small follicles (P<0.001). Moreover, we found an increased content of all investigated mRNAs in oocytes isolated from large follicles as compared with small follicles (P<0.001). Western-blot analysis demonstrated a higher level of pZP3 protein in oocytes isolated from large and medium follicles as compared with small follicles (P<0.001). Our results suggest that the levels of transcripts and proteins for selected molecules contributing to the fertilization ability of oocytes are associated with follicular size in puberal gilts.
In this study, we evaluated the effect of different concentrations of cysteine in in vitro maturation (IVM) medium during IVM under low oxygen tension (5% O2) of porcine oocytes on the intracellular content of glutathione (GSH) and subsequent in vitro fertilization (IVF) and development. Cumulus oocyte complexes (COCs) were collected from ovaries obtained at a local slaughterhouse, cultured in IVM medium supplemented with 0 (control), 0.05, 0.1, 0.2 or 0.6 mM cysteine for 44-46 h, fertilized in vitro and subsequently cultured for 6 days in total. The GSH content of the IVM oocytes exposed to 0, 0.05, 0.1, 0.2 or 0.6 mM cysteine increased significantly (P<0.05) as the concentration of cysteine increased (12.2, 14.0, 15.1, 16.4 and 16.4 pmol/oocyte, respectively). However, the rates of oocyte maturation, sperm penetration, male pronuclear formation, monospermy and even cleavage on Day 2 (the day of IVF was defined as Day 0) and blastocyst formation on Day 6 did not differ among the groups. Moreover, the cell numbers of blastomeres in blastocysts were uniform among the groups. These results indicate that supplementation with 0.05-0.6 mM cysteine during IVM under 5% O2 tension significantly increased the intracellular GSH contents of IVM oocytes; however, it had no promoting effects on nuclear maturation, fertilization, male pronucleus formation and subsequent embryonic development to the blastocyst stage.
The present study investigated effects of three semen extenders and storage temperatures on post-thaw characteristics of Bryde's whale spermatozoa. Spermatozoa were collected from the vasa deferens of three mature Bryde's whales captured during the Japanese whale research in the north-west Pacific (May to August 2007) after death. The three semen extenders used for freezing were 1) a commercialized synthetic extender (AndroMed: AM), 2) Tris-based + 10% bovine serum albumin (BSA) and 3) Tris-based + egg yolk (EY). The sperm samples from the three whales were frozen with the three extenders, and the post-thaw spermatozoa were stored at three different temperatures (35 C; 20-25 C, room temperature; and 5 C) for 0, 6, 12, 24, 48 and 96 h. At each time-point, total and progressive motility (PM), viability (live or dead), the hypo-osmotic test, defective acrosomes and malformation were examined. Immediately after thawing, AM resulted in similar recovery rates (60.4 and 83.3%) in 2 of the 3 whales examined and had comparable post-thaw recovery rates to those obtained using the EY and BSA extenders. Immediately after thawing, the proportion of PM in EY (17.6%) was higher (P<0.05) than that in BSA (15.0%). In the hypo-osmotic test, the proportions of AM (26.0%) and BSA (25.2%) were higher (P<0.05) than that of EY (17.3 %). The three extenders had similar viabilities (36.7, 37.9 and 32.1%, respectively), but the viability of BSA was higher (P<0.05) than that of EY. The present study showed that a synthetic semen extender, AndroMed, could be used for cryopreservation of whale spermatozoa in addition to Tris-based extenders containing bovine serum albumin or egg yolk. Storage of the post-thaw Bryde's whale spermatozoa was better at 5 C than at room temperature or 35 C. The frozen-thawed Bryde's whale spermatozoa maintained their motility and viability for at least two days at room temperature and for four days at 5 C.
Mammalian oocytes can undergo artificial parthenogenesis in vitro and develop to the blastocyst stage. In this study, using real-time PCR, we analyzed the expression of genes representative of essential events in development. In vitro matured oocytes were either fertilized or activated with ionomycin + 6-DMAP and cultured in simple medium. The pluripotency-related gene Oct3/4 was downregulated in parthenotes, while the de novo methylation DNMT3A gene was unchanged. Among the pregnancy recognition genes, IFN-t was upregulated, PGRMC1 was downregulated and PLAC8 was unchanged in parthenotes. Among the metabolism genes, SLC2A1 was downregulated, while AKR1B1, COX2, H6PD and TXN were upregulated in parthenotes; there was no difference in SLC2A5. Among the genes involved in compaction/blastulation, GJA1 expression increased in parthenotes, but no differences were detected within ATP1A1 and CDH1. Expression of p66shc and the Bax/Bcl2 ratio were higher in parthenotes, and there was no difference in p53. Parthenotes and embryos may differ in the way they stimulate apoptosis, with a preponderant role for p66shc within parthenotes. Differentially affected functions may also include pluripotency, de novo methylation and early embryonic signalling.
Apoptosis plays an essential role in normal spermatogenesis, but deregulations of this biological process, which is closely associated with male infertility, have been found. Whereas calcium homeostasis is a key regulator of cell survival, sustained elevation of intracellular calcium plays a role in apoptosis. The aim of this research was to determine the role of two different calcium mobilizing agents, hydrogen peroxide (H2O2) and the physiological agonist progesterone, on the apoptosis process of human ejaculated spermatozoa. Translocation of membrane phosphatidylserine was examined with an annexin V binding assay, DNA damage was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL assay) and caspase-3 activity was assessed using a fluorometric assay. After incubation of spermatozoa for 1 h with either 10 μM H2O2 or 20 μM of progesterone, there was a significant increase in both caspase-3 activity and the percentage of annexin V-positive cells. Similarly, the TUNEL results were significantly higher 1 h after incubation with either 10 μM H2O2 or 20 μM of progesterone. In fact, progesterone-treated cells showed a three-fold increase (from 17.6 to 52.9%) of TUNEL-positive cells compared to untreated cells, while H2O2-treated cells exhibited a two-fold increase (from 17.6 to 37.9%). In sum, our results suggest that spermatozoa treated with calcium mobilizing agents, such as H2O2 and progesterone, seem to undergo an apoptosis process that is dependent on caspase-3 activation.
The aim of this study was to investigate the effect of luteinizing hormone (LH) surge on expression of nerve growth factor (NGF), trkA, p75 and inhibin α-subunit in ovarian interstitial cells of golden hamsters after human chorionic gonadotropin (hCG) treatment. NGF, two NGF receptors (trkA, p75) and inhibin α-subunit were immunolocalized by immunohistochemistry, and gonadotropins, steroid hormones and immunoreactive (ir-) inhibin concentrations were measured by radioimmunoassay. Stronger positive staining of NGF, trkA and p75 were found in interstitial cells at 6, 12 and 18 h after hCG injection in the treated group compared with the control groups. Inhibin α-subunit staining was found in interstitial cells at 12 and 18 h after hCG injection in the treatment group, but not in the control group. Plasma concentrations of progesterone increased significantly from 6 to 18 h after hCG treatment, whereas plasma concentrations of estradiol-17β significantly decreased compared with the control group. An increased plasma concentration of FSH and decreased concentrations of ir-inhibin from 6 to 18 h after hCG treatment verified the negative relationship. There were no significant changes in the concentrations of LH in the hCG-treated group. In contrast, the typical preovulatory LH surge was found at 1700 h on day 4 (proestrus) in the control group. These results suggested that LH surge can induce expression of NGF, trkA, p75 and inhibin α-subunit in ovarian interstitial cells and that NGF, trkA, p75 and inhibin α-subunit may have a paracrine or autocrine role in modulation of ovarian interstitial cell function in golden hamsters.
Our previous studies have demonstrated that prenatally administered diethylstilbestrol (DES) impairs testicular endocrine function in male offspring. The present study examined whether maternal DES treatment influences testicular steroidogenesis and spermatogenesis. DES was injected subcutaneously at 0.5 or 1.5 μg/kg/day (DES 0.5 and 1.5 groups, respectively) into pregnant SD rats on days 7-21 of gestation. Male offspring in the DES 0.5 and 1.5 groups were autopsied at 1, 3, 6 and 15 weeks after birth. At 1 week, DES treatment did not lead to a change in the volume of P450scc-positive cells (Leydig cells), suggesting that DES has no inhibitory effect on the development of Leydig cells. DES administration disrupted luteinizing hormone receptor (LHr) expression and exerted inhibitory effects on signal transduction from LHr to steroidogenic acute regulatory protein (StAR) in testicular steroidogenesis (P<0.05), although there were no changes in the mRNA expression levels of steroidogenic enzymes, such as P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD) and P45017α, which may have caused a decrease in the plasma testosterone level. DES treatment did not disrupt the cycle of spermatogenesis but did upregulate the expression levels of androgen receptor (AR) mRNA in both DES groups at 15 weeks (P<0.05). These results indicate that maternal DES treatment disrupts steroidogenesis but induces a high level of AR mRNA expression to counteract the low levels of testosterone during spermatogenesis.
Successful cloning by somatic cell nuclear transfer (SCNT) requires a reprogramming process in which the epigenetic state of a differentiated donor nucleus must be converted into an embryonic totipotent state. However, this epigenetic reprogramming is incomplete in SCNT embryos, causing low production efficiency. Recently, it has been reported that trichostatin A (TSA), an inhibitor of histone deacetylase, potentially enhances cloning efficiency. The aim of the present study was to optimize the TSA treatment for miniature pig SCNT embryos and investigate the effect of the acetylation level of histone on developmental competence of SCNT embryos. In order to optimize the TSA treatment, we examined the developmental competence of SCNT embryos under various exposure times (0-50 h) and concentrations (0-500 nM). Treatment with 5 nM TSA for 15 and 20 h beginning at the start of activation significantly increased the blastocyst formation rate (34.6 and 32.4 vs. 18.2%, respectively) and mean cell number (57.0 ± 2.7 and 56.6 ± 2.7 vs. 43.5 ± 2.1, respectively) as compared with the non-treated group (0 h). We then investigated the acetylation levels of histone H3 in SCNT embryos treated with or without TSA (TSA (+) or TSA (-)) as compared with in vitro- fertilized (IVF) embryos. The acetylation levels of the TSA (-) SCNT embryos at the pseudo-pronuclear and 2-cell stages were significantly lower than those of the IVF embryos at the same developmental stages. In contrast, the acetylation levels of the TSA (+) SCNT embryos were similar to those of the IVF embryos. There was no difference in the acetylation levels of all groups at the blastocyst stage. Our data therefore suggests that the acetylation level of histone H3 at the pseudo-pronuclear and 2-cell stages is positively correlated with subsequent development of SCNT embryos, which may be an important event for the vital development of SCNT embryos in miniature pigs.
Supplementation of semen extender with caffeine and CaCl2 for artificial insemination (AI) of fresh spermatozoa has been demonstrated to reduce recruitment of uterine polymorphonuclear leukocytes (PMNs) and the activity of phagocytosis. Here, we determined if addition of caffeine and CaCl2 to semen extender improves the fertility of frozen-thawed boar semen. In experiment 1, gilts were cervically inseminated twice with froxzen-thawed boar spermatozoa (25 × 108 cells per dose) suspended in Modena solution (n=7) or modified Beltsville Thawing Solution supplemented with caffeine and CaCl2 (BCC, n=7). The gilts were slaughtered 4 h later, and their oviducts and uterine horns plus the body of the uterus were flushed to recover PMNs and non-phagocytosed spermatozoa. There was no difference in the total number of uterine PMNs between gilts inseminated with Modena solution and those inseminated with BCC (3.8 × 108vs. 1.5 × 108 cells, respectively); however, the total number of uterine spermatozoa was higher when gilts were inseminated with BCC (40.6 × 106 cells) compared with those inseminated with Modena solution (1.4 × 106 cells, P<0.05). In experiment 2, gilts and sows were subjected to intrauterine insemination twice with frozen-thawed spermatozoa suspended (25 × 108 sperm per dose) in Modena (n=21) or BCC (n=21). The overall pregnancy and farrowing rates were higher in females inseminated with BCC (71.4 and 61.9%, respectively) compared with those inseminated with Modena solution (38.1 and 28.6%, respectively, P<0.05). However, no significant difference in litter size of piglets was observed between treatments (7.2 ± 1.6 piglets for Modena solution vs. 8.2 ± 0.9 piglets for BCC solution). In conclusion, we demonstrated that use of BCC solution for frozen-thawed boar semen produced better pregnancy and farrowing rates following AI than Modena solution, probably by reducing the phagocytosis of spermatozoa.
The aims of the present study were to clarify the effect of kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and growth hormone (GH) in prepubertal male and female cattle. The experiments were performed from May to June using five male (4-6 months old) and five female (5-6 months old) Japanese Black calves. A single intravenous (iv) injection of Kp10 (5 μg/kg body weight (b.w.): 3.85 nmol/ kg b.w.) significantly stimulated the release of LH and FSH in male and female calves (P<0.05). A single intramuscular injection of Kp10 (5 μg/kg b.w.) also significantly stimulated the release of LH and FSH in male calves (P<0.05), though the response was smaller than that to the iv injection. The injection of Kp10 did not alter the basal plasma concentration of GH in male or female calves. The area under the curve (AUC) of both LH and FSH for a 120-min period after the iv injection of Kp10 was significantly greater in the males than females (P<0.05). These results show that Kp10 can stimulate the release of LH and FSH in calves of both sexes and that the response to the peptide is greater in males at this age. They also show that Kp10 has no effect on the release of GH in male and female calves and that the LH- and FSH-releasing effect of Kp10 is greater after an iv injection than after an im injection in calves.
The aim of this study was to investigate the effects of the presence and the numbers of corpora lutea (CL) in porcine ovaries on in vitro oocyte maturation and embryonic development following intracytoplasmic sperm injection (ICSI). At oocyte collection, the ovaries of non-delivered and delivered pigs were classified into four groups by CL presence. The effect of the number of CL was also investigated following re-division of the non-delivered groups into four groups. In addition, the progesterone (P4) concentrations in follicular fluid (FF) of all the groups were measured to confirm the relationship between the presence and numbers of CL. Throughout the present study, the oocytes recovered from the CL-holding ovaries showed high (P<0.05) oocyte maturation rates, blastocyst rates and P4 concentrations in FF. Furthermore, in the non-delivered groups, the blastocyst rates and P4 concentrations in FF seemed to coincide with the CL numbers in each ovary. From these findings, we concluded that the presence and number of CL in the ovary can be used as an indicator for estimation of the developmental competence of porcine oocytes. Additionally, the present study suggests that P4 in FF influences in vitro oocyte maturation and embryonic development in porcine in vitro production.
The present study was carried out to develop a noninvasive monitoring system for evaluation of Oct-3/4 promoter gene status in miniature pig somatic cell nuclear transfer (SCNT) embryos during in vitro development. Miniature pig fetal fibroblasts (MPFFs) were transfected with a gene construct consisting of two expression units, a mouse Oct-3/4 promoter-driven enhanced green fluorescent protein (EGFP) gene (EGFP expression only detected in Oct-3/4-expressing cells) and a neomycin resistance gene. After neomycin selection, MPFFs that did not express EGFP were fused with enucleated pig oocytes, cultured in vitro and assessed for EGFP expression. EGFP expression was detectable in all morulae (at 4-6 days of culture) and 50.0% of blastocysts (at 5-6 days of culture), whereas none of the 1-cell to 16-cell embryos at 1-5 days of culture expressed EGFP. On the other hand, EGFP expression was not maintained in all blastocysts at 7 days of culture. The reactivity with anti-Oct-3/4 antibodies also peaked from the morula to blastocyst stages at 5 days of culture. The results showed that reactivation of the Oct-3/4 promoter gene of donor nuclei occurs in the morula to blastocyst stages at 4-6 days after SCNT and that this noninvasive monitoring system using Oct-3/4 promoter-driven EGFP gene would be useful for evaluation of the reprogramming status of donor nuclei.
Tetraploid embryos normally develop to the blastocyst stage before implantation, but fail to survive after implantation. To better understand these characteristics of the tetraploid embryo, we produced tetraploid embryos by electrofusion and analyzed expressed genes that participated in mammalian embryogenesis using a DNA microarray analysis and a publicly available bioinformatics analysis of hatched tetraploid and diploid blastocysts. Transcriptome analysis with the DNA microarray revealed that the expression level of most genes was almost the same between diploid and tetraploid blastocysts. We found that the expression levels 2,800 genes were increased, but the expression levels over 1,600 genes were decreased in tetraploid blastocysts, which have a genomic composition identical to that of diploid blastocysts. In tetraploid blastocysts, the levels of 15 genes were decreased more than two-fold compared with the levels in diploid blastocysts. Among these downregulated genes, Ccnb1 (cyclin B1), which was decreased by 3-fold, seemed to play a particularly important role in the cellular organization of the tetraploid blastocyst. To classify the major functional classes of all the genes differentially expressed between diploid and tetraploid blastocysts, we employed a publicly available bioinformatics database, the VisuaL Annotation Display (VLAD). VLAD revealed several altered pathways in tetraploid blastocysts. Some of the enhanced biological processes were moderately involved with chromosome organization, while the suppressed processes were significantly involved with cell division and the mitotic cell cycle, metabolic processes and protein localization and transport. Taken together, our results revealed a large population of downregulated genes in tetraploid hatched blastocysts, and our convergent data suggest that the downregulation might primarily individualize the tetraploid phenotype in hatched blastocysts.
N-Acetyl-D-glucosamine (GlcNAc) is a major component of glycosaminoglycan, which is involved in sperm-oocyte interactions. We examined the effect of adding GlcNAc and other monosaccharides, D-mannose and D-fucose, to the in vitro fertilization (IVF) medium on bovine sperm-oocyte interactions. In medium in which sperm and a zona pellucida (ZP) were co-incubated with monosaccharides for 5 min, addition of GlcNAc (5 or 25 mM) significantly reduced the number of sperm that attached to the ZP. Pretreatment of gametes with GlcNAc (5 mM) prior to co-incubation also suppressed sperm-ZP attachment. Addition of GlcNAc (5 or 25 mM) to the medium in which sperm and a ZP were co-incubated for 5 h, however, significantly increased the number of sperm binding to and penetrating the ZP in a concentration-related manner. The other monosaccharides, D-fucose and D-mannose, did not have this effect. Supplementation of the sperm-oocyte co-incubation medium with 5 mM GlcNAc also enhanced the rate of polyspermic fertilization. When the ZPs were removed from the oocytes, GlcNAc did not affect the fertilization rate. Furthermore, incubation of sperm with 5 mM GlcNAc induced sperm membrane destabilization and an acrosome reaction, as evidenced by the hypo-osmotic swelling test and fluorescein isothiocyanate-labeled peanut agglutinin/propidium iodide (FITC-PNA/PI) staining. Finally, GlcNAc suppressed ZP hardening following fertilization, as determined by measuring the time required for pronase to dissolve the ZP. In conclusion, supplementation of IVF medium with GlcNAc has various effects on sperm-oocyte interactions including suppression of initial attachment, induction of sperm membrane destabilization and acrosome reaction, increase in the number of sperm secondarily bound to and penetrating the ZP, suppression of ZP hardening following sperm-oocyte co-incubation and increase in the rate of polyspermic fertilization.
Nanoparticle technology refers to research and technology developed at the atomic or molecular level for materials of approximately 1-100 nm in length. Through accidental or involuntary exposure, nanoparticles are potentially toxic to the body, including reproductive organs. Ovarian granulosa cells play a major role in maintaining ovarian function, health, and female fertility. Since these cells are involved in steroidogenesis, we wished to evaluate whether nanoparticles affected them after traversing their membranes. Cells were co-incubated with 10 nm gold particles for up to 24 h. Transmission electron micrographs were taken of GC treated with 10 nm gold particles in order to compare and contrast ultrastructural locations of nanoparticles with treatment. From micrograph comparisons of treated vs. untreated GC at various culture times, it appeared that some intracellular organelles involved in steroidogenesis were infiltrated and/or altered due to the presence of the nanogold particles. Medium samples were taken in order to determine estradiol-17beta (E2) accumulation/secretion by untreated vs. treated cells. GC incubated with 10 nm nanogold particles for 1, 3, or 5 h were found to accumulate significantly increased amounts of estrogen compared with untreated cells. Conversely, at 24 h there was a significant attenuation with respect to controls. The data presented here provide insight into the toxicologic effects gold nanoparticles elicit on ovarian granulosa cells.