The presence of RNase A activity was studied in vitro in homogenates of quail oocytes and early embryos using [3H]poly(U) as a substrate. The activity was measured by adsorption of the undegraded substrate onto DE-81 filter paper discs and by chromatographic separation on a Sephadex G-50 column. RNase A activity examined by these methods was almost undetectable in quail previtellogenic, vitellogenic and ovulated oocytes as well as in the embryos from laid eggs. It is estimated to be about 1.1 × 10−5 Kunitz units per ovulated oocyte. Higher activity starts to appear in gastrulating embryos. These findings are discussed in relation to other studies demonstrating the high stability of maternal RNA during early development, especially in growing oocytes.

The present study was undertaken to determine the effect of prolactin (Prl) on Bufo arenarum oocyte maturation and ovulation, two characteristic events of the breeding period, the stage of the sexual cycle in which gamete growth is complete. We observed that Prl, at the doses assayed, did not affect nuclear maturation per se. In addition, when follicles were pretreated with Prl and progesterone was later added to the medium as a physiological nuclear maturation inducer, the percentage of germinal vesicle breakdown obtained with the steroid was unaffected by Prl. The analysis of the in vitro ovulation process demonstrated that pituitary homologous homogenate (PHH) produced a dose- and month-dependent stimulating effect. The maximum percentage of ovulated oocytes was obtained from the end of July to October, the period in which oviposition naturally occurs. Prl by itself did not affect the ovulation process, but when both the hormone and PHH were present in the incubation medium, a significant increase in ovulated oocytes was observed. The results suggest that Prl does not participate in oocyte maturation; however, its presence in the incubation medium would increase oocyte sensitivity to the action of the physiological ovulation inducers.

This study tested whether feeding Rasa Aragonesa ewes certified organic feed, from 15 days before mating until lamb weaning, improved oocyte quality and in vitro maturation (IVM) and fertilization (IVF) performances of the offspring. In a second experiment, ovaries from ewe lambs that were bred on an organic farm and were of the same breed were compared with those from conventionally bred animals. The number (± standard error of the mean) of healthy oocytes per ewe lamb did not differ significantly between organic (12.2 ± 3.3) and conventionally (13.6 ± 4.0) fed ewes. Ovaries from ewe lambs born on an organic farm had significantly (P < 0.0001) more healthy oocytes per ewe lamb (39.6 ± 5.2) than did those born on a conventional farm (25.0 ± 4.2), and higher IVM (76.5% vs. 53.1%, P < 0.0001) and IVF (97.3 vs. 91%, P < 0.05) rates. In conclusion, this preliminary approach to the study of the effect of organic procedures on the sheep oocyte quality indicates that the total integration in the complete organic system improved the oocyte quality of ewe lambs, although organic feeding alone was insufficient to improve quality.

The tubulin cofactors TBCD and TBCE play an essential role in regulation of the microtubule dynamics in a wide variety of somatic cells, but little information is known about the expression of these cofactors in human sperm and oocytes. In this study, we focused on the investigation of the presence of, and the differential distribution of, the tubulin cofactors TBCD and TBCE in human sperm and during human oocyte maturation. We performed expression assays for TBCD and TBCE by reverse transcription-polymerase chain reaction (RT-PCR), western blot and immunofluorescence and verified the presence of both cofactors in human gametes. TBCD and TBCE were located mainly in the middle region and in the tail of the sperm while in the oocyte the localization was cytosolic. The mRNA of both tubulin cofactors were present in the human oocytes but not in sperm cells. This finding gives a first insight into where TBCD and TBCE could carry out their function in the continuous changes that the cytoskeleton experiences during gametogenesis and also prior to fertilization.

This study aimed to analyze the influence of different temperatures on the embryonic and larval development of discus fish Symphysodon aequifasciatus and determine the time required for the beginning of exogenous feeding. Eggs and larvae were obtained from natural spawns and distributed in five treatments: 24.0, 26.0, 28.0, 30.0, and 32.0 °C. To assess the developmental stages and embryonic structures, samples were taken at regular intervals and checked under an optical microscope. At the end of the experimental period, statistical analysis was performed, followed by Tukey’s test. As a result, it was possible to observe the significant effects of temperature on the variables. It was noted that the temperature accelerated the embryonic and larval development of the discus and also contributed to a reduction in the time between the incubation period and the feeding transition. It was also noted that the incubation of eggs and larvae at a temperature of 24.0 °C can cause damage to embryos, such as malformation of the body as well as anomalies in the circulatory system.

Spermatogenesis is an androgen-dependent event, and testosterone is the major androgen source. The enzyme 5-alpha reductase converts testosterone to dihydrotestosterone (DHT) in testicular and peripheral tissues. Polymorphisms in genes encoding 5-alpha reductase may be associated with impaired male fertility. The present study aimed to investigate the relationship between 5-alpha reductase type 2 (SRD5A2) gene rs523349 polymorphism and non-obstructive azoospermia (NOA) in Turkish patients. The study included 75 NOA patients and 43 fertile men from Turkey. No significant relationship was found between SRD5A2 gene rs523349 polymorphism and male infertility (P = 0.071). There was a statistically significant difference in total testosterone level and total testis volume between NOA patients and the control groups, however there was no significant difference between serum follicle-stimulating hormone and luteinizing hormone levels. Our results showed that SRD5A2 gene rs523349 polymorphism was not associated with NOA in Turkish patients.

Both the content and composition of polar and neutral lipids from the mitochondrial fraction of ovarian full-grown Bufo arenarum oocytes were analysed in the present study. Triacylglycerols (TAG) represent 33% of the total lipids, followed by phosphatidylcholine (PC), free fatty acids (FFA) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) or cardiolipin, a specific component of the inner mitochondrial membrane, represents about 4% of the total lipid content. Palmitic (16:0) and arachidonic (20:4n6) acids are the most abundant fatty acids in PC and PE, respectively. DPG is enriched in fatty acids with carbon chain lengths of 18, the principal component being linoleic acid. In phosphatidylinositol (PI), 20:4n6 and stearic acid (18:0) represent about 72 mol% of the total acyl group level. The main fatty acids in TAG are linoleic (18:2), oleic (18:1), and palmitic acids. The fatty acid composition of FFA and diacylglycerols (DAG) is similar, 16:0 being the most abundant acyl group. PE is the most unsaturated lipid and sphingomyelin (SM) has the lowest unsaturation index.

The goal of this study was to determine the distribution of preantral follicles in bovine ovaries. Follicular distribution in the ovaries (n = 12) was evaluated in the region of the greater curvature of the ovary (GCO) and the region close to the ovarian pedicle (OP) of Bos taurus indicus heifers of the Nelore breed. Two fragments were obtained from each region of the ovary (GCO and OP). The mean weight of the ovaries was 4.04 ± 0.32 g. The mean antral follicle count (AFC) was 54.58 ± 3.55 follicles (minimum and maximum variation of 30 and 71 follicles, respectively). In total, 1123 follicles were visualized in the region of the GCO; 949 (84.5%) of them were primordial follicles and 174 (15.5%) were developing follicles. The region close to the OP contained 1454 follicles, of which 1266 (87%) were primordial follicles and 44 (12.9%) were developing follicles. The OP region showed a higher proportion of intact follicles in the primordial (P < 0.0001) and primary (P = 0.042) stages compared with the GCO region. The proportion of secondary follicles was similar in the OP and GCO regions. The ovaries of two bovine females (16%; 2/12) contained multi-oocytes follicles, which were characterized as primary follicles. Therefore, the distribution of preantral follicles in the bovine ovary was heterogeneous, with the region close to the OP containing a greater number of preantral follicles compared with the GCO region (P < 0.05).

The maintenance and preservation of strains of mice used in biomedical research presents a unique challenge to individual investigators and research institutions. The goal of this study was to assess a comprehensive system for mouse strain conservation through a combination of natural mating, sperm cryopreservation and assisted reproductive technology. Our strategy was based on the collection and cryopreservation of fresh epididymal sperm from male mice by semi-vasectomy; these mice were then naturally mated for breeding purposes. If no satisfactory results were obtained from natural breeding, then the cryopreserved sperm were used for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI); resultant embryos were then transferred into pseudopregnant-recipient female mice. Our results show that some semi-vasectomized mouse strains can be conserved by natural breeding, and that sterile males can be compensated for through the use of IVF and ICSI technology. As such, we believe this system is suitable for the purpose of strain conservation, allowing the continuation of natural breeding with the safeguard of assisted reproduction available.

Wagyu bulls are known to have a highly exacerbated libido, as shown by the intense sexual interest of young calves. Therefore we believe that Wagyu male animals have specialized Sertoli and Leydig cells that are directly involved with the sexual precocity in this breed as mature bulls have a small scrotal circumference. This study aimed to evaluate whether there were differences in the hormone and sperm characteristics of Wagyu bulls compared with the same characteristics of subspecies Bos indicus and Bos taurus sires. Frozen–thawed semen from Wagyu, Nellore, and Angus sires were analyzed for sperm kinetics (computer-assisted sperm analysis), plasma membrane integrity, chromatin integrity, acrosome status, mitochondrial activity, lipid peroxidation and hormone [luteinizing hormone (LH) and testosterone] serum concentration. The results showed that Wagyu had lower total motility and an increased number of sperm with no motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from those breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation or hormone (LH and testosterone) concentrations. We concluded that Wagyu sires had lower total motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from these breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation, or hormone (LH and testosterone) concentrations.

This study was conducted to monitor the cellular and molecular changes of buffalo cumulus–oocytes complexes (COCs) cultured under high or low oxygen levels. Morphologically good quality COCs (n = 1627) were screened using brilliant cresyl blue (BCB) staining and placed into three groups (BCB+, BCB− and control). All groups of COCs were cultured under low (5%) or high (20%) oxygen tensions. Intracellular and molecular changes including oocyte ultrastructure, lipid contents, mitochondrial activity and transcript abundance of genes regulating different pathways were analyzed in the matured oocyte groups. The results revealed that oxygen tension did not affect cumulus expansion rates, however the BCB+ group had a higher (P ≤ 0.05) expansion rate compared with the BCB− group. BCB− oocytes recorded the lowest meiotic progression rate (P ≤ 0.05) under high oxygen levels that was linked with an increased level of reactive oxygen species (ROS) compared with the BCB+ oocytes. Ultrastructure examination indicated that BCB+ oocytes had a higher rate of cortical granules migration compared with BCB− under low oxygen tension. In parallel, our results indicated the upregulation of NFE2L2 in groups of oocytes cultured under high oxygen tension that was coupled with reduced mitochondrial activity. In contrast, the expression levels of MAPK14 and CPT2 genes were increased (P ≤ 0.05) in groups of oocytes cultured under low compared with high oxygen tension that was subsequently associated with increased mitochondrial activity. In conclusion, data from the present investigation indicated that low oxygen tension is a favourable condition for maintaining the mitochondrial activity required for nuclear maturation of buffalo oocytes. However, low-quality oocytes (BCB−) responded negatively to high oxygen tension by reducing the expression of gene-regulating metabolic activity (CPT2). This action was an attempt by BCB− oocytes to reduce the increased levels of endogenously produced ROS that was coupled with decreased expression of the gene controlling meiotic progression (MAPK14) in addition to nuclear maturation rate.

Interspersed RNA is an abundant class of cytoplasmic poly(A)+ RNA which contains repetitive elements within mostly heterogeneous single copy sequences. In spite of its quantitative importance in oocytes or eggs (two-thirds of the total poly(A)+ RNA), very little is known about its synthesis, its interaction with other molecules, and its functional significance. Here, we analysed a prevalent family of interspersed RNa (XR family) during Xenopus oogenesis. We found that XR interspersed RNA, unlike extracted interspersed RNA, did not form RNA duplexes in vivo. Im small oocytes (stage III), XR RNA interacted with proteins forming rapidly sedimenting ribonucleoprotein particles (RNPs) with a median sedimentaion constant of 80S. However, towards the end of oogenesis (stage VI), these XR RNPs changed into smaller particles with a median sedimentaion constant of 40S. By analysing the proteins associated with XR RNA sequence, we have identified a 42 kilodalton protein in small oocytes, which was replaced by a 45 kilodalton protein at stage V of oogenesis.

In this study, nuclear transfer (NT) embryos were produced by using C57Bl/6 mouse morula blastomeres and Kunming mouse metaphase II (MII) oocytes as donors and recipients, respectively, to investigate the effects of sucrose treatment of MII oocytes with different concentrations on the manipulation time of NT, electrofusion and the in vitro and in vivo development of reconstructed embryos. The results demonstrated that: (i) when the oocytes were enucleated with 1, 2 and 3% sucrose treatment, respectively, the enucleating rates were not affected by the different sucrose concentrations, but the manipulation time had significant difference and the mean nuclear transfer manipulation times of every oocyte were 180 ± 10 s, 130 ± 10 s and 120 ± 10 s, respectively; (ii) different sucrose concentrations had no significant effects on the fusion rate and the in vitro developmental potential of the NT embryos (p > 0.05). Furthermore, 59 embryos were transplanted into the oviducts of two recipients. In the end, three dead full-term developed fetuses were obtained on 21 days post coitus (dpc). These results suggested that the mouse MII oocytes enucleated via sucrose treatment might be an alternative source for mouse cloning and could support the embryonic NT embryos developed to term in vivo.

While all ascidians (Urochordata) are hermaphroditic, some, including Halocynthia roretzi (Fuke, 1983) and Ciona intestinalis (Rosati & De Santis, 1978) are strictly self-sterile because of a self–nonself recognition system in the interaction between the sperm and the vitelline coat of the eggs. However, immature oocytes (Fuke & Numakunai, 1996) and acidic-seawater-treated mature oocytes (Morgan, 1939; Kawamura et al., 1991) are self-fertile. It is thought that a putative self–nonself recognition molecule, which is detached or modified by treatment with acidic seawater, may be attached to the vitelline coat during oocyte maturation. Although the existence of a self–nonself recognition system in the fertilisation process is well known, the molecular entity has yet to be conclusively identified. However, there have been several attempts to identify such a molecule in Ciona (Marino et al., 1999). In the present study, we have isolated and analysed a molecule which appears to be responsible for allorecognition in the interaction between sperm and eggs of the ascidian Halocynthia roretzi.

Biologicals. A solitary ascidian Halocynthia roretzi Type C was used in this study. The fertilisation experiment was carried out as described previously (Sawada et al., 1982).

Isolation and N-terminal Sequencing of Hr VC70. Vitelline coats were isolated from immature and mature oocytes of the ascidian by homogenisation and repeated washing with 5× diluted artificial seawater. The isolated vitelline coats were subjected to SDS-PAGE, followed by blotting to a PVDF membrane. The N-terminal amino acid sequence of the 70 kDa main component (HrVC70) was determined by a protein sequencer.

The ingression of primary mesenchyme cells (PMCs) in the sea urchin embryo is initiated with local degradation of the basal lamina at the vegetal plate epithelium (e.g. Katow & Solursh, 1980). The ingressed PMCs encounter pamlin, a cell adhesion protein in the basal lamina (Katow, 1995), which guides PMC migration to a particular embryonic region to form a ring pattern (Katow & Komazaki, 1996; Katow et al, 2000). Thus extracellular matrix (ECM) provides a necessary guidance cue to the migratory cells, and this implicates the occurrence of intracellular signalling to promote not only cell locomotion but also orientation for the migration. Using embryos of the sea urchin, Hemicentrotus pulcherrimus, I report the temporal expression of P35, a PMC surface protein, during the very early stages of PMC ingression that is downregulated with SUp62 protein in the cytoplasm, and tyrosine phosphorylation of SUp62 as a consequence of PMCs encountering pamlin in light of ECM/cell signal transduction.

Chemical oocyte enucleation holds the potential to ease somatic cell nuclear transfer (SCNT), although high enucleation rates remain limited to micromanipulation-based approaches. Therefore, this study aimed to test mitomycin C (MMC) for use in bovine functional chemical oocyte enucleation. Incubation of denuded eggs in 10 µg ml−1 MMC for different periods did not affect most maturation rates (0.5 h: 85.78%A, 1.0 h: 72.77%B, 1.5 h: 83.87%A, and 2.0 h: 82.05%A) in comparison with non-treated controls (CTL; 85.77%A). Parthenogenetic development arrest by MMC was efficient at cleavage (CTL: 72.93%A, 0.5 h: 64.92%A,B, 1.0 h: 60.39%B,C, 1.5 h: 66.35%A,B, and 2.0 h: 53.84%C) and blastocyst stages (CTL: 33.94%A, 0.5 h: 7.58%B, 1.0 h: 2.47%C, 1.5 h: 0.46%C, and 2.0 h: 0.51%C). Blastocysts were obtained after nuclear transfer (NT) using MMC enucleation [NT(MMC): 4.54%B] but at lower rates than for the SCNT control [NT(CTL): 26.31%A]. The removal of the meiotic spindle after MMC incubation fully restored SCNT blastocyst development [NT(MMC+SR): 24.74%A]. Early pregnancies were obtained by the transfer of NT(MMC) and NT(MMC+SR) blastocysts to synchronized recipients. In conclusion, MMC leads to functional chemical oocyte enucleation during SCNT and further suggests its potential for application towards technical improvements.

This study attempted to investigate and validate whether epididymis cold storage could be a suitable alternative for short-term preservation of spermatozoa. Mouse cauda epididymides and spermatozoa were preserved at 4–8°C from 1 day to 6 weeks. From days 1 to 10, motility and fertility were daily examined when motility loss occurred. From week 1, spermatozoa were used for intracytoplasmic sperm injection (ICSI) at weekly intervals to test their fertility, and spermatozoa DNA integrity was determined by comet assay. We found that motility and progressive motility scores gradually decreased with storage time. In nearly all spermatozoa, DNA integrity was maintained from days 1 to 10, but the percentage of spermatozoa with damaged DNA significantly increased from week 2 to week 6. Spermatozoa retained fertility until day 6, although fertility gradually decreased after day 3. From week 1 to week 5, fertilization rates by ICSI were more than 82.69% but decreased gradually after week 3. We found that spermatozoa preserved in the epididymis at 4–8°C had progressively lower motility, fertility and proportion of undamaged DNA, but could still fertilize oocytes. However, all the parameters of cold-preserved spermatozoa were completely inferior to that from cold-preserved cauda epididymides. The results imply that cold storage of cauda epididymides could be conducive to short-term preservation of spermatozoa, and the cold-stored spermatozoa can resist DNA denaturation, which is necessary for maintaining reproductive ability.

Knowledge of the sperm–oocyte ratio in fish fertilization serves as the basis for studies on artificial reproduction and gamete manipulation. The aim of this study was to determine the minimum insemination dose for Brycon amazonicus oocyte fertilization. Female and male gametes were used and tested with the following doses of spermatozoa oocyte–1 ml–1: 10,000, 20,000, 40,000, 60,000 and 80,000 (in triplicate). Fertilization rates were calculated and estimated from the regression equation by applying the segmented regression model ‘Linear Response Plateau’ to determine the appropriate proportion of gametes. Based on the equation Ŷ = 14.3415 + 0.0007836X, the fertilization rate increased up to 63.34% as it reached a plateau with a proportion of 62,524 spermatozoa oocyte–1 ml–1, which is the minimum insemination dose recommended for artificial insemination of the species.

Polyclonal antibodies that cross-react with rodent metallothionein I (MT I) and metallothionein II (MT II) were microinjected in 1-cell and 2-cell mouse embryos, into either the cytoplasm or the nucleus. Regardless of the experimental treatment, mouse embryo development in vitro was not affected and most of the embryos cleaved normally until the morula stage. The results suggest that metallothionein is not essential for normal mouse early preimplantational development, in agreement with recent studies in mice with inactivated MT I and MT II genes.