This study investigated the effect of three different culture media (α minimum essential medium (α-MEM), McCoy or TCM199 during the in vitro culture (IVC) of bovine isolated pre-antral follicles. Pre-antral follicles greater than 150 μm in size were isolated and cultured for 0 (control), 8 or 16 days in one of the abovementioned culture media. Follicles were evaluated for survival, growth and antrum formation at days 8 and 16. The results showed that TCM199 was the most suitable medium to preserve follicular viability and ultrastructure, resulting in the highest rates of antrum formation. In conclusion, TCM199 promotes the in vitro development of isolated pre-antral follicles without hampering follicular functionality by sustaining in vitro growth and antrum formation.

The ‘maternal age effect’ in human reproduction, characterized by a negative relationship between maternal age and reproductive efficiency, remains a poorly understood phenomenon. Current data suggest that oocyte physiology determines this relationship. In this review, we present a hypothesis of a mitochondrial role in the physiology of ageing in human oocytes. We suggest that the efficiency of oxidative phosphorylation in the ageing human oocyte is degraded by free radical attack on the primordial oocytes residing in the ovary. Although deficiencies in oxidative phosphorylation can be accounted for in the short term by anaerobic respiration, we suggest that, in the long term, the level of oxidative phosphorylation strongly influences oocyte quality.

Tail-cut bovine spermatozoa were microinjected into ooplasmic lipid polarised, in vitro matured bovine oocytes using a piezomicropipette-driving system. No exogenous oocyte activation treatment was used. Of the sperm-injected oocytes, 86.3% were activated, 71.8% cleaved and 22.7% developed to the blastocyst stage. The average cell count of the blastocysts was 122.5 ± 15 and a majority (81.8%) of the blastocysts were cytologically normal (diploid). When transferred to recipient cows, 5 of 8 blastocysts developed to fetuses and 4 of 7 recipients became pregnant. Normal offspring were born.

Constant progress in the diagnosis and treatment of cancer disease has increased the number and prognosis of cancer survivors. However, the toxic effects of chemotherapy and radiotherapy on ovarian function have resulted in premature ovarian failure. Patients are, therefore, still expecting methods to be developed to preserve their fertility successfully. Several potential options are available to preserve fertility in patients who face premature ovarian failure, including immature or mature oocyte and embryo cryopreservation. However, for children or prepubertal women needing immediate chemotherapy, cryopreservation of ovarian tissue is the only alternative. The ultimate aim of this strategy is to implant ovarian tissue into the pelvic cavity (orthotopic site) or in a heterotopic site once oncological treatment is completed and the patient is disease free. Transplantation of ovarian tissue with sufficiently large numbers of follicles could potentially restore endocrine function and allow multiple cycles for conception. However, the success of ovarian tissue transplantation still has multiple challenges, such as the low number of follicles in the graft that may affect their longevity as well as the survival of the tissue during ex vivo processing and subsequent transplantation. Therefore, this review aims to summarize the achievements of ovary grafting and the potential techniques that have been developed to improve ovarian graft survival.

In the hatchery-bred tambaqui Colossoma macropomum, spontaneous semen release does not occur, and hand-stripping produces reduced semen volume. The goal of this work is to evaluate the effects of hormonal induction with carp pituitary extract (CPE) on both qualitative (visual aspect, pH, motility, viability and morphological abnormalities) and quantitative (volume, concentration and number of spermatozoa per ejaculate) traits of tambaqui semen. Eleven males were treated with CPE (induced), and 11 were left untreated as a control (non-induced). All analysed parameters except motility and percentage of viable spermatozoa presented significant differences (p < 0.05) between the induced and non-induced treatments. CPE induction resulted in a 25-fold increase in semen volume and a 10-fold increase in the number of spermatozoa collected. However, both sperm concentration and the frequency of sperm with morphological abnormalities (commonly detached heads or bent tails) were significantly lower in CPE-induced fish. The hormonal induction of tambaqui males with CPE is efficient and positively influences some qualitative and quantitative properties of semen. Additionally, semen collection via gentle abdominal massage occurs more readily in CPE-induced fish.

Nerve growth factor (NGF) is a prototype member of the neurotrophins family and has important functions in the maintenance of viability and proliferation of neuronal and non-neuronal cells, such as certain ovarian cells. The present review highlights the role of NGF and its receptors on ovarian follicle development. NGF initiates its multiple actions through binding to two classes of receptors: the high affinity receptor tyrosine kinase A (TrkA) and the low-affinity receptor p75. Different intracytoplasmic signalling pathways may be activated through binding to NGF due to variation in the receptors. The TrkA receptor activates predominantly phosphatidylinositol-3-kinase (PI3K) and mitogenic activated protein kinase (MAPK) to promote cell survival and proliferation. The activation of the phospholipase type Cγ (PLCγ) pathway, which results in the production of diacylglycerol (DAG) and inositol triphosphate (IP3), culminates in the release of calcium from the intracytoplasmic cellular stocks. However, the details of activation through p75 receptor are less well known. Expression of NGF and its receptors is localized in ovarian cells (oocyte, granulosa, theca and interstitial cells) from several species, which suggests that NGF and its receptors may regulate some ovarian functions such as follicular survival or development. Thus, the use of NGF in culture medium for ovarian follicles may be of critical importance for researchers who want to promote follicular development in vitro in the future.

The optimum oxygen tension for culturing mammalian embryos has been widely debated by the scientific community. While several laboratories have moved to using 5% as the value for oxygen tension, the majority of modern in vitro fertilization (IVF) laboratory programmes still use 20%. Several in vivo studies have shown the oxygen tension measured in the oviduct of mammals fluctuates between 2% and 8% and in cows and primates this values drops to <2% in the uterine milieu. In human IVF, a non-physiological level of 20% oxygen has been used in the past. However, several studies have shown that atmospheric oxygen introduces adverse effects to embryo development, not limited to numerous molecular and cellular physiology events. In addition, low oxygen tension plays a critical role in reducing the high level of detrimental reactive oxygen species within cells, influences embryonic gene expression, helps with embryo metabolism of glucose, and enhances embryo development to the blastocyst stage. Collectively, this improves embryo implantation potential. However, clinical studies have yielded contradictory results. In almost all reports, some level of improvement has been identified in embryo development or implantation, without any observed drawbacks. This review article will examine the recent literature and discusses ongoing efforts to understand the benefits that low oxygen tension can bring to mammal embryo development in vitro.

This study was designed to examine the effects of extracellular reduced glutathione on development of pig embryos, produced by in vitro maturation (IVM) and in vitro fertilisation (IVF), in a chemically defined North Carolina State University (NCSU) 23 medium or in NCSU 23 medium with bovine serum albumin (BSA). Microfilament distribution, as a marker of embryo quality, was also examined by immunocytochemical staining and confocal microscopy. When the inseminated oocytes were cultured in the defined medium containing 0-0.5 mM glutathione, blastocyst formation as observed only in the media with glutathione (8.5-16.0%). Increased numbers of blastomeres were observed in the blastocysts as the concentration of glutathione was increased (18.8±7.2 to 31.0±8.6). In NCSU 23 medium with 4 mg BSA/ml, addition of glutathione at concentrations of 0.125-0.5 mM significantly increased the proportions of oocytes that developed to blastocysts (39.2-52.5%) compared with the control (29.5%). However, no difference was observed in the average cell number in the blastocysts (41.9±15.6 to 49.1±15.5). There were no significant differences in the microfilament distribution in the embryos produced in the defined medium and in the BSA-containing medium. These results indicate that pig embryos produced by IVM/IVF can develop to the blastocyst stage in a defined medium. BSA and glutathione have a synergistic effect on pig embryo development.

The decrease in maturation-promoting factor (MPF) activity precedes that in mitogen-activated protein kinase (MAPK) activity after egg activation, but the cellular functions of this delayed inactivation of MAPK are still unclear. The present study was conducted to examine the essential role of MAPK activity for supporting the transition from metaphase to interphase in porcine oocytes matured in vitro. The increases in the phosphorylated forms of MAPK and the activities of MAPK and histone H1 kinase (H1K) were shown in oocytes arrested at the metaphase II (MII) stage. After additional incubation of MII-arrested oocytes in medium with added U0126, a specific inhibitor of MAPK kinase, 24% of oocytes completed the second meiotic division and underwent entry into interphase with pronucleus (PN) formation, but not second polar body (PB-2) emission. The intensities of the phosphorylated forms of MAPK and the activities of MAPK and H1K in matured oocytes treated with U0126 were significantly decreased by the treatment with U0126. Electrostimulation to induce artificial activation caused both H1K and MAPK inactivation; the inactivation of H1K preceded the inactivation of MAPK and sustained high levels of MAPK activity were detected during the period of PB-2 emission. However, the time sequence required for MAPK inactivation was significantly reduced by the addition of U0126 to the culture medium following electrostimulation, resulting in the dramatic inactivation of MAPK distinct from that of H1K. In these oocytes, PB-2 emission was markedly inhibited but little difference was found in the time course of PN formation compared with oocytes not treated with U0126. These findings suggest that the decrease in MAPK activity is partly involved in driving matured oocytes out of metaphase to induce PN development, and that the delayed MAPK inactivation after the onset of MPF inactivation in activated oocytes has a crucial role for PB-2 emission to accomplish the transition from meiosis to mitosis.

To determine the minimal amount of cytoplasm necessary to support normal development of mouse oocytes, mature unfertilised oocytes were reduced in size into approximately 1/2, 1/4 and 1/8 by removing the cytoplasm, then inseminated. More than 80% of 1/2-size oocyte were fertilised normally and almost all fertilised eggs developed to blastocysts. When transferred to foster females, 31% of the blastocysts developed into normal offspring. In contrast, 1/4- and 1/8-size oocytes, although they were penetrable by spermatozoa and extruded the second polar bodies, could not reach the 2-cell stage. In these oocytes, sperm nuclei did not develop into full-sized pronuclei. These results suggest that an oocyte can develop to full term after losing about half its cytoplasm, but not more.

The objective of the present study was to evaluate the influence of heat stress on bovine oocyte maturation. Both nuclear stage and distribution of cortical granules (CG) were simultaneously evaluated in each oocyte. Oocyte overmaturation under standard conditions of culture was also evaluated. For this purpose, logistic regression procedures were used to evaluate possible effects of factors such as heat stress, overmaturation, replicate, CG distribution and metaphase II (MII) morphology on oocyte maturation. Based on the odds ratio, oocytes on heat stressed (HSO) and overmaturated (OMO) oocyte group were, respectively, 14.5 and 5.4 times more likely to show anomalous MII morphology than those matured under control conditions (CO). The likelihood for an oocyte of showing the CG distribution pattern IV (aging oocyte) was 6.3 and 9.3 times higher for HSO and OMO groups, respectively, than for the CO group. The risk of undergoing anomalous oocyte maturation, considering both nuclear stage and distribution of CG was 17.1 and 18 times greater in oocytes cultured in HSO and OMO groups, respectively, than those in the CO group. In conclusion, heat stress proved to be valuable in aging oocytes. Heat stress advanced age for nuclear and cytoplasmic processes in a similar form to that of oocyte overmaturation.

The aim of the present study was to assess the role of follicle stimulating hormone (FSH), epidermal growth factor (EGF) or a combination of EGF and FSH on the in vitro growth of porcine preantral follicles, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. Porcine preantral follicles were cultured for 3 days in the absence or in the presence of FSH or EGF. Oocytes from these follicles were then matured, fertilized in vitro and embryos were cultured. Estradiol secretion and histological analysis of cultured follicles were also carried out. The results showed that when FSH, or a combination of EGF and FSH, was added to the culture medium, most of preantral follicles grew to antral follicles with high estradiol secretion and the oocytes from these antral follicles could mature, fertilize and develop to the blastocyst stage. Without FSH, or a combination of EGF and FSH, preantral follicles were unable to develop to the antral stage. Histology demonstrated that the resulting follicles were nonantral, estradiol production was reduced and none of their oocytes matured after in vitro maturation. The results indicate the essential role of FSH in promoting in vitro growth of porcine preantral follicle, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. EGF with FSH treatment of porcine preantral follicles improves the quality of oocytes, shown by a higher frequency of embryonic development.

The black armoured catfish Rhinelepis aspera is an important commercial species found in the São Francisco River (Brazil) that has great potential for pisciculture. This paper presents, for the first time, information on the embryogenesis and larval development of this species. The breeder fish were subjected to spawning induction with crude carp pituitary extract. Eggs were kept in incubators at 24°C and embryogenesis was observed with a stereomicroscope until hatching. Ovary fragments, samples of recently extruded oocytes and larvae were fixed in Bouin's solution and subjected to routine histology techniques. Recently extruded oocytes were also analyzed with a scanning electron microscope (SEM). Females (80%) had a positive response to hypophysation. Oocyte extrusion was performed at 8 h and 30 min after the second hormone injection and the fertilization rate achieved 72.4 ± 8.8%. Recently extruded oocytes had a mean diameter of 1360.80 ± 40.87 μm and presented a conspicuous jelly coat covering the zona pellucida. Histochemical reactions indicated the presence of neutral glycoproteins in the yolk globules and in the zona pellucida. These reactions also indicated the presence of neutral glycoproteins and carboxylated acid glycoconjugates in the follicular cells and the jelly coat. These compounds form mucosubstances that can be related to the adhesiveness of the eggs. Under the SEM the oocytes presented a micropyle disc and a thick jelly coat over their surface. The closure of the blastopore occurred 9 h and 40 min after fertilization and embryogenesis was completed at 45 h and 50 min after fertilization, when the embryos were incubated at 24°C. The total absorption of the yolk sac occurred during the fifth day indicating the need for an exogenous food supply. These results provide essential information for the successful breeding and management of the promising R. aspera.

Fertilisation is a highly programmed process by which two radically different cells, sperm and egg, unite to form a zygote, a cell with somatic chromosome numbers. Development of the zygote begins immediately after sperm and egg haploid pronuclei come together, pooling their chromosomes to form a single diploid nucleus with the parental genes. Mammalian fertilisation is the net result of a complex set of molecular events which allow the capacitated spermatozoa to recognise and bind to the egg's extracellular coat, the zona pellucida (ZP), undergo the acrosome reaction, and fuse with the egg plasma membrane. Sperm-zona (egg) interaction leading to fertilisation is a species-specific carbohydrate-mediated event which depends on glycan-recognising proteins (glycosyltransferases/glycosidases/lectin-like molecules) on sperm plasma membrane (receptors) and their complementary glycan units (ligands) on ZP. The receptor-ligand interaction event initiates a signal transduction pathway resulting in the exocytosis of acrosomal contents. The hydrolytic action of the sperm glycohydrolases and proteases released at the site of sperm-egg interaction, along with the enhanced thrust generated by the hyperactivated beat pattern of the bound spermatozoon, are important factors regulating the penetration of egg investments. This review focuses on sperm molecules believed to be important for the interaction with the female genital tract, passage through cumulus oophorus and attachment to ZP, induction of the acrosome reaction, secondary binding events, and passage through the ZP. An understanding of the expression and modifications of molecules thought to be important in multiple events leading to fertilisation will allow new strategies to block these modifications and alter sperm function.

This study was conducted to determine the effects of l-carnitine (LC), as an antioxidant, in preventing spermatozoa damage during the freezing–thawing process in both astheno- and normozoospermic human semen samples. Seventy semen samples (37 asthenozoospermic and 33 normozoospermic) were involved in this study. Cryopreservation medium supplemented with 1.0 g/l LC was mixed with semen at a ratio of 1:1 (v/v). Controls were cryopreserved with freezing medium only. Assessment of motility, viability (VIA), mitochondrial membrane potential (MMP) and DNA fragmentation index (DFI) were performed on aliquots of fresh semen, frozen–thawed control and frozen–thawed LC treated samples. Supplementation of the cryopreservation medium with LC induced a significant improvement in post-thaw sperm parameters in both the asthenozoospermic and normozoospermic semen samples, compared with those of the control, regarding sperm fast forward motility, forward motility, total motility and VIA. LC showed better protective effects towards asthenozoospermia for DFI (F = 115.85, P < 0.01) and VIA (F = 67.14, P < 0.01) than did normozoospermic semen samples. We conclude that supplementation with LC prior to the cryopreservation process reduced spermatozoa cryodamage in both asthenozoospermic and normozoospermic semen samples. LC had better protective effects for asthenozoospermic human semen samples. Future research should focus on the molecular mechanism for and the different protective effects of LC between asthenozoospermic and normozoospermic semen samples during cryopreservation.

Tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are cytokines that are involved in the development, proliferation and apoptosis of ovarian follicular cells in domestic mammals. The expression of these cytokines in various follicular compartments, depending on the stage of follicle development, demonstrates their involvement in the control of primordial follicle growth up to the preovulatory stage. The mechanism of action of these factors depends on the presence of their receptors that transduce their biological actions. This review shows the expression sites of TNF-α, IL-1β and their receptors in ovarian follicles, and discusses the mechanism of action of these cytokines during follicle development, oocyte maturation and ovulation in domestic animals.

Several ultrastructural changes occur during dehydration and subsequent rehydration of Arabidopsis thaliana pollen. The cytoplasmic channels, present in the outer part of the intine of the mature, dehydrating pollen grain, degenerate and develop into electron-dense inclusions. At the same time a large quantity of electron-dense material is deposited in the cavities of the exine. A large number of vesicles is produced in the vegetative cell, and they become predominantly located in the peripheral region near the intine. Starch of amyloplasts is consumed and the lipid bodies which originally surround the sperm cells become randomly distributed. In addition, the individual lipid bodies become enveloped by single rough endoplasmic reticulum cisterns.

During the early preimplantation stages of human embryos, pyruvate and lactate, but not glucose, are the preferred energy substrates. Transport of these monocarboxylates is mediated, in mammalian cells, by a family of transporters, designated as monocarboxylate transporters (MCTs). Human and mouse genetic expression of MCT members 1, 2, 3, 4 and basigin, a chaperone protein of MCT1 and MCT4, was qualitatively analysed using the reverse transcription nested polymerase chain reaction (RT-nested PCR) in immature oocytes (germinal vesicle stage; GV), in non-fertilised metaphase II (MII) oocytes and in embryos from 2-cell stage to blastocysts. Transcripts encoding for MCT1 and MCT2 were present, under a polyadenylated form, in the majority of the human and mouse oocytes and early embryos. MCT3 transcripts were not detected in either human or mouse. MCT4 mRNA was not detected in human oocytes and embryos, but was present in mouse oocytes and embryos. This fact could imply differences in lactate transport and regulation of intracellular pH between human and murine early embryos. Basigin transcripts were present in mouse and human MII oocytes and preimplantation embryos, but were not detected at GV stage. However, using 3' end-specific primers in the RT reaction instead of Oligo(dT)12-18 primers, transcripts encoding for this protein were then detected at GV stage in both species. This result suggests that a regulated polyadenylation process occurs during oocyte maturation for these transcripts. Thus, basigin mRNA can be considered as a marker of oocyte cytoplasmic maturation in human and mouse species.

Heat shock may disrupt oocyte function by increasing the generation of reactive oxygen species (ROS). We evaluated the capacity of the antioxidant melatonin to protect oocytes using two models of oxidative stress – heat shock and the pro-oxidant menadione. Bovine cumulus–oocyte complexes (COC) were exposed in the presence or absence of 1 µM melatonin to the following treatments during maturation: 38.5°C, 41°C and 38.5°C+5 µM menadione. In the first experiment, COC were matured for 3 h with 5 µM CellROX® and analyzed by epifluorescence microscopy to quantify production of ROS. The intensity of ROS was greater for oocytes exposed to heat shock and menadione than for control oocytes. Melatonin reduced ROS intensity for heat-shocked oocytes and oocytes exposed to menadione, but not for control oocytes. In the second experiment, COC were matured for 22 h. After maturation, oocytes were fertilized and the embryos cultured for 7.5 days. The proportion of oocytes that cleaved after fertilization was lower for oocytes exposed to heat shock and menadione than for control oocytes. Melatonin increased cleavage for heat-shocked oocytes and oocytes exposed to menadione, but not for control oocytes. Melatonin tended to increase the developmental competence of embryos from heat-shocked oocytes but not for embryos from oocytes exposed to menadione or from control oocytes. In conclusion, melatonin reduced production of ROS of maturing oocytes and protected oocytes from deleterious effects of both stresses on competence of the oocyte to cleave after coincubation with sperm. These results suggest that excessive production of ROS compromises oocyte function.

Pronuclear formation and intracellular content of glutathione, containing reduced and oxidised forms, in porcine oocytes matured in vitro were determined following insemination and/or electrical stimulation. After insemination, sperm penetration had occurred as early as 3 h and female pronuclei had formed by 6 h with complete development by 12 h. Male pronuclear formation occurred, primarily, between 9 and 12 h after insemination. Glutathione content of the oocytes decreased following sperm penetration and remained at a depressed level until 12 h. After electrical stimulation, oocyte activation had occurred and female pronuclei had formed by 3 and 6 h, respectively. Oocyte glutathione content did not change as a result of oocyte activation. When oocytes were exposed to an electrical pulse and then spermatozoa, female pronuclear formation was observed by 3 h after stimulation/insemination. Sperm penetration was observed between 3 and 9 h. However, the incidence of male pronuclear formation observed at 12 h was extremely low, although sperm decondensation had occurred in some oocytes. Oocyte glutathione content had not decreased by 6 h following electrical activation. These results demonstrate that the changes in glutathione content in porcine oocytes following fertilisation in vitro differ from those due to electrical activation. Further, the decreased intracellular glutathione content in oocytes activated by sperm penetration appears to be due to the presence of a sperm factor.