Efects of the pine needle abortifacient, isocupressic acid, on bovine oocyte maturation and preimplantation embryo development

Isocupressic acid (ICA) [15-hydroxylabda-8 (17), 13E-dien-19-oic acid], a labdane diterpene acid, isolated from ponderosa pine (Pinus ponderosa), Lodgepole pine (Pinus contorta), common juniper (Juniperus communis) and Monterey cypress (Cupressus macrocarpa), induces abortion in pregnant cows when ingested primarily during the last trimester. The objective of this study was to investigate the effects of isocupressic acid on bovine oocyte maturation (in vitro maturation (IVM)-Experiment I) and preimplantation embryo development (in vitro culture (IVC)-Experiment II) using in vitro embryo production techniques and to subsequently evaluate viability and developmental competence of ICA-cultured embryos via embryo transfer to recipient heifers (Experiment III). A complete randomized block experimental design was used. In Experiment I and II, isocupressic acid was added to IVM or IVC media at 0 (TRT1, control), 1.3 (TRT2), and 2.6 microg/ml (TRT3) Results from Experiment I and II indicated that ICA did not inhibit oocyte maturation and did not adversely affect preinpiantation embryo development. Furthermore, results from Experiment II demonstrated that isocupressic acid enhanced bovine preimplantation embryo development in vitro in a dose dependent manner. Subsequently, Day 8 (Day 0 = IVF) blastocysts cultured in vitro in the medium containing 2.6 microg/ml ICA were transferred to recipient heifers and resulted in normal pregnancies as determined by ultrasound imaging. Subsequently, all but two births were normal as evaluated by post natal veterinary examination. In conclusion, ICA showed no adverse effects on oocyte maturation and preimplantation embryo development in vitro or subsequent viability in vivo using the ICA concentrations and in vitro culture parameters of this study.     

Charles Thibault and assisted reproduction in France

Charles Thibault was liked by French gynaecologists. There was not a year that Charles Thibault did not attend clinician gynaecology conferences. He made great strides in research on in vitro fertilisation, being the first to perform in vitro fertilised (IVF) oocyte transfers in rabbits. Later, in 1978 the first human pregnancy following IVF was achieved in the UK when Louise Brown was born. In 1980, two French teams,one at the Sèvres hospital and the other at the Clamart University Teaching Hospital, carried out egg retrievals in patients with natural cycles, after determination of the urinary LH peak, under general anaesthesia and by laparoscopy. The Clamart team developed LH SIR, which enabled a more accurate determination of the ideal time for egg collection. In 1983, the same team reported the first ambulatory oocyte retrievals by ultrasound, under local anaesthesia. This new technique did not require general anaesthesia. Finally, in 1983, the rate of births, per transfer, for the Sèvres team rose to 5.31%. 1984 showed considerable improvement: 13.83%. The first step in establishing IVF in France was completed with the Carghese symposium, in September 1984, where Charles Thibault pleaded for animal experimentation before human clinical trials. It was only later that ART developed significantly, necessitating a legislative framework and organisations such as GEFF and FIVNAT.     

Effects of leptin on in vitro maturation, fertilization and embryonic cleavage after ICSI and early developmental expression of leptin (Ob) and leptin receptor (ObR) proteins in the horse

Background  

The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity after fertilization by IntraCytoplasmic Sperm Injection (ICSI).     

Potential researchable areas in ARTs--oocyte maturation and embryo development

Infertility is a commonly encountered situation occurring equally in both sexes. In vitro fertilization and embryo transfer (IVF-ET) and other assisted reproductive technologies (ARTs) have enhanced the possibilities for successful treatment to tackle infertility. However, ARTs currently face limitations due to the fact that although success rate is high for the initial stages such as ovulation induction and fertilization, it dwindles progressively so that the success rate of a take home baby is as low as 15-20%. Research centred around various stages in an IVF programme is therefore necessary to devise protocols that ensure a higher success rate. This review takes a look at the potential areas currently under research in the field of ARTs, such as, in vitro oocyte maturation, oocyte/embryo cryopreservation, embryo culture, preimplantation genetic diagnosis. Their applications, in clinical conditions such as cancer, have been discussed.     

Meiosis-activating sterol promotes the metaphase I to metaphase II transition and preimplantation developmental competence of mouse oocytes maturing in vitro

The objective of this study was to determine the effects of a sterol found in ovarian follicular fluid, known as meiosis-activating sterol (FF-MAS), on the maturation of mouse oocytes in vitro. Possible effects of FF-MAS in promoting the metaphase I (MI) to metaphase II (MII) transition (nuclear maturation) and the competence of oocytes to complete preimplantation embryo development to the blastocyst stage (cytoplasmic maturation) were assessed. Cumulus cell-enclosed oocytes that were compromised in their ability to undergo nuclear maturation and subsequent development because of the age or genotype of the female were isolated at the germinal vesicle stage and matured in vitro using media supplemented with 0 to 20 microM FF-MAS. Oocytes that progressed to MII were inseminated in vitro, and the percentages developing to the 2-cell and blastocyst stages were determined. The sterol was omitted from the media used for oocyte insemination or preimplantation development. FF-MAS promoted a significantly higher percentage of oocytes in all groups to progress to MII in vitro. Moreover, FF-MAS treatment of oocytes maturing in vitro dramatically increased the competence of all but one of the groups of oocytes to complete preimplantation development. Therefore, FF-MAS improved mouse oocyte quality by promoting both nuclear and cytoplasmic maturation in vitro.     

Intrafollicular amino acid concentration and the effect of amino acids in a defined maturation medium on porcine oocyte maturation, fertilization, and preimplantation development

The objective of this study was to determine the intrafollicular concentrations of free amino acids in pigs and to examine the effect of amino acids in a chemically defined maturation medium on oocyte maturation, in vitro fertilization (IVF), and embryo development in vitro. Pooled follicular fluid aspirated separately from small (<3mm in diameter), medium (3-8mm), and large follicles (>8mm) in three pairs of ovaries was analyzed for amino acid concentration. In addition, oocyte maturation, fertilization, and embryo development were examined after in vitro maturation (IVM) of oocytes in a defined maturation medium supplemented individually with glutamate (GLU), glutamine (GLN), glycine (GLY), aspartate (ASP), asparagine (ASN), arginine (ARG), alanine (ALA), leucine (LEU), lysine (LYS), proline (PRO), and valine (VAL). Irrespective of follicle size, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pig follicular fluid (pFF). Sperm penetration was not altered by amino acid treatment during IVM, but monospermic fertilization was increased (P<0.05) by GLN, ASP, and VAL. All amino acids except ASP and ASN stimulated (P<0.05) male pronuclear formation after IVF. ARG and ALA treatment during IVM improved (P<0.05) blastocyst formation. In conclusion, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pFF and amino acids in a defined medium improved porcine monospermic fertilization, male pronuclear formation, and preimplantation development.     

Maternal supply of omega-3 polyunsaturated fatty acids alter mechanisms involved in oocyte and early embryo development in the mouse

Despite the well-known benefits of omega-3 (n-3) polyunsaturated fatty acid (PUFA) supplementation on human health, relatively little is known about the effect of n-3 PUFA intake on fertility. More specifically, the aim of this study was to determine how oocyte and preimplantation embryo development might be influenced by n-3 PUFA supply and to understand the possible mechanisms underlying these effects. Adult female mice were fed a control diet or a diet relatively high in the long-chain n-3 PUFAs for 4 wk, and ovulated oocytes or zygotes were collected after gonadotropin stimulation. Oocytes were examined for mitochondrial parameters (active mitochondrial distribution, mitochondrial calcium and membrane potential) and oxidative stress, and embryo developmental ability was assessed at the blastocyst stage following 1) in vitro fertilization (IVF) or 2) culture of in vivo-derived zygotes. This study demonstrated that exposure of the oocyte during maturation in the ovary to an environment high in n-3 PUFA resulted in altered mitochondrial distribution and calcium levels and increased production of reactive oxygen species. Despite normal fertilization and development in vitro following IVF, the exposure of oocytes to an environment high in n-3 PUFA during in vivo fertilization adversely affected the morphological appearance of the embryo and decreased developmental ability to the blastocyst stage. This study suggests that high maternal dietary n-3 PUFA exposure periconception reduces normal embryo development in the mouse and is associated with perturbed mitochondrial metabolism, raising questions regarding supplementation with n-3 PUFAs during this period of time.     

Update on reproductive biotechnologies in small ruminants and camelids

Recent advances in reproductive biotechnologies in small ruminants include improvement of methods for in vitro production of embryos and attempts at spermatogonial stem cell transplantation. In vitro production of embryos by IVM/IVF, intra-cytoplasmic sperm injection (ICSI), or nuclear transfer (NT) has been made possible by improvements in oocyte collection and maturation techniques, and early embryo culture systems. However, in vitro embryo production still is not very efficient due to several limiting factors affecting the outcome of each step of the process. This paper discusses factors affecting in vitro embryo production in small ruminants and camelids, as well as preliminary results with the technique of spermatogonial stem cell transplantation.     

Maternal diabetes and oocyte quality

Maternal diabetes has been demonstrated to adversely affect preimplantation embryo development and pregnancy outcomes. Emerging evidence has implicated that these effects are associated with compromised oocyte competence. Several developmental defects during oocyte maturation in diabetic mice have been reported over past decades. Most recently, we further identified the structural, spatial and metabolic dysfunction of mitochondria in oocytes from diabetic mice, suggesting the impaired oocyte quality. These defects in the oocyte may be maternally transmitted to the embryo and then manifested later as developmental abnormalities in preimplantation embryo, congenital malformations, and even metabolic disease in the offspring. In this paper, we briefly review the effects of maternal diabetes on oocyte quality, with a particular emphasis on the mitochondrial dysfunction. The possible connection between dysfunctional oocyte mitochondria and reproductive failure of diabetic females, and the mechanism(s) by which maternal diabetes exerts its effects on the oocyte are also discussed.     

Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development

alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.     

The mycotoxin beauvericin induces oocyte mitochondrial dysfunction and affects embryo development in the juvenile sheep

Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus‐oocyte‐complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase‐mediated dUTP nick‐End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short‐term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long‐term carry‐over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late‐stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3–0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.     

Responses of canine oocytes to in vitro maturation and in vitro fertilization outcome

The potential benefits of assisted reproduction techniques, such as in vitro maturation (IVM) and in vitro fertilization (IVF) in canids, are linked to the protection and saving of species threatened by extinction due to worldwide habitat destruction and pollution. In both domestic and wild species, these technologies will form the basis for the next leap in reproductive performance by improving fertility rates in valuable middle-aged females, by improving pregnancy rate in infertile or sub-fertile populations and by rescuing biological material to replenish populations of endangered species. In vitro techniques are supposed to answer the reproductive questions of canids, to introduce new methods for contraception and to compete with artificial insemination (AI) as the major or predominant method of embryo production, oocyte- and embryo cryopreservation and cloning. The causes affecting in vitro meiosis of dog oocytes are likely to be diverse. Incomplete understanding of the events associated with oocyte developmental competence are imputed to species reproductive physiology, medium composition and source of ovarian oocyte population used for in vitro maturation. This review addresses some issues on the current state of in vitro maturation and in vitro fertilization of canine oocytes.     

家猫的胚胎工程

家猫是惟一一种没有被列为珍稀或濒危的猫科动物。通过家猫的胚胎工程研究,对保护其它濒危猫科物种有重要的借鉴意义。本文描述了家猫的一般生殖特点,着床前的胚胎在体内的发育概况;综述了近年来对家猫的超数排卵,卵母细胞的体外成熟,体外受精,胚胎的体外培养,胚胎移植,冷冻保存和胚胎克隆等方面的研究进展。     

Effects of in vitro maturation of monkey oocytes on their developmental capacity

The study of in vitro maturation (IVM) of rhesus monkey oocytes has important implications for biomedical research and human infertility treatment. In vitro-matured rhesus monkey oocytes show much less developmental potential than IVM oocytes of other species. Since about 1980 when rhesus monkey IVM, in vitro fertilization (IVF) and in vitro embryo culture (IVC) systems were established, numerous efforts have been made to improve the developmental competence of oocytes and to understand the mechanisms regulating oocyte maturation. This review describes recent progress in this area, particularly the effects of factors such as steroid hormones, energy substrates, amino acids, ovarian follicle status, maternal age and breeding season on the developmental competence, gene expression patterns and genome integrity of rhesus IVM oocytes.     

Environment of oocyte and embryo determines health of IVP offspring

In vitro embryo production (IVP) enhances the number of offspring from a single female and offers the possibility of accelerated genetic progress in animal husbandry. However, it also leads to a low but unacceptable percentage of anomalies in the offspring. The aim of this paper is to introduce the three speakers at this afternoon session who will speak about the demands of culture conditions and the endometrial environment to support normal embryonic development without effects on the embryonic genome. It will be argued that the in vitro conditions should mimic precisely the oviductal contributions to homeostatic mechanisms within the embryo. The further normal development can be guaranteed at synchrony in development of both endometrium and embryo. If that is not the case one can expect disturbances of gene expression, in particular of imprinted genes. However, it cannot be excluded that some processes might have started already in the cytoplasm of the oocyte. Since the oocyte was not planned to be a separate subject in this symposium, this introduction is also aimed to ask attention for the selection of cumulus-oocyte-complexes (COCs) and the conditions around oocyte maturation in vitro. The optimal quality of both the oocyte and maturation medium are prerequisites for an undisturbed cytoplasmic maturation. It has been argued that the exclusion of COCs from atretic follicles, the abjuration of the use of serum and high O2 tension in the gas phase might help to reduce the proportion anomalies in the offspring after synchronous transfers. In human IVF, in vivo matured oocytes are used with no great problems. But before IVP, including oocyte maturation in vitro (IVM) and a longer lasting embryo culture (IVC), will be introduced into the human assisted reproduction, it is important to think about the ins and outs of the potential causes for deviations.     

Microorganisms within Human Follicular Fluid: Effects on IVF

Our previous study reported microorganisms in human follicular fluid. The objective of this study was to test human follicular fluid for the presence of microorganisms and to correlate these findings with the in vitro fertilization (IVF) outcomes. In this study, 263 paired follicular fluids and vaginal swabs were collected from women undergoing IVF cycles, with various causes for infertility, and were cultured to detect microorganisms. The cause of infertility and the IVF outcomes for each woman were correlated with the microorganisms detected within follicular fluid collected at the time of trans-vaginal oocyte retrieval. Microorganisms isolated from follicular fluids were classified as: (1) ‘colonizers’ if microorganisms were detected within the follicular fluid, but not within the vaginal swab (at the time of oocyte retrieval); or (2) ‘contaminants’ if microorganisms detected in the vagina at the time of oocyte retrieval were also detected within the follicular fluid. The presence of Lactobacillus spp. in ovarian follicular fluids was associated with embryo maturation and transfer. This study revealed microorganisms in follicular fluid itself and that the presence of particular microorganisms has an adverse affect on IVF outcomes as seen by an overall decrease in embryo transfer rates and pregnancy rates in both fertile and infertile women, and live birth rates in women with idiopathic infertility. Follicular fluid microorganisms are a potential cause of adverse pregnancy outcomes in IVF in both infertile women and in fertile women with infertile male partners.     

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.     

Transient exposure to sodium butyrate after germinal vesicle breakdown improves meiosis but not developmental competence in pig oocytes

Oocyte maturation is a complex process during which epigenetic modifications are dramatically changed, especially histone acetylation and phosphorylation. We have investigated the effects of NaBu (sodium butyrate), a natural HDAC (histone deacetylase) inhibitor, on porcine oocyte maturation at different stages and subsequent embryonic development to improve IVF (in vitro fertilization) and embryo production. COCs (cumulus oocyte complexes) were cultured, IVM (in vitro maturation) supplemented with 1 mM NaBu before or after GVBD [GV (germinal vesicle) breakdown] during maturation. NaBu delayed oocyte meiosis in the GV and GVBD stages in an exposure-dependent manner. However, the short treatment with 1 mM NaBu after GVBD significantly improved the meiotic competence. No positive effects of NaBu on GSH levels and subsequent embryonic development following IVF were seen. Transient exposure to NaBu after GVBD improves meiotic competence, but not subsequently, probably by having an effect on histone acetylation during oocyte maturation.     

A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.