The secretion and metabolism of cumulus cells support fertilization in the bovine model.

The cumulus oophorus is a structure that surrounds the mammalian egg and plays a key role in fertilization. However, very little is known with regards to how secretions from the cumulus cells can specifically promote fertilization. We hypothesized that secretions from bovine cumulus cells, and the reduction of oxygen stress by metabolic change, would enhance the fertilization capacity of sperm during in vitro fertilization (IVF) procedures. To prove our hypothesis, sperm were pre-incubated in chemically defined capacitation media containing methyl-beta-cyclodextrin and used to inseminate cumulus cell oocyte complexes, or denuded oocytes, with some components. While sperm capacitation was induced in capacitation media, fertilization was impeded by the removal of cumulus cells from cumulus cell oocyte complexes. Secretions from cumulus cells promoted the formation of two pronuclei via a filter and the fertilization of denuded oocytes was dramatically enhanced with hyaluronate, low oxygen concentration, or progesterone in fertilization media (P < 0.05). This demonstrates that these factors-maintained sperm motility and capacitation or enhanced the hyper-activation of capacitated sperm (P < 0.05). We conclude that cumulus cells secrete progesterone, hyaluronate and undergo metabolic events to reduce oxidative stress in fertilization media. These phenomenons help to improve the fertilization capacity of sperm. We believe that this study makes a significant contribution to our understanding of the function of cumulus cells during fertilization in animals and humans.

ACRBP (Sp32) is involved in priming sperm for the acrosome reaction and the binding of sperm to the zona pellucida in a porcine model.

In boar sperm, we have previously shown that capacitation is associated with the appearance of the p32 tyrosine phosphoprotein complex. The principal tyrosine phosphoprotein involved in this complex is the acrosin-binding protein (ACRBP), which regulates the autoconversion of proacrosin to intermediate forms of acrosin in both boar and mouse sperm. However, the complete biological role of ACRBP has not yet been elucidated. In this study, we tested the hypothesis that tyrosine phophorylation and the presence of the ACRBP in the sperm head are largely necessary to induce capacitation, the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding, all of which are necessary steps for fertilization. In vitro fertilization (IVF) was performed using matured porcine oocytes and pre-capacitated boar sperm cultured with anti-phosphotyrosine antibodies or antibodies against ACRBP. Anti-ACRBP antibodies reduced capacitation and spontaneous AR (P<0.05). Sperm-ZP binding declined in the presence of anti-phosphotyrosine or anti-ACRBP antibodies. The localisation of anti-ACRBP antibodies on the sperm head, reduced the ability of the sperm to undergo the AR in response to solubilized ZP or by inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase. These results support our hypothesis that tyrosine phosphorylated proteins and ACRBP are present upon the sperm surface in order to participate in sperm-ZP binding, and that ACRBP upon the surface of the sperm head facilitates capacitation and the AR in the porcine.

Detrimental effects of oxidative stress in bovine oocytes during intracytoplasmic sperm injection (ICSI).

Intracytoplasmic sperm injection (ICSI) is an essential technology in animal and human reproduction. However, the developmental competence and pregnancy rate of embryos derived from ICSI are still lower than that from the conventional in vitro fertilization technique. In this report, we focused on reactive oxygen species (ROS) as a potential detrimental factor for ICSI. Experiment 1 was conducted to evaluate the effect of oxidative stress by two different oxygen concentrations (20%: control vs. 5%) in ICSI on the developmental competence (blastocyst rate: day 7, DNA fragmentation rate: day 4) and, ROS concentration and mitochondrial membrane potential of oocytes in ICSI. In the 5% O2 group, the blastocyst rate (29.5%) was higher and DNA fragmentation rate (4.8 ±â€¯1.0%) was lower than those in the control group significantly (12.7% and 18.2 ±â€¯2.4%, respectively, P < 0.05). Also, ROS concentration in the 5% O2 group (12.8 ±â€¯0.7) was significantly lower than that in the control group (47.8 ±â€¯6.9, P < 0.05). In experiment 2, we examined the supplementation of media with reduced glutathione (GSH) during ICSI procedure in an attempt to reduce the oxidative stress. The addition of GSH to the culture medium improved the blastocyst rate (17.6% vs. 30.4%, P < 0.05), and decreased the ROS levels in the oocytes (70.0 ±â€¯7.4 vs. 23.9 ±â€¯4.0, P < 0.05). In conclusion, our present study revealed that oocytes are under oxidative stress in ICSI procedure. Reduction of the oxygen concentration to 5% in the culture environment, or the addition of GSH in to the medium during ICSI procedure can promote the normal embryo development following the ICSI.

Changes in Sperm Motility and Capacitation Induce Chromosomal Aberration of the Bovine Embryo following Intracytoplasmic Sperm Injection.

Intracytoplasmic sperm injection (ICSI) has become the method of choice to treat human male infertility. One of the outstanding problems associated with this technique is our current lack of knowledge concerning the effect of sperm capacitation and motility upon the subsequent development of oocytes following ICSI. In the present study, we first examined the capacitation state of sperm exhibiting normal motility, along with sperm that had been activated, and examined the effect of reactive oxygen species (ROS) produced by these sperm types upon embryogenesis following bovine in vitro fertilization (IVF) and ICSI. Data showed that activated sperm reduced the chromosomal integrity of IVF/ICSI embryos at the blastocyst stage, while capacitated sperm produced ROS in capacitation media. Secondly, we treated sperm with carbonyl cyanide m-chlorophenyl hydrazine (CCCP), a chemical known to uncouple cell respiration within the mitochondria, and investigated the effect of this treatment upon blastocyst formation and chromosomal integrity at the blastocyst stage. Activated sperm in which the mitochondria had been treated with CCCP reduced levels of chromosomal aberration at the blastocyst stage following ICSI, by reducing mitochondrial activity in activated sperm. In conclusion, these findings suggest that capacitated sperm exhibiting activated motility induced chromosomal aberration during development to the blastocyst stage following ICSI. The injection of sperm exhibiting normal motility, or activated sperm in which mitochondrial activity had been reduced, improved the quality of ICSI-derived embryos. Therefore, the selection of sperm exhibiting progressive motility may not always be better for early embryo development and fetal growth following human ICSI, and that the use of a bovine model may contribute to a deeper understanding of sperm selection for human ICSI embryo development.

The developmental potential of oocytes is impaired in cattle with liver abnormalities.

Here, we investigated the effect of liver abnormality on the developmental potential of bovine oocytes. Good quality oocytes from healthy cows and from animals with a liver abnormality were matured and fertilized in vitro and then cultured to the blastocyst stage. On day 7 after fertilization, embryo cleavage and development were assessed. The concentrations of glucose, nonesterified fatty acid (NEFA), γ-glutamyl transpeptidase (γ-GTP), ß-hydroxybutyrate (BHBA) and glutathione were measured in follicular fluids (FF). The proportion of good quality oocytes and the frequency of development to the blastocyst stage were lower in the liver anomaly group than those of the control group (P<0.05). The concentrations of γ-GTP and BHBA in the FF of the liver anomaly group were higher than those of the control group (P<0.05). The concentration of glutathione in the FF of the liver anomaly group was lower than that of the control group (P<0.05). Moreover, there was a negative correlation between these concentrations and the proportions of oocytes that developed to the blastocyst stage (P<0.05). Supplementation of the culture medium with γ-GTP or BHBA did not affect the rate of oocyte maturation but did cause a concentration-dependent reduction in the frequency of fertilized oocytes that developed to the blastocyst stage. Our findings indicate that the quality of oocytes and their potential for development are lower in cattle with liver disorders than those in healthy cattle; one possible cause may be the high concentration of γ-GTP and/or BHBA in their FF.

Effect of polyvinylpyrrolidone on sperm function and early embryonic development following intracytoplasmic sperm injection in human assisted reproduction.

The objective here was to review the effects of polyvinylpyrrolidone (PVP) upon sperm function and embryonic development in humans. PVP has been used successfully in intracytoplasmic sperm injection (ICSI) to facilitate the handling and immobilization of sperm for both domestic animals and humans. In our previous reports, PVP solution exists locally in embryos injected during the early developmental period, and also exerts influence over the developmental capacity of such embryos. In other reports, PVP causes significant damage to sperm membranes that can be detected by transmission electron microscopy, and has been associated with chromosomal abnormalities in pregnancy derived from ICSI embryos. In some Japanese clinics, PVP-free media has been used for sperm immobilization in order to optimise safety. Consequently, it is strongly suggested that the success rate of fertilization and clinical pregnancy could be improved by using PVP-free solution for human ICSI. In conclusion, our interpretation of the available data is to perform ICSI without PVP or select a lower concentration of PVP solution in order to reduce safety for pregnancy and children born via ICSI.

Capacitation status of activated bovine sperm cultured in media containing methyl-ß-cyclodextrin affects the acrosome reaction and fertility.

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. One of the key processes involved in capacitation is the activation of sperm motility. Here, we investigated the capacitation and fertility status of activated sperm which had been cultured in media containing methyl-ß-cyclodextrin (MBCD). In order to do this, single activated sperm were caught using a micropipette and stained with chlortetracycline (CTC). Firstly, we investigated the effects of preincubation upon motility, capacitation of activated sperm and fertility. Culture in preincubation media supplemented with MBCD increased the rates of activation and fertilization compared with sperm cultured by control methods (p < 0.05). Following capture, individual activated sperm mostly exhibited a pattern characteristic of capacitation.Secondly we examined the effects of culturing sperm in media with or without glucose (G) and pyruvate acid (P) upon activated motility, the capacitation of activated sperm and fertility. Supplementation of culture media with G and P resulted in higher proportions of activated sperm and increased fertilization rates compared to culture without G and P (p < 0.05). Most of the sperm activated by culture in G and P exhibited patterns characteristic of capacitation. Without G and P, individual activated sperm mostly exhibited patterns characteristic of the acrosome reaction (p < 0.05). In conclusion, activated sperm exhibited patterns characteristic of capacitation. In addition, sperm activated in media containing an energy source (glucose and pyruvate acid) appeared to exhibit acrosome reactiveness and fertility.

Antioxidant treatment during preservation of bovine ovaries increased the development potential of embryos.

The overnight preservation of bovine ovaries would be highly useful in the subsequent harvest of viable oocytes for reproductive study. The present study aimed to optimize conditions for overnight preservation of bovine ovaries by examining the effects of temperature, solution and supplementation. In Experiment 1, the rate of development to the blastocyst stage of oocytes derived from ovaries preserved at 15°C was higher than that at either 5 or 25°C (p < 0.05). In Experiment 2, the rate of development to the blastocyst stage of oocytes derived from ovaries preserved in University of Wisconsin solution was higher than when PBS or saline was used (p < 0.05). In Experiment 3, oocytes preserved in saline supplemented with 0.3 mM glutathione (GSH) exhibited an increase in the rate of blastocyst formation compared with oocytes supplemented with 0 or 3 mM GSH (p < 0.05). In Experiment 4, supplementation with 10 µM epigallocatechin gallate during ovary preservation increased the rate of blastocyst formation (p < 0.05). The blastocysts derived from ovaries stored in saline supplemented with GSH at 15°C for 24 h were shown to develop into normal offsprings following transfer to recipient heifers. Our studies indicate that bovine IVM/IVF embryos derived from ovaries preserved in saline supplemented with an antioxidant at 15°C for 24 h can successfully develop to the blastocyst stage and result in offspring.

The effects of methyl-ß-cyclodextrin on in vitro fertilization and the subsequent development of bovine oocytes.

In this study, we investigated the effects of methyl-ß-cyclodextrin (MBCD) on in vitro fertilization and the subsequent development of bovine oocytes. Bovine oocytes matured in serum-free medium were inseminated with frozen-thawed sperm pre-incubated in protein-free modified Brackett and Oliphant medium (BO) containing various concentrations of MBCD for various periods. MBCD decreased the frequency of live sperm, however enhanced the capacitation and acrosome reaction of the live sperm. Pre-incubation of sperm with 0.5, 1.0 and 1.5 mM MBCD for 2 and 4 h increased the frequency of normal fertilization. Embryos derived from oocytes fertilized with spermatozoa pre-incubated with MBCD developed normally to the blastocyst stage and term. There were individual differences and similar tendencies in four different sires in terms of the effects of MBCD upon fertilization. These results indicate that the pre-incubation of bovine sperm with MBCD affects viability and capacitation status of the sperm and promotes fertilization in vitro. Embryos derived from oocytes fertilized with sperm pre-incubated with MBCD developed normally to the blastocyst stage and term.