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C. Vincent, S. J. Pickering, and M. H. Johnson

Summary. When mouse ovulated oocytes were exposed to 1·5 m-dimethylsulphoxide (DMSO) the resultant hardening of the zona pellucida was not a direct effect but required the presence of an oocyte. The hardening of the zona pellucida when zonae used were aged in vitro was also dependent upon the presence of the oocyte. Protocols of DMSO exposure that induce zona-hardening also caused depletion of the numbers of cortical granules underlying the oocyte surface, whereas protocols without effect on the zona did not reduce significantly the cortical granule count. It is proposed that the effects of DMSO may be mediated by a release of cortical granule contents.

Keywords: oocyte; zona pellucida; dimethylsulphoxide; fertilization; cryopreservation; cortical granule; ageing; mouse

Free access

Kimberly A Preis, George Seidel Jr, and David K Gardner

In vitro maturation of oocytes has enormous potential in assisted reproductive technology, but its use has been limited due to insufficient knowledge of oocyte physiology during this dynamic period and lack of an adequate maturation system. The aim of this study was to characterize the metabolic profiles of three groups of oocytes throughout maturation: cumulus–oocyte complexes (COCs), denuded oocytes, and denuded oocytes co-cultured with cumulus cells. Mouse oocytes were collected from 28-day-old unstimulated females and matured in a defined medium. Oocytes were matured individually and transferred into fresh 0.5 μl drops of medium at 4 h intervals until 16 h. Ultramicrofluorimetry was used to quantitate carbohydrate consumption from and metabolite release into the medium. Glucose consumption and lactate production of COCs increased (P < 0.001) over the maturation interval (0–16 h). Glucose consumption by COCs that subsequently fertilized was higher between 8–12 h of maturation than by COCs that did not fertilize (38 versus 29 pmol/COC per h, respectively; P < 0.01). Lactate production by COCs that subsequently fertilized was higher between 8–16 h of maturation, than by oocytes that did not fertilize (8–12 h, 66 versus 46 pmol/COC per h, P < 0.01; 12–16 h, 56 versus 40 pmol/COC per h, respectively; P < 0.05). These data indicate that the final hours of maturation may hold a unique marker of oocyte competence, as during this time fertilizable COCs take up more glucose and produce more lactate than those not subsequently fertilized.

Free access

Chong Li, Eiji Mizutani, Tetsuo Ono, and Teruhiko Wakayama

In mammals, ICSI is now a very important tool for both assisted reproductive technology and studying the mechanisms of fertilization. In the latter experiments, it is important to use spermatozoa that have lost their oocyte activation capacity but still retain their developmental potential. In this study, we used high-concentration NaOH to remove oocyte activation potential from spermatozoa, and examined whether normal offspring could be generated from these spermatozoa after ICSI. The spermatozoa were treated with different concentrations of NaOH (1–100 mM) for 1 h and then neutralized with equal amounts of same concentration of HCl. In 10 mM NaOH-treated spermatozoa, the cell membrane was broken and most of them failed to activate oocytes after their injection into the oocytes. However, these spermatozoa did not show strong damage, and after artificial activation with SrCl2, all of the zygotes were judged as normal by immunostaining to check the methylation status of histone H3 lysine 9, low chromosome damage by karyotype assay and staining with DNA double-strand breaks marker, γH2AX. Moreover, after transferring those embryos into recipient females, 106 (36.7%) live and healthy offspring were delivered, which is similar to the rate in the fresh control group. By contrast, spermatozoa treated with lower NaOH concentrations retained their oocyte activation capacity and those treated with higher concentrations lost their developmental potential. This suggests that 10 mM NaOH for 1 h is the best treatment to completely destroy the cell membrane and activation capacity of spermatozoa without injuring their developmental potential.

Free access

J Wu, L Zhang, and X Wang

The purpose of this study was to determine the rates of maturation, fertilization and embryo development of ultrarapidly frozen immature oocytes (immature cumulus-oocyte complexes; COCs) obtained from antral follicles in ovaries of patients with chocolate ovarian cysts. The COCs were cryopreserved by a vitrification method using 5.5 mol ethylene glycol l (-1) plus 1.0 mol sucrose l (-1) in Dulbecco's PBS (DPBS). The survival, maturation and fertilization rates, and the percentage of embryos developing to the two-cell stage were 59, 64, 70 and 71%, respectively. No significant differences were noted in the rates of maturation, fertilization and embryo development between control and cryopreserved oocytes. Two embryos that developed from cryopreserved oocytes of the oocyte donor programme were selected for transfer into the uterus of a recipient with premature ovarian failure, after the recipient had received steroid replacement. A biochemical pregnancy occurred in the recipient after embryo transfer. These results indicate that immature oocytes can survive after cryopreservation and subsequently can be cultured to mature oocytes that are capable of undergoing fertilization in vitro and developing into embryos.

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Ming-Wen Li, Brian Baridon, Amanda Trainor, Esi Djan, Amanda Koehne, Stephen M Griffey, John D Biggers, Mehmet Toner, and K C Kent Lloyd

Apolipoprotein E (Apoe)-deficient knockout mice were used to test the hypothesis that mutant mice preserved as evaporatively dried (ED) spermatozoa, stored at −80 °C for 6 months, and then recovered by ICSI will exhibit the same phenotype as before preservation. The birth rate of mice recovered by ICSI of evaporatively dried spermatozoa was lower than that of fresh spermatozoa (17.5 vs 38.0%). Progeny of mice preserved using evaporatively dried spermatozoa were reproductively sound. From these, the second generation of mice produced by natural mating showed lesions typical of APOE deficiency, including severe hypercholesterolemia, hypertriglyceridemia, markedly increased plasma low-density lipoprotein level, and extensive and severe atherosclerotic lesions in the aorta. We conclude that the expected phenotype caused by an induced genetic mutation can be faithfully recapitulated and sustained in subsequent generations of mice preserved and stored as ED spermatozoa and recovered using ICSI. Because it is simpler, faster, and cheaper than conventional (cryopreservation) and nonconventional (freeze–drying) preservation procedures, evaporative drying is a viable, cost-effective, and efficient method for preserving and storing valuable mutant mouse strains.

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E. A. McLaughlin, W. C. L. Ford, and M. G. R. Hull

The proportion of human spermatozoa from 28 ejaculates to lose their acrosomes during cryopreservation was measured and correlated with the number that became immotile or lost the integrity of their plasma membrane. The ability of washed spermatozoa to acrosome react in response to A23187 before and after cryopreservation was compared. Motility was assessed by time-lapse photography; intact acrosomes were stained with fluorescein conjugated Pisum sativum agglutinin and dead spermatozoa were stained with bisbenzimide (H33258). Twenty-four per cent of spermatozoa lost their acrosomes during freezing and thawing, but the number that did so was not correlated with the number that became immotile or non-viable. Frozen spermatozoa exhibited fewer spontaneous acrosome reactions than did fresh spermatozoa (5 versus 13% after 4 h), but they responded to A23187 in a similar way. Although frozen spermatozoa were significantly more likely to die during the incubation, the data do not suggest that degenerative acrosome loss had a major influence on the results. In the hamster egg test frozen–thawed spermatozoa achieved more penetrations than did fresh spermatozoa when stimulated with 0 or 1 μmol A23187 l−1 but considerably fewer when stimulated with 4 μmol A23187 l−1. The following conclusions were made. First, cryopreservation damage to the acrosome, the plasma membrane and the flagellum can occur independently. Second, acrosome function is maintained after cryopreservation as long as the organelle remains mechanically intact. Third, some spermatozoa that lose their acrosomes during cryopreservation remain viable and can fuse with zona-free hamster eggs.

Free access

Florencia Ardon and Susan S Suarez

Artificial insemination with frozen semen allows affordable, worldwide dissemination of gametes with superior genetics. Nevertheless, sperm are damaged by the cryopreservation process. Elucidating the molecular effects of cryopreservation on sperm could suggest methods for improving fertility of frozen/thawed semen. This study was undertaken to examine the effect of cryopreservation on the coating of sperm by binder of sperm (BSP) proteins in seminal plasma. BSP proteins are secreted by the seminal vesicles and coat the surface of sperm by partially intercalating into the outer leaflet of the sperm plasma membrane. The BSP proteins are known to play roles in the formation of the oviductal sperm storage reservoir and in sperm capacitation. We investigated the effects of cryopreservation on the sperm BSP protein coat using Bovipure to separate live sperm from extended semen and then assaying the amounts of BSP proteins on sperm using quantitative western blotting with custom-made antibodies against unique sequences of each BSP protein. Greater amounts of all three BSP proteins (BSP1, BSP3, and BSP5) were detected on frozen/thawed sperm than on fresh sperm. Furthermore, the reduction of BSP3 from 15 to 13 kDa in mass, which occurs during incubation of sperm under mild capacitating conditions, was enhanced by cryopreservation. We concluded that freezing alters the BSP protein coating on sperm, which could account in part for reduced fertility of cryopreserved semen samples.

Free access

Cengiz Yildiz, Palma Ottaviani, Napoleon Law, Renise Ayearst, Ling Liu, and Colin McKerlie

Efficient freezing, archiving, and thawing of sperm are essential techniques to support large scale research programs using mouse models of human disease. The purpose of this study was to investigate the effects of variable combinations and concentrations of cryoprotectants on sperm-assessment parameters of frozen–thawed mouse sperm in order to optimize cryopreservation protocols. Sperm was frozen using combinations of 3% skim milk + 0.2 or 0.3 M nonpermeating raffinose with either permeating glucose, fructose, propylene glycol, ethylene glycol, glycerol, or sodium pyruvate in CD-1, C3FeB6F1/J, B6129SF1, C57BL/6NCrIBR, 129S/SvPaslco, and DBA/2NCrIBR mice. Sperm-assessment parameters included progressive motility, plasma membrane integrity (SYBR-14 + PI), in vitro fertilization rate, and in vitro embryo development rate to blastocyst. DNA content analysis of sperm was measured by the sperm chromatin structure assay (SCSA). 0.3 M raffinose with 0.1 M fructose significantly improved post-thaw sperm-assessment parameters for CD-1, C3B6F1, B6129SF1 mice (P < 0.05–0.01), whereas 0.2 M raffinose with 0.1 M glycerol or 0.1 M fructose enhanced sperm assessment values for C57BL/6 and 129S mice (P < 0.01), compared to 0.3 M raffinose alone. DNA fragmentation during cryopreservation was significantly increased in all strains evaluated when compared with fresh control sperm in a strain-dependent manner (P < 0.01). Supplementation with permeating glycerol or fructose to the cryoprotectant (CPA) solution showed a significant protective effect to DNA integrity when cryopreserving sperm from C57BL/6 and 129S mice. Damage to sperm DNA significantly decreased the rate of in vitro embryo development to blastocyst in C57BL/6 mice. The type of monosaccharide sugar or polyols, CPA molarity, and combination of permeating and nonpermeating cryoprotectant are significant factors for improving progressive motility, plasma membrane integrity, DNA integrity, in vitro fertilization rate, and in vitro embryo development rate to blastocyst in cryopreserved mouse sperm.

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Megan Lim, Jeremy G Thompson, and Kylie R Dunning

The ovarian follicle provides the oocyte with the ideal environment for growth and development in preparation for ovulation and fertilisation. The follicle undergoes many structural changes as it grows, including changes in vasculature, cell proliferation and differentiation and the formation of a fluid-filled antrum. These changes collectively create a low oxygen environment within the follicle. Thus, the oocyte itself develops in a potentially hypoxic environment. The survival of hypoxic tissues is controlled by hypoxia-inducible factors (HIFs) that are activated in a low oxygen state. The understanding of HIF pathways is growing across all fields of biology, and its role in ovarian development is steadily gaining clarity. One of the genes upregulated by HIF is a vascular endothelial growth factor, the main inducer of angiogenesis which is required for follicle development and corpus formation. Ovulation is also intrinsically linked to HIF activity through the ovulatory luteinising hormone surge increasing HIF expression. The role for HIF in oocyte maturation is less understood, as efforts to replicate the low oxygen environment of the in vivo follicle are not achievable by culturing in low oxygen alone. There is potential for other factors present in vivo, but lost in vitro, to be involved in oxygen regulation. One factor of interest is haemoglobin, the oxygen-binding protein, which brings the exciting possibility of sensitive oxygen regulation, consequently affecting HIF-regulated gene expression. A thorough understanding of oxygen regulation within the follicle would provide vital applications for the field of assisted reproductive technologies, in particular in vitro oocyte maturation.

Free access

M Arias-Álvarez, R M García-García, L Torres-Rovira, A González-Bulnes, P G Rebollar, and P L Lorenzo

Extreme body mass indexes may impair reproductive outcome in assisted reproductive technologies. Leptin reflects the amount of body fat and could act as a modulator of oocyte quality through activation of specific transcription factors. The aim of this work was to establish whether: 1) leptin influences meiotic and cytoplasmic oocyte maturation; 2) STAT3 and MAPK mediate the effects of leptin and 3) leptin modulates steroid secretion by cumulus–oocyte complexes (COC) during in vitro maturation (IVM). We confirmed immunolocalisation of leptin receptor in oocytes, cumulus/granulosa cells during the peri-ovulatory period. The confocal study showed that COC supplemented with 1, 10 and 100 ng/ml leptin had a significantly higher metaphase II (MII) percentage than those IVM without leptin (P<0.05) and a similar MII index compared to the group supplemented with 10% FCS. Leptin did not increase the percentage of cytoplasmically matured oocytes in terms of cortical granule migration rate, whereas a significantly higher index was found in the FCS group (P<0.001). Oestradiol concentrations in spent media were higher in the FCS group compared to other treatments (P<0.001). Leptin-stimulated nuclear oocyte maturation was significantly impaired when leptin-induced JAK2/STAT3 and MEK 1/2 activation was suppressed by the inhibitors (P<0.001). Steroid secretion of COC was not affected by leptin activation of JAK2/STAT3 or MEK 1/2 pathways. In conclusion, JAK2/STAT3 and MEK 1/2 pathways mediate the enhancement of nuclear oocyte maturation by leptin; however, neither cytoplasmic oocyte maturation nor steroidogenic response of COC were improved in the present rabbit model.