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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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M Lin, X Zhang, R Murdoch and RJ Aitken

A medium modified from eutherian systems was used to culture epididymal epithelial cells of the brushtail possum (Trichosurus vulpecula) for more than 2 months. Epididymal tubule fragments from the caput, corpus and cauda epididymides were used to generate cell monolayers. All three epididymal cell culture systems supported maturational changes in marsupial spermatozoa and enabled immature possum spermatozoa to differentiate from a T-shape to a streamlined shape, accompanied by the development of progressive motility after co-culture with 7-day-old cultured epididymal cell monolayers. This epididymal cell and sperm co-culture system for marsupial species may facilitate the identification of specific epithelial factors that affect sperm maturation, particularly in a species in which morphological maturation is readily visible.

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M. Lin, X. Zhang, M. Wade, M. Harris and M. Nickel

Recent studies indicate that subacrosomal proteins are necessary for the attachment of the acrosome onto the nucleus during sperm formation, and for the stability of the nuclear membrane during fertilization. For the first time, subacrosomal proteins have been isolated from a marsupial species, the tammar wallaby (Macropus eugenii), using a method developed in our laboratory. Whole ejaculated spermatozoa were fractionated into head and tail sections by ultrasonication to extract subacrosomal proteins. The heads (> 95% purity) were then isolated from tail sections using centrifugation with a three-step discontinuous sucrose gradient (35, 68 and 75% (w/v)). The heads were treated with 0.1% (v/v) Triton X-100 which stripped off the acrosome, but not the subacrosomal proteins, from the head. The proteins were finally extracted by 100 mmol NaOH l−1. Four prominent subacrosomal polypeptides, with molecular masses of 45, 38, 33 and 29 kDa, were recognized from the SDS-PAGE gel. The localization of these polypeptides (particularly the 45 kDa polypeptide) was confirmed by fluorescent and immunogold labelling with polyclonal antibodies raised in mice against the obtained polypeptides. In wallaby testes, the 45 kDa polypeptide was detected as early as at the step 3 spermatid and was mainly associated with the membrane of the newly formed acrosome vesicle. This polypeptide was also found on the acrosomal membrane of all older spermatids. The 45 kDa polypeptide was found on the acrosomal region of the spermatozoa collected from the caput, corpus and cauda of the epididymis. The similarity of the sperm anatomy of the tammar wallaby with that of other marsupials, such as the brushtail possum, implies that this procedure could be applied effectively to other marsupial species with minor modification.

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X. Zhang, G. M. Kidder, C. Zhang, F. Khamsi and D. T. Armstrong

Plasminogen activator has been implicated in tissue invasion and remodelling because of its role in the degradation of the extracellular matrix. Its activity can be detected in mouse embryos as early as day 6 of pregnancy, suggesting that plasminogen activator is involved in the process of implantation. The present study determined the time course of expression of the genes encoding tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) during the preimplantation period in rats by the sensitive mRNA phenotyping procedure of reverse transcription–PCR. The tPA mRNA was present in rat oocytes and two-cell embryos, but was not detected between the four-cell and blastocyst stages. The uPA mRNA was first detected in two-cell rat embryos, and was present through to the blastocyst stage. In chromogenic assays, plasminogen activator activity was detected in oocytes and embryos between two-cell and blastocyst stages. Most plasminogen activator activity present in preimplantation embryos appeared to be uPA, as it could be inhibited by anti-uPA antibody and a specific uPA inhibitor, amiloride, but not by anti-tPA antibody. The present data demonstrate the expression of uPA gene and uPA activity in preimplantation rat embryos, suggesting that embryonic uPA may be involved in early embryo development and implantation.

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J. M. Wang, L. Tao, X. L. Wu, L. X. Lin, J. Wu, M. Wang and G. Y. Zhang

Summary. Concentrations of (+) and (−) gossypol were measured by high performance liquid chromatography after they were incubated with plasma proteins in vitro. The concentration of (−) gossypol decreased more than the concentration of (+) gossypol. A similar decrease in free gossypol concentrations in the blood plasma of rats was observed after intravenous infusion of gossypol enantiomers. The concentration of (−) gossypol was also found to be lower than the concentration of (+) gossypol at the blood–testis barrier. The biological effect of (−) gossypol probably results from its stereospecific binding to extra- and intracellular proteins in vivo and inhibition of the biological activity of some proteins.

Keywords: gossypol enantiomers; HPLC; protein binding; blood–testis barrier; rat

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X. Zhang, G. M. Kidder, A. J. Watson, G. A. Schultz and D. T. Armstrong

The sensitive mRNA phenotyping technique of reverse transcription–polymerase chain reaction was used to demonstrate that insulin receptor mRNA is present in rat embryos during the preimplantation period. In addition, mRNA encoding insulin-like growth factor (IGF) type I and type II receptors have also been detected in rat preimplantation embryos. IGF-I mRNA was not detected in preimplantation embryos but was found in oviducts and uteri of prepubertal and early pregnant rats. IGF-II mRNA was present in both embryos and in oviducts and uteri during the preimplantation period. These findings suggest that insulin and IGF-I could influence early embryo development in endocrine or in paracrine fashions, whereas IGF-II may have an additional autocrine mode of action in affecting preimplantation embryos in rats.

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H T Nie, Y X Guo, X L Yao, T W Ma, K P Deng, Z Wang, G M Zhang, L W Sun, Z Y Wang, H C Wang and F Wang

This study aimed to determine if short-term nutrient alteration affects (1) ovarian morphology, (2) plasma and ovarian antioxidant capability and (3) cell apoptosis and AKT signaling within the ovary. After estrus synchronization, 24 Hu sheep were assigned to three groups based on the nutrient requirement recommended for maintenance (M): 1 × M (Control), 1.5 × M (S) and 0.5 × M (R) during days 7–14 of their estrous cycle. The results indicated that undernourishment significantly increased the counts and volume of follicles <2.5 mm and decreased the counts and volume of follicles ≥2.5 mm (P < 0.05). Feed restriction altered the plasma and follicular redox balance within follicles ≥2.5 mm by inhibiting total antioxidant capacity, increasing malondialdehyde concentration (P < 0.05) and reducing the mRNA expression levels of superoxide dismutase 2 (SOD2) and glutathione peroxidase (GSH-PX), as well as the activities of total SOD and GSH-PX. Feed restriction also attenuated B-cell lymphoma-2 (BCL2) but enhanced Bcl-2-associated X protein (BAX) and BAX/BCL2 transcription and translation levels in granulosa cells (P < 0.05). Uniform staining intensities of AKT and P-AKT-Ser473 were observed in each follicle stage, whereas weaker P-AKT-Thr308 staining in the antral follicle than in the pre-antral follicle suggested possible involvement of P-AKT-Thr308 during the beginning of follicle development. P-AKT-Ser473 levels in follicles ≥2.5 mm was significantly reduced in the R group (P < 0.05). The results presented in this study demonstrate that suppressed folliculogenesis caused by feed restriction might be associated with attenuated AKT signaling, reduced follicular antioxidant capacity and enhanced granulosa cells apoptosis.

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F Guo, B Yang, Z H Ju, X G Wang, C Qi, Y Zhang, C F Wang, H D Liu, M Y Feng, Y Chen, Y X Xu, J F Zhong and J M Huang

The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.

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Y Du, C S Pribenszky, M Molnár, X Zhang, H Yang, M Kuwayama, A M Pedersen, K Villemoes, L Bolund and G Vajta

The purpose of the present study was to improve cryotolerance using high hydrostatic pressure (HHP) pretreatment of porcine in vitro matured (IVM) oocytes, to facilitate their further developmental competence after parthenogenetic activation. A total of 1668 porcine IVM oocytes were used in our present study. The pressure tolerance and optimal duration of recovery after HHP treatment were determined. Oocytes were treated with either 20 or 40 MPa (200 and 400 times greater than atmospheric pressure) for 60 min, with an interval of 10, 70, and 130 min between pressure treatment and subsequent vitrification under each pressure parameter. Oocytes from all vitrification groups had much lower developmental competence than fresh oocytes (P<0.01) measured as cleavage and blastocyst rates. However, significantly higher blastocyst rates (P<0.01) were obtained in the groups of 20 MPa pressure, with either 70 (11.4±2.4%) or 130 (13.1±3.2%) min recovery, when compared with the vitrification control group without HHP treatment where no blastocysts were obtained. The influence of temperature at HHP treatment on further embryo development was also investigated. Treatments of 20 MPa with 70 min recovery were performed at 37 °C or 25 °C. Oocytes pressurized at 37 °C had a significantly higher blastocyst (14.1±1.4%) rate than those treated at 25 °C (5.3±1.1%; P<0.01). Our results demonstrate that HHP pretreatment could considerably improve the developmental competence of vitrified pig in vitro matured (IVM) oocytes. The HHP pretreatment will be tested as a means to improve survival and developmental competence at different developmental stages in different species including humans.