Analysis of ubiquitination in vivo using a transgenic mouse model.

The primary pathway for the proteolytic destruction of cellular proteins is through ubiquitin-mediated targeting to the proteasome. This pathway is pivotal not only in the elimination of damaged or misfolded proteins but also in the temporal, developmental, or signal-mediated destruction of normal cellular substrates. The list of known substrates of the ubiquitin/proteasome pathway is long, but most substrates have been identified in yeast or, more recently, in cultured mammalian cells. It is likely that many mammalian substrates with developmental or disease relevance have yet to be identified because their ubiquitination occurs in tissue or organ systems that cannot be adequately modeled in vitro. We have developed a transgenic mouse model that will allow the isolation and identification of these substrates. The human UbC promoter was used to drive expression of a hexahistidine-tagged version of human ubiquitin in a variety of mouse tissues from early embryonic stages, as assessed by a green fluorescent protein marker. Cleavage of the fusion protein by endogenous enzymes produced epitope-tagged ubiquitin that was detected both in monomeric form and conjugated to cellular proteins. This mouse model should facilitate in the analysis of normal and disease-related ubiquitination events in vivo.

Protective effects of mutant ubiquitin in transgenic mice.

The K48R mutant ubiquitin can exert profound in vivo protective effects against a variety of insults, including agents of direct clinical relevance. The manipulation of the ubiquitin/proteasome pathway has enormous potential for clinical benefit, and it is not unreasonable to expect that such benefits will include diseases of aging.

Percutaneous epididymal sperm aspiration and intracytoplasmic sperm injection in the management of infertility due to obstructive azoospermia.

OBJECTIVE: To evaluate the recovery rate of spermatozoa from the epididymis using a percutaneous aspiration technique and to examine the fertilization rate after intracytoplasmic sperm injection. DESIGN: Prospective observational study. SETTING: Private infertility clinic, London. SUBJECTS: Twenty patients with obstructive azoospermia who each had an attempt at IVF. The sperm used for intracytoplasmic sperm injection was retrieved by percutaneous epididymal sperm aspiration in 16 patients. In one patient, microepididymal sperm aspiration was performed in addition because the quality of the sperm obtained by percutaneous epididymal sperm aspiration was not considered suitable for microinjection. In the remaining three patients, neither percutaneous epididymal sperm aspiration nor microepididymal sperm aspiration resulted in the recovery of sperm, which was obtained by testicular biopsy in one of them. INTERVENTION: Assisted fertilization with intracytoplasmic sperm injection. MAIN OUTCOME MEASURES: Normal fertilization and pregnancy rates. RESULTS: A total of 179 eggs were collected and 157 subsequently were microinjected. Normal fertilization occurred in 22 oocytes (14%) and the total number of embryos cleaved was 30. Twelve patients underwent ET in which three conceived (pregnancy rate 25% per transfer). The implantation rate was 10% and failed fertilization occurred in four cycles. CONCLUSION: Percutaneous epididymal sperm aspiration can be used successfully to recover sperm in men with obstructive azoospermia for use in assisted fertilization IVF cycles. The technique is simple, effective, and less traumatic compared with an open microsurgical operation.

Late intracytoplasmic sperm injection in unexpected failed fertilization in vitro: diagnostic or therapeutic?

OBJECTIVE: To evaluate fertilization potential of 24-hour-old unfertilized oocytes using intracytoplasmic sperm injection and the pregnancy potential of resultant embryos. DESIGN: Prospective observational study. SETTING: Private infertility clinic, London, United Kingdom. PATIENTS: Fifteen patients with a history of infertility who underwent treatment with IVF and showed failure of fertilization on the day after oocyte retrieval. INTERVENTION: Assisted fertilization with intracytoplasmic sperm injection was carried out at 24 hours after oocyte retrieval. RESULTS: A total of 121 metaphase II oocytes were subjected to intracytoplasmic sperm injection. Of these, 9 were damaged (7%), 2 were polyploidic (2%), and 58 showed normal fertilization (48%). Of the latter, 47 cleaved normally (81%). Forty embryos were transferred and three were cryopreserved. One patient conceived (7%) but in this case only one of three embryos transferred was from intracytoplasmic sperm injection. CONCLUSION: Late (24 hours) intracytoplasmic sperm injection can give good fertilization and cleavage rates but the potential of the generated embryos to achieve pregnancy seems to be low.

Assisted fertilization with intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the significance of intracytoplasmic sperm injection in severe male factor infertility and previous failed fertilization. DESIGN: Prospective observational study. SETTING: Private infertility clinic, London. SUBJECTS: Sixty-nine patients with a long-standing history of infertility of which 48 had previous failed fertilization, 15 had < 1 million progressive motile sperm per ejaculate, and the remaining 6 had obstructive azoospermia. INTERVENTION: Assisted fertilization with primary intracytoplasmic sperm injection was carried out in 69 IVF. OUTCOME: Normal (two pronuclei [2PN]) fertilization and pregnancy rates. RESULTS: A total of 967, oocytes were collected and 785 were subsequently microinjected. Normal fertilization (2PN) occurred in 410 oocytes (52%) and 90.5% of those cleaved. Sixty-four patients underwent ET, with a total of 181 embryos transferred. Twenty-five patients conceived with a pregnancy rate of 39% per transfer. The implantation rate was 16% and the total pregnancy loss rate 24%. Failed fertilization after intracytoplasmic sperm injection occurred in four cases. CONCLUSIONS: Intracytoplasmic sperm injection is increasingly becoming the treatment of choice in infertile couples where assisted fertilization is indicated. The high fertilization and pregnancy rates observed with this technique, together with a low risk of abnormalities, has revolutionized treatment of male factor infertility.

Pregnancies after assisted fertilization for "extreme" male factor: report on three cases.

Three cases of extreme male factor infertility, not acceptable for conventional IVF, are presented. In the first case, only 30 sluggishly motile spermatozoa were obtained in two ejaculates but treatment with SUZI resulted in a viable singleton pregnancy. In the second case, a combination of microepididymal sperm aspiration, for vasal obstruction after failure of reversal, with intracytoplasmic sperm injection resulted in a viable singleton pregnancy. In the third case, the husband had a vasal reservoir for distal vasal blockage. Sperm from the reservoir with extremely low motility and high antisperm antibody were microinjected by intracytoplasmic sperm injection and this resulted in a triplet pregnancy.

Delayed spinocerebellar ataxia in transgenic mice expressing mutant ubiquitin.

Spinocerebellar ataxia type 1 (SCA1) is an incurable neurodegenerative disease resulting from loss of Purkinje neurones within the cerebellum. The ubiquitin proteasome pathway (UPP) has been implicated in SCA1 but the role of proteolysis in the disease is still poorly understood. To further investigate this issue in vivo, genetic crosses were performed between an established mouse model of SCA1 and novel strains expressing elevated levels of wild type or mutant isoforms of ubiquitin. The K48R mutant isoform of ubiquitin (a dominant negative inhibitor of proteolysis) was found to significantly delay the deterioration of Purkinje neurones as evidenced by behavioural, morphological, and molecular indicators. This delay was accompanied by stabilization of p300/CBP, transcriptional mediators whose abundance and activity would otherwise decline in the course of the SCA1 disease, and persistence of protein kinase C gamma (PKCgamma), a protein involved in Purkinje cell dendritic development that is mutated in one form of spinocerebellar ataxia. Whereas the stabilization of p300/CBP was found to occur at the post-translational level the modulation of PKCgamma was at the level of transcription. These results are consistent with transcriptional dysregulation as a key mechanism in neurodegeneration through loss of p300/CBP. Further, the results suggest that the UPP is a potentially useful target for the development of novel therapies for the treatment of neurodegenerative disease.

Generation of superoxide anion and lipid peroxidation in different cell types and subcellular fractions from rat testis.

Mitochondria and microsomes from whole rat testis, seminiferous tubules and Leydig cells were investigated with respect to their capacity to generate superoxide anion. In addition, lipid peroxidation by whole testis mitochondria and microsomes was measured. In the presence of NADH and various respiratory inhibitors all three mitochondrial preparations catalyzed the formation of superoxide anion at a rate of 0.27-1.67 nmol/min.mg. This formation was concluded to be confined mainly to the NADH dehydrogenase region of the respiratory chain. Addition of NADPH to whole testis or Leydig cell mitochondria, but not tubule mitochondria, caused an additional formation of superoxide anion, which was unrelated to the respiratory chain, accelerated several-fold by menadione, and presumably catalyzed by NADPH-cytochrome c reductase and cytochrome P-450. Microsomes isolated from whole testis, seminiferous tubules, and Leydig cells generated superoxide anion at rates between 0.19 and 0.44 nmol/min.mg. These rates were also strongly stimulated by menadione. It is likely that both NADPH-cytochrome c reductase and cytochrome P-450 were involved in the microsomal generation of superoxide. Free radical scavengers of various types inhibited both the mitochondrial and microsomal formation of superoxide anion. Lipid peroxidation in whole testis essentially paralleled superoxide anion generation. However, the rate of mitochondrial lipid peroxidation was twice that of the microsomal rate. It is concluded that seminiferous tubules and Leydig cells generate superoxide anion at different rates and by different mechanisms. Together with cytochrome P-450-dependent hydroxylases, e.g., BP and DMBA hydroxylases, this superoxide generation may reflect a potential for cell-specific peroxidative damage in the testis.

Testicular needle aspiration as an alternative to biopsy for the assessment of spermatogenesis.

The technique of fine needle aspiration (FNA) may have a role as a reliable, quick and easy method of obtaining testicular tissue. Recent advances in the management of male subfertility and, in particular, the finding that spermatozoa recovered from the epididymis and testis can result in embryo generation after intracytoplasmic sperm injection (ICSI), question the traditional role of open testicular biopsy for the assessment of spermatogenesis. FNA of the testis was performed on 19 cases of male subfertility and histological and cytological preparations obtained were assessed by light microscopy. FNA provided intact testicular tubules adequate for the histological assessment of spermatogenesis in all cases. There was good correlation with the cytological preparations which gave an indication of the number of mature spermatozoa present. FNA should be considered as a simple alternative to open testicular biopsy in the current investigation of male subfertility and as a method of retrieving spermatozoa for assisted conception using ICSI.

The value of single versus repeated insemination in intra-uterine donor insemination cycles.

Pregnancy rates per cycle of intra-uterine donor insemination following ovulation induction were compared retrospectively for those patients having a single, and those having repeated insemination using frozen donor semen. Single insemination was performed in 69 cycles in which 15 women became pregnant (pregnancy rate = 22%). Of 65 cycles in which repeated insemination was performed, 16 women became pregnant (pregnancy rate = 25%). This difference in pregnancy rates was not statistically significant (chi 2 = 3.6, P = 0.84). We conclude that cycle fecundity may not be increased by repeating insemination.

T cell non-Hodgkin's lymphoma presenting in the first trimester of pregnancy.

This case report details of the presentation of a young woman in the first trimester of her pregnancy with lethargy, weakness, vomiting, pyrexia and lymphadenopathy. Extensive investigation revealed an advanced T cell lymphoma and only the second reported case in pregnancy to our knowledge. We discuss her management and subsequent chemotherapy in the context of a short review of the literature spanning the last decade.

Cumulative experience of percutaneous epididymal sperm aspiration (PESA) with intracytoplasmic sperm injection.

OBJECTIVE: Our objective was to evaluate the recovery rate of spermatozoa from the epididymis using a percutaneous aspiration technique and to assess the fertilisation rate after intracytoplasmic sperm injection. MATERIALS AND METHODS: Fifty-four patients with azoospermia had a total of 59 cycles at IVF with intracytoplasmic sperm injection (ICSI). The cause of the azoospermia was failed vasectomy reversal in 23 cases, congenital absence of the vas in 22 cases, partial testicular failure in 5 cases, and retrograde ejaculation in 2 cases, while the remaining 2 patients had erectile disorders. RESULTS: A total of 741 oocytes was collected and 521 metaphase II oocytes were subsequently microinjected. Normal fertilisation occurred in 274 oocytes (52.6%), and of these, 234 cleaved (85.4%). In 54 cycles, embryo transfer of more than one embryo occurred (91.5%) and a total of 155 embryos was replaced. The pregnancy rate was 30.5% per cycle and 33.3% per embryo transfer. The implantation rate was 14.2%; failure of fertilisation occurred in two cycles, while in three other cycles the embryos did not cleave. CONCLUSIONS: Percutaneous epididymal sperm aspiration can be used successfully to retrieve sperm in men with azoospermia due to obstructive, or nonobstructive, disorders. The technique is simple, cost-effective, and associated with fewer complications than an open microsurgical operation.

Sensitivity of mammalian cells expressing mutant ubiquitin to protein-damaging agents.

There is convincing evidence from studies in yeast that a functional ubiquitin/proteasome pathway is required to degrade misfolded or oxidatively damaged proteins but for technical reasons, it has been difficult to perform comparable studies in mammalian cells. To investigate the possibility that the ubiquitin/proteasome pathway is cytoprotective for mammalian cells, we have introduced epitope-tagged wild-type ubiquitin or dominant-negative mutant versions of ubiquitin into mouse HT4 neuroblastoma cells. Cells expressing mutant versions of ubiquitin were found to be sensitive to cadmium, an agent that causes oxidative damage to cellular components, and to canavanine, an amino acid analog that generates misfolded proteins. The greatest sensitivity to canavanine was observed in cells expressing a mutant version of ubiquitin unable to support the formation of Lys(48) linkages. Substrates of the proteasome were found to accumulate in these cells, suggesting a general deficit in proteolysis. Our data suggest that defects in the ubiquitin-mediated proteolytic system predispose mammalian cells to the toxic effects of abnormal protein.

Experience with subzonal insemination (SUZI) and intracytoplasmic sperm injection (ICSI) on unfertilized aged human oocytes.

OBJECTIVE: The aim of this study was to assess the fertilizability of unfertilized aged human oocytes from failed in vitro fertilization (IVF) cycles using SUZI and ICSI. METHODS: A total of 363 oocytes which showed no fertilization after conventional IVF was subjected to assisted fertilization using SUZI or ICSI. The microinjected oocytes which were derived from 72 patients undergoing their first IVF treatment had an intact polar body and no signs of degeneration. SUZI was carried out in 265 oocytes and ICSI in the remaining 98. RESULTS: Significantly more oocytes were damaged after ICSI (9 vs 0.3%, P < 0.01). Normal fertilization rates were higher at 24 hr in both groups and occurred more frequently after ICSI, although the difference did not reach statistical significance. Abnormal fertilization occurred significantly more often after SUZI at 48 hr (P < 0.005), but not at 24 hr. Cleavage rates were significantly higher after ICSI (94.4 vs 57.1%, P < 0.025) at 24 hr, but this was not observed at 48 hr, although the ICSI group still showed better cleavage rates (33.3 vs 19.1%). There was no difference in embryo quality in either group. CONCLUSIONS: Our results indicate that micromanipulation rather than reinsemination should be carried out on unfertilized human oocytes from failed IVF attempts. Both techniques can be used to achieve fertilization which occurs more often after ICSI. However, the trauma from the former technique on the microinjected oocytes may impair the potential of the generated embryos to achieve pregnancy compared to SUZI. Prospective randomized trials are necessary to address the problem.

Simplified sperm retrieval and intracytoplasmic sperm injection in patients with azoospermia.

OBJECTIVE: To evaluate the rate of recovery of spermatozoa from the epididymis using a percutaneous aspiration technique and to assess the fertilization rate following intracytoplasmic sperm injection (ICSI). PATIENTS AND METHODS: Forty-two patients with azoospermia underwent a total of 46 treatment cycles of in vitro fertilization (IVF) and ICSI. The sperm used for ICSI was retrieved percutaneously by fine-needle aspiration and syringe suction (percutaneous epididymal sperm aspiration, PESA) from the epididymis in 28 patients (mean age 34.9 years), over 32 cycles. Six patients underwent microsurgical sperm aspiration (MESA) and in the remaining eight patients, neither percutaneous aspiration nor MESA yielded suitable sperm and spermatozoa extracted from testicular biopsy were used. RESULTS: A total of 362 oocytes were collected and of those, 286 (79%) were subjected to ICSI. Of the injected oocytes, 49 (17.2%) were damaged, 138 (48.3%) achieved normal fertilization and, of those, 112 (81.2%) cleaved. A total of 67 embryos were transferred and 18 more were suitable for cryopreservation. Of the 25 cycles with embryo transfer, eight resulted in a pregnancy and of these, one miscarried. The pregnancy rate was 25% per cycle and 32% per embryo transfer. The implantation rate was 12%. CONCLUSIONS: This extensive series of PESA/ICSI cycles indicates that PESA can be used successfully to retrieve spermatozoa in patients with azoospermia. The technique is simple, cost-effective and is associated with fewer complications compared to an open microsurgical procedure.

Factors influencing the outcome of in-vitro fertilization with percutaneous aspirated epididymal spermatozoa and intracytoplasmic sperm injection in azoospermic men.

In-vitro fertilization (IVF) by intracytoplasmic sperm injection (ICSI) with spermatozoa retrieved by percutaneous epididymal sperm aspiration (PESA) is a novel, simple and effective treatment for azoospermic men. In all, 38 azoospermic men had an IVF/PESA/ICSI cycle. A total of 42 cycles were performed. The aetiology of azoospermia was classified as failed vasectomy reversal (12 patients/16 cycles), inflammatory obstruction (five patients/five cycles), partial testicular failure (five patients/five cycles) and bilateral congenital absence of vas (16 patients/16 cycles). Adequate sperm preparations for ICSI were obtained from 38 of the 42 treatment cycles (90%). The mean fertilization rate was 32.7%, and fertilization occurred in 35 cycles (92.0%). Embryo transfer was performed in 13 out of 14 cycles (93%) in men with a failed vasectomy reversal, four out of five cycles in men with an inflammatory obstruction (80%), four out of four cycles in men with a partial testicular failure (100%), and 14 out of 15 cycles in men with a bilateral congenital absence of vas (93%). The overall pregnancy rate per two or three embryos transferred was 28.6 and 26.3% per treatment cycle respectively. The sperm parameters of the final pooled sperm aspirate preparations varied widely among the four aetiological groups. These parameters were of no value in predicting the fertilization or pregnancy rates (P > 0.05), and neither was the embryo cleavage rate.

Significant bacteriuria has prognostic significance in primary biliary cirrhosis.

Significant bacteriuria in women has been found to be associated with increased mortality in community-based studies. We have previously reported a high prevalence of significant bacteriuria with a high recurrence rate in females with primary biliary cirrhosis (PBC), particularly those with late stage disease on liver biopsy. During a 5-year period we prospectively screened for significant bacteriuria in 187 women with primary biliary cirrhosis, (median follow-up of 47 months, range 1-83). Significant bacteriuria was found in 30 (17%) in their first urine (index bacteriuria), 90 (48%) died and 15 (8%) had liver transplants. Cox's proportional hazard models showed that age, serum bilirubin, ascites and cirrhosis were independent prognostic variables. Index bacteriuria added significantly to this model (P = 0.069) being independent from other variables, with an increased relative hazard for death of 1.65 (65% increase in risk of death) compared to non-bacteriuric patients. This effect was due mainly to non-cirrhotic patients with significant bacteriuria as shown by using multiplicative variables for histological stage and significant bacteriuria. An index of recurrent bacteriuria was significantly increased in patients with index bacteriuria (P less than 0.001) and in those who died or underwent transplantation (P less than 0.001). In this study, significant bacteriuria defined a specific sub-group of PBC patients with an increased risk of death.

Experience with assisted fertilization in severe male factor infertility and unexplained failed fertilization in vitro.

We present results of in-vitro fertilization (IVF) cycles using assisted fertilization at our centre. Assisted fertilization was performed in those couples who had failed to fertilize oocytes with conventional IVF, or where this was predicted by the presence of severe male factor infertility. In 20 consecutive assisted fertilization cycles 223 oocytes were subjected exclusively to subzonal insemination (SUZI). Subsequently in 32 consecutive assisted fertilization cycles 418 oocytes were subjected to intra-cytoplasmic sperm injection (ICSI). More oocytes were damaged by ICSI (8.9%) than by SUZI (2.3%) (P = 0.03), but normal fertilization resulted more often after ICSI (56.9%) than SUZI (35.8%) (P = 0.004). Sperm parameters, other than sufficient numbers to perform the procedures, had no effect on fertilization or pregnancy rates. Every cycle led to the transfer of at least one embryo. Pregnancy resulted from eight of the SUZI cycles (40%) and nine of the ICSI cycles (28%). Implantation rates were calculated as 25 and 12% for SUZI and ICSI respectively. The presence of living spermatozoa is the only semen parameter limiting assisted fertilization. At present more centres are able to perform SUZI than ICSI and we feel it is premature to abandon SUZI altogether. Local conditions and success rates should be considered when decisions are made in assisted fertilization cycles.