Ann A. Kiessling

Sperm-mediated gene transfer in mice

Sperm‐mediated DNA transfer to offspring has the potential to markedly simplify the generation of transgenic animals, but the efficiency in mice has been controversial. To determine the basis of the variability of the procedure in mice, we undertook a large, collaborative study of sperm‐mediated DNA transfer to mouse eggs in well‐established laboratory conditions for in vitro fertilization and offspring development following embryo transfer. Sperm were incubated with plasmid DNA during the capacitation period and then added to freshly ovulated mouse oocytes for fertilization; cleaved embryos were then transferred to the oviducts of pseudopregnant recipients for gestation. From a total of 75 experiments, 13 produced 130 transgenic offspring, amounting to 7.4% of total fetuses. In five experiments, more than 85% of offspring were transgenic, but the factors leading to this high success rate were not discovered. Clustering of such a low frequency event could account for the disparate reports of transgenic success with sperm‐mediated DNA transfer to mouse offspring. Discovering the factors important to success would not only allow this simplified approach to become an important tool in the generation of transgenic mice, but could also lead to important insights into natural protective mechanisms against sperm‐mediated transfer of foreign DNA. Mol. Reprod. Dev. 50:406–409, 1998.

HIV-1 in semen : an isolated virus reservoir

/L, no known infected, andnormal genitourological examination at the time ofspecimen donation. Protease-gene sequences weredetermined for 11 blood and eight semen virus clones frompatient A who had not received antiretroviral therapy, andfrom nine blood and nine semen virus clones from patient Bwho had been on antiretroviral therapy for several years,including a protease inhibitor for 4 months. The sequenceswere aligned for maximum homology and phylogeneticanalysis were done (figure).Protease-gene sequences of all clones contained severalmutations relative to the reference virus, hxb2. Thephylogenetic analyses of the sequences from each patientrevealed two distinct families of viruses, one in the bloodand one in the semen. The differences between the familiesranged from four to seven or from four to eight aminoacidsubstitutions for patients A and B, respectively. Bloodclones from patient B contained mutations at aminoacidresidues 36(I), 54(V), 63(P), and 82(A), characteristic ofemerging resistance to the protease inhibitor. By contrast,protease resistance conferring mutations were not found inthe semen virus clones from patient B.These findings support the concept that semen HIV-1arises from a distinct reservoir of HIV-1 infection whichmay be isolated from antiretroviral therapy and mayfunction independently in the pathobiology of HIV-1disease. This suggests that consideration of the specialisedfeatures of the semen compartment needs to be included indisease monitoring and the design of treatment strategies.

Semen leukocytes: friends or foes? *

OBJECTIVE To test the hypothesis that male reproductive tract leukocytes function in the elimination of abnormal spermatozoa from ejaculated semen. DESIGN Semen specimens with > or = 2 x 10(6) nonspermatozoal cells/mL were examined for leukocytes and for mature sperm with ideal morphology. SETTING Andrology laboratory of a Center of Assisted Reproductive Technology. RESULTS Semen specimens with elevated concentrations of leukocytes contained a significantly higher frequency of sperm with ideal morphology than semen specimens with elevated numbers of immature germ cells and low numbers of leukocytes. CONCLUSIONS The direct correlation between leukocyte density and sperm with ideal morphology supports the concept that sperm surveillance is a normal function of male reproductive tract leukocytes. Understanding such germ cell-leukocyte interactions may provide valuable new insights into immunologic control mechanisms in male reproductive tract tissues.

Rapid Communication: Somatic Cell Nuclear Transfer in Humans: Pronuclear and Early Embryonic Development

Human therapeutic cloning requires the reprogramming of a somatic cell by nuclear transfer to generate autologous totipotent stem cells. We have parthenogenetically activated 22 human eggs and also performed nuclear transfer in 17 metaphase II eggs. Cleavage beyond the eight-cell stage was obtained in the parthenogenetic-activated eggs, and blastocoele cavities were observed in six. Three somatic cell-derived embryos developed beyond the pronuclear stage up to the six-cell stage. The ability to create autologous embryos represents the first step towards generating immune-compatible stem cells that could be used to overcome the problem of immune rejection in regenerative medicine.

Analysis of human immunodeficiency virus in semen: indications of a genetically distinct virus reservoir

It is well established that HIV is found in semen, either as cell-free or cell associated virus, yet many questions remain about the source of the virus. A number of factors, including anatomic features of the male reproductive tract, the restricted access of the immune system to the germ cell compartment, and the results from sexually transmitted virus studies, suggest that the source of HIV in semen may be different from that in the peripheral blood. In this study, we examine the HIV in the infected cells of semen as indicators of the virus producing reservoir. The frequency of HIV positive leukocytes in semen is compared to that of concurrent blood samples from eight donors and these values are found to be highly variable and frequently discordant. The protease gene sequences of HIV strains isolated from semen cells and blood cells were determined and phylogenetic analyses were performed which indicate the virus populations in the two sources are genetically distinct. In one patient receiving anti-HIV protease inhibitor therapy, gene sequences indicative of protease inhibitor resistance were found in the blood, but not the semen cell compartment. These results suggest that HIV in the semen and blood compartments are distinct, and further, may respond differently to antiviral therapy.

Multiple drug resistance mutations in human immunodeficiency virus in semen but not blood of a man on antiretroviral therapy

The concept that the male reproductive tract harbors isolated reservoirs of human immunodeficiency virus (HIV) infection has now been widely accepted. The significance of semen viral burden to sexual transmission of HIV is obvious; however, its contribution to disease progression is unknown. We report a case study that demonstrates the emergence of resistance-conferring mutations to antiviral therapy in infected seminal leukocytes from a man with asymptomatic prostatitis associated with leukospermia. This finding demonstrates the potential importance of male reproductive tract organs to the development of therapy resistance in HIV-infected men.

Parameters that predict success for natural cycle in vitro fertilization-embryo transfer.

OBJECTIVES To analyze our natural cycle IVF-ET experience in order to identify parameters predictive for success. DESIGN Retrospective study. SETTING Private-based reproductive center. PATIENTS All patients who presented for IVF-ET interested in unmedicated cycles. MAIN OUTCOME MEASURES Routine monitoring for IVF-ET was performed. In addition, serum values for FSH, PRL, E2, and P were analyzed. RESULTS Day 3 FSH values were not useful, whereas elevated day 3 LH values adversely correlated with outcome. Age was the single most important predictor of success. With two cycles, 87.5% of pregnancies occurred. CONCLUSION With proper patient selection, natural cycle IVF-ET results can be substantially improved.

Detection and identification of bacterial DNA in semen

OBJECTIVE To detect and identify bacteria in semen by sequencing polymerase chain reaction (PCR)-amplified ribosomal RNA gene regions (rDNAs). DESIGN Bacterial rDNAs were detected by PCR amplification of semen DNA. Conditions were adjusted to detect only abundant organisms, no fewer than 20,000 bacteria/mL of semen. SETTING Clinical andrology laboratory and academic research laboratories. PATIENT(S) Men undergoing fertility evaluation (n = 29) or vasectomy (n = 5). INTERVENTION(S) None. MAIN OUTCOME MEAURE(S): Frequency of bacterial rDNA-positive specimens, relationship of rDNAs to bacteria in GenBank, and correlation with semen cells. RESULT(S) Twenty-five (56%) of the specimens from 22 (65%) of the men were positive. A total of 141 bacterial rDNA sequences were compared with GenBank data for identification. The largest group matched gram-positive anaerobic cocci (Peptoniphilis, Anaerococcus, Finegoldia, Peptostreptococcus spp.) in 13 specimens, followed by Corynebacterium spp. in 10 specimens, Staphylococcus, Lactobacillus, and Streptococcus spp. in 7 specimens each, Pseudomonas spp. in 4 specimens, and Haemophilus and Acinetobacter spp. in 2 specimens each. The rDNA-positive specimens averaged 59 +/- 13 million sperm/mL, 46 +/- 5% of which were motile, not statistically different from the rDNA-negative specimens (77 +/- 16 million/mL, 47 +/- 5% motile). Normal sperm forms were lower in the rDNA-positive (10 +/- 1.1%) than in the rDNA-negative specimens (22 +/- 2%), and lymphocytes/monocytes were fivefold lower in the rDNA-positive specimens (0.4 +/- 0.2 million/mL) than in the negative specimens (1.9 +/- 0.7 million/mL). CONCLUSION(S) Abundant bacteria in semen are not commensal, arise from infection in the male genitourinary tract, may influence fertility, and may reflect an inadequate cellular immune response.

Expression and potential function of the c-mos proto-oncogene in human eggs.

OBJECTIVE To investigate the expression and possible function of the c-mos proto-oncogene in human eggs. DESIGN Eggs obtained as discarded material from assisted reproductive technology procedures were analyzed for c-mos messenger RNA by reverse transcriptase-polymerase chain reaction. As an approach to investigating c-mos function, we measured maturation-promoting factor (MPF) activity (histone H1 kinase) in eggs without and with inhibition of protein synthesis. Detection of RNA transcripts of c-raf was included as control. RESULTS Transcripts of c-mos were detected in small fractions of individual eggs, indicating that c-mos is abundantly transcribed. Inhibition of protein synthesis resulted in loss of MPF, leading to chromatin decondensation and reformation of a nucleus. C-raf maternal messages were also detectable in individual human eggs. CONCLUSION The c-mos proto-oncogene is an abundant maternal message in human eggs as in other species. The effects of inhibiting protein synthesis in human eggs are similar to those obtained in mouse and Xenopus eggs, either as a consequence of protein synthesis inhibition or specific ablation of c-mos RNA by injection of anti-sense oligonucleotides. The c-mos gene product is thus likely to play a critical role in human oocyte meiosis by regulating the activity of MPF.

Genome-wide microarray evidence that 8-cell human blastomeres over-express cell cycle drivers and under-express checkpoints

PurposeTo understand cell cycle controls in the 8-Cell human blastomere.MethodsData from whole human genome (43,377 elements) microarray analyses of RNAs from normal 8-Cell human embryos were compiled with published microarrays of RNAs from human fibroblasts, before and after induced pluripotency, and embryonic stem cells. A sub database of 3,803 genes identified by high throughput RNA knock-down studies, plus genes that oscillate in human cells, was analyzed.ResultsThirty-five genes over-detected at least 7-fold specifically on the 8-Cell arrays were enriched for cell cycle drivers and for proteins that stabilize chromosome cohesion and spindle attachment and limit DNA and centrosome replication to once per cycle.ConclusionsThese results indicate that 8-cell human blastomere cleavage is guided by cyclic over-expression of key proteins, rather than canonical checkpoints, leading to rapidly increasing gene copy number and a susceptibility to chromosome and cytokinesis mishaps, well-noted characteristics of preimplantation human embryos.