Chromatin structure in globozoospermia: a case report.

Sperm nuclear abnormalities in patients with globozoospermia have not been well characterized and may lead to the high rates of fertilization failure and embryo loss reported in patients with this form of teratozoospermia. This study used transmission electron microscopy (TEM), the sperm chromatin structure assay (SCSA), and single cell gel eletrophoresis assay (COMET) to assess if globozoospermia is associated with sperm chromatin structure abnormalities, DNA fragmentation, or both. The flow cytometric SCSA measures abnormal chromatin structure based on the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ. COMET measures DNA fragmentation in individual sperm nuclei based upon gel electrophoretic patterns. Although sperm concentration (113 million/mL) and motility (66%) were normal in the patient, there was complete acrosome deficiency. TEM and SCSA data confirmed light microscopic examination that showed that sperm populations included a mixture of round and elongated sperm heads. Even though 100% of sperm had abnormal head morphology, only 13% demonstrated DNA denaturation (COMPalpha(t)), which is below our threshold of 15% COMPalpha(t), and consistent with high-fertility patients. Of interest, 13% of the sperm were also positive in the COMET assay, supporting our previous observations that SCSA-positive cells are also positive for DNA fragmentation. It was unexpected but of great interest that a human sperm population with 100% sperm morphology abnormalities had a chromatin integrity at the molecular level that is equivalent to sperm populations shown in previous studies to be highly fertile. These data are the first reported using SCSA and COMET assays to evaluate a patient with globozoospermia and support previous reports that intracytoplasmic sperm injection of globozoospermia may result in fertility/pregnancy. Lower success rates seen in some patients may be due to unrelated factors.

Sperm chromatin structure assay parameters as predictors of failed pregnancy following assisted reproductive techniques.

The predictive value of sperm chromatin integrity for pregnancy outcome following in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) was studied in 24 men attending a university-based assisted reproductive techniques laboratory using the flow cytometric sperm chromatin structure assay (SCSA). The SCSA is a measure of the susceptibility of sperm DNA to low pH-induced denaturation in situ. The mean percentage of spermatozoa in the neat sample demonstrating DNA denaturation was significantly lower in the seven men that initiated a pregnancy (15.4 +/- 4.6, P = 0.01) than in the 14 men who did not initiate a pregnancy (31.1 +/- 3.2). No pregnancies resulted if > or =27% of the spermatozoa in the neat semen sample showed DNA denaturation. These data demonstrate that SCSA parameters are independent of conventional semen parameters. Furthermore, the SCSA may allow physicians to identify male patients for whom IVF and ICSI will be unlikely to result in pregnancy initiation.

Lack of effect of a high-fat meal on the bioavailability of 17 beta-estradiol/norgestimate in healthy postmenopausal women.

The effect of a high-fat meal on the absorption and pharmacokinetics of 17 beta-estradiol (E2), estrone (E1), estrone sulfate (E1S), and 17-deacetylnorgestimate (17d-NGM) were determined in this two-way complete crossover study of a single dose of E2/NGM (2 mg/180 micrograms) in 24 postmenopausal women. Equal numbers of subjects were randomly assigned to two treatment sequences indicated by the order of fed and fasting treatments. Serial blood samples were collected before and after dosing and assayed using validated methods. Food had no effect on the pharmacokinetics of E2, the pharmacologically active estrogen species. Food increased the rates of formation of E1 and E1S and slowed the formation of 17d-NGM. However, because E1 and E1S are pharmacologically less active metabolites of E2, and since the pharmacokinetic alterations in 17d-NGM were observed over a short time period, these results are probably of no clinical relevance. The extent of formation of all analytes, as measured by AUC, was not affected by food. In conclusion, administration of a tablet containing 17 beta-estradiol/norgestimate (2 mg/180 micrograms) was safe and well tolerated by healthy postmenopausal women and may be given without regard to the timing of meals in relation to dosing.

Density gradient centrifugation and glass wool filtration of semen remove spermatozoa with damaged chromatin structure.

The ability of double-layered density gradient centrifugation (DGC) or glass wool filtration (GWF) of semen to remove spermatozoa with damaged chromatin structure was assessed by the flow cytometric sperm chromatin structure assay (SCSA), which measures the susceptibility to sperm nuclear denaturation in situ. Ejaculates from 26 men attending a university-affiliated assisted reproduction laboratory were processed by DGC and GWF. Unprocessed, DGC- and GWF-processed specimens were assessed by the SCSA and by conventional semen parameters. Changes in chromatin structure were compared with conventional semen parameters. Both sperm preparation techniques yielded sperm suspensions with improved sperm chromatin structure as well as motility (%), forward progression (1-4) and viability (%). DGC was superior to GWF in the efficiency of recovering motile, morphologically normal, mature sperm suspensions. However, GWF produced improved chromatin integrity (SDalpha(t)) and viability. Moderate correlations between SCSA and conventional sperm parameters were observed. Nevertheless, the SCSA provides additional information about the biochemical integrity of sperm DNA and may be used in future studies to provide insight into assisted reproduction technology outcomes not explained by conventional sperm parameters.

Meiotically induced rec7 and rec8 genes of Schizosaccharomyces pombe.

The Schizosaccharomyces pombe rec7 and rec8 genes, which are required for meiotic intragenic recombination but not for mitotic recombination, have been cloned and their DNA sequences determined. Genetic and physical analyses demonstrated that the cloned fragments contained the rec genes rather than rec mutation suppressors. A 1.6-kb DNA fragment contained a functional rec7 gene, and a 2.1-kb fragment contained a functional rec8 gene. The nucleotide sequences of these fragments revealed open reading frames predicting 249 amino acids for the rec7 gene product and 393 amino acids for the rec8 gene product. Northern hybridization analysis showed that both rec gene mRNAs were detectable only at 2-3 hr after induction of meiosis. The absence of these mRNAs in mitosis and their disappearance at 4 hr and later in meiosis suggest that the rec7 and rec8 gene products may be involved primarily in the early steps of meiotic recombination in S. pombe.

Influence of template strandedness on in vitro replication of mutagen-damaged DNA.

We analyzed the ability of DNA polymerases to bypass damage on single- and double-stranded templates. In vitro DNA synthesis was studied on UV-irradiated and polyaromatic hydrocarbon reacted (benzo[a]pyrenediol epoxide and oxiranylpyrene) double-stranded templates by a protocol involving initiation on a uniquely nicked circular double-stranded template. The template was prepared by treating single-stranded (+)M13mp2 circular strands with mutagen and then hybridizing with restricted M13 RFmp2, followed by isolation of the nicked RFII forms. The protocol permits either (+), (-), or both strands to carry lesions. We found that the rules for termination and bypass of lesions previously observed with single-stranded DNA templates also hold for double-stranded templates. Termination of synthesis occurs primarily one nucleotide 3' to the lesion in the template strand. Bypass of UV-induced lesions can be followed in a series of three partial reactions in the presence of Mn2+ and dGMP, which relax the specificity of nucleotide insertion and 3'----5' exonuclease activity, respectively. There is no evidence for greater permissivity of bypass in double-as opposed to single-stranded templates. As with single-stranded templates, purines and preferentially deoxyadenosine (dA) are inserted opposite lesions. Lesions in the nontemplate strand elicit neither termination nor pausing. The addition of Rec A protein resulted in a measurable increase of bypass in this system.