Estrogenic pyrethroid pesticides regulate expression of estrogen receptor transcripts in mouse Sertoli cells differently from 17beta-estradiol.

Studies suggested that exposure to agricultural pesticides may affect male fertility. Pyrethroids are widely used pesticides due to their insecticidal potency and low mammalian toxicity. A recombinant yeast assay system incorporating the human alpha-estrogen receptor was used to analyze the estrogenicity of a range of readily available pyrethroid pesticides. The commercial product Ripcord Plus showed estrogenic activity by this assay. To determine whether pyrethroid compounds might exert an effect on male fertility, mouse Sertoli cells were exposed in vitro to the endogenous estrogen, 17beta-estradiol, and selected estrogenic pyrethroids. Following exposure, transcript levels of the alpha- and beta-estrogen receptors were assessed. Exposure of Sertoli cells to the pyrethroid compounds, both at high and at low published serum concentrations, affected the expression of the two estrogen receptors; however, the influence on estrogen receptor gene expression was different from the effect from exposure to 17beta-estradiol. These results from our model systems suggest that (1) estrogenic pyrethroid pesticides affect the estrogen receptors, and therefore potentially the endocrine system, in a different manner from that of endogenous estrogen, and (2) should cells in the male testes be exposed to pyrethroid pesticides, male fertility may be affected through molecular mechanisms involving estrogen receptors.

Molecular analysis of polymerase gamma gene and mitochondrial polymorphism in fertile and subfertile men.

CAG trinucleotide repeat length in the nuclear polymerase gamma gene (POLgamma) has been shown to be associated with men with reduced fertility. The present study investigated the frequency of CAG repeat length genotypes and three exonuclease motifs of the POLgamma in relation to the frequency of mitochondrial nucleotide substitutions. DNA from semen samples of 93 normozoospermic men and 192 non-normozoospermic men was isolated and the specific regions of the genes were amplified by polymerase chain reactions (PCR) and sequenced to identify mutations. The genotypic frequencies of pooled POLgamma CAG repeat lengths, =10/ not equal 10 heterozygotes and not equal 10/ not equal 10 homozygotes, were significantly different between normozoospermic and non-normozoospermic men (p < 0.05), with non-normozoospermic men having a slightly higher frequency of the =10/=10 genotypes. The allelic frequency for =10 is 0.79 and not equal10 is 0.21 for normozoospermic men and 0.85 and 0.15, respectively, for non-normozoospermic men (p < 0.025). There was no mutation detected in the exonuclease motifs in all the samples tested. Eighty normozoospermic and 124 non-normozoospermic semen samples were analysed for nucleotide substitutions in mitochondrial genes by PCR and sequencing. Heteroplasmic mutations were found in one azoospermic man, four asthenozoospermic men and two normozoospermic men. Only one asthenozoospermic man was heterozygous for the POLgamma genotype. Of the 17 men with non-synonymous nucleotide substitutions, 14 were homozygous for the POLgamma genotype. Non-normozoospermic men had twice as many nucleotide substitutions than normozoospermic men. However, there were no significant differences in the frequencies of nucleotide substitution and POLgamma genotypes in the two groups of men.

Molecular cloning and characterisation of a novel membrane receptor gene from the lobster Jasus edwardsii.

The eyestalk of the lobster, Jasus edwardsii, is an important source for hormones involved in the regulation of growth and reproduction. How these hormones transfer their messages to the cell and nucleus is not known. This paper describes the cloning, characterization and expression analyses of two genes that code for two membrane-associated peptides that may be involved in signal transduction. These genes, peJK2 and peJK3, were isolated from a cDNA library derived from lobster eyestalk mRNAs. The two clones shared 96.6 % sequence homology, and code for putative proteins of 110 and 113 amino acids, respectively. These were likely to be two allelic forms of the same gene. Northern blot analysis using these clones as probes detected the same mRNA from eyestalk, muscle and epithelial extracts, but with greater intensity in the eyestalk extract. In situ hybridisation also indicated the predominant expression of these genes in the eyestalk. Analysis of the putative protein sequences showed that they contained two transmembrane (TM) helices, a short amino acid sequence sharing high homology with the G-protein-coupled receptor (GPCR) motif in the second TM, a signal sequence between the TMs, and a protein kinase phosphorylation site at the C termini. Sequence analyses therefore suggested that the deduced peptides may function in signal transduction.

High incidence of single nucleotide substitutions in the mitochondrial genome is associated with poor semen parameters in men.

Single nucleotide polymorphisms (SNPs) in 7000 bp of the mitochondrial genome, encompassing 15 coding regions from COI to ND5, were characterized by single strand polymorphism analysis and confirmed by DNA sequencing. About 2.4% of normozoospermic men and 8.4% of men with poor semen quality had at least one nucleotide substitution. Most of the substitutions occurred in the third codon and did not change the amino acid. Hydrophobicity plots of the proteins with changes in an amino acid as a result of a nucleotide substitution suggested that they did not affect the function of the protein. The two most common substitutions at nucleotide (nt) 9055 and 11719 had significantly higher frequencies in men with reduced sperm motility. Eleven percent of the men with poor semen parameters and 1.3% of normozoospermic men had a 9055 substitution, 12% of the men with poor semen parameters had a substitution at nt 11719, but none of the normozoospermic men had this substitution. All the patients with these substitutions had reduced sperm motility and/or low sperm count. These SNPs in the mitochondrial genome were in a homoplasmic state. Thus, we propose that possessing these mitochondrial mutations compromises the semen quality of these men.

Electroporation of salmon sperm for gene transfer: efficiency, reliability, and fate of transgene.

Uptake of exogenous DNA by electroporated salmon sperm for gene transfer is being investigated. Our studies show that electroporated salmon sperm cells were more efficient and more reliable than untreated sperm in picking up exogenous DNA and subsequently transferring the DNA into salmon embryos. Indirect evidence suggest that some of the exogenous DNA was internalized in the sperm nuclei. The taken up DNA retained its integrity as demonstrated by PCR. The foreign DNA was detected in 15-month-old fish, and had a mosaic pattern of distribution. Integration of the foreign DNA occurred infrequently, and the expression of the foreign genes was poor. The potential of sperm-mediated gene transfer as a routine protocol for mass gene transfer in salmon will be dependent on the improvement of integration and expression of the foreign gene.

Frequency of microdeletions in the azoospermia factor region of the Y-chromosome of New Zealand men.

AIM: To determine the frequency of microdeletions in the azoospermic factor (AZF) genes on the Y-chromosome of New Zealand men attending the Fertility Centre. METHODS: World Health Organisation criteria were used to classify men as normospermic, oligozoospermic, severely oligozoospermic, and azoospermic. Microdeletions were detected from DNA of semen samples by the sequence-tagged site polymerase chain reaction. RESULTS: Microdeletions were detected in 20% (3/15) of azoospermic men, 4% (2/50) of severely oligozoospermic men, 3.2% (2/62) of oligozoospermic men, and 0.7% (1/141) normospermic men. One azoospermic man had multiple non-contiguous deletions. Overall, 5.5% of infertile men had at least one microdeletion in the long arm of the Y-chromosome. One severely oligozoospermic man and one oligozoospermic man had produced unassisted pregnancies. CONCLUSION: New Zealand men attending a Christchurch fertility centre have a similar frequency of microdeletions in the Y-chromosome to other populations. Azoospermic men have a higher frequency of microdeletions than men with less severe spermatogenic failure. Men with microdeletions can have reduced fertility, but are not necessarily sterile.

Association of a novel human mtDNA ATPase6 mutation with immature sperm cells.

This study reports the first clearly defined heteroplasmic mutation in immature human sperm cells. The human sperm mitochondrial genome from residue 8186-9341 was analysed with the aim of identifying point mutations which may be associated with human male infertility. The semen samples analysed were obtained from 88 fertile men, 19 with oligozoospermia, and 12 with severe oligozoospermia. Using single strand conformation polymorphism analysis a heteroplasmic T to C transition was detected in the ATPase6 gene, at nucleotide position 8821, in semen samples from one out of 12 (8%) severely oligozoospermic men, but not in oligozoospermic men or normospermic men. This mutation changed the amino acid serine to proline at residue 99 of the mitochondrial ATPase6 in a region which is highly conserved in other vertebrates including rat, bovine, chicken, salmonids and Xenopus. The mutation was detected in semen samples collected from the same man 9 months apart and in peripheral blood lymphocytes. Single sperm cell analyses did not find this mutation in the mature sperm, but the mutation was detected in 7% of immature spermatids. Our finding suggests that immature spermatids with this mutation fail to develop fully.

The role of JH III and ecdysterone in the regulation of the left colleterial gland activities in Periplaneta americana during the reproductive cycle.

We studied the in vitro effects of JH III and ecdysterone on RNA synthesis and secretory activities of the left colleterial gland (LCG) in mature females of Periplaneta americana during the reproductive cycle. We found that RNA synthesis is a cyclic and highly dynamic process. The major increases in poly(A)- RNA synthesis occurred 8 hr before the peak leucine incorporation into colleterial polypeptides, whereas high poly(A)+ RNA synthesis coincided with high leucine incorporation. Depending on the stage of the cycle JH III exhibited stimulatory or inhibitory effects, or caused no change in RNA synthesis. JH III stimulated RNA synthesis in glands from stages in the reproductive cycle with high endogenous RNA synthesis. The pattern of protein synthesis was not affected by JH III. Ecdysterone lowered RNA synthesis in the LCG and induced secretory activity in LCGs isolated at stages far removed from ovulation. JH III reduced the inhibitory effect of ecdysterone on RNA synthesis and inhibited the ecdysterone-induced secretory activity. The presence of ovarioles isolated at 62 hr of the reproductive cycle (onset of in vivo LCG secretory activity) in the incubation medium inhibited RNA synthesis in 32 hr LCG but induced secretory activity. We suggest that changes in synthetic and secretory activities are coordinated with ovulation and ootheca formation through the interactions between JH III and ovarioles, and ecdysterone may also play a role.

The effect of ecdysterone and juvenile hormones on protein synthesis and development of imaginal wing discs of Drosophila melanogaster.

The effect of ecdysterone and juvenile hormone on protein synthesis and development of imaginal wing discs of Drosophila melanogaster has been studied. It is found that juvenile hormone apparently does not inhibit the synthesis of the ecdysterone-inducible proteins, although wing disc development is inhibited to various extent by different juvenile hormones. It is suggested that the ecdysterone-inducible proteins are not involved directly in the initiation of wing disc evagination, it is possible that some of these proteins are involved in the maintenance of chromatin activities or they are involved in gene activation.

Puromycin enhances 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster.

The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the 'early' and 'late' proteins. However, when puromycin was applied after hormone treatment, only the 'late' proteins were induced. The possible implication of these observations is discussed.

Relationship between 'early' and 'late' protein induced by 20-hydroxyecdysone in imaginal wing discs of Drosophila melanogaster.

The insect moulting hormone, 20-hydroxyecdysone, induces the synthesis of two groups of proteins in the imaginal wing discs of Drosophila melanogaster. The early induced group appears about 4 h after hormone treatment, the late induced group appears about 12 h after treatment. Studies using alpha-amanitin to inhibit mRNA synthesis during the period of hormonal treatment showed that the induction of the late proteins is dependent on mRNA synthesis and possibly the synthesis of the early proteins.

Nucleolar silver-staining patterns related to cell cycle phase and cell generation of PHA-stimulated human lymphocytes.

Silver staining (Ag-I) was used to investigate changes in the nucleolar structure of PHA-stimulated human lymphocytes through the phases of the cell cycle, G1, S and G2. Ag-I patterns and cell cycle phases of individual cells were assessed by sequential silver staining, Feulgen staining, DNA microdensitometry and 3H-thymidine autoradiography. The morphology and number of Ag-I nucleoli in a particular cell depended upon the phase of the cell cycle reached and on the number of generations the cell had passed through in culture. Resting, unstimulated cells usually had one small silver positive nucleolus. During blast transformation, the silver stained nucleoli increased in number and size, and then fused to form one very large, rounded or irregular-shaped nucleolus which was present through all cell cycle phases of the first reproductive cycle. Many lymphocytes developed a band-shaped nucleolus during their first S phase in culture. Lymphocytes at all cell cycle stages of the second and third generations after PHA-stimulation had multiple nucleoli whose combined areas approximated that of the single large nucleolus observed in first generation cells.