Effects of follicle stimulating hormone (FSH) on follicular development, oocyte retrieval, and in vitro fertilization (IVF) in ewes during breeding season and seasonal anestrus

Administration of FSH increases the number of developing follicles, and affects oocyte health and cleavage rate. To determine the optimal level of FSH treatment, studies were conducted during the normal breeding season and seasonal anestrus. In Experiment 1, ewes were implanted with SyncroMate-B (SMB; norgestomet) for 14 days during the breeding season. Beginning on day 12 or 13 after SMB implantation, ewes were treated with saline (control; n=10), or treated with FSH for two days (2D; n=9) or three days (3D; n=10). In Experiment 2, conducted during seasonal anestrus, ewes were implanted with SMB for 14 days (n=23) or were not implanted (n=26). The SMB-implanted and nonimplanted ewes were assigned to one of three treatments as in Experiment 1: control (n=13), 2D (n=21) or 3D (n=15). In Experiments 1 and 2, ewes were laparotomized to count the number of follicles < or = 3 mm and > 3 mm and to retrieve oocytes. Healthy oocytes from each treatment were used for IVF. In Experiment 3, ewes (n=6) were implanted twice with SMB for 14 days during seasonal anestrus. Ewes were injected with FSH for 2 days, and the oocytes were collected and fertilized as in Experiments 1 and 2. In Experiment 1, FSH-treatment increased (P < 0.05) the number of follicles > 3 mm, the number of oocytes retrieved from follicles < or = 3 mm and > 3 mm, the proportion of healthy oocytes, and the number of oocytes used for IVF. Oocytes from control and 2D ewes had greater (P < 0.01) cleavage rates than 3D ewes (68% and 71% vs. 42%). In Experiment 2, implanted and nonimplanted ewes had similar (P > 0.05) numbers of follicles, total oocytes, and healthy oocytes; therefore, data were combined. The FSH treatment increased (P < 0.01) the number of follicles > 3 mm, and the number of oocytes recovered from follicles > 3 mm. The recovery rate of oocytes and the percentage of healthy oocytes were similar for control and FSH-treated ewes. The cleavage rate in Experiment 2 ranged from 4 to 16%. In Experiment 3, the cleavage rate for ewes treated twice with SMB was 27% which tended to be greater (P < 0.07) than for the 2D ewes that received one SMB implant in Experiment 2. These data indicate that FSH increased the number of developing follicles and the number of healthy oocytes retrieved from ewes during the breeding season and seasonal anestrus. However, cleavage rates during seasonal anestrus were lower than during the normal breeding season in both FSH-treated and control ewes. Treatment of ewes for 2 days with FSH resulted in a greater cleavage rate than treatment of ewes for 3 days.     

The frequency of heteroplasmy in the HVII region of mtDNA differs across tissue types and increases with age

An immobilized sequence-specific oligonucleotide (SSO) probe system consisting of 16 SSO probes that detect sequence polymorphisms within five regions of the mtDNA control region was used to investigate the frequency of heteroplasmy in human mtDNA. Five regions of hypervariable region II (HVII) of the control region were studied in blood-, muscle-, heart-, and brain-tissue samples collected from 43 individuals during autopsy. An initial search for heteroplasmy was conducted by use of the SSO probe system. Samples in which multiple probe signals were detected within a region were sequenced for the HVII region, to verify the typing-strip results. The frequency of heteroplasmy was 5 of 43 individuals, or 11.6%. The frequency of heteroplasmy differed across tissue types, being higher in muscle tissue. The difference in the frequency of heteroplasmy across different age groups was statistically significant, which suggests that heteroplasmy increases with age. As a test for contamination and to confirm heteroplasmy, the samples were sequenced for the HVI region and were typed by use of a panel of five polymorphic nuclear markers. Portions of the tissues that appeared to be heteroplasmic were extracted at least one additional time; all gave identical results. The results from these tests indicate that the multiple sequences present in individual samples result from heteroplasmy and not from contamination.     

Identification of a locus for autosomal dominant polycystic liver disease, on chromosome 19p13.2-13.1

Polycystic liver disease (PCLD) is characterized by the growth of fluid-filled cysts of biliary epithelial origin in the liver. Although the disease is often asymptomatic, it can, when severe, lead to complications requiring surgical therapy. PCLD is most often associated with autosomal dominant polycystic kidney disease (ADPKD); however, families with an isolated polycystic liver phenotype without kidney involvement have been described. The clinical presentation and histological features of polycystic liver disease in the presence or absence of ADPKD are indistinguishable, raising the possibility that the pathogenetic mechanisms in the diseases are interrelated. We ascertained two large families with polycystic liver disease without kidney cysts and performed a genomewide scan for genetic linkage. A causative gene, PCLD, was mapped to chromosome 19p13.2-13.1, with a maximum LOD score of 10.3. Haplotype analysis refined the PCLD interval to 12.5 cM flanked by D19S586/D19S583 and D19S593/D19S579. The discovery of genetic linkage will facilitate diagnosis and study of this underdiagnosed disease entity. Identification of PCLD will be instrumental to an understanding of the pathogenesis of cyst formation in the liver in isolated PCLD and in ADPKD.     

Comparative mapping reveals a complex relationship between the pearl millet genome and those of foxtail millet and rice

Comparative genetic maps were constructed of the pearl millet genome with foxtail millet and used to describe the homoeology between the genomes of pearl millet, foxtail millet and rice. Despite the close taxonomic relationship of pearl and foxtail millet, their genomes were highly, rearranged. A comparison of the millet and rice genomes indicated that most of these rearrangements were likely to have taken place in pearl millet. Two duplications were identified in pearl millet. A duplication between the distal segments of linkage groups 1 and 4 corresponds to the ancient duplication previously identified between rice chromosome arms 11S and 12S and foxtail millet chromosomes VII and VIII. The other putative duplication, also between regions of linkage groups 1 and 4, is likely to be species-specific. The exploitation of the comparative maps in pearl millet research is discussed. Received: 10 February 1999 / Accepted: 6 July 1999     

The Evolution of Sex Ratio Distorter Suppression Affects a 25 cM Genomic Region in the Butterfly Hypolimnas bolina

Symbionts that distort their host''s sex ratio by favouring the production and survival of females are common in arthropods. Their presence produces intense Fisherian selection to return the sex ratio to parity, typified by the rapid spread of host ‘suppressor’ loci that restore male survival/development. In this study, we investigated the genomic impact of a selective event of this kind in the butterfly Hypolimnas bolina. Through linkage mapping, we first identified a genomic region that was necessary for males to survive Wolbachia-induced male-killing. We then investigated the genomic impact of the rapid spread of suppression, which converted the Samoan population of this butterfly from a 100∶1 female-biased sex ratio in 2001 to a 1∶1 sex ratio by 2006. Models of this process revealed the potential for a chromosome-wide effect. To measure the impact of this episode of selection directly, the pattern of genetic variation before and after the spread of suppression was compared. Changes in allele frequencies were observed over a 25 cM region surrounding the suppressor locus, with a reduction in overall diversity observed at loci that co-segregate with the suppressor. These changes exceeded those expected from drift and occurred alongside the generation of linkage disequilibrium. The presence of novel allelic variants in 2006 suggests that the suppressor was likely to have been introduced via immigration rather than through de novo mutation. In addition, further sampling in 2010 indicated that many of the introduced variants were lost or had declined in frequency since 2006. We hypothesize that this loss may have resulted from a period of purifying selection, removing deleterious material that introgressed during the initial sweep. Our observations of the impact of suppression of sex ratio distorting activity reveal a very wide genomic imprint, reflecting its status as one of the strongest selective forces in nature.     

Direct evidence for the role of LGL in the inhibition of experimental tumor metastases

The role of NK cells in the control of the metastatic spread of tumor cells was studied. Rats pretreated with rabbit anti-asialo GM1 (anti-asGM1) serum exhibited a diminished ability to destroy circulating MADB106 mammary adenocarcinoma cells, which in turn caused an increased incidence of experimental pulmonary metastasis. The anti-asGM1 treatment caused a selective inhibition of NK activity without detectable effect on T cell-mediated immunity, and overall had no effect on the cytotoxic activity or numbers of alveolar macrophages (alv.M phi) or monocytes. The suggestion of a role for NK cells in resistance to metastases from the MADB106 tumor cells was confirmed by the adoptive transfer of 5 X 10(6) highly purified large granular lymphocytes (LGL) into NK-depressed animals 2 hr before tumor challenge. This transfer of LGL, highly enriched in NK activity, partially or fully restored the ability of these rats to inhibit the development of pulmonary metastases. This ability to adoptively transfer resistance to metastases appeared to be confined to the LGL population, because transfer of the same number of mature peripheral blood T cells had no effect on tumor development. These results provide the first unequivocal evidence that LGL, with high NK activity, are involved in in vivo resistance to tumors, particularly in the elimination of potentially metastatic tumor cells from the circulation and capillary beds.     

Thermoregulatory behavior and diel activity of yearling winter flounder,Pseudopleuronectes americanus (Walbaum)

Synopsis Sixteen yearling winter flounder, tested individually for 3-day periods in electronic shuttleboxes, voluntarily occupied an 8–27°C range of temperature, with a modal final thermal preferendum of 18.5°C (mean 18.7°C, median 19°C, midpoint 17.5°C, s.d. 1.9°C, S _k + 0.33). The locomotor activity pattern of the yearling fish in the laboratory was markedly nocturnal, with mean hourly nocturnal activity exceeding mean hourly diurnal activity by a factor of 3.4. Maximum activity occurred at 0300 EST, minimum activity at 1400 EST. While activity generally increased with temperature, a local activity minimum occurred at 18.7°C, coinciding with the 24-hour mean final thermal preferendum. Comparison of these laboratory data with previously published field data suggest that behavioral responses to temperature and light play significant roles in determining age- and size-specific differences in seasonal depth stratification and onshore-offshore distributions in this species.     

T47D Cells Expressing Myeloperoxidase Are Able to Process,Traffic and Store the Mature Protein in Lysosomes: Studies in T47D Cells Reveal a Role for Cys319 in MPO Biosynthesis that Precedes Its Known Role in Inter-Molecular Disulfide Bond Formation

Among the human heme-peroxidase family, myeloperoxidase (MPO) has a unique disulfide-linked oligomeric structure resulting from multi-step processing of the pro-protein monomer (proMPO) after it exits the endoplasmic reticulum (ER). Related family members undergo some, but not all, of the processing steps involved with formation of mature MPO. Lactoperoxidase has its pro-domain proteolytically removed and is a monomer in its mature form. Eosinophil peroxidase undergoes proteolytic removal of its pro-domain followed by proteolytic separation into heavy and light chains and is a heterodimer. However, only MPO undergoes both these proteolytic modifications and then is further oligomerized into a heterotetramer by a single inter-molecular disulfide bond. The details of how and where the post-ER processing steps of MPO occur are incompletely understood. We report here that T47D breast cancer cells stably transfected with an MPO expression plasmid are able to efficiently replicate all of the processing steps that lead to formation of the mature MPO heterotetramer. MPO also traffics to the lysosome granules of T47D cells where it accumulates, allowing in-depth immunofluorescent microscopy studies of MPO trafficking and storage for the first time. Using this novel cell model we show that formation of MPO’s single inter-molecular disulfide bond can occur normally in the absence of the proteolytic events that lead to separation of the MPO heavy and light chains. We further demonstrate that Cys³¹⁹, which forms MPO’s unique inter-molecular disulfide bond, is important for events that precede this step. Mutation of this residue alters the glycosylation and catalytic activity of MPO and blocks its entry into the endocytic pathway where proteolytic processing and disulfide bonding occur. Finally, using the endocytic trafficking of lysosomal hydrolases as a guide, we investigate the role of candidate receptors in the endocytic trafficking of MPO.