Influence of estradiol treatment on bone marrow cell differentiation in collagenase-induced arthritis.

OBJECTIVE AND DESIGN: Estrogen is one of the important regulators of the balance between bone formation and bone resorption that can modulate the levels and activity of certain growth factors and cytokines. In this study, we investigated the effect of 17ß-estradiol (ED) on bone marrow (BM) cell differentiation in vivo and ex vivo in a mouse model of collagenase-induced osteoarthritis (CIOA). SUBJECT: ICR (CD-2) female mice were used in present experiments (total number = 75) and bone marrow cells were used for in vitro studies. TREATMENT: Mice were orally fed under different schemes with 17ß-estradiol at a dose of 2 µg or 4 µg for 30 days. METHODS: The effect of estradiol was estimated by histopathological, flow cytometry, and ELISA assays. Statistical differences were determined by one-way ANOVA. RESULTS: Estradiol treatment ameliorated cartilage destruction and osteophyte formation if started from day 0 of CIOA induction, attended with a decrease of uterine and ovarian weights. Long time treatment lowered the percentage of megakaryocyte/platelet (CD62P+) populations and osteoclast (RANK+) populations in BM. Cells obtained from estradiol-treated CIOA mice showed inhibited capacity to differentiate into RANK+ and mesenchymal cells under osteoclastogenic conditions in vitro. Estrogen decreased serum IL-6 levels. CONCLUSION: Results indicate a potential protective role for estrogen against the development of OA.

Lupus progression deteriorates oogenesis quality in MRL/lpr mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of the immune response against self antigens. Numerous reproductive complications, including reduced birth rate and complications for the mother and the fetus during pregnancy, have been observed in women with SLE. In the present study, we aimed to investigate the effect of SLE development on oocyte meiosis in lupus-prone mice. Lupus-prone MRL/lpr mice were used for the experiments: disease-free (4 weeks of age) and sick (20 weeks of age, virgin and postpartum). The immune response was monitored by flow cytometry, ELISpot, ELISA, and histology. Oocytes were analyzed by fluorescence microscopy based on chromatin, tubulin, and actin structures. The lupus-prone MRL/lpr mice developed age-dependent symptoms of SLE with increased levels of various autoantibodies, proteinuria, and renal infiltrates and a tendency for the immune response to worsen with changes in cell populations and the cytokine profile. The number and quality of oocytes were also affected, and the successful pregnancy rate of MRL/lpr mice was limited to only 60%. Isolated oocytes showed severe structural changes in all studied groups. Systemic alterations in immune homeostasis in SLE affect the quality of developing oocytes, which is evident from a young age. The data obtained is in line with the trend of reduced fertility in lupus-prone MRL/lpr mice. The phenomenon can be explained by changes in the microenvironment of the relevant organs and close connection between ovulation and inflammatory processes.

Follicle stimulating hormone and estradiol alter immune response in osteoarthritic mice in an opposite manner.

Although a number of studies have shown that the occurrence and progression of osteoarthritis (OA) is related to endocrine system dysfunction, there is limited evidence about what roles sex hormones play. The aim of the present study was to examine the capacity of 17ß-estradiol (ED) and follicle stimulating hormone (FSH) to alter the differentiation of bone marrow (BM) cells in arthritic mice. The experiments were conducted in collagenase-induced osteoarthritis in mice. Cartilage degradation was observed by safranin and toluidine blue staining. Flow cytometry was used to define different BM and synovial cell populations. The influence of FSH and ED on osteoclastogenesis was studied in BM cultures and on the osteoblastogenesis in primary calvarial cultures. The levels of IL-8, TNF-α, FSH, and osteocalcin were estimated by ELISA. FSH increased cartilage degradation and serum osteocalcin levels, while ED abolished it and lowered serum osteocalcin. FSH elevated the percentage of monocytoid CD14+/RANK+ and B cell CD19+/RANK+ cells in contrast to ED which inhibited the accumulation of these osteogenic populations. Also, ED changed the percentage of CD105+/F4/80+ and CD11c+ cells in the synovium. FSH augmented and ED suppressed macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast (OC) formation, and this correlated with a respective increase and decrease of IL-8 secretion. FSH did not influence osteoblast (OB) formation while ED enhanced this process in association with changes of TNF-α, IL-8, and osteocalcin production. ED reduced osteoclast generation in bone. The key outcome of the current study is that both hormones influenced BM cell differentiation, with FSH favoring osteoclast formation and ED favoring osteoblast accumulation.

Osteoarthritis Affects Mammalian Oogenesis: Effects of Collagenase-Induced Osteoarthritis on Oocyte Cytoskeleton in a Mouse Model.

Known as a degenerative joint disorder of advanced age affecting predominantly females, osteoarthritis can develop in younger and actively working people because of activities involving loading and injuries of joints. Collagenase-induced osteoarthritis (CIOA) in a mouse model allowed us to investigate for the first time its effects on key cytoskeletal structures (meiotic spindles and actin distribution) of ovulated mouse oocytes. Their meiotic spindles, actin caps, and chromatin were analyzed by immunofluorescence. A total of 193 oocytes from mice with CIOA and 209 from control animals were obtained, almost all in metaphase I (M I) or metaphase II (MII). The maturation rate was lower in CIOA (26.42% M II) than in controls (55.50% M II). CIOA oocytes had significantly larger spindles (average 37 µm versus 25 µm in controls, p < 0.001), with a proportion of large spindles more than 64% in CIOA versus up to 15% in controls (p < 0.001). Meiotic spindles were wider in 68.35% M I and 54.90% M II of CIOA oocytes (mean 18.04 µm M I and 17.34 µm M II versus controls: 11.64 µm M I and 12.64 µm M II), and their poles were approximately two times broader (mean 6.9 µm) in CIOA than in controls (3.6 µm). CIOA oocytes often contained disoriented microtubules. Actin cap was visible in over 91% of controls and less than 20% of CIOA oocytes. Many CIOA oocytes without an actin cap had a nonpolarized thick peripheral actin ring (61.87% of M I and 52.94% of M II). Chromosome alignment was normal in more than 82% in both groups. In conclusion, CIOA affects the cytoskeleton of ovulated mouse oocytes-meiotic spindles are longer and wider, their poles are broader and with disorganized fibers, and the actin cap is replaced by a broad nonpolarized ring. Nevertheless, meiotic spindles were successfully formed in CIOA oocytes and, even when abnormal, allowed correct alignment of chromosomes.

Defective oogenesis in mice with pristane-induced model of systemic lupus.

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by generation of autoantibodies and severe damage of various organs. The hormonal changes associated with pregnancy and especially estrogen might lead to damage of reproductive function and ovarian quality. We employed a pristane-induced lupus model of Balb/c mice which resembles human lupus in an attempt to follow oogenesis disruption during the disease progression. The integrity of cytoskeletal and chromatin structures was estimated in oocytes derived by hormonally stimulated ovulation in lupus mice and the results were compared with those from healthy mice. Chromatin, tubulin and actin structures in oocytes were detected by Hoechst 33258, anti-alpha-tubulin antibody and rhodamine-labeled phalloidin, respectively. All available meiotic spindles were analyzed - in immature (metaphase I) and mature oocytes (metaphase II). The total number of mature oocytes obtained from lupus mice was lower compared to healthy controls. The maturation rate was 9.8 % for lupus mice, 12.7 % for 7-month old controls, and 14.3 % for the young control mice (4 weeks old). Another major difference between the studied groups was the higher percentage of defective metaphase I spindles registered in oocytes derived from lupus mice (60 % normal spindles), while for the young and older controls this proportion was 86 % and 81 %, respectively. No such difference was registered for metaphase II spindles. For both metaphase I and metaphase II oocytes, the proportions of normal actin cap and chromosomal condensation were similar between the experimental groups.

Analysis of human unfertilized oocytes and pronuclear zygotes--correlation between chromosome/chromatin status and patient-related factors.

OBJECTIVE: The objective was to evaluate the relationship between ploidy and chromatin status of human unfertilized oocytes/zygotes and infertility history, female age, and stimulation regimens. STUDY DESIGN: Two hundred and eighty-nine unfertilized oocytes and 63 zygotes were subjected to cytogenetic analysis: karyotyping for oocytes and fluorescent in situ hybridization (FISH) analysis for zygotes. Ploidy and chromosome/chromatin status were analyzed according to stimulation regimen, female age, and infertility history. The correlation coefficient was estimated and data were interpreted using a five-grade scale. RESULTS: Aneuploidy in karyotyped oocytes (19.7% hyperhaploidy, 18.8% hypohaploidy, and 6.3% haploid abnormal) was associated with chromosome fragmentation and lesions due to chromosome aging in culture. Premature chromosome condensation and cytoskeletal defects were significantly higher in unexplained infertility (34.7% and 52.9%, respectively; p<0.05). Chromatin quality was most important for successful ploidy analysis of zygotes. FISH analysis of abnormal zygotes elucidated genetic aspects of pronuclear number aberrations and raised questions about the current selection criteria. Abnormalities were found to correlate moderately with stimulation strategy and female age and significantly with infertility history. CONCLUSION: Genetic analysis of human oocytes and zygotes showed that poor chromatin quality and patient-related factors contribute to aneuploidy and pronuclear number aberrations.

Chromosomal disorders and nuclear and cell destruction in cleaving human embryos.

Three types of defects of preimplantation embryogenesis contribute to the developmental arrest of cleaving human embryos: blastomere fragmentation, abnormal nuclear status and chromosomal disorders. Data concerning the relation and succession of these abnormalities during first mitotic cycles of the human zygote are controversial and mainly empirical at present. In this study we have performed simultaneous evaluation of blastomere fragmentation, nuclear apoptotic changes and the ploidy of four chromosomes (1, 5, 19 and X or 18, 21, X and Y) in 193 human embryos. Another group of 28 embryos was subjected to TUNEL for confirmation of apoptosis in blastomere nuclei. Nuclei with apoptotic chromatin were seen in nearly 1/10 of blastomeres of embryos with good morphology and in more than 1/5 of blastomeres of embryos with more than 20% fragmentation. The correct number of investigated chromosomes was registered in 85.2% of successfully tested embryos. Chromatin apoptotic changes are the only limiting factor for the success of chromosomal FISH tests. Nearly 1/2 of embryos with at least one apoptotic nucleus were chromosomally abnormal. For the embryos that contain only normal nuclei, the rate of ploid normality was more than 89%. The rate of euploidy was higher (66%) in embryos with a significant degree of cell fragmentation. Moderate cell fragmentation was not related to significant increase of chromatin and chromosomal disorders. In a substantial portion of abnormal blastomeres, chromatin damage preceded cell fragmentation. Nuclear destruction in human blastomeres was illustrated by fluorograms of different stages of chromatin lesions.

Ploidity and chromatin status of human oocytes after failed in vitro fertilization.

OBJECTIVE: To investigate the chromatin status of oocytes and their ploidity in order to consider possible genetic reasons for their unsuccessful fertilization in vitro. STUDY DESIGN: Eighty-six unfertilized in vitro human oocytes were scored in six groups according to their chromatin status. Analysis was performed by cytogenetics and by fluorescent in situ hybridization (FISH). RESULTS: 73.26% of oocytes were successfully analyzed: 44.19% by classical cytogenetics and 17.44% by FISH. 11.63% of the analyzed cells contained sperm chromosomes with premature chromosome condensation (PCC). Among karyotyped oocytes, a high aneuploidy rate was found (5.26% haploid abnormal, 21.05% hypohaploid, 36.84% hyperhaploid and 7.89% diploid). FISH for chromosomes X, 1, 5 and 19 performed on unsuitable for karyotyping metaphase plates revealed 13.33% aneuploid cells. CONCLUSION: Analysis of fertilization failure could be more informative when a combination of methods is applied.

Possible cytoskeletal structures of rainbow trout sperm revealed by electron microscopic observation after detergent extraction.

Despite the considerable research interest in fish sperm ultrastructure, little is known about the functions of different sperm cell components. Our electron microscopic study was aimed at identifying possible tissue-specific cytoskeletal structures in spermatozoa of rainbow trout Oncorhynchus mykiss (Teleostei, Salmoniformes, Salmonidae; formerly Salmo gairdneri). Based on the known resistance of the cytoskeleton to nonionic detergents, we compared the ultrastructure of unextracted and Triton-extracted sperm cells. Besides the nucleus, the centrioles and the axoneme, there were also other structures preserved in Triton-treated spermatozoa: the lateral extensions (sidefins) and a thin layer corresponding in position to the membrane-like structure underlying the midpiece plasma membrane in intact cells. Because of their stability, it could be hypothesized that these cytoplasmic components are likely to have cytoskeletal nature. They are possibly analogous to the well known tissue-specific cytoskeletal components of mammalian spermatozoa with periaxonemal and submitochondrial localization.