IL-6 and Mouse Oocyte Spindle

Interleukin 6 (IL-6) is considered a major indicator of the acute-phase inflammatory response. Endometriosis and pelvic inflammation, diseases that manifest elevated levels of IL-6, are commonly associated with higher infertility. However, the mechanistic link between elevated levels of IL-6 and poor oocyte quality is still unclear. In this work, we explored the direct role of this cytokine as a possible mediator for impaired oocyte spindle and chromosomal structure, which is a critical hurdle in the management of infertility. Metaphase-II mouse oocytes were exposed to recombinant mouse IL-6 (50, 100 and 200 ng/mL) for 30 minutes and subjected to indirect immunofluorescent staining to identify alterations in the microtubule and chromosomal alignment compared to untreated controls. The deterioration in microtubule and chromosomal alignment were evaluated utilizing both fluorescence and confocal microscopy, and were quantitated with a previously reported scoring system. Our results showed that IL-6 caused a dose-dependent deterioration in microtubule and chromosomal alignment in the treated oocytes as compared to the untreated group. Indeed, IL-6 at a concentration as low as 50 ng/mL caused deterioration in the spindle structure in 60% of the oocytes, which increased significantly (P<0.0001) as IL-6 concentration was increased. In conclusion, elevated levels of IL-6 associated with endometriosis and pelvic inflammation may reduce the fertilizing capacity of human oocyte through a mechanism that involves impairment of the microtubule and chromosomal structure

Morphology and function of cryopreserved whole ovine ovaries after heterotopic autotransplantation

<p>Abstract</p> <p>Background</p> <p>The objective of this study was to perform complex characterization of cryopreserved and then autotransplanted ovaries including determination of the ability to respond to in vivo follicle stimulating hormone (FSH)-treatment, fertilizability of retrieved oocytes, and morphology, vascularization, cellular proliferation and apoptosis in sheep.</p> <p>Methods</p> <p>Mature crossbred ewes were divided into two groups; an intact (control) group (n = 4), and autotransplanted group (n = 4) in which oophorectomy was performed laparoscopically and ovaries with intact vascular pedicles frozen, thawed and transplanted back into the same animal at a different site. Approximately five months after autotransplantation, estrus was synchronized, ewes were treated with FSH, and ovaries were collected. For all ovaries, number of visible follicles was determined, and collected cumulus oocyte complexes (COC) were matured and fertilized in vitro. Remaining ovarian tissues were fixed for evaluation of morphology, expression of factor VIII (marker of endothelial cells), vascular endothelial growth factor (VEGF; expressed by pericytes and smooth muscle cells), and smooth muscle cell actin (SMCA; marker of pericytes and smooth muscle cells), and cellular proliferation and apoptosis. Two fully functional ovaries were collected from each control ewe (total 8 ovaries).</p> <p>Results</p> <p>Out of eight autotransplanted ovaries, a total of two ovaries with developing follicles were found. Control ewes had 10.6 +/- 2.7 follicles/ovary, oocytes were in vitro fertilized and developed to the blastocyst stage. One autotransplanted ewe had 4 visible follicles from which 3 COC were collected, but none of them was fertilized. The morphology of autotransplanted and control ovaries was similar. In control and autotransplanted ovaries, primordial, primary, secondary, antral and preovulatory follicles were found along with fully functional vascularization which was manifested by expression of factor VIII, VEGF and SMCA. Proliferating cells were detected in follicles, and the rate of apoptosis was minimal in ovaries of control and autotransplanted ovaries.</p> <p>Conclusion</p> <p>These data demonstrate successful autotransplantation of a portion of frozen/thawed ovaries manifested by restoration of selected ovarian function including in vitro maturation of collected oocytes, presence of follicles from several stages of folliculogenesis and blood vessels expressing specific markers of vascularization, and proliferation and apoptosis of ovarian cells. Thus, heterotopic autotransplantation of a whole frozen/thawed ovary allows for development of preovulatory follicles, oocyte growth, and for restoration of vascularization and cellular function. However, additional improvements are required to enhance the efficiency of autotransplantation of frozen/thawed ovaries to produce more oocytes.</p

Confocal images of oocytes.

<p><b>A</b>. Unexposed control; <b>B</b>. Effect of IL-6 at 50 ng/mL; <b>C</b>. Effect of IL-6 at 100 ng/mL; and <b>D</b>. Effect of IL-6 at 200 ng/mL Scale – 1 pixel, 5 mm.</p

Percentage of oocytes with “Poor” scores (score 3, 4) in microtubule (MT) and chromosomal alignment (CH) at different concentrations of IL-6.

<p>Percentage of oocytes with “Poor” scores (score 3, 4) in microtubule (MT) and chromosomal alignment (CH) at different concentrations of IL-6.</p

Algorithm of scoring alterations in microtubule and chromosomal alignment on the Metaphase-II mouse oocyte based on previously published data.

<p>Algorithm of scoring alterations in microtubule and chromosomal alignment on the Metaphase-II mouse oocyte based on previously published data.</p

Percentage of oocytes with “Good” (score 1, 2) and “Poor” (score 3, 4) score in microtubular (MT) and chromosomal (CH) alignment at different concentrations of IL-6.

<p>Percentage of oocytes with “Good” (score 1, 2) and “Poor” (score 3, 4) score in microtubular (MT) and chromosomal (CH) alignment at different concentrations of IL-6.</p