Ovarian tissue cryopreservation and subsequent transplantation can restore fertility in cancer patients. This study used a mouse ovarian grafting model to investigate whether the graft site (bursal cavity, the kidney capsule or subcutaneous) influences the number, fertilization rate and developmental potential of oocytes recovered from grafts and whether using a standard gonadotrophin stimulation protocol would increase oocyte yield from the grafts. Mouse ovarian tissue was grafted into four week old mice and collected three weeks later. Graft recipients were treated either with or without exogenous gonadotrophin stimulation prior to graft collection. Grafted ovaries yielded oocytes that were either at the germinal vesicle (GV) stage or mature metaphase II (MII) stage at collection. These GV oocytes were matured before in vitro fertilization (IVF), while the MII oocytes underwent IVF immediately. Oocytes collected from the oviducts of non-grafted superovulated mice of the same age served as controls. Two-cell embryos were transferred to pseudopregnant recipients and recovered at day 15 of gestation or left to go to term. Graft retrieval and the number of oocytes from each graft were lowest from the subcutaneous graft site. The number of two-cell embryos produced was significantly higher for oocytes from the grafts to the bursa as compared with the other sites. All graft sites gave rise to embryos with comparable implantation rates and developmental potential to fetuses and offspring following transfer. However, the oocytes from grafted ovaries had a significantly lower developmental potential when compared with the control group. Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation and development. In conclusion, graft site affects the number and quality of oocytes produced from ovarian grafts.
Industrial deployment of magnesium alloys in most instances requires anti-corrosion coatings. Distinct from conversion coatings, the electro/electroless plating techniques are emerging as the common means of coating magnesium; however, more research is needed. A state-of-the-art review was undertaken with respect to aqueous plating systems (including pretreatment, under-coating, and electroplating), electroless plating (being treated individually, though it is used as undercoating for subsequent plating in some cases), non-aqueous plating systems (including high-temperature molten salts and ionic liquids), and novel plating methods. In addition to the performance assessment of various plated coatings on magnesium alloys, merits and demerits of existing plating techniques are discussed. Based on the literature to date, the practical issues faced in magnesium plating are raised, and possible advances discussed, providing some instructive guidelines for future work.
Ovarian tissue grafts commonly contain only limited numbers of follicles. The functional life span and ability to retrieve as many mature oocytes as possible from ovarian grafts is important when grafting is used to restore fertility. This study aimed to determine whether ovarian grafts responded to exogenous hormones in a similar manner to that of in situ ovaries. Ovaries of C57BlxCBA F1 mice were cut in half and grafted to one of three different graft sites in females of the same F1 line; bursal capsule (BC, n = 12), kidney capsule (KC, n = 6), subcutaneous tissue (SC, n = 24). Three weeks after grafting, half of the graft recipients in each group were treated with 5IU PMSG followed by 5IU hCG 48 hours later. Oocytes were collected directly from the grafted ovaries 10 hours after the hCG injection and fertilized in vitro. Oocytes from the ovaries of superovulated normal mice (n = 4) of the same hybrid strain were used as controls. Two-cell embryos were transferred to pseudopregnant recipients and collected at day 15 of gestation or the animals were allowed to go to term. Mature fertilisable MII oocytes were retrieved from stimulated grafts from all graft sites, however, the number (BC 9, KC 5, SC 2 oocytes per ovary) and proportion of two-cell embryos in each grafted group (BC 52%, KC 32%, SC 32%) was significantly (P < 0.05) lower than in the in vivo matured control (16 oocytes, 85% two-cell). The fetal and placental weights of fetuses produced from graft-derived oocytes were not significantly different to the control group. Phenotypically normal pups were born in each of the graft and control groups. In conclusion, ovarian grafts treated with exogenous gonadotrophins produce significantly fewer mature oocytes and two cell embryos compared to in situ ovaries. Work supported by ARC and NIH RFA.
Cystic ovarian follicle (COF) is one of the most frequently diagnosed ovarian diseases and a major cause of reproductive failure in cattle. Despite an abundance of reports on this subject, the exact pathogenesis of COF still remains unclear. It is generally accepted that disruption of the hypothalmo-pituitary-gonadal axis, by endogenous and/or exogenous factors, causes cystic formation. We here examined whether ion concentration and expression of ion channels are altered in the follicle fluid derived from a Korean native cow with COF. In an ovary with a cystic follicle, granulosa cell layers were exfoliated; the theca interna was thinner than that in an ovary without cystic follicle, based on histological examination. Concentrations of K+, Na+, and Cl- in COF fluid (COFF) were 10.4 � 3.5 mM, 138 � 12 mM, and 104.9 � 7.0 mM, respectively. In COFF, K+ concentration showed a significant difference from the value observed in normal follicle fluid (NFF) (P < 0.05; NFF: 10.4 � 3.5 mM vs. COFF: 6.2 � 0.8 mM). The total numbers of follicles observed (normal, 3–5 mm in diameter vs. COF, 20–30 mm in diameter) were 200 and 20 in normal and COF, respectively. To compare mRNA expression of K+ channels, we performed semiquantitative RT-PCR using follicle fluid and ovaries with or without cystic follicles. RT-PCR showed that mRNA levels of TASK channels (TASK-1, TASK-3, and TASK-5) decreased by 50% in COFF and an ovary with cystic follicles compared to NFF and a normal ovary. TASK channels are involved in apoptosis of mammalian cells. Our results suggest that potassium may play an important role in the pathogenesis of COF.
The rate at which grafts become revascularized differs at different sites. The number of follicles which survive grafting also differs at different graft sites, it is, however, not known whether the quality of oocytes grown at different sites differ in their capacity to form fetuses and live young. To investigate this question ovaries of C57Bl×CBA F1 mice were cut in half and grafted to either of three different graft sites (ovarian bursa, kidney capsule, subcutaneous tissue) in females of the same F1 line. Oocytes were collected 3 weeks after grafting directly from the grafted ovaries and then both matured and fertilized in vitro. As controls, we investigated oocytes collected from the ovary (these mature to MII after overnight in vitro maturation) and ovulated, mature MII, oocytes. Results: graft recovery (no. recovered/no. grafted) was higher for the kidney capsule (46/48, 96%) and bursa (35/40, 88%) than for subcutaneous grafts (60/96, 63%). Subcutaneous grafts gave the lowest oocyte recovery (55 oocytes from 60 grafts). All oocytes recovered from the grafts were matured and fertilized in vitro. The fertilization of the control IVM IVF group was equivalent to the IVM IVF grafted groups, but all IVM IVF groups were below the in vivo matured, control (81-85% 2-cells). Two-cell embryos were transferred to pseudopregnant recipients and collected at day 15 of gestation. Embryos derived from grafts to the bursa were also transferred and left to go to term. Oocytes collected from grafts to the ovarian bursa gave rise to fetuses (4/14, 28%) and live young (2/8, 25%) with the same efficiency as normal IVF controls (5/20, 30% and 3/12, 25% respectively). We also obtained fetuses from the kidney capsule (2/20) and subcutaneous (1/1) grafts. Embryos derived from grafts to the kidney capsule were associated with a large number of fetal resorptions (9 of 11 implanted embryos), but the weights of the two remaining fetuses were comparable to that in the IVF control group (both were 0.26 ± 0.01 g). Further studies are being conducted to ascertain whether the observed differences in oocyte developmental potential are a result of the effect of the graft site on the developing oocyte.
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