Tag Archives: follicle (Part 3)

Methoxychlor-Induced Atresia: MATERIALS AND METHODS(1)

Chemicals

MXC (99% pure) was purchased from Chemservice (West Chester, PA) in a powdered form. Stock solutions were prepared by mixing the appropriate amount of MXC with a constant volume of sesame oil. For the 8 mg/kg dose, 50 mg of MXC was mixed with 10 ml sesame oil; for the 16 mg/kg dose, 100 mg MXC was mixed with 10 ml sesame oil; for the 32 mg/kg dose, 200 mg MXC was mixed with 10 ml sesame oil; for the 64 mg/kg dose, 400 mg MXC was mixed with 10 ml sesame oil. Thus, the amount of chemical was concentrated with each subsequent dose so that mice received comparable injections based on their weights. This eliminated the need to give a greater volume injection to mice receiving higher doses than those receiving lower doses.

Methoxychlor-Induced Atresia: INTRODUCTION(3)

INTRODUCTION(3)

Antral follicles are major producers of estrogen in the ovary and are also responsive to gonadotropin stimulation. These follicles will begin to die without proper hormonal stimuli. Specifically, follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol are major survival factors for antral follicles. Thus, it is possible that antral follicles may undergo an increased rate of atresia due to an alteration in hormones or because fewer antral follicles are available for estrogen production, which might in turn lead to altered levels of gonadotropins.

Methoxychlor-Induced Atresia: INTRODUCTION(2)

The Bcl-2 and Bax proteins are well characterized, and studies have shown that they are important regulators in the ovary. Yang and Rajamahendran demonstrated that progesterone-induced antral follicle atresia was associated with an increased ratio of Bax levels to Bcl-2 levels in bovine follicles. Another study showed that overexpression of Bcl-2 during embryonic life resulted in a greater number of primordial follicles at birth. Similarly, a Bcl-2 knockout mouse model was found to have fewer than the normal complement of primordial follicles at birth. Mice deficient in Bax, a proapoptotic protein, were found to have greater numbers of ovarian primordial follicles that were maintained into advanced chronological age.

Methoxychlor-Induced Atresia: INTRODUCTION(1)

INTRODUCTION(1)

Methoxychlor (MXC) is an organochlorine pesticide that was developed to replace dichlorodiphenyltrichloroethane (DDT). MXC is less persistent than DDT; nevertheless, it is considered a potent environmental toxicant. We have previously shown that MXC affects the ovary by increasing the rate of ovarian atresia and decreasing the number of healthy antral follicles. Atresia is thought to begin with apoptosis in the granulosa cells.

Vascular Remodeling and Angiogenesis: DISCUSSION(3)

Survival of follicles was spatially related to presence of pericytes rather than to endothelial cells in the area of the graft (Fig. 1, G-I; Fig. 2, A-O; Fig. 3, G-R). The ovarian cortex showed better follicular maintenance, probably due to sufficient blood supply (see Fig. 1I). Interestingly, Dissen et al. showed that the mRNA expression of the two angiogenic factors, VEGF and TGFp-1, is upregulated mainly at the ovarian cortex 48 h after transplantation. In the subcutaneous implants, no recovery of the initial damage was observed, whereas in the intramuscular grafts, improvement was observed within 6-7 days postimplantation, showing healthy follicular morphology and vascular integrity, including endothelial cells, pericytes, and SMC.

Vascular Remodeling and Angiogenesis: DISCUSSION(2)

DISCUSSION(2)

Ovarian fragments implanted intramuscularly showed much better follicular maintenance. Many follicles, from the primordial through the antral stages of development, could be found in the implant, suggesting recovery of ovarian function. At 6 days after implantation, the vasculature in the implant was very similar to that of control ovaries, with normal morphology of blood vessels. Despite this favorable outcome, necrotic regions in the medulla of the graft were detected at the first days (1-3 days) after transplantation, suggesting that early transitory necrosis may be overcome and the maintenance of the grafts improved at a later time (6-7 days). The medullar damage during the first hours after transplantation was accompanied by regression of pericytes and SMC, which was symptomatic of insufficient blood supply. generic geodon

Vascular Remodeling and Angiogenesis: DISCUSSION(1)

The role of vascularization in ovarian transplantation was studied here on a model system in which fresh fragments of rat ovaries (donor) were xenografted into female CD-1 nude mice (recipient). Two transplantation sites were compared, subcutaneous and intramuscular. Ovarian maintenance was markedly better in the intramuscular transplants. The rich blood supply within the muscle provided superior graft reception compared with that observed for the relatively poor supply of blood in the subcutaneous region. Substantial necrotic areas were detected in all the subcutaneous ovarian grafts, mainly in the medullar regions of the graft.

Vascular Remodeling and Angiogenesis: RESULTS(6)

RESULTS(6)

Changes in signal intensity reflective of vascular leakage were followed for 32 min (Fig. 6C). The accumulation of contrast agent was restricted to the graft and was not observed in the surrounding muscle (Fig. 6D). Histological staining of the biotinylated contrast agent was performed using avidin-FITC. Consistent with the MRI data, FITC staining was restricted to blood vessels within the muscle, whereas it leaked into the graft (Fig. 6E). The contrast agent was also detected in the follicular fluid. This leakage of the contrast agent in the ovarian grafts was similar to that observed in intact rats that were injected with the contrast agent intravenously 45 min prior to retrieval of the ovaries. Accordingly, the vascular properties of the ovarian grafts in the intramuscular transplantation appeared to be similar to those of the intact ovary.

Vascular Remodeling and Angiogenesis: RESULTS(5)

Dividing the intramuscular grafts into three groups according to the time after transplantation reveals that, during the short time periods of 1-3 days after transplantation, the ovaries scored —7.7 (n = 11), after 6-7 days, the score was 25.5 (n =;8), and at 28-31 days, the score was 8.0 (n = 7; Fig. 5). This scoring method reflects ovarian graft survival and was the lowest in the subcutaneous grafts throughout the 2-24 days after transplantation. The state of the intramuscular grafts was suboptimal during the first 3 days after transplantation; however, it improved after 6-7 days. After a period of about a month, a decrease in the follicular state was observed, although the grafts remained viable. The number of the small primordial follicles per square millimeter in the intramuscular site remained similar during the entire period examined (Fig. 4, D-F). canadian health & care mall

Vascular Remodeling and Angiogenesis: RESULTS(4)

RESULTS(4)Ovarian Preservation and Follicle Growth in the Grafts

We determined the state of grafted ovaries by the size and integrity of the follicles. Follicles were divided into seven groups according to their size, and both the viable and the atretic follicles were counted and graded. Grafts of half ovaries were assessed. In control ovaries, fixed immediately after the animal was killed (n = 3), all stages of follicular development were detected. In the controls, small atretic follicles were only rarely found at early stages of follicular development (Fig. 4A). In the subcutaneous ovarian grafts (n = 6; 2-24 days after transplantation), the largest viable follicles that were detected reached stage d (three layers of granulosa cells). A large number of degenerated primordial and primary follicles were detected (Fig. 4B).

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