Is radiation-induced ovarian failure in rhesus monkeys preventable by luteinizing hormone-releasing hormone agonists?: Preliminary observations.

With the advent of cancer therapy, increasing numbers of cancer patients are achieving long term survival. Impaired ovarian function after radiation therapy has been reported in several studies. Some investigators have suggested that luteinizing hormone-releasing hormone agonists (LHRHa) can prevent radiation-induced ovarian injury in rodents. Adult female rhesus monkeys were given either vehicle or Leuprolide acetate before, during, and after radiation. Radiation was given in a dose of 200 rads/day for a total of 4000 rads to the ovaries. Frequent serum samples were assayed for estradiol (E2) and FSH. Ovariectomy was performed later. Ovaries were processed and serially sectioned. Follicle count and size distribution were determined. Shortly after radiation started, E2 dropped to low levels, at which it remained, whereas serum FSH level, which was low before radiation, rose soon after starting radiation. In monkeys treated with a combination of LHRHa and radiation, FSH started rising soon after the LHRHa-loaded minipump was removed (after the end of radiation). Serum E2 increased after the end of LHRHa treatment in the nonirradiated monkey, but not in the irradiated monkey. Follicle counts were not preserved in the LHRHa-treated monkeys that received radiation. The data demonstrated no protective effect of LHRHa treatment against radiation-induced ovarian injury in this rhesus monkey model.

Reproductive performance of female rats treated with cyclophosphamide and/or LHRH agonist.

Previous studies addressing the ovarian protective effects of luteinizing hormone releasing hormone agonists (LHRHa) against adverse effects of chemotherapy have examined histologic and/or hormonal parameters without evaluating reproductive performance. In this report, we initially established a model for chronic treatment of female rats with cyclophosphamide (CTX) that allowed long term survival. In a second experiment, thirty seven female cycling rats (age: 70 days) were divided into 4 treatment groups. They were given either CTX (4 mg/kg/day, 5 days/week) for a total of 76 days (217 mg/kg) and/or the LHRHa leuprolide (Lupron) 5 micrograms/day by subcutaneous minipump (Alza) for 98 days. LHRHa was started 10 days before CTX and ended 12 days after the last CTX injection. All LHRHa-treated rats entered persistent diestrus. At the end of treatment, most rats treated with CTX only were in persistent estrus. Breeding was started at 218 days of age. A laparotomy to count implantation sites was performed 15 to 16 days after vaginal plug/sperm was demonstrated. All nonpregnant rats were remated. Chi square and ANOVA were used for statistical analysis. The data presented demonstrate that: 1. LHRHa given before and after CTX increased the pregnancy rate/mating (from 4/11 to 9/10; P < 0.05), the number of implantations/mated rat (from 2.5 +/- 1.4 to 13.7 +/- 1.7; P < 0.01), and reduced the need for remating (from 7/11 to 1/10; P < 0.05); 2. LHRHa-treated rats performed better than controls. We conclude that LHRHa protects against chemotherapy-induced fertility reduction in female rats.

Genetic structure of natural populations of the free-living amoeba, Naegleria lovaniensis. Evidence for sexual reproduction.

The genetic structure of two populations of Naegleria lovaniensis, comprising 71 isolates collected from the same local geographical area was investigated by isoenzyme analysis. Allelic variation at seven polymorphic enzymatic loci allowed identification of 45 distinctive genotype associations. Analysis of single locus variation reveals that most of them are close to Hardy-Weinberg equilibrium, which indicates segregation and free recombination between alleles. The recovery of a relatively high number of distinct genotypic associations (most of them being unique), and the absence of linkage disequilibrium between genotypes at the different loci also support the existence of recombination. Although we have no idea about the process involved, the results clearly indicate that genetic exchanges occur, at least occasionally, in natural populations of N. lovaniensis.

The effect of "activated" cyclophosphamide on human and rat ovarian granulosa cells in vitro.

We investigated the mechanism of cyclophosphamide (CTX) induced ovarian toxicity by studying the effect of an activated form, 4-hydroperoxycyclophosphamide (PCTX), on human and rat granulosa cell function in vitro. In previous experiments we demonstrated that in short-term incubations with rat granulosa cells, high (greater than or equal to 100 micrograms/mL) but not low (less than or equal to 10 micrograms/mL) PCTX concentrations inhibited progesterone accumulation in vitro. In this study, human granulosa cells were obtained from patients undergoing follicle puncture for in vitro fertilization. PCTX at 10, 100, and 500 micrograms/mL, but not at 1 micrograms/mL, resulted in a dose-related reduction in progesterone accumulation in short-term human granulosa cell cultures. Rat granulosa cells were obtained from PMSG-primed immature rats and incubated for 24 h with PCTX concentrations of 1, 5, and 10 micrograms/mL. Ovine LH (10 ng/mL) was added in selected tubes. In comparison to control, progesterone accumulation was significantly reduced by PCTX concentration of 10 micrograms/mL. The above findings demonstrate that cyclophosphamide metabolites, at concentrations achievable in vivo during CTX therapy, decrease rat and human granulosa cell function in vitro. The effect of PCTX on granulosa cells is dependent on PCTX concentration and duration of exposure, as well as the species from which granulosa cells are obtained.

Effect of cyclophosphamide on the immature rat ovary.

To investigate the early ovarian changes after cyclophosphamide treatment, immature rats primed for 48 h with pregnant mare serum gonadotropin were given injections i.p. of cyclophosphamide (100 mg/kg) at 1, 2, 4, 16, and 24 h before decapitation. Serum estradiol dropped significantly after 24 h of exposure to cyclophosphamide (P less than 0.001). Following 16 and 24 h of cyclophosphamide exposure, (a) the number of granulosa cells expressed from each ovary decreased (P less than 0.05 and P less than 0.01, respectively); (b) the number of nucleated bone marrow cells decreased (P less than 0.01 and P less than 0.01), and their median nuclear size was significantly reduced (P less than 0.05 and P less than 0.05) as measured by Coulter Counter and C-256 channelyzer (Hialeah, FL); and (c) the mean follicular diameter and the number of follicles with diameters greater than 300 microns were significantly lower than in control. After 4, 16, and 24 h of exposure, median granulosa cell nuclear size significantly increased (P less than 0.05, P less than 0.01, and P less than 0.01, respectively). DNA cross-links in granulosa cells, measured by alkaline elution, reached a maximum at 2 h of exposure and decreased thereafter. The above findings demonstrate that cyclophosphamide has significant effects on the rat ovary structure and function and that the granulosa cell is an important target of cyclophosphamide-induced ovarian toxicity.

The effect of "activated" cyclophosphamide on rat granulosa cells in vitro.

We investigated the mechanism of cyclophosphamide (CTX)-induced ovarian toxicity by studying the effect of an activated form, 4-hydroperoxycyclophosphamide (PCTX), on rat granulosa cells in vitro. Cells were obtained from PMSG-primed immature rats and incubated with PCTX at concentrations of 1, 10, 100, and 500 micrograms/mL. Ovine LH (10 ng/mL) was added in selected tubes. Cell viability before and after seven hours incubation was determined. Progesterone and prostaglandin E accumulation were measured by radioimmunoassay. Granulosa cell viability was significantly decreased at PCTX concentrations of 10 micrograms/mL or higher in a dose-related manner. PCTX at concentrations of 100 micrograms/mL and 500 micrograms/mL significantly decreased basal and LH-induced progesterone and prostaglandin E accumulation. The above findings demonstrate that cyclophosphamide metabolites decrease granulosa cell survival and function in vitro. These direct effects suggest a possible mechanism for CTX-induced premature ovarian failure.