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.

Effect of cyclophosphamide on mouse oocyte in vitro fertilization and cleavage: recovery.

To investigate the mechanism of cyclophosphamide (CTX)-induced ovarian failure, we previously reported that CTX metabolites added in vitro inhibit mouse oocyte fertilization and embryo development. In this study, we injected CTX (100 mg/kg) intraperitoneally in female mice at 0, 1, 4, 14, 18, and 24 h before sacrifice. Mice were superovulated with PMSG and hCG. Oocytes were recovered, washed, and fertilized with sperm obtained from nontreated proven breeders, and incubated for 3 days in 5% CO2 in air. CTX reduced oocyte fertilization and early cleavage rates. To examine the recovery process, CTX was injected at 0, 1, 3, 7, and 14 days before sacrifice. The most pronounced adverse effects on oocyte number and function were observed 1 and 3 days after exposure to CTX. Evidence of partial recovery was observed one week after CTX treatment. The data demonstrate that exposure of oocytes to CTX metabolites in vivo adversely effects oocyte function. This process, however, appears to be partially reversible. The oocytes may be involved in the mechanism of CTX-induced ovarian failure.

The uptake and metabolism of cyclophosphamide by the ovary.

The uptake of cyclophosphamide (CTX) and its metabolites was evaluated by injecting adult female rats with 14C-CTX on the morning of metestrous or proestrus. Rats were sacrificed 1 and 5 hours after 14C-CTX injection. The ovary, uterus, spleen, thymus, liver, kidney, anterior pituitary, duodenum, skeletal muscle and whole blood were isolated from each rat. Samples were combusted using a biological material oxidizer and the resulting CO2 was absorbed and counted. Liver and kidney had the highest uptake of 14C-radioactivity. The ovary appears to have 14C uptake comparable to the thymus and other tissues. Metabolism of CTX by the ovary was investigated by incubating 14C-CTX with human and rat granulosa cells and other ovarian cells obtained from pregnant mares' serum gonadotropin (PMSG)-primed immature rats, in separate experiments. The conversion of CTX to two marker metabolites, 4-ketocyclophosphamide and 4-hydroxycyclophosphamide was negligible and did not change in the presence of luteinizing hormone (LH). It is concluded that 1) following 14C-CTX injection, the ovary takes up a proportion of 14C-radioactivity comparable to other target tissues (e.g. thymus) and 2) the ovary is not capable of activating CTX in vitro in our system.

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 "activated" cyclophosphamide on mouse oocyte in vitro fertilization and cleavage.

To investigate the involvement of oocytes in the mechanism of chemotherapy-induced ovarian failure, ovulation was induced in mice using pregnant mares' serum gonadotropin and human chorionic gonadotropin. Oocytes (with cumulus) were incubated for 4 hours with "activated" cyclophosphamide, 4-hydroperoxycyclophosphamide (PCTX) at 0, 1, 10, 100, and 500 micrograms/mL. Oocytes were then washed, fertilized with sperm obtained from nontreated male proven breeders, and incubated for 4 days. "Activated" cyclophosphamide inhibited dissolution of the cumulus and reduced fertilization and early cleavage rates in a dose-related manner. The data demonstrate that exposure of oocytes to cyclophosphamide metabolites in vitro adversely affects oocyte function. Oocytes may be involved in the mechanism of cyclophosphamide-induced ovarian failure.

The use of hybridoma antibodies to probe the antigenic determinants of thyroglobulin.

Using monoclonal antibodies we have begun to define the epitopes of the murine thyroglobulin molecule that elicit autoimmune responses. Based on the principle of complementation, 18 monoclonal antibodies were classified into 8 groups, defined by their reactions with the same or neighboring determinants. Further distinctions between the monoclonals were drawn from comparisons of their cross-reactions with thyroglobulins of other species and their patterns in isoelectric focusing. One low molecular weight fragment of bovine thyroglobulin, which cross-reacts extensively with thyroglobulins of other species, has been isolated and partially characterized.