Phillip A. Fields

Glial cell-specific differences in response to alkylation damage

Oligodendrocytes are preferentially sensitive to the toxic, carcinogenic, and teratogenic effects of methylnitrosourea (MNU). The mechanisms responsible for this enhanced sensitivity have not been fully elucidated. One of the most vulnerable cellular targets for this chemical is mitochondrial DNA (mtDNA). To determine if differences in mtDNA damage and repair capacity exist among the different CNS glial cell types, the effects of MNU exposure on oligodendroglia, astroglia, and microglia cultured separately from neonatal rat brain were compared. Quantitative determinations of mtDNA initial break frequencies and repair efficiencies showed that whereas no cell type‐specific differences in initial mtDNA damage were detected, mtDNA repair in oligodendrocytes, oligodendrocyte progenitors, and microglia was significantly reduced compared to that of astrocytes. In astrocytes, and all other cell types previously evaluated in our laboratory, >60% of N‐methylpurines were removed from the mtDNA by 24 hr. In contrast, only 35% of lesions were removed from mtDNA of oligodendrocytes, oligodendrocyte progenitors, and microglia during the same time period. Mitochondrial perturbations by a variety of xenobiotics have been linked to apoptosis. In the present study, apoptosis, as determined by DNA laddering and ultrastructural analysis, was clearly induced by MNU treatment of cultured oligodendrocyte progenitors and microglia, but not in astroglia. These data demonstrate a correlation between diminished mtDNA repair capacity and the induction of apoptosis. However, further experimentation is necessary to determine if a causal relationship exists and contributes to the vulnerability of oligodendroglia following exposure to N‐nitroso compounds in the environment or in chemotherapeutic regimen. GLIA 24:304–312, 1998.

Regulation of PKC δ expression by estrogen and rat placental lactogen-1 in luteinized rat ovarian granulosa cells.

Protein kinase C (PKC) delta is dramatically upregulated in the corpus luteum in the second half of pregnancy in the rat. To gain insight into the hormonal regulation of PKC delta expression, studies were undertaken to analyze the regulation of PKC delta expression in a luteinized rat granulosa cell model. PKC delta protein expression was evaluated in luteinized granulosa cells, isolated from human (h)CG-treated immature female rats 7 h after the injection of an ovulatory dose of hCG and cultured up to 12 days. Cytochrome P450 cholesterol side chain cleavage enzyme expression was observed throughout the culture period, and a majority of the cells expressed steroidogenic acute regulatory protein and responded to rat placental lactogen (rPL)-1 by exhibiting hypertrophy, consistent with maintenance of the luteal phenotype. Both PKC delta protein and mRNA expression increased 3.5-4-fold with time of culture, and PKC delta mRNA expression could be eliminated by treatment of cells with the PKC inhibitor GF109203X. E(2) caused a specific dose- and time-dependent increase in expression of PKC delta protein of twofold, whereas PKC delta mRNA was unaffected by E(2) over a 12-day culture period. Treatment of cells with 500 ng/ml rPL-1 for the final 4 days of a 12-day culture in the absence of E(2) had no effect on PKC delta protein or mRNA expression, while treatment with 500 or 3000 ng/ml rPL-1 in the presence of E(2) significantly enhanced both PKC delta protein and mRNA expression (up to threefold). These results show that two of the major regulators of luteal function in the second half of pregnancy in the rat, E(2) and rPL-1, cooperate to regulate PKC delta expression in luteinized granulosa cells.

Relaxin and Other Luteal Secretory Peptides: Cell Localization and Function in the Ovary

The corpus luteum (CL) plays a critical role in reproductive processes of all mammals. Its secretory products amplify fertility by a) providing a uterine environment for implantation, b) maintaining pregnancy if implantation occurs, c) enhancing nontraumatic fetal delivery, and d) assuring a new opportunity for pregnancy should there be a failed pregnancy. This is accomplished by initiating regression of the CL for subsequent development of a new follicle for ovulation.

Potential Binding Sites for Relaxin in Pregnant Rabbits

Abstract: The purpose of this study was to identify tissues in the day 25 pregnant rabbit that bind relaxin. First, the clearance half‐life of relaxin from the rabbit (n= 6) was determined by injecting 10 g porcine relaxin via the marginal ear vein. One‐milliliter blood samples were collected via a cannula in the central ear artery. Samples were collected at 10‐ and 5‐min pre‐relaxin and at 1‐min intervals post‐relaxin injection, and the relaxin concentrations determined by radioimmunoassay. The clearance half‐life was 4 min. Next, pregnant rabbits were infused with [125I]‐relaxin. Control rabbits (n= 3) received 10 g radio‐inert relaxin via the marginal ear vein in order to saturate endogenous receptors. Ten minutes later, 10 ng [125I]‐relaxin was similarly injected. Treated rabbits (n= 3) received only [125I]‐relaxin. After allowing sufficient time for clearance (24 min), a 1‐mL blood sample was removed via the central ear vein. Rabbits were euthanized, tissues of maternal and fetal origin excised, and cpm/mg of tissue divided by cpm/mL of blood was determined. Differences in uptake of [125I]‐relaxin between control and treated animals, using the Student paired t test, were found for the uterus (P<.05), uterine cervix (P <.03), and mammary gland (P <.05). These data suggest potential rabbit tissues with the LGR‐7 receptor.

Immunohistochemical localization of relaxin-like factor/insulin-like peptide-3 in the bovine corpus luteum.

Abstract: Relaxin‐like factor/insulin‐like peptide (INSL)‐3 is highly expressed in the bovine corpus luteum throughout the estrous cycle and pregnancy. Demonstration of translation of the relaxin‐like factor message was previously shown for the follicle but not the corpus luteum. In this study, relaxin‐like factor mRNA was highly expressed in the corpus luteum on days 7 and 14 of pregnancy. Tissues were fixed in 10% neutral buffered formalin, and utilizing two different antibodies to relaxin‐like factor, W3 rabbit anti‐bovine and 2‐8F mouse anti‐bovine, relaxin‐like factor was localized in fibroblast‐like cells. Staining was also observed in the Leydig cells of bovine testicular tissue. No staining was observed in small and large steroidogenic luteal cells, indicating a nonsteroidogenic source of luteal relaxin‐like factor. Definitive cell type identification is currently being determined via electron microscopy.

Is relaxin a calcium transporter/buffer?

Abstract: Relaxin inhibits smooth muscle contractions by binding Ca2+ and making it unavailable to the contractile elements. Rat uterus sarcoplasmic reticulum (SR) was isolated, incubated with 45Ca in the presence and absence of porcine relaxin, and filtered. In the presence of relaxin, only 4% of the 45Ca was on the filter with the SR as compared to that measured on the filter with SR alone or in the presence of proteins of similar isoelectric point (cytochrome C) and size (insulin). A Sephadex G‐25 column was equilibrated with buffer containing 45Ca, and 0.5 mL fractions were collected. Each fraction contained 240‐250K cpm. Relaxin was diluted in the buffer, added to the top of the column, and eluted. A peak of 45Ca (>600K cpm) was found with the eluted relaxin. No peak of 45Ca was associated with cytochrome C or insulin under the same conditions. The eluted fractions containing 45Ca and relaxin were combined. A sample was incubated with rabbit anti‐porcine relaxin serum and goat anti‐rabbit serum and centrifuged, and 45Ca in the pellet and supernatant was determined. Ninety‐eight percent of the 45Ca was found in the supernatant. Thus, 45Ca was not tightly bound to relaxin or the antibodies knocked the 45Ca off. Is relaxin a calcium transporter or buffer?

Clinical Use of Relaxin to Facilitate Birth: Reasons for Investigating the Premise

Abstract: In the United States, both medical and nonmedical factors have driven the cesarean section rate to over 26% of all deliveries. In addition to questions of increased cost associated with operative delivery, some have questioned the ethics of performing cesarean section for nonmedical reasons. Reduction of both the duration and the pain associated with vaginal delivery would likely bring about a decline in the rate of both medical and nonmedical cesarean sections. This chapter summarizes recent findings that support the premise that through its growth‐promoting and softening effects on the cervix, short‐term subcutaneous administration of pharmacologic amounts of relaxin to women at term holds promise as a means of reducing the duration and discomfort associated with delivery. Two recent studies conducted in pregnant rats demonstrated that the cervix is highly responsive to relaxin during the antepartum period and that short‐term subcutaneous administration of the hormone to relaxin‐deficient animals not only promotes growth and softening of the cervix, but also reduces the duration of labor and delivery. Moreover, recent human clinical trials examining the influence of 24 weeks of continuous subcutaneous administration of recombinant human relaxin for the treatment of scleroderma provided evidence not only that the human reproductive tract is responsive to relaxin, but also that the administration of the hormone does not cause serious adverse side effects. It is concluded that recent findings provide an impetus for an investigation into relaxins potential for cervical remodeling and facilitating birth in women.

Conceptus numbers do not affect blood concentrations of relaxin in the rabbit.

On day 18 of pregnancy, conceptuses were surgically removed in order that rabbits would be left with seven, five, three, or one conceptus. During pregnancy, blood concentrations of relaxin were determined by radioimmunoassay. There was no difference between relaxin concentrations in rabbits carrying one versus seven conceptuses. The results indicate that conceptus number does not influence the blood concentration of relaxin in the rabbit.

The Effect of Prostaglandin F2α on Mitochondrial Election Dense Inclusions and Secretory Granules of the Bovine Large Luteal Cell During Late Pregnancy

The bovine corpus luteum has been shown to contain two populations of electron dense granules which are morphologically distinct (Fields et al., 1985). One population consists of small (100–300 nm diameter) membrane bounded granules that are readily observed to undergo exocytosis (Singh, 1975; Heath et al., 1983; Fields et al., 1985). The product of these small luteal granules has not been identified in the pregnant cow. However, in the nonpregnant cow luteal secretory granules have been shown to contain oxytocin associated neurophysin (Fields and Fields, 1986).