Identification of novel GH-regulated genes in C2C12 cells.

Growth hormone (GH) is the main regulator of longitudinal growth before puberty, and treatment with human recombinant (rh) GH can increase muscle strength. Nevertheless, molecular mechanisms responsible remain mostly unknown. Many physiological effects of GH require hormone-mediated changes in gene expression. In an attempt to gain insight into the mechanism of GH action in muscle cells we evaluated the effects of rhGH on gene expression profile in a murine skeletal muscle cell line C2C12. The objective of the work was to identify changes in gene expression in the murine skeletal muscle cell line C2C12 after rGH treatment using microarray assays. C2C12 murine skeletal muscle cell cultures were differentiated during 4 days. After 16 h growing in serum-free medium, C2C12 myotubes were stimulated during 6 h with 500 ng/ml rhGH. Four independent sets of experiments were performed to identify GH-regulated genes. Total RNA was isolated and subjected to analysis. To validate changes candidate genes were analyzed by real-time quantitative polymerase chain reaction. One hundred and fifty-four differentially expressed genes were identified; 90 upregulated and 64 downregulated. Many had not been previously identified as GH-responsive. Real-time PCR in biological replicates confirmed the effect of rGH on 15 genes: Cish, Serpina3g, Socs2, Bmp4, Tnfrsf11b, Rgs2, Tgfbr3, Ugdh, Npy1r, Gbp6, Tgfbi, Tgtp, Btc, Clec3b, and Bcl6. This study shows modifications in the gene expression profile of the C2C12 cell line after rhGH exposure. In vitro and gene function analysis revealed genes involved in skeletal and muscle system as well as cardiovascular system development and function.

Differential gonadotropin releasing hormone (GnRH) expression, autoregulation and effects in two models of rat luteinized ovarian cells.

GnRH has been suggested to participate in corpus luteum function. Here we studied the expression of GnRH mRNA and peptide in two models of rat luteinized tissues: ovarian cells from PMSG-hCG treated prepubertal rats (SPO) and from intrasplenic ovarian tumors (Luteoma). A GnRH autoregulatory effect was evaluated as well as its action on cell proliferation and apoptosis. GnRH mRNA was present in SPO, isolated corpora lutea from SPO and Luteoma from 1 week to 7 months of development. In vitro cultures of Luteoma cells expressed 2-fold higher GnRH mRNA and 10-fold higher GnRH peptide than SPO cells. Buserelin (GnRH analog) increased GnRH mRNA and peptide expression in SPO but not in Luteoma cells. While basal proliferation was very low in Luteoma cells, SPO cells showed a significant increase in cell number by both the thymidine and the MTS methods after 72 h in culture. Buserelin induced a decrease in cell number in both cell types to a similar degree. Although basal apoptosis levels were higher in SPO than in Luteoma cells, Buserelin-induced apoptosis was only detected in Luteoma cells after 48 h treatment. These results show that the two types of rat, luteinized tissues, Luteoma and SPO, markedly differed in some intrinsic properties and in their local GnRH systems. Luteoma cells proliferate very weakly, express and secrete high amounts of GnRH, do not show an autoregulatory effect and respond to the decapeptide with apoptosis stimulation. In contrast SPO cells proliferate significantly, secrete low levels of GnRH but possess a positive, autoregulatory mechanism and respond to GnRH stimulation with impairment of proliferation.

Gonadotropins and inhibins along the development of a luteinized rat ovarian tumor.

Luteinized intrasplenic ovarian tumors develop in response to high circulating gonadotropins. The relationship between tumor development, gonadotropins and inhibins was studied. Tumor-bearing animals were sacrificed weekly along the first 6 weeks of development. Inhibins were measured by enzyme-linked immunosorbent assay (ELISA), serum gonadotropins, GH and IGF-1 by RIA. Inhibin subunit mRNAs were determined by Northern blot. Tumor histology was examined. Ovarian grafts grew significantly along development. LH increased ten-fold on week 1; a further significant increment was observed on week 3. FSH peaked on weeks 1 and 2 and fell significantly thereafter. Serum inhibins markedly increased on weeks 3-5. Tumor inhibin A content and mRNA levels for alpha and beta A subunits also increased on week 3. Inverse correlations between inhibins and FSH and direct correlations between inhibins and LH were observed. Tumor inhibin A and IGF-1 contents correlated significantly. Increasing levels of luteinization were observed along tumor development. These luteinized tumors develop mainly in response to LH, since growth continues under FSH inhibition. The active inhibin secretion and the positive correlation between inhibins and LH suggests that LH may be the main driving force behind this production, while growth factors produced by the gonads may also participate in their regulation.

Development of an experimental ovarian tumor: immunocytochemical analysis.

OBJECTIVE: The aim of the present work was to study whether immunocytochemical parameters present in the normal ovary were altered after tumor development under high gonadotropin levels. METHODS: Ovarian tumors (luteoma): castrated female rats had an ovary grafted into the spleen; tumors were left to develop for 1, 2, 3 or 7 months. The presence of apoptotic cells (TUNEL method) and the expression of proliferating cell nuclear antigen (PCNA), gap junction protein (Cx43), steroidogenic acute regulatory protein (StAR), aromatase and synaptosome-associated protein of 25 kDa (SNAP-25) were determined by immunocytochemistry. Some of these findings were confirmed by RT-PCR (Cx43, StAR, SNAP-25). Inhibin subunit mRNAs were investigated by Northern blot. RESULTS: PCNA staining of tumors was mainly found in granulosa cells of transforming follicles and was absent from luteinized follicles. A nearly complete absence of apoptosis was observed. Cx43 was mainly found in follicles, while it was very weakly expressed or absent in luteinized follicles. StAR protein expression, indicating active steroidogenesis, was demonstrated only in luteinized follicles and in thecal cells, but was absent from granulosa cells. Aromatase immunoreactivity was very intense in granulosa and also present in luteal cells. Membrane-associated and cytoplasmic SNAP-25 immunostaining was determined in patches of endocrine cells in the follicles, as well as in the luteinized follicles. The expression of mRNAs for Cx43, StAR and SNAP-25 (RT-PCR) and inhibin subunits (Northern blots) were confirmed in 1-, 3- and 7-month-old tumors. CONCLUSIONS: These results indicated that luteoma most likely develop from unruptured follicles by hypertrophy and proliferation of follicular cells. Circulating gonadotropins seem to play a fundamental role in maintaining the expression of proteins typically expressed in normal ovary, while avoiding apoptosis in this tissue.

Actions of immunosuppressor drugs on the development of an experimental ovarian tumor.

Immunosuppression has been related to the incidence of tumor apparition, including endocrine tumors. The intrasplenic ovarian tumor (luteoma) is a typical benign endocrine tumor that develops under high gonadotropin stimulation and, from the immunological perspective, is located in a critical organ involved in immune response. To establish if immunosuppression could alter the development of this experimental tumor, the effects of cyclosporin A (CsA) and dexamethasone (Dex) were evaluated. After surgery, tumor-bearing and sham animals were kept without treatment for 4 weeks; thereafter, they were distributed into CsA (25 mg/kg), Dex (0.1 mg/kg), or vehicle (75:25 castor oil:ethanol) groups and were injected on alternate days for 50 days. Body weight was evaluated weekly. Animals were sacrificed after a jugular vein blood sample was obtained. Thymi were weighed. Tumors were measured and placed in formaline for histological studies. Serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin (PRL), and estradiol were measured by radioimmunoassay. Hematological parameters were determined. CsA induced a significant decrease in survival rates both in tumor-bearing and sham animals (P < 0.01). Dex significantly impaired weight increase in both groups of animals. CsA induced a significant weight loss in sham animals, not observed in tumor-bearing animals. Dex induced thymus weight loss in both groups, whereas CsA induced thymus weight loss only in sham animals. Only Dex induced a decrease in lymphocyte number in both groups. CsA induced an increase in monocyte number only in sham animals. Treatments did not alter LH, FSH, or estradiol, whereas PRL was increased by CsA only in sham rats. Neither Dex nor CsA induced any significant variations in tumor volume, nor did they alter tumor histology. In addition, no visible metastases or alterations in other organs were observed. We conclude that, though immunological parameters were altered by the treatments, immunosuppressor drugs did not condition tumor development. In addition, tumors secrete one or more factor/s that counteract CsA effect.

Function and structure of rat hepatic coproporphyrinogen oxidase.

Rat hepatic coproporphyrinogen oxidase, the sixth enzyme in the heme biosynthetic pathway, was purified 1340-fold with a yield of 39.7%. To obtain the soluble enzyme, different methods were applied to disrupt mitochondria, with sonication giving the highest yield (85%). The minimum catalytic form of enzyme was a dimer with a molecular mass of 77 +/- 4 kDa. The existence of aggregated forms was possible since in fractions of gel filtration elution activity was observed with higher molecular mass. We determined a Stokes radius of 36.3 A, a sedimentation coefficient (S20,w) of 5.06 S, and frictional ratio of 1.29, suggesting a nearly globular shape of the protein. Regardless of the type of salt, high ionic strength inhibits the enzyme, probably modifying its native structure. Experiments with amino acid modifiers showed that histidine, arginine, and tryptophan are involved in the catalytic process. Non-ionic detergents and phospholipids activated the enzyme, probably because they reproduce its natural hydrophobic environment. The present study describes a simple method for the purification of rat liver coproporphyrinogen oxidase, introducing for the first time data on the structure and function of the protein in a tissue often used as a laboratory model in biological studies, and contributing to the study of human hereditary coproporphyria.

Simple and rapid method for the determination of coproporphyrinogen oxidase activity.

Coproporphyrinogen oxidase, the sixth enzyme in the biosynthetic heme pathway, catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX. A reversed-phase high pressure liquid chromatography method was developed to measure coproporphyrinogen oxidase enzymatic activity in rat liver. With this method, the separation, identification and quantification of coproporphyrin III (oxidized substrate) and protoporphyrin IX (oxidized product) present in the assays could be carried out with no need of derivatization and in less than 15 min. Rat and human liver coproporphyrinogen oxidase basal activities determined using this method were 0.41+/-0.05 nmol of protoporphyrin IX/h per mg of hepatic protein and 0.87+/-0.06 protoporphyrin IX/h per mg of hepatic protein, respectively. Kinetic studies showed that optimum pH for rat CPGox is 7.3, and that its activity is linear in the range of protein concentrations and incubation times assayed. The present paper describes a sensitive, specific and rapid fluorometric high performance liquid chromatography method to measure coproporphyrinogen oxidase, which could be applied to the diagnosis of human coproporphyria, and which is also suitable for the study of lead and other metal poisoning that produce alterations in this enzymatic activity.

Development of an experimental ovarian tumor over a year in the rat.

Tumor growth, possible malignant transformation or metastatic propagation and hormonal patterns were evaluated over a year in luteoma induced by introducing an ovary into the spleen of ovariectomized 60 day-old rats. Sham castrated animals had a piece of muscle inserted into the spleen. Jugular blood samples were taken monthly. After a year animals were cycled and decapitated. Troncal blood was collected, autopsies were performed and luteoma were measured and fixed in 10% buffered formalin. Serum LH, FSH, PRL, estradiol and progesterone were measured. Serum inhibin content was determined in one month-old tumors-bearing animals and estrous rats as controls. After one year no external changes in tumor-bearing rats were observed, nor differences in body weight or mortality rates compared to Sham animals. Metastatic propagation was absent. Routine histological examination showed two types of tumors according to either granulosa or luteal cell predomination, tumor type did not determine hormonal patterns. However, a clear relationship between gonadotropin levels and tumor size was established. Low gonadotropins: Small tumors, 18.7% of cases and high gonadotropins: Large tumors, 81.3%. In Sham animals gonadotropins attained castrate levels and remained elevated until the end of the experiment. In the Small group no increases in gonadotropins or estradiol were detected, progesterone and PRL fluctuated. In the Large tumor group LH increased to Sham titers until month 7, then fell to initial levels, FSH augmented significantly as from month three and remained high up to month 5. No variations in either estradiol, progesterone or PRL were observed. Serum inhibin of one month-old tumor-bearing rats was significantly elevated, justifying the lack of FSH increase at this time point. We conclude that these luteoma do not suffer malignant transformation or induce metastases. They appear in two histological types. Tumor size depends on hormonal patterns. The delay in the initial increase and the sharp decrease observed in FSH in animals bearing Large tumors suggest a possible role for inhibin in this regulation.