Expression of receptors for luteinizing hormone,gastric-inhibitory polypeptide,and vasopressin in normal adrenal glands and cortisol-secreting adrenocortical tumors in dogs

Hypercortisolism caused by an adrenocortical tumor (AT) results from adrenocorticotropic hormone (ACTH)-independent hypersecretion of glucocorticoids. Studies in humans demonstrate that steroidogenesis in ATs may be stimulated by ectopic or overexpressed eutopic G protein-coupled receptors. We report on a screening of 23 surgically removed, cortisol-secreting ATs for the expression of receptors for luteinizing hormone (LH), gastric-inhibitory polypeptide (GIP), and vasopressin (V_1a, V_1b, and V₂). Normal adrenal glands served as control tissues. Abundance of mRNA for these receptors was quantified using quantitative polymerase chain reaction (QPCR), and the presence and localization of these receptors were determined by immunohistochemistry. In both normal adrenal glands and ATs, mRNA encoding for all receptors was present, although the expression abundance of the V_1b receptor was very low. The mRNA expression abundance for GIP and V₂ receptors in ATs were significantly lower (0.03 and 0.01, respectively) than in normal adrenal glands. The zona fasciculata of normal adrenal glands stained immunonegative for the GIP receptor. In contrast, islands of GIP receptor-immunopositive cells were detected in about half of the ATs. The zona fasciculata of both normal adrenal glands and AT tissue were immunopositive for LH receptor; in ATs in a homogenous or heterogenous pattern. In normal adrenal glands, no immunolabeling for V_1bR and V₂ receptor was present, but in ATs, V₂ receptor-immunopositive cells were detected. In conclusion, QPCR analysis did not reveal overexpression of LH, GIP, V_1a, V_1b, or V₂ receptors in the ATs. However, the ectopic expression of GIP and V₂ receptor proteins in tumorous zona fasciculata tissue may play a role in the pathogenesis of canine cortisol-secreting ATs.     

Expression of messenger ribonucleic acid encoding for steroidogenic acute regulatory protein and enzymes, and luteinizing hormone receptor during the spring transitional season in equine follicles

The period of spring transition, from the anovulatory to the ovulatory season, is characterized in many mares by cyclical growth and regression of large dominant follicles. These follicles produce only low concentrations of estradiol and it is thought that acquisition of steroidogenic competence by large follicles during spring transition is prerequisite in stimulating LH prior to first ovulation. In situ hybridization was used to localize and quantify expression of factors that play a key role in follicular steroidogenesis: StAR, P450scc (CYP11A1), P450c17 (CYP17), P450arom (CYP19), and LH receptor (LHr). One ovary was obtained from mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle (defined as the transitional follicle), and the remaining ovary was removed at the third estrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter (defined as the preovulatory follicle). Messenger RNAs encoding StAR, CYP11A1, and CYP17 were detected only in theca cells and CYP19 mRNA was confined to the granulosa layer. There was significantly lower expression of mRNAs for the steroidogenic enzymes, StAR (P<0.001) and LHr (P<0.05) in transitional follicles than in preovulatory follicles. In conclusion, large equine follicles during spring transition have low levels of mRNA encoding steroidogenic enzymes, StAR and LHr which will contribute to the steroidogenic incompetence of dominant follicles during spring transition and their subsequent regression.     

Melanocortin 2 receptor antagonists in canine pituitary-dependent hypercortisolism: in vitro studies

Canine hypercortisolism is most often caused by an ACTH-secreting pituitary adenoma (pituitary-dependent hypercortisolism; PDH). An interesting target for a selective medical treatment of PDH would be the receptor for ACTH: the melanocortin 2 receptor (MC2R). In this study we investigated whether two peptide compounds, BIM-22776 (#776) and BIM-22A299 (#299), are effective MC2R antagonists in vitro. Their effects on cortisol production and mRNA expression of steroidogenic enzymes, MC2R and melanocortin 2 receptor accessory protein (MRAP) were evaluated in primary adrenocortical cell cultures (n?=?8) of normal canine adrenal glands. Cortisol production stimulated by 50 nM ACTH was dose-dependently inhibited by #299 (inhibition 90.7?±?2.3% at 5 μM) and by #776 (inhibition 38.0?±?5.2% at 5 μM). The ACTH-stimulated mRNA expression of steroidogenic enzymes, MC2R and MRAP was significantly inhibited by both compounds, but most potently by #299. These results indicate that canine primary cell culture is a valuable in vitro system to test MC2R antagonists, and that these compounds, but especially #299, are effective MC2R antagonists in vitro. To determine its efficacy in vivo, further studies are warranted. Antagonism of the MC2R is a promising potential treatment approach in canine PDH.     

Expression of steroidogenic enzymes and synthesis of steroid hormones during development of ovarian follicles in prepubertal goats

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 -hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) were characterized by the RT-PCR technique and concentrations of progesterone (P4), testosterone (T0) and estradiol (E2) were measured by radioimmunoassay during follicular development of prepubertal goats. Synthesis of mRNAs encoding P450scc and P450c17 began in preantral follicles, but mRNA encoding P450arom was not detectable until early antral formation. While mRNA for P450scc was expressed in both theca and granulosa cells, mRNA for P450c17 was expressed only in theca cells while P450arom mRNA only in granulosa cells. In nonatretic follicles from prepubertal ovaries, the relative quantity of mRNA expression of all the three enzymes increased with follicle size; however, while the concentration of P4 and E2 increased, that of T0 decreased with follicle size. While expression of mRNA encoding P450scc was unaffected, that of P450c17 mRNA decreased to the lowest level and mRNA for P450arom became undetectable following atresia; accordingly, while the concentration of P4 increased in the atretic medium follicles, that of T0 and E2 decreased to the lowest level after atresia. While the adult follicular stage follicles showed a similar cytochrome expression as the nonatretic follicles of prepubertal goats, the former contained higher levels of E2 and P4 than the latter. The presence of corpus luteum in an ovary decreased expression of P450scc, significantly in large follicles while it increased concentration of P4. These findings indicated that (1) similar to other species, changes in follicular steroid production in goats were explained in large measure by changes in steroidogenic enzyme expression; (2) while mRNA expression was similar, activities of some of the steroidogenic enzymes may differ between sexually mature and immature goats.     

Immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation

To elucidate the relationship between steroidogenic hormones and developing adrenal glands, we investigated the immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation. Fetal adrenal glands were obtained from three horses at 217, 225 and 235 days of gestation. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Histologically, cortex and medulla cells were clearly observed in the three fetal adrenal gland tissue samples. P450scc and P450c17 were identified in cortex cells close to medulla cells and in some medulla cells in the fetal adrenal glands. P450arom was present in both cortex and medulla cells in the fetal adrenal glands. However, 3betaHSD was not found in any of the equine fetal adrenal gland tissue samples. These results suggest that equine fetal adrenal glands have the ability to synthesize androgen and estrogen, which may play an important physiological role in the development of equine fetal adrenal glands.     

Expression of inhibins, activins, insulin-like growth factor-I and steroidogenic enzymes in the equine placenta

In this study, the expression patterns of inhibins, activins, insulin-like growth factor-I (IGF-I) and steroidogenic enzymes in equine placentae recovered during the latter two-thirds of gestation were examined. Concentrations of inhibin A and inhibin pro-alphaC in endometrial and fetal placental tissue homogenates were very low during the period examined, whereas these tissues contained high concentrations of activin A. In both maternal endometrial and fetal placental tissues, activin A levels decreased as pregnancy progressed. Expression of inhibin alpha-subunit was not observed in the placenta using either immunohistochemistry or in situ hybridization. Inhibin/activin betaA-subunit and its mRNA were confined to maternal endometrial glands, whereas immunopositive betaB-subunit was not detected in either endometrial glands or microcotyledons. Cytochrome P450 side chain cleavage enzyme was detected by immunohistochemistry in both endometrial glands and microcotyledons, whereas cytochrome P450 17alpha-hydroxylase/lyase was absent in these tissues. Immunopositive signals for 3beta-hydroxysteroid dehydrogenase and cytochrome P450 aromatase were localized in microcotyledons but not in endometrial glands. Immunohistochemistry revealed that IGF-I was highly expressed in microcotyledons around Day 130, and decreased as pregnancy progressed. Changes in the expression of IGF-I were correlated with the number of PCNA positive cells in the placenta. The present study demonstrated the presence and localized the site of expression of activin, IGF-I and steroidogenic enzymes in equine placental tissues during the latter two-thirds of gestation; the results suggest that activin and IGF-I may be involved in the regulation of placental development.     

Changes in Testicular Steroidogenic Acute Regulatory (STAR) Protein,Steroidogenic Enzymes and Testicular Morphology Associated with Increased Testosterone Secretion in Bulls Receiving The Luteinizing Hormone Releasing Hormone Agonist Deslorelin

Testosterone secretion and the expression and relative contents of steroidogenic acute regulatory (StAR) protein and steroidogenic enzymes cholesterol side-chain cleavage cytochrome P450 (P450_scc), 3β-hydroxysteroid dehydrogenase /Δ⁵ → Δ⁴ − isomerase (3β-HSD), and 17α-hydroxylase cytochrome P450/C_17–20 lyase (P450_17α) were determined in testicular tissues of bulls treated with a LHRH agonist. Testis morphology and spermatogenesis were also examined. In Experiment 1, bulls (30-mo-old) received no treatment (control, n = 7) or were implanted for 10 days with the LHRH agonist deslorelin (n = 7). Bulls were castrated on Day 10 and testis tissues prepared for Western and Northern blotting. At castration, bulls implanted with deslorelin had greater plasma testosterone (5-fold) and testis content of testosterone (10-fold) compared with control bulls. Relative content (per μg total testis protein or RNA) of StAR protein, 3β-HSD, P450_scc, and mRNA for P450_17α in bulls treated with deslorelin ranged from 3- to 6-fold that of control bulls. In Experiment 2, bulls (20-mo-old) were left untreated (control, n = 6) or implanted with deslorelin (n = 12) for 120 days. On Day 120, bulls were castrated and right testis tissues prepared for morphology. Testis volume and weight were increased (P < 0.01) in bulls treated with deslorelin compared with control bulls. Stereological analysis revealed that this increase occurred in all compartments (seminiferous epithelium, lumen and interstitium) studied, but was significant (P < 0.01) only for the seminiferous epithelium. Absolute numbers of round spermatids per testis were increased (P < 0.05) in bulls treated with deslorelin compared with control bulls. Increased testosterone secretion in bulls treated with deslorelin was associated with increased testicular StAR protein and steroidogenic enzymes. Bulls treated long-term with deslorelin had a faster rate of testis growth and increased daily sperm production at the end of the experiment.     

Immunolocalization of steroidogenic enzymes and their expression during the breeding season in the testes of wild raccoon dogs (Nyctereutes procyonoides)

The objective of this study was to investigate immunolocalization of steroidogenic enzymes 3βHSD, P450c17 and P450arom and their expression during the breeding season in wild male raccoon dogs. The testicular weight, size and seminiferous tubule diameters were measured, and histological and immunohistochemical observations of testes were performed. The messenger RNA expression (mRNA) of 3βHSD, P450c17 and P450arom was measured in the testes during the breeding season. 3βHSD was found in Leydig cells during the breeding and non‐breeding seasons with more intense staining in the breeding season. P450c17 was identified in Leydig cells and spermatids in the breeding season, whereas it was present only in Leydig cells in the non‐breeding season. The localization of P450arom changed seasonally: no immunostaining in the non‐breeding season; more extensive immunostaining in Leydig cells, Sertoli cells and elongating spermatids in the breeding season. In addition, 3βHSD, P450c17 and P450arom mRNA were also expressed in the testes during the breeding season. These results suggested that seasonal changes in testicular weight, size and seminiferous tubule diameter in the wild raccoon dog were correlated with spermatogenesis and immunoreactivity of steroidogenic enzymes and that steroidogenic enzymes may play an important role in the spermatogenesis and testicular recrudescence and regression process.     

Effect of spray-dried plasma and lipopolysaccharide exposure on weaned pigs: II. Effects on the hypothalamic-pituitary-adrenal axis of weaned pigs

A study was conducted with 20 weaned barrows (14 d, 4.98 +/- 0.21 kg) to determine the effect of feeding spray-dried plasma (SDP) after weaning on the pig's stress response to a lipopolysaccharide (LPS) challenge. After weaning, pigs were fed a diet containing 0 or 7% SDP for 7 d. On d 6 after weaning, all pigs were nonsurgically fitted with a jugular catheter. On d 7 after weaning, the pigs were given i.p. injections of either saline or LPS (150 microg/kg BW) followed by serial blood collection every 15 min for a 3-h period. Following the 3-h blood collection, all pigs were killed and tissue was collected for mRNA analysis. Pig weight on d 7 after weaning was not affected by dietary treatment (P > 0.21). Pigs fed the diet with SDP had lower (P < 0.05) levels of hypothalamic corticotropin-releasing hormone (CRH) mRNA, pituitary gland CRH receptor mRNA, and adrenal gland adrenocorticotropin-releasing hormone (ACTH) receptor mRNA. Dietary treatment did not affect pituitary gland proopiomelanocortin (POMC) mRNA. No effect of LPS treatment was observed in any of the mRNA levels examined. For both serum ACTH and cortisol, there was a significant diet x LPS treatment interaction (P < 0.01) such that both the ACTH and cortisol responses to the LPS challenge were greater in the pigs fed the diet with SDP than in the pigs fed the diet without SDP. For pigs given the saline injection, diet did not affect basal serum cortisol concentration; however, basal serum ACTH concentration was lower in those pigs fed the diet with SDP (P < 0.0001). A diet x LPS treatment interaction (P < 0.024) was observed for adrenal gland mRNA expression for steroidogenic acute regulatory (StAR) protein such that the LPS-induced increase in StAR mRNA was greater in the pigs fed SDP than in pigs fed the diet without SDP. These results demonstrate that pigs fed a diet with SDP have an increased activation of the pituitary-adrenal axis following an LPS challenge compared to pigs fed a diet without SDP.     

Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues

Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.     

Administration of ACTH to restrained, pregnant sows alters their pigs' hypothalamic-pituitary-adrenal (HPA) axis

This study was designed to examine the physiology and behavior of pigs whose dams were snared and then injected with ACTH during gestation. Administration of ACTH to dams during pregnancy has been shown to replicate the effects of prenatal stress in other species. Control sows (n = 8) were given no treatment, whereas the treatment sows (ACTH, n = 8) were immobilized by snaring the snout and then administered an i.v. injection of ACTH (1 IU/kg BW) weekly from 6 to 12 wk of gestation. A pig was killed from each sow at 1, 30, and 60 d of age. The hypothalamus, pituitary gland, adrenal glands, and liver were immediately obtained to determine the amounts of corticotropin-releasing hormone (CRH), beta-endorphin, and mRNA for pro-opiomelanocorticotropin (POMC), the ACTH receptor (ACTH-R), and insulin-like growth factor I (IGF-I). Pituitary corticotrope and somatotrope cell numbers and adrenal cortex-to-medulla area ratios (CORT:MED) were also determined. Pigs' behaviors were recorded at 6 and 8 wk of age. At 75 d of age, a blood sample was taken and a biopsy puncture was created on one pig from each litter, then pigs were stressed by mixing. Blood was sampled every other day for 10 d to determine plasma cortisol concentrations and differential leukocyte counts. Biopsy damage was evaluated for healing. At 1 d of age, control pigs tended to weigh more (P = .09), have a lower expression of ACTH-R mRNA (P = .01) and IGF-I mRNA (P = .01), and a lower CORT:MED (P = .04) than ACTH pigs. At 30 d of age, control pigs had a greater concentration of beta-endorphin (P = .01) and tended to have a lower concentration of CRH (P = .09) and IGF-I mRNA (P = .10) than ACTH pigs. At 60 d of age, control pigs tended to have lighter pituitary glands (P = .08), a lower expression of POMC mRNA (P = .02), and a CORT:MED (P = .003) than ACTH pigs. At 8 wk of age, control pigs performed a higher frequency of belly nosing (P = .07) and oral vice behaviors (P = .01) than ACTH pigs. In response to mixing stress, control pigs had lesser concentrations of plasma cortisol (P = .03) and healed faster (P = .006) than ACTH pigs. Thus, exogenous ACTH and restraint during gestation alters the HPA axis of the sow's offspring, and during stressful situations later in life health, and therefore welfare, may be compromised.     

Trilostane-induced inhibition of cortisol secretion results in reduced negative feedback at the hypothalamic–pituitary axis

Cushing's disease caused by pituitary corticotroph adenoma in dogs is usually treated by medical treatment, and the efficacy of this treatment has been reported. However, controversy remains as to whether reduced negative feedback through the inhibition of cortisol secretion, similar to Nelson's syndrome, may appear as an adverse effect. The purpose of this study was to investigate the effect of reduced negative feedback through the inhibition of cortisol secretion by daily trilostane administration on the pituitary–adrenal axis in clinically normal dogs. Dogs were administered 5 mg/kg trilostane twice a day every day for 8 weeks (n = 8) or 16 weeks (n = 3). After the initiation of trilostane administration, plasma adrenocorticotropic hormone (ACTH) concentrations were increased remarkably. As assessed by magnetic resonance imaging (MRI) during administration, the pituitary became enlarged. After trilostane administration, the cytoplasmic areas of the pituitary corticotrophs were increased and the ratio of pituitary corticotrophs to all cells in the anterior lobe was greater in the trilostane-treated dogs than that in untreated animals. In addition, histological examinations revealed bilateral adrenal cortical hyperplasia. Using real-time PCR quantification, the expression of proopiomelanocortin (POMC) mRNA in the pituitary and ACTH receptor (ACTH-R) mRNA in the adrenal gland was greater in the dogs treated with trilostane than in untreated dogs. These results indicate that reduced negative feedback induced hyperfunction of the pituitary corticotrophs and pituitary enlargement in healthy dogs. These changes suggest that the inhibition of cortisol secretion by trilostane may increase the risk for accelerating the growth of corticotroph adenomas in dogs with Cushing's disease.     

Indicators of malignancy of canine adrenocortical tumors: histopathology and proliferation index

Tumors of the adrenal cortex account for 10-20% of the naturally occurring Cushing's syndrome diagnosed in dogs. Differentiating between adrenocortical adenoma and carcinomas is often difficult. The purposes of this study were to determine which histopathologic criteria can be used as markers for malignancy in canine adrenocortical tumors and the relevance of the proliferation marker, Ki-67, for differentiation between cortical adenomas and carcinomas. Twenty-six adrenocortical carcinomas, 23 adenomas, and 11 normal adrenal glands were examined. Morphologic criteria significantly associated with adrenocortical carcinomas included a size larger than 2 cm in diameter, peripheral fibrosis, capsular invasion, trabecular growth pattern, hemorrhage, necrosis, and single-cell necrosis, whereas hematopoiesis, fibrin thombi, and cytoplasmic vacuolation were significantly associated with adrenocortical adenomas. The mean (+/- SD) proliferation index, measured by immunohistochemistry for the Ki-67 antigen, was 9.3 +/- 6.3% in carcinomas, 0.76 +/- 0.83% in adenomas, and 0.58 +/- 0.57% in normal adrenal glands. The Ki-67 proliferation index was significantly higher in carcinomas compared with adenomas and normal adrenal glands. A threshold value of the proliferation index of 2.4% reliably separated carcinomas from adenomas. Based on these results, it appears that thorough evaluation of morphologic features combined with immunohistochemical assessment of the proliferation index is extremely useful for differentiating between adrenocortical adenomas and carcinomas in dogs.     

Correlations among antral follicular echotexture,apoptosis and expression of key steroidogenic enzymes in sheep

Nineteen cycling ewes underwent transrectal ultrasonography of ovaries followed by ovariectomies during the growth phase of the first follicular wave of the interovulatory interval or the proestrus/estrus phase of the cycle. Quantitative ultrasonographic characteristics of the antrum and follicular wall in a total of forty-three ovine antral follicles were examined for correlations with the protein expression of three steroidogenic enzymes (cytochrome P450 17α-hydroxylase, CYP17; cytochrome P450 aromatase, CYP19; and 3β-hydroxysteroid dehydrogenase, 3β-HSD) determined by densitometric analysis of immunohistochemical slides, follicular dimensions, granulosa layer thickness and the percentage of apoptotic granulosa cells. Significant correlations were found between echotextural attributes of ovine antral follicles and the percentage of apoptotic granulosa cells, CYP17 expression (theca), CYP19 expression (granulosa) and 3β-HSD expression (theca cells). Computer-aided analyses of ultrasonographic images can be beneficial to the development of assisted reproductive technologies and diagnosis of hormonal imbalances without the need for ovarian biopsies or hormone assays.     

Effects of the β2 -agonist clenbuterol on testicular steroidogenic acute regulatory protein mRNA expression in adult rats

Ma, J.‐K., Zhu, W.‐J. Effects of the β₂‐agonist clenbuterol on testicular steroidogenic acute regulatory protein mRNA expression in adult rats. J. vet. Pharmacol. Therap. 33 , 558–563. This study was carried out to investigate the effects of clenbuterol (CLB) on the testicular (steroidogenic acute regulatory, StAR) protein mRNA expression in rats. Thirty adult male rats were administered CLB by gavage daily at the doses of 0.4, 2.0 and 18.5 mg/kg bw for 14 days in the subacute experiment, whereas 20 rats received a single treatment with CLB at the doses of 20 and 40 mg/kg bw in the acute experiment and 20 rats were treated with 0.9% NaCl solution as vehicle groups. Testicular tissues were collected and snap‐frozen in liquid nitrogen and stored at ?70 °C until use. The levels of StAR mRNA were detected by RT–PCR. The levels of StAR mRNA were markedly increased (P < 0.05) at both dosages of 20 and 40 mg/kg bw but the effects were not dose‐dependent and the mRNA levels of StAR were returned to near normal level after 7 days of CLB withdrawal, compared with the control animals. In the subacute experiment, CLB induced a dose‐dependent but no statistical significant reduction (P > 0.05) in the expression levels of StAR mRNA, and the mRNA levels were recovered to near normal level in the groups treated with CLB at dosages of 0.4 and 2.0 mg/kg bw/day following a 7‐day withdrawal period, compared with the control animals. The mRNA levels of StAR showed a significant decrease in the groups treated with CLB at the dosage of 18.5 mg/kg bw/day (P < 0.05) after a 1‐ or 7‐day withdrawal period with respect to the control animals. These results demonstrated transient stimulative effects of CLB on testicular StAR mRNA levels and inhibitory effects after treatment with CLB for 14 consecutive days.     

Administration of estradiol-3-benzoate down-regulates the expression of testicular steroidogenic enzyme genes for testosterone production in the adult rat

ABSTRACT. To study the role of estrogen in the testes, testosterone and testicular steroidogenic enzyme mRNA levels were investigated in male Sprague-Dawley rats 24 hr after intramuscular administration of a single dose of estradiol-3-benzoate (EB). EB administration resulted in a greater decrease in intra-testicular and serum testosterone in 10-week-old rats than in 3- or 5-week-old rats. A dose of 2 microg EB/kg had the lowest observed effect. The level of serum luteinizing hormone (LH) was unchanged at any dose. Semiquantitative RT-PCR analysis revealed that, of the four major testicular steroidogenic enzymes, mRNA levels of cytochrome P450 side-chain cleavage and 17beta-hydroxysteroid dehydrogenase type-III were significantly reduced, and mRNA levels of cytochrome P450 17alpha-hydroxylase/ C17-20 lyase (P450c17) were reduced severely and significantly, by EB administration. However, the level of 3beta-hydroxysteroid dehydrogenase type-I mRNA was not changed. In addition, the P450c17 mRNA level in EB-treated rats was much lower than that in the testes of hypophysectomized rats, with the level in the latter being equal to that in control rats. LH is secreted into blood periodically, the effects of estrogen on the LH secretion pattern of the pituitary gland, for example, in frequency and amplitude of LH pulse, were difficult to detect with the methods of the present study. The results indicated, at least, that EB administration down-regulates P450c17 gene expression predominantly, resulting in the inhibition of testosterone production. From the differences in the steroidogenic enzyme expressions between hypophysectomized and EB-treated rats, it was suggested that EB acts on the testis directly or indirectly though not via alteration of LH secretion and induces reduction of P450c17 mRNA level.     

The expression of carbonic anhydrase in canine mammary gland and mammary gland tumor

Carbonic anhydrase (CA), a metallo-enzyme containing zinc, broadly distributes in mammalian tissues and participates in physiological regulation such as respiration, acid-base balance, ion transport, bone resorption, as well as the development of tumor by the reversible hydration of carbon dioxide. However the expression of CA in the tissue of mammary gland tumor was not documented. In this study we examine the histolocalization and gene expression of CA in both normal canine mammary gland tissue and mammary gland tumor by histochemical examination, and RT-PCR. Four mRNA expression of CA isoenzymes, such as CA II, IV, VI and IX were found under RT-PCR analysis and different band patterns were found between normal canine mammary tissue and canine mammary gland tumor tissue. CA II, IV, VI and IX gene mRNA expression were found in the normal mammary gland tissue, indicating CA II, IV, VI and IX are likely to be the essential enzymes to maintain the normal physiological condition of canine mammary gland tissue cells. However the expression of CA IV was not found in the tissue of malignant mammary gland tumor that may become the marker for the prognostic recognition of canine mammary gland tumor.