Expression of the ACTH receptor, steroidogenic acute regulatory protein, and steroidogenic enzymes in canine cortisol-secreting adrenocortical tumors

Studies of human adrenocortical tumors (ATs) causing Cushing's syndrome suggest that hypersecretion of cortisol is caused by altered expression of steroidogenic enzymes and that steroidogenesis can only be maintained when there is expression of the ACTH receptor (ACTH-R). Here we report the screening for the mRNA expression of the ACTH-R, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase, 21-hydroxylase (all in 38 cortisol-secreting ATs), 17α-hydroxylase, and 11β-hydroxylase (both in 28 cortisol-secreting ATs). Real-time PCR (RT-PCR) was applied in all samples and was compared with that in normal canine adrenal glands. Messenger-RNA encoding StAR, steroidogenic enzymes, and ACTH-R were present in both normal adrenal glands and cortisol-secreting ATs. The amounts of mRNA encoding StAR and enzymes of the steroidogenic cluster needed for cortisol production did not differ significantly between either adenomas or carcinomas and normal adrenal glands. The amount of mRNA encoding ACTH-R was significantly lower in carcinomas than in normal adrenal glands (P = 0.008). In conclusion, RT-PCR analysis revealed no overexpression of StAR and steroidogenic enzymes in canine cortisol-secreting ATs. Significant downregulation of ACTH-R in carcinomas might be associated with the malignant character of the AT.     

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.     

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.     

Changes in plasma follicle-stimulating hormone, luteinizing hormone, estrogen and progesterone during growth of ovulatory follicles in the pig

This experiment was conducted to determine the changes in secretion of LH, FSH, estrogen and progesterone during follicle maturation. Ovaries were recovered from 11 non-treated (control) gilts, three on day 13, four on day 16, and four on day 19 of the estrous cycle, and from four altrenogest-treated gilts on day 19. Altrenogest, a progesterone agonist, was fed at a dose of 20 mg once daily from days 13 to 18 to block spontaneous follicle maturation. Gilts were bled daily from day 12 until slaughter. For control gilts, the number of follicles/gilt 1-6 mm in diameter decreased (P less than .05) from 93.5 on day 13 to 21.5 on day 19, and the number of large (greater than 6 mm) follicles increased (P less than .05) from 5.3 to 13.2. Altrenogest treatment blocked loss of small follicles and growth of large follicles between days 13 and 19. Plasma progesterone decreased (P less than .001) between days 12 and 16 in both control and altrenogest-treated gilts. Plasma FSH decreased (P less than .05) between days 12 and 16 only in control gilts. Plasma LH was not significantly affected by day or altrenogest treatment. Plasma estrogen increased (P less than .05) between days 15 and 19 only in control gilts. These results indicate that 1) no increased LH secretion was detected in conjunction with emergence of ovulatory follicles, and 2) atresia of nonovulatory follicles was associated with decreased secretion of FSH. Both atresia and decreasing FSH secretion began before estrogen concentration increased in the systemic circulation.     

Expression of calbindin-D9k messenger ribonucleic acid in the gastrointestinal tract of dairy cattle

The calcium demands of pregnancy and lactation are known to up-regulate expression of Calbindin-D9k (CaBP-9k) mRNA in the intestines. The gastrointestinal CaBP-9k mRNA expressions has not been studied in dairy cows, which are bound to experience several pregnancies and lactation stages. In this study, the CaBP-9k mRNA expression were examined in the gastrointestinal tract of Holstein dairy cattle by Northern blot analysis. Detectable expression of CaBP-9k mRNA was localized in the proximal portion of the small intestines. These expressions were higher at the most proximal region of the duodenum and gradually decreased distally. The duodenal CaBP-9k mRNA was detected in all dairy cattle from 0.4 to 83.4 months old, but was not detectable in foetuses. There were no significant correlations between the age and the levels of CaBP-9k mRNA expression or between the plasma 1,25-(OH)2D3 concentrations and the levels of CaBP-9k mRNA expression.     

性腺外FSH受体和LH受体表达研究进展

雌性动物的性腺是卵巢,性腺外的生殖组织器官主要包括:输卵管、子宫颈、子宫肌层等,其中子宫是内分泌器官,除有局部内分泌功能外,还可能对下丘脑-垂体-卵巢轴有调节作用.促卵泡素(FSH)和促黄体素(LH)的生理功能是通过其FSHR、LHR来介导的,其受体在性腺和性腺外均有表达.本文主要对雌性性腺外生殖组织器官上两种受体的表达加以综述.     

Elicitation of release of luteinizing hormone by N-methyl-d,l-aspartic acid during three paradigms of suppressed secretion of luteinizing hormone in the female pig.

Two experiments were conducted to determine the minimal effective dose during lactation and site of action of N-methyl-d,l-aspartic acid (NMA) for elicitation of release of luteinizing hormone (LH) in female pigs. In the first experiment, three doses of NMA were given to lactating primiparous sows in which endogenous LH was suppressed by suckling of litters. In the second experiment, ovariectomized gilts were pretreated with estradiol benzoate or porcine antisera against GnRH to suppress LH and then given NMA to determine if it elicited secretion of LH directly at the anterior pituitary or through release of GnRH. In experiment 1, 3 lactating sows (17 +/- 1.5 d postpartum) were each given three doses of NMA (1.5, 3.0 and 5.0 mg/kg body weight [BW]; IV) on 3 consecutive days in a Latin Square design. Blood samples were collected every 10 min from -1 to 1 hr from injection of NMA. NMA at 1.5 and 3.0 mg/kg did not affect (p greater than .5) secretion of LH; however, 5 mg NMA/kg elicited a 114% increase (p less than .001) in circulating levels of LH during 1 hr after treatment. In experiment 2, 8 ovariectomized gilts were given either estradiol benzoate (EB; 10 micrograms/kg BW; IM n = 4) to suppress release of GnRH or porcine antiserum against GnRH (GnRH-Ab; titer 1:8,000; 1 ml/kg BW; IV; n = 4) to neutralize endogenous GnRH. Gilts infused with GnRH-Ab were given a second dose of antiserum 24 hr after the first. Gilts were then given NMA (10 mg/kg BW; IV) 33 hr after EB or initial GnRH-Ab. Blood samples were drawn every 6 hr from -12 to 24 hr from EB or GnRH-Ab treatments, and every 10 min from -2 to 2 hr from NMA. Serum LH declined (p less than .001) after EB (from 1.87 +/- .2 ng/ml at 12 hr before EB to 0.46 +/- .02 ng/ml during 24 hr after EB) and GnRH-Ab (from 1.97 +/- .1 to 0.59 +/- .02 ng/ml). In gilts treated with EB, the area under the curve (AUC) for the LH response (ng.ml-1.min) 1 hr after NMA (38.7 +/- 3) was significantly greater (p less than .01) than the 1 hr prior to NMA (21.3 +/- 1.5). Treatment with NMA had no effect (p greater than .5) on secretion of LH in gilts infused with GnRH-Ab.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Changes in the peripheral concentrations of inhibin, follicle-stimulating hormone, luteinizing hormone, progesterone and estradiol-17beta during turnover of cystic follicles in dairy cows with spontaneous follicular cysts

Several studies have clarified that the follicular cysts degenerate and are replaced by newly growing follicles that develop into new follicular cysts without ovulation, i.e., turnover of ovarian follicular cysts in cows. However, the relativity of endocrinological changes, including the inhibin profile during turnover of spontaneous follicular cysts in dairy cows, is still unclear. In the present study, the relationship between turnover of follicular cysts and changes in the peripheral blood concentrations of progesterone (P), estradiol-17beta (E(2)), luteinizing hormone (LH), follicle stimulating hormone (FSH) and inhibin were examined in lactating dairy cows. Five cows diagnosed with follicular cysts (follicles of more than 25 mm in diameter in the absence of a corpus luteum) were investigated. Their ovarian dynamics were monitored using ultrasonography, and blood samples were collected at 2- or 3- day intervals throughout the experiment. The day when a follicle fated to become a follicular cyst reached more than 8 mm in diameter was defined as the start of a cystic follicular wave. Four of the 5 cows exhibited a similar patterns of cystic follicular changes and hormone profiles. The data from the 4 cows was used for analysis of the relationships between turnover of cystic follicles and the hormone profiles. Two or three new cystic follicular waves occurred in each cow during the experimental period. The mean diameter of the cystic follicles was more than 25 mm 13 to 15 days after the start of the cystic follicular wave, and it began to decrease 1 to 6 days before the start of the subsequent cystic follicular wave. The levels of E(2) and inhibin tended to decrease for 7 to 9 days before the start of a new cystic follicular wave and to increase concomitantly with new follicular cyst growth. The levels of FSH rose for 1 to 3 days before the start of a new cystic follicular wave. The present study clarified the relationship between FSH and inhibin during turnover of spontaneous follicular cysts in dairy cows and found that it was very similar to previous results for cows. The present results suggest that an increase in FSH secretion following a reduction in inhibin secretion triggers turnover of cystic follicles in cows with spontaneous follicular cysts.     

Effects of heat stress on proliferation, protein turnover, and abundance of heat shock protein messenger ribonucleic acid in cultured porcine muscle satellite cells

It is well established that heat stress (HS) negatively affects growth rate in swine. Although reduced feed intake undoubtedly plays a significant role in this reduction, studies in laboratory animals and other nonswine species indicate muscle growth also is affected by HS-related alterations in muscle physiology. Evidence is now emerging that heat shock proteins (Hsp), produced in response to HS and other types of cellular stress, may play an important role in regulating the rate and efficiency of muscle growth. Because muscle satellite cells play a crucial role in postnatal muscle growth, the effects of HS on rates of satellite cell proliferation, protein synthesis, and protein degradation play an important role in determining the rate and extent of muscle growth. Consequently, in the current study we have examined the effects of mild HS (40.5°C for 48 h) on the rates of proliferation, protein synthesis, and protein degradation and on quantities of Hsp90, Hsp70, and Hsp25/27 mRNA and protein in cultured porcine muscle satellite cells (PSC). Mild HS of PSC cultures resulted in 2.5-, 1.4-, and 6.5-fold increases (P < 0.05) in the abundance of Hsp90, Hsp70, and Hsp25/27 mRNA, respectively, relative to control cultures. Abundance of Hsp 90, 70, and 25/27 proteins was also increased in HS PSC cultures compared with those in control cultures. Proliferation rates in HS PSC cultures were 35% less (P < 0.05) than those in control cultures. Protein synthesis rates in HS-fused PSC cultures were 85% greater (P < 0.05) than those in control cultures, and protein degradation rates in HS-fused PSC were 23% less (P < 0.05) than those in control cultures. In light of the crucial role satellite cells play in postnatal muscle growth, the HS-induced changes we have observed in rates of proliferation, protein turnover, and abundance of Hsp mRNA and Hsp protein in PSC cultures indicate that mild HS affects the physiology of PSC in ways that could affect muscle growth in swine.     

Protein and mRNA expression of follicle‐stimulating hormone receptor and luteinizing hormone receptor during the oestrus in the yak (Bos grunniens)

Follicle‐stimulating hormone (FSH) and luteinizing hormone (LH) have a central role in follicle growth, maturation and oestrus, but no clear pathway in the seasonal oestrus of yak (Bos grunniens) has been found. To better understand the role of FSH and LH in seasonal oestrus in the yak, six yaks were slaughtered while in oestrus, and the pineal gland, hypothalamus, pituitary gland, and gonads were collected. Using real‐time PCR and immunohistochemical assays, we determined the mRNA and protein expression of the FSH and LH receptors (FSHR and LHR) in these organs. The analysis showed that the FSHR mRNA expression level was higher in the pituitary gland tissue compared with LHR (p < .01) during oestrus. By contrast, there was low expression of FSHR and LHR mRNA in the pineal gland and hypothalamus. FSHR mRNA expression was higher than that of LHR (p < .05) in the ovary, whereas LHR mRNA expression was higher than that of FSHR (p < .01) in the uterus. FSHR and LHR proteins were located in the pinealocyte, synaptic ribbon and synaptic spherules of the pineal gland and that FSH and LH interact via nerve fibres. In the hypothalamus, FSHR and LHR proteins were located in the magnocellular neurons and parvocellular neurons. FSHR and LHR proteins were localized in acidophilic cells and basophilic cells in the pituitary gland, and in surface epithelium, stromal cell and gland epithelium in the uterus. In the ovary, FSHR and LHR protein were present in the ovarian follicle. Thus, we concluded that FSHR and LHR are located in the pineal gland, hypothalamus, pituitary and gonad during oestrus in the yak. However, FSHR was mainly expressed in the pituitary gland and ovaries, whereas LHR was mainly expressed in the pituitary gland and uterus.     

Immunolocalization of steroidogenic cells in small follicles of the chicken ovary: Anatomical arrangement and location of steroidogenic cells change during follicular development

Previous anatomical and histochemical studies suggested that interstitial cells were the only steroidogenic cells in the theca layer of small follicles of the chicken ovary. However, the precise cellular site of steroid production in the small follicles is not certain. Therefore, our goal was to identify steroidogenic cells in small follicles (< 10 mm in diameter) of the chicken ovary which have not entered the follicular hierarchy by localizing steroidogenic enzymes with immunocytochemistry. Polyclonal antisera used were anti-cholesterol side-chain-cleavage cytochrome P450 (P450scc), anti-17-hydroxylase cytochrome P450 (P450c17), and anti-aromatase cytochrome P450 (P450arom) for pregnenolone-, androgen-, and estrogen-producing cells, respectively. Ovaries were collected 2 hr after oviposition and embedded in Paraplast after fixation with 4% paraformaldehyde, 10% formaldehyde, or Bouin's solution. Tissues were sectioned (4–6 μm) and sections were mounted on poly-L-lysine coated slides. Sections were incubated overnight at room temperature with each specific antiserum raised in rabbits against cytochrome P450 steroidogenic enzymes or normal rabbit serum as a control and were immunostained with an avidin-biotin-peroxidase complex. Immunoreactivity for the P450 enzymes was absent in the granulosa layer but was present in the theca layer of the small follicles (< 10 mm in diameter). Interstitial cells in the single theca layer of cortical follicles embedded in the ovarian cortex (less than 1 mm in diameter) contained P450scc and P450c17. Cells which contained P450arom, identified as aromatase cells, surrounded the interstitial cells in the theca layer. In small white follicles (approximately 1 mm in diameter), large white follicles (approximately 2–4 mm in diameter), and small yellow follicles (approximately 5–10 mm in diameter) which protruded from the surface of the ovary, the theca layer is divided into the theca interna and the theca externa. P450scc and P450c17 were localized in interstitial cells in the theca interna and externa whereas P450arom was localized in aromatase cells of the theca externa. With follicular development, more interstitial cells staining for P450scc and P450c17 appeared in the theca interna than in the theca externa whereas aromatase cells staining for P450arom were localized only in the theca externa. The distance between interstitial cells and aromatase cells within the theca layer increased as the follicles matured, resulting in a change in the anatomical relationship of steroidogenic cells. Our results of immunolocalization of cytochrome P450 steroidogenic enzymes in developing small follicles suggest that: 1) granulosa cells in small follicles are steroidogenically inactive; 2) steroids are produced in two distinct cell populations in the theca layer of small follicles, namely interstitial cells and aromatase cells; and 3) the anatomical relationship and location of interstitial cells and aromatase cells in the theca layer change with follicular maturation (a two-cell model for steroidogenesis in small follicles during follicular development).     

Concentrations of prolactin, luteinizing hormone and follicle stimulating hormone in pituitary and serum of horses: effect of sex, season and reproductive state

Pituitary and serum from 86 male or female horses of various reproductive states were collected in the normal breeding season (summer) and in the nonbreeding season (winter) at a commercial slaughterhouse. Concentrations of prolactin (PRL), luteinizing hormone (LH) and follicle stimulating hormone (FSH) were measured by radioimmunoassay. Concentrations of pregnant mare serum gonadotropin and reproductive steroids in serum and gross appearance of the reproductive tract and gonads were used to catagorize reproductive state. Concentrations of PRL were higher (P less than .01) in summer than in winter in pituitary and serum of mares, stallions and geldings. In summer, mares had higher (P less than .01) concentrations of PRL in serum than stallions. In mares, concentrations of LH in pituitary were higher (P less than .05) in summer than in winter. Concentrations of LH in serum were higher (P less than .01) in summer than in winter in mares and geldings, higher (P less than .01) in mares than in stallions in summer, higher (P less than .01) in geldings than in stallions in summer and higher (P less than .01) in mares with low serum progesterone (P) concentrations than in mares with high P concentrations in summer. Concentrations of FSH in pituitary and serum did not differ between summer and winter for any type of horse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Patterns of secretion of luteinizing hormone, follicle stimulating hormone and testosterone in stallions during the summer and winter

Samples of jugular blood were drawn from each of five stallions every 15 min for 12 h during the summer and winter to determine the short-term fluctuations in plasma concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone. Concentrations of LH and FSH were generally not pulsatile, although one stallion exhibited three distinct pulses in these hormones during the winter. In general, patterns of secretion of all three hormones were similar in both seasons and the number of significant rises in hormonal concentrations did not differ between seasons. Concentrations of LH and FSH were positively correlated (P less than .05) for eight of the ten sampling periods, indicating a close relationship between the secretion rates of these two gonadotropins. Testosterone concentrations varied in an episodic manner during the 12-h period, and all stallions exhibited at least one episode of high testosterone secretion regardless of the pattern of LH concentrations. The response in testosterone concentrations to the three LH pulses exhibited by the one stallion in winter was not the same for each pulse. The correlations between a single random sample and mean concentrations over the 12-h period were high (r between .88 and .99) for all three hormones, indicating that a single sample of blood would be representative of overall concentrations. It appears that the stallion differs from males of other domestic species in that concentrations of gonadotropins and testosterone vary in a much less pulsatile manner.     

能量对初情期前母猪卵巢LHR和FSHR mRNA表达的影响

对9头50日龄、体质量为30 kg的初情期前杜×长×大三元杂交母猪进行长期日粮能量差异饲养试验.饲养结束后屠宰,取卵巢液氮保存.半定量RT-PCR检测每头母猪卵巢LH和FSH受体mRNA的含量.结果,高能组卵巢LH和FSH受体mRNA的含量显著高于中能组和低能组(P<0.05);而低能组显著低于中能组和高能组(P<0.05).表明,能量水平高的日粮可以显著地促进初情期前卵巢上LH和FSH受体在mRNA水平的表达,而能量水平不足的日粮则不利于这种表达.本文首次报道了日粮能量水平对母猪卵巢LH和FSH受体mRNA表达的影响.