2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) blocks ovulation by a direct action on the ovary without alteration of ovarian steroidogenesis: lack of a direct effect on ovarian granulosa and thecal-interstitial cell steroidogenesis in vitro

The main purpose of this study was to investigate the direct effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on ovarian function including ovulation and steroidogenesis. In vivo effects of TCDD were investigated on ovulation and alteration of circulating and ovarian steroid hormones in immature hypophysectomized rats (IHR) primed with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In addition, in vitro effects of TCDD on the steroidogenesis of granulosa cells (GC), theca-interstitial cells (TIC), and whole ovarian dispersates derived from the ovary of IHR were investigated. In the ovulation model, rats were hypophysectomized on Day 23 of age. On Day 26, the IHR were given 20 μg TCDD/kg by gavage. The next day eCG (10 IU) was injected sc to stimulate follicular development. Fifty-two hours after eCG, 10 IU hCG was given to induce ovulation. TCDD (20 μg/kg) blocked ovulation and reduced ovarian weight in IHR. Concentrations of progesterone (P4), androstenedione (A4), and estradiol (E2) in sera and ovaries were not altered by TCDD at 12, 24, 48, and 72 h after eCG, except for a two-fold increase in ovarian concentration of A4 at 48 h after TCDD. However, this higher concentration of A4 at 48 h after TCDD did not reflect that of A4 in sera and did not correlate with E2 in either sera or ovaries. In isolated GC from untreated IHR, TCDD (0.1 to 100 nM) had no significant effect on P4 and E2 after stimulation by LH or FSH. In TIC and whole ovarian dispersates containing GC, TIC, and other ovarian cells, TCDD (0.1 to 800 nM) had no effect on A4 and P4 secretion stimulated by LH. Using RT-PCR, AhR mRNA was shown to be expressed constitutively in the whole ovary of IHR with maximum down-regulation at 6 h after TCDD (20 μg/kg). Ovarian CYP1A1 was induced maximally at 6 h after TCDD, whereas CYP1B1 could not be detected. The induction of AhR related genes by TCDD in the ovary implies the existence of AhR-mediated signal transduction pathways. In summary, these results indicate that TCDD does not affect ovulation in IHR by altering ovarian steroidogenesis. It seems that inhibition of ovulation by TCDD is due to processes related to follicular rupture.     

多聚物PVP载体人生长激素基因在去垂体鼠体内表达的实验研究

目的以人工合成多聚物5%聚乙烯吡咯烷酮(PVP)为基因治疗载体,研究人生长激素基因骨骼肌肌肉注射方法在生长激素(GH)缺乏症模型鼠中的表达.方法选用人GH基因的真核表达载体pCEP-hGH与PVP混合,采用不同剂量胫前肌肌肉注射方法,1～4周时取注射部位肌肉,匀浆后采用PCR、RT-PCR方法分别检测hGH载体、hGH mRNA,采用放射免疫法检测血清中hGH.结果构建的真核表达载体经酶切、PCR、测序鉴定正确,各组hGH载体骨骼肌肌肉注射后PCR、RT-PCR均为阳性,100μg、300μg剂量组4周内血清均得到表达,呈缓慢上升趋势.300μg剂量组表达大于100 μg剂量组,差异有极显著意义.结论应用PVP作载体,在去垂体鼠内骨骼肌肌肉注射hGH基因,能获得较长期的表达.     

DNA重组hGH的生物测定方法的探讨：Ⅱ.胫骨法

本文对ＤＮＡ重组ｈＧＨ的另一生物测定方法即未成年去垂体大鼠胫骨骨骺软骨增宽法进行了探讨，摸索了胫骨染色切片后软骨宽度测量方法。实验结果表明在一定剂量范围内ｌｏｇ剂量与胫骨反应呈直线关系，胫骨法（λ＝０．２８）比体重法（λ＝０．４５）更精密。通过对多批进口和国产ｒｈＧＨ产品用胫骨法进行生物测定，得出本法具有灵敏、精密、成功率高等优点。     

Intermittent hyperinsulinaemia and arterial glycosaminoglycans in dogs

Summary The effect of insulin on the glycosaminoglycan content of the arterial ground substance was compared in age-matched alloxan-diabetic dogs, hypophysectomized dogs and normal controls. Hyaluronic acid and the three sulphated components, heparan, dermatan and isomeric chondroitin sulphates were quantitatively determined in the arch, thoracic and abdominal aorta, and in the carotid, coronary, iliac, renal and mesenteric arteries. Diabetic animals were either well or poorly controlled. Six well controlled dogs with a fasting mean plasma glucose level of 8.5 mmol/l were given around 30 units of porcine insulin/day and showed fluctuations in plasma insulin concentrations between 24 and 175 mU/l. Four poorly controlled dogs with a mean fasting plasma glucose level of 18.5 mmol/l received an average of 14 units of insulin/day, and the fluctuations ranged from 8 to 113 mU/l. Eight normal, untreated controls showed mean fluctuations between 15 and 22 mU/l. Within 14 weeks of insulin treatment, well controlled animals displayed glycosaminoglycan alterations in five arterial segments, usually involving more than one glycosaminoglycan constituent. In poorly controlled animals the number of segments that showed glycosaminoglycan alterations was the same, but abnormalities were limited to one of the sulphated components only. The coronary arteries displayed identical glycosaminoglycan alterations in the two groups of diabetic dogs, namely a significant rise in dermatan sulphate content (p < 0.01, mean±SEM) from the normal value of 1.12±0.03 mg/g dry defatted tissue to 1.31±0.03 mg/g in well controlled animals and to 1.37±0.08 mg/g in poorly controlled animals. In order to ascertain that the abnormalities in the glycosaminoglycan chemistry were related to hyperinsulinaemia rather than hyperglycaemia, six hypophysectomized dogs were treated with 1.5 U/day of porcine protamine zinc insulin, for 3 weeks. Average plasma glucose levels were in the order of 4.3 mmol/l and plasma insulin levels fluctuated between 9 and 27 mU/l; the latter represented twice the peak value seen in five untreated hypophysectomized dogs. The resulting chemical changes in the glycosaminoglycan content were in line with those encountered in diabetic animals, including the rise in dermatan sulphate content of the coronary arteries. These results indicate that: (1) hyperinsulinaemia produced by injections of insulin causes alterations of the arterial glycosaminoglycan content; (2) not all segments of the arterial tree are equally responsive to insulin and (3) the coronary arteries have a particularly insulin-sensitive dermatan sulphate metabolism.     

Hormonal regulation of renal multidrug resistance-associated proteins 3 and 4 (Mrp3 and Mrp4) in mice

Multidrug resistance-associated proteins 3 and 4 (Mrp3 and Mrp4) are expressed at much higher levels in female than male kidney. Sex steroids and sex-specific growth hormone (GH) secretion patterns often mediate gender-predominant gene expression. Thus, three models were used to investigate potential endocrine regulation of Mrp3 and Mrp4: (1) gonadectomized (GNX) mice with 17beta-estradiol (E2) or 5alpha-dihydroxytestosterone (DHT) replacement; (2) hypophysectomized (HPX) mice receiving E2, DHT, or simulated male-pattern (MP) or female-pattern (FP) GH secretion; (3) lit/lit mice, which have a spontaneous mutation in the growth-hormone releasing-hormone (GHRH) receptor, with simulated MP- or FP-GH secretion. GNX and HPX decreased Mrp3 mRNA levels compared with intact females. In both respective models E2 administration increased Mrp3 expression in GNX and HPX mice. DHT markedly repressed Mrp3 from GNX+placebo levels, however, this was not observed in the HPX model. In lit/lit mice, Mrp3 expression was lower than in wild-type controls, and MP-GH and FP-GH simulation slightly increased Mrp3 expression. Whereas GNX increased Mrp4 in males to female levels, HPX actually increased Mrp4 expression in both genders +375% and +66%, respectively. In both models DHT markedly repressed Mrp4. Furthermore, Mrp4 was higher in lit/lit than wild-type male mice, and simulation of MP-GH secretion suppressed female-predominant Mrp4 expression. In conclusion, these data indicate that E2 contributes to higher Mrp3 mRNA expression in females, yet a role for androgens in Mrp3 repression cannot be discounted. In contrast, Mrp4 mRNA is higher in females due to repression by both DHT and MP-GH secretion in males.     

Specificity of the direct effect of an LHRH agonist on testicular 17-hydroxylase but not on 5 alpha-reductase activity in hypophysectomized adult rats

The direct effect of treatment with a potent LHRH agonist on testicular steroidogenesis was studied by incubation of radioactive steroids with a testicular homogenate or with a suspension of interstitial cells obtained following 7 days of treatment of adult hypophysectomized male rats. The animals received [D-Ser(tBu)6,des-Gly-NH2(10)]LHRH ethylamide (25 micrograms) administered 3 times a day, hCG (5 or 25 IU) once daily or a combination of both drugs. The metabolism of tritiated progesterone into delta 4-metabolites by a suspension of interstitial cells was markedly reduced by treatment with the LHRH agonist (LHRH-A) alone or following combined treatment with hCG and LHRH-A. No formation of 5 alpha-reduced steroids was detected in the medium following incubation with testicular homogenate or interstitial cells. Similar findings were obtained by measurement of testicular steroid content. The present data demonstrate that the direct effect of the LHRH agonist is limited to the Leydig cells on 17-hydroxylase activity. This inhibitory effect is reflected by an accumulation of testicular pregnenolone and progesterone content and a marked inhibition of progesterone metabolism into delta 4-androgens. However, no stimulation of 5 alpha-reductase, an enzyme localized in seminiferous tubules, could be detected. Such data show clear differences between the direct and the pituitary-mediated effects of treatment with LHRH agonists on testicular steroidogenesis. While the LHRH agonist administered at high doses in the rat can directly inhibit 17-hydroxylase activity, the stimulatory effect on 5 alpha-reductase activity is regulated by another mechanism.     

Steroid and FSH action on LH receptors and LH-sensitive testicular responsiveness during sexual maturation of the rat.

This study examines the effect of FSH, testosterone and estradiol on testicular LH receptors and in vitro testicular responsiveness to LH in immature rats under various conditions. FSH treatment of 15-day-old immature rats significantly increased the number of LH receptors but did not alter testicular responsiveness. FSH treatment of hypophysectomized immature rats increased the number of LH receptors and markedly increased testicular responsiveness. Treatment of hypophysectomized rats with testosterone proprionate for 4 days, followed by a 5-day treatment with FSH, enhanced the effect of FSH on the number of LH receptors but did not increase the effect of FSH on testicular responsiveness. In contrast, treatment with estradiol for 4 days before FSH treatment had no effect on the FSH-induced increase in LH receptors but completely inhibited the FSH-induced increase in testicular responsiveness. These observations suggest that during male sexual maturation (1) regulation of LH receptors is distinct from regulation of testicular responsiveness to LH, (2) estradiol may be a factor in the regulation of testicular responsiveness to LH, and (3) testosterone may enhance the FSH-induced increase of LH receptors.     

Role of the hypophysis in the regulation of vitamin D metabolism

Hypophysectomy of animals given maintenance levels of vitamin D and adequate levels of dietary calcium and phosphorus brings about a marked reduction in plasma 1,25-dihydroxyvitamin D₃ levels and a significant elevation in plasma 24,25-dihydroxyvitamin D₃ levels. The hypophysectomy, as expected, results in reduced growth and lowered plasma levels of inorganic phosphorus. The injection of growth hormone markedly increases plasma levels of 1,25-dihydroxyvitamin D₃ in hypophysectomized animals while bringing about a reduction in 24,25dihydroxyvitamin D₃ levels. These results support the idea that the hypophysis plays a role in the regulation of vitamin D metabolism and that growth hormone either directly or indirectly is one of the hypophyseal factors bringing about this regulation.     

重组人生长激素体内生物活性测定中美药典方法对比研究

目的： 对重组人生长激素（rhGH）中国药典与美国药典的体内生物活性测定方法进行试验比较与梳理分析。方法： 按照《中国药典》2015年版四部通则1219和《美国药典》40版通则126的rhGH去垂体大鼠体内生物测定法,分别测定3批rhGH生物活性。结果与结论： 应用二种方法测定3批rhGH原料生物效价的结果相近。通过分析比较两种测定方法的结果及各个环节,可以得出结论,中国药典与美国药典的rhGH体内生物活性测定方法基本相同。