Long‐term feeding of hydroalcoholic extract powder of Lepidium meyenii (maca) enhances the steroidogenic ability of Leydig cells to alleviate its decline with ageing in male rats

This study examined whether feeding hydroalcoholic extract of Lepidium meyenii (maca) to 8‐week‐old (sexually maturing) or 18‐week‐old (mature) male rats for more than a half year affects serum testosterone concentration and testosterone production by Leydig cells cultured with hCG, 22R‐hydroxycholesterol or pregnenolone. Testosterone concentration was determined in the serum samples obtained before and 6, 12, 18 and 24 weeks after the feeding, and it was significantly increased only at the 6 weeks in the group fed with the maca extract to maturing rats when it was compared with controls. Testosterone production by Leydig cells significantly increased when cultured with hCG by feeding the maca extract to maturing rats for 27 weeks (35 weeks of age) and when cultured with 22R‐hydroxycholesterol by feeding it to mature rats for 30 weeks (48 weeks of age). Overall testosterone production by cultured Leydig cells decreased to about a half from 35 to 48 weeks of age. These results suggest that feeding the maca extract for a long time to male rats may enhance the steroidogenic ability of Leydig cells to alleviate its decline with ageing, whereas it may cause only a transient increase in blood testosterone concentration in sexually maturing male rats.     

Effect of two different extracts of red maca in male rats with testosterone-induced prostatic hyperplasia

Aim： To determine the effect of two different extracts of red maca in male rats. Methods： Prostatic hyperplasia was induced in male rats with testosterone enanthate （TE）. The study comprised six groups： one control group （group 1）, one group treated with TE （group 2）, two groups treated with TE and aqueous extract of red maca （groups 3 and 4）, one group treated with hydroalcoholic extract of red maca （group 5） and one group treated with finasteride （0.1 mg, group 6）. Differences in the aqueous extract dependent on the length of time of boiling, whether for 2 or 3 hours, for groups 3 and 4 was assessed. Extracts of red maca contained 0.1 mg of benzylglucosinolate. Thereafter, a doseresponse effect of different doses of benzylglucosinolates （0.02-0.08 mg） in red maca extracts was assessed. Results： Prostate weight was similar in rats treated with freeze-dried aqueous extract of red maca prepared after 2 and 3 hours of boiling. Freeze-dried aqueous extract of red maca, hydroalcoholic extract of red maca and finasteride reduced prostate weight in rats with prostatic hyperplasia. No difference was observed between the data obtained from aqueous extract or hydroalcoholic extract of red maca. A dose dependent reduction of prostate weight was observed with the increase of the dose of benzylglucosinolates in red maca extracts. Conclusion： The present study showed that hydroalcoholic or aqueous extract of red maca containing 0.1 mg of benzylglucosinolate can reduce prostate size in male rats in which prostatic hyperplasia had been induced by TE.     

淫羊藿总黄酮对雄性大鼠生殖功能影响的初步研究

目的 研究淫羊藿总黄酮对雄性大鼠生殖内分泌功能的影响。方法 (1)观察淫羊藿总黄酮对未成年大鼠生殖器官重量的影响。淫羊藿总黄酮经灌胃给药7天后。腺垂体、睾丸、附睾及精囊腺称重；(2)观察相同剂量的淫羊藿总黄酮对不同年龄大鼠睾酮、雌二醇和黄体生成素的影响。睾酮、雌二醇和黄体生成素水平用放免法测定；(3)观察淫羊藿总黄酮在大鼠睾丸间质细胞体外培养中对睾酮的影响。结果 淫羊藿总黄酮可增加未成年大鼠腺垂体、附睾及精囊腺重量。提高睾酮、雌二醇和黄体生成素水平。能明显促进离体大鼠间质细胞睾酮基础分泌。结论研究表明。淫羊藿总黄酮对雄性生殖系统和生殖内分泌的功能具有促进作用。     

Atrazine effects on testosterone levels and androgen-dependent reproductive organs in peripubertal male rats

Previous studies have reported that atrazine, a widely used herbicide that selectively inhibits photosynthesis in broadleaf and grassy weeds, has adverse effects on reproductive function in the male, suggesting a direct effect of atrazine on the hypothalamicpituitary-testicular axis. As yet, however, no studies have critically examined the doses of atrazine that elicit such effects, and few have focused on the mechanism by which atrazine acts. Herein we report a dose-response study of the effects of atrazine ingestion on reproductive function in male Sprague-Dawley rats during a critical developmental period, the peripubertal period. Atrazine was administered by gavage to rats from day 22 to day 47 of age, at doses of 1-200 mg/kg body weight per day. Atrazine administration of up to 50 mg/kg per day had no effect on any of the measured variables. Serum testosterone concentration was reduced by atrazine at doses of 100 and 200 mg/kg per day, as were seminal vesicle and ventral prostate weights. Intratesticular testosterone concentration was reduced in parallel with serum testosterone, suggesting that the reductions in serum testosterone resulted from reduced testosterone production by Leydig cells or from changes in testosterone metabolism within the testis, or both. Serum luteinizing hormone (LH) concentration was reduced despite the reduced serum testosterone, suggesting an effect on the hypothalamus, the pituitary gland, or both. At the termination of the study, the average body weight of rats receiving atrazine at 100 mg/kg per day was found to be reduced by approximately 9%. This suggested the possibility that the effects of atrazine on the reproductive tract may not be direct, but rather, the noted deficits of the male reproductive tract resulted from reduced food intake by the treated rats. We tested this by feeding control (vehicle-gavaged) rats amounts of food equivalent to that consumed by the atrazine-fed rats, and then assessing reproductive tract endpoints. Even mild food restriction resulted in reductions in serum testosterone concentration, in the weights of androgen-dependent organs, and in serum LH concentration; the same deficits that were seen in atrazine-gavaged rats. Indeed, the effects of atrazine on the male reproductive tract seen in rats receiving atrazine at greater than 50 mg/kg per day could not be distinguished from the effects of reduced food consumption. These results suggest that caution must be exercised before concluding that atrazine (or any potentially toxic chemical) has direct, detrimental effects.     

Protective effects of Eugenia jambolana extract versus N‐acetyl cysteine against cisplatin‐induced damage in rat testis

To assess the protective effects of Eugenia jambolana extract (EJE) or N‐acetyl cysteine (NAC) on testis, cisplatin (CIS, 5 mg kg^?1 bw, single dose) was administered either alone or along with EJE (25 mg kg^?1 bw, alternate day) or NAC (150 mg kg^?1 bw, Day 1 and 4) for 7 days. Significant alterations in serum LH, FSH and testosterone were observed in CIS group which were effectively modulated by EJE or NAC supplementation. Upregulation of 3β‐HSD gene indicated the rise in functional Leydig cells. This was further confirmed from the identical improvement in hCG‐stimulated testosterone production in isolated Leydig cells. Reduction in oxidative stress was associated with restoration of total antioxidant capacity and glutathione levels, and activation of antioxidant enzymes, SOD, catalase, glutathione s‐transferase (GST) and glutathione reductase (GR). CIS‐induced apoptosis of germ and Leydig cells was contained by both NAC and EJE intervention by effective modulation of apoptotic markers in the extrinsic, intrinsic and other pathways of metazoan apoptosis. Taken together, the study findings establish the potential of EJE as a therapeutically better antioxidant than NAC for use in curtailing the adverse effects of anticancer drugs on testicular function.     

Development of Diabetic-Infertility induced by Streptozotocin in the Male Rat

Diabetes was induced in male rats by streptozotocin (60 mg/kg BW). Rats were sacrificed at 0, 1, 2, 3, 4, 5, 6, 8, 11 and 14 weeks after induction of diabetes. On autopsy the prostate, coagulating glands, seminal vesicles, epididymides and testes were isolated and weighed. Sperm isolated from the epididymis were characterized. The endocrine function of the testes was evaluated by counting number of Leydig cells per testes, testicular and serum testosterone and the testosterone production by the isolated Leydig cells.
The following changes were found; testicular and serum testosterone decreased significantly starting the 2nd week of the diabetes, the atrophy of the accessory glands began shortly after diabetic induction and reached maximal values after 8 weeks, testicular weight began to decrease from the 8th week of the diabetic induction as well as the Leydig cells, the function of the epididymis was depressed as reflected in its weight and sperm quality.
It can be concluded that the axis LH – Leydig cells is much more sensitive than FSH – tubuli to the block of LHRH secretion following streptozotocin induced diabetes.     

Effect of Typha capensis (Rohrb.)N.E.Br. rhizome extract F1 fraction on cell viability,apoptosis induction and testosterone production in TM3‐Leydig cells

Typha capensis (Rohrb.)N.E.Br. (bulrush) is used by traditional healers in Southern Africa to treat male reproductive problems. This study aimed at investigating the effects of T. capensis on TM3‐Leydig cells. T. capensis rhizome crude extract obtained from autumn, winter, spring and summer harvest was fractionated using HPLC into four fractions, and TM3‐Leydig cells were incubated with different concentrations of the F1 fraction (0.01, 0.02, 0.1, 1, 10 and 100 μg/ml) for 24, 48 and 96 hr respectively. The following parameters were evaluated: cell morphology, viability (MTT assay), testosterone production (testosterone ELISA test), apoptosis (Annexin V‐Cy3 binding) and DNA fragmentation (TUNEL assay). Results revealed that the summer harvest obtained the highest amount of extract. The F1 fraction of all harvests was the most effective. This fraction significantly enhanced testosterone production in TM3 cells in a dose‐dependent manner with maximum effect at 0.1 μg/ml. At higher concentrations, lower testosterone production was observed. Cell viability including apoptosis was not affected at concentrations used by the traditional healers to treat patients. This study shows that T. capensis enhanced testosterone production and might be useful to treat male infertility and ageing male problems.     

Prolactin and Leydig cells: biphasic effects of prolactin on LH-, T3- and GH-induced testosterone/oestradiol secretion by Leydig cells in pubertal rats

The effect of rat prolactin (rPRL) on basal and LH-, GH- and T3-mediated testosterone and oestradiol secretion was studied in pubertal rat Leydig cells. Purified Leydig cells were cultured for 24 h at 37 degrees C in a medium containing 4% foetal calf serum (FCS). The medium was then replaced with fresh medium containing different concentrations of rPRL (5-400 ng/mL) for 48 h at 34 degrees C without FCS. rPRL increased testosterone secretion by Leydig cells at doses of 50-400 ng and maximum stimulation was observed at a dose of 200 ng. Oestradiol secretion was parallel to that of testosterone except at low doses (5-50 ng/mL). To assess the modulatory effect of rPRL on LH-, GH- and T3-induced Leydig cell testosterone and oestradiol secretion, minimum (50 ng) and maximum (200 ng) effective doses of rPRL were co-administered with LH (25/100 ng), GH (10/50 ng) and T3 (25/50 ng). Co-administration of rPRL (50/100 ng) with T3 (25/50 ng) decreased testosterone secretion. While co-administration of T3 (25 ng) decreased rPRL-induced oestradiol secretion, the latter was unaltered at a dose of 50 ng T3. A minimum effective dose of rPRL (50 ng) plus LH (25 ng) stimulated both testosterone and oestradiol secretion. While a maximum effective dose of rPRL (200 ng) did not alter LH (25 ng)-induced testosterone and oestradiol secretion, it inhibited testosterone secretion induced by 100 ng LH and increased oestradiol secretion. Both doses of rPRL (50, 200 ng) plus GH (10/50 ng) inhibited testosterone secretion when compared with testosterone secretion induced by either GH or PRL alone and stimulated oestradiol secretion. The present in vitro study indicates that rPRL stimulates both testosterone and oestradiol secretion by Leydig cells and that this effect can be modulated by LH, GH and T3.     

Effects of flavonoids from Semen Cuscutae on the reproductive system in male rats

Aim: To evaluate the effects of the flavonoids extracted from the Semen Cuscutae (FSC) on the reproductive and endocrine functions in male rats. Methods: (1) FSC were obtained from the semen of Cuscuta sinensis l_;am through solvent extraction and polyamide columnar chromatography; (2) Effect of FSC on the reproductive organs was assessed in immature rats. Rats were administered FSC through gastric garage at a dose of 300 mg/kg per day for 7 days and the weights of testis, epididymis, seminal vesicle and pituitary gland were then observed; (3) To observe the effect of FSC on the reproductive endocrine function: same dose level of FSC was given to male rats of different age groups for 7days; on day 8, the plasma testosterone (T), estradiol (E2) and LH were determined by RIA, the specific binding of LH was estimated and the testes were weighed. (4) Effect of FSC on LH secretion was assessed in vitro on cultured adenohypophysis. (5) Effect of FSC on T secretion was assessed in vitro on Leydig cell culture. Results: FSC increased the weights of testis, epididymis and pituitary gland, and stimulated T and LH secretion both in vitro and in immature rats. Conclusion: FSC invigorates the reproductive system and reproductive endocrine function in male rats.     

Effects of 3-methyl-4-nitrophenol in diesel exhaust particles on the regulation of testicular function in immature male rats

We investigated the effects of 3-methyl-4-nitrophenol (4-nitro-m-cresol, PNMC) isolated from diesel exhaust particles (DEP) on the reproductive functions of male rats. Twenty-eight-day-old rats were injected subcutaneously with PNMC (1, 10, or 100 mg/kg) daily for 5 days. The weights of the epididymis, seminal vesicle, and Cowper gland were significantly decreased in rats treated with 10 mg/kg PNMC. The plasma concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were significantly increased by PNMC at 100 mg/kg. However, the plasma concentrations of testosterone and immunoreactive (ir)-inhibin were significantly decreased by PNMC at 100 mg/kg. The testosterone content of the testicles was significantly decreased in the group treated with 100 mg/kg PNMC compared with the control group. Furthermore, testicular concentration of ir-inhibin was significantly decreased by PNMC at 1 mg/kg or 100 mg/kg. To investigate the direct effects of PNMC on the secretion of LH and FSH from the anterior pituitary gland, and on the secretion of testosterone from the testes, we exposed cultured anterior pituitary and interstitial Leydig cells to PNMC (10(-6), 10(-5), 10(-4) M) with or without gonadotropin-releasing hormone (GnRH; 10 nM) (for the LH and FSH tests) and human chorionic gonadotropin (hCG; 0.1 IU/mL) (for the testosterone test) for 24 hours. PNMC did not change either the basal or GnRH-stimulated levels of FSH and LH secretion. However, PNMC significantly inhibited both basal and hCG-stimulated testosterone production. These findings suggest that PNMC has a direct effect on the testes of immature male rats, causing a reduction in testosterone secretion.     

Protective effects of the hydroalcoholic extract of Fumaria parviflora on testicular injury induced by torsion/detorsion in adult rats

This study was designed to determine the effects of daily oral administration (250 mg/kg) of the hydroalcoholic extract of Fumaria parviflora (FP) for 14 days on the sperm parameters, oxidative stress parameters, serum testosterone levels, expression of Bax and Bcl‐2 genes, and apoptosis index of germ cells after testicular torsion–detorsion (ischaemia–reperfusion, IR) injury model in rats. Twenty‐eight adult male Wistar rats were divided randomly into four groups of seven each: sham operation, torsion–detorsion (TD), TD plus the hydroalcoholic extract FP (TDFP) and only FP without TD application (FP). Testicular torsion was created by rotating the left testis 720° in a counterclockwise direction; then, after 4 hr, detorsion was performed. The Johnson's score, mean seminiferous tubule diameter (MSTD) and height (thickness) of seminiferous tubule epithelium (HST) were significantly increased in TDFP and FP groups as compared to TD group. The gene expression of Bcl‐2, level of serum testosterone hormone and antioxidant parameters—GPx and SOD—were significantly higher in TDFP and FP groups than TD group. The index of apoptosis, the gene expression of Bax and the level of MDA were significantly higher in TD group than TDFP and FP groups. Therefore, F. parviflora could decrease oxidative stress induced by testicular torsion–detorsion.     

Cell-cell interactions in the testis of adjuvant-induced arthritic rat

Rats with adjuvant-induced arthritis (AA) have low levels of serum testosterone, and production of testosterone reportedly is influenced by macrophage secretory products. This study was undertaken to understand the mechanism mediating this hypoandrogenism. Testicular macrophages from AA and nonarthritic (NA)rats were cultured, and conditioned media was added to testicular interstitial cells and Percoll-purified cells from NA rats. Testosterone production by interstitial cells stimulated with luteinizing hormone (LH) and incubated with adjuvant-induced arthritic macrophage conditioned medium (AAMCM) was significantly lower than in cells incubated with nonarthritic macrophage conditioned medium (NAMCM). However, there was no difference in testosterone production by Percoll-purified Leydig cells and those stimulated with LH when incubated with AAMCM or NAMCM. To determine whether an intermediary cell type was involved in mediating inhibition of testosterone production, AAMCM and NAMCM were added to a reconstituted preparation of testicular interstitial cells. Addition of AAMCM restored the inhibitory effect, suggesting that arthritic hypoandrogenism is mediated by cell-cell interaction. These results suggest that a factor produced by macrophages from AA rats appears to mediate testosterone production by acting in conjunction with other cells in the testicular interstitium.     

Effect of STS-557 (17α-cyanomethyl 17β-hydroxy-estra-4, 9(10)-dien-3-one) on blood hormone levels, the testis, accessory sex organs and fertility of rats

Treatment with STS-557 (17 alpha-cyanomethyl 17 beta-hydroxy-estra-4,9(10)-dien-3-one; 10 mg kg-1 daily s.c.) for 4 weeks induced atrophy of the seminiferous tubules in adult rats with a reduction in tubule diameter and in the number of round spermatids at stage VII. Elongated spermatids were not detected. Leydig cells were atrophied from the second week of treatment with a concomitant decrease in blood levels of testosterone. The blood levels of FSH and LH were reduced from the third week of treatment. The weight of the reproductive organs was reduced after STS-557 treatment. The treatment induced sterility in 50% of rats after 2 weeks of treatment but after 4 weeks none of the treated males mated. Normal fertility and normal levels of testosterone and FSH were restored after 6 weeks and LH after 4 weeks of withdrawal of treatment. All other parameters studied recovered to pretreatment levels 6 weeks after withdrawal of treatment. STS-557 could act on the pituitary-gonadal axis (reducing gonadotrophin secretion) as well as directly affecting the Leydig cells. The consequent reduction in the blood levels of testosterone in combination with reduced gonadotrophins was presumably responsible for the suppression of spermatogenesis.     

PADAM动物模型进行同种异体睾丸间质细胞移植的研究

目的老年SD大鼠进行同种异体睾丸间质细胞移植探讨中老年男性雄激素部分缺乏症(PADAM)动物模型的可行性和疗效。方法选10只符合PADAM模型标准的老年SD大鼠作为受体，用成年SD大鼠的睾丸进行体外分离和培养，将获得的高纯度和高活力的睾丸间质细胞移植到老年SD大鼠的大腿内侧肌群内，定期检查其移植前后血清睾酮和游离睾酮的水平变化，并观察移植部位的情况。结果未应用免疫抑制剂，移植后的睾丸间质细胞保持良好的分泌功能，老年SD大鼠的血清睾酮和游离睾酮水平均显著升高，并大约于移植后的7～12d开始稳定在一定的水平，可持续27d以上。移植部位未见异常。结论睾丸问质细胞同种异体移植安全、有效、无明显排斥反应。     

银杏叶提取物对2型糖尿病大鼠睾丸间质细胞雄激素合成的影响

目的:研究银杏叶提取物(EGB)对2型糖尿病大鼠睾丸间质细胞雄激素合成的影响。方法:雄性SD大鼠30只,随机均分成3组:正常对照组、2型糖尿病组、EGB治疗组。用光镜和透射电镜观察EGB对2型糖尿病大鼠睾丸的形态学改变;ELISA法检测血清LH、T水平;半定量RT-PCR检测睾丸间质细胞类固醇激素合成急性调节蛋白(StAR)、细胞色素P450侧链裂解酶(P450scc)、细胞色素P45017a-羟化酶(P450c17)、3β-羟基类固醇脱氢酶Ⅰ型(3β-HSD1)、17β-羟基类固醇脱氢酶Ⅲ型(17β-HSD3)的mRNA水平。结果:糖尿病组光镜下睾丸间质细胞较正常对照组缩小;透射电镜下见到间质细胞核固缩,胞质内内质网减少。糖尿病组血清LH及T水平显著低于正常对照组(P<0.05);睾丸组织P450scc的mRNA水平显著低于正常对照组(P<0.01),StAR、17β-HSD3及3β-HSD1的mRNA水平也明显低于正常对照组(P<0.05),P450c17基因表达呈下降趋势(P>0.05);EGB治疗12周与糖尿病组比较睾丸组织病理改变较轻,血清LH、T含量升高(P<0.05),StAR、P450scc的mRNA水平升高(P<0.05),P450c17、17β-HSD3及3β-HSD1的mRNA水平呈上升趋势(P>0.05)。结论:EGB能减轻糖尿病大鼠睾丸损害并使2型糖尿病大鼠睾丸间质细胞合成和分泌T能力增强。     

Changes in binding of iodomelatonin to membranes of Leydig cells and steroidogenesis after prolonged in vitro exposure to melatonin

The aim of the present study was to investigate the effects of prolonged exposure to melatonin (MLT) on the binding of iodomelatonin to membranes of rat Leydig cells and the subsequent modulation of testosterone and cyclic adenocine monophosphate (cAMP) secretion from these cells by MLT itself. Leydig cells were Percoll-purified from adult rats and cultured in vitro with MLT (1--100 nmol/L) for 16 h. Binding assays with 2(125I)iodomelatonin were then performed; moreover, testosterone and cAMP secretion during an acute challenge with lutenizing hormone (LH) (20 mIU/mL for 3 h) was assayed by RIA. As a result of prolonged MLT administration, a decrease in maximum binding density (Bmax) and equilibrium dissociation constant (Kd) of the binding of 2(125I)iodomelatonin to purified cell membranes was noted. Higher testosterone and cAMP secretion during LH challenge were recorded in cells pre-incubated with MLT; notwithstanding, the inhibitory effect of acutely administered MLT on LH-challenged secretions was not only retained but also reinforced, as the IC50 was 30% lower in cells pre-treated with the higher concentration of MLT (100 nM). Cycloheximide administration (10 microg/mL for 16 h) did not prevent hyper-sensitization to LH challenge or to acute MLT administration on LH challenge. Pertussis toxin (180 ng/mL for 16 h) prevented hyper-sensitization to LH, but not to acutely administered MLT. Forskolin (10 nmol/L) administration abolished either phenomena. In conclusion, prolonged exposure to MLT modulates the secretion of testosterone by cultured rat Leydig cells. Although MLT receptors were reduced, hyper-sensitization to LH challenge and to acutely administered MLT on LH challenge were observed with the higher concentration of MLT. Reduction in intracellular cAMP as a result of prolonged administration of MLT, could be the primary cause of both phenomena. On the one hand, reduced cAMP could start re-arrangement of the G-proteins and thus LH-dependent adenylate cyclase sensitization. On the other hand, reduced cAMP could render the Leydig cells more responsive to MLT itself through a mechanism which does not involve G-protein re-arrangement.     

Decreased steroidogenesis and cAMP production in vitro by leydig cells isolated from rats made hypothyroid during adulthood

The Leydig cell function of adult male rats made hypothyroid with 6-propyl-2-thiouracil (6-PT, 0.1% w/v in drinking water for 1 month) was studied and compared with that of age-matched controls. After 6-PT treatment, a slight, non-significant decrease in serum testosterone was observed, but no changes in testis weight or number of Leydig cells were noted. The in vitro function of Leydig cells was therefore investigated during incubation for 3 h in the presence or absence of several stimuli: LH (30 mIU/mL), forskolin (FK 1 microM), isobutylmethylxanthine (IBMX, 100 microM), GnRH (100 nM) or FK 1 microM + IBMX 100 microM. Irrespective of the stimulus, cells from hypothyroid rats secreted less cyclic AMP, 17-hydroxyprogesterone, androstenedione and testosterone. No differences in LH receptors were noted between the groups. Prolonged incubation with triiodothyronine (5-250 ng/mL) or thyroxine (5-250 ng/mL) for 3, 16, 24 or 48 h did not affect testosterone secretion in either group; however, administration of IGF-I (8 ng/mL for 24 h) resulted in increased spontaneous and stimulated testosterone production in both groups. However, when hypothyroid animals were supplemented in vivo with thyroxine a full recovery of Leydig cell function in vitro was noted. In conclusion: (1) Leydig cells from rats made hypothyroid during adulthood produce less testosterone in vitro, both spontaneously and in response to cAMP and non-cAMP-mediated stimuli; (2) this is due to a reduction in cAMP production and in the activity of the enzymes in the androgen biosynthetic pathway, and not to changes in LH receptors; (3) direct administration of thyroid hormones did not improve testosterone secretion in either group, while incubation with IGF-I did.