Hormonal regulation of spermatogenesis

Proper functioning of the mammalian testis is dependent upon an array of hormonal messengers acting through endocrine, paracrine, and autocrine pathways. Within the testis, the primary messengers are the gonadotrophins, follicle stimulating hormone and luteinizing hormone, and the androgens. Abundant evidence indicates that the role of the gonadotrophins is to maintain proper functioning of testicular somatic cells. It is the androgens, primarily testosterone, which act through the somatic cells to regulate germ cell differentiation. Despite extensive research in this area, little is known about the cell-specific requirements for androgens and even less is understood about the downstream effectors of androgen signalling. However, recent work using cell-specific ablation of androgen receptor function has demonstrated a clear requirement for androgen signalling at multiple, discrete time points during spermatogenesis. These models also provide useful tools for identifying the targets of androgen receptor activity. The purpose of this review is to provide a brief overview of recent advances in our understanding of hormonal regulation of spermatogenesis, with an emphasis on the role of testosterone within the testis, and to pose important questions for future research in this field.     

精子发生的激素调节

长期以来一致公认 ,垂体促性腺激素——卵泡刺激素 ( FSH)和黄体生成素 ( L H)所介导的睾酮 ( T)是所有哺乳动物和人类精子发生的主要调节物。然而 ,近年来大量的动物实验和一些临床研究结果对上述观点提出了至少两方面的挑战。一方面 ,睾丸产生的相当数量的雌激素也是精子发生和成熟的必须激素。因为敲除雌激素两种受体基因 ( ERαKO、ERβKO)或敲除芳香化酶基因 ( Ar KO)的雄性小白鼠均出现多种生殖功能障碍或不育。临床上也出现了因 ERα基因突变而不育的个别报道 ;另一方面 ,发现了至少已有 5例因 FSH受体 ( FSHR)基因或FSH…     

Mechanism of protection of rat spermatogenesis by hormonal pretreatment: stimulation of spermatogonial differentiation after irradiation

Pretreatment of rats with hormones that suppress testosterone levels and sperm production enhances the recovery of spermatogenesis from stem cells after a cytotoxic insult. It is not known whether the enhanced recovery results from an increase in the numbers of surviving stem cells or whether their ability to differentiate is enhanced. In this study, untreated rats and rats pretreated with testosterone plus estradiol-17beta (T + E) were irradiated with 3.5 or 6 Gy, and the recovery of spermatogenesis from surviving stem cells was assessed at 6, 10, and 20 weeks after irradiation. T + E pretreatment did not significantly affect the numbers of A spermatogonia remaining in the tubules at 6 weeks after irradiation. In rats that were given irradiation alone, spermatogenesis steadily declined after 6 weeks because the stem cells lost their ability to differentiate. However, when rats were treated with T + E before irradiation, this decline was prevented, and in fact, at least at the lower dose of radiation, there was a progressive recovery of spermatogenesis. Given the similar spermatogonial counts at 6 weeks after irradiation in the irradiated-only and T + E-treated, irradiated rats, the hormone treatment appears not to protect stem cells from being killed by the cytotoxic agent. Rather, the later enhancement of spermatogenic recovery results from prevention of an injury-induced change in spermatogonia or in their environment, which would have otherwise resulted in failure of spermatogonial differentiation.     

Paracrine factors and the regulation of spermatogenesis

Summary Main problem: Although the gonadotropins and testosterone are required for normal spermatogenesis, it is believed that local control factors regulate spermatogenesis. For many years these regulatory factors had not been identified. Over the past five years, a number of growth factors have been identified in testis or isolated testicular cell types or secretions. Growth factors are key regulatory molecules which affect cell proliferation, meiosis, and differentiated function. These factors usually act in an autocrine (acting upon the cell which secreted it) or paracine (affecting another cell) manner and thus are involved in intercellular communications. Methods: Growth factor secretion by testicular cell types or testis tissue has been analyzed using a variety of assays measuring cell proliferation in vitro, as well as assays using immunocytochemicals. Growth factor gene expression in testis has been analyzed by Northern blot analysis and in situ hybridization, which gives information concerning the stage and cell specific expression of the gene. Inbred strains of mice with mutations or deletions in a growth factor gene has been used to suggest the function of two specific factors in testicular development and growth. Results: Among the growth factors expressed or secreted by testicular cell types, most are common to some other cell types in the body, such as transforming growth factors alpha and beta, epidermal growth factor, fibroblast-like growth factors, insulin-like growth factors, interleukins, endorphins, inhibin and activin, while others may be more testis specific such as mullerian inhibiting substance (anti-mullerian hormone) and Sertoli cell secreted growth factor. A variety of proto-oncogenes are expressed at discrete stages of spermatogenesis, as well as by the somatic cells of the testis. Many of these encode growth factors, receptors or other proteins involved in signal transduction. Conclusion: With the exception of the kit ligand and the c-kit proto-oncogene, which have been demonstrated to play a role in the survival of the primordial germ cell in the testis during embryogenesis, little is known of the direct role of the other growth factors in spermatogenesis. It is likely that in the near future that the function of many of these proteins in the regulation of spermatogenesis will be identified. Eventually, this information will be used to develop specific therapies and diagnostic procedures for the infertile male.Supported in part by NIH grant DK37919 (to D.J.L.) and a grant from the Methodist Hospital (to D.J.L.) The senior author (C.S.N.) is an American Foundation to Urologic Diseases Scholar and a Serono/American Fertility Society Fellow     

The renal nerves in the newborn rat

Immunocytochemical methods were used to investigate the distribution of afferent [calcitonin gene-related peptide-(CGRP) immunoreactive and substance P-immunoreactive] nerves and efferent (neuropeptide Y-immunoreactive and dopamine -hydroxylase-immunoreactive) nerves in the kidneys of rats within the 1st day of life. The newborn rat kidney possesses an afferent and efferent innervation. Both afferent and efferent nerves reach the kidney in the same bundles. The afferent sensory fibers predominate overwhelmingly in the renal pelvis and ureter while the efferent fibers clearly predominate in the vasculature. The corticomedullary connective tissue contains both types of innervation with a more prominent afferent innervation (CGRP immunoreactive). Only afferent arterioles of perihilar nephrons were innervated by efferent sympathetic fibers. The distribution and extent of afferent and efferent innervation is consistent with the renal nerves playing a significant role in the transition from fetal to newborn life. The close proximity between afferent and efferent fibers suggests a possible interaction between the two systems.     

Possible involvement of proteases in the regulation of spermatogenesis

Summary.  Proteolytic enzymes, which are synthesized and secreted by cells of the seminiferous tubule of the testis, have important functions in spermatogenesis. We performed metabolic studies using small peptide hormones as a substrate to investigate the activity of proteases in cultured Sertoli cells of the rat. High-performance liquid chromatographic analysis of the cell culture supernatants showed cleavage of met- and leu-enkephalin, substance P, and bradykinin. No peptidolysis was observed for the cyclic peptide oxytocin. The hormone cleavage pattern and the use of specific protease inhibitors in peptide degradation experiments demonstrated activities of several proteases in Sertoli cells. These are mainly metalloproteinases including neutral metalloendo-peptidases, angiotensin-converting enzyme and aminopeptidases. In addition, activities of serine and aspartic proteases were detected. Only marginal proteolytic activities were observed in Sertoli cell conditioned supernatants, indicating that the investigated proteases are mainly located on Sertoli cell membranes. The peptide hormones used in this study have been found to play a potential role in the endocrine, paracrine or autocrine regulation of testicular cells. The membrane-associated proteases reported here may therefore be involved in the metabolism and inactivation of these peptides.     

磷脂酰乙醇胺结合蛋白在精子发生过程中的特异表达

目的运用蛋白质组学方法，寻找大鼠精子发生相关蛋白。方法用重力沉降法（STAPUT法）从9d龄雄性大鼠睾丸中分离出A型精原细胞，从成年的雄性大鼠睾丸中分离出粗线期精母细胞、圆形精子细胞。分别提取这3种细胞的总蛋白，进行双向电泳。对所得到的双向电泳图谱进行分析，找出差异蛋白。对挑选出的差异蛋白做质谱分析。结果发现磷脂酰乙醇胺结合蛋白（RKIP）在这3种不同细胞的表达上有明显差异。在双向电泳图谱中，RKIP在圆形精子细胞中表达量较高，并且形成分子量相同但等电点不同的并排4个蛋白质点；而在A型精原细胞和粗线期精母细胞的双向电泳图中，RKIP仅为一个单独的蛋白质点。结论RKIP在精子发生过程中有明显的差异表达，可能对精子发生起重要调节作用。     

Predictors for partial suppression of spermatogenesis of hormonal male contraception

Aim： To analyze factors influencing the efficacy of hormonal suppression of spermatogenesis for male contraception. Methods： A nested case-control study was conducted, involving 43 subjects, who did not achieve azoospermia or severe oligozoospermia when given monthly injections of 500 mg testosterone undecanoate （TU）, defined as partial suppressors compared with 855 subjects who had suppressed spermatogenesis （complete suppressors）. Sperm density, serum testosterone, luteinizing hormone （LH） and follicle stimulating hormone （FSH） concentrations at the baseline and the suppression phase were compared between partial and complete suppressors. Polymorphisms of androgen receptor （AR） and three single nucleotide variants and their haplotypes of FSH receptor （FSHR） genes determined by polymerase chain reaction （PCR） and DNA sequencing technique were compared between 29 partial and 34 complete suppressors. Results： Baseline serum LH level was higher and serum LH as well as FSH level during the suppression phase was less suppressed in partial suppressors. Additionally, in a logistic regression analysis larger testis volume, higher serum FSH concentrations alone, or interaction of serum LH, FSH, testosterone and sperm concentrations were associated with degree of suppression. The distribution of polymorphisms of AR or FSH receptor genes did not differ between partial and complete suppressors. In cases with incomplete FSH suppression （FSH 〉 0.2 IU/L）, the chances of reaching azoospermia were 1.5 times higher in the subjects with more than 22 CAG triplet repeats. Conclusion： Partial suppression of spermatogenesis induced by 500 mg TU monthly injections is weakly influenced by hormonal and clinical features but not polymorphism in AR and FSHR genes.     

The role of C-type natriuretic peptide in rat testes during spermatogenesis

C-type natriuretic peptide (CNP) is a 22-amino acid peptide and act as a local paracrine or autocrine regulator. There is growing evidence that CNP is involved in male reproductive processes. To investigate the role of CNP during spermatogenesis, we measured the mRNA expression of CNP and its specific membrane-bound natriuretic peptide receptor-B (NPR-B) using real-time RT-PCR in the testes of normal rats on different postnatal days. After that spermatogenesis dysfunction model induced by ornidazole was established with the aim to study the correlation of CNP with spermatogenic dysfunction. Then, Sertoli cells from 18- to 22-day-old healthy male rats were cultured in the presence of different CNP concentrations (1×10(-6), 1×10(-7) and 1×10(-8) mol l(-1)), and the mRNA expression levels of androgen-binding protein, inhibin B and transferrin were examined at 0 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h. During the postnatal development of rat testes, the highest mRNA expression levels of CNP and NPR-B were found at postnatal D(0), and the levels then declined gradually, with a second CNP peak at postnatal D(35). In the ornidazole-induced infertile rat testes, CNP gene expression was lower than in the uninduced rats (P<0.05), while NPR-B gene expression was greater (P<0.05). In cultured Sertoli cells, supplementation with CNP stimulated the gene expression of androgen-binding protein/inhibin B/transferrin, particularly at 12 h, and 1×10(-7) mol l(-1) CNP had the highest upregulation effect. The gene expression levels of CNP/NPR-B in rat testes at different postnatal stages and in infertile rat testes indicated that CNP may participate in the physiology and/or pathology related to spermatogenesis. Moreover, CNP regulated endocrine function in Sertoli cells. Taken together, these results showed that CNP is closely tied to spermatogenesis.     

The effects of diabetes on male fertility and epigenetic regulation during spermatogenesis

The effects of diabetes mellitus include long-term damages, dysfunctions, and failures of various organs. An important complication of diabetes is the disturbance in the male reproductive system. Glucose metabolism is an important event in spermatogenesis. Moreover, glucose metabolism is also important for maintaining basic cell activity, as well as specific functions, such as motility and fertilization ability in mature sperm. Diabetic disease and experimentally induced diabetes both demonstrated that either type 1 diabetes or type 2 diabetes could have detrimental effects on male fertility, especially on sperm quality, such as sperm motility, sperm DNA integrity, and ingredients of seminal plasma. Epigenetic modifications are essential during spermatogenesis. The epigenetic regulation represents chromatin modifications including DNA methylation, histone modifications, remodeling of nucleosomes and the higher-order chromatin reorganization and noncoding RNAs. If spermatogenesis is affected during the critical developmental window, embryonic gonadal development, and germline differentiation, environmentally-induced epigenetic modifications may become permanent in the germ line epigenome and have a potential impact on subsequent generations through epigenetic transgenerational inheritance. Diabetes may influence the epigenetic modification during sperm spermatogenesis and that these epigenetic dysregulation may be inherited through the male germ line and passed onto more than one generation, which in turn may increase the risk of diabetes in offspring.     

Effects of gestational hyperglycemia on glucose metabolism and its hormonal control in the fasted, newborn rat during the early postnatal period

To evaluate the effects of gestational hyperglycemia on glucose metabolism and its regulation in the fasted rat during the early postnatal period, unrestrained rats were continuously infused with glucose during the last week of pregnancy. Control rats were infused with distilled water. Newborns were studied during the first six postnatal hours. At birth, newborns from glucose-infused rats, compared with controls, showed higher plasma glucose levels, increased plasma insulin, and lower plasma glucagon and catecholamine concentrations. Between birth and 2 h postpartum, newborn rats from both groups exhibited a marked hypoglycemia, which was, however, more severe in newborns from glucose-infused rats (15 mg/dl) than in controls (26 mg/dl). During the first four postnatal hours, plasma insulin concentration remained higher, while plasma glucagon and catecholamine concentrations remained lower in newborns from hyperglycemic rats. At 6 h, the glycemia reached normal values and the concentrations of the different hormones were similar in controls and newborns from glucose-infused mothers. Concurrently, in the newborns from glucose-infused rats, hepatic glucose production was altered, as they were unable to mobilize liver glycogen stores during the six postnatal hours. Despite slightly delayed phosphoenolpyruvate carboxykinase induction, the rate of gluconeogenesis from 10 mmol/L lactate estimated on isolated hepatocytes was higher in newborns from hyperglycemic mothers than in controls. These results show that gestational hyperglycemia compromises the metabolic and hormonal adaptation of the newborn rat to early extrauterine life; the striking feature of these neonates is the absence of mobilization of liver glycogen stores, which can probably be explained by fetal and neonatal hyperinsulinism associated with the defect of counterregulatory hormones.     

The role of adenosine in the regulation of coronary blood flow in newborn lambs

Adenosine is a metabolic vasodilator of the coronary vessels in the adult. Whether it plays a similar role in the regulation of coronary blood flow (CBF) in the newborn is not known. We evaluated changes in adenosine release during periods of decreased oxygen supply (hypoxia) and increased oxygen demand (dobutamine infusions). In anesthetized open-chest lambs (age 1 to 8 days), aortic and coronary sinus adenosine concentrations, circumflex CBF, and myocardial oxygen consumption (MVO2) were measured. Adenosine was assayed by high-performance liquid chromatography, and the release of adenosine was calculated as the product of the aortic-coronary sinus plasma level difference and CBF in milliliters per minute per 100 gm myocardial tissue. Control values were obtained when the lambs were ventilated with 60% oxygen. In the first series of experiments, hypoxemia resulted in an increase in CBF from 120 +/- 5 to 171 +/- 8 ml/min/100 gm (p less than 0.01). This was associated with sixfold increase in adenosine release. In a second set of experiments the intravenous infusion of dobutamine resulted in parallel increases in MVO2 and CBF. Concomitantly, adenosine release increased by fivefold. There were significant linear relationships between MVO2 and CBF (r = 0.96; p less than 0.01), MVO2 and adenosine release (r = 0.69; p less than 0.002), and adenosine release and CBF (r = 0.71; p less than 0.002). These data support the hypothesis that adenosine may play an important role in the regulation of CBF in the newborn lamb.     

The effects of caffeine on the maxillary composition in the newborn rat

Summary The possible influence of caffeine on maxillary structure was studied. Seventeen pregnant rats at days 9 of gestation were randomly divided into two groups. The dams of group 1 received a 20% protein diet ad libitum throughout the experimental period. The dams of group 2 were pair-fed, with group 1, a 20% protein diet supplemented with 2 mg/100 g body weight (B.W.) caffeine. At birth, pups were mixed within the same group and 8 randomly selected pups were assigned to each dam and continuously fed the respective diet. On day 22, 11 male pups from the control and 12 males from the caffeine group were randomly selected, separated from the dams, and continued to be fed their respective diets. On day 44, a rubber elastic band was inserted between the first and second maxillary right molars. The size of the elastic band was increased throughout the next 5 days. Animals were sacrificed at day 49 and the composition of the maxillas was analyzed. After pulverization, organic and inorganic contents of the bones were measured. Zinc (Zn) and hydroxyproline concentration of the caffeine group showed a significant decrease when compared with those of the controls. However, Ca, P, Mg, and hexosamine concentration showed no difference between the groups. The interdental space measured occlusally and laterally with the visual method, and occlusally in histological sections showed no significant difference between the control and caffeine groups, although variation of the space in the caffeine group was less than in the control group. The present study suggests that caffeine intake during the gestational and lactational period by their dams and the growing period of pups affect the maxillary composition of their offspring.     

大鼠精子发生过程中热休克蛋白70-2特异表达的研究

目的运用蛋白质组学方法,寻找大鼠精子发生相关蛋白质,找到目标蛋白质后,进一步用免疫组织化学方法做定位研究。方法用重力沉降法(STAPUT法)从9 d龄雄性大鼠睾丸中分离出A型精原细胞,从成年的雄性大鼠睾丸中分离出粗线期精母细胞、圆形精子细胞。分别提取这3种细胞的总蛋白质,进行双向电泳。对所得双向电泳凝胶扫描,分析并找出差异蛋白质。对挑选出的差异蛋白质做质谱分析,发现在这3种不同细胞的表达上有明显差异的蛋白。进一步做睾丸免疫组织化学组织定位、定量研究。结果双向电泳结果显示,HSP70-2在粗线期精母细胞、圆形精子细胞中表达量较高, 在A型精原细胞则表达量极少。HSP70—2在粗线期精母细胞中表达量最大。用HSP70—2抗体对大鼠睾丸组织切片行免疫组织化学研究,发现粗线期精母细胞、Sertoli细胞、Leydig细胞有阳性颗粒反应。HSP70—2主要表达于细胞质。结论 HSP70—2在精子发生过程中有明显的差异表达,推测其对精子发生起重要调节作用。     

Effect of epidermal growth factor on spermatogenesis in the cryptorchid rat

PURPOSE: Epidermal growth factor (EGF) is secreted mainly from the submandibular glands. Submandibular gland ablation causes a marked decrease in male fertility, which suggests that EGF influences spermatogenesis. We investigated the effect of EGF in combination with orchiopexy on cryptorchid rat testes in which tubular deterioration had become partially irreversible. MATERIALS AND METHODS: Unilaterally cryptorchid rats were obtained by daily administration of 7.5 mg flutamide (Nihonkayaku, Tokyo, Japan), an androgen receptor antagonist, to pregnant rats. At age 10 weeks the unilaterally cryptorchid rats underwent orchiopexy with or without EGF administered into the cryptorchid testis. EGF solution (10 microg/ml) was delivered into the seminiferous tubules by retrograde perfusion through the rete testis. At 14 days testicular recovery was assessed based on the maturity of spermatogenesis using a modified Johnsen score and from the number of apoptotic germ cells per seminiferous tubule. RESULTS: Mean Johnsen score +/- SEM was significantly higher in the orchiopexy with EGF than in the orchiopexy without EGF group (7.85 +/- 0.12 vs 7.12 +/- 0.13, p <0.001). The number of apoptotic germ cells tended to be smaller in the orchiopexy with EGF group than in the orchiopexy without EGF group (0.16 +/- 0.05 vs 0.28 +/- 0.08). CONCLUSIONS: Although orchiopexy for cryptorchidism partly improved spermatogenesis, recovery was limited. EGF administered in combination with orchiopexy was more effective for spermatogenesis than orchiopexy alone. This may be applicable in patients with cryptorchidism.     

提睾肌在无张力疝修补术后对大鼠生精功能的保护意义

【摘要】 目的 探究提睾肌在无张力疝修补术后对大鼠生精功能的保护意义。方法〓选择成年SD大鼠建立实验动物模型,分正常对照组(NC组)、假手术组(Sham组)、手术组,手术组又分为OP1组和OP2组,前者保留提睾肌的完整性后者完全切断提睾肌。于术后90天分别将各组大鼠术侧睾丸组织HE染色观察病理变化,流式细胞仪测定各级生精细胞数量变化。结果〓OP1组生精过程轻度异常,生发上皮紊乱,生精细胞层数减少,可见大量的精子细胞和精子,各级生精细胞与Sham组相比差异无统计学意义。OP2组大鼠睾丸呈退行性变化,生发上皮紊乱,生精细胞层数减少,单倍体细胞及二倍体细胞数量减少,与OP1组及Sham组比较P<0.05。结论〓提睾肌对雄性大鼠的生精功能具有保护作用,男性腹股沟疝患者行无张力修补术无张力修补术时,应尽可能保护提睾肌的连续性和完整性。     

The effects of duodenal-jejunal exclusion on hormonal regulation of glucose metabolism in Goto-Kakizaki rats

BACKGROUND: The antidiabetic effect of bariatric surgery has been interpreted as a conceivable result of surgically induced weight loss and decreased caloric intake. However, glycemic control often occurs within days, before significant weight loss has been reached. The aim of our work was to investigate the hormones that control glycemic status in diabetes mellitus after a duodenal-jejunal exclusion in an animal model of nonobese type 2 diabetes. METHODS: Twelve (12- to 14-week-old) rats (Goto-Kakizaki) randomly underwent one of the following procedures: gastrojejunal bypass (group 1, n = 6) or no intervention (controls) (group 2, n = 6). Both groups were fed with the same type and amount of diet. At basal time (preoperative) and after intervention (1 week and 1 month), weight and fasting glycemia were measured. An oral glucose tolerance test (OGTT) was realized at same times. Hormone levels (insulin, glucagons-like peptide 1 [GLP-1], glucose-dependent insulinotropic peptide [GIP], glucagon, and leptin) were measured after 20 minutes of oral glucose overload. Age-matched Goto-Kakizaki rats were used as controls for all variables. RESULTS: Rats in group 1 and group 2 remained with the same weight during the protocol. The OGTT showed an improvement in glycemic levels in group 1; glucose levels were better at 1 week and 1 month after the surgery in all times of OGTT (basal, 10 minutes, and 120 minutes). Basal glucose levels at time 0 in basal time, at 1 week, and at 1 month were lower in group 1 than group 2. Postoral glucose overload levels of glucagon, insulin, GLP-1, and GIP remained unchanged during the treatment in both groups. In group 1, leptin levels had a significant decrease at 1 week and 1 month after surgery (basal time (6.1 +/- 1.6 ng/mL) versus 1 week (0.9 +/- 0.9 ng/mL) versus 1 month (0.7 +/- 0.6 ng/mL) (P < .05). CONCLUSION: Gastrojejunal bypass in a nonobese diabetic model improves glycemic control with a significant decrease in leptin levels, without changes in enteroinsular axis (GLP-1, GIP, glucagons, and insulin levels).     

Effect of Cd transferred via food product on spermatogenesis in the rat

The aim of this work was to assess the effect of organic Cd on the pituitary-testicular axis in rats given diet containing Cd incorporated in radish bulb. A control group was assigned a diet containing ordinary radish for 12 week and second received for the same period a diet containing Cd-contaminated radish at the rate of 20 + 2 μg g⁻¹ of dry weight. At the end of treatments, the rats were anaesthetized, blood samples were collected and epididymides were removed for establishing sperm count. Circulating FSH, LH and testosterone levels were determined by electrochemiluminescence using automate (Elecsys 2110; Rochediagnostics). A decrease in FSH levels was observed in Cd-exposed animals. This seems to be at the origin of the large drop in the number of spermatozoa. Concerning the plasma testosterone levels we observed a significant increase in contaminated animals. Surprisingly, LH does not exhibit any variation, leading to the conclusion that the feedback control between testosterone and LH was disrupted by the use of Cd. In conclusion, our data indicate that the hypothalamic-pituitary axis is the principal target of Cd toxicity.