Size of rat spermatozoa during maturation along the epididymis.

An electrical sizing apparatus based on the Coulter Counter was used to measure rat spermatozoa from the proximal (caput) and distal (caudal) ends of the epididymis and from the ejaculate. The typical size distribution is unimodal with a positive skew, the crescent shape of the cells precluding absolute volume determination. During their passage through the epididymis, spermatozoa decrease in size as part of maturation. Saponin causes cell lysis and chymotrypsin cell shrinkage, both effects being more pronounced in the proximal region. It would seem that, during the maturation process within the epididymis, changes occur in the spermatozoon membrane that make the cells more stable.     

Modification of the rat sperm flagellar plasma membrane during maturation in the epididymis

Previously, we demonstrated that surface radiolabeling of rat epididymal spermatozoa by lactoperoxidase-catalyzed iodination reveals a major component with an apparent molecular weight of 26,000 to 28,000 daltons (26 kDa) on spermatozoa from the cauda but not the caput epididymidis. To characterize this surface component further, sperm surface constituents radiolabeled by lactoperoxidase-catalyzed iodination were separated by 2-D PAGE. The 26 kDa component was localized by autoradiography and appeared as the major labeled acidic spot on cauda spermatozoa, but neither a radiolabeled spot nor a corresponding stained spot was present on caput spermatozoa. The 26 kDa spot was excised from 2-D gels of plasma membranes from cauda spermatozoa and utilized for immunization. The monospecific antiserum stained a single band of 26 kDa on Western blots of SDS-PAGE-separated plasma membranes from cauda spermatozoa and in a 100,000 X g supernatant fluid of the luminal contents of the cauda epididymidis. Immunohistochemical staining of cauda spermatozoa revealed antigen exclusively on the flagellar domain; the antigen was not seen on caput spermatozoa but first appeared in spermatozoa from the proximal corpus epididymidis. Immunoelectron microscopy confirmed the 26 kDa component was localized to the external face of the flagellar plasma membrane. Immunohistochemical staining of caput spermatozoa incubated in vitro with cauda epididymal luminal fluid revealed the 26 kDa component specifically bound the flagellar domain of immature spermatozoa.     

Serotonin concentration, synthesis, cell origin, and targets in the rat caput epididymis during sexual maturation and variations associated with adult mating status: morphological and biochemical studies

The caput epididymis of some mammals contains large quantities of serotonin whose origin, targets, and physiological variations have been poorly studied. We combined morphological and biochemical techniques to begin approaching these aspects of serotonin in the rat caput epididymis. Serotonin immunostaining was detected in mast, epithelial, and neuroendocrine cells. Epithelial cells displayed immunoreactivity to 5HT(1A), 5HT(2A,) and 5HT(3) serotonin receptors. Endothelial and mast cells labeled positive for 5HT(1B) serotonin receptors and spermatozoa displayed 5HT(2A) and 5HT(3) serotonin receptor immunoreactivity. Epithelial, endothelial, and mast cells stained positive for serotonin transporters. Only epithelial cells showed tryptophan hydroxylase immunoreactivity; this enzyme catalyzes the limiting step in the serotonin synthetic pathway. In addition, Western blot analyses of caput homogenates documented the presence of 2 protein bands ( approximately 51 kd and approximately 48 kd) that were immunoreactive for tryptophan hydroxylase. Chromatographic analyses documented the presence of tryptophan hydroxylase in the caput, and showed that both its activity and serotonin availability increased with sexual maturation and decreased following p-chlorophenylalanine treatment, an inhibitor of tryptophan hydroxylase activity. Interestingly, serotonin concentration and tryptophan hydroxylase activity tended to be higher in breeding males than in those with no mating experience. We think that these results support the existence of a local serotoninergic system in the rat caput epididymis that might regulate some aspects of male reproductive function.     

Testosterone and dihydrotestosterone levels in the epididymis, vas deferens and preputial gland of mice during sexual maturation

The levels of testosterone (T) and dihydrotestosterone (DHT) in the epididymis, vas deferens and preputial gland were assessed in mice from 1 to 90 days. The weight increase of these 3 organs was proportionately greater than that of the whole body until 50 or 60 days, and they attained their adult histological appearance approximately 20 days prior to puberty. Expressed in ng/g, the concentration of androgens (T+DHT) in the epididymis (14.3 to 36.5), vas deferens (6.6 to 24.0) and preputial gland (1.5 to 4.7) were higher than in plasma (0.2 to 3.6 ng/ml). The concentration of either androgen varied little during sexual maturation and was not correlated with circulating levels. The highest concentration of androgen (T+DHT) was observed at birth suggesting that the neonatal period is crucial for development of the accessory sexual organs. In the epididymis and preputial gland T was the predominant androgen during the infantile phase of development, whilst DHT predominated thereafter. In the vas deferens concentrations of T were always equal to or higher than those of DHT. These results suggest that the ability of the accessory sexual organs to accumulate androgens appears to be more important than the circulating concentration of androgens in determining their growth and differentiation.     

Differences in nonhistone protein changes in rat ventral and dorsolateral prostate during sexual maturation

Age-related changes of chromosomal proteins in the dorsolateral and ventral prostates of rats from 6 to 31 weeks of age were studied by SDS-polyacrylamide gel electrophoresis. A nonhistone protein having a molecular weight of about 20,000 (20K-NHP), abundantly localized in the dorsolateral prostate, increased rapidly in content during the early stage of sexual maturation (6-11 weeks of age) in association with increases of serum testosterone concentration and prostatic tissue weight. Serum testosterone concentration decreased after week 11 and then remained constant until week 31. In contrast, the 20K-NHP content continued to increase after 11 weeks of age in the dorsolateral prostate, but not in the ventral prostate. The rapid increase of 20K-NHP in the dorsolateral prostate during the early stage of sexual maturation could not be attained in immature rats (5 weeks of age) by injection of excess amounts of androgens and/or prolactin for a week. But the 20K-NHP content in the ventral prostate of rats treated with testosterone propionate was almost the same as that of mature rats.     

Separation of phospholipase A2 in the testis and cauda epididymis of the adult rat by chromatofocusing

Phospholipase A2 (PLA2) activity was measured in the reproductive organs of adult male rats. Phosphatidylethanolamine and phosphatidylcholine labelled with 14C linoleic (lino-PE, lino-PC) and arachidonic acid (ara-PE, ara-PC) at the 2-position were used as substrates. Lino-PE was hydrolysed most strongly by homogenates of the distal cauda epididymis but the testis, vas deferens and caput and corpus epididymis also contained hydrolytic activity. Ara-PC and lino-PC were hydrolysed by homogenates of the cauda epididymis and testis. No hydrolysis of ara-PE was detected using homogenates of reproductive tissues. Chromatofocusing of testis homogenate resulted in the appearance of two active forms of PLA2 with different pl-values (6.5 and 5.6) when lino-PE was used as substrate. Maximum activities of both enzymes with 1 mM Ca2+ were observed at pH 9.5. These isoenzymes have marked differences in response to Cu2+, N-ethylmaleimide and p-bromophenacyl bromide (p-BPB). Cu2+ and N-ethylmaleimide had almost no effect on PLA2 activity with a pl value of 6.5, but inhibited the other isoenzyme strongly; the latter was almost more resistant to p-bromophenacyl bromide. Both enzymes hydrolysed lino-PE most strongly. Chromatofocusing of an homogenate of cauda epididymis also revealed two isoenzymes of PLA2 with different pl-values (6.0 and 5.0). The latter form was resistant to p-bromophenacyl bromide but was more sensitive to Triton X-100 and sodium deoxycholate than was other isoenzyme. The pH optimum of the isoenzyme with a pl value of 6.0 ranged from 6.25 to 8.75 whilst the other isoenzyme was most active at pH 8.0-8.75.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of gossypol on the concentration of sodium and potassium in the rat epididymis

The effects of administration of gossypol acetic acid (7.5 mg/kg daily for 4 weeks) on the concentration of Na+ and K+ in the rat epididymis was assessed. Epididymal fluid samples, collected by micropuncture, from the caput, corpus, proximal cauda and distal cauda epididymis from gossypol-treated and control animals were analysed for Na+ and K+ concentrations. Gossypol-treated males failed to impregnate healthy females, presumably because their sperm were immotile. In gossypol-treated rats, Na+ levels decreased significantly (P less than 0.01) in the caput, corpus, proximal and distal cauda epididymis. In contrast, the K+ concentration was increased significantly (P less than 0.05) only in the caput and corpus epididymis. This altered electrolyte milieu may be responsible, to some extent, for immotility and hence infertility.     

S-adenosyl-L-methionine decarboxylase activity in the rat epididymis: ontogeny and androgenic control

The authors describe the occurrence of high levels of S-adenosyl-L-methionine decarboxylase (SAMDC) activity in the rat epididymis, and its ontogeny and androgenic control. As early as 15 days of age, SAMDC activity exists, although a peak of activity is observed at 25 days. Bilateral orchidectomy resulted in a decline of epididymal SAMDC activity. However, an androgen-independent fraction, accounting for 34% of total activity, appears to exist in the epididymis. In 45-day-old orchidectomized rats, SAMDC activity was stimulated by testosterone treatment in a dose-dependent manner. However, treatment of 45-day-old intact animals with a high dose of the androgen failed to modify SAMDC activity, indicating that, at this age, the enzyme is maximally stimulated by endogenous androgens. The observed effect of testosterone on castrated rats was completely abolished by concomitant treatment with the antiandrogen flutamide. This compound was ineffective on the androgen-insensitive fraction. To assess the contribution of circulating and luminal androgens to the maintenance of epididymal SAMDC, rats were unilaterally orchidectomized and activity was determined in both epididymides after 7 days. The SAMDC activity was identical in epididymides from both sides, suggesting circulating androgens suffice to maintain normal levels of activity. It was concluded that androgens regulate epididymal SAMDC activity, although an androgen-independent fraction appears to exist.     

Seminal emission by electrical stimulation of the spermatic nerve and epididymis

The spermatic nerve and epididymis were stimulated electrically in dogs to elucidate the possibility of artificial seminal emission after bilateral transection of the hypogastric nerves and sympathetic trunks. Before transection, electrical stimulation of a distal end of the severed spermatic nerve caused a trace amount of emission in two dogs and no emission in the remaining four. In contrast, 1 month after the transection, stimulation of a distal end of the severed spermatic nerve caused seminal emission in all six dogs examined, with full seminal volume in four dogs and partial volume in the remaining two. Anatomically, sympathetic nerves originating from the upper portion of the lumbar sympathetic ganglia descended along the spermatic arteries to the testes as spermatic nerves. The present results indicate that spermatic nerves have the potential to generate seminal emission as a compensatory pathway after bilateral transection of the hypogastric nerves. Both direct and percutaneous electrical stimulation of epididymal tails resulted in a full volume of seminal emission in all dogs with transection of both hypogastric nerves and lumbosacral sympathetic trunks as well as in unoperated controls, while high voltage (8 V vs 40-80 V) was required to cause seminal emission by electrical stimulation on the skin surface. Direct stimulation of epididymal tails in men undergoing orchidectomy as treatment for prostatic carcinoma or during biopsy of the contralateral testis in a patient with a testicular tumour, resulted in seminal emission in all five epididymides examined either from the end of the severed vas deferens or in the posterior urethra if the vas deferens was not severed.     

热休克蛋白70-2基因沉默对大鼠精子附睾成熟的影响

目的 观察热休克蛋白(HSP)70-2基因沉默对大鼠附睾中精子成熟的影响.方法 构建HSP70-2特异的短发夹RNA(shRNA)质粒载体,注入大鼠附睾中,1周后处死大鼠,无菌切取附睾,用逆转录-聚合酶链反应(RT-PCR)方法 检测附睾中HSP70-2 mRNA表达,Western blot观察HSP70-2蛋白表达,应用苏木素-伊红(HE)染色检测附睾管内精子密度.结果 空白对照组HSP70-2蛋白表达量为1.31±0.10,附睾管内成熟精子密度为(63.46±14.43)%.基因沉默组HSP70-2蛋白表达量为0.93±0.13,附睾管内成熟精子密度为(31.51±10.67)%,与对照组比较均显著减少,差异有统计学意义(P＜0.01).结论 HSP70-2基因沉默影响附睾中精子成熟.     

犬马尾神经受压后腰骶髓中一氧化氮合酶活性与神经细胞凋亡的相关性

目的：研究马尾神经受压后一氧化氮合酶（NOS）活性变化与腰骶髓神经细胞凋亡的关系。方法：将27只健康家犬随机分为3组：A组21只，水囊置于椎管内并注水加压制成马尾神经压迫模型（马尾神经压迫组）；B组3只，置入水囊但未注水加压（假手术组）；C组3只（正常组）。A组再分为7组，即马尾神经持续受压4h、8h、12h、24h、48h、72h和168h组（n=3）。检测各组腰骶髓组织中的NOS活性变化，用TUNEL法标记凋亡神经元和神经胶质细胞，光镜（HE染色）和透射电镜观察细胞形态学改变。结果：B组和C组腰骶髓神经细胞形态未见异常，A组光镜及电镜观察结果均提示神经细胞发生凋亡。B组和C组未见凋亡神经元，A组于压迫后12h可见凋亡神经元，24～48h神经元凋亡最多。B组与C组腰骶髓组织中NOS活性比较无显著性差异（P〉0．05），A组于压迫12h后，NOS活性即明显增高，与B组和C组比较有显著性差异（P〈0．05），24～48h达到高峰，72h后开始下降。结论：犬马尾神经受压后腰骶髓组织中NOS活性增高，与相应神经元凋亡存在正相关。     

Effect of androgens on the activity of acid hydrolases in rat epididymis

The enzymatic activity of 6 acid hydrolases was studied in rat epididymal homogenates following castration, testosterone replacement and during postnatal growth. Acid phosphatase and N-acetyl-β-D-glucosaminidase activity decreased after castration and increased with hormonal treatment as well as during growth. β-Glucuronidase and cathepsin D activity increased during the involution of the organ and decreased or did not change with hormone treatment or during sexual maturation. Arylsulphatase and deoxyribonuclease did not recover normal activity after hormonal treatment. Their activities were particularly high in epididymal and rete testis fluid of normal animals.     

The development of vascular supply of normal rat prostate during the sexual maturation: an angiographic study.

Vascular distribution of small blood vessels and capillaries using India ink angiography was studied in normal rat prostate from puberty up to full sexual maturation. The study included both macroscopic observation of the lobular vascular irrigation and the histological assessment of the periacinar capillary network. The topographical distribution of the main prostate branches was found to be different among the rat prostate lobes. The ventral lobe seems to be better irrigated than the dorsal one. The former being supplied by two parallel vascular systems, one irrigating the median two-thirds of the ventral lobe, whereas the remaining external one-third was found to be conjointly irrigated from the pericapsular branches of the fat pads. The blood vessels of the dorsal and lateral lobes emerged radially from a periurethral circle, with the dorsal branches ending blindly in the connective tissue of the pelvic cavity, whereas the lateral prostate was also conjointly irrigated by a dual vascularization from the pericapsular fat pad and the periurethral circle branch. The histological study revealed quantitative differences between the periacinar capillaries of both ventral and dorsal lobes. The capillary density was found to be age dependent and directly proportional to the acinar size. Small acini were less well irrigated by capillaries than were the larger ones. The number of capillaries per acinar area increased progressively toward the maturation (day 90); thereafter, their number remained constant. Both prostate lobes showed identical patterns. The heterogeneous vascular networks among the rat prostatic lobes might offer an additional clue for their distinctive morphophysiological characteristics, which most probably play a role in various pathogenetic processes.     

Implantation of cauda equina nerve roots through a biodegradable scaffold at the conus medullaris in rat

Background contextTraumatic injuries occurring at the conus medullaris of the spinal cord cause permanent damage both to the central nervous system and to the cauda equina nerve roots.PurposeThis proof-of-concept study was to determine whether implanting the nerve roots into a biodegradable scaffold would improve regeneration after injury.MethodsAll experimental works involving rats were performed according to the approved guidelines by the Mayo Clinic Institutional Animal Care and Use Committee. Surgical procedures were performed on 32 Sprague-Dawley rats. Four ventral cauda equina nerve roots were reimplanted either directly into the ventral cord stump or through a poly(lactic-co-glycolic acid) (PLGA) scaffold. These experimental groups were compared with a control group in which the nerves were inserted into a muscle fascia barrier that was placed between the spinal cord and the nerve roots. Animals were sacrificed at 4 weeks.ResultsThere was no difference in motor neuron counts in the spinal cord rostral to the injury in all treatment groups, implying equal potential for the regeneration into implanted nerve roots. One-way analysis of variance testing, with Tukey post hoc test, showed a statistically significant improvement in axon regeneration through the injury in the PLGA scaffold treatment group compared with the control (p<.05, scaffold n=11, control n=11).ConclusionsThis pilot study demonstrated that a PLGA scaffold improved regeneration of axons into peripheral nerve roots. However, the number of regenerating axons observed was limited and did not lead to functional recovery. Future experiments will employ a different scaffold material and possible growth factors or enzymes to increase axon populations.     

Leptin secretion from the epididymal fat pad is increased by the sexual maturation of the male rat

Although leptin has been implicated as an important factor in triggering the onset of puberty in females, much less is known about the role of this adipose tissue hormone in the sexual maturation of males. Previous work in the rat has suggested that the peripubertal rise in testosterone precedes an increase in leptin secretion, and it has been suggested that the testosterone rise induces the leptin increase. These studies examined some of the interactions between leptin secretion and the peripubertal testosterone rise in male rats. Serum leptin concentrations were significantly elevated in young adult male rats compared with immature rats. Cultured epididymal fat pads obtained from adult animals secreted significantly more leptin than did those obtained from immature rats. Castration of immature rats with or without testosterone replacement for 1 week did not result in a significant change in either the serum leptin concentrations or the ability of the epididymal fat pad to secrete leptin. Exposure of epididymal fat to 5 ng/mL of testosterone in vitro resulted in a significantly enhanced secretion of leptin into the media compared with plain media controls. These results confirmed that there is an increase in serum leptin concentrations with sexual maturation in the male rat. They also suggest that this increase is due to an enhanced ability of adipose tissue to secrete leptin. Within a normal physiologic range, testosterone may play a role in inducing this increased ability to secrete leptin.     

Temporal relation between leptin and various indices of sexual maturation in the male rat.

Recent studies in humans and rhesus monkeys have suggested the possibility that the adipose tissue hormone leptin has a stimulatory and/or permissive effect on the onset of puberty in the male. We evaluated this hypothesis by measuring leptin in groups of male rats between the ages of 26 days and 96 days. A statistically significant positive correlation was present between serum leptin and age, body weight, prostate, seminal vesicle, and testes weight (both absolute and as a function of body weight). A statistically significant negative correlation was present between leptin and serum FSH and alpha-inhibin. There was not a statistically significant correlation between leptin and testosterone or LH. There was a statistically significant increase in the serum leptin concentrations at day 47. This rise was coincident with the peripubertal growth spurt in the secondary sexual organs and the peripubertal testosterone rise but occurred after the prepubertal rise in testicular weight, the appearance of elongating spermatids in the testes, and the start of the decline in FSH. In animals in which the peripubertal testosterone rise was delayed by the administration of EDS, serum leptin showed statistically significant differences from control. These data do not support the hypothesis that leptin provides a trigger for the onset of puberty in the male rat. They do suggest that leptin may be involved in the secondary sexual organ growth spurt and are consistent with the hypothesis that testosterone stimulates leptin synthesis during puberty.     

Strain and excursion in the rat sciatic nerve during a modified straight leg raise are altered after traumatic nerve injury.

PURPOSE: This study investigated the biomechanics of the sciatic nerve with hind limb positioning in live and euthanized Sprague-Dawley rats after traumatic nerve injury. METHODS: With radiographic analysis, sciatic nerve excursion and strain were measured in situ during a modified straight leg raise, which included sequential hip flexion and ankle dorsiflexion. Comparisons were made between nerves in uninjured, sham-injured and mild crush-injured rats at the 7-day and 21-day recovery times. RESULTS: Significant strain and proximal excursion of the sciatic nerve were observed in all groups during hip flexion, and additional increased strain was noted during dorsiflexion. Seven days after nerve injury, strain increased significantly during hip flexion (17.64+/-14.12%; p=0.0091) and dorsiflexion (22.56+/-15.47%; p=0.0082) compared to the sham-injured controls. At 21 days after injury, the strains were similar between the injured and sham-injured groups. CONCLUSIONS: Nerve bed elongation during straight leg raise causes sciatic nerve strain and excursion towards the moving joint with the greatest movement nearest the moving joint. In the first week after injury, the maximal strain exceeded the level previously shown to impair nerve conduction and circulation.     

Gamma-glutamyl transpeptidase activity in the epididymis during fetal and postnatal development in rats.

The histochemical and biochemical distributions of gamma-glutamyl transpeptidase (gamma-GT) were investigated in the epididymis of rats during fetal and postnatal development. In the epididymal homogenates, gamma-GT activity was detected on the fifth day after birth. A sharp increase was observed after 30 days of life in the caput homogenates. Moderate levels of the enzyme were found in the cauda epididymis. Gamma-GT is histochemically detected from the 15th day of gestation in Wolffian ducts and in 17- to 18-day-old fetuses in newly differentiated epididymal tubules. Enzyme activity, was associated with the plasma membranes (apical, lateral, and basal), was preponderant on the apical part of the epithelial cells. During the first 15 days of the postnatal life, the histochemical reaction intensities were identical from the caput to the cauda epididymidis. From the 18th day onwards, enzyme activity decreased in the corpus and in the cauda, while gamma-GT increased in the caput epididymidis, and a strong activity was found on the apical surface of epithelial cells. Weak or moderate gamma-GT activity of spermatozoa in the caput tubules, increasing steadily from caput to cauda epididymidis, suggests that gamma-GT may be related to the functional maturation of spermatozoa.     

Ontogeny of the secretory pattern of LH and FSH in male mice during sexual maturation

Plasma LH and FSH concentrations were measured in male mice at 10-day intervals from 1 to 90 days and at 2-day intervals between 20 and 40 days. The skewed distribution and variability of LH concentrations observed in mice aged 20 to 90 days suggests that LH is released in an episodic fashion. Mean levels of LH and baseline concentrations increased significantly from infantile (1–20 days) to adult age (50–90 days). Pulsatile discharges of LH appeared to start at 22 days, and their frequency increased from 9.6% (prepubertal stage: 20–30 days) to 31.6% (pubertal stage: 30–40 days). In contrast, for FSH no evidence of pulsatile secretion was found, and mean levels increased 5-fold from the infantile to the peripubertal stage, and adult levels were then attained.