Sitthichai Iamsaard

Induction of the Acrosome Reaction in Black Tiger Shrimp (Penaeus monodon) Requires Sperm Trypsin-Like Enzyme Activity

Abstract Trypsin-like enzymes in egg water (EW), a natural acrosome reaction (AR) inducer, are known for their importance in shrimp AR. In this report, we describe a unique phenomenon of the AR of black tiger shrimp (Penaeus monodon) sperm. It was completed within 45–60 sec and comprised only the acrosomal exocytosis and depolymerization of the sperm head anterior spike. We used peptidyl fluorogenic substrates to show the presence of trypsin-like enzymes in P. monodon EW and sperm, but minimal activities of chymotrypsin-like enzymes. In sperm, these trypsin-like enzymes existed both on the sperm surface and in the acrosome. The acrosomal enzyme was revealed as a 45-kDa band by fluorogenic substrate in-gel zymography. Although EW possessed high trypsin-like enzyme activities, they were not essential for the AR induction; EW pretreated with an irreversible trypsin inhibitor, or heat-inactivated EW (HI-EW), to abolish the trypsin-like activities could still induce the AR. The HI-EW-induced AR was inhibited by the presence of a membrane impermeant soybean trypsin inhibitor (SBTI) in the sperm suspension, indicating the significance of sperm-borne trypsin-like enzymes (on the surface and/or in the acrosome) in this AR process. However, pretreatment of sperm with SBTI followed by its removal from the suspension still allowed the AR to occur within 5 min of sperm exposure to HI-EW. Since trypsin-like activity of the SBTI-pretreated sperm surface at 5 min after SBTI removal was at the minimal level, our results suggest the importance of the acrosomal trypsin-like enzyme in the AR process.

Sperm surface arylsulfatase A can disperse the cumulus matrix of cumulus oocyte complexes

Cumulus cell layers of expanded cumulus oocyte complexes (COCs) are interlinked with networks of hyaluronic acid, chondroitin sulfate B proteoglycans and link proteins, and they can be dispersed by sperm surface hyaluronidases. In this report, we showed that arylsulfatase A (AS‐A), existing on the sperm head surface, also had this dispersion action. Purified AS‐A free of protease, hyaluronidase and chondroitinase activities could disperse the cumulus matrix of expanded COCs. However, this COC dispersion action was not associated with AS‐A desulfation activity, assayed by using p‐nitrocatecholsulfate (artificial substrate). COCs incubated for 1 h with sperm pretreated with anti‐AS‐A IgG in the presence of apigenin (a hyaluronidase inhibitor) did not exhibit matrix dispersion, whereas several cumulus layers were already dispersed in COCs incubated with sperm pretreated with preimmune IgG. Furthermore, sperm from AS‐A null mice showed a significant delay in COC dispersion, compared with wild‐type sperm. Within 1 h of sperm‐COC co‐incubation, the size of COCs incubated with AS‐A null sperm was 65% of the original dimension, whereas that of COCs inseminated with wild‐type sperm was only 17%. A further delay in COC dispersion by AS‐A(−/−) mouse sperm was observed when apigenin was present in the co‐incubation. We also showed for the first time that AS‐A had a specific affinity for chondroitin sulfate B, a component of cumulus matrix proteoglycan networks; this might provide a mechanism of cumulus matrix destabilization induced by sperm surface AS‐A. J. Cell. Physiol. 213: 201–211, 2007.

Moringa oleifera extract enhances sexual performance in stressed rats

Aphrodisiacs are required to improve male sexual function under stressful conditions. Due to the effects of oxidative stress and dopamine on male sexual function, we hypothesized that Moringa oleifera leaves might improve male sexual dysfunction induced by stress. Therefore, the effects on various factors playing important roles in male sexual behavior, such as antioxidant effects, the suppression of monoamine and phosphodiesterase type 5 (PDE-5) activities, serum testosterone and corticosterone levels, and histomorphological changes in the testes, of a hydroethanolic extract of M. oleifera leaves were investigated. Various doses of extract including 10, 50, and 250 mg/kg body weight (BW) were given orally to male Wistar rats before exposure to 12 h-immobilization stress for 7 d. The results demonstrated that the extract showed both antioxidant and monoamine oxidase type B (MAO-B) suppression activities. At 7 d of treatment, the low dose of extract improved sexual performance in stress-exposed rats by decreasing intromission latency and increasing intromission frequency. It also suppressed PDE-5 activity, decreased serum corticosterone level, but increased serum testosterone, numbers of interstitial cells of Leydig and spermatozoa. The increased numbers of interstitial cells of Leydig and spermatozoa might have been due to the antioxidant effect of the extract. The increased sexual performance during the intromission phase might have been due to the suppression of MAO-B and PDE-5 activities and increased testosterone. Therefore, M. oleifera is a potential aphrodisiac, but further research concerning the precise underlying mechanisms is still needed.摘要目的探讨辣木叶提取物对改善应激条件下雄性性功能障碍的作用。创新点根据氧化应激和多巴胺对雄性性功能的影响, 推测具有抗氧化和单胺调节功能的辣木叶能改善应激条件下的雄性性功能障碍。方法本文以雄性Wistar大鼠作为实验对象, 通过对影响雄性性功能的各种因素进行测量及统计学分析, 包括抗氧化性、 B型单胺氧化酶(MAO-B)和5型磷酸二酯酶(PDE-5)活性、 血清睾酮和皮质甾酮水平、 睾丸内组织形态学变化。结论研究结果表明, 辣木叶提取物作为一种抗氧化剂具有抑制MAO-B和PDE-5活性的作用, 同时能降低血清皮质酮水平, 提高血清睾酮水平, 增加睾丸间质细胞和精子的数量。 睾丸间质细胞和精子的数量的增加可能是由于辣木叶提取物的抗氧化作用, 插入阶段性功能增强可能是由于MAO-B和PDE-5活性的抑制和睾酮水平的增加。 因此, 辣木可以作为一种潜在的壮阳药。

Phenolic contents and antioxidant capacities of Thai-Makham Pom (Phyllanthus emblica L.) aqueous extracts *

概要通过对余甘子不同部位(叶、 枝和树皮)水提物的总酚含量的测定及体外抗氧化活性的研究, 为其体内及临床试验研究奠定了基础。 余甘子叶、 枝和树皮的水提物皆具有抗氧化活性, 其中树皮中的总酚含量最高, 抗氧化活性最强。

Anethum graveolens Linn. (dill) extract enhances the mounting frequency and level of testicular tyrosine protein phosphorylation in rats

ObjectiveTo investigate the effect of Anethum graveolens (AG) extracts on the mounting frequency, histology of testis and epididymis, and sperm physiology.MethodsMale rats induced by cold immobilization before treating with vehicle or AG extracts [50, 150, and 450 mg/kg body weight (BW)] via gastric tube for consecutive 1, 7, and 14 d were examined for mounting frequency, testicular phosphorylation level by immunoblotting, sperm concentration, sperm acrosome reaction, and histological structures of testis and epididymis, respectively.ResultsAG (50 mg/kg BW) significantly increased the mounting frequency on Days 1 and 7 compared to the control group. Additionally, rat testis treated with 50 mg/kg BW AG showed high levels of phosphorylated proteins as compared with the control group. In histological analyses, AG extract did not affect the sperm concentration, acrosome reaction, and histological structures of testis and epididymis.ConclusionsAG extract enhances the aphrodisiac activity and is not harmful to sperm and male reproductive organs.

Changes of testicular phosphorylated proteins in response to restraint stress in male rats.

ObjectiveTo investigate male reproductive parameters via changes of potential testicular protein markers in restraint-stress rats.MethodsMale Sprague-Dawley rats were divided into two groups (non-immobilized control and restraint-immobilized/stress groups, n=8 each group). The stress animals were immobilized (12 h/d) by a restraint cage for 7 consecutive days. All reproductive parameters, morphology and histology were observed and compared between groups. In addition, the expression of steroidogenic acute regulatory (StAR) and phosphotyrosine proteins (previously localized in Sertoli and late spermatid cells) in testicular lysate was assayed by immuno-Western blotting.ResultsTestosterone level, sperm concentration and sperm head normality of stress rats were significantly decreased while the corticosterone level was increased as compared with the control (P<0.05). Histologically, stress rats showed low sperm mass in epididymal lumen and some atrophy of seminiferous tubules. Although the expression of testicular StAR protein was not significantly different between groups, changed patterns of the 131, 95, and 75 kDa testicular phosphorylated proteins were observed in the stress group compared with the control group. The intensity of a testicular 95-kDa phosphorylated protein was significantly decreased in stress rats.ConclusionsThis study has demonstrated the alteration of testicular phosphorylated protein patterns, associated with adverse male reproductive parameters in stress rats. It could be an explanation of some infertility in stress males.中文概要目 的寻找雄性大鼠应激模型中潜在的睾丸蛋白标记物,探索评估男性生殖能力新的参数。创新点压力可以影响男性生育能力,但其具体机制尚未明了。睾丸组织中酪氨酸磷酸化蛋白在精子形成过程中扮演着重要的角色。本研究首次利用动物模型说明了压力对睾丸组织中酪氨酸磷酸化蛋白表达的影响,进而探索评估男性生殖能力新的参数。方 法雄性SD 大鼠被随机分为对照组和束缚应激的实验组。实验组大鼠每天禁足12 小时,持续7 天。观察比较两组类固醇激素、精子密度、生殖系统形态学和组织学的差别,同时利用免疫印迹方法比较两组睾丸组织裂解产物中急性类固醇调节蛋白和酪氨酸的表达差别(早期在支持细胞中表达,后期在精子细胞中表达)。结 果实验组大鼠的睾酮水平和精子密度较对照组均显著降低,而精子畸形率和皮质酮显著升高。组织学比较发现实验组大鼠精子团数量减少并且出现输精管萎缩。两组大鼠睾丸组织中酪氨酸磷酸化蛋白的表达具有显著差异,实验组95 kDa 的磷酸化蛋白表达量显著降低,但两组急性类固醇调节蛋白的表达没有显著差异。结 论本研究证明束缚应激可使大鼠睾丸组织磷酸化蛋白表达降低,并相应伴随着生殖能力参数的降低。这也许可以从一个方面解释压力对男性不育的影响。

The sensitivity of male rat reproductive organs to monosodium glutamate.

OBJECTIVE This study aimed to investigate the sensitivity of the testis, epididymis, seminal vesicle, and sperm acrosome reaction (AR) to monosodium L- glutamate (MSG) in rats. MATERIALS AND METHODS Rats were divided into four groups and fed with non-acidic MSG at 0.25, 3 or 6 g/kg body weight for 30 days or without MSG. The morphological changes in the reproductive organs were studied. The plasma testosterone level, epididymal sperm concentration, and sperm AR status were assayed. RESULTS Compared to the control, no significant changes were discerned in the morphology and weight of the testes, or the histological structures of epididymis, vas deferens and seminal vesicle. In contrast, significant decreases were detected in the weight of the epididymis, testosterone levels, and sperm concentration of rats treated with 6 g/kg body weight of MSG. The weight loss was evident in the seminal vesicle in MSG-administered rats. Moreover, rats treated with MSG 3 and 6 g/kg exhibited partial testicular damage, characterized by sloughing of spermatogenic cells into the seminiferous tubular lumen, and their plasma testosterone levels were significantly decreased. In the 6 g/kg MSG group, the sperm concentration was significantly decreased compared with the control or two lower dose MSG groups. In AR assays, there was no statistically significant difference between MSG-rats and normal rats. CONCLUSION Testicular morphological changes, testosterone level, and sperm concentration were sensitive to high doses of MSG while the rate of AR was not affected. Therefore, the consumption of high dose MSG must be avoided because it may cause partial infertility in male.

Variant insertion of the teres major muscle.

The teres major (TerMa) muscle has a clinical significance for tendon transfer procedures in patients with massive rotator cuff tears. Individually, it originates from the dorsum of the inferior angle of scapula and inserts into the medial lip of bicepital groove of the humerus. Functionally, TerMa in cooperation with latissimus dorsi (LD) adducts arm, medially rotates arm, and assists in arm extension. The variation of TerMa insertion is very rare. In the shoulder and axillary regions of a 33-year-old Thai male cadaver, the variant insertion of the right TerMa was found. The muscle fibers of TerMa are directly attached at the supero-medial border of LD tendon. Notably, there was no terminal tendon of TerMa. To explain an unusual movement of the arm, this rare variation of the TerMa insertion is necessary to be recognized. This case report is very important for surgeons to preoperatively consider using the terminal tendon of TerMa for tendon transfer in treating patients with irreparable cuff tears.

Antioxidant activity and protective effect of Clitoria ternatea flower extract on testicular damage induced by ketoconazole in rats

Background: Ketoconazole (KET), an antifungal drug, has adverse effects on the male reproductive system. Pre-treatments with antioxidant plant against testicular damage induced by KET are required. The flowers of Clitoria ternatea (CT) are proven to have hepatoprotective potential. However, the protective effect on KET-induced testicular damage has not been reported. Objective: To investigate the protective effect of CT flower extracts with antioxidant activity on male reproductive parameters including sperm concentration, serum testosterone level, histopathology of the testis, and testicular tyrosine phosphorylation levels in rats induced with KET. Methods: The antioxidant activity of CT flower extracts was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Male rats were treated with CT flower extracts (10, 50, or 100 mg/kg BW) or distilled water via a gastric tube for 28 d (preventive period: Days 1–21) and induced by KET (100 mg/kg BW) via intraperitoneal injection for 7 d (induction period: Days 22–28). After the experiment, all animals were examined for the weights of the testis, epididymis plus vas deferens and seminal vesicle, serum testosterone levels, sperm concentration, histological structures and diameter of testis, and testicular tyrosine phosphorylation levels by immunoblotting. Results: The CT flower extracts had capabilities for DPPH scavenging and high reducing power. At 100 mg/kg BW, the extract had no toxic effects on the male reproductive system. Significantly, in CT+KET groups, CT flower extracts (50 and 100 mg/kg BW) alleviated the reduction of reproductive organ weight parameters, testosterone levels, and sperm concentration. In addition, CT flower extracts gave protection from testicular damage in KET-induced rats. Moreover, in the CT100+KET group, CT flower extracts significantly enhanced the expression of a testicular 50-kDa tyrosine phosphorylated protein compared with that of other groups. Conclusions: C. ternatea flower extracts possessing antioxidant activity are not harmful to the male reproductive system and can protect against testicular damage in KET-induced rats.

Immunohistochemical analysis of sustentacular cells in the adrenal medulla, carotid body and sympathetic ganglion of mice using an antibody against brain-type fatty acid binding protein (B-FABP)

Little attention has been paid to adrenal sustentacular cells, and several major histology textbooks do not even describe them. This study presents a detailed morphological description of sustentacular cells using immuno‐light microscopy and an antibody against brain‐type fatty acid‐binding protein. The immunopositive sustentacular cells and processes formed lattices with holes of various sizes and compactnesses or openness. In addition, weakly immunostained sheet‐like structures with ill‐defined contours were often associated with the processes and lattices. In the carotid body, which has traditionally been classified under the name of paraganglia in common with the adrenal medulla, immunostained sustentacular cell processes formed lattices in association with the weakly immunostained sheet‐like structures, but the lattices with sheets were more compact and rigid than the adrenal medulla, and appeared like individually distinct compartments. In the ganglion, the immunostained satellite cell processes with the sheets tightly enclosed individual neurons. As a result, the immunostained sheet‐like structures were regarded as en‐face views of thinly flattened sustentacular cytoplasmic envelopes partially covering the chromaffin cells in the adrenal medulla, and widely in the carotid body in a way rather similar to the satellite cells in the ganglion. In brief, the terminal enclosing portions of adrenal sustentacular cell processes, in cut‐views, were too thin/flat to be recognized as distinct lines in immuno‐light microscopy because of its resolution limit. They are recognized in en‐face views as entities of a substantially spacious extension in immuno‐light microscopy.