Quanwei Wei

Cell-specific expression and immunolocalization of nitric oxide synthase isoforms and the related nitric oxide/cyclic GMP signaling pathway in the ovaries of neonatal and immature rats

ObjectiveThe present study is designed to investigate the cellular expressions and immunolocalizations of three different nitric oxide synthase (NOS) isoforms and the related nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway in the ovaries of neonatal and immature rats.MethodsThe ovaries were obtained from ICR (Institute for Cancer Research) female Sprague-Dawley rats at postnatal days 1, 5, 7, 10, and 19. Then we carried out the histologic examination, immunohistochemistry, measurement of NOS activity, and modifications within the NO/cGMP pathway.ResultsDuring postnatal days 1, 5, 7, 10, and 19, all three isoforms of NOS were mainly localized to the oocytes and expressed as a gradual increase in granulosa cells and theca cells within the growing follicle. The ovarian total NOS activities and NO levels were increased at postnatal days 7 and 10 compared with other days.ConclusionsOur findings suggest that the locally produced NO and the NO/NOS signaling systems are involved in the follicular development to puberty.

Effects of thyroid hormones on the antioxidative status in the uterus of young adult rats

Thyroid hormones and oxidative stress play significant roles in the normal functioning of the female reproductive system. Nitric oxide (NO), a free radical synthesized by nitric oxide synthases (NOS), participates in the regulation of thyroid function and is also a good biomarker for assessment of the oxidative stress status. Therefore, the purpose of this study was to investigate effects of thyroid hormones on uterine antioxidative status in young adult rats. Thirty immature female Sprague-Dawley rats were randomly divided into three groups: control, hypothyroid (hypo-T) and hyperthyroid (hyper-T). The results showed the body weights decreased significantly in both the hypo-T and hyper-T groups and that uterine weights were decreased significantly in the hypo-T group. The serum concentrations of total triiodothyronine (T3) and thyroxine (T4), as well as estradiol (E2), were significantly decreased in the hypo-T group, but increased in the hyper-T group. The progesterone (P4) concentrations in the hypo- and hyperthyroid rats markedly decreased. Immunohistochemistry results provided evidence that thyroid hormone nuclear receptor α/β (TRα/β) and three NOS isoforms were located in different cell types of rat uteri. The NO content and total NOS and inducible NOS (iNOS) activities were markedly diminished in the hypo-T group but increased in the hyper-T group. Moreover, the activities of both glutathione peroxidase (GSH-Px) and catalase (CAT) exhibited significant decreases and increases in the hypo-T and hyper-T groups, respectively. The malondialdehyde (MDA) contents in both the hypo-T and hyper-T groups showed a significant increase. Total superoxide dismutase (T-SOD) activity in the hypo- and hyper-T rats markedly decreased. In conclusion, these results indicated that thyroid hormones have an important influence on the modulation of uterine antioxidative status.

Nitric oxide and thyroid hormone receptor alpha 1 contribute to ovarian follicular development in immature hyper- and hypo-thyroid rats

Thyroid dysfunction can cause ovarian cycle and ovulatory disturbances, however, the molecular link(s) between these two disorders remains largely unknown. In the current study, we examined the roles of nitric oxide synthase (NOS) and thyroid hormone receptor alpha 1 (TRα1) in these disorders using immature hyper-thyroid (hyper-T) and hypo-thyroid (hypo-T) rats. In comparison to controls, hyper-T rats had higher serum concentrations of triiodothyronine (T3) and thyroxine (T4), whereas hypo-T rats had lower serum T3 and T4. Serum estradiol (E2) level was decreased in both hyper-T and hypo-T animals and serum E2 in hyper-T rats were lower than in hypo-T rats. We found that neuronal NOS (nNOS) and TRα1 were present in oocytes, granulosa cells and theca cells of all examined rat groups. Ovarian nitric oxide (NO) content and the constitutive NOS (cNOS) activity in hyper-T rats were significantly decreased compared with control or hypo-T rats. Moreover, the number of large antral follicles was reduced in hyper-T rats, and number of primordial follicles was decreased in hypo-T rats compared with control rats. In conclusion, we observed an association between thyroid hormone and NO signaling pathways during the process of ovarian follicular development in immature rats. In hyperthyroidism, thyroid hormones induced an estrogen deficiency that inhibited the function of nNOS, resulting in the inhibition of NO synthesis and suppressed development of large antral follicles, while in hypothyroidism only development of primordial follicles was inhibited.

Expression patterns of taste receptor type 1 subunit 3 and α-gustducin in the mouse testis during development.

Taste receptor type 1 subunit 3 (T1R3) and its associated heterotrimeric G protein α-gustducin (Gα) are involved in sweet and umami sensing in taste cells. They are also strongly expressed in the testis and sperm, but their expression patterns and potential roles involved were previously unknown. In present study, we investigated the expression patterns of T1R3 and Gα in the mouse testis at critical stages of postnatal life, and throughout the spermatogenic cycle. Our results indicated that T1R3 and Gα exhibited a stage-dependent expression pattern during mouse development, and a cell-specific pattern during the spermatogenic cycle. Their expressions have been increased significantly from prepubertal to pubertal periods (P<005), and decreased significantly in aged mice (P<005). The changes were mainly attributed to the differential expression of T1R3 or Gα in elongated spermatids and Leydig cells at different stages of the spermatogenic cycle. In addition, the expression of T1R3 and Gα were first observed in residual bodies of spermatozoa and endothelial cells of blood vessels at post-pubertal mice, while Gα was located in apoptotic spermatogonia of postnatal mice. These novel expression patterns suggest a role of T1R3 and Gα in the onset of spermatogenesis, pace of spermatogenic cycle, and aging of the testis.

Prostaglandin F2α induces expression of activating transcription factor 3 (ATF3) and activates MAPK signaling in the rat corpus luteum

The current study was conducted to evaluate the expression of ATF3, in association with the activation of mitogen-activated protein kinases (MAPK) during prostaglandin F2α analog (PGF)-induced luteal regression in rats. A sequential PMSG/hCG treatment paradigm was used to obtain a single, well-defined generation of corpora lutea (CL) in rats. Rats were treated with PGF for 0-4h on day 7 of pseudopregnancy. Results showed that serum progesterone (P4) concentrations declined in a time dependent manner. Western blot results revealed that ATF3 increased within 2h post-PGF injection. Phosphorylated ERK1/2 (p-ERK) and JNK (p-JNK) increased within 30min and then were gradually reduced in response to PGF. In contrast, the levels of phosphorylated p38 MAPK (p-p38) were not significantly altered. The immunostaining density for p-ERK decreased from the periphery to the center of the corpus luteum following treatment with PGF, while ATF3 was expressed uniformly in the nuclei of luteal steroidogenic cells. These results indicated that treatment with PGF in vivo could induce increases in MAPK phosphorylation, especially in p-ERK, which might be correlated with the increases in ATF3 expression and the decline in P4 concentrations. To our knowledge, this is the first study to provide evidence for temporal relationships between MAPK activation and ATF3 expression during PGF-induced luteal regression in the rat.

Protective effects of Big-leaf mulberry and physiological roles of nitric oxide synthases in the testis of mice following water immersion and restraint stress

Big-leaf mulberry is a new hybrid plant from the application of cell engineering technology, but its effect in stress-induced testicular dysfunction is unknown. Nitric oxide (NO) is a tiny, highly reactive lipophilic molecule produced by nitric oxide synthases (NOS). Three isoforms of NOS (neuronal NOS, inducible NOS and endothelial NOS) have been identified. Our aim was to investigate the effect of water immersion and restraint stress (WIRS) on NOS in the testis, and the effect of Big-leaf mulberry to protect against WIRS. The activity and expression of NOS, and total antioxidant capacity (T-AOC) in the mouse testis of different treatment groups (non-WIRS, 3 h-WIRS, WIRS-recovery) were examined. Histological analysis of WIRS-induced testicular damage and immunohistochemical staining of NOS were also analyzed. Results demonstrated that WIRS-exposed mice produced several injuries and showed an increased iNOS and eNOS mRNA expression in testes, whereas pretreatment with Big-leaf mulberry down-regulated iNOS and eNOS mRNA expressions and up-regulated T-AOC activities. Immunohistochemical studies showed that both iNOS and eNOS were localized in germ cells, spermatozoa and blood vessels in addition to Leydig cells and Sertoli cells, but nNOS was not present in these areas. In conclusion, our results suggested that Big-leaf mulberry exerted a protective effect on WIRS-induced testicular dysfunction, and iNOS and eNOS appeared to exert an important action in mouse testes exposed to WIRS.

Expression of bone morphogenetic protein 2, 4, and related components of the BMP signaling pathway in the mouse uterus during the estrous cycle

The objective was to investigate the expression of bone morphogenetic protein (BMP) family members in the mouse uterus during the estrous cycle by real-time polymerase chain reaction (PCR) and immunohistochemistry. Uterine samples from Swiss ICR mice were collected and dissected free of surrounding tissue. One uterine horn was snap frozen in liquid nitrogen immediately after collection and stored at −80 °C for RNA extraction, and the other was fixed in 40 mg/ml paraformaldehyde at room temperature for immunolocalization of BMP2 protein. Real-time PCR analysis showed that the expression level of Bmp2 was significantly higher at proestrus than at estrus and metestrus (P<0.05). The relative abundance of Bmp4 exhibited significant fluctuations, but there were no statistically significant differences between the expression levels of Bmp2 and Bmp4 (P>0.05). The expression levels of Bmpr1a and Bmpr2 remained unchanged during estrous cycles. However, the level of Bmpr1b mRNA decreased significantly at estrus (P<0.05), increasing subsequently at metestrus. Furthermore, the level of Bmpr1b mRNA was significantly lower than those of Bmpr1a and Bmpr2 mRNA at the corresponding stages (P<0.05). All three receptor-regulated Smads (R-Smads) detected were differentially expressed in the mouse uterus and the expression levels of Smad1 and Smad5 were significantly higher than that of Smad8 (P<0.05). In addition, the expression level of Smad4 did not change substantially throughout the estrous cycle. Immunohistochemical experiments revealed that BMP2 protein was differentially expressed and localized mainly in the uterine luminal and glandular epithelial cells throughout the estrous cycle. In conclusion, our results provide information about the variation in the mRNA levels of Bmp2 and Bmp4 and related components of the BMP signaling pathway. The data provide quantitative and useful information about the roles of endometrial BMP proposed and demonstrated by others, such as the degradation and remodeling of the endometrium.

Roles of poly (ADP-ribose) polymerase (PARP1) cleavage in the ovaries of fetal, neonatal, and adult pigs

Poly(ADP-ribosylation), which occurs rapidly in cells following DNA damage and is regulated by poly (ADP-ribose) polymerase 1 (PARP1), is a post-translational modification of proteins playing a crucial role in many processes, including DNA repair and cell death. Although PARP1 has recently been implicated in a variety of physiological and pathological processes, its role in the process of follicular development and atresia is not yet completely defined. This study was designed to investigate the cellular expression pattern and immunolocalization of PARP1, cleaved PARP1, caspase 3, and cleaved caspase 3 in fetal, neonatal, and adult porcine ovaries. Our results showed that in fetal and neonatal pigs, PARP1 cleavage is involved in the process of oocyte nest breakdown, primordial follicle formation, and transition to primary follicles. The results of immunohistochemistry indicated that PARP1 cleavage was involved in the process of follicular development and atresia, which was in accordance with our previous study; however, it was noted that cleaved caspase 3 was mainly localized in and around the nucleus of apoptotic granulosa cells (GCs), whereas cleaved PARP1 was mainly localized in the nucleus of the apoptotic GCs. RIA data showed increased serum progesterone and estradiol concentrations with age after birth. Collectively, our findings suggest that the PARP1 signaling pathway is involved in oocyte nest breakdown and primordial follicle formation in fetal and neonatal porcine ovaries, but is different from follicular atresia in adult porcine ovaries that involves cellular apoptosis.

Effects of concomitant diabetes mellitus and hyperthyroidism on testicular and epididymal histoarchitecture and steroidogenesis in male animals.

This study evaluated the effects of comorbid disorders of diabetes and hyperthyroidism in the adult male mice. In total, 32 ICR strain mice were equally distributed into four groups: control (C), diabetic (D), diabetic-plushyperthyroid (DH), and hyperthyroid (H). Mice allocated for diabetes received a single intraperitoneal injection of streptozotocin (STZ) at 200 mg/kg body weight. At the onset of diabetes, one group of mice was concomitantly injected levothyroxine (LT4; 0.3 mg/kg body weight) and the other set of animals received the same treatment independently on a daily basis. The body weight, as well as the testicular and epididymal weights, was reduced markedly in D and DH mice. Higher trends of blood glucose levels were seen in the DH group, in comparison to euthyroid diabetic mice. Thyroid hormones could exert a transient effect on blood glucose homeostasis by altering the serum blood glucose level in diabetic patients. Histomorphometric analysis showed increased luminal sizes of seminiferous tubules, along with decreased epithelial height and atrophic changes in germinal stem cells in the testis of DH and H mice. Caput epididymis of DH mice showed extensive compaction of principal cells, loss of stereocilia, lipid vacuolization, and inflammatory infiltrations; however, damaged tubular integrity, packed clear cells, exfoliated cells, and round spermatids were profoundly noticed in the cauda epididymis. Hyperthyroidism elevated the serum testosterone levels in H and DH mice and produced critical damages to the histoarchitecture of the epididymis. Collectively, this experiment endeavored to mimic the polyglandular autoimmune syndrome, which will be helpful to better understand the reasons for male infertility in diabetic-cum-hyperthyroid patients.概要目 的评估糖尿病和甲状腺功能亢进对雄性动物睾丸和 附睾组织形态学及类固醇激素合成的影响, 并初 步探讨其作用机制。创新点以小鼠为模型, 首次研究并发糖尿病和甲状腺功 能亢进对雄性哺乳动物睾丸、附睾发育和类固醇 激素合成的影响。方 法32 只ICR 品系小鼠分为四组: 对照组(C)、糖 尿病组(D)、糖尿病+甲亢组(DH) 和甲亢组 (H)。D 组小鼠以200 mg/kg 剂量单次腹膜内注 射链脲佐菌素(STZ), 诱导糖尿病成功。另对 其中一半以0.3 mg/kg 剂量每天注射甲状腺素, 组成DH 组。小鼠试验结束后, 采集睾丸、附睾 和血液, 并离心分离获得血清。睾丸和附睾用4% (0.04 g/ml) 多聚甲醛固定, 并用苏木精-伊红染 色法(H & E) 观察睾丸和附睾组织形态学变化, 用放射免疫测定(RIA) 试剂盒检测血清中睾酮、 促甲状腺激素(TSH)、胰岛素、甲状腺素(T4) 和三碘甲状腺原氨酸(T3) 的含量并进行分析。结 论D 和DH 组小鼠的体重、睾丸和附睾的重量显著 降低。相比于正常甲亢或糖尿病小鼠, DH 组中 血糖水平显著升高。甲状腺激素可能是通过改变 糖尿病患者的血清血糖水平对血糖稳态产生瞬 时影响。 组织形态学分析结果显示, 在DH 和H 组小鼠睾丸中, 输精管管腔增大, 上皮厚度减少, 睾丸生殖干细胞发生萎缩性变化。DH 组小鼠的 附睾头呈现主细胞压实、纤毛、脂质空泡化和炎 症浸润现象。在附睾尾部观察到了小管完整性受 损、透明细胞聚积和细胞脱落, 并发现圆形精子。 对于DH和H 组, 甲亢提高了小鼠血清睾酮水平, 并损害了附睾的组织形态。总之, 本试验模拟了 多腺体自身免疫综合征对雄性繁殖的影响, 这将 有助于更好地了解男性并发糖尿病和甲亢患者 不育的原因。

Effects of Daily Exposure to Saccharin and Sucrose on Testicular Biologic Functions in Mice

ABSTRACT Saccharin sodium consumption is considered safe and beneficial, owing to its very intense sweetness without any associated calories, but supporting scientific data remain sparse and controversial. Herein, we demonstrate that dose-response relationships existed with regard to administration of saccharin or sucrose to mice for 35 days, and this association involved testis-expressed sweet-tasting molecules (taste receptor type 1 subunit 3 [T1R3]; G protein alpha-gustducin [Galpha]). Mouse body weights and testis weights in middle- and low-dose saccharin-treated groups were increased with up-expressions of molecules involved in testicular sweet taste and steroidogenic (middle saccharin: steroidogenic acute regulatory protein [StAR]; P450 cholesterol side-chain cleavage enzyme [CYP11A1]; 17-alpha-hydroxylase/C17,20-lyase [CYP17A1]; low saccharin: StAR). Moreover, a high-dose saccharin-related decline in reproductive hormone levels and injuries to testis and sperm were observed to be associated with suppression of testicular T1R3 and Galpha, as well as steroidogenic-related factors (StAR; 3-beta-hydroxysteroid dehydrogenase [3-beta-HSD]; CYP11A1; CYP17A1; 17-beta-hydroxysteroid dehydrogenase [17-beta-HSD]), and activation of cleaved caspase-3. However, abnormalities of the testis and sperm in high- and middle-dose sucrose-exposed mice were related to the increased-cleaved caspase-3, but independent of T1R3 and/or Galpha. Collectively, our results clearly suggest that saccharin-induced physiologic effects on testis are associated with testicular T1R3 and Galpha, which differed from sucrose. We hence call for a reassessment of the excessive use of sweeteners in daily life, especially artificial ones, considering their potential side effects.