应激是机体对应激源（例如环境变化）所产生的反应。动物可以在机体或心理上对这些应激源做出反应，并且对这些应激作出调整或应对。慢性应激会引起身体的自然防御，从而产生多种生理症状，即身体的自主神经系统会产生综合的应激反应，进而引起机体的生理变化，从而使身体能够应对应激。应激还会导致生殖系统功能性损伤及障碍，有研究表明，应激会提高应激相关激素（例如皮质醇）的水平，从而抑制身体的性激素，促性腺激素释放激素（GnRH），进而减少精子数量，抑制性活动和排卵。值得注意的是，母体的应激也会对后代的健康和发育产生有害影响；即使在最初没有新生儿其他并发症，也可能会造成低的初生重且发育迟缓。而且，所产生的影响可能会在出生后一段时间持续对个体产生影响，这与其胎儿期间生理及病理进程存在一定的关联。尽管先前已经进行了广泛的研究，但应激对动物生殖的影响及其机制仍不清楚。应激影响任动物下丘脑-垂体-性腺轴的任何部分都可能会影响到其生殖机能；应激影响动物生殖的机制可能涉及雄性中枢和外周途径之间的复杂相互作用。为了理解这些机制，确定由特定应激源触发的应激途径来进行研究非常重要，且需要进一步确定这些应激是如何影响GnRH的分泌并进一步影响到动物的生殖。

束缚应激（RS）是使用最广泛的应激模型之一，它是固定应力的一种修正类型。应用该模型可以解释应激对心理和身体健康的影响机制，此外，该模型还表明，应激发挥作用与机体所处的阶段也存在关联。因此，对束缚应激的研究不仅仅要集中在个体对应激的反应，而且还要考虑个体所处的生长发育阶段。而且，将动物放入塑料管中以防止其活动会引起不必要的身体损伤。

白藜芦醇（RES）是一种植物化合物，可作为抗氧化剂。该化合物主要存在于葡萄和浆果的皮和种子中。对于白藜芦醇作为药物高剂量使用方面的研究，很多研究都是在动物模型和实验室中进行的。一项新的研究表明，红酒中的白藜芦醇通过阻止与大脑应激调节有关的酶的表达来发挥抗应激作用，它对雄性不育症中的精子具有保护作用；而且，白藜芦醇可以调节细胞对细胞损伤的反应，并减少小鼠和大鼠的生殖细胞凋亡。在生物学层面，白藜芦醇发挥作用的浓度远低于先前研究中使用的浓度；应用动物模型的研究表明，束缚应激会造成动物体身体及心理损伤，例如增加了心理病理性进程以及生殖相关疾病；因此，需要应用不同种类的动物模型才能更清楚地了解应激对机体特定器官的影响。进行本研究的目的是探究白藜芦醇在成年（束缚应激）和新生（母体应激）小鼠束缚应激条件下对睾丸发育和精子形成影响的潜在作用。

1.动物束缚应激模型的建立

在动物生理学、病理学和药理学研究中使用束缚应激模型有着悠久的历史。该技术的主要用途是作为诱导动物应激反应综合症的“应激源”。但有关束缚应激对动物生长发育的影响仍有待进一步研究。本研究中，我们对小鼠进行了束缚应激处理（每天进行4小时/天束缚应激），并在第3、9、15和18天研究束缚应激的影响。我们发现，在第15天和第18天的束缚应激组中，小鼠表现出应激状态，血糖水平升高，脾脏重量增加且日饮水量增加，而在第15天和第18天则没有任何显着差异；但在第 3和第 9天差异不显著。另一方面，我们发现在第 15和18天，小鼠体重，采食量和睾丸重量显着减少，而在第 15和 18天，精子数量，精子活率异常情况也显著增加，然而，尽管在第3和第 9天的束缚应激处理组没有影响。因此，我们的研究表明，束缚应激可能对小鼠的生长发育造成影响，而本研究能够确立研究成年小鼠束缚应激处理的最有效时期，从而为以后的进一步研究提供参考依据。

2.白藜芦醇改善了束缚应激小鼠的睾丸组织病理学

目前，寻找有效的抗应激药物仍具有挑战性。白藜芦醇是一种植物抗毒素，其功能尚待阐明。因此，本研究的目的是探究束缚应激引起的小鼠生殖功能紊乱以及白藜芦醇是否起抗应激作用。我们的结果证实，白藜芦醇在减轻小鼠应激中可能起保护作用。我们在30天大的未成熟雄性小鼠中连续15天评估了束缚应激对小鼠生殖的影响以及白藜芦醇处理后的作用。将50只ICR小鼠分为5组（每组10只）：对照组，束缚应激（RS），束缚应激+媒介物（RS + V），RS + 2 mg/kg和RS + 20 mg/kg。我们根据苏木精和曙红（HE），“高碘酸-希夫”染色以及TUNEL测定结果进行分析。结果表明，与对照组相比，束缚应激显着降低了体重，睾丸重量和水/食物摄入量。白藜芦醇改善了上述指标。RS处理后，小鼠生精小管直径，管腔直径和生精小管面积，小管内腔面积，上皮高度，间质细胞数和白膜宽度均降低了。在RS + 2 mg/kg组中，这些参数恢复正常。根据我们的评估评分系统，RS，RS + V和RS + 20 mg组的精子发生发展显著延迟。但是，我们观察到RS + 2mg组与对照组相比无显著差异。RS + 2mg/kg组的TUNEL阳性细胞数量也明显减少。总之，我们发现2mg / kg的给药量是对抗小鼠束缚应激的有效剂量。

3. 白藜芦醇通过影响小鼠睾丸中凋亡诱导因子途径调节束缚应激

本部分将研究不同剂量白藜芦醇（2mg/kg和20mg/kg）是否对束缚应激小鼠的生殖方面产生影响。首先对成年雄性小鼠进行束缚应激（4小时/天），我们的结果表明，与对照组和白藜芦醇 2mg / kg组相比，束缚应激处理显著降低了睾丸重量，附睾精子数以及精子活力从而引起精子生成异常。我们还观察到束缚应激增加了曲精细管管腔的直径并造成了生殖细胞的离散；而在RS + RES 20mg / kg的组中，与对照组和RS + RES 2mg / kg的组相比，生殖细胞的离散显著改善。凋亡诱导因子（AIF）在束缚应激处理组中呈现明显的着色和定位，而在白藜芦醇 2mg / kg组则着色较轻。蛋白质印迹（WB）结果表明， caspase-9的表达上调，Bcl-2的表达下调；因而， 2mg / kg剂量的白藜芦醇处理降低了束缚应激小鼠Bcl-2，Bas和caspase-9的表达。我们的研究结果表明，白藜芦醇对束缚应激下雄性小鼠睾丸损伤具有一定的保护作用。

4.白藜芦醇在小鼠睾丸生殖细胞抗母体束缚应激中的作用

研究报道白藜芦醇在对抗应激中起到某种改善的作用。怀孕期间的母体应激会影响其神经内分泌调节的变化，从而影响胎儿大脑的早期编程，并且有文献中也证明了束缚应激后其后代行为方面的变化。然而，母体应激如何影响睾丸发育以及白藜芦醇是否具有潜在的保护作用尚不清楚。本研究旨探究白藜芦醇处理下小鼠母体应激对后代的影响；将24只小鼠分为4组：对照组（C），母体束缚应激（MRS），母体束缚应激+白藜芦醇（MRS + RES）2 mg和母体束缚应激+白藜芦醇（MRS + RES）20 mg。从妊娠12-18天开始，每天进行束缚应激4个小时，从而建立模型。结果显示，MRS的雄性幼鼠的睾丸重量，肛门生殖器距离，生精小管面积，生精小管直径，管腔面积，管腔直径和生精小管上皮高度均明显降低。然而，在束缚应激下产生的生殖道异常通过使用白藜芦醇 2 mg / kg得以恢复。在MRS和MRS + RES 20mg/kg组中，TUNEL染色阳性生殖细胞有显着差异，而在MRS + RES 2mg/kg组和对照组之间无显着差异。此外，MRS和MRS + RES 20mg / kg组的血糖水平升高，而MRS + RES 2mg/kg组的实验动物恢复明显。这些结果表明，较低剂量的白藜芦醇可以抵抗早产雄性子代的母体应激所造成的不良影响。因此，我们的研究表明，在母体应激小鼠的第一周龄中可以将较低剂量的白藜芦醇 2mg/kg用作安全有效的药物，我们的结果对将来的研究具有参考价值。

总之，我们的研究结果表明，束缚应激损害了雄性动物身体健康和生殖系统。应激是导致雄性生育力下降并影响到雄性生育率水平的重要因素，并且其对人类和动物以后的生育力产生重要影响，因而成为生殖生物学研究的重要课题，但有关机制仍有待进一步的研究。本研究是首次尝试通过不同应激时间长度的束缚应激进行处理和研究，我们的模型有助于理解动物对束缚的反应。因此，我们的结果可以对束缚应激对睾丸影响机制的研究提供有价值的参考。此外，有研究表明抗氧化剂在应激导致的雄性功能障碍（尤其是雄性不育症）中存在有益的作用，这说明抗氧化剂在减少应激对生殖影响方面具有应用前景，但仍需要进行进一步充分的研究，从而提供更多的可靠证据。我们的结果表明白藜芦醇对抗应激方面的有益作用，有助于减少应急造成的雄性生殖障碍，并减少母体应激引起的新生儿遗传疾病。

Stress is an organism's response to a stressor such as an environmental change. Animals can be physically, mentally, or emotionally react to those changes, and requires an adjustment or response. Chronic stress can cause the natural defenses of the body resulting in a variety of physical symptoms, i.e., the autonomic nervous system of the body has an integrated stress response which causes physiological changes to allow the body to act on stressful situations.  Stress causes also false standards and hypocrisy for the reproductive systemof mice. Scientists proved that stress increases the levels of stress hormones-glucocorticoids such as cortisol-that inhibit the body's main sex hormone, such asgonadotropin releasing hormone (GnRH), and subsequently reduce sperm count, sexual activity and ovulation. Likewise, maternal stress can also have a harmful effect on offspring’s health and development. It could enhance the risk of developmental delay among offspring with low birth weight, even if they had no initial neonatal complications. It also may be recurrent, associated with ongoing events in a fetus life. The mechanisms by which stress influences reproduction remain unclear, in spite of extensive researches. Activation of stress mechanisms at any stage of the hypothalamo-pituitarygonadal axis may potentially affect reproduction. The mechanisms by which stress affects reproduction are likely to involve complex interactions between a variety of central and peripheral pathways in males.

In order to understand these mechanisms, it is important to determine the stress pathways triggered by particular stressors and to determine how those pathways influence GnRH's secretion and behavior.

One of the most widely used models is restraint stress (RS), which is a modified type of stress on immobilization. As well as explaining the mechanism by which stress contributes to mental and physical health problems, the RS model suggests that each step in this phase can be influenced by individual trait. This dimension of the model has become very significant, particularly in recent years, since a lot of research on RS has centered on how individual differences affect the stress cycle. In this process, unnecessary physical and mental stress is caused by putting the animals in a plastic tube to prevent their movement.This is an established experimental stressor that has simultaneous physical and psychological effects.

Resveratrol (RES) is a compound of plants which acts as an antioxidant. This compound appears to be found mostly in grape and berry skins and seeds. However, with high amounts of the drug, much of the RES research was done in animals and test tubes. New research suggests that the plant compound RES used in red wine exhibits anti-stress effects by blocking the expression of an enzyme linked to stress regulation in the brain, according to a buffalo-led study by a university. It has a protective effect on the role of spermatozoa in RS in male infertility.  Numbers of studies have reported that RES can regulate cell response to various stimuli that cause cell injury, as well as decrease germ cell apoptosis in mice and rats. This stress response reflects a layer of biology that has been widely ignored, and RES helps in triggering it at concentrations far lower than those used in previous studies. Many lines of studies on animal subjects have shown that RS contributes too many negative outcomes like increased psychopathologic, physiological, and reproductive incidence adversely. Especially different kind of animal models are required to make it more clearly about depressions.

For RS, special experiment modeling and to understand different kind of factors which are necessary for the stress response should be designed for research objectives. The present study was conducted to investigate the potential role of RES in testing adult (stressed)and new born (maternal stressed) mice against the impact of RS on testicular development and spermatogenesis.

1. Development of animal restraint stress models

The usage of restraint for animal physiology, anatomy, and pharmacology investigations has a long history. The primary application of this technique has been for the induction of stress response syndromes in animals as a "stressor. Such long- or short term impaction of a single restraint stress (RS) has not been extensively explored. In this study, we subjected mice to different days for restraint to simulate the confine episode, and investigated 3, 9, 15, and 18 days after the restraint for 5 hour/day impacts. We found that the mice displayed depression-like behaviors, increased blood glucose level, spleen weight, and water intake, in the RS groups of days (D) 15 and days 18, while there was no any significant difference in the groups of D 3 and D 9 days of same parameters. On other hand, we found significant decreased body weight, feed intake, and testes weight in the groups of D 15 and 18. Results for sperm count, percentage of viability, and abnormality were also significantly increased at D 15, and D 18 in order to RS, while no any significant results were found during giving RS at D 3 and D 9. These findings demonstrated that a single severe stressful process could produce harm full effects for entire life of an organism. The present study establishedto investigate the most effective period of RS in mature mice which makes an easy understand pathways for future studies.

2. Resveratrol ameliorates testicular histopathology of mice exposed to restraint stress

The search for effective medicines is challenging. Resveratrol is a phytoalexin, and its function remains elucidated. Therefore, we undertook the present study to investigate reproductive disturbances due to restraint stress in mice and whether resveratrol plays an anti-stress role. We evaluated immobilization stress and resveratrol supplementation in immature male mice at 30 days of age for 15 consecutive days. Fifty Swiss mice were divided into five groups (10 mice each): Controls, restraint stress (RS), restraint stress + vehicle (RS + V), RS + 2 mg/kg, and RS + 20 mg/kg5 hours back to back days. We determined results on the basis of hematoxylin and eosin (HE), “Periodic acid-Schi” staining, and TUNEL assay. The results indicated that immobilization stress significantly decreased body weight, testis weight, and water/food intake compared to the control; while resveratrol ameliorated these effects. The quantitative histologic evaluation of the seminiferous tubule diameter, luminal diameter, and area of seminiferous tubules, area of tubule lumen, epithelial height, Leydig cell number, and the width of the tunica albuginea were similarly decreased after exposure to RS. These parameters recovered back to normal in the RS+2 mg/kg group. The development of spermatogenesis was significantly delayed in the RS, RS+V, and RS+20 mg groups based upon our evaluation score system. However, we observed no significant differences in the RS+2mg group compared with the control group. The number of TUNEL-positive cells also significantly decreased in the RS+ 2mg/kg group. In conclusion, we found that the administration of 2mg/kg was an effective dose against immobilization stress in mice.

3. Mitigation of stress from testis injuries by resveratrol may occur via AIF signaling in mice

This study was undertaken to investigate whether restraint stress (RS) interferes with low 2mg/kg and high 20mg/kg doses of resveratrol (RES) -induced to check reproductive betterment in mice in comparison to control (C) groups. Mature male mice were subjected to RS (5 hr/day) and maintained feed and water for 15 days. In our results, testes weight, epididymal sperm count, sperm viability, and sperm abnormality was significantly decreased with RS compared to C and RES 2mg/kg groups. We also observed that RS increased lumen size and separations of germ cells, while, in the group of RS+RES 20mg/kg there was dramatically separations of germ cells compared to C and RS+RES 2mg/kg groups. Results of the apoptosis-inducing factor (AIF) antibody was significantly presented in the RS groups, while it was moderated in RES 2mg/kg group. In western blotting (WB), we found up-regulated expression of caspase-9, and down-regulated Bcl-2 expression, while RES 2mg/kg treatment reduced the effects of RS on the expression Bcl-2, Bax, and caspase-9. These significant results of our study shows a positive and healing effects of Res against RS in mature male mice.

4. Positive roles of resveratrol in early development of testicular germ cells against maternal restraint stress in mice

Maternal stress during pregnancy affected the early programming of the brain in the fetus with changes in neuroendocrine regulation, and offspring behavior was proven in the literature. In addition, resveratrol was reported to play positive roles against stress. However, how maternal stress affects testicular development and whether RES has potential protecting roles is unknown. Our present study was designed to evaluate the effects of resveratrol (RES) in Swiss mice by exposing them to prenatal stress. Twenty-four Swiss mice were divided into four groups: control (C), maternal restraint stress (MRS), maternal restraint stress + resveratrol (MRS + RES) 2mg/kg, and maternal restraint stress + resveratrol (MRS+RES) 20mg/kg.

Dams were exposed to stress by restraint in plastic tubes for four hours a day from 12–18 days of gestation. The results showed that male pups of MRS were significantly decreased in the testis weight, anogenital distance, area of seminiferous tubules, diameter of seminiferous tubules, area of the lumen, diameter of the lumen, and epithelial height of seminiferous tubules. However, the anomalies of the reproductive tract produced under restraint stress were neutralized by the use of RES 2 mg/kg. A significant difference was observed between terminal deoxynucleotidyl transferase dUTP nick ends labeling (TUNEL) - positive germ cells in MRS and MRS+RES 20mg/kg groups, while it was non-significant between MRS+RES 2mg/kg and C groups. Apart from these effects, blood glucose levels were increased in MRS and MRS+RES 20mg/kg groups, while experimental animals of the MRS+RES2mg/kg group significantly recovered. These results suggested that a lower dose of RES could cure the adverse effects of prenatal stress in early age male progeny. Thus, our study suggests, for the first time, practical values for a lower dose of RES 2mg/kg as a safe and effective agent in the first week age of prenatally stressed mice.

In conclusions, overall our results indicate that male health and reproductive systems have been impaired by RS stressor in the studies. The decline in male fertility due to especially stressful environment conditions plays a significant role in fertility levels, and its effect on the future of humans and animals population also makes this a significant topic of public health in this era, and there is no general agreement on how to quantify it objectively. This research is among the first to experiment with the different stress cycles across which RS stressor interact to affect reproduction. We demonstrate models that are doubtless important when it comes to understanding how animals react to RS, whether adaptive or not. Therefore, we urge further analysis of the adaptive nature and mechanisms that mediate the effect of RS on testicular histology and other parameters for evidence.

Several studies have reported a beneficial role for antioxidants in stress-induced germ dysfunction especially in male infertility, their usage should be considered, but not before diagnostic research, as a reliable proof on this is a difficult process. We feel confident in suggesting a specific factor for RES administration to reduce the exposure to stressors and systematically increase the chances of good male fertility and reduce the possibility of possible newborn genetic disorders in females.