Intervention of Xuduan Zhongzi Formula on spermatogenesis epididymal morphological changes in a mice model of oligospermia

LIU Jin-yao, HE Ming, WANG Quan-sheng, TANG Zhen-yu, YANG De-fen

1.Guangxi University of Chinese Medicine, Nanning 530200, China

2.Guilin Hospital of Traditional Chinese Medicine, Guilin 541002, China

3.The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China

Keywords:

ABSTRACT Objective: To investigate the intervention of Xuduan Zhongzi Formula on the epididymis structure of model of oligospermia mice.Methods: Ten of the 45 male mice were labeled as the normal group, and the remaining 35 mice were injected with chloral hydrate for five consecutive days, and the randomly selected five mice were anesthetized with chloral hydrate.The left epididymis was removed, and a few sperm were found in the epididymis under the optical microscope, indicating successful construction of the model.They were randomly divided into three groups: the normal group, the L-carnitine group and the Xuduan group with 10 rats in each group.The the L-carnitine group and the Xuduan group were administrated with equivalent dose of human solvent, and the normal group and the model group were administrated with normal saline.After 8 weeks of intragastric administration, all experimental mice were killed, and the left epididymis was extracted for sperm detection, seminal plasma biochemical detection, HE staining, and electron microscopy.Results: In the model group,enlarged epididymal epithelial cells, vacuolar degeneration of primary and basal cells, and edema of interstitial cells and vascular dilation were found.Compared with the normal group,semen concentration, activity, SOD, A-glucosidase and fructose in the model group were significantly decreased, and the difference was statistically significant (P<0.01).After eight weeks of drug intragastric treatment, the semen concentration, activity, SOD, A-glucosidase and fructose in the Xuduan group were significantly increased compared with the model group,and the difference was statistically significant (P<0.01).Conclusion: Xuduan Zhongzi Formula can significantly improve the epididymal structure, the microenvironment of epididymal sperm maturation and the stability of epididymal epithelial structure of oligospermia model mice,creating conditions for sperm maturation in the epididymis.

1.Introduction

According to statistics, more than 65% of the causes of male infertility and its related pathogenesis have not been discovered.According to the relevant data of the European Society of Urology in 2017, the proportion of male infertility patients caused by oligospermia is increasing[1].Infertility due to male factors accounts for about 50%, and there are at least 50 million patients with oligospermia[2].As one of the types of male infertility, oligospermia is caused by many factors: abnormal spermatogenic function,epididymal function, vas deferens obstruction, genetic and living environment factors[3,4], among which the change of epididymal structure and its microenvironment is one of the important causes of oligospermia[5].The development of sperm from spermatogonial cells to mature sperm has undergone a complex process, and the epididymis is an important place for sperm maturation and storage.At present, the study on oligospermia of epididymis in Chinese medicine is rarely reported.After summing up the actual clinical experience, our research group believes that male infertility is mostly caused by “Yin qi failure, kidney essence deficiency”, and the treatment is mostly Xuduan seed, and relevant experimental studies have shown that the treatment is indeed effective[6].The experiment intended to create a mouse model of experimental oligospermia.By observing the local microscopic and ultrastructure of the mouse epididymal structure, the intervention effect of interrupted seed formula on the epididymal structure of the mouse model of oligospermia was studied, so as to provide a breeding site for sperm maturation.

2.Materials and methods

2.1 Experimental instrument

MACRO sperm counting board and WLJY-9000 color sperm quality detection system (Beijing Weili New Century Technology Development Co., LTD.); TDL-4ZB low speed automatic balancing centrifuge (Hunan Xingke Scientific Instrument Co., LTD.); Electron optical microscope, Department of Pathology, the First Affiliated Hospital of Guangxi University of Traditional Chinese Medicine.

2.2 Experimental reagent

Baixiaoan injection, lot number: K1625007, purchased from Aladdin Company; TUNE Kit, Product number :11684817910,Beijing Zhongshan Jinqiao Biotechnology Co., LTD.α-glucosidase detection kit, Nanjing Xindi Biopharmaceutical Engineering Co,LTD., Lot number: A10740102.

2.3 Laboratory animal

Forty-five male mice aged 6 to 8 weeks and weighing 24 to 28 g were purchased from the Laboratory Animal Center of Guangxi Medical University with animal license number [SCXK(GUI)2009-0002].All the mice were fed separately in cages and kept at 20-25 ℃for 7 days.Replace dry bedding in three days.This experiment was approved by the Animal Ethics Committee of Guangxi University of Traditional Chinese Medicine.

2.4 Animal model making and grouping

After 7 d of feeding, the experimental mice showed no abnormal activities, and the oligospermia mouse model was prepared.The method was as follows [7]: Except for 10 mice in the normal group,the remaining mice were treated with Baixiaoan (Sigma product)combined with 5 mL 0.09% normal saline and injected intravenically with Baixiaoan (10 mg/kg) for 5 consecutive days, and the normal group was injected with the same amount of normal saline.5 of the 35 mice were selected to be killed and the epididymis of the mice was removed laparotomy, and a small number of sperm were observed under the optical microscope.The results indicated that oligospermia mouse model was successfully created.The remaining 30 mice were divided into model group, left card group and Xuduan group with 10 mice each according to random number table method.

2.5 Experimental drug

2.5.1 Xuduan Zhongzi Formula

Drugs include (Fructus nigrifolia, yam, bone, cuscuta, red vine,achyrania, Eucommia, codonopsis, white art, Horny medium,Wolfberry, continuous 10 g), single Chinese medicine granules(non-decoction Chinese medicine, Jiangsu Jiangyin Tianjiang Pharmaceutical Co., Ltd.production, product batch number 1203002).

2.5.2 The L-carnitine group

L-carnitine Oral Liquid (Shenyang First Pharmaceutical Co., LTD.,SINopol H19990372)

2.6 Methods

2.6.1 Intervention method

After successful modeling, the patients were treated by intragastric administration with 0.9% normal saline (model group and normal group).Xuduan group was given 9 g∙kg-1∙d-1Xuduan seed formula plus normal saline.Leucca group was given 75 mg∙kg-1∙d-1leuccarnitine oral solution combined with normal saline.Once a day for 8 weeks.

2.6.2 Sampling method

24 h after the end of the last gavage, the head and neck of the mice were sacrificed, their abdominal epididymis was excised, and relevant tissues of one side of the epididymis of each group were removed and quickly placed in Bouin solution for fixation.After fixation, ethanol was dehydrated, xylene was transparent, paraffin was embedded, and hematoxylin-Eosin staining was hematic.HE staining and electron microscopy were used to detect epididymis tissue.The other side of the epididymis was crushed with ophthalmic scissors in a container with 2 mL culture solution, and sperm were fully free and swimming (36.5 ℃ , 30 min) for semen analysis and seminal plasma biochemical preparation.

2.6.3 Automatic semen detection

Sperm concentration and motility were measured with sperm counting plates and sperm examination system, and some parameters of sperm were determined according to WHO’s 《Manual for Laboratory Testing of Human Semen Examination and Processing》[8].

2.6.4 Detection of seminal plasma biochemical indexes

The semen after 3 000×g centrifugation was liquefied for 10 min and the seminal plasma was placed at -20 ℃ for testing.Serum NAG activity was determined by α-glucosidase assay kit.The concentration of sugar in refined berry was determined by resorcinol colorimetry.

2.6.5 HE staining method

HE staining was used to stain 10 sections of epididymis tissue in each group, and 2 visual fields were observed in each section.Epithelial high evaluation was performed at the head and tail of the epididymis using 15 transverse sections of the epididymal tubules with a rounded shape (×200 times).Tubular (epithelial, luminal septum, and smooth cell) proportions were measured at the head and tail of the epididymis, and 10 images (×200) were randomly captured per animal.A grid containing 100 intersections was superimposed on the image, and these points were classified as: epithelium, lumen,smooth muscle and stroma, with a total of 1 000 intersections per animal, and the volume proportion of each group was calculated.The diameter and epithelial thickness of the epididymal tubules in the head and tail were measured by optical microscopy.The diameter of the epididymal tubule is determined by the mean of the longitudinal and transverse cross sections.

2.6.6 Ultrastructure detection

Appropriate epididymal tissues of each group were selected and placed in a mixture of 1% paraformaldehyde and 2% glutaraldehyde.Changes of epididymal tissues were observed under electron microscopy, and subcellular structures such as epididymal epithelium, mitochondria, endoplasmic reticulum and epididymal duct were visible in the visual field.

2.6.7 Statistical processing

The data were expressed as mean±standard deviation (±s)and were processed by SPSS21.0 statistical software.P<0.05 was considered statistically significant.

3.Results

3.1 Correlation of mice after modeling

The drinking water and eating of the mice in each group were reduced compared with before, and the mental condition was poor, the autonomic activity was decreased, the body weight was decreased, the hair was lost, and the reaction was not sensitive.The above symptoms gradually improved after 1 week of drug administration.During the period, 2 mice died due to inaccurate intragastriction dose (1 in each of the model group and the L-carnitine group), and 2 mice died due to factors such as improper drug intragastriction operation leading to puncturing of some organs(1 in each of the Xuduan group and model group).

3.2 Effects of epididymis sperm quality, A-glucosidase,fructose and SOD

Compared with normal group, semen concentration, activity,superoxide dismutase (SOD), A-glucosidase and fructose of mice in model group were significantly decreased (P<0.01).Compared with model group, semen concentration, activity, SOD, A-glucosidase,fructose and SOD in Xuduan group were significantly increased,and the difference was statistically significant (P<0.01).Compared with Leucca group, semen concentration, vitality and fructose in the Xuduan group were significantly increased (P<0.05), and A-glucosidase and SOD were significantly increased (P<0.01), as shown in Table 1.

Tab 1 Effects on epididymal sperm quality, A-glucosidase, fructose and SOD(±s)

Tab 1 Effects on epididymal sperm quality, A-glucosidase, fructose and SOD(±s)

Note: Comparison with model group,**P<0.01; Compared with the L-carnitine group, #P<0.05, ##P<0.01.

Group n Sperm concentration(106/mL) Activity rate(%) α- glucosidase (mU/g) levulose (mg/mL) SOD(nmoL/mg)Normal group 10 47.53±5.28 61.71±6.03 16.51±3.42 111.81±4.47 106.04±15.69 Model set 8 17.29±4.83 20.17±6.22 10.56±2.62 83.23±13.22 77.52±21.42 L-carnitine group 9 30.05±5.25# 36.10±6.48# 14.45±5.24## 114.32±12.08# 107.32±17.63##Xuduan seed set 9 34.37±5.08** 41.49±7.53** 13.81±4.57** 106.53±12.65** 118.04±12.18**F 52.2 53.1 22.7 48.9 137.8 t 0.29 0.21 0.18 0.56 0.22

3.3 Effect on the height and proportion of epididymal duct epithelium, interventricular septum and smooth muscle

Compared with normal group, epithelial height and proportion,smooth muscle proportion and tube spacing in model group were significantly increased (P<0.01).Compared with the model group,the height and proportion of epithelium, smooth muscle proportion and tube spacing in the Xuduan group were significantly decreased(P<0.01).Compared with the left card group, the proportion of epithelium in the Xuduan group was decreased (P<0.05), the proportion of smooth muscle and tube spacing were significantly decreased (P<0.01), and the differences were statistically significant.See Table 2.

Tab 2 Effects on height and proportion of epididymal tube epithelium, interventricular septum and smooth muscle(±s)

Tab 2 Effects on height and proportion of epididymal tube epithelium, interventricular septum and smooth muscle(±s)

Note: Comparison with model group,**P< 0.01; Compared with the L-carnitine group, #P<0.05, ##P<0.01.

Group n Epithelial height Epithelial ratio Tube spacing(%) Smooth muscle ratio(%)Normal group 10 23.94±2.47 20.39±0.68 18.31±4.80 3.71±0.25 Model set 8 28.97±2.19 29.53±2.01 29.16±2.96 6.19±0.50 L-carnitine group 9 23.54±2.52 21.90±0.94# 31.23±2.86## 4.70±0.24##Xuduan seed set 9 24.12±2.55** 17.80±0.43** 28.23±8.33** 4.59±0.27**F 31.2 19.3 21.8 5.8 t 0.28 0.86 0.52 0.33

3.4 Effect on the structure of mouse epididymis

Under light microscope observation, it can be seen that the relevant tissues of the epididymis of mice in the normal group were intact,the sperm were evenly distributed in the lumens, and the relevant tissue structures such as normal fibers and small vessels existed in the interstitium of the epididymis (Figure 1A).In the model group, more shedding germ cells were observed in the epididymal lumen, spermatozoa decreased, some spermatozoa denaturated and fused together.The epididymal duct epithelial cells increased, the number of halo cells and bright cells in the epithelium increased significantly, and the inflammatory cells and fibrous tissue hyperplasia were observed between the epididymal duct.Epididymal interstitial edema, lumen contains a small number of sperm remains,immature spermatogenic cells, deformed sperm, etc.(Figure 1B).In the interrupted group, only a single detached germ cell could be seen in the lumen of the epididymis head, while the other two epididymis tissues (the epididymis body and the tail of the epididymis) were structurally intact, and epididymal duct epithelial cells, basal membrane cells, interstitial cells, and vascular endothelial cells were intact, and a large number of sperm cells were stored in the lumen(Figure 1C).In the left card group, only a single detached germ cell could be seen in the lumen of the head of the epididymis, and the other two epididymis tissues (the epididymis body and the tail of the epididymis) were structurally intact, with a little edema in the interstitium of the epididymis, small vasodilation, and vacuolated lumen of the main and basal cells (Figure 1D).

Fig 1 Changes of structure of epididymis related tissues in each group under light microscope(HE×200)

3.5 Effect on ultrastructure of mouse epididymis

Under electron microscope, the endoplasmic reticulum and Golgi complex were observed in the epididymal cells of normal control group.The closely linked complex between epididymal cells constitutes the blood-testis barrier, on which the barrier mechanism is based (Figure 2A).In the model group, the endoplasmic reticulum and Golgi complex were scattered in the epididymis, and the epididymal tubular epithelial cells showed degenerative changes.The cytoplasm of the lumen with disordered structure was scattered in large quantities, and the number of cytoplasm in the vacuolated basal part increased obviously, and some were locally absent.Spermrelated structures break down, showing coils of varying sizes and shapes (Figure 2B).The endoplasmic reticulum and mitochondria of the Golgi complex in the main cells of the epididymis group were orderly distributed, and the tissue morphology was complete, and many mature sperm existed in the epididymal lumen (Figure 2C).In the left Ka group, more neatly distributed tubular epithelial cells of the epididymis were observed, the endoplasmic reticulum and Golgi complex of the epididymis were in normal shape, the degraded mitochondria were significantly changed, and mature sperm and malformed sperm interspersed in the lumen (Figure 2D).

Fig 2 Ultrastructure of epididymis of mice in each group under electron microscope (EM×3 000)

4.Discussion

As a common clinical disease of male infertility, oligospermia has become one of the hotspots of male infertility research due to its complex pathogenesis and various etiologies.The epididymis structure and microring mirror are crucial to the development and maturation of sperm, and the epididymis can also accelerate the secretion of carnitine, glycoprotein and various enzymes, and the maturation, movement and routine physiological functions of sperm are closely related to the above substances[9].The content of androgens in the epididymal cavity is very high, and the epididymal epithelium itself can secrete a small amount of androgens.Studies have shown that testosterone is essential for spermatogenesis and one of the factors that modulate epididymal contraction and luminal fluid viscosity[10].The destruction of epididymis structure can lead to oligospermia, asthenospermia and sperm malformation.Therefore,the research group established oligospermia model mice to study the effects of Xuduan seed formula on the epididymal tube structure and microenvironment of model mice, so as to provide a breeding base for sperm development and maturity.

The mouse epididymis is composed of a single elongated flat tubule with high curl, which can be divided into three parts from top to bottom: head, body and tail of the epididymis.Each region has a special intracranial microenvironment, which is crucial for the maturation of testicular sperm when they reach the tail.Studies have shown that the head and body are the microenvironments that provide sperm maturation, while the tail area mainly serves as the storage of reticular sperm[11].The epididymal epithelium is composed of different cell types, including master cells, basal cells, clear cells, apical cells, and halo cells, forming a single layer around the inner cavity[12].In the initial part, the tall columnar master cell is responsible for water absorption; Ions and small organic molecules are also absorbed.The master cells in the head are involved in the secretion of proteins that attach to the sperm membrane and change its protein composition.The main cells of the body have an abundance of lipids in their supranuclear regions,and these cells may help modify the lipid components of the sperm plasma membrane.The epithelium of the tail is much shorter than that of the head segment, and clear cells are prevalent, which engulf cytoplasmic droplets shed from mature sperm and may also engulf other lumen debris.In the tail, excess lumen proteins are absorbed,while fixed proteins are secreted to keep the sperm still.These cell types within the epididymal epithelium have separate functions,while major cells form tight connections between adjacent cells to generate blood-epididymal barrier protection against immunogenic responses to prevent sperm from entering the epididymal epithelium,which is necessary for a stable and specific microenvironment within the epididymal cavity.Regulation of cell connections is considered to be a key determinant of sperm maturation in the epididymis[13].Sperm maturation involves a highly coordinated interaction between the lamina propria surrounding the epididymal epithelium, the epididymal epithelium, the sperm in the sperm cavity and the sperm itself to provide conditions for sperm maturation.This segmented feature determines the different physiological functions of each epididymis segment, and also provides a microenvironment for the development and maturation of sperm.

Baixiaoan is an alkylating agent, which is commonly used as a palliative treatment for diseases such as leukemia and malignant lymphoma in clinical practice, and is also one of the classic drugs used to establish oligospermia and azoospermia animal models[14].It can destroy differentiated spermatogonium cells in testis, resulting in spermatogenic dysfunction, and have toxic and damaging effects on morphology, structure and function of epididymis, especially forward movement and fertilization ability of mature sperm[3,15].It has been reported that leucoanil can increase the epithelial cells of the head and tail of the epididymis of mice, reduce the golgi apparatus and lipids contained in them, narrow the epididymal tube chamber, and cause sperm damage caused by the persistent toxicity in the epididymal tube[16].Promote the formation of reactive oxygen species (ROS), and the increase of ROS can stimulate lipid peroxidation (LP) and signal transduction changes in the epididymis,resulting in DNA damage and destruction of spermatogenic epithelial cells[17,18], LP and oxidative stress induction, resulting in changes in the structure and internal environment of the epididymis.The epididymis is damaged, epithelial secretion is reduced, the cells contain vesicles and cytoplasm is degraded, its storage of substances associated with sperm maturation is reduced.The increase in dense vesicles and lipid droplets, which in some cases occupy the cytoplasm of the entire cell, promotes cell swelling,increased epithelial cells and instability, and vesicle accumulation may trigger altered secretion patterns of the epididymis, possibly leading to insufficient sperm maturation and decreased motility.In the experimental observation, the epididymal epithelial cells in the model group were scattered and the tubular epithelial cells showed degenerative changes.Lumen diameter narrowed, epithelial height increased, local histiocytic edema, the number of vacuolated basal cytoplasm increased significantly, and local deletion was observed.The structure of the sperm is broken, and it is coiled in different sizes and shapes.After treatment, the endoplasmic reticulum and mitochondria of Golgi complex in the main cells of epididymal epithelium were intact and neatly arranged, the epithelial height and lumen diameter were normal, and more mature sperm existed in the lumen of epididymal epithelium.It has been reported in the literature that the increase of epithelial cells and the accumulation of vesicles may cause changes in the absorption function of the epididymis, hinder the normal operation of sperm in the epididymal chamber[19], and shorten the acceleration of sperm transit time in the head and tail of the epididymis.The results of experiments show that the concentration, motility, microscopic, ultrastructure and serum biochemical enzymology of epididymal sperm in oligospermia model mice are closely related to the changes of microscopic,ultrastructure and microenvironment in the epididymal tissue of mice.

The treatment of male infertility in Chinese medicine mainly starts from the aspects of kidney, spleen kidney, liver and kidney.Through relevant literature exploration and the summary of actual clinical experience, our research group found that “Ying qi failure, kidney essence deficiency” is an important factor leading to male infertility, among which “Ying Qi failure” is the local qi and blood dysfunction of testis; Local Qi-blood movement of testis epididymis is blocked, Yingwei is disharmony, vascular permeability is disordered, meridians are blocked, nutrients cannot be exchanged, kidney is displaced, and hair is sterile.Treat with“tonifying spleen and strengthening kidney, activating collaterals and channelling stagnation” the Xuduan seed formula.This recipe is found in the book “Medical Zhengyin”, in which Yue family[20] believes that this recipe is the fastest way to obtain seeds and produce essence.Prescription in the Xuduan, bone broken supplement, the combination of the two drugs to tonic, tonifying kidney essence to remove stasis, so that the newborn essence can go smoothly without blockage.Codonopsis, yam, Baizhu soil tonifying spleen and stomach, spleen and stomach strong acquired qi is nourishing, blood is active, blood is full of kidney essence nourishing; Wolfberry, cuscuta and Ligustrine can promote the formation and transformation of the essence in the kidney and replenish the essence in the kidney.Radix achyranthes filling essence is good for carrying medicine down, red vine entering blood branch stasis, Eucommia warm kidney is proficient in removing the evil gas of lower jiao; Epimedium strong kidney aphrodisiac;The combined use of all kinds of drugs, a total of tonifying the spleen, strengthening the kidney and producing essence, activating the collaterals and removing stasis.Pharmacological studies have shown that wolfberry and cuscuta can restore damaged testicular cells and reduce the occurrence of apoptosis; total flavonoids can inhibit the generation of oxidative stress-related indicators, improve semen quality, regulate the secretion of sex hormones, and inhibit the apoptosis of spermatogenic epithelial cells[21,22].Ligustrum Ligustrum contains rich trace element Zn, which can improve oxidation activity, accelerate spermatogenic cell proliferation, and provide conditions for sperm development and maturation[23].Total achyranthes saponins can inhibit the secretion of IL-6 and Th17 and reduce the content of inflammatory factors[24].Eucommia percha enhances the synthesis ability of testosterone, promotes the secretion of sex hormones, reduces sperm oxidation reaction, and improves sperm motility[25,26].

In summary, the present study showed that the recipe of the seed can effectively improve the epididymal structure of oligospermia model mice, maintain the stability of epididymal epithelial structure,improve the microenvironment of epididymal sperm maturation, and create favorable conditions for epididymal sperm maturation.Further research is needed on the specific mechanism of spermatozoa protection of epididymis in oligospermia model mice.

Author’s contribution

LIU Jin-rao: Animal modeling, data collation, paper writing; HE Ming: Data analysis and related experimental index analysis; TANG Zhen-yu: Experimental index and data collation; YANG De-fen:Literature search; WANG Quan-sheng: Paper review.

There is no conflict of interest between the authors.