Expression of the NSE, SP, NFH and DβH in Normal and Cryptorchid Testes of Bactrian Camel


 Background: Neuroendocrine substances play important roles in regulating the normal physiological functions of testicles.The purpose of this study is to explore the localization and effects of four neuroendocrine markers(NSE, SP, NFH and DβH) in normal and cryptorchid testes of Bactrian camels.Methods: The cryptorchid testes were located in the abdominal cavity and were collected by orchiectomy. Fresh testes tissues were processed into small pieces and then divided into three samples. One sample was frozen in liquid nitrogen for western blotting hybridization reaction, while the other sample was fixed with 4% paraformaldehyde solution for histochemical analysis, and the third fixed in glutaraldehyde for transmission electron microscopy observation.Results: The results showed that cryptorchidism caused a reduction in layers of spermatogenic epithelium and decreased glycogen positivity in the basement membrane.The ultrastructure revealed that macrophages were always found around the Leydig cells, which were crowded with swelling mitochondria in cryptorchidism. Expression of NSE in the Leydig cells of cryptorchidism was significantly weakened compared to that in the normal group(p<0.01). We found that SP was always distributed along the nerve fibers in normal testes and was expressed in the Leydig cells of cryptorchidism. However, expression of NFH in the cryptorchidic tissue was strongly positive in spermatogenic epithelium, with limited expression in Leydig cells and no expression in peritubular myoid cells. Therefore, expression of DβH in the Sertoli cells was comparatively strong in both the normal and cryptorchidism groups.NFH and DβH expression was significantly increased in the cryptorchidism group compared with the nomal group(p<0.01).Conclusions: These findings indicated that the underdeveloped seminiferous epithelium and pathological changes in cryptorchid tissue in Bactrian camels were potentially related to a disorder in glycoprotein metabolism.Our results suggest that NSE and SP could helpful to judge the pathological changes of cryptorchidism. The present study provides the first evidence at the protein level for the existence of NFH and DβH in Sertoli and Leydig cells in Bactrian camel cryptorchidism and provides a more in-depth understanding of neuroendocrine regulation is crucial for animal cryptorchidism.


Background
Gametogenesis in mammals is a very complex process in which paracrine and autocrine mechanisms in addition to the hypothalamic-pituitary-testicular endocrine axis in uence are intimately involved in complicated feedback loops [1,2].Leydig cells originate in the adult testis by differentiation from embryonic neuroectoderm precursor cells and mesenchymal cells and express a variety of neural and neuroendocrine markers [3,4].Studies have suggested that in addition to steroid hormones the Leydig cells of different rodents and humans produce and possess a number of neuronal markers such as 5hydroxytryptamine(5-HT),neural cell adhesion molecule(N-CAM),and microtubule-associated protein(MAP-2)catecholamine and norepinephrine protease [3,5,6].The distribution and localization of PGP9.5,neuropeptide Y,neuron-speci c enolase(NSE),substance P(SP),dopamine-β-hydroxylase(DβH),and tyrosine hydroxylase-related peptides in the testicles of mammals,such as cats [7],donkeys [8],ruminants [9],dromedaries [10]and humans [11]were studied by immunohistochemistry,and results showed that such neuroendocrine substances play important roles in regulating the normal physiological functions of the testicles.Previous researches have shown that the expression of NSE,SP and NFH (High Neuro laments ) in the testicular tissues of adult rats is regulated by serum sex hormone concentrations [12].The occurrenc-e of numerous marker substances,such as SP,NFP-200,NSE and DβH characterized human Leydig cells as new members of the "diffuse neuroendocrine system" or the family of paraneurons [1,4].Normal levels of neuroendocrine markers are indispensable for spermatogenesis,and malfunction of one or more of the neuroendocrine regulated processes within the testis is closely related to the occurrence of sterility [13,14,15].However,the neuroendocrine nature of camel infertility is not well characterized.Here,we present histochemical results and the distribution characteristics of four neuroendocrine markers(NSE,SP,NFH and DβH)in cryptorchidism of camels.These result are necessary to clarify the capacity of Leydig cells to synthesize these substances,and to establish their functional signi cance in reproduction.
The Bactrian camel(Camelus Bactrianus)is an important domestic animal in some of the desert and semidesert areas of the world.Its ability to reproduce in very poor living conditions is related to its unique breeding mechanism.Cryptorchidism is reproductive system disease that causes sterility in camels.A few studies have reported the pathological characteristics of testes in infertile male Bactrian camels,such as abnormalities in the seminiferous tubule and the decrease in the synthetic ability of Leydig cells was a meaningful index for the further research of the steroid biosynthesis [16,17].However,there is no detailed report about the the neuroendocrine characteristics of cryptorchid testes in Bactrian camels.Additionally,we have compared the expression of the PGP9.5 and neuropeptide Y in Leydig cells between the normal and cryptorchid testes of Bactrian camels,and reported for the rst time the role of neural markers on Bactrian camel infertility [15].Based on these ndings,the aim of this study was to compare the distribution of NSE, SP,NFHand DβH in Leydig cells from the normal and cryptorchid testicular tissues of Bactrian camels,we also investigated the microstructural characteristics and markers of Leydig cells to explore the changes of the Leydig neuroendocrine characteristics,suggest that the young adult cryptorchid camels is innervated by NFH and DβH,which may serve as major endocrine markers for testicular functions at this age.The expression of SP was signi cantly decrease in cryptorchidism provide a morphological basis for further research on the roles in testes descent.The innervation of Bactrian camels testis by cholinergic and adrenergic nerves present are summarized in this study and include evidence of indirect,as well as rare direct neuroendocrine character of cryptorchidism Bactrian camels.

Methods
All protocols involving the use of animals were performed in accordance with the approved Guidelines for Animal Experiments of Gansu Agricultural University,and were approved by the Animal Experimental Ethical Inspection Committee of Gansu Agricultural University.

Experimental animals and test reagent
A total of 11 male unmated about two-year-old Bactrian camels were collected in winter fr om breeding farmers in Laohe County,Wuzhong City,Ningxia Hui Autonomous Region.Th ese home-bred animals were divided into two groups:the normal( ve of samples)and cryptorchidism grou ps(six of samples)for comparative analyses.The cryptorchid testes were located in the abdominal cavity (Intra-abdominal cryptorchidism).Tissue specimens were collected by orchiectomy.
The samples were treated according to different test schemes.Fresh testes were processed into small pieces and then divided into three samples.O n e sample was frozen in liquid nitrogen for western blotting hybridization reaction,while the other sample was xed with 4% paraformaldehyde solution for histochemical analysis,and the third xed in glutaraldehyde for transmission electron microscopy observation.The study desi gn was approved by the academic committee of the College of Veterinary Medicine of Gansu Agricultural University.
All antibodies were purchased from commercial suppliers. Rabbit polyclonal antibody NSE(bs-10445R-HRP,anti-NSE/HRP), SP(bs-0065R-HRP, anti-SP/HRP), NFH(bs-10680R-H RP, anti-NFH/HRP) and DβH(bs-0596R-HRP,anti-DβH /HRP), anti-rabbit IgG antibody(bs-0295G-AF488) and DAB chromogenic kit(ZLI-9018)w ere purchased from Beijing ZSGB-BIO Co.,Ltd., Beijing,China,Immunohistochemical stai n ing kit(SP-0023),produced by ZYMED Laboratories Co.,Ltd.,San Diego,USA, purchas e d f r om Jiamay Biotech Co.,Ltd.,Beijing,China.And other chemicals were obtained comm e rcially and of reagent grade. Experiment 1: H&E staining,histochemical staining,and observation of tissue samples Testicular tissue samples (1×1× 0.6 cm) were xed with paraformaldehyde and rinsed in running water for 24h before conv e ntional g r adient ethanol dehydration. Afterwards, they were made transparent with xylene, e mbedded i n para n,and 5μm serial sections were cut every veth section was mounted and stain e d either with standard haematoxylin and eosin(H&E) to examine the general m orphology, f ollowed by Alician blue(AB pH=2.5,30 min)and periodic acid Schiff reaction(PAS) and examined for the presence of positive staining cells,the acidic mucin and prote oglycan w e re blue,with the nuclei being reddish[18].

Experiment 2: transmission electron microscopic technique
The testes were cut into 0.1cm×0.1cm×0.1cm, xed in the 2.5% glutaraldehyde in 0.1M phosphatebuffered saline(pH=7.4) or 48h,and then post xed in 1% osmium tetr oxide at 4°C for 1h.The pieces were dehydrated with a graded ethanol series,and then e mbed d ed in epoxy resin (Epon812, American). Ultrathin sections were cut,stained with ura nyl ac etate and lead citrate,and then examined under a JEM-100CX electron microscope (NEC, J a pan).

Experiment 3: immuno uorescence staining
Green immuno uorescence staining:Para n sections of testicular tissues were prepared following the same procedure as demonstrated in section 1.2.1.Slice thickness was 5μm,high-pressure antigen retrieval was performed,30g/L H 2 O 2 aqueous solution was added to block peroxidase for 10min,Sections were incubated with goat serum albumin for 15min.A volume of 50μL of primary antibody(i.e.,rabbit polyclonal anti-NSE,anti-SP,anti-NFH and anti-DβH)was added dropwise at a dilution ratio of 1:1,000.For the negative control group,0.01mol.L-1 phosphate buffer solutionbuffered saline(PBS)was used as the substitute for the primary antibody.Incubation was carried out for 2h at 37°C before t he uorescent secondary antibody was added dropwise.Alexa Fluor488-labeled goat anti-r abbit IgG antibody(1:1,000,bs-0295G-AF488) was used as the secondary antibody.After incubation at 37°C for 2h,observations were made directly under the microscope.Positive c ells displayed uorescent green and the immuno uorescence images were collected by a n ECHO uorescence microscope(REVOLVE RVL-100-G).

Experiment 4: immunohistochemical staining
The streptavidin-peroxidase method was used for immunohistochemical staining.The p ara n sections were routinely dewaxed and differentiated by gradient ethanol;3% H 2 O 2 in methanol solution was used to block peroxidase for 10min.Normal goat serum album i n was incubated for 15min; 50μL of rabbit polyclonal anti-mouse NSE(bs-10445R),SP (bs-0065R),NFH(bs-10680R)or DβH(bs-0596R) antibodies were diluted at 1:400 and added dropwise t o e ach section separately.After incubation at 37°C for 2h and washing by PBS shaking,50μL of biotinylated goat anti-rabbit IgG working solution was added dropwise to each section,followed by the addition of 50μL of horseradish-labeled streptavidin working sol u tion.T-he sections were then incubated at 37°C for 15min.Freshly prepared DAB substr a te solute i on was then added for microscopic examination,followed by routine dehydration,making tr ansparent,and mounting.Positive cells displayed as brown-yellow,with nuclei sho wing as blue after counterstaining.The negative control group was stained with 0.01mo l·L-1 o f P BS instead of the primary antibody.

Experiment 5: western blot analysis
Total protein was extracted from testicular tissue was extracted.100mg of liquid nitrogen-pres e r ved testicular tissue was weighed and crushed.Radioimmunoprecipitation assay buffer c o n t aining phenylmethylsulfonyl uoride was added proportionally.The mixture was then h o m ogenized with a glass homogenizer until fully lysed,and then subjected to hypothermal centrifugation at 14,000×g for 5min.The supernatant was transferred to another tube,a nd r eagent with half of the volume of the solution was used to dissolve the precipitate.After standing still at 25°C for 30 min,the mixture was then centrifuged at 4°C and 1 9,575×gfor 20min.The supernatant was analyzed for protein concentration according to the instru ctions of the BCA protein assay kit(Beyotime Biotechnology Research Instit ute),dispense d,and stored at -70°C for use.
The protein was extracted and loaded following the routine procedure:the protein w a s separated by polyacrylamide gel electrophoresis(SDS-PAGE);5% stacking gel and 12% separating gel were prepared.30μg of protein was loaded to the gel sample well for ele c t rophoresis,and the protein in the gel with the target band was subjected to wet transfer t o the membrane.The membrane was incubated with primary antibody NSE,SP,NFH or D βH(1:1,000)at 4°C overnight,and then was washed by Tris-buffered saline+Tween 20(T B ST).The horseradish peroxidase-labeled cow anti-rabbit IgG was used as the secondary a nt i body for incubation for 2h at 25°C,and TBST was used to wash the membrane three ti mes for 10min each.The polyvinylidene uoride membrane was then subjected to color d evelopment.The chemiluminescent substrate solutions A and B were mixed at a ratio of 1:1,and were allowed to react at 25°C.The transfer membrane was photographed for an a l ysis.β-actin was used as the internal reference.

Measurement and statistical analysis
Tissue sections were imaged using a Nikon Eclipse 80i microscope camera sy s tem.Leydig cell characteristic index:ten sections were randomly selected from each group,and six non-repeating elds were randomly selected from each section(bar=20μm,400×).T he transverse and longitudinal diameters of Leydig nuclei,as well as the mean area of Leydig nuclei in the triangular and quadrangular mesenchymal tissues of each eld were randomly counted(n=40).Statistical analyses were performed by Image Pro Plus 6.0 soft ware.
The sections were imaged using a NIKON ECLIPSE 80i microscope camera s ystem.Five sections were selected randomly from each group for immunohistochemical s t aining.Six non-repetitive elds(bar=20μm,400×)were randomly selected for each section.T he mean positive signal intensity and positive area of each eld were statistically analyzed by Image Pro Plus 6.0 software to evaluate the average light absorbance.A total of 30 statistical data were collected for each group,and the results were expressed as mean±standard deviation(mean ± SD).SPSS15.0 software was used for statistical analysis,and the expression difference of NFH,SP,NSE and DβH between normal and cryptorchid testes was analyzed by one-way analysis of variance.Paired t-tests were carried out,and the lev e l of statistical signi cance was set at p<0.05.
The western Blot expression band was rst selected,the gray curve of which was t hen analyzed using Image J 1.48.The area under the peak was calculated as the band d ensity value.The density of β-actin was taken as the base value,and the relative densities were obtained by comparing the densities of NSE,SP,NFH or DβH expression bands in the normal testis group(Simply marked as N-NSE,N-SP,N-NFH or N-DβH)and the crypto r chidism group(Simply marked as C-NSE,C-SP,C-NFH or C-DβH).The relative density v a l ues were then statistically processed by SPSS 21.0 statistical software.All data were ex pressed as mean±standard deviation.Difference between variables were analyzed by t-tests difference between variables were analyzed using paired t-tests,and the level of statistical s igni cance was set at p< 0.05.

Results
Comparison of the histochemical characteristics between normal and cryptorchid testes of Bactrian camels As shown in Fig. 1, H&E staining revealed that the seminiferous epithelium in normal testes of Bactrian camels was developed well with seminiferous cells located on both sides of Sertoli cells,and round,oval,or irregular-shaped Leydig cells of considerably large size (Fig.a).We performed a histochemical analysis to identify the cellular changes underpinning the changes in cryptorchidism.We detected glycogen by PAS staining and distinct purple-red glycogen-positive bands in the lamina propria and interstitial capillary walls was observed (Fig.b);Acid mucins was also clearly visible in the interstitial capillary wall and lamina propria as demonstrated by AB staining (Fig.c);Examination of the H&E images revealed that cryptorchidism causes a reduction in layers of spermatogenic epithelium,the Leydig cells lacked distinct structural characteristics and the nucleus was round,elliptical,or irregular,with relatively large cell bodies (Fig.d);We next examined whether the reduction in seminiferous tubule cross-section was caused by a decrease in glycoprotein metabolism.PAS staining of the cryptorchid testes showed positive expression in the basement membranes and the wall of interstitial blood vessels (Fig.e),while the AB blue positive band was almost invisible in the interstitial tissue of the cryptorchid group (Fig.f).
Statistical analyses indicated that the transverse and longitudinal diameters of the nuclei in Leydig cells of cryptorchid Bactrian camels were signi cantly increased compared to those in normal testicles(p < 0.05).The average areas of the Leydig cells were signi cantly larger in cryptorchid testes than in normal testes(p < 0.01, Table.1).

Comparison of the ultrastructure of Leydig cells between normal and cryptorchid Bactrian camels
Electron micrographs showed that Leydig cells of the normal testis with the typical features of granular reticulum in the cisternae in direct continuity with this type of agranular reticulum.The granular reticulum were also sparse,the Golgi apparatus and mitochondria were dispersed,and lipid droplets were not obvious (Fig. 2a).Furthermore, collagen bers were distributed around the interstitial capillaries,which revealed endothelial cells with at nuclei and clear basement membranes in normal testicular tissue (Fig. 2b).The villi protrusions of the Leydig cell was unobvious and the swelling mitochondria and indistinct reticulum crowded in the cytoplasm which also consists of few lipid droplets.Moreover,the macrophages around Leydig cells exhibited obvious pseudopodia,but many lysosomes of different sizes and residual bodies were observed in cytoplasm (Fig. 2c).The capillary vessel was smaller with mostly clear pithelial basal lamina,and scattered particles were observed between adjacent microvessel endothelial cells,while perivascular collagen brils were richer in the cryptorchid group (Fig. 2d).
Comparisons of the distributions of NSE, SP, NFH and DβH in normal and cryptorchid testes of Bactrian camels In normal tissues (Fig. 3A a-d),the expression of NSE was primarily observed in Leydig cells and vascular endothelial cells,not in seminiferous epithelial cells( Fig. 3A-a),while the obvious SP immunopositive nerve bers in the interstitial tissue surrounding the Leydig cells( Fig. 3A-b);In addition,almost no NFH expression was observed in testicular tissues (Fig. 3A-c),whereas DβH was positively expressed in Sertoli cells, but not in seminiferous cells,Leydig cells or the surrounding tissues (Fig. 3A-d).No negative control had a positive reaction (Fig. 3A-e).Compared to normal testes,the expressions of NSE was obvious in Sertoli cells,peritubular myoid cells,and Leydig cells in cryptorchid tissues (Fig. 3A-f),but SP expression in the seminiferous epithelium and Leydig cells was signi cantly decreased( Fig. 3A-g),and NFH was clearly expressed in Leydig and seminiferous cells (Fig. 3A-h).In cryptorchid tissues,DβH was highly expressed in cytoplasm rich primary spermatocytes,with occasional expression observed in atypical nuclear shaped Sertoli cells (Fig. 3A-i).No expression of endocrine markers was detected in the negative control group (Fig. 3A-j).
In addition, no signi cant difference in NSE was observed in testicular tissues between normal and cryptorchid testes in Bactrian camels,and both the SP and NFH average absorbance were increased in cryptorchid testicular tissues.A much higher abundance of DβH was observed in the cryptorchid group than in the normal group (Fig. 3B).
Expressions of neuroendocrine markers was further examined in normal and cryptorchid testes of Bactrian camels by western blotting.As shown in Fig. 3C and D,the protein levels of NFH and DβH were signi cantly increased in cryptorchid testicular tissues(p < 0.01).
Comparisons of the localization of NSE, SP, NFH and DβH between normal and cryptorchid testes of Bactrian camels.
Green immuno uorescence analysis indicated that NSE was mainly expressed in Leydig cells and weakly expressed in the seminiferous epithelium (Sertoli cells,peritubular myoid cells and spermatogenic cells) of the normal testicular tissues (Fig. 4a),and SP was prominently expressed in Leydig cells and nerve bers in interstitial tissue (Fig. 4b).NFH was weakly stained in Leydig cells of normal testes (Fig. 4c).Similarly,DβH was weakly expressed in Leydig cells,whereas strong staining was detected in Sertoli cells,and no expression was detected in peritubular myoid cells of the normal group (Fig. 4d, Table 2).
As shown in Fig. 4e-f,in cryptorchid tissues,weak staining of NSE was observed in the whole testicular tissues (Fig. 4e),and SP was weakly expressed in the interstitial tissue (Fig. 4f).NFH protein was highly expressed in cytoplasm rich primary spermatocytes and Leydig cells,with occasional expression observed in atypical nuclear shaped Sertoli and peritubular myoid cells (Fig. 4g).DβH protein was strongly stained in the Sertoli and spermatogenic cells,while no expression was observed in peritubular myoid cells or Leydig cells (Fig. 4h,Table 2).

Discussion
Cryptorchidism is often associated with testicular heat stress,which leads to pathological changes,including decreased sperm motility,delayed development of adult stem cells,meiotic disorders,and reduced number of germ cells [20,21].Cryptorchidism of six-month-old Ziwuling Black goats caused a reduction in the number of spermatogenic epithelial cells,a signi cant decrease in the average diameter of the seminiferous tubules,abnormal differentiation of Sertoli cells,and retarded sperm development in the testes [22].Male Bactrian camels reach sexual maturity at approximately four years old and breeding starts at ve years old.The age of Bactrian camels studied here was two-year-old haven't eached mating age yet,the seminiferous tubules were developed with three to ve layers of seminiferous cells,but lack of obvious sperm in the seminiferous epithelia.and the cryptorchidism caused a reduction in layers of spermatogenic epithelium,which only comprised one to three layers.Therefore,the next experiments were designed to explore the underlying mechanisms of spermatogenic defects in cryptorchidism.
The interstitial connective tissue of mammalian testes is composed of different ingredients,such as blood capillaries,lymphoid vessels and Leydig cells.Leydig cells in testicular sections play crucial roles in male development and the maintenance of reproductive functions [23].We have demonstrated that the swelling of mitochondria in Leydig cells was related to oxidative stress in cryptorchid yaks [24]. Novo et al. [25] revealed that oxidative stress mediates mitochondrial dysfunction and programmed cell death.In the present study,the villus protrusions of Leydig cells were not obvious and with swelling mitochondria and indistinct reticulum crowding in the cytoplasm of Leydig cells in cryptorchidism.A previous study focused on the similarities between aging-associated and cancer-associated oxidative stress and mitochondrial dysfunction as their common phenotype [26].In our study,the pathological changes of cryptorchidism in camel should be closely related to the decrease in oxidative capacity of swollen mitochondria in Leydig cells.
There are two distinct populations of Leydig cells,fetal and adult,that arise at different times during the development of the testis [27].The adult population,appearing at or just before puberty,is responsible for spermatogenesis and the maintenance of male secondary sex characteristics [27].Leydig cells are rst recognized morphologically 2 days after birth with the appearance of lipid droplets in the cytoplasm of certain interstitial cells.The lipid content closely matches the steroid content of the developing testis and marks the maturation of the steroid synthesis pathway in tammar testis [28].Research has shown that the minimal concentration of testosterone in testicular tissues occurs in 2 year-old camels during the nonbreeding season,which was lower than that in 1.5 year-old and 3 year-old young camels and then increased with advancing age [29].Furthermore,the onset of puberty coincides with a dramatic increase in the average Leydig cell size,which accompanied by a peak in the steroid-producing capacity per Leydig cell [30].The present results show that the lipid droplets in Leydig cells were non-signi cant in cryptorchidism tissue compared to the normal camel,which was still at the early stage of pre-pubertal and sexual activity was not obvious and Leydig cells were in the process of development.Much evidences suggested that there is a signi cant correlation between the testosterone concentration and the size and number of Leydig cells [31,32].Our results showed that the average area of Leydig nuclei in cryptorchid tissues was signi cantly lower than that in normal testes,it was consistent with that of cryptorchid goats [22]. Ezeasor [33] demonstrated that the increase of the accumulation of hormone precursors in Leydig cells in cryptorchid goats stimulates immune stress due to decreases in the synthetic activity of Leydig cells.Therefore,our ndings regarding the ultrastructural alterations in the Leydig cell indicate defects in the steroid synthesis pathway in cryptorchidism,and also we observed that cryptorchidism caused obvious phagocytic characteristics of the macrophages near Leydig cells,it may be an clinical cue for degraded testes immune microenvironmental system.
Glycoprotein are important biological macromolecules that present at the cell membrane,intercellular substance,plasma,and mucus.Glycogen and glycoprotein are abundantly expressed in neurons,endocrine, and neuroendocrine cells [34,35].Prior studies showed that the positive bands of PAS were primarily distributed in the vascular wall and the basement membrane of seminiferous tubules in the testes of dromedary camels.It was clearly indicate that the camel testis contains a wide range of glycoconjugates and speci c carbohydrate structures are required for spermatogenesis during periods of rutting season and particularly apparent in the breeding period [36,37]. Methods for the detection of mucin are considered with the AB staining,acidic mucus containing hydroxyl and sulfate groups forms an insoluble complex which stains blue [18,38].The levels of acidic mucus increase with establishment of the Leydig cell groups and the maturity of spermatogenic cells [39].Our previous study observed PAS-positive bands distributed in the vascular wall and lamina propria of seminiferous tubules in the normal goat testes,while cryptorchidic goat caused a decrease in glycoproteins,which may indicate that saccharides function as essential components of basic nutrition metabolism,and play crucial roles in spermatogenesis [22].Here,we observed the same results in cryptorchidic Bactrian camel.The sulfated glycoproteins protect the normal activity of sperm [40].Our studies revealed that the cryptorchidism decreased the diameter of the leydig cell (p < 0.05) and induced glucose metabolism disorders in the surrounding connective tissue,which may contributed to the abnormal distribution of local neurohormones in testis.
Soluble brain protein 14-3-2 rst described by Moore and McGregor in 1965 is now known to be a cell speci c isoenzyme of the glycolytic enzyme enolase,designated neuron speci c enolase (NSE) [41].It is not only a marker for all types of neurons,but also for all neuroendocrine or paraneuronal cells [42].It has been proven that Leydig cells of human testis possess neuroendocrine properties and are therefore a member of the diffuse neuroendocrine(paraneuron)system,and NSE is immunopositive in the cytoplasm of human Leydig cells [43].Comparative immunocytochemical studies have demonstrated that NSE was detected in Leydig cells of the golden hamsters,guinea pigs,and rats at all stages studied: fetal,neonatal and adult.NSE may be synthesized by Leydig cells and was involved in the synthesis of steroids [43,44].Furthermore,the expression intensity of NSE in the testes of young rats and hamsters was higher than that of adults,and NSE is positively expressed not only in Leydig cells,but also in sperm cells and Sertoli cells in adult guinea pig testes [44].The demonstration that tumors of the nervous system and of neuroendocrine origin contain NSE was promoted the NSE as a possible tumor marker.Immunohistochemical staining revealed positive expression of NSE in human testicular tumors [45].NSE was predominantly detected in Sertoli cells caused by Leydig cell tumors in canine cryptorchidism [46].Here,the expression of NSE was only observed in Leydig cells and vascular endothelial cells of normal testes.More importantly,NSE was expressed not only in Leydig cells but also in Sertoli cells in cryptorchidism.Although there was no signi cant difference between the two groups,this difference in NSE expression is helpful to judge the pathological changes of testicular tumors.Our results suggested that cryptorchidism of the Bactrian camel has a tendency toward malignant transformation.
SP was accidentally isolated from the intestinal plain muscle and brain of horses in the 1930s by Euler and Gaddum,which lowers arterial blood-pressure and stimulates the tone and rhythm of the rabbit's isolated intestine [47].Euler demonstrated that human semen and extracts of the prostate glands of various animals have pharmacological actions due to SP [48].SP immunoreactivity can be detected in Leydig cells,particularly human testes,and to a lesser degree in mouse Leydig cells,it could be potentially paracrine substance regulating intratesticular function [49].SP is present in Leydig and Sertoli cells of humans,mice,hamsters,marmosets,and rats,and evidence suggests that it has potential regulatory roles regarding in spermatogenesis,spermatozoa function and motility,and testicular steroidogenesis [50].The SP immunopositive bers in camel testes were mainly distributed in the caput and cauda of testes [10].Expression of SP in cremaster muscle was decreased in cryptorchid tissues,resulting in a decrease in testicular autonomic nerve regulation,and testicular retention in the groin [51].Our study showed that immunostaining of SP in Leydig cells and peripheral nerve bers in normal testes was strongly positive,however,its distribution in cryptorchid tissues was signi cantly decreased.Therefore,our results suggest the distribution differences of SP in cryptorchidism may reducing the local autonomic innervation and related to steroidogenesis of Bactrian camel.
Neuro laments(NFs)are composed of light(NF-L),medium(NF-M),and heavy(NF-H)subunits of approximately 68,145,and 200 kDa,respectively.All three subunits are phosphorylated and most of the phosphorylation sites are located in the tail domain of NFH [52]New evidence that NFH exists within axons and in uences neurotransmission suggests that NFH might contribute to normal synaptic function and neuropsychiatric disease states [53].A correlation was found between the expression of NFH immunoreactivity in Sertoli cells and the stage of spermatogenesis;in human cryptorchidism,Sertoli cells exhibited strong NFH immunoreactivity whereas Sertoli and Leydig cells showed no or only weak reactivity in normal testes [54].NFH immunoreactivity in perivascular locations,intermingled with interstitial cells and close to the wall of seminiferous tubules was found only in the testes from immature rhesus monkeys.Furthermore,it show a marked degree of plasticity during development,especially around the time of puberty,and in cells of the tubular wall and vascular cells directly and/or indirectly via intermediation of mast cells [55].Immunohistochemical analyses showed that expression of NFH was signi cantly increased in Sertoli cell only syndrome and germ cell arrest biopsies in males [56].The results showed that during the recovery of rat testicular Leydig cells following ablation with ethane dimethanesulfonate,expression of NFH in Leydig cells increased,suggesting that NFH contributes to the proliferation of Leydig cells [57].We also discovered that immature of Leydig cells,the abnormal proliferation of the Sertoli cells and arrested spermatogenesis were the main characteristics in cryptorchid testes of Bactrian camels [5,16].The present study provides the rst evidence at the protein level for the existence of NFH in Sertoli and Leydig cells of the Bactrian camel cryptorchidism,and also western blot studies demonstrated that NFH was signi cantly increased in cryptorchid testicular tissues.Our study demonstrates that NFH correlates with developmental abnormalities of Sertoli and Leydig cells in Bactrian camel cryptorchidism.
Dopamine β-hydroxylase(DβH) is an adrenergic monoamine enzyme.DβH immuneoreactivity in the cytoplasm of interstitial Leydig cells in human testes provides strong evidence for the neuroendocrine nature of the Leydig cells [1].Immunohistochemical studies in pigs suggest that DβH-positive neurons should be considered elements of highly testosterone-dependent autonomic circuits involved in the regulation of urogenital function [58].A previous study identi ed Leydig cells as the presumed sites of catecholamine production in both fetal and mature human testes and further supported the recognized neuroendocrine characteristics of this cell type [59]. Romeo et al[60]found that DβH is a neurotransmitter involved in the regulation of testicular function and activates Leydig cell receptors,especially,during psychogenic or somatic stress environments,which could contribute to the high concentration of catecholamines and to suppression of testicular functions.Considering the controversial data presented in previous reports,compared to the control cases,Leydig cell-speci c expression of DβH was signi cantly weaker in human cryptorchidism and suggested that Leydig cells are not essentially involved in the pathogenesis of the disturbances[61].These results are in accordance with our immunohistochemical and western blotting data for DβH,which was weakly expressed in Leydig and peritubular myoid cells in Bactrian camel cryptorchidic tissue.We suggest that DβH expression in Leydig cells has little to do with whether they are normal.However,more importantly,the results presented here provide evidence that DβH is strongly expressed in the Sertoli and spermatogenic cells,which is very different from other reports.

Conclusions
The current study demonstrated that the underdeveloped seminiferous epithelium and pathological changes in cryptorchid tissue in Bactrian camels are potentially related to disorders in glycoprotein metabolism. Therefore, our results suggest that distribution differences ofNSE, NFH, SP and DβH in normal and cryptorchid testes of Bactrian camels are very important to analyze the neuroendocrine mechanism of cryptorchidism. NSE and SP were correlate with developmental abnormalities of Leydig cells in Bactrian camel cryptorchidism. The present study provides the rst evidence at the protein level for the existence of NFH and DβH in Sertoli and Leydig cells in Bactrian camel cryptorchidism and indicates a close relationship between DβH and Sertoli cells.These results provide a more in-depth understanding of neuroendocrine regulation that is crucial in animal cryptorchidism. Nevertheless, the mechanisms of neuroendocrine activity in Sertoli cells need to be further investigated.