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Electroacupuncture enhances spermatogenesis in rats after scrotal heat treatment

Spermatogenesis is regulated by a cascade of steroid regulated genes in the testis. Recent studies suggested that acupuncture may improve fertility in men with abnormal semen parameters. Yet, the underlying mechanisms in which acupuncture enhances spermatogenesis remain largely unknown. Here we used a scrotal heat-treated rat model to study the effect of electroacupuncture (EA) on recovery of spermatogenesis. In this model, spermatogenesis was disrupted by 30 min scrotal heat treatment at 43°C. Ten sessions of EA were given at Baihui (GV20), Guanyuan (CV4), Zusanli (ST36) and Sanyinjiao (SP6) from day 9 to day 36 post-treatment. Sperm motility and production, morphology of the germinal epithelium by Johnsen’s scoring, germ cell apoptosis by TUNEL staining, proliferation by proliferating cell nuclear antigen (PCNA) staining, as well as serum testosterone and inhibin B levels by immunoassays were evaluated on day 0, 1, 9, 25, 37, 46, 56 and 79. When compared with the heat-treated (H) group, the heat-treated plus EA (H+EA) group showed a significant increase (p < 0.05) in PCNA-positive cells and inhibin B levels on days 37 and 46, and a higher Johnsen’s score till day 56. On day 79, motile spermatozoa could be found in the vas deferens of H+EA group only. Consistently, there was a trend of improved motility and increased number of motile epididymal spermatozoa in the H+EA group than the H group; while apoptosis of germ cells and serum testosterone levels were similar between the two groups. Taken together, EA enhanced germ cell proliferation through improvement of Sertoli cell functions. This may facilitate the recovery of spermatogenesis and may restore normal semen parameters in subfertile patients.


Introduction
Subfertility affects 15% of couples worldwide, and half of the cases are attributed to male factors including impaired sperm production, activity and transport. 1,2 The causes of male subfertility are multi-factorial. Genetic, epigenetic, environmental and lifestyle-related factors, as well as gene-environmental interactions are known to affect human fertility. 3 Intervention includes surgical correction of varicoceles and obstructions of the male reproductive ductal system, hormonal therapy, antioxidant therapy, antibiotics, corticosteroids, methylxanthines, vitamins, minerals and amino acids, but only a few of them have been confirmed to be effective by randomized controlled studies. 4,5 Acupuncture has a history of 3000 years in China and is increasingly used throughout the world especially in the UK, 6 US, 7 Australia 8 and Japan, 9 for pain relief and a variety of disorders, including subfertility. [10][11][12] It is an effective alternative for pain relief during oocyte retrieval. 10,13 Acupuncture on the day of embryo transfer may improve the live birth rate of IVF patients. 10,14,15 Accumulating evidence suggests that acupuncture may improve sperm count, [16][17][18] motility, 19,20 morphology, 21 ultra-structural integrity 22 and fertilization rate after ICSI 19 in subfertile men, partly by modulation of FSH, LH and testosterone levels. 23 Acupuncture has been postulated to increase antioxidant supply by vasodilation 21 and regulate immune defense and local inflammation. 17 However, the underlying mechanism on how acupuncture improves male fertility remains largely unknown.
The heat-treated testis model in rats was successfully used to study spermatogenesis. [24][25][26] Heating of the rat testes in a 43°C water bath for 15 min significantly reduced the testicular weight and increased serum FSH and LH levels on day 9. 25 The decrease in testicular weight and sperm count was partially recovered by day 97. 26,27 Heat-treatment caused apoptosis of germ cells in the testis. The number of apoptotic cells increased rapidly in a stage and cell specific manner one day after treatment but returned to normal level by day 9. 25 The number of proliferating cells as detected by proliferating cell nuclear antigen (PCNA) staining decreased after heat-treatment 28 or in experimentally induced cryptorchid testis in mice. 29 In humans, a transient increase in scrotal temperatures by occupational exposure, lifestyle, clothing or cryptorchidism was correlated with low sperm concentrations, 30,31 reduced sperm motility 32 and reduced percentage of normal morphology, 33 although some of the associations remained controversial. 34,35 We hypothesize that electroacupuncture (EA) increases proliferation and decreases apoptosis of the germ cells in the heat-treated rat testis, thereby facilitates the recovery of spermatogenesis. Therefore, the objective of the present study is to investigate the changes in spermatogenesis and sperm count in heat-treated rats with or without EA treatment.

Results
Body weight, weight of testis, epididymis, seminal vesicle and coagulating gland. The weight of the rat, epididymis, testis and seminal vesicle plus coagulating gland were compared among the untreated control (C), heat-treated (H) and heat-treated plus electroacupuncture (H + EA) groups. No significant differences were found in the body weight and the weight of seminal vesicle plus coagulating gland among the three groups from day 37 onwards. The weights of the testis and the epididymis were significantly reduced (p , 0.05) on day 25 after heat treatment (H25). The weight of the testis, but not of the epididymis increased gradually from day 46 onward in both H and H + EA groups. However, there was no significant difference on the weight of the testis or the epididymis between the H and H + EA groups till day 79 ( Table 1).
Histology of the seminiferous tubules. The representative histology of the seminiferous tubules of the three groups was shown in Figure 2. There was a drastic change in histology one day after heat exposure (H1). These changes included a decrease in the epithelial thickness, loosening and vacuolization of the germinal epithelium, presence of cellular debris in the lumen, appearing of multinucleated giant cells, and pyknotic cells with fragmented nucleus. On day 9 (H9) and 25 (H25), majority of the seminiferous tubules contained spermatogonia with or without an incomplete layer of primary spermatocytes and no spermatids. Spermatogenesis recovered steadily from day 37 onwards. There were an increasing proportion of normal tubules containing all types of germ cells. Compared with the H group, the H + EA group exhibited a faster recovery in spermatogenesis, though both groups showed variation in the extent of recovery among individual animals within the same treatment group and among seminiferous tubules within the same testis manifested as the presence of some poorly-recovered tubules scattering among normal tubules. The Johnsen's score ( Table 2) was used to quantify the difference in spermatogenesis in the three groups. There was a significant decrease in Johnsen's score on day 9 (H9) and 25 (H25) after heat treatment when compared with the control (C0). On day 37, the Johnsen's score of the H + EA group (H + EA37) was comparable to that of the control group (C37) indicating a recovery of spermatogenesis, while the Johnsen's score of the H group did not reach a normal value until day 79. From day 37 to day 79, the Johnsen's score was higher in the H + EA group than the H group, albeit the differences were not statistically significant.
Germ cell proliferation. The proliferation of germ cells was detected by PCNA staining (Fig. 3). The number of PCNApositive cells per tubule was significantly reduced on day 25, 37 and 46 after heat treatment (H25, H37 and H46) (p , 0.05, Table 2) when compared with the controls (C0, C37 and C46). On day 37 and 46, the number of PCNA-positive cells of the H + EA group was comparable to that of the control group (C37 and C46), but was significantly higher than that of the H group (H37 and H46, p , 0.05). Interestingly, the germ cell proliferation was comparable among the three groups from day 56 onwards. Results were expressed as mean ± SD. a-b is significantly different (p , 0.05) between treatment groups of the same day. * is significantly different (p , 0.05) from C0.
Germ cell apoptosis (TUNEL assay). The number of apoptotic cells per tubule increased from 0.2 in the control group on day 0 (C0) to 63.7 one day after heat exposure (H1) (p , 0.05, Table 2 and Figure 3). The value returned to normal on day 9 due to the depletion of germ cells, and remained low during the recovery of spermatogenesis. No statistical difference (p . 0.05) in the number of apoptotic cells was observed among the control, H and H + EA groups from day 37 to day 79.
Serum inhibin B and testosterone concentration. As shown in Table 2, serum inhibin B levels decreased significantly one day after heat treatment (H1). The inhibin B levels were higher in the H + EA group than the H group on day 37 (p = 0.053) and 46 (p , 0.05). No difference was detected in serum testosterone levels after heat treatment or among the three groups throughout the study ( Table 2).
Number of motile spermatozoa in the vas deferens. Scrotal heat treatment significantly decreased the number of motile spermatozoa in the vas deferens as no motile spermatozoa were detected in both H and H + EA groups from day 25 to day 56 ( Table 3). Interestingly, some motile spermatozoa appeared in the vas deferens of the H + EA group from day 79, and the number was not significantly different from that of the control group (p . 0.05). No motile spermatozoa were found in the H group till the end of the study on day 79 ( Table 3).
Motility of epididymal spermatozoa. No motile spermatozoa were found in the epididymis by day 25 post-heat treatment. Newly produced motile spermatozoa were found in the H + EA group from day 46 and in the H group from day 56, but the percentage of progressive motile (a+b) and non-progressive motile (c) spermatozoa in these groups were significantly lower than that of the control group (p , 0.05). On day 79, the H + EA group had a percentage of non-progressive motile spermatozoa comparable to while that of the H group remained significantly lower (p , 0.05) than that of the control group. Yet, the difference in the percentages was not statistically significant between the H and H + EA group (p . 0.05).

Discussion
In the present study, the effects of acupuncture in spermatogenesis and semen parameters were studied using a rat model with heat-treated testes. When compared with the H group, the H + EA group exhibited a faster recovery of spermatogenesis as shown by a higher Johnsen's score and an improved sperm production and motility. This improvement by EA is associated with an enhanced germ cell proliferation (PCNA staining) and Sertoli cell function (inhibin B level), but not with germ cell apoptosis and Leydig cell function.
The heat-treated testis model is commonly used for spermatogenesis study. A single heat exposure of the rat scrotal testis at 43°C for 30 min significantly reduced the number of motile spermatozoa on day 25 post-treatment. This is consistent with others that the number of spermatozoa in the heat-treated testis and epididymis decreased significantly by day 35 and gradually recovered thereafter. 26,27 In a surgical cryptorchid rat model, the number of intact spermatozoa in the testis and epididymis decreased remarkably and no intact spermatozoa was found on day 15. 36 In addition, the percentage of motile spermatozoa in the epididymis decreased from day 3 after the surgery, and the sperm motility was significantly impaired from day 5. 37 In mice, scrotal heat-treatment at 40°C for 60 min increased the percentage of abnormal spermatozoa in cauda epididymis from day 7 to day 35. 38 Heat treatment decreased the number of PCNA positive cells in the testis. It was likely that the total number of spermatogonia and spermatocytes was reduced. In sheep, heat treatment reduced the production of A 1 spermatogonia after 20 d, 39 and the spermatogonia were arrested. 40 Moreover, heat treatment compromised DNA synthesis and reduced DNA polymerases activities in the germ cells of a cryptochid rat model. 28 In the present study, almost all secondary spermatocytes and round spermatids were depleted on day 9. The depletion of germ cells was mediated by apoptosis and formation of multinucleated giant cells. 25, 36 We also found that the proliferation of germ cells was reduced at day 25, but returned to normal level on day 56 onward after heat treatment.
The Johnsen's score in the H + EA group was comparable to that of the control group on day 37 after treatment, indicating a full recovery of spermatogenesis after 10 sessions of EA treatment, while the scores in the H group on days 37, 46 and 56 were significantly lower than that of the control. Although the differences in the Johnsen's score and the percentage of advanced tubules between the H + EA and H groups were not statistically significant, probably due to the small sample size and the large sample variation within the same group, a trend of faster recovery in the H + EA group was consistently seen throughout the study.
We observed an increase in germ cell proliferation in the H + EA group when compared with the H group, and a positive correlation between the proliferation index and the Johnsen's score (Spearman's rank correlation coefficient: 0.716, p , 0.001). Although there is no evidence on a stimulatory effect of EA on germ cell proliferation, EA facilitates neurogenesis and maturation of newborn neurons in the striatum after transient global ischemia. 41 Moreover, EA induces differential expression of genes related to cell differentiation, cell proliferation, muscle repair and hyperplasia in mouse skeletal muscles. 42 Furthermore, pre-treatment with moxibustion at one of the acupoints, Zusanli (ST36), of this study, was reported to promote proliferation of gastric mucosal cells in stress-induced gastric ulcer. 43 EA facilitates the recovery of spermatogenesis by increasing the motility and production of spermatozoa in the epididymis. Clinical evidence suggests that EA increases sperm count in oligozoospermic patients. [16][17][18]22 In the present study, sperm production was measured by the number of motile spermatozoa in the vas deferens as the immotile spermatozoa lost their fertilization ability in vivo. On day 79, a small amount of motile spermatozoa were only present in the H + EA group but not in the H group. Yet, the increase in sperm motility may result from an enhanced epididymal function by EA, since epididymis is the major place for sperm maturation and storage. 44 It may be worthwhile to investigate whether the improved sperm motility after EA was related to changes in the water, pH value or protein profiles in the epididymal fluid that affect sperm maturation and storage. 45 How EA stimulates spermatogenesis and increases spermatogonial proliferation remains obscure. It is possible that EA modulates the hypothalamus-pituitary-gonadal axis by altering FSH and LH secretion. Increased FSH secretion induced the production of follistatin and inhibin, which facilitated the transition of gonocytes to spermatogonia in culture of 3-d-old rat testicular fragments. 46 Furthermore, FSH was considered as a prerequisite for the completion of spermatogonial mitosis and the differentiation of spermatocytes into spermatids. 47 In the heat-treated testis model, the elevation of FSH level from day 9 to day 28 25 is possibly a response to facilitate spermatogonial recruitment and proliferation when most of the apoptotic cells have been removed. Thus, the improved basal cell proliferation in the present study may be partly mediated via EA-induced upregulation of FSH. Accumulating evidence suggest that acupuncture restored the lowered FSH to normal level in subfertile men. 23 However, whether EA played a similar role in the present study on regulation of FSH needs further investigations.
The serum testosterone levels as well as the weight of the androgen-responsive glands (seminal vesicles and coagulating glands) did not change with or without EA. It was reported that heat treatment did not affect plasma and testicular testosterone concentrations, 25 although an increased in serum LH may drive the Leydig cells to produce enough testosterone to maintain normal spermatogenesis. 48,49 In line with this, both acupuncture and Chinese herbs were found to restore the reduced LH and testosterone concentrations in subfertile men and rats. 23,50 It is possible that GnRH may be involved in the regulation of FSH and LH by EA. In fact, EA was reported to increase the number of GnRH neurons, the GnRH mRNA level in the hypothalamus, and the GnRH-receptor mRNA level in the pituitary in the ovariectomized rats. 51 In normal female rats, the expression of hypothalamic GnRH was higher after EA treatment. 52 It may be worthwhile to investigate the GnRH level if the FSH and LH levels are confirmed to be changed after EA.
EA affects ovarian blood flow (OBF) and it was found that low-frequency (2 or 10 Hz) EA increased the OBF by the ovarian sympathetic nerves via supra-spinal pathways in normal rats. 53 Similarly, heat treatment of rat testes to 43°C for 30 min resulted in a significant reduction in blood flow per testis. 49 Since the testes and ovaries share similar innervations, EA with a frequency of 3 Hz/9 Hz in the present study may also improve the testicular blood flow, facilitating the delivery of nutrients for germ cell repopulation and tissue repair. Future studies should be Results were expressed as mean ± SEM for normally distributed data and median (range) for skewed data. a-b is significantly different (p , 0.05) between treatment groups of the same day. * is significantly different (p , 0.05) from C0.
www.landesbioscience.com Spermatogenesis performed to examine the effect of EA on testicular blood flow with a laser-Doppler flow probe. 54 Although manual acupuncture is more common in clinical practice, EA was mostly used in research for the sake of standardization of stimulation. The present EA protocol was chosen based on its positive effect on spermatogenesis, semen parameters and hormone levels. 16,17,19 The selected acupoints were commonly used in both research and clinical practice related to the reproductive system. According to the Chinese medicine theory, Baihui belongs to the Du meridian and Guanyuan to the Ren meridian. Acupuncture on the two points helps to restore the balance between Yin and Yang. Acupuncture also nourishes the Kidney Yin at Sanyinjiao and improves production of Qi and blood at Zusanli, which are regarded as necessaries for fertility. Although a Deqi sensation (i.e., a feeling of soreness, numbness, distension or pain by patients) represents adequacy of stimulation in humans, the sensation cannot be assessed in rats. A widely accepted criterion for successful stimulation in animal acupuncture is the local muscle twist, which reflects the activation of muscle-nerve afferents. [51][52][53] The adequacy of EA stimulation in animals may also be confirmed by the blood β-endorphine level, which was elevated by 30 min and remained high even at 24-h after acupuncture in humans. 55 The dosage of EA (10 sessions) may be inadequate since in standard clinical practice, it usually takes several months to treat subfertility. 56 The effect of EA on spermatogenesis may be more prominent if additional sessions are given.
In conclusion, EA improves sperm production after scrotal heat treatment. EA facilitates the recovery of spermatogenesis by enhancing germ cell proliferation and restoring normal inhibin B levels. The underlying mechanism may be related to the changes in hormone levels, testicular blood flow as well as Sertoli cell and Leydig cell functions after EA and warrants further investigations.

Materials and Methods
Animals. Adult SD rats at 9-11 weeks old were used. The rats were housed at the Laboratory Animal Unit of the University of Hong Kong and were acclimatized for at least 7 d before experimentation. They were kept under controlled temperature (22°C) and 12 h light-dark cycles, with free access to water and food. The experiment protocol was approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong (No. 1554-07), and was conducted according to its guidelines for the use of experimental animals.
Scrotal heat treatment and electroacupuncture. Rats were divided into 3 groups (C: control; H: heat-treated; H + A: heattreated plus electroacupuncture, n = 3-6 at each time point). Before heat treatment, the rats were anesthetized. The lower body and scrota containing testes were immersed in a thermostatically controlled water bath (Grant Instruments, model SS40-2) at 43°C (H and H + EA) or 22°C (C) for 30 min. Animals in the C and H group were sacrificed on day 0, 1, 9, 25, 37, 46, 56 and 79 after heat exposure (Fig. 1A). Animals in the H + EA group received additional EA under general anesthesia every 3 d from day 9 to day 36 (10 sessions in total) after heat treatment, and were sacrificed on day 37, 46, 56 and 79 (Fig. 1A). Each EA session lasted for 25 min. Disposable stainless steel needles (0.25 ¾ 40 mm) (Suzhou Medical Appliance, catalog number: AY1981) were inserted into 6 acupoints in the scalp, abdomen and hind limbs [Baihui (GV20), Guanyuan (CV4), Sanyinjiao (SP6; bilateral), Zusanli (ST36; bilateral)], which were then connected to an electrical stimulator (ITO, Model ES-160) (Fig. 1B). The acupoints were electrically stimulated with alternating frequencies of 3 Hz and 9 Hz to prevent desensitization of tissue to stimulation. Individual pulses were symmetric, bi-phasic square wave pulses with alternating polarities and a pulse duration of 0.25 ms. The intensity was the minimal level to induce local muscle contractions (normally 1.5-2 mA), which indicated the activation of muscle-nerve afferents. 53 Animals in the control and H group received anesthesia without EA.
Blood collection, tissue preparation and release of spermatozoa from the vas deferens and epididymis. The serum from scarified rats was obtained from clotted blood by centrifugation at 4000 rpm for 30 min and stored at -80°C until used. The serum concentration of testosterone and inhibin B was measured by the enzyme linked immunosorbent assay (Diagnostic Systems Laboratories, catalog number: DSL-10-4000 for testosterone and DSL-10-84100i for inhibin B). Each vas deferens was removed with a length of 3 cm from the end joining the prostate gland, and was placed in a Petri dish containing 0.2 ml pre-warmed (37°C) modified Biggers, Whitten, and Whittingham (BWW) medium. 57 The spermatozoa were flushed out with 0.8 ml pre-warmed Table 3. Vas deferens sperm count and motility of epididymal spermatozoa of control (C), heat-treated (H) and heat-treated plus electroacupuncture (H + EA) rats on day 37, 46, 56 and 79     Results were expressed as median (range). a-b is significantly different (p , 0.05) between treatment groups of the same day. * is significantly different (p , 0.05) from C0.
www.landesbioscience.com Spermatogenesis modified BWW from a syringe fitted with a blunt ended needle. The sperm suspension from vas deferens of both sides was then pooled in a 2 ml eppendorf tube. Meanwhile, the left testis was weighed, cut transversely from the center and fixed in 4% paraformaldehyde. The right testis and epididymis, bilateral seminal vesicles plus coagulating glands were also weighed before frozen at -80°C. The distal half of the left cauda epididymis was cut into 3 portions and incubated in 2 ml modified BWW at 37°C for 10 min to facilitate release of spermatozoa and recovery of sperm motility. 58 Histological evaluation of spermatogenesis by the Johnsen's score. Paraffin-embedded testis were cut in 5 mm and stained with hematoxylin (Sigma-aldrich) and eosin (Sigma-aldrich, E6003). More than 100 cross-sections of the seminiferous tubules from each animal were scored from 1 (no cells in the tubule) to 10 (complete spermatogenesis), according to the most advanced cell type in the tubule. The Johnsen's score was calculated as the mean of these scores. 59 The assessor was blinded to the assignment of treatment.
Immunohistochemical staining for PCNA. The assay was performed as published with minor modification. 60,61 Briefly, after deparaffinization and antigen retrieval, the sections were quenched with 3% H 2 O 2 for 30 min, followed by blocking with 10% rabbit serum (Sigma-aldrich, R9133) in phosphate-buffered saline for 30 min. The sections were then incubated sequentially with PCNA primary antibody (PC-10, DAKO, catalog number: M0879) at 1:5000 in the blocking solution for 1 h, biotinylated rabbit anti-mouse IgG (DAKO, catalog number: E0354) at 1:3000 in the blocking solution for 30 min and ABC (Vector Laboratories, Inc., catalog number: PK-6100) for 30 min. The peroxidase activity was visualized with 3,3'-diaminobenzidine-4 HCl (DAB) (DAKO). The slides were counterstained with hematoxylin, dehydrated, and mounted with the Permount TM (Electron Microscopy Sciences, catalog number: 17986-01). In the negative control, blocking solution was used in place of the primary antibody.
The proliferation index was calculated as the total number of PCNA-positive cells (spermatogonia and preleptotene primary spermatocytes) per tubule. Only basal germ cells were counted because they are the cells at the cycle of active DNA synthesis. 60 Twenty randomly chosen tubules were evaluated for each slide, because our unpublished data showed that the proliferation index from 20 tubules was representative of that from more tubules. The assessor was blinded to the assignment of the treatment.
In situ terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. The TUNEL assay was performed on the deparaffinized sections with the In Situ Cell Death Detection Kit, AP (Roche Diagnostics), according to the manufacturer's instructions. The sections were then counterstained with hematoxylin (Sigma-aldrich) and mounted with DAKO mounting solution (DAKO). Positive control sections were pretreated with deoxyribonuclease I (40 IU/mL) (USB, 78411) for 10 min at 37°C before labeling. Sections for the negative control were incubated in the staining solution free of the terminal deoxynucleotidyl transferase.
The level of germ cell apoptosis was expressed as the total number of TUNEL-positive germ cells (red signal) per tubule. 62 More than 100 randomly chosen, round tubules from each specimen were evaluated in a blinded manner. Since the aim was to assess germ cell apoptosis during recovery, tubules from day 37 to day 79 were evaluated only if more than 5 spermatocytes or more advanced cells were present; this would exclude the condition in which the low incidence of apoptosis was due to loss of germ cells. The staging of seminiferous tubules was performed according to the criteria previously published. 63 Sperm motility and number. Eight microliter of sperm suspension was placed in a Cell VU chamber with a chamber depth of 20 mm (Millennium Sciences Inc.) on a heated (37°C) microscope stage to facilitate free movement of the spermatozoa. The assessment was performed in line with the WHO guidelines for semen analysis 64 and ESHRE guidelines for sperm motility. 65 The concentration of spermatozoa from the vas deferens was determined with the improved Neubauer hemocytometer according to the WHO criteria. 64 Sperm number was calculated as sperm concentration ¾ 2 ml / 6 cm (million per cm vas deferens). The number of motile spermatozoa was obtained by multiplying the sperm count with the percentage of motile spermatozoa in the vas deferens.
Statistical analysis. The results were expressed as mean ± SEM for normally distributed data and median (range) for skewed data. Statistical analysis was done by One Way ANOVA for normally distributed data and Kruskal-Wallis One Way ANOVA on Ranks for skewed data with SigmaStat (Version 3.10, Systat Software, Inc.). Differences with a p value less than 0.05 were statistically significant.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.