Studies on biochemical, oxidative and genotoxicity alterations following vas blockage with reversible inhibition of sperm under guidance and reversal in rats.

CONTEXT: Vas obstruction with reversible inhibition of sperm under guidance (RISUG) for contraception and its reversal, may cause oxidative stress or inimical effects on male reproductive functions.
OBJECTIVE: To evaluate the biochemical and genotoxicity at the level of reactive oxygen species (ROS) following vas occlusion with RISUG and its reversal by Dimethyl sulphoxide (DMSO) and 5% NaHCO3 in Wistar albino rats. SETTINGS AND
DESIGN: Animals were divided into seven groups (n = 10), namely sham-operated control, short-term vas occlusion with RISUG for 90 days, reversal with DMSO and 5% NaHCO3, long-term vas occlusion with RISUG for 360 days, reversal with DMSO and 5% NaHCO3.
MATERIALS AND METHODS: Biochemical markers in reproductive tissues, hematology, serum biochemistry, serum electrolytes and ROS measuring indicators, e.g., lipid peroxidation, superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase were examined. STATISTICAL ANALYSIS USED: One-way analysis of variance test was performed for analyses of data obtained in this study using the SPSS 10.0 software (SPSS Inc., Chicago, IL, USA).
RESULTS: The tissue and clinical chemistry did not show appreciable alterations in RISUG injected and reversal Groups (II-VII) as compared to sham control. The genotoxicity and various ROS markers fluctuated within control limits following short- and long-term vas occlusion and reversal.
CONCLUSIONS: The study suggested that the reversal procedures, following RISUG contraception, were not associated with any kind of toxicological manifestations.

Introduction

Reactive oxygen species (ROS) levels may play an imperative role in capacitation, hyper-activation, and acrosome response towards fertilizing ability in the male reproductive system. The augmented production of ROS in tissues by endogenous or exogenous sources is detrimental and overwhelms the antioxidant defenses at the cellular level. In addition, raised free radical production deteriorates the antioxidant defense system ultimately causes oxidative stress (OS). It concurrently causes lipid peroxidation (LPO), DNA impairment, and apoptosis. An endogenous enzymatic defense system in the form of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST) exists in the body which tends to stabilize the cellular changes caused by ROS generation.[1] The OS has been concerned greatly as ROS and their metabolites can damage DNA, lipids and proteins; alter enzymatic machinery; produce irreparable changes resulting into cell death and finally, lead to recession in the reproductive ability.[2]

Reversible inhibition of sperm under guidance (RISUG®), through extensive preclinical and clinical studies, has been developed as an intravasal injectable male contraceptive.[345] Currently, advanced studies in the form of Phase III clinical trials are ongoing in India.[4] A major challenge for an ideal male contraceptive method is the reestablishment of fertility upon requirement. RISUG® has demonstrated functional reversibility in rats, rabbits, and langurs.[5] RISUG® blockage can be rid off using administration of Dimethyl sulphoxide (DMSO) or NaHCO3 in the lumen of the vas deferens.[6] As vas deferens is exposed to RISUG® and reversal solutions, effects on testis and epididymis become a matter of concern. Towards exploring possible effects on RISUG® reversal in terms of OS on the reproductive system we examined clinical biochemistry, alterations in genotoxicity, and generation of ROS in reproductive tissues, following short-and long-term reversal of RISUG® blockage by means of two solvent systems (DMSO and 5% NaHCO3) in rats, as an animal model.

Materials and Methods

Test materials

RISUG® was used as test material in the present investigation. Prof. S. K. Guha, Indian Institute of Technology (IIT), New Delhi kindly provided the drug. DMSO and 5% NaHCO3 were used for blockage reversal.

Animal model

The 10–12 weeks old Rattus norvegicus (Male Wistar albino rats), weighing from 150 g to 180 g with established fertility were maintained and reared in rat cages with normal room temperature, humidity, and light facility in the experimental animal house of the University Department of Zoology, Jaipur. Free access of safe drinking water and standard rat pellets were provided to animals. CPCSEA guidelines on scientific experiments on animals were followed to maintain animals with veterinary supervision.[7] The Institutional Animal Ethics Committee approved all experimental protocols vide letter No. UDZ/2013/IAE/03 dated January 28, 2013.

Experimental protocol

A total of 70 animals were allocated in the present study. Groups I–VII contained 10 animals each. In Group I, the animals were undergone for bilateral vas occlusion, but without administration of RISUG®(served as sham-operated control); Group II: The animals were vas occluded with 5–7 μL of RISUG®, bilaterally, and sacrificed after 90 days; Groups III and IV: The animals were undergone for vas blockage reversal, respectively, with DMSO and 5% NaHCO3 after 90 days of blockage; Group V: The animals were vas occluded with 5–7 μL of RISUG®, bilaterally, and sacrificed after 360 days; and Groups VI and VII: The animals were undergone for vas blockage reversal, respectively, with DMSO and 5% NaHCO3 (250–500 μL) after 360 days of blockage.

Vas occlusion

Bilateral vas blockage was performed in rats and postoperative care with confirmation of vas blockage by mating studies was carried out as reported earlier.[6]

Vas occlusion reversal

Short-term (90 days) and long-term (360 days) vas blockage reversal and its assessment of success were done as reported earlier.[6]

Sacrification schedule

Animals were sacrificed as per the completion of the experimental schedule.

Homogenate preparation

The target tissues (testis and epididymis) were homogenized, the resultant supernatant obtained following centrifugation at 10,000 × g for 20 min at 4°C and used for tissue biochemistry and the levels of ROS.

Parameters

Tissue biochemistry

In testis, cholesterol, glycogen,[8] lactate dehydrogenase (LDH)[9] and protein[10] were estimated. In the epididymis, sialic acid,[11] L-carnitine,[12] neutral α-glucosidase activity, and protein were measured. Fructose and acid phosphatase were also assayed in the seminal vesicle and ventral prostate, respectively, according to the WHO manual.[13] All markers were estimated spectrophotometrically (Model No. 108, Systronics, Ahmedabad, India).

Clinical chemistry

Hematology

A part of blood was obtained in vials containing Ethylenediaminetetraacetic acid via cardiac puncture during scheduled sacrification. Estimations of red blood cell (RBC), white blood cell (WBC), hemoglobin, packed cell volume, and standard hematological indices, namelymean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and MCH concentration (MCHC) were made by Automated Hematology Analyzer (CBC-360Plus, Accurex Biomedica Pvt. Ltd., Mumbai, India).

Serum biochemistry

Remaining blood was received in normal, without the coating of any anticoagulant, blood collection tubes and kept for clotting at room temperature for separation of serum following centrifugation at 3000 rpm for 10 min. Various serum biochemical parameters, viz., glucose, protein, albumin, bilirubin, serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase, creatinine, urea, creatine phosphokinase, cholesterol, high-density lipoprotein, and Triacylglycerol Lipase were analyzed by assay kits (Transasia Biomedical Ltd., Mumbai, India) with the help of automatic biochemical analyzer (Erba Smartlab, Mumbai, India).

Serum electrolytes

With the above referred automatic biochemical analyzer various electrolytes in serum were also analyzed by assay kits supplied by Span Diagnostic Ltd., Daman, India, Accurax Biomedical Ltd., Thane, India, Biorex Diagnostic Ltd., Antrin and United Kingdom, and Centronic GmbH, Wartenberg, Germany.

Genotoxicity

Micronucleus test

At the time of sacrification of animals, femurs were dissected out, flushed with normal saline to harvest bone marrow cells in vials containing 5% v/v fetal bovine serum (FBS). The collected fluid was spined and the pellet of bone marrow cells was resuspended in FBS to wash and again centrifuged. The prepared smear on glass slides were processed for fixation and staining, respectively, in methanol for 15 min and May-Gruenwald and Giemsa.[14] The polychromatic (PC) and normochromatic (NC) erythrocytes were counted and the ratio of both types of cells was estimated. In a separate set of animals, mitomycin C obtained from Acros Organics, New Jersey, USA was administered (3 μg/g b.wt., i.p.) before 48 h of scarification schedule to serve as positive control.

Chromosomal aberration test

The cell division arrested at the metaphase stage was obtained by injecting 0.04% colchicine prior to 2 h of sacrification schedule. Bone marrow cells were collected by flushing the femur of rats in a test tube. After centrifugation, the pellet processed in 0.56% potassium chloride hypotonic solution was fixed in Carnoy's fluid. This cell suspension was dropped on precooled clean slides for staining with 5% Giemsa. Under x100 oil immersion, the chromosomal aberration was counted in 100 metaphase arrested cells. Likewise, mitotic activity was also observed in 100 bone marrow cells. Estimation of cytotoxic activity was made by using mitotic index. Acrylamide purchased from Himedia Lab Pvt. Ltd., Mumbai, India was administered orally at the dose of 50 mg/kg body weight for 5 days in animals to serve as positive control.[15]

In vitro DNA damage in spermatozoa

The sperm samples collected from cauda epididymis were washed two times in 0.9% normal saline. The samples were smeared on clean glass slides, air-dried, fixed in Carnoy's solution and stained in 0.1% acridine orange. After washing in deionized water, the slide was air-dried and observation was made for green (normal) and red or orange (disrupted) sperm heads under a fluorescent microscope with the setting of excitation filter at 490 nm and barrier filter at 530 nm (Labomed, Labo America Inc., USA) for calculation of DNA damage percentage.[16]

Reactive oxygen species and antioxidant system

Fresh homogenate supernatant (described above) was used for estimations of LPO,[17] superoxide dismutase,[18] CAT,[19] GPx,[20] and GST[21] in testis and epididymis.

Statistical analysis

The values are depicted as mean ± standard deviation and subsequently comparison was made between different experimental groups for calculation of statistically significant variations. The analysis of variance was executed for data obtained in the present investigation with SPSS software version 10.0 (SPSS Inc., Chicago, IL, USA). To analyze the inter-group statistical difference between means the multiple comparison test of Holm-Sidak was applied. The obtained P ≤ 0.05 were used as significant.

Results

Tissue biochemistry

Concentrations of cholesterol, glycogen, LDH and protein in testis were observed in the range of 4.35 ± 2.02 mg/g to 5.58 ± 2.90 mg/g; 2.00 ± 0.92 mg/g to 2.90 ± 0.95 mg/g; 13.83 ± 1.24 U/g to 17.25 ± 1.12 U/g; and 3.60 ± 0.58 mg/g to 4.28 ± 1.31 mg/g, respectively [Figure 1a–d].

Figure 1

(a-d) Biochemical markers in testes of sham control, vas occluded and reversed male albino rats

The levels of biochemical markers in the epididymis, namely sialic acid, L-carnitine, neutral α-glucosidase, and protein remained unaltered following 90 and 360 days of RISUG® injection and after its reversal [Figure 2a–d].

Figure 2

(a-d) Biochemical markers in epididymis of sham control, vas occluded and reversed male albino rats

The fructose concentration produced by seminal vesicle and activity of acid phosphatase in ventral prostate ranged from 6.04 ± 1.02 mg/g to 7.84 ± 1.21 mg/g; and 0.36 ± 0.04 to 0.44 ± 0.02 U/mg, respectively. The tissue biochemistry was marked without any significant difference in both short- and long-term vas blockage with RISUG® and after the reversal (Data not shown).

In Groups I-VII, the range of RBC (5.27 ± 0.21 106/mm 3–5.45 ± 0.23 106/mm3), WBC (6.00 ± 0.26 103/mm 3–7.17 ± 0.90 103/mm3), Hb (13.35 ± 0.67 g per 100 mL–13.93 ± 0.31 g per 100 mL), PCV (44.71 ± 2.02%–48.74 ± 4.52%), MCV (77.92 ± 7.42 fL–90.71 ± 6.80 fL), MCH (23.78 ± 3.77 pg–26.07 ± 1.00 pg) and MCHC (28.31 ± 2.51%–32.04 ± 3.14%) was recorded, respectively (Data not shown). The hematological parameters did not indicate any alterations in experimental groups as compared to sham control.

The levels of various biochemical parameters were observed without any significant changes as compared to Group I (Data not shown).

Serum electrolytes of experimental groups were remained unaltered as compared to Group I (Data not shown).

The presence of micronucleated PC erythrocytes (PCEs) was determined among 1000 PCEs in total RBCs. Among animals of all Groups (I– VII) micronuclei were found in between 0.07 and 0.11% and ratios of PCEs/NCEs were observed to lie in between 24.51 and 26.80%. No significant variations were found within the groups under study.

In animals, after vas occlusion and reversal no change was detected in the incidence of micronuclei in PC and NC erythrocytes as compared to Group I. Total micronuclei frequencies in erythrocytes in positive control animals, however, exhibited 0.74%, a marked significant increase (P < 0.001) when compared to all experimental groups (Data not shown).

Chromosomes with the least abnormalities were observed in both single and double chromatids with no significant variation in chromosomal aberrations among different experimental groups. Considerable enhancement (P < 0.05) in total aberrant cells was noticed in positive control in comparison to animals of other groups. Mitotic indices as well as frequency of aberrant cells were remained unaltered [Table 1].

Table 1

Frequency of aberrant cells* of bone marrow in sham control, vas occluded and reversed male albino rats

Treatment groups	Cells with single aberration	Cells with more than one aberration	Total aberrant cells
Group I	21.2±1.30	0.8±0.44	22.0±1.00
Group II	22.0±0.67**	-	22.0±0.67**
Group III	24.0±0.92**	-	24.0±0.92**
Group IV	20.0±0.68**	-	20.0±0.68**
Group V	18.6±1.14**	1.2±0.20**	19.8±1.30**
Group VI	18.8±1.09**	1.2±0.44**	20.0±1.41**
Group VII	20.0±1.22**	1.6±0.54**	21.6±1.48**
Positive control (acrylamide @50 mg/kg body weight)	46.4±3.64#	23.8±2.86#	70.2±5.16#

Values are mean±SD (n=10). *100 metaphase cells/animal, # Statistically significant (P<0.05) when compared with all groups, **Statistically non-significant when compared with Group I. SD: Standard deviation

Values of DNA damage in Groups II-VII were found to be in the range of 2.54% ± 0.78%–3.02% ± 0.54% in comparison to animals of Group I, indicating no detectable significant changes (Data not shown).

Antioxidant system and reactive oxygen species in testis

The various ROS markers, namely LPO, SOD, CAT, GPx, and GST levels in testes of experimental groups did not show marked variations as compared to sham control [Figure 3a–e].

Figure 3

(a-e) Reactive oxygen species and antioxidant system in testes of sham control, vas occluded and reversed male albino rats

Antioxidant system and reactive oxygen species in epididymis

Animals of all study groups did not show any marked variation in the aspect of different markers of ROS in epididymis after vas blockage and reversal upon comparison to animals of sham-operated control [Figure 4a–e].

Figure 4

(a-e) Reactive oxygen species and antioxidant system in epididymis of sham control, vas occluded and reversed male albino rats

Discussion

Male infertility due to OS is considered as one of the eminent causes. In vas obstructive methods, production of free radicals and their metabolism require balance for normal germinal cells function. The failure in detoxification and repair in the biological system may lead into serious damages in cells and tissues. This cell damage can be escaped by the interaction of antioxidants with free radicals to prevent their formation in the cells. The relevant antioxidant enzymes such as SOD, CAT, GPX, and GST evade oxidation by reducing the rate of chain formation. The oxidation chain can halt everlasting through these enzymes by finding primer free radicals.[22] Traditional and most opted vas-based contraception through vasectomy and its reversal has been observed with elevated levels of ROS which may lead to sub-fertility.[23] As RISUG® is substantially injected into vas deferens followed by its reversal through solvents, it is necessary to examine the postresponsive effect to correlate the previous studies. In this article, thereby, we presented the alterations in biochemical markers, ROS levels and genotoxicity, if any, as subsequent effects on the reproductive function, following short-and long-term exposure to RISUG® blockage and its reversal by both solvents in rats.

The accumulation of cholesterol in the testis is associated with the inhibition of spermatogenesis or disturbance in testicular metabolism. Glycogen is the main source of energy for the maturation of germ cells. It has been observed that glycogen stores get depleted towards the end of the spermatogenic cycle in a number of species. Elevated testicular glycogen concentration has been reported with germinal cell aplasia, impaired testicular metabolism and inhibition of spermatogenesis. LDH, a carbohydrate metabolism marker of spermatogenesis, plays an essential role in the energy supplementation process for spermatozoa motility. Any modifications in the enzyme will undoubtedly influence their respective physiological functions in the testis, which may lead to poor fertility.[24] Testicular necrosis ensuing antispermatogenic drug administration lowers the testicular protein contents chiefly due to lack of postspermatogenesis stages.[25] In the present study, lack of significant variations in testicular biochemistry were observed following vas blockage with RISUG® as well as its reversal in rats which suggests normal functioning of spermatogenesis in the testis.[6]

Sialic acid either free or bound to proteins is secreted by the epididymis. Its concentration in sperms and plasma of lumen may create an impact on epididymal sperm maturation and participate in the maintenance of ionic balance, epididymal and sperm antigenic interactions, acrosome stabilization and intact structure of spermatozoa.[26] In sperm metabolism, L-carnitine provides freely available energy to spermatozoa for use which directly affects motility, maturation, and overall spermatogenic phenomena of sperms as well as protection against ROS by exerting antioxidant properties.[27] Neutral α-glucosidase plays a substantial role in epididymal spermatozoa maturation and diagnosis of epididymal dysfunction.[28] The secretory function of the seminal vesicle is reflected by fructose that has been regarded as source of energy for sperm motility. The acid phosphatase as a prostatic biomarker analyses the glandular functions and their secretions. Currently, the epididymal, seminal vesicle, and prostatic biochemical markers showed no detectable changes following vas blockage with RISUG® and its reversal in this study also indicates normal secretory activity.

In serum clinical biochemical study, the levels of different serum biochemical and serum electrolyte parameters were recorded unaltered due to vas blockage with RISUG® followed by its reversal. Studies in langur monkeys also indicated that clinical biochemical parameters did not exhibit any significant alterations due to RISUG® administration followed by its reversal.[4]

Blood as a fundamental benchmark for biological studies, is the most important body fluid that governs vital functions. Values of hematological parameters in experimental groups fall within the normal range as of Group I and hence, in agreement with previous results observed in langur monkey, rhesus monkey and human.[3]

The present study, for the first time, also reports whether the reproductive organ membranes are susceptible to ROS generation or not. Increased or decreased levels of LPO in testis after the administration of a drug may also contribute to the suggested vulnerability of these membranes to OS.[17] The SOD converts highly reactive superoxide radicals to less reactive free radicals, namely hydrogen peroxide (H2O2).[1] CAT also plays an important role in cellular detoxification by converting H2O2 to water and oxygen molecule. The GPx and GST function for detoxification of reactive lipid peroxidases. In our results, the values of ROS markers fluctuated within the limits of sham control indicating that a balance was maintained between ROS and antioxidants even after RISUG® administration and its reversal in both short-and long-term studies. Advanced well-planned investigations are warranted to elucidate the particular role of OS facilitated by cells.

In the recent past decade, the role of DNA integrity in males has been crucially highlighted which is important for the transmission of genetic information. The concept of the study relies upon the existence of RISUG® plug in the vas deferens till requirement of contraception as well its reversal upon need. Therefore, the chronic effects on chromosomes need to be examined to assess the possible mutagenic hazard. The ratio of PC to NC erythrocytes was proved its toxic effect on bone marrow components. Presently, observed results indicated no significant alterations in erythrocytes with micro-nuclei incidences of bone marrow proves noncytogenetic consequence. The observed values on the mitotic index remained unchanged in all groups, simultaneously. A study indicated remarkably smaller micronucleus frequencies despite tested styrene maleic anhydride (SMA) doses (LD50:240 mg/day body weight; Maximum tolerated dose: 166.6 mg/day body weight) in the treatment groups was not found clastogenic at the level.[29] Presently, random frequency of chromosomal aberration with comparable results to the corresponding control was observed that suggested no selectivity, without any consequence on the chromosomal aberration incidences. Previously, a study related to bone marrow chromosomal aberration was investigated in mice with administration of SMA at 2–10 mg/kg body weight doses. Significant chromosomal aberrations were not seen in these investigations. Our present study also revealed outcomes without significant DNA loss in cauda epididymal spermatozoa. Therefore, past and present investigations further confirm that reversal following RISUG® administration does not alter the chromosomal structure and generate micronucleus suggesting free from any genotoxicity.

Conclusions

The success of any vas-based contraceptive relies on the long-term efficacy, safety, and reversibility upon need. In the light of all the above results, it is concluded that the overall procedure of vas obstruction and its reversal does not cause acute or chronic stress and toxicity with unaltered biochemistry and normal glandular secretions. Extensive studies with observations on relevant parameters suggested that OS do not lead to any toxicological manifestations following RISUG® induced contraception reversal. Therefore, this approach will be a preferable addition in available contraceptives for men in near future.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.