Taurine and N-acetylcysteine reverse reproductive and neuroendocrine dysfunctions in levetiracetam-treated epileptic male rats

ABSTRACT Reproductive neuroendocrine dysfunction is becoming common among patients with epilepsy, Notably, possible indication of taurine and N-acetylcysteine (NAC) in the management of reproductive impairment has increased over the years. However, scientific evidence to supplement our understanding of the impact of taurine and NAC in protecting reproductive neuroendocrine cells is lacking. Hence, this study aimed to investigate the possible effects of taurine and NAC on levetiracetam-induced reproductive neuroendocrine dysfunction in epileptic rats. The study was done in two experimental protocols: drug alone and reversal. In the drug-alone protocol, rats in groups 1–6 received treatment orally for 21 days, respectively. In the reversal-protocol, the animals in group 1, received saline (2.5 mL/kg) and served as normal control, while epilepsy was induced in animals in groups 2–5, after which groups 3–5 received levetiracetam (150 mg/kg), levetiracetam (150 mg/kg) + NAC (100 mg/kg), levetiracetam (150 mg/kg) + taurine (150 mg/kg) respectively for 14 days. Pilocarpine alone or in combination with levetiracetam significantly decreased neuro-endocrine related factors, hormonal profile, and spermatogenesis, whereas treatment with NAC and taurine reversed the effects induced by pilocarpinethrough neuro-endocrine-related factors and hormonal modulation mechanism.


Introduction
Epilepsy is a common brain disorder characterized by the recurrence of unprovoked seizures caused by abnormally excessive or synchronous neuronal activity in the brain, usually with two unprovoked seizures >24 h apart.Over 70 million people are affected globally, and it is bimodally distributed, with the highest risk in infants and older age groups [1,2].
The search for the etiology and mechanisms of action of epilepsy has led to the use of animal models in epilepsy research, thus implicating the use of drugs such as pilocarpine in the induction of epilepsy in animal model [3,4].Consequently, epilepsy related to the drugs used in treatment, as well as the disease itself have been reported to play a role in the etiology of infertility [5].Clinical studies have shown that epilepsy itself and antiepileptic drugs alter the hypothalamicpituitary-gonadal (HPG) axis, thus affecting secretion of pituitary hormones and levels of the peripheral sex steroid hormones [6][7][8][9][10].In both sexes, sexual problems and endocrine changes have been frequently associated with the disorder [11][12][13].
Levetiracetam (LEV) is the most common first-line antiepileptic drug (AED) that is widely and frequently utilized in the treatment of epilepsy during childhood, adolescence, young adulthood, and adulthood periods [14,15].However, its utility has been implicated in the pathogenesis of male infertility [10,16].Therefore, the search for natural therapeutic agents such as N-acetylcysteine (NAC) and taurine might be a core value in the management of reproductive impairment associated with levetiracetam therapy.
Taurine and NAC are endogenous molecules produced by cysteine, and increase gonadal function via hormonal enhancing mechanisms and spermatogenesis [17,18].Because of the widespread clinical use of LEV in the treatment of epilepsy, LEVinduced alteration in HPG axis has been substantially reported in large group of patients with epilepsy [10,16].19 Given the lack of scientific investigation of the reproductive neuroendocrine mechanisms involving taurine or NAC in the management of pilocarpine-induced epilepsy, it is therefore necessary to investigate the therapeutic effects of taurine and NAC in LEV-treated epileptic male Wistar rats.

Experimental animal
Approved Research Committee on the Ethical Use of Animals (DSUA Care), with reference number REC/FBMS/DELSU/18/06, was used for this study.Fifty-five adult male Wistar rats of 10-12 weeks old, weighing 160 ± 10 g were procured from the animal house in University of Medical Sciences, Ondo, Ondo State.The animals were cared for and maintained in the animal facility in accordance with regulation, guidelines and policies governing use of animals in research as described in public health service policy on human care and use of laboratory animals approved by Institute of Laboratory Animal Resource, National Research Council (2011).

Animal care
The animals were kept in a ventilated plastic cages, cushioned with wood shavings and housed in the animal facilities of University of Medical Sciences, Ondo, Ondo State, Nigeria.The animals were monitored under standard laboratory conditions: at 35.5-37.0•Cand 12 h light: 12 h darkness cycle.Rats were fed with standardized pellet (vital feed) and clean water.Proper hygiene was also observed in their housing.

Drugs administration
Pilocarpine, NAC, taurine and LEV were purchased from Sigma-Aldrich (USA).Pilocarpine was administered at 30 mg/kg intraperitoneally, as established by Wang et al. [3].NAC was administered at 100 mg/kg orally following the dose established by Emojevwe et al. [19].Taurine and LEV were administered at 150 mg/kg orally as established by Oyovwi et al. [20,21] and Baysal et al. [10], respectively.NAC, taurine and LEV were administered orally using normal saline as a vehicle.

Induction of seizure and grading of epilepsy symptoms
Epileptic-like symptoms was induced by single intraperitoneal administration of pilocarpine (30 mg/kg) as established previously [3].Within an hour after pilocarpine administration, epilepsy symptoms exhibited by the animals were graded based on the Racine grading criteria, as recently adopted by Wang et al. [3] and Ahmed and Che [4].Grade 0: No response; Grade I: Facial clonus (blinking, moving, rhythmic chewing, etc.); Grade II: plus rhythmic nod in addition to facial clonus; Grade III: Rhythmic nod, facial clonus plus forelimb myoclonus without upright hind limbs; Grade IV: forelimb myoclonus without upright hind limbs plus upright hindlimb; Grade V: generalized tonic, a burst of seizures, and loss of position control.Grade IV and above was used for confirmation of epilepsy.

Sample collection
At the end of the experimental duration, the animals were fasted overnight and euthanized under low-level ether anesthesia in order to prevent marked chemical changes and blood samples were collected from each rats via the retro-orbital sinus into plain bottle.The testes, hypothalamus, pituitary gland and cerebrum were carefully dissected out, weighed and used for histological evaluation, while epididymis were collected and used for physiological assays.Blood samples were collected from retro-orbital sinus for biochemical assays.

Serum hormone analysis
The concentrations of GnRH, follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone and Kisspeptin were determined using standard laboratory kits involving the enzyme-linked immunosorbent assay (ELISA) technique using a ELISA machine, according to the manufacturer's recommended protocol as described previously [18,19,22].

Semen analysis
From the caudal epididymis of each rat, sperm fluid was squeezed onto a microscope slide.Sperm motility was assessed by counting the number of motile spermatozoa per unit area and was expressed as motility per unit area.Sperm counts were made with the aid of a hemocytometer and expressed as millions/ ml of suspension.Sperm viability was determined by preparing a uniform smear of spermatozoa on the slides using eosin-nigrosin stain according to the method of Omirinde et al. [23] and Yu et al. [24].Sperm morphology was evaluated by aniline blue staining method [23,24].

Sperm plasma Integrity
This was done by the hypo-osmotic swelling test (HOST) method following the description of Oyovwi et al. [25], in which a hypo-osmotic solution was prepared by adding 0.735 g of sodium citrate dehydrate and 1.351 g of fructose to 100 ml of distilled water.After which, 0.1 mL of epididymis sperm suspension was added to 1.0 mL of hypo-osmotic solution and incubated for 30-60 min at 37°.A drop of the mixture was then added to a glass slide and a cover slip was applied.The slide was then viewed with an ×40 lens to observe swellings.Sperms with a swollen tail were regarded as host positive, while those without a swollen tail were regarded as host negative.

Sperm capacitation, acrosome reaction and chromatin integrity
Sperm capacitation and acrosome reaction were estimated using the Coomassie brilliant blue staining technique earlier described by Oyovwi et al. [17].Sperm chromatin integrity was examined as by the toluidine blue staining technique as used by Selvam and Agarwal [26], and repeated by Emojevwe et al. [19].

Histological study
The right testicle was harvested and immediately fixed in Bouin's fluid for at least 24 h before a standard histological technique.Each sample was dehydrated using ascending grades of alcohol (70%, 80%, 90%, 95%, 100%).
It was cleared with two changes of xylene, embedded in paraffin wax, trimmed, nicked and sectioned using a microtome and stained using hematoxylin and eosin (H&E) for the purpose of determining the general morphology changes at ×10 and ×40 magnifications using a Tecnai G2 (FEI, USA) transmission electron microscopy.All changes were confirmed by a histology specialist

Statistical analysis
Version 8 of GraphPad prism was used for the analysis of data obtained from the study.The data was expressed as Mean ± standard error of mean (SEM) (n = 5 per group).The differences in mean were compared by a one-way analysis of variance (one-way ANOVA) followed by Bonferroni post hoc test for multiple comparison, while p < 0.05 was considered statistically significant.

Effect of NAC, taurine, and LEV treatments on sperm indices on naïve and pilocarpineinduced epilepsy in rats
Figures 1 and 2 represent the effects of NAC, TAU and LEV on the sperm indices in naïve and pilocarpine-induced epilepsy in rats.
As represented in Figure 1 (drug-alone treatment protocol), treatment with LEV shows a significant (p < 0.05) decreased in sperm motility (Figure 1a), sperm count (Figure 1c) with corresponding increase in abnormal sperm morphology (Figure 1d) when compared with normal control groups receiving normal saline.However, co-administration of LEV + NAC and LEV+TAU individually increased sperm motility, count, and decreased abnormal sperm morphology.No significant changes were seen in sperm viability (Figure 1b).
administered alone and in combination with LEV significantly decreased sperm motility (Figure 2a) and sperm count (Figure 2c); and increased abnormal sperm morphology (Figure 2d) when compared with normal control groups.However, LEV + NAC and LEV + TAU respectively significantly reversed the decrease caused by pilocarpine-induced epilepsy relative to pilocarpine-alonetreated rats.More so, combined treatment with LEV + TAU revealed a highly significant decrease in abnormal sperm morphology when compared with pilocarpine-treated rats and LEV + NAC.But, no significant changes were noticed in sperm viability (Figure 2b) in all the treated groups

Effects of NAC, taurine and LEV treatments on sperm membrane integrity in naïve and pilocarpine-induced epilepsy in rats
Figure 3 represents the effects of NAC, TAU and LEV on sperm membrane integrity in naïve, while Figure 4 shows the effects of NAC, TAU and LEV on sperm membrane integrity in pilocarpineinduced epilepsy following the used of HOST.As presented in Figure 3 in the drug-alone treatment protocol, LEV-treated rats showed a significant (p < 0.05) decreased in HOST value (Figure 3) when compared with normal control group.
Accordingly, NAC and TAU administered alone increased sperm HOST values in the rats treated with LEV relative to LEV-treated rats alone.
In the reversal treatment protocol shown in Figure 4, pilocarpine administered alone and in combination with LEV significantly decreased HOST value (Figure 4) when compared with normal control groups.Notwithstanding, LEV + NAC and LEV + TAU respectively significantly reversed the decreased caused by pilocarpine-induced epilepsy relative to pilocarpine-treated rats alone.More so, combined treatment with LEV + TAU revealed a highly significant increased HOST value when compared with pilocarpine-treated rats and LEV + NAC.

Effects of NAC, taurine and LEV treatments on sperm chromatin integrity in naïve and pilocarpine-induced epilepsy in rats
Figure 5 represents the effects of NAC, TAU and LEV on sperm chromatin integrity in naïve rats using toluidine blue staining (TBS).As presented in Figure 4 in the drug-alone treatment protocol, NAC-, TAU-and LEV-treated rats show no significant (p < 0.05) alteration in sperm chromatin integrity when compared with normal control groups.In the reversal treatment protocol, as shown in Figure 6, pilocarpine administered alone and in combination with LEV significantly decreased sperm chromatin integrity (Figure 4b) when compared with normal control groups.Meanwhile, coadministration treatment, LEV + NAC and LEV + TAU respectively significantly reversed the decrease caused by LEV in pilocarpine-induced epilepsy.

Effects of NAC, taurine and LEV treatments on sperm acrosomal reaction and capacitation in naïve and pilocarpineinduced epilepsy in rats
Figures 7 and 8 represent the effects of NAC, TAU and LEV treatments on sperm acrosomal reaction and capacitation in naïve and pilocarpine-induced epilepsy in rats using commassie brilliant blue staining (CBBS).As represented in Figure 7 (drugalone treatment protocol), treatment with LEV shows a significant (p < 0.05) decreased in acrosome-reacted capacitated sperms (Figure 7a), acrosome intact capacitated sperms (Figure 7b) and acrosome intact uncapacitated sperms (Figure 7c) when compared with normal control groups.However, co-administration of LEV + NAC and LEV +TAU individually increased sperm acrosome reaction and capacitation.
In the reversal treatment protocol, shown in Figure 8, one-way ANOVA followed by post hoc test showed that pilocarpine administered alone and in combination with LEV significantly decreased acrosome reacted capacitated sperms (Figure 8a), acrosome intact capacitated sperms (Figure 8b) and acrosome intact uncapacitated sperms (Figure 8c) when compared with normal control groups.But, LEV + NAC and LEV + TAU respectively significantly reversed the decrease in sperm acrosome reaction and capacitation caused by pilocarpine-induced epilepsy relative to pilocarpine-treated rats alone.More so, combined treatment with LEV + TAU revealed a highly significant higher sperm acrosome reaction and capacitation when compared with pilocarpine-treated rats and LEV + NAC.reproductive hormone and kisspeptin in naïve and pilocarpine-induced epilepsy , respectively.As represented in Figure 9 (drug alone protocol), treatment with TAU showed a significant (p < 0.05) increase in the level of LH (Figure 9a) relative to normal controls were as no changes were observed in NAC-alone-treated rats.Furthermore, LEV-treated rats revealed a significant drop in LH level when compared to the control group.The combined treatment with LEV + NAC and LEV + TAU elevated LH concentration as compared to LEV-treated rats alone.As shown in Figure 9 b, treatment with NAC showed a significant (p < 0.05) increase in the level of FSH relative to normal controls, meanwhile no changes was observed in TAU-alone-treated rats.Furthermore, LEV-treated rats revealed no significant changes in FSH level when compared to control groups.Figure 9c treatment with both NAC, TAU and LEV shows no significant (p < 0.05) changes in the level of testosterone relative to normal controls.Furthermore, NAC, TAU also did not show any significant (p ≤ 0.05) changes in the level of GnRH (Figure 9d) relative to normal controls.However, LEV a significant (p ≤ 0.05) decrease in the level of GnRH. Figure 9e, treatment with both NAC, TAU and LEV produced significant (p < 0.05) changes in the level of kisspeptin relative to the LEV group, but however did not show any significant changes when compared with the normal control, respectively.Combination of LEV and NAC also shows significantly higher values when compared with LEV alone.

Effects of NAC, taurine and LEV treatments on serum reproductive hormones and kisspeptin in naïve and pilocarpine-induced epileptic male Wistar rats
In the reversal protocol (Figure 10), pilocarpine administered alone and in combination with LEV significantly declined the level of serum LH (Figure 10a) [F (4, 15) = 6.740, p = 0.025] when compared with normal control group.However, NAC and TAU coadministered with LEV respectively significantly reversed the decrease in LH concentration caused by LEV alone or in combination with pilocarpine.Figure 10b shows that pilocarpine administered alone and in combination with LEV significantly declined the level of serum FSH [F (4, 15) = 17.96, p < 0.0001] when compared with normal control groups.However, LEV + NAC and LEV + TAU respectively significantly reversed pilocarpine-induced decrease in FSH concentration relative to pilocarpine- treated rats.More so, combined treatment with LEV + NAC revealed a highly significant increase in serum FSH level when compared with pilocarpine-treated rats.In Figure 10c, pilocarpine administered alone and in combination with LEV significantly declined the level of serum testosterone [F (4, 15) = 29.49,p < 0.0001] when compared with normal control groups.Interestingly, LEV + NAC and LEV + TAU respectively significantly reversed pilocarpineinduced decrease in testosterone concentration relative to pilocarpine-treated rats.Moreover, combined treatment with LEV + TAU revealed a highly significant increase in serum testosterone level when compared with pilocarpinetreated rats.A similar trend was also shown in serum GnRH (Figure 10d) as pilocarpine administered alone and in combination with LEV significantly decreased serum GnRH level [F (4, 15) = 11.65,p = 0.002] when compared with normal control groups.However, LEV + NAC and LEV + TAU respectively significantly reversed pilocarpine-induced decrease in GnRH level relative to pilocarpine-treated rats.Additionally, combined treatment with LEV + NAC revealed a significant increase in serum GnRH when compared with pilocarpinetreated rats.Figure 10e shows a significant reduction in the pilocarpine group and the group treated with pilocarpine and LEV when compared with the control group.However, the group treated with pilocarpine + LEV + TAU have a significantly higher level of kisspeptin when compared with the pilocarpine and the pilocarpine +LEV-treated groups, respectively.

Effects of NCA, taurine and LEV treatments on pilocarpine-induced histological alteration in the testes of male Wistar rats
The effects of the various treatments on testicular architecture are shown in Plate 1. Accordingly, testicular tissues from the control containing normal and completely developed germinal cells and few seminiferous tubules exhibiting maturation arrest.pilocarpine+LEV +TAU-treated group shows several normal seminiferous tubules containing normal and completely developed germinal cells and few seminiferous tubules exhibiting maturation arrest.

Effects of treatment on pilocarpine-induced histological alteration in the cerebral cortex, anterior pituitary and hypothalamus of male Wistar rats
The effects of NAC and taurine on levetiracetam (LEV) and pilocarpine-induced histomorphological alteration in the cerebral cortex, pituitary and hypothalamus of male Wistar rats is shown in plates 2-4.Accordingly, tissues from the control, NAC and taurine groups showed normal cerebral cortex (plate 2), pituitary cells (plate 3) and hypothalamic neuronal cells (plate 4).In the LEV-treated group however, there was mildly necrotic stroma of the pituitary.The groups treated with LEV+NAC and LEV+TAU respectively did not show any changes in the cerebral cortex (plate 2), pituitary (plate 3) and hypothalamus (plate 4).But pilocarpine-treated group shows mild cerebral congestion while the pituitary and hypothalamus appeared normal.Furthermore, the brain section from the group treated with pilocarpine + LEV shows cerebral cortex with some neuronal cells appearing chromatolyzed, pituitary cells look necrotized while neuronal cells of the hypothalamus exhibiting chromatolysis.The photomicrographs of brain section from the group treated with pilocarpine + LEV + NAC and the group treated with pilocarpine + LEV + TAU respectively show high levels of improvement in architecture of the cerebral cortex (plate 2) as compared to the PIL+LEV group, while the pituitary (plate 3) and hypothalamus (plate 4) appear normal in both groups.

Discussion
The therapeutic implications of NAC and taurine on LEV-treated pilocarpine-induced epileptic rats, linked to reproductive neuro-endocrine dysfunction, were evaluated in this study.Accordingly, we reported that treatment with NAC and TAU is safe for use as they did not cause any harmful effects as compared to the control, while LEV caused neuroendocrine and reproductive dysfunctions in the non-epileptic rats.In the rats exposed to pilocarpine-induced epilepsy, there was neuroendocrine and testicular dysfunctions, as evidenced by the decreased neuro-endocrine related factors (Kisspeptin and GnRH), decreased reproductive hormone (testosterone, FSH, and LH) levels, reduced spermatogenesis, reduced sperm membrane integrity as indicated by a decrease in HOST value, and increased abnormal chromatin integrity following toluidine blue staining analyses.LEV administered to pilocarpine-induced epileptic rats aggravated the negative impacts of pilocarpine and further caused a decrease in testicular acrosome-reacted sperm capacitation, alteration in testicular architecture, as well as increased brain neuronal degeneration.However, treatment with NAC or taurine reversed the reproductive neuro-endocrine dysfunction induced by LEV, pilocarpine or their combined effects.
The Pilocarpine animal model of epilepsy has been well used for the study of the disease [27,28].The model intends to mimic temporal lobe epilepsy (TLE), and therefore, rodents display a similar 'clinical history' as seen in patients with epilepsy [28,29].The mechanism of action of pilocarpine in this regard was previously reported to occur through a primary peripheral effect on white blood cells, thereby resulting in elevated serum levels of IL-1β, which alters blood-brain barrier (BBB) permeability.Notably, this alteration causes ionic imbalance due to K + accumulation in the extracellular space and causing convulsive activity by stimulating muscarinic receptors in the brain [30].However, the mechanism of action of LEV remains unclear, but it is believed to induce seizures by reducing the release of glutamate, the primary excitatory neurotrnamitter in the brain, thereby inhibiting musculoskeletal receptor activity.Furthermore, some reports have also suggested that the mechanisms of the antiepileptic and neuroprotective actions of LEV seem to be mediated, at least in part, through the combination of inhibitory effects on depolarization-induced and Ca 2± induced neurotransmitter release [31].
In the present study, pilocarpine caused vascular congestion and chromatolysed cells in the cerebral cortex, confirming its effect on the cerebral cortex, possibly via the mechanisms highlighted above.LEV administered was able to resolve the vascular congestion, justifying its usage in the management of epilepsy.Also, in the naïve rats, TAU and NAC did not cause any negative alterations in the semen quality and reproductive hormonal status, and testicular and brain architecture.In fact, they were found to be beneficial as they caused a significant rise in the levels of LH, FSH and kisspeptin as well sperm count in these animals when compared to their control counterpacts, possibly due to their cyto-protection potentials.Male reproductive hormones are essential for initiating reproductive function [32][33][34].It is known that kisspeptin has an oversight function in the release of GnRH, while the normal pulsatile GnRH levels is known for the cyclic release of LH and FSH in negative feedback control fashion [35].
A fall in the level of kisspeptin indicates a disorder in the kiss1 gene as well as functions of the hypothalamus, and thus could cause a reduction in the levels of reproductive hormones in males [35].In the present study, treatment with pilocarpine and LEV caused a reduction in the level of kisspeptin.This could be as a result of alteration in the nuclei of the hypothalamus responsible for the release of this substance.Although, much has not been reported on the effects of pilocarpine and LEV on kisspeptin, this could be the major pathway by which they instigate reproductive dysfunction.Fortunately, NAC and taurine were able to reverse the effects of pilocarpine and LEV on the kisspeptin-GnRH system.
Although pilocarpine administered alone caused some levels of derangements, treatment with LEV however significantly decreased serum kisspeptin, GnRH, LH, FSH, and testosterone in this study.This implies that, while LEV may be a potent AED, it may at the same time exert an anti-fertility effect through depletion of the kisspeptin-GnRH system as well as the alteration of the HPG axis, accounting for its neuro-endocrine and reproductive dysfunction activities.Although most AEDs have been associated with profound reproductive impairment [36], the link between LEV-induced endocrine system derangement and hormonal imbalance has not been well characterized.However, NAC or taurine significantly reversed LEV or pilocarpine-induced decreases in GnRH, kisspeptin, LH, FSH, and testosterone levels when coadministered in the present study.Remarkably, most AEDs are known to inhibit the reproductive system through diverse mechaniams such as oxidative stress, inflammation, and apoptosis as well as homonal dernagements [36].
Sperm parameters (count, morphology, motility, and viability), sperm capacitation, acrosome reaction, sperm membrane integrity, and sperm DNA are all important indicators of testicular function and sperm ability to fertilize the ovum [19].Therefore, a derangements in these markers is a pointer to weak testicular function [37].
Notably, the lower sperm count, sperm viability, motility, as well as the increased number of abnormal sperm cells, fall in sperm capacitation and acrosome reaction, abnormal sperm membrane integrity, and sperm DNA observed in this study suggest that LEV affects spermatogenesis, spermatozoa plasma membranes, as well as sperm DNA, possibly due to changes in intracellular redox state, as indicated by previous reports [38,39].Our results also showed that the use of NAC or taurine brought about an increase in semen quality and quantity.
The reduced sperm levels in LEV-treated rats could be due to the down-regulation of steroidogenesis resulting from a fall in testosterone, preventing sperm production in the testes and maturation in the epididymis, respectively [40].Also, the reduced sperm motility by LEV could be attributed to its ability to impair sperm flagellum, due to its known effects on Ca 2+ channels [4,31].Interestingly, this arrest in spermatogenesis also corresponds to the reduced FSH, which is an indicator of germ cell survival and functions.This is as a result of the alteration in the kisspeptin-GnRH system as observed in this study.This finding is consistent with the increasing body of evidence pointing to the toxic effects of AEDs on male reproductive function [41].
Furthermore, co-administration of LEV with taurine or NAC for 14 days in pilocarpineinduced epilepsyin rats was able to reverse the effects of both pilocarpine and LEV on semen quality and quantity.This may be via the cytoprotective effects of NAC and taurine, also established in this study, suggesting that either of both supplements may protect against LEV assault in a clinical setting.
The testes comprise seminiferous tubules lined with a layer of cells (germ cells) that grow into sperm cells as well as the sertoli cells that nurture the germ cells.Between the seminiferous tubule are the interstitial cells of Leydig that are steroidogenic in nature, which provides the enabling environment for spermatogenesis to coccur.Hence, any alteration in the normal testicular architecture is harmful to the functions of the testis [41], and this is one mechanism by which reprotoxic agents with the capacity to permeate the BBB or blood testes barrier (BTB), interfere with the functions of the testis [41,41].Notably, our histopathological findings on testicular architecture in the present study revealed seminiferous tubules lined with distorted spermatogenic series cells containing distended and congested vessels with aggregates of red blood cells, and Sertoli cell degeneration.This outcomes suggest involvement of intrinsic mechanisms in the action of LEV on the germ cells, probably due to its ability to pass through the BTB.
Recent investigation carried out by Oyovwi et al. [20] revealed that rats treated with LEV-induced abnormal testicular architecture as observed in this study.Interestingly, LEV-induced deleterious effects on pilocarpine-induced epilepsy were attenuated by co-administration of taurine or NAC.The fact that treatment with taurine or NAC was able to reverse the effects of pilocarpine and LEV on testicular architecture indicates a possible restorative potential of these compounds on testicular architecture and reproductive capacity [19,21].Furthermore, pilocarpine caused vascular congestion and chromatolysed cells in the cerebral cortex confirming its pro-epileptic effect on the cerebral cortex possibly via the mechanisms highlighted above, but LEV was able to resolve the vascular congestion, justifying its usage in the management in the management of epilepsy.
Lastly, histopathological findings also showed chromatolized and necrotized neuronal cells of the hypothalamus and pituitary gland in the group treated with pilocarpine +LEV, which revealed the negative impacts of LEV on hypothalamus and pituitary gland.This confirms the alteration observed in the kisspeptin-GnRH system as well as the reduction in LH and FSH concentrations earlier noted in this study.However, treatments with NAC or taurine were able to reverse the effects.

Conclusion
Conclusively, this study established that NAC and taurine reversed reproductive neuroendocrine dysfunction in levetiracetam-treated pilocarpine-induced epilepsy in rats through normalization of neuro-endocrine related factors and hormonal modulatory mechanisms, thereby supporting the therapeutic potential of NAC and taurine as adjunct therapies in the management of epilepsy.

Figure 3 .
Figure 3. Effects of NAC, TAU and LEV treatments on sperm membrane integrity in naïve rat model following the used of hypo-osmotic swelling test (HOST).Bars represent Mean ± SEM (n = 5) (one-way ANOVA followed by Bonferroni post hoc test).a P < 0.5 relative to controls (normal saline); b P < 0.05 relative to pilocarpine + LEV.

Figure 4 .Figure 5 .
Figure 4. Effects of NAC, TAU and LEV treatments on sperm membrane integrity in naïve and pilocarpine-induced epilepsy following the used of hypo-osmotic swelling test (HOST).Bars represent Mean ± S.E.M. (n = 5) (one-way ANOVA followed by Bonferroni post hoc test).aaaaa P < 0.0001 relative to controls (normal saline); *p < 0.05 relative to the pilocarpine group, bbbb P < 0.0001 relative to pilocarpine + LEV.

Figures 9 Figure 6 .
Figures 9 and 10 represent the effects of NAC, TAU and LEV on the concentration of male