Modulation of Testicular and Whole Blood Trace ElementConcentrations in Conjunction with Testosterone ReleaseFollowing Kisspeptin Administration in Male Rabbits(Oryctolagus cuniculus)

Irfan Zia Qureshi & Qamar Abbas

Received: 5 April 2013 /Accepted: 29 May 2013 /Published online: 30 June 2013# Springer Science+Business Media New York 2013

Abstract The present study investigated the role of kisspeptin-10 on reproductively significant trace elements in relation totestosterone release in male rabbits, Oryctolagus cuniculus.Groups of rabbits were exposed to single 1 μg kisspeptin dose(i.v., saphenous vein), while simultaneous groups werepretreated with a kisspeptin antagonist, peptide-234 (50 μg)20 min before administering kisspeptin. Sequential blood sam-pling was done through marginal ear vein puncture at staggeredtime intervals: 0, 0.5, 1, 2, 4, and 24 h to determine serumtestosterone. Testes and whole blood were collected at 4 and24 h post dosage to determine trace element concentrationsthrough atomic absorption spectrophotometry. In testes, zinc(Zn), manganese (Mn), and Fe concentrations showed signifi-cant increases at 24 h, while copper (Cu) concentration wasfound elevated at 4 and 24 h both (P<0.001). In whole blood,Zn and Cu concentrations were significantly elevated at 4 and24 h, while Mn and cobalt (Co) concentrations showed in-creases only at 24 h (P<0.001). Blood iron concentration wasnot altered in the blood. In contrast, no change occurred intesticular Co, and chromium or nickel concentrations in eithertestes or blood. Compared to control and predose groups, serumtestosterone levels increased gradually and peaked at 2 h(P<0.001) post kisspeptin treatment but declined thereafter.Pretreatment with antagonist abolished all increases in traceelements and testosterone concentrations. The present study

provides first evidence that reproduction- and fertility-relatedpeptide “kisspeptin” modulates testicular and blood trace ele-ments and that this action is likely GPR54-dependent.

Keywords Traceelements .Essentialminerals .Kisspeptin .

Animal reproduction . Testes . Male rabbits

Introduction

Production of healthy spermatozoa with seminal plasma richin necessary constituents is the key to successful reproduc-tion. Besides strong regulation by hormones and major elec-trolytes, minor mineral elements, the so called “trace ele-ments,” play pivotal roles in testicular development, mainte-nance, and spermatogenesis. For instance in humans, zinc(Zn) being major constituent of seminal plasma is necessaryfor the formation and maturation of spermatozoa and forfertilization [1]. Zinc-deficient animals, chiefly males, havebeen shown to have lower concentrations of FSH and LH [2],whereas oligospermia is a sensitive indicator of Zn deficien-cy [3]. Males deficient in Zn show atrophy of seminiferoustubules and inefficient testicular development in young onesleading to reduced testicular size, lack of libido, and adverse-ly affected spermatogenesis [4]. Manganese (Mn) is essentialfor reproductive processes [5] and acts as a cofactor forbiosynthesis of sex steroids like progesterone, estrogen,and testosterone [6]. In males, dietary deficiencies of Mnlead to absence of libido, decreased motility of spermatozoa,and reduced number of sperms in the ejaculate [7]. Severalenzymes activated by Mn contribute to the metabolism ofcarbohydrates, amino acids, and cholesterol [4]. Similarly,copper (Cu) protects the testes from oxidative stress.

I. Z. Qureshi (*) :Q. AbbasLaboratory of Animal and Human Physiology, Department ofAnimal Sciences, Faculty of Biological Sciences, Quaid-i-AzamUniversity, 45320, Islamabad, Pakistane-mail: irfanzia@qau.edu.pk

I. Z. Qureshie-mail: irfanziaqureshi@gmail.com

Biol Trace Elem Res (2013) 154:210–216DOI 10.1007/s12011-013-9720-x

Accordingly in male animals, Cu deficiency leads to de-creased libido, lower semen quality, and severe damage oftesticular tissue which may render the animal sterile [8, 9].Likewise, studies on rats indicated that nickel (Ni) deficiencyleads to impaired reproductive performance. Its deprivationsignificantly decreases density of spermatozoa in epididy-mis, epididymal transient time of spermatozoa, testis spermproduction rate, and the weights of seminal vesicles andprostate glands [10]. Much is known about the toxicity ofcobalt (Co), chromium (Cr) on sexual behavior, altered fer-tility and problems with sperm shape or count reduction insperm acrosome integrity, remarkable degeneration ofSertoli cells, spermatocytes and spermatids, and prematurerelease of germ cells into the tubular lumen [11], but theirdefinitive physiological role in male reproduction is unclear.Some evidence, however, indicates that dietary Cr deficiencyin male rats leads to decreased sperm cell production andimpaired fertility [12].

Kisspeptin-GPR54 system discovered almost a decade agohas revolutionized the reproductive biology of male and fe-male mammalian and submammalian vertebrates. Kisspeptins(KP) act as gatekeepers of puberty, regulators of thehypothalamo–pituitary–gonadal axis (HPG axis), andmaintainreproduction, fertility, and pregnancy. Kisspeptin/GPR54 sys-tem regulates the HPG axis through modulation of the GnRH,LH, and FSH release in adult mice [13], rats [14], cows [15],primates [16], and humans [17]. Central or peripheralkisspeptin administration leads to potent LH and FSH releasein rats, mice, sheep, pigs, horses, and subhuman primates [18,19]. Kisspeptin is a member of the Arg-Phe (RF)-amidesuperfamily and was initially identified as a tumor suppressorpeptide in melanoma cells and was termed “metastin”(Kisspeptin-54) [20]. The native protein transcribed from theKiSS-1 gene is a 145 amino acid protein whose proteolyticcleavage gives rise to 54-amino acid peptide namelykisspeptin-54 (KP-54). Further enzymatic cleavage of KP-54gives rise to even shorter fragments namely: KP-14, KP-13,and KP-10 with the KP-10 fragment showing maximal bind-ing affinity with the G-protein coupled receptor GPR54. Bothkisspeptin and its receptor GPR54 are expressed in a variety oftissues including the central nervous system, testis, ovary,pancreas, intestine, liver, pituitary, and placenta [21].

Through a plethora of studies done over the past few yearsat molecular, genetic, biochemical, pharmacological, andfunctional level [19, 22], it has now been very wellestablished that kisspeptins act as master molecular switchfor onset of puberty and critically regulate reproduction; andincluding humans, act as powerful stimulants for gonadotro-pin release in a number of mammalian species andsubmammalian vertebrates for consequent regulation of sexsteroids to maintain gonads and production of gametes.However, the role of kisspeptin in regulating or modulatingconcentration of the trace elements crucial to testicular well

being and spermatogenesis is still unknown. Presently, welaunched a preliminary but pioneering investigation to eval-uate the role of kisspeptin-10 on trace element concentrationusing domestic rabbits as a model system.

Materials and Methods

The present study was designed and conducted at the De-partment of Animal Sciences, Quaid-i-Azam University, Is-lamabad, Pakistan. Guidelines provided by the “Ethics com-mittee of the Department of Animal Sciences on AnimalHandling and Use for Scientific Research” were followed.

Animals and Maintenance Thirty healthy adult male domes-tic rabbits (Oryctolagus cuniculus) with an average weight of1,490±65 g were purchased from local suppliers through theNational Institute of Health Islamabad. Rabbits were acclimat-ed for 2 weeks before experimentation. They were maintainedin the Animal House Facility under constant conditions of12:12 light/dark photoperiod and a room temperature of26±1 °C. The animals were fed with fresh vegetables, grams,and grasses. Drinking water was provided ad libitum. Rabbitswere currently used as a model system because they arereadily available, have shorter gestation periods, relativelydocile and easy to handle [23], and a large volume of bloodcould be easily taken, as in the present case where sequentialblood sampling had to be done, while blood was also requiredfor atomic absorption spectrophotometry. Other rodents likemice and rats are metabolically more active and handlingstress could be least avoided. Moreover, there exists to dateno report on the effect of kisspeptin in rabbits.

Experimental Design and Dose Preparation Rabbits weredivided into control and treatment groups, each containingfive individuals. The following groups were formulated:

Group I: Control (0.9 % saline treated)Group II: (a) Alone kisspeptin treated (1 μg), killed after 4 hGroup III: (b) Alone kisspeptin treated (1 μg), killed after 24 hGroup IV: (a) Antagonist pretreatment (50 μg) + kisspeptin

(1 μg) treated, killed after 4 hGroup V: (b) Antagonist pretreatment (50 μg) + kisspeptin

(1 μg) treated, killed after 24 h

Kisspeptin-10 (metastin; 112–121) was purchased fromCalbiochem (USA); kisspeptin-234 trifluoroacetate salt, akisspeptin antagonist, was obtained from Sigma-Aldrich(USA). Stock solutions were prepared according to the man-ufacturer’s instructions. These were further diluted withultrapure distilled water to prepare appropriate dosage con-centrations. The doses were injected intravenously (saphenousvein). Final experimental doses of kisspeptin (1 μg) andkisspeptin antagonist (50 μg) were worked out empirically

Trace Elements Modulation by Kisspeptin 211

through trial experiments. Following the standard method ofear vein puncture, sequential blood sampling was done at0 min (before dose administration), and at 0.5, 1, 2, 4, and24 h post dose administration. Serum was prepared and storedat −20 °C until analyzed.

Determination of Serum Testosterone Standard enzyme im-munoassay was performed to determine the testosteroneconcentration using the commercially available kits obtainedfrom Amgenix (USA) on an ELISA plate reader (MicrowellPlate Reader 6XR BIORAD, Tokyo, Japan).

Determination of Trace Element Concentrations For traceelement determination, only statistically necessary numberof rabbits (n=5) were killed at 4 and 24 h out of abovegroups. Whole blood was obtained through cardiac puncturewhile testes were removed. Wet weight of tissues (testes) wasrecorded. About 0.5 g of tissue or 1 ml of whole blood weretaken and digested with 5 ml 69 % pure concentrated nitricacid (Merck, Germany) using the microwave digestion sys-tem (MARS, CEM, USA). The maximum temperature wasset at 200 °C, and the power was 1,200 W. Samples weredigested for 5 min, filtered, and diluted to 20 ml with deion-ized water. Digests were subjected to air/acetylene fast se-quential flame atomic absorption spectrophotometer (Varian,AA240 FS, USA). Instrument conditions were according tothe manufacturer. Wavelengths for the elements were: Mn(279.5 nm), Zn (213.9 nm), Fe (248.3 nm), Co (240.7 nm),Cu (324.7 nm), Ni (232.0 nm), and Cr (357.9). Calibrationcurves were made using the standard solutions of varyingconcentrations for each trace element. Elemental concentra-tions that were obtained in parts per million (milligrams perliter) were converted into micrograms per gram of tissue ormicrograms per milliliter of blood.

Statistical Analysis Data are expressed as mean±SEM (stan-dard error of mean). Data were analyzed through one-wayanalysis of variance (ANOVA) followed by post hoc Tukey’stest using the statistical software (GraphPad Prism Inc, Ver. 5.0for windows); where normality test failed, one-way ANOVAon Ranks followed by post hoc Dunnett’s test was used.P<0.05 was considered statistically significant difference.

Results

Serum Testosterone Testosterone concentration began to in-crease at 30 min post kisspeptin dose and demonstrated apeak value at 2 h post treatment (p<0.001) as compared topretreatment samples and control non-treated animals. Theconcentration of testosterone finally declined toward 4 h.Testosterone concentration remained unaltered in the grouppretreated with kisspeptin antagonist (Fig. 1).

Trace Element Concentrations in Testes As compared tocontrol, a highly significant increase (F=9.788; p<0.001)occurred in Zn concentration at 24 h after kisspeptin treat-ment, which abolished on treatment with the antagonist(Fig. 2a). Mn concentration increased significantly at 24 h(F=3.872; p<0.017) as compared to control samples, whileits concentration remained near control values after treat-ment with the kisspeptin antagonist (Fig. 2b). Cu concentra-tion increased highly significantly at both 4 h (H=18.47;p<0.001) and 24 h (p<0.001) after kisspeptin treatment butthe concentration approached near control values on antag-onist pretreatment (Fig. 2c). Fe concentration elevated highlysignificantly (F=12.239; P<0.001) at 24 h post kisspeptintreatment when compared with the control but valuesremained neared control upon antagonist treatment (Fig. 2d).Cr, Co, and Ni concentrations did not respond to kisspeptintreatment (Fig. 3a–c). Between groups comparisons demon-strated statistically significant difference (p<0.001) between4 and 24 h groups, whereby Zn, Mn, Cu, and Fe concentra-tions elevated further at 24 h.

Trace Element Concentrations in Whole Blood Blood Znconcentration showed significant increases at both 4 and24 h (F=31.712; p<0.001) post kisspeptin treatment. An-tagonist pretreatment could not elevate Zn concentrationupon kisspeptin treatment (Fig. 2a). Kisspeptin treatmentled to significant elevation of blood Mn concentration at24 h (F=4.526; p<0.013), while pretreatment with thekisspeptin antagonist abolished this effect (Fig. 2b). BloodCu concentration increased highly significantly (H=18.795;p<0.001) at 4 and 24 h both after kisspeptin treatment butnormalized to near control values upon pretreatment with theantagonist (Fig. 2c). Blood Co concentration increased(F=3.361; p<0.029) only at 24 h after treatment with thekisspeptin but neared control values upon pretreatment with

0 0.5 1 2 4 240

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Fig. 1 Serum testosterone levels in rabbits following kisspeptintreatment. Testosterone concentration gradually increased andshowed peak concentration at 2 h (***P<0.001) and then declinedat 4 h. KP = kisspeptin; Ant = antagonist

212 Qureshi and Abbas

the antagonist (Fig. 2d). Cr, Fe, and Ni concentrations werenot altered upon kisspeptin treatment (Fig. 3a-c). Again,trace element concentrations determined at 24 h showedsignificantly greater elevations than at 4 h (p<0.001).

Discussion

The present study on male domestic rabbits (O. cuniculus),although preliminary, addressed a very important issue ofkisspeptin-GPR54 system in regulating and/or modulatingmale reproduction-related trace element concentrations fol-lowing an exogenous intravenous bolus dose of kisspeptin-10.

Although as expected, significant increase in serum testos-terone concentration (from 6.01±0.23 to 8.86±0.26 ng/mlwithin 2 h) in rabbits was in line with previous studies on avariety ofmale mammals [24], ours is the first report on rabbitson regulation of the GnRH–gonadotropin–gonadal loop sys-tem by kisspeptin in lagomorphs. An increase in serum tes-tosterone occurred within 1 to 2 h of 1 μg kisspeptin admin-istration which was followed by a gradual decline as evidentby the samples taken at 4 and 24 h. Importantly, artificialtestosterone elevation was abolished upon application ofkisspeptin antagonist. This observation appears parallel tokisspeptin-10 tested on rats where increase in serum testoster-one levels occurred within 20–60min [24], and alsomice [25];however, in the present case of rabbits, it occurred a little late.

The results of the present study demonstrated for the firsttime that kisspeptin/GPR54 system is a potent modulator oftrace elements essential to male reproduction and spermato-zoa. Significant elevation occurred in testicular Zn, Mn, Cu,and Fe concentrations, while in the whole blood; Zn, Mn,Cu, and Co concentrations were increased on kisspeptinadministration. Fe concentration was not affected in the

blood while no alterations occurred in Cr or Ni concentra-tions in either testes or blood.

Relatively little data exist on the effect of testosterone ontrace elements, some evidence however indicates that exog-enous testosterone administration increases serum zinclevels [26]. Since kisspeptin is a much potent peptide thatworks to regulate the HPG axis through the GnRH loopsystem at extremely low concentrations even when givenextracentrally [27], it is presumed that in the current inves-tigation, kisspeptin altered blood and testicular trace elementconcentrations via elevation of testosterone release. Never-theless, the occurrence of both kisspeptin and its GPR-54receptor in testes [28, 29] also points to a direct effect ofkisspeptin on male gonads.

It is very well known that Zn, Cu, Mn, and Se are involvedin testicular development, the process of spermatogenesis, andactivity of sperms and are found in testes, epididymis, andsemen [30]. Importantly, the occurrence of antioxidant en-zymes such as superoxide dismutase (SOD), glutathione per-oxidase, and catalase has been reported in human seminalplasma and several other animal species for protection of pro-tections of sperms that likely occurs from oxidative damagecaused by free radicals and reactive oxygen species [31, 32].Several trace elements like Zn, Mn, Cu, Fe, Ni, and Se are partof these metalloenzymes. For instance, Cu/Zn are part of SOD,Mn is a component of mitochondrial SOD2, and Fe formsheme group in catalases. Selenium is required for glutathioneperoxidase, Co is required for oxidoreductases, while nickel isan activator of dehydrogenases and carboxylases [7]. Super-oxide free radicals are converted into molecular oxygen andhydrogen peroxide by SOD, while glutathione peroxidase andcatalase convert hydrogen peroxide to water and oxygen [33].

As it is an established fact now that kisspeptin is a criticalregulator of reproduction, elevation caused by kisspeptin in

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Fig. 2 In testes, significantincrease occurred in Zn, Mn,Cu, and Fe concentrations;while Zn, Mn and Cuconcentrations were elevated inwhole blood, followingkisspeptin administration inrabbits at 4 and 24 h (*p<0.05;**p<0.01; ***p<0.001). n=5in each group. KP = kisspeptin;Ant = antagonist

Trace Elements Modulation by Kisspeptin 213

the levels of Zn, Cu, Mn, and Fe appears to be very logical.Increases in testicular Zn, Mn, Fe, and Cu concentrationsfollowing kisspeptin administration, while waning in thelevels of these metal concentrations upon pretreatment ofrabbits with the kisspeptin antagonist indicate regulatory role

of kisspeptin on gonadal trace element concentration throughthe G-protein coupled receptor GPR54. Trace element con-centrations were found altered in both 4 and 24 h groups ofrabbits but a highly significant increase was noticeable in the24-h treatment group. This observation indicates that due to

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Fig. 3 Co concentration increased only in the whole blood at 24 h, whereas Cr and Ni concentrations were neither affected in testes nor whole bloodfollowing kisspeptin administration in rabbits. (*p<0.05). n=5 in each group. KP = kisspeptin; Ant = anatgonist

214 Qureshi and Abbas

the complex metabolic processes, it took a little while forkisspeptin to alter trace element concentration as opposed toserum testosterone that elevated within 1 to 2 h afterkisspeptin injection. For obvious reasons, it would have beendue to the fact that many trace elements act as cofactors forthe enzymes required in metabolic processes. The presentobservations bear relevance to reproduction as many of thesetrace elements are part of the antioxidant metalloenzymesand play crucial roles in testicular development and overallprocesses of steroidogenesis and spermatogenesis [34].

Of several trace elements, Zn being major constituent ofseminal plasma is of much significance and is a componentpart of an antioxidant enzyme the SOD. Zinc is a constituentpart of >300 enzymes that also includes the DNA polymer-ase [35]. Zinc is required for normal functioning of malereproductive system and production of spermatozoa. It isalso required for the production of testosterone. Deficiencyof seminal plasma Zn levels may lead to hypogonadism,inadequate testosterone concentration, decrease in testicularvolume, and atrophy of seminiferous tubules [36]. Morespecifically, deficiency of Zn may cause degeneration ofcells involved in processing of after spermatozoa meioticdivision [37]. Also, increase in seminal plasma pH occursupon decreases in Zn concentration [38]. Moreover, Zn in P2protamines plays a role in stabilization of sperm chromatinand in inhibition of transcription [39]. Poor Zn nutrition maybe an important risk factor for low quality of sperm andidiopathic male infertility [40]. Consequently, human semi-nal plasma contains relatively higher levels of Zn [41, 42].Although we did not focus on seminal plasma at present,significant elevation in testicular Zn and blood Zn concen-trations indicate strong regulation by the kisspeptin peptidenot only in the testes but also in the semen.

Like Zn, Mn concentration was also elevated significantlyin both testes and blood. Again,Mn is a cofactor in cholesterolsynthesis which is necessary for progesterone, estrogen, andtestosterone production [6]. Manganese provides antioxidantactivity and protects sperms from free radical damage and isan effective antioxidant for sperm cryoprotection [43]. Thus, itis obvious to presume as to why would kisspeptin upregulatethe Mn testicular concentration?

Presently, both Fe and Cu concentrations also increasedsignificantly but only in the testes. This increase indicatesthat kisspeptin might be regulating oxidative functions intestes through Fe and Cu metabolism. As cellular Fe isinvolved in oxidative phosphorylation and DNA synthesisand both Fe and Cu protect the testes from oxidative stress[8], Zn in combination with Fe takes part in oxidation andreduction processes, and Zn together with Cu prevents del-eterious effects of reactive oxygen species on spermatozoa ascofactors of Cu–Zn superoxide dismutase [44, 45]. Thuselevation of Fe and Cu concentrations as observed presentlyin rabbits following kisspeptin administration indicates

regulation of essential minerals metabolism for ultimate tes-ticular maintenance and for keeping up with the productionand healthy status of spermatozoa. However, by whichmechanism kisspeptin would have caused increases in tes-ticular and blood Zn, Mn, Fe, and Cu is remaining to beworked out. Similarly consequent increases in blood Zn, My,and Co concentrations on kisspeptin exposure appears to beof immense significance.

Although Co, Cr, and Ni also play important roles insperm motility and in maintaining the antioxidant status ofsemen [30], in contrast to Zn, Mn, Cu, and Fe, the concen-trations of Co, Cr, and Ni in rabbit testes were not affected bykisspeptin, raising the possibility that regulation of the traceelement metabolism by kisspeptin is even more complexthan one can assume. It is therefore equally important toexplore which mechanisms inhibited kisspeptin to alter tes-ticular Co, Cr, and Ni levels. At other end, elevation of serumCo concentration indicates regulation of important vitaminsby the kisspeptin whilst Co is known to be an essentialcomponent of vitamin B12 [46]. It is very well known thatmany vitamins play essential roles in fertility. B-12 deficien-cy reduces sperm motility and sperm count and has benefi-cial roles for spermatozoa. Vitamins A, B Vitamins (B6,B12, and folate), C, D, and E are all known to be powerfulantioxidants for sperm healthiness, sperm counts and motil-ity, maintenance of the male genital tract and spermatogen-esis and overall increase of fertility [47].

In conclusion, the present findings indicate that kisspeptinmight be playing an important role in the maintenance ofgrowth, reproduction, and energy metabolism through mod-ulation of trace element concentrations. This obviously war-rants in-depth investigation of trace element levels in testes,seminal plasma, and accessory sex glands. Since kisspeptinis very well known to regulate the fertility status [18], regu-lation and preservation of normal trace elements and vitaminbalance in the reproductive organs and seminal fluids ap-pears to be of particular significance for both human andanimal fertility and to treat infertility problems.

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