REPRODUCTIONRESEARCH

Kisspeptin regulates steroidogenesis and spermiation in anuran amphibian

Rosanna Chianese1, Vincenza Ciaramella2, Silvia Fasano1, Riccardo Pierantoni1 and Rosaria Meccariello3 1Dipartimento di Medicina Sperimentale sez ‘F. Bottazzi’, Università degli Studi della Campania ‘Luigi Vanvitelli’, Napoli, Italy, 2Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale ‘F. Magrassi-L. Lanzara’, Università degli Studi della Campania ‘Luigi Vanvitelli’, Napoli, Italy and 3Dipartimento di Scienze Motorie e del Benessere, Università di Napoli Parthenope, Napoli, Italy Correspondence should be addressed to R Meccariello; Email: [email protected]

Abstract

Kisspeptin (Kp) system has a recognized role in the control of gonadotropic axis, at multiple levels. Recently, a major focus of research has been to assess any direct activity of this system on testis physiology. Using the amphibian anuran, Pelophylax esculentus, as animal model, we demonstrate – for the first time in non-mammalian vertebrate – that testis expresses both Kiss-1 and Gpr54 −6 proteins during the annual sexual cycle and that ex vivo 17B- (E2, 10 M) increases both proteins over control group. Since the interstitium is the main site of localization of both ligand and receptor, its possible involvement in the regulation of steroidogenesis has been evaluated by ex vivo treatment of testis pieces with increasing doses of Kp-10 (10−9–10−6 M). Treatments have been carried out in February – when a new wave of spermatogenesis occurs – and affect the expression of key enzymes of steroidogenesis inducing opposite effects on and estradiol intratesticular levels. Morphological analysis of Kp-treated testes reveals higher number of tubules with spermatozoa detached from Sertoli cells than control group and the expression of connexin 43, the main junctional protein in testis, is deeply affected by the treatment. In spite of the effects on spermatozoa observed ex vivo, in vivo administration of Kp-10 has been unable to induce sperm release in cloacal fluid. In conclusion, we demonstrate Kp-10 effects on steroidogenesis with possible involvement in the balance between testosterone and estradiol levels, and report new Kp-10 activities on spermatozoa–Sertoli cell interaction. Reproduction (2017) 154 403–414

Introduction Kp/GPR54 signaling regulates gonadal physiology (Leòn et al. 2016). Accordingly, as in the hypothalamus, The gonadotropic axis is under the control of in testis a similar Kp/GPR54 signaling system has -releasing hormone (GnRH) whose been reported in primates, rodents and amphibians release is upstream regulated by kisspeptins (Kps) (Meccariello et al. 2014, Chianese et al. 2016 for review), (Messager et al. 2005). Kps are a group of becoming part of the intricate intratesticular network of derived from Kiss1 gene (Kotani et al. 2001) able to regulators governing testis physiology (Pierantoni et al. activate Kiss1 receptor (GPR54, Thompson et al. 2004). 2002a). Although several studies have reported the The central Kp/GPR54 signaling system has been deeply expression of both ligand and receptor in the male characterized in mammals, especially as a potent trigger reproductive organs of several vertebrates, humans of GnRH secretion during puberty onset (Pinilla et al. included (Meccariello et al. 2014, Chianese et al. 2016, 2012, Herbison 2016). Conversely, gene knockout Wabab et al. 2016), the possible physiological role of studies have revealed that kiss/gpr54 signaling is not intragonadal Kp signaling is still controversial (Mei et al. absolutely required for zebrafish reproduction in both 2013, Meccariello et al. 2014, Chianese et al. 2016). sexes (Tang 2015), due to compensation mechanisms In testis, the interstitial compartment is mainly capable to stimulate the gonadotropic axis in the involved in testosterone production thanks to the absence of Kiss1 signaling (Liu 2017). contribution of Leydig cells (Chen et al. 2009) in which However, in Gpr54 knockout mice, the re-expression Kp has been localized (Anjum et al. 2012, Salehi et al. of GPR54 exclusively in GnRH neurons reactivates the 2015). Studies carried out on the Leydig cell line neuroendocrine axis, but does not completely restore MA-10 – that also expresses Gpr54 mRNA – do not gonadal functions, strongly suggesting that peripheral reveal any effect of Kp-10 (Mei et al. 2013), the active

© 2017 Society for Reproduction and Fertility DOI: 10.1530/REP-17-0030 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/28/2021 03:10:44PM via free access

10.1530/REP-17-0030 404 R Chianese and others metabolite produced by the proteolytic cleavage of the Materials and methods largest precursor protein Kp-145 (Oakley et al. 2009). Chemicals and antibodies Unlike rodents, in rhesus monkey in vivo administration of Kp-10 stimulates testosterone production and Kp-10 (Metastin 45-54 (H-YNWNSFGLRF-NH2)) of human exhibits synergistic activity with human chorionic origin, a Kp form with a potency similar to Xenopus-Kp-10 gonadotropin (hCG) on steroid biosynthesis in acyline- for bullfrog Gpr54 (Moon et al. 2009), was purchased from treated monkeys, thus suggesting a direct effect on DBA Italia, Milan, Italy; Kp-234, a specific Gpr54 antagonist testis (Irfan et al. 2014). Conversely, the in vitro Kp-10 (Roseweir et al. 2009) was purchased from DBA Italia; treatment fails to have any effect (Tariq & Shabab 2016). 17B-estradiol (E2) was purchased from Sigma-Aldrich S.R.L.; In germ cell compartment, many traits of a Kp-dependent ICI 182780, a high-affinity receptor antagonist, was physiological autocrine/paracrine network are still purchased from AstraZeneca, UK; and , a GnRH elusive, especially in mammals, since the only clear agonist, was a gift from Dr J Sandow, Hoechst, Frankfurt, Germany. evidence has been provided in spermatozoa (SPZ), with For Western blot and immunohistochemistry the following a suggested role of Kp in sperm motility and fertilization antibodies were used: goat polyclonal anti-Kiss-1 antibody et al et al. capacity (Pinto . 2012, Hsu 2014). (raised against a mapping at the C-terminus of Kiss-1 Interestingly, studies in non-mammalian of human origin; sc-18134) and rabbit polyclonal anti-Gpr54 vertebrates have been devoted to clarify Kp role in antibody (raised against amino acids 141–342 mapping within both spermatogenesis and steroidogenesis (Tena- an internal region of Gpr54 of human origin sharing 78% Sempere et al. 2012). In the prepubertal teleost Scomber similarity with P. esculentus Gpr54; sc-134499); both were japonicus, peripheral injection of Kp-15 accelerated purchased from Santa Cruz Biotechnology and were validated spermatogenesis and induced sperm production for Western blot and immunohistochemistry (present data, (Selvaraj et al. 2013a,b). Accordingly, in the anuran Supplementary Figs 1 and 2, see section on supplementary amphibian Pelophylax esculentus, Kp role has been data given at the end of this article; Hsu et al. 2014, deeply investigated in testis. P. esculentus shows a very Ciaramella et al. 2016). Mouse monoclonal anti-α-tubulin peculiar organization of testis, since germ cells at the (T6199) was purchased from Sigma-Aldrich. same maturational stage are clustered in cysts – formed by Sertoli cell cytoplasmic protrusions – and develop during the annual sexual cycle (Rastogi et al. 1976, Animal and tissue collection Pierantoni et al. 2002b). In particular, in March–April Adult P. esculentus male frogs (n = 5) were collected monthly (breeding season), SPZ are released and spermatogonia in the neighborhood of Naples (Italy) except in August. To (SPG) start proliferating after the winter stasis (December– minimize the stress, the animals were anesthetized in ethyl February), when environmental and hormonal conditions 3-aminobenzoate methanesulfonate salt, MS222 (Sigma- are not advantageous to reproduction. Meiotic Aldrich) and immediately killed after capture. Testes were stages only appear in May–July (post-reproductive removed, rinsed in Krebs Ringer Buffer for amphibians (KRB, period), when spermatogenesis actively proceeds; 68 mM NaCl, 1 mM KCl, 1.17 mM MgSO4, 1.2 mM KH2PO4, lastly post-meiotic cells richly populate testis during 25 mM NaHCO3, 2.5 mM CaCl2, 10 mg/L gelatin, pH 7.4) and resumption (September–November) (Rastogi et al. 1976, processed for qPCR, Western blot and immunohistochemistry, or in vitro incubated with Kp-10 and E as reported below. Pierantoni et al. 2002b). Interestingly, the use of this non- 2 mammalian animal model has contributed to unravel Brain was also removed from frogs of February and processed for validation of antibody as positive control. Experiments important aspects of Kp-dependent intratesticular were performed under the guidelines established in the signaling. Notably, gpr54 mRNA was localized in the National Institute of Health Guide for Care and Use of interstitial compartment and in proliferating germ Laboratory Animals and were approved by the Italian Ministry cells and its expression was indicated to be estradiol- of Education, University and Research. dependent (Chianese et al. 2013). Furthermore, Kp-10 was able to modulate the expression of estrogen receptor alpha (era) (Chianese et al. 2013), erb, proliferating Ex vivo incubation of testis cell nuclear antigen (pcna) and GnRH system – both Experiment 1: Male frogs (n = 15) collected in February were ligands and receptors (Chianese et al. 2015), since the used for treatment with E2. Testes were cut into halves and suggested idea of Kp as a testicular bioregulator of germ were incubated for 1 h in KRB alone (control group, C), in KRB/ cell progression. −6 −5 E2 10 M (E2 group), in KRB/ICI 182780, 10 M for 30 min – Starting from this evidence, we localized both Kiss-1 −6 −5 preliminarily – then in KRB/E2 10 M and ICI 182780 10 M ligand and Gpr54 receptor in frog testis during the (EI group). E2 and ICI 182780 doses and incubation times were annual sexual cycle. Then, at the onset of a new cycle of chosen on the basis of previous studies in frogs (Cobellis et al. spermatogenesis (February), we investigated a possible 1999). After the treatment, testes were stored at −80°C and role exerted by Kp-10 on steroidogenesis and evaluated then processed for protein extraction. the possibility that Kps may regulate the detachment of Experiment 2: Testes were removed from n. 60 animals SPZ from Sertoli cells. collected in pre-reproductive period (February) and divided

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Downloaded from Bioscientifica.com at 09/28/2021 03:10:44PM via free access Kisspeptin in steroidogenesis and spermiation 405 into halves taking care to group together all left and right testes AB284119.2); Ciaramella et al. 2016 for cytochrome P450 that were incubated in KRB alone or in KRB and increasing 19A/aromatase (cyp19; GenBank KM983560); Izzo et al. doses of Kp-10 (10−9–10−6 M with 10 fold increments) for 1 h 2006 for connexin 43 (cx43; GenBank AM040672.1)). Primers (left testis) and 4 h (right testis). Doses and incubation times designed on Rana rugosa nucleotide were used to amplify were chosen on the basis of previous experiments in frogs receptor (ar) sequence: S: 5′-ccagaggatggcagttcatt-3′ (Chianese et al. 2013, 2015). For each dose and time point, 4 and AS: 5′-agcagaagcgtcctccataa-3′, predicted amplificate testes were stored at −80°C and used for expression analysis; size 156 bp. The cloning of ar partial cDNA was carried out 4 halves of testes were fixed in Bouin’s fluid and processed for in pGEM-T Easy Vector (Promega), transforming DH5a high standard histological analysis; and 4 halves of testes were used efficiency competent cells and sequencing the insert on both for steroid dosage. strands by Primm Sequence Service (Primm srl, Naples Italy). P. esculentus AR sequence revealed 96% and 98% nucleotide and amino acid identity, respectively, vs R. rugosa androgen In vivo treatments and protein extraction from SPZ receptor sequences. T (°C) annealing for all primers was 60°C. Male frogs (n = 3 each treatment) collected in December were A negative control in which cDNA was replaced by water was injected in the dorsal sac with 100 µL of KRB (control group, also included. Signals were normalized toward the reference C); KRB/Kp-10 at 10−6 M (treated group, Kp-10); KRB/Kp-234 gene fp1 whose boundary as reference gene was previously at 10−5 M and after 30 min with KRB/Kp-10 at 10−6 M (treated reported (Chianese et al. 2011, 2012). Assay included a group, Kp-10 + A); KRB/GnRH agonist/buserelin at 10−5 M melting curve analysis for which all samples displayed single (treated group, bus), a well-known treatment capable to induce peaks for each primer pairs. Data were then reported as mean spermiation in frogs (Minucci et al. 1989). After 2 h from the fold change ± s.d. over the minimal value arbitrarily assigned injection, cloacal fluid was collected and analyzed under to control group. light microscopy (CTR500; Leica) for the detection of SPZ. Frogs were then sacrificed as previously described and testes Protein extraction and Western blot analysis were removed for histological analysis. Two positive controls were used: human and rat SPZ. Human semen samples were Total proteins were extracted from testes and brain (n = 5), provided by one healthy volunteer donor and processed for from E2 or E2/ICI-treated testes, and then processed for Western SPZ isolation as previously described (Meccariello et al. blot analysis as previously reported (Ciaramella et al. 2016). 2008); rat SPZ were collected in PBS pH 7.4 from epididymis Anti-Kiss-1 and anti-Gpr54 antibodies were both diluted conveniently removed and cut into few pieces and centrifuged 1:500 and validated as shown in Supplementary Figs 1 and 2; at 1000 g for 15 min at 4°C. Freshly collected SPZ from frog, rat mouse monoclonal anti-α-tubulin diluted 1:15,000 was used and human were immediately processed for protein extraction to quantify protein content. Western blot signals were scanned as previously described (Meccariello et al. 2007, 2008). and protein levels were plotted as quantitative densitometry analysis of signals. Data were expressed as mean of Kiss-1 or Gpr54/tub ratio fold increase s.d. Total RNA extraction and cDNA preparation ± The extraction of total RNA from frog testis (n = 4) was performed using Trizol reagent (Life Technologies) following the Immunohistochemistry analysis manufacturer’s instructions. DNaseI (10 U/sample) (Amersham Frog testes (n = 5) collected in February, July and November Pharmacia Biotech, UK) treatment at 37°C for 30 min ensured were fixed in Bouin’s fluid for histological observation and the elimination of any genomic DNA contaminations. RNA embedded in paraffin using standard procedures. Thus, purity and integrity were determined by spectrophotometer sections (5 µm) were used to analyze Kiss-1 and Gpr54 analyses at 260/280 nm and by electrophoresis. distribution in testis. Endogenous peroxidase activity was

The reverse transcription of a total RNA pool was then carried inhibited in methanol and 0.3% H2O2 for 20 min. To prevent out using 5 μg total RNA, 0.5 μg oligo dT(18), 0.5 mM dNTP background staining, sections were washed in PBS 0.01 M, mix, 5 mM DTT, 1× first strand buffer (Life Technologies), 40 U pH 7.1, and incubated in PBS containing 3% BSA and 1:30 RNase Out (Life Technologies), 200 U SuperScript-III RnaseH− goat serum (PBS/BSA/serum, PBS-BS) in a moist chamber at Reverse Transcriptase (Life Technologies) in a final volume of room temperature. Slides were incubated with the previously 20 μL, following the manufacturer’s instructions. As negative described anti-Kiss-1/GPR54 antibodies diluted 1:100 in control, total RNA not treated with reverse transcriptase PBS-BS overnight at 4°C in a moist chamber. Another section was used. on the same slide was incubated only with PBS-BS as an immunohistochemical control. After three washes in PBS and Quantitative real-time RT-PCR (qPCR) 0.3% Triton X-100, sections were incubated with biotinylated rabbit anti-goat IgG (E0466) or goat anti-rabbit IgG (E0432), for In brief, cDNA was diluted 1:5 in water and subjected to Kiss-1 and Gpr54 detection, respectively (DAKO), and diluted qPCR using 10 pmol of oligonucleotide primers designed on 1:100 in PBS-BS for 1 h at room temperature. Subsequently, P. esculentus nucleotide sequence as previously described the sections were incubated with Vectastain ABC Reagents (Chianese et al. 2014 for 3β-hydroxysteroid dehydrogenase/D-5-4 (Vector Laboratories, Inc., Burlinghane, CA, USA) for 30 min isomerase (3β-HSD; GenBank AB284117.1) and cytochrome at room temperature. Immunoreaction products were P450 17alpha-hydroxylase/17,20 lyase (cyp17; GenBank detected with 3,3′-diaminobenzidine tetrahydrochloride www.reproduction-online.org Reproduction (2017) 154 403–414

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(Sigma-Aldrich) in Tris–HCl 0.05 mM buffer according to the Results manufacturer’s protocol. To check the specificity of the immunoreactions, controls Frog testis expresses both Kiss-1 and Gpr54 were treated omitting incubation with primary or secondary With the aim to shed light on the role of Kp system in antiserum or incubating section with PBS-BS alone during frog spermatogenesis we analyzed the expression of the procedures. For antiKISS-1, preadsorption of antiserum Kiss-1 and Gpr54 in testis during the annual sexual with the corresponding epitope was also carried out (details cycle by Western blot. Kiss-1 signal shows high levels in Supplementary Figs 1 and 2). The sections were observed in March–April (P < 0.01) in conjunction with a new under a light microscope, and images were captured using a spermatogenic wave (Rastogi et al. 1976), and then high-resolution digital camera (DC300F; Leica). it decreases in summer to increase again during the resumption (September–November) and the winter stasis Histological analysis and count of tubules containing (December–February) (P < 0.01) (Fig. 1A). The profile detached and released SPZ of Gpr54 shows clear peaks of expression in April, during the reproductive period, when SPZ are released Testes from Kp-10 dose–response experiments and in vivo (P < 0.01), in June–October – with the appearance of treatment were fixed in Bouin’s fluid and embedded in paraffin following standard procedures, and slides (5 µm) were then stained with standard hematoxylin–eosin and observed under a light microscope. Therefore, one section/ treatment/time point/animal approximately every 50 µm, except 10−9 M Kp-10 treatment, was analyzed and the following elements were counted: the number of total cross sections of seminiferous tubules; the number of cross sections of seminiferous tubules containing only SPZ detached from Sertoli cells; and the number of cross sections of seminiferous tubules containing SPZ completely released in the lumen. Data were reported as the % of tubules with released SPZ or tubules with detached SPZ over the total of tubules counted for animals ± s.d.

Steroid detection Steroid extraction from testis Testes (n = 5) were homogenized in 70% methanol and extracted with 2 × 7 mL diethyl ether. After drying, each extract was dissolved in PBS (0.1 M, pH 7)–0.2% gelatine for hormone determination (Pierantoni et al. 1984).

EIA assay

Intratesticular E2 and testosterone (TS) levels were quantified using commercially available EIA kits according to the directions provided by the manufacturer (Cayman and DRG Diagnostics GmbH, Germany, respectively). The detection limits of kits were approximately 20 pg/mL and 83 pg/mL, for

E2 and TS, respectively. The intra- and inter-assay coefficients of variation, determined at multiple points, were 7.4% and

6.3%, and 5.7% and 1.6%, for E2 and TS, respectively. All determinations were made in triplicate from each sample, expressed in pg steroid/mg/weight of testes. Then, for each experimental group we calculated the ratio E2/TS and normalized each ratio vs control. Figure 1 Kiss-1, Gpr54 and α-tubulin (tub) expression in frog testis during the annual sexual cycle analyzed by Western blot (A). Signals were quantified from by densitometry analysis of Kiss-1 (B) or Gpr54 Statistical analysis (C) vs tubulin. Data are representative of three separate experiments and are expressed as mean fold increase (FI) calculated on the One-way ANOVA followed by Duncan’s test for multi-group minimal value observed ± s.d. Different letters indicate statistically comparison or Student’s t test were used where appropriate. significant differences P( < 0.01). M–F: consecutive months of the year, from March to February.

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Downloaded from Bioscientifica.com at 09/28/2021 03:10:44PM via free access Kisspeptin in steroidogenesis and spermiation 407 meiotic and early post-meiotic stages – and in February spermatogonia (IISPG) (Fig. 2G and H). In July, Gpr54 again – when spermatogonia proliferation resumes. was localized in cysts of primary spermatocytes (ISPC) Lowest expression rates have been detected during and IISPC (Fig. 2I and J), whereas in November the the winter stasis (Rastogi et al. 1976, Pierantoni et al. immunolabeling was detected in post-meiotic germ 2002b) (Fig. 1B). cells such as spermatids (SPT) and SPZ (Fig. 2K and L). By immunohistochemistry Kiss-1 and Gpr54 Immunohistochemical signals completely disappeared proteins were localized in frog testis of February, July in the control sections obtained by omitting one and November animals (Fig. 2). The analysis revealed step of reaction to demonstrate the specificity of the the presence of Kiss-1 in the interstitial compartment immunoreactivity (Fig. 2B, D, F, H, J and L insets). in all the observed periods (Fig. 2A, B, C, D, E and F), To corroborate Gpr54 immunolocalization in post- whereas in November a weak immunolabeling was also meiotic cells, we carried out a Western blot analysis detected in germinal compartment especially in cysts of on proteins extracted from frog and rat SPZ: the latter secondary spermatocytes (IISPC) (Fig. 2E and F). In all the used as positive control. Gpr54 signal appeared in both analyzed periods, Gpr54 protein was mainly detected in lanes, even if with greater intensity in rat SPZ protein the interstitium (Fig. 2G, H, I, J, K and L). In February, extracts (Fig. 2M). Gpr54 was also localized in peritubular myoid cells, Further details concerning antisera validation for both Sertoli cells surrounding actively proliferating primary Western blot and immunohistochemistry are shown in spermatogonia (ISPG), ISPG and cysts of secondary Supplementary Figs 1 and 2.

Figure 2 Sections of P. esculentus testis collected in February (A, B, G, H), July (C, D, I, J) and November (E, F, K, L) analyzed by immunocytochemistry for Kiss-1 (A, B, C, D, E and F) or Gpr54 (G, H, I, J, K and L). Kiss-1 signal almost exclusively localized in the interstitial compartment (i). Instead, Gpr54 also followed the progression of spermatogenesis since the signal marked ISPG and IISPG cysts (*), but also Sertoli (s) and peritubular myoid cells (m) (February G, H), cysts of ISPC and IISPC (Δ) (July I, J), and post-meiotic cells such as SPT and SPZ (▲) (November K, L). The specificity of the immunolocalization was carried out by omitting one step of reaction (Kiss-1: B, D, F insets; Gpr54: H, J, L insets). Scale bars = 20 µm. Expression of Gpr54 protein in frog and rat SPZ analyzed by Western blot (M).

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E2 modulates the expression of both Kiss-1 and Gpr54 in frog testis A possible estrogen regulation of both Kiss-1 and Gpr54 protein expression was assayed in frog testis by in vitro treatment with E2. Interestingly, such a treatment increased the expression levels of both ligand and receptor vs control (P < 0.01). A combined treatment with the estrogen receptor antagonist ICI 182780 reduced Kiss-1 and Gpr54 levels to control values (Fig. 3).

Kp effects on key enzymes of steroidogenesis To give insight in the direct functional role of Kp system in basic testicular activities we evaluated in vitro the possible effect of increasing doses of Kp-10 on the expression of 3β-HSD, cyp17 and cyp19, key enzymes in steroidogenesis. After 1 h treatment (Fig. 4A) the expression of 3β-HSD significantly decreased at all Kp-10 doses compared to the control (P < 0.01), whereas the expression of cyp17 and cyp19 significantly increased with major effects at high Kp-10 doses (P < 0.01). After 4 h treatment (Fig. 4B) 3β-HSD expression was also strongly stimulated by Kp-10. Differently from 1 h observations, the expression of cyp17 only increased at high Kp-10 doses (10−7 and 10−6 M) (P < 0.01). Cyp19 expression increased at all Kp-10 doses with a major effect at 10−7 M (P < 0.01). Default expression rate of 3β-HSD, cyp17 and cyp19 throughout the treatment has been detected comparing the expression rate of the selected genes between 1 and 4 h KRB-incubated control group (Fig. 4C). The expression rate of 3β-HSD and cyp17 in 1 h control group was significantly higher than that in 4 h control group (P < 0.01); conversely, the expression rate of cyp19 in 1 h control group was significantly lower than that in 4 h control group (P < 0.01).

Kisspeptin regulates the expression of ar and the intratesticular levels of testosterone and E2 Figure 3 Direct actions of estradiol (E2) on the expression of Kiss-1 and Possible effect of Kp-10 treatment has been analyzed Gpr54 in frog testis of February analyzed by Western blot. C, control, −6 on ar expression (Fig. 5A). After 1 h treatment ar testis incubated with KRB; E2, testis incubated with 10 M E2; EI, testis −6 −5 expression significantly decreased at all Kp-10 doses incubated with both E2 (10 M) and ICI 182780 (10 M). ‘a’ letter (P < 0.01); conversely, 4 h treatment dose-dependently indicates statistically significant differences P( < 0.01). Incubation time: 1 h. Data, normalized against -tubulin profile, are reported as mean increased ar levels compared to the control (P < 0.01). α FI ± s.d. over the value one arbitrarily assigned to the C and are Default expression rate of ar during the treatment was representative of three separate experiments at least (n = 5). significantly higher in 1 h control group than in 4 h −9 control group (P < 0.01). on the other hand, E2 levels increased at 10 M Kp-10 In support to these data, we measured intratesticular (P < 0.05 vs control) and then decreased at higher Kp-10 levels of both testosterone and E2 after Kp-10 in vitro doses (P < 0.05 vs control). Conversely, 4 h treatment treatment by EIA method (Fig. 5B). After 1 h treatment, failed to have any significant effect on both testosterone Kp-10 significantly decreased testosterone levels and E2. The ratios between E2 and testosterone at 1 h compared to the control at all tested Kp-10 doses normalized vs control group were as follows: control (P < 0.01 vs control), with major effects at lower doses group 1; Kp-10 (10−9 M) 3.21; Kp-10 (10−8 M) 1.19; (P < 0.01 10−9 and 10−8 M vs 10−7 and 10−6 M Kp-10); Kp-10 (10−7 M) 0.46; Kp-10 (10−6 M) 0.65.

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Figure 4 Direct actions of Kp-10 on the expression of 3β-HSD, cyp17 and cyp19 in frog testis of February by qPCR. Kp-10 incubation time were 1 h (A) and 4 h (B). Default expression rate of 3β-HSD, cyp17 and cyp19 throughout the treatment in control groups incubated for 1 h (C1h) or 4 h (C4h) with KRB (C). C, control testis incubated with KRB; −9/−6, Kp-10 concentrations (−logM). Different letters indicate statistically significant differences (P < 0.01). Quantitative PCR data, normalized against fp1, are reported as normalized fold expression (nfe) ± s.d. over the value one arbitrarily assigned to the sample C. Data are representative of three separate experiments at least (n = 4).

Possible role of Kp-10 in spermiation being characterized by ISPG and SPZ, after 1 h treatment 10−8, 10−7 and 10−6 M Kp-10 doses strongly increased In February, at the end of the winter stasis, frog testis the number of tubules with detached SPZ (Fig. 6A5) that is populated by ISPG that start to proliferate forming appeared untidily distributed in the seminiferous tubule germinal cysts and by SPZ that have to be released (P 0.01 vs control) (Fig. 6A2–A4). In addition, 10−7 M during the breeding season (Rastogi et al. 1976). Usually, < Kp-10 also induced the formation of a bolus of SPZ during spermiation, SPZ are detached from Sertoli cells (P 0.01 vs control) in parallel to the increase of tubules and concentrate in the lumen of seminiferous tubules. In < containing detached SPZ (P 0.01 vs control) (Fig. 6A3). testis treated with increasing doses of Kp-10 for 1 and 4 h < Similarly, after 4 h treatment all Kp-10 doses significantly (Fig. 6A1–A4 and B1–B4) a massive detachment of SPZ induced the detachment of SPZ (Fig. 6B2–B5) compared from Sertoli cells was observed, but most of them were to the control section (Fig. 6B1), but in 10−7 and 10−6 M just detached from Sertoli cells and do not concentrate Kp-10 treated testis many tubules appeared enriched in the middle lumen of seminiferous tubules. Thus, we with SPZ ready to be released (P 0.01 vs control) counted the number of tubules with released SPZ or < (Fig. 6B3, B4 and B5), in parallel to a significant increase with detached SPZ over the total tubules counted for of B tubules (Fig. 6B5, P 0.01 vs control). each section, after 1 and 4 h treatment (Fig. 6A5 and B5). < Kp-10 treatment also affected the expression of While in the control sections the lumen of seminiferous cx43, reported to have a prominent role to maintain the tubule cross sections appeared empty (Fig. 6A1 and B1)

Figure 5 Direct actions of Kp-10 on the expression of ar in frog testis of February (A) by qPCR. Quantitative PCR data, normalized against fp1, are reported as normalized fold expression (nfe) ± s.d. over the value one arbitrarily assigned to the sample C. Intratesticular androgen (testosterone, TS: bars)

and estrogen (estradiol, E2: dark line) levels measured by EIA and expressed in pg/mg tissue (B). Kp-10 incubation time were 1 h and 4 h. C, control testis incubated with KRB; −9/−6, Kp-10 concentrations (−logM). Different letters indicate statistically significant differences (P < 0.01) (A). For hormone dosage histograms (B) a, b letters indicate statistically significant differences P( < 0.01) for TS levels and a′, b′ letters indicate statistically significant differences P( < 0.01; a′ vs control P < 0.05) for E2 levels. Data are representative of three separate experiments at least (n = 4). www.reproduction-online.org Reproduction (2017) 154 403–414

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Figure 6 Histological analysis of Kp-10-treated frog testis of February, after staining with standard hematoxylin–eosin. Incubation time were 1 h (A1–A4) and 4 h (B1–B4). Representative histological analysis of control testis (A1, B1), 10−8 M Kp-10 (A2, B2), 10−7 M Kp-10 (A3, B3) and 10−6 M Kp-10 (A4, B4) incubations. White arrows, fully released SPZ; black arrows, detached SPZ. Count of tubules with released SPZ (white bars) or detached SPZ (black bars) over the total number of tubules/section/animal at 1 h (A5) and 4 h treatment (B5). Control testis incubated with KRB (C); −8/−6, Kp-10 concentrations (−logM). Data are reported as the % of tubules containing released (detached) SPZ over the total of tubules counted for animals ± s.d. (n = 3–4); at each treatment dose, asterisks (letters) indicate the statistically significant differences P( < 0.01) among tubules containing released SPZ (detached SPZ) vs control. Direct actions of Kp-10 on the expression of cx43 in frog testis of February after 1 h (A6) and 4 h treatment (B6) by qPCR. Quantitative PCR data, normalized against fp1, are reported as normalized fold expression (nfe) ± s.d. over the value one arbitrarily assigned to the sample C. Different letters indicate statistically significant differences P( < 0.01). Data are representative of three separate experiments at least (n = 4). connections between Sertoli and germ cells – including the testes of control group showed empty tubules with spermatozoa – in P. esculentus (Palmiero et al. 2003). prevalence of quiescent ISPG and SPZ ready to be After 1 and 4 h treatment, 10−8 M Kp-10 dose significantly released under suitable hormonal and environmental decreased cx43 compared to the control (P < 0.01), conditions (Fig. 7A1). In buserelin-treated animals whereas both 10−7 and 10−6 M Kp-10 increased it most tubules were devoid of SPZ attached to Sertoli (P < 0.01) (Fig. 6A6 and B6). cells, and the number of tubules containing released To ascertain any role of Kp on SPZ, during the SPZ in the middle of the lumen was higher than that winter stasis (December) Kp-10 effects were observed in control group (Fig. 7A4 and A5) (buserelin vs in vivo in parallel to those of buserelin, a well- control P < 0.01); by contrast, in Kp-10-treated frogs known GnRH agonist capable to induce spermiation most tubules showed detached SPZ (Fig. 7A2 and (Minucci et al. 1989). While SPZ were collected in A5) (P < 0.01 vs control group). Kp-10 effect was cloacal fluid of buserelin-injected animals, no SPZ completely counteracted by the combined treatment were collected from the cloacal fluid of Kp-10- with Kp-234 (Kp-10 + A), suggesting a direct effect on injected animals (Fig. 7). Morphological analysis of Gpr54 receptor (Fig. 7A3 and A5).

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Downloaded from Bioscientifica.com at 09/28/2021 03:10:44PM via free access Kisspeptin in steroidogenesis and spermiation 411

April, when the expression rate of both proteins is high, throughout the annual cycle the expression peaks of Kiss-1 precede those of Gpr54, with opposite expression profile detected in post-reproductive period (May–July) and in late resumption – early winter stasis (November– December). Nevertheless, Kiss-1 and Gpr54 expression profiles parallel the fluctuations of steroid hormones that characterize the sexual cycle of frogs (Fasano et al. 1993, Cobellis et al. 1997, Polzonetti et al. 1998). In fact, overlapping profiles between Kiss-1 and testosterone levels occur throughout the annual cycle with highest levels registered in reproductive period and resumption, when testosterone drives sperm release and post-meiotic stages maturation, respectively (Rastogi et al. 1976). The expression profile of Gpr54, instead, parallels the biosynthesis of estradiol, except in the breeding season (April) when its expression peak parallels the increase of testosterone levels that supports sperm release. The high expression levels of both Kiss-1 and Gpr54 at the end of the winter stasis (February), a critical period for the onset of a new reproductive cycle under the hormonal control of estradiol that recruits quiescent spermatogonia for proliferation in cooperation with intratesticular activity of GnRH (Cobellis et al. 2002, 2003) and Kp-10 (Chianese et al. 2015), deserve particular attention. These observations corroborate the evidence that in frog, in the same timeframe, Kp-10 has the ability to promote the transcription of its own receptor similar to estradiol (Chianese et al. 2013). Consistently, in vitro treatment of frog testis collected in February just revealed estradiol- dependent expression of both Kiss1 and Gpr54 proteins, according to previous observations concerning the Figure 7 Histological analysis of frog testis injected in December with expression rate of gpr54 mRNA in frog (Chianese et al. KRB alone (C; A1), Kp-10 (10−6 M; A2), Kp-10 + A (frogs were firstly 2013) and data from mammalian brain (Pinilla et al. −5 injected with 10 M Kp-234; after 30 min they were injected with 2012, Herbison 2016). However, estradiol effects were 10−6 M Kp-10; A3) and buserelin (10−5 M; A4) after staining with standard hematoxylin–eosin. White arrows, fully released SPZ; black fully counteracted by ICI 182780, thus involvement of arrows, detached SPZ. Count of tubules with released SPZ (white nuclear estrogen receptors may be suggested. bars) or with detached SPZ (black bars) on the total number of At present, most Kp functions in reproduction, tubules/section/animal. Data are reported as the % of tubules including puberty onset and the switch on/off of steroid containing released (detached) SPZ over the total of tubules counted biosynthesis, have been supposed to be the direct for animals ± s.d. (n = 3–4). Asterisks (letters) indicate the statistically consequence of its hypothalamic activity on GnRH- significant differences P( < 0.01) among tubules containing released secreting neurons (Pinilla et al. 2012). Nevertheless, (detached SPZ) vs control. the localizations of Kiss1 and Gpr54 proteins suggest autocrine and paracrine activities inside the testis due Discussion to intratesticular Kp production. In fact, interstitial Although the characterization of Kp system in the brain is Leydig cells are the main producers of Kiss1 throughout one of the major finding in reproductive endocrinology the annual sexual cycle, as reported in mouse and of the last 20 years, data concerning the synthesis and primates (Anjum et al. 2012, Mei et al. 2013, Hsu et al. the activity of Kp inside testis are really poor. Here we 2014, Salehi et al. 2015, Wang et al. 2015, Irfan et al. demonstrate, for the first time in a non-mammalian 2016). In parallel, immunoreactivity for Gpr54 has vertebrate, the intratesticular production of Kiss-1 been detected in interstitial compartment, confirming protein during the annual sexual cycle and confirm the previous data concerning the localization of gpr54 presence of Gpr54 previously reported at mRNA level mRNA by in situ hybridization (Chianese et al. 2013). only (Chianese et al. 2013). The fluctuations of Kiss-1/ Thus, autocrine activity on Leydig cells may occur. Gpr54 proteins during the annual sexual cycle of frogs Conversely, Gpr54 immunoreactivity has been observed open new perspective in understanding the possible role in the germinal compartment in both somatic and germ in testis physiology. With the exception of February and cells suggesting the involvement of Kp signaling in www.reproduction-online.org Reproduction (2017) 154 403–414

Downloaded from Bioscientifica.com at 09/28/2021 03:10:44PM via free access 412 R Chianese and others intratesticular paracrine communication (i.e. Leydig vs do not fully parallel the expression profile of3 β-HSD Sertoli cells, Leydig vs germ cells and Sertoli vs germ and ar mRNA, that are comparable at all doses or cells). The localization of Gpr54 in Sertoli cells is not decreased, respectively. Similarly, for estradiol levels, surprising since it has been detected in adult testes the expression of cyp17, cyp19, era and erb mRNA of rhesus monkey and common marmoset monkey increases (present data and Chianese et al. 2013, 2015) (Irfan et al. 2016) but not in rodents (Mei et al. 2013, in parallel to the decrease of estradiol levels observed Hsu et al. 2014). Conversely, at present, the frog is the at 10−8–10−6 M doses. Conversely, at longer treatment only specie that expresses Gpr54 – both protein and time (4 h), steroid levels do not display any statistically mRNA – in peritubular myoid cells and spermatogonia significant difference, whereas the expression rates (present data and Chianese et al. 2013). This observation of all the considered steroidogenesis enzymes and confirms recent data concerning the requirement of Kp of ar, era and erb mRNA are higher than those in signaling for spermatogenesis onset and progression control group (present data and Chianese et al. 2013, in frog (Chianese et al. 2013, 2015), a situation that in 2015). Intriguingly, data in frog revealed a possible mammals mimics puberty onset. However, Kp half-live involvement of Kp in the balance between testosterone in biological tissues has been reported to be very low tone and its irreversible conversion into estradiol. In (Liu et al. 2013), and the presence of different shorter fact, (i) at each dose, Kp-10 effects on testosterone Kp fragments, with specific inter- and intra- cellular and estradiol are opposite; (ii) estradiol–testosterone localizations and expression profiles (and functions ratio revealed that 10−9 and 10−8 M Kp-10 doses −7 as consequence), has been reported in the brain move hormonal balance toward E2 whereas 10 and (Franceschini et al. 2013). At present, the biochemical 10−6 M Kp-10 doses toward testosterone ones. Thus, characterization and purification of any Kiss-1 cleavage lower Kp-10 doses may promote estradiol-dependent peptides have never been reported from the gonad of intratesticular activities and highest doses the amphibians. Furthermore, the anti-Kiss1 antiserum used testosterone-dependent ones. Accordingly, in the same in this study is directed towards the C-terminal common time frame, Kp-10 treatment induced the progression portion of all Kiss-1 products, including both the larger of spermatogenesis (Chianese et al. 2015) with lowest form and the cleavage products, raising the possibility doses increasing mitotic stages – well-known estradiol- that different Kiss-1 peptides may exert different dependent stages (Cobellis et al. 2002) – and highest biological activities throughout the annual cycle of frogs. doses promoting the progression toward meiotic stages Since the major site of localization of Gpr54 is the (Chianese et al. 2015) and the release of spermatozoa interstitium, the possible role of Kp system in the direct in the lumen (present data), well-known testosterone- regulation of steroidogenesis has been examined by ex dependent process in mammals as for the progression vivo treatment of testis with increasing doses of Kp-10. of germ cells beyond meiosis (Sharpe 1994). Interestingly, we demonstrated that the expression of Frog SPZ expressed Gpr54, a condition functionally key enzymes for the biosynthesis of testosterone and correlated to fertilization process and sperm estradiol were differently regulated by Kp-10 after hypermotility in human and mouse SPZ, respectively 1 h of treatment, with direct consequences on the (Pinto et al. 2012, Hsu et al. 2014). In frog, pituitary intratesticular levels of both testosterone and estradiol. and estradiol signaling are critical to Consistently, Kp-10 administration in adult male rhesus gain successful spermiation and sperm transport toward monkeys treated with GnRH-antagonist sustains the cloaca (Cobellis et al. 2005, 2008). Here, morphological hCG-induced production of testosterone (Irfan et al. analysis of Kp-10-treated testes revealed a large number 2014); by contrast, no effects on testosterone biosynthesis of tubules with SPZ detached from Sertoli cells, and the have been detected following in vitro treatment of adult detection of Gpr54 in both Sertoli cells and peritubular testis in monkey (Tariq & Shabab 2016) nor in cultured myoid cells supports a possible involvement in sperm Leydig cells (Mei et al. 2013, Wang et al. 2015) or testis release. Accordingly, fully released SPZ occur in the explants in mice (Mei et al. 2013). In this study we lumen of animals treated at higher Kp-10 doses. Indeed, demonstrate that at 1 h of treatment, Kp-10 directly in vivo Kp-10 caused the detachment of SPZ from decreases intratesticular testosterone levels vs control Sertoli cells, but failed to induce the transport of SPZ group with higher effects observed at the lowest doses in cloacal fluids as buserelin, used as positive control, (10−9–10−8 M) and have double effects on estradiol did. Thus, Kp-10 involvement in the reshuffle of junction levels, being stimulatory at 10−9 M dose only and proteins between Sertoli and sperm cells may be inhibitory at the highest ones. In parallel, decreased hypothesized. Consistently, Kp-10-dependent changes expression levels of 3β-HSD and increases of cyp19, in the expression of cx43, the main junction protein the enzyme that irreversibly converts testosterone connecting Sertoli cells to germ cells and spermatozoa into estradiol, have been observed. Interestingly, at in P. esculentus (Palmiero et al. 2003), have been here 10−7 and 10−6 M Kp-10 doses, testosterone levels are reported. In particular, 10−8 M Kp-10 dose decreases the significantly lower than those in control group, but expression of cx43 whereas higher Kp-10 doses increase are higher than those in 10−9 and 10−8 M doses and it. This aspect fully fits the steroid-dependent expression

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Downloaded from Bioscientifica.com at 09/28/2021 03:10:44PM via free access Kisspeptin in steroidogenesis and spermiation 413 of cx43 (Izzo et al. 2009). In fact, 10−8 M dose decreases Chianese R, Ciaramella V, Scarpa D, Fasano S, Pierantoni R & Meccariello R the intratesticular level of estradiol, a steroid that usually 2012 Anandamide regulates the expression of GnRH1, GnRH2 and GnRHRs in frog testis. American Journal of Physiology: Endocrinology exerts positive effects on cx43 expression. By contrast, and Metabolism 303 E475–E487. (doi:10.1152/ajpendo.00086.2012) highest Kp-10 doses may promote the testosterone- Chianese R, Ciaramella V, Fasano S, Pierantoni R & Meccariello R 2013 dependent activities, including the expression of cx43 Kisspeptin receptor, GPR54, as a candidate for the regulation of testicular activity in the frog Rana esculenta. Biology of Reproduction 73 1–11. (Izzo et al. 2009). In addition, the involvement of local Chianese R, Ciaramella V, Fasano S, Pierantoni R & Meccariello R 2014 GnRH signaling system – that in frog comprises two Hypothalamus-pituitary axis: an obligatory target for endocannabinoids ligands and three receptors (Chianese et al. 2011) and to inhibit steroidogenesis in frog testis. General and Comparative has a recognized role in spermiation (Minucci 1989) – Endocrinology 205 88–93. (doi:10.1016/j.ygcen.2014.02.010) Chianese R, Ciaramella V, Fasano S, Pierantoni R & Meccariello R 2015 may be suggested. In fact, in February, gnrhs and gnrhrs Kisspeptin drives germ cell progression in the anuran amphibian mRNA have been detected in SPZ attached to Sertoli Pelophylax esculentus: a study carried out in ex vivo testes. General cells (Chianese et al. 2012) and high Kp-10 doses and Comparative Endocrinology 211 81–91. (doi:10.1016/j. ygcen.2014.11.008) positively affect the expression rates of GnRH-II and Chianese R, Cobellis G, Chioccarelli T, Ciaramella V, Migliaccio M, GnRH-Rs (Chianese et al. 2015). Fasano S, Pierantoni R & Meccariello R 2016 Kisspeptins, estrogens and Taken together, our data demonstrate the existence of male fertility. Current Medicinal Chemistry 23 4070–4091. (doi:10.2174 a complete Kp system in the testis of a non-mammalian /0929867323666160902155434) Ciaramella V, Meccariello R, Chioccarelli T, Sirleto M, Fasano S, specie and a double functional role in the modulation Pierantoni R & Chianese R 2016 Anandamide acts via kisspeptin in of spermatogenesis in frog. First, as autocrine factors the regulation of testicular activity of the frog, Pelophylax esculentus. for Leydig cells, Kp signaling modulates steroid Molecular and Cellular Endocrinology 420 75–84. (doi:10.1016/j. biosynthesis. Second, as paracrine factor, Kp produced mce.2015.11.011) Cobellis G, Pierantoni R & Fasano S 1997 c-fos- and c-jun-like mRNA by the interstitium targets spermatogonia and sperm expression in frog (Rana esculenta) testis during the annual reproductive cells to affect spermatogenesis onset/progression and cycle. General and Comparative Endocrinology 106 23–29. (doi:10.1006/ sperm functions, respectively. gcen.1996.6846) Cobellis G, Pierantoni R, Minucci S, Pernas-Alonso R, Meccariello R & Fasano S 1999 c-Fos activity in Rana esculenta testis: seasonal and estradiol-induced changes. Endocrinology 140 3238–3244. (doi:10.1210/endo.140.7.6790) Supplementary data Cobellis G, Meccariello R, Fienga G, Pierantoni R & Fasano S 2002 Cytoplasmic and nuclear Fos protein forms regulate resumption of This is linked to the online version of the paper at http://dx.doi. spermatogenesis in the frog, Rana esculenta. Endocrinology 143 org/10.1530/REP-17-0030. 163–170. (doi:10.1210/endo.143.1.8567) Cobellis G, Meccariello R, Minucci S, Palmiero C, Pierantoni R & Fasano S 2003 Cytoplasmic versus nuclear localization of fos-related proteins in Declaration of interest the frog, Rana esculenta, testis: in vivo and direct in vitro effect of a gonadotropin-releasing hormone agonist. Biology of Reproduction 68 The authors declare that there is no conflict of interest that 954–960. (doi:10.1095/biolreprod.102.008938) could be perceived as prejudicing the impartiality of the Cobellis G, Lombardi M, Scarpa D, Izzo G, Fienga G, Meccariello R, Pierantoni R & Fasano S 2005 Fra1 activity in the frog, Rana esculenta, research reported; there is any financial or other potential testis: a new potential role in sperm transport. Biology of Reproduction conflict of interest. 72 1101–1108. 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Reproduction (2017) 154 403–414 www.reproduction-online.org

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