Annual Sexual Cycle Research Articles

Динамика морфологических и физиологических показателей промысловых рыб Каспийского моря на разных стадиях полового цикла

Serious changes in the ecological situation of а number of valuable fish species functioning in the Caspian Sea lead to a significant transformation of their breeding conditions, which contributes to significant changes in the entire fish fauna of the aquatic ecosystem of the region. Therefore, the purpose of the study was to monitor the morphological and physiological parameters of commercial fish of the Caspian Sea. The experiment was carried out on the material of 1200 species of economically valuable commercial fish species belonging to different ecological groups. The results of the study showed that the nature of the change in the content of reserve fat in the body of fish is closely related to the development and maturation of reproductive products during the annual sexual cycle. For example, it was shown that in September, in the gonads of the studied fish, trophoplasmic growth of oocytes and the transition of the ovaries to the third stage of maturity begin. From this moment, a noticeable increase in the maturity coefficient is observed, which by November in some fish species approaches its maximum value. And fatness and fat content at the same time are noticeably reduced (except for perch). A violation of the usual dynamics of the formation of fat and its distribution in the body during the year, caused by violations of environmental conditions due to human economic activity, was noted. Keywords: COMMERCIAL FISH, AQUACULTURE OF THE CASPIAN SEA, MORPHOLOGICAL AND PHYSIOLOGICAL PARAMETERS

Sexual segment of the kidney of the lizard, Eutropis carinata: A light microscopic and ultrastructural seasonal study.

We studied seasonal variation of the secretory granules in the epithelial cells of the sexual segment of the kidney (SSK) during the annual sexual cycle in the lizard, Eutropis carinata using light and electron microscopy in correlation with measurements of androgen levels. During the breeding phase, the epithelium of the SSK consists of simple columnar cells with basal nuclei. The cytoplasm contains numerous eosinophilic secretory protein and carbohydrate granules, but lacks glycosaminoglycans. These secretory granules develop during the regenerative phase when the circulating testosterone level increase. During the breeding phase, when the circulating testosterone levels are high, three types of secretory granules can be differentiated in the cytoplasm based on size and opacity; electron translucent type I, electron dense type II, and biphasic type III granules. Type II granules are found at various stages of maturity and degeneration/utilization. All types of secretory granules are released through an apocrine process. Microvilli and tight junctions are prominent at the apical portion of the cell. The cytoplasm contains, Golgi complexes, an abundant network of rough endoplasmic reticulum, numerous tubular mitochondria, condensing, mucus filled and empty vacuoles. Intercellular canaliculi are narrow and indistinct during the regenerative and breeding phases, respectively. During the regressed phase, when the circulating testosterone levels are lowest, the cells are found regressed with wide intercellular canaliculi and devoid of secretory granules. Then the cytoplasm contains a few round mitochondria, Golgi and scanty endoplasmic reticulum.

Determination of annual reproductive cycle in male sterlet, Acipenser ruthenus using histology and ultrasound imaging.

The aim of this study was to evaluate seasonal testicular development in the cultured sterlet, Acipenser ruthenus. During annual sexual cycle of male sterlet, stages of gonad maturity were examined using histology and ultrasonography approaches. The histology identified males at different stages of maturity among fish sampled monthly. According to the seasonal changes in the testes, reproductive cycle was divided into four stages including resting, pre-spawning, spawning, and post-spawning. The histology examination revealed considerable variation in testicular developmental stages. These changes were identified based on persistent spermatogenesis and asynchronous gonad development in testes, showing that regulation of annual gonadal cycle is influenced by season. Also, the results obtained using ultrasound suggested that reproductive stages can be identified based on morphology and tissue echogenicity. At each phase of testicular development, gonadosomatic index (GSI) and number of spermatogenic cysts were variable. The present study focused on determination of annual reproductive development in cultured male sterlet which clearly identifies reproductive stage in each season as valuable guide for future researches on reproductive biology of sterlet. This study presents basic knowledge about reproductive biology in sterlet contributing to optimal broodstocks management that allows comparison of reproductive development among sturgeon species.

Kisspeptin regulates steroidogenesis and spermiation in anuran amphibian.

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 proteins during the annual sexual cycle and that ex vivo 17B-estradiol (E2, 10-6 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 testosterone 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.

Cryptic virulence and avirulence alleles revealed by controlled sexual recombination in pea aphids.

Although aphids are worldwide crop pests, little is known about aphid effector genes underlying virulence and avirulence. Here we show that controlling the genetics of both aphid and host can reveal novel recombinant genotypes with previously undetected allelic variation in both virulence and avirulence functions. Clonal F1 progeny populations were derived from reciprocal crosses and self-matings between two parental genotypes of pea aphid (Acyrthosiphon pisum) differing in virulence on a Medicago truncatula host carrying the RAP1 and RAP2 resistance genes. These populations showed Mendelian segregation consistent with aphid performance being controlled largely by a dominant virulence allele derived from only one parent. Altered segregation ratios on near-isogenic host genotypes differing in the region carrying RAP1 were indicative of additional heritable functions likely related to avirulence genes originating from both parents. Unexpectedly, some virulent F1 progeny were recovered from selfing of an avirulent parent, suggesting a reservoir of cryptic alleles. Host chlorosis was associated with virulence, whereas necrotic hypersensitive-like response was not. No maternal inheritance was found for any of these characteristics, ruling out sex-linked, cytoplasmic, and endosymbiotic factors. Our results demonstrate the tractability of dissecting the genetic basis of pest-host resistance mechanisms and indicate that the annual sexual cycle in aphids may lead to frequent novel genotypes with both increased and decreased virulence. Availability of genomes for both pest and host can facilitate definition of cognate gene-for-gene relationships, potentially leading to selection of crop genotypes with multiple resistance traits.

Diversity of sexual strategies of fish species caught by bottom trawl off the western Iberian Peninsula

Samples of boarfish Capros aper, small red scorpionfish Scorpaena notata and comber Serranus cabrilla were collected from monthly by-catch samples on board commercial trawlers off the western coast of Portugal. A histological approach was undertaken to study each species’ reproductive strategy and annual sexual cycle. Temporal variations of the gonadosomatic index and of two condition indices (hepatosomatic and Fulton's K) were also analysed. Results showed that C. aper and S. notata are gonochoristic whilst S. cabrilla is a simultaneous hermaphrodite. Fertilization is external and development of secondary growth follicles is asynchronous in the three species. Mass atresia characterized the end of the spawning season in S. notata and S. cabrilla. In C. aper a high prevalence of atresia was observed throughout the year and was not attributed to regressing or regenerating phases or skip spawning events. The main spawning season of the three species was in the summer and it is suggested that spawning is related to upwelling. During the summer, the spawning interval was 1.09, 1.23 and 1.84 days for S. cabrilla, S. notata and C.aper, respectively. The results of this study indicate potential plasticity and environmental adaptation of sexual strategies.

Kisspeptin Receptor, GPR54, as a Candidate for the Regulation of Testicular Activity in the Frog Rana esculenta1

Kisspeptins, acting via GPR54, are new players in the control of reproductive axis. They have the ability to communicate with GnRH neurons sending environmental, metabolic, and gonadal signals, with the induction of GnRH and LH secretion as final effect. At present, the physiological significance of kisspeptin signaling in the gonad is poorly investigated. We cloned GPR54 receptor from the anuran amphibian Rana esculenta testis and investigated its expression in several tissues (brain, spinal cord, ovary, muscle, and kidney). In particular, the expression analysis was carried out in pituitary and testis during the annual sexual cycle. Pituitary and testicular GPR54 mRNA increased at the end of the winter stasis (February) and reached high levels during the breeding season (April). The analysis of GPR54 expression in testis was reinforced by in situ hybridization that revealed GPR54 presence in the interstitial compartment and in proliferating germ cells. Testicular GPR54 expression in February and in June was indicated to be estradiol dependent. Furthermore, in February, kisspeptin-10 (Kp-10) induced the testicular expression of both GPR54 and estrogen receptor alpha (ERalpha) in a dose-dependent manner. Conversely, in March, Kp-10 had a biphasic effect on the expression of ERalpha, being inhibitory at short (1 h) and stimulatory at longer (4 h) incubation time. In conclusion, our results demonstrate that frog testis expresses GPR54 in an estradiol-dependent manner and that Kp-10 modulates the testicular expression of ERalpha; thus, the kisspeptin/GPR54 system might be locally involved in the regulation of estrogen-dependent testicular functions such as germ cell proliferation and steroidogenesis.

Anandamide regulates the expression of GnRH1, GnRH2, and GnRH-Rs in frog testis

Gonadotropin-releasing hormone (either GnRH1 or GnRH2) exerts a local activity in vertebrate testis, including human testis. Relationships between endocannabinoid (eCB) and GnRH systems in gonads have never been elucidated in any species so far. To reveal a cross-talk between eCBs and GnRH at testicular level, we characterized the expression of GnRH (GnRH1 and GnRH2) as well as GnRH receptor (GnRH-R1, -R2, and -R3) mRNA in the testis of the anuran amphibian Rana esculenta during the annual sexual cycle; furthermore, the corresponding transcripts were localized inside the testis by in situ hybridization. The possible endogenous production of the eCB, anandamide (AEA), was investigated in testis by analyzing the expression of its biosynthetic enzyme, Nape-pld. Incubations of testis pieces with AEA were carried out in the postreproductive period (June) and in February, when a new spermatogenetic wave takes place. In June, AEA treatment significantly decreased GnRH1 and GnRH-R2 mRNA, stimulated the transcription of GnRH2 and GnRH-R1, and did not affect GnRH-R3 expression. In February, AEA treatment upregulated GnRH2 and GnRH-R3 mRNA, downregulated GnRH-R2, and did not affect GnRH1 and GnRH-R1 expression. These effects were mediated by type 1 cannabinoid receptor (CB1) since they were fully counteracted by SR141716A (Rimonabant), a selective CB1 antagonist. In conclusion, eCB system modulates GnRH activity in frog testis during the annual sexual cycle in a stage-dependent fashion.

Life history parameters as possible discriminators of bluemouth Helicolenus dactylopterus (Delaroche, 1809) populations in Portuguese waters

The age, growth and reproductive biology of bluemouth, Helicolenus dactylopterus were studied in order to assess the use of these life history parameters as possible population discriminators in Portuguese waters. Fish were obtained from commercial landings in mainland Portugal (Peniche), Azores, and Madeira and supplementary samples were obtained from research surveys in the Portuguese continental slope. Significant differences in mean total-length and mean length-at-age were found between the three areas. The reproductive biology was analysed using the annual sexual cycle, three bionergetic indexes and the maturity ogives. Individuals presented a similar reproductive strategy in Portuguese waters with asynchrony of sexual cycles, similar female spawning season in the three areas and a sperm retention period in the ovaries of 1 to 6–7 months. The bionergetic indexes showed significant differences through the maturative stages and among areas. No significant differences were obtained when comparing the length at first maturity between mainland Portugal and Azores for both sexes. The results of this study will provide a comprehensive understanding of this species life history and presented useful evidence that can support the presence of different populations in the Portuguese waters.

Fission, regeneration, gonad production and lipids storage in the pyloric caeca of the sea star Allostichaster capensis

Allostichaster capensis is a fissiparous sea star that displays an annual sexual and asexual reproductive cycle. After fission, individuals spend the rest of the year regenerating the missing arms. To analyze feeding rates, we conducted three experiments with sea stars in different states of regeneration: recently split, regenerating and arms with advanced regeneration. Three different treatments of food availability (high, intermediate and low) were carried out during each experiment. We found that food availability and regeneration state had an important effect on feeding rates. Recently split individuals showed the lowest values of feeding rates, amount of lipids in the pyloric caeca and gonad production, regardless of the food treatment. Regenerating individuals had the highest feeding rates, allocated of resources in the pyloric caeca and produced gametes. Individuals with arms in an advanced regeneration state showed intermediate feeding rates and no deposition of lipids in the pyloric caeca. These individuals spawned during the experiment, thus indicating that the gonads were mature. Low food availability produced the lowest feeding rates regardless of the regeneration state. Regenerating individuals died only in low food availability. No death was observed in individuals with advance regenerated arms. On the other hand, individuals that had recently undergone fission died regardless of the food availability. Since fission and regeneration are part of the life-history of A. capensis, regenerating individuals may have high feeding rates to achieve a good nutritional state to support regeneration, gamete production and survival after fission.

Anandamide modulates the expression of GnRH-II and GnRHRs in frog, Rana esculenta, diencephalon

In the hypothalamus, endocannabinoids affect neuroendocrine activity by means of Gonadotropin-Releasing-Hormone-I (GnRH-I) inhibition. Since most vertebrates, human included, possess at least two GnRH molecular forms, the aim of this work was to investigate the effect of endocannabinoids on GnRH molecular forms other than GnRH-I and on GnRHRs.Thus, we cloned GnRH precursors as well as GnRH receptors (GnRHR-I, GnRHR-II, GnRHR-III) from the diencephalons of the anuran amphibian, Rana esculenta. GnRH-II expression was evaluated in pituitary, whole brain, spinal cord, hindbrain, midbrain and forebrain during the annual sexual cycle. Then, in post-reproductive period (May), GnRH-I, GnRH-II and GnRHRs expression was evaluated by quantitative real time (qPCR) after incubation of diencephalons with the endocannabinoid anandamide (AEA). AEA significantly decreased GnRH-I and GnRH-II expression, up regulated GnRHR-I and GnRHR-II mRNA and it had no effect upon GnRHR-III expression. These effects were counteracted by SR141716A (Rimonabant), a selective antagonist of type I cannabinoid receptor (CB1).In conclusion our results demonstrate a CB1 receptor dependent modulation of GnRH system expression rate (both ligands and receptors) in frog diencephalons. In particular, we show that AEA, besides GnRH-I, also acts on GnRH-II expression.

Reproductive phase‐related variations in cocaine‐ and amphetamine‐regulated transcript (CART) in the olfactory system, forebrain, and pituitary of the female catfish, Clarias batrachus (Linn.)

The annual sexual cycle of Clarias batrachus is divisible into resting (December-January), preparatory (February-April), prespawning (May-June), spawning (July- August), and postspawning (September-November) phases. The gonosomatic indices rose steadily through the preparatory and prespawning phases, peaked in the spawning phase, and were greatly reduced during the postspawning and resting phases. A clear pattern of change was also identified in the immunocytochemical profile of the luteinizing hormone (LH) cells in the pituitary. These changes were correlated with the cocaine- and amphetamine-regulated transcript (CART)-immunoreactive system in the forebrain and pituitary. In the olfactory bulb, CART immunoreactivity in the terminal fields of the mitral cell layer, granule cells, and medial olfactory tracts gradually decreased during the resting through prespawning phases. However, it was considerably augmented during spawning (P < 0.001) and showed highest activity in the postspawning phase (P < 0.001). A different pattern was noticed in the fibers and/or neurons of the lateral part of ventral telencephalic area, the entopeduncular nucleus, and the dorsal part of the nucleus preopticus periventricularis. In these areas, intense immunoreactivity seen in preparatory phase, declined during prespawning (P < 0.01) then through spawning, and was partially augmented during the postspawning and resting phases (P < 0.05). A similar pattern was also seen in the nucleus preglomerulosus lateralis and medialis, nucleus dorsalis posterioris of thalamus, lobobulbar nucleus, and the nucleus of posterior recess. CART was transiently expressed in LH cells in the pituitary during the preparatory period. We suggest that the CART system may play a role in triggering the brain-pituitary-ovary axis at the onset of the preparatory phase.

The reproductive activity in the testis of Podarcis s. sicula involves d-aspartic acid: A study on c-kit receptor protein, tyrosine kinase activity and PCNA protein during annual sexual cycle

The current study provides substantial evidence that the pattern of synthesis of d-aspartic acid ( d-Asp) in the testes of lizard Podarcis s. sicula throughout the reproductive cycle is in parallel with seasonal variations of testosterone, c-kit receptor protein, tyrosine kinase activity, and proliferating cell nuclear antigen (PCNA) protein. Although the trend is the same in all phases of the sexual cycle, the peaks of these three molecules are detectable only during the reproductive period. Using Western blot technique, we demonstrated that both polyclonal c-kit and PCNA antibodies specifically recognized bands with molecular mass of ∼150 and ∼36 kDa, respectively. By immunocytochemical methods, d-Asp immunopositivity appeared spread in the germinal epithelium as well as in the interstitial compartment of the testes. We also found specific c-kit labeling in I and II spermatogonia (SPG), in I and II spermatocytes (SPC), in the elongated spermatides, in spermatozoa, in Sertoli and Leydig cells. Like c-kit, PCNA positivity was located in the germinal epithelium pattern. Furthermore, we investigated the relationship between testosterone, c-kit receptor, tyrosine kinases activity and PCNA following treatment with d-Asp. In vivo experiments, entailing a single injection of d-Asp (2.0 μmol/g body weight), demonstrated that this amino acid significantly accumulated in the testes. After 3 h, its uptake was accompanied by an increase in testosterone levels and in the expression and intensity of immunostaining of c-kit receptor protein. Furthermore, at 6 h, exogenous d-Asp affected the phosphorylation of tyrosine kinases, whose activation was positively correlated with the temporal uptake of both d-Asp and testosterone detected in the testes. Thereafter, between 6 and 15 h, the expression of PCNA was induced and an increase in its immunolabeling intensity was observed. Taken all together, these results provide new insights into the testicular activity during the reproductive cycle of Podarcis s. sicula, suggesting that a sequential cascade of a functional relationship between testosterone levels, c-kit receptor protein, tyrosine kinase activity and PCNA could be partly mediated by d-aspartic acid.

Biochemical composition of body compartments during the reproductive cycle of the starfish Allostichaster capensis in Patagonia, Argentina

Allostichaster capensis (Perrier, 1875) (= inaequalis) is a fissiparous starfish inhabiting the South Atlantic Ocean coasts. On the Patagonian coast, it displays an annual sexual and asexual reproductive cycle. Fission is most frequent during the austral summer (November-January) and arm regeneration takes place during the rest of the year. In this article, seasonal changes in biochemical composition of body compartments are presented for a population of this fissiparous starfish in Bahia Kaiser, Golfo Nuevo, Chubut, Argentina, from June 2000 to May 2002. During the annual cycle, an inverse relationship between gonad and pyloric caeca indices suggests nutrient transfer. Biochemical analysis revealed that only lipids are translocated from pyloric caeca to gonads at the beginning of gametogenesis. Rev. Biol. Trop. 56 (Suppl. 3): 351-360. Epub 2009 January 05.

Human follicle-stimulating hormone modulation of adrenal gland activity in the Italian crested newt, Triturus carnifex (Amphibia, Urodela)

The aim of the present study was to verify if human FSH influences the adrenal gland of the newt, Triturus carnifex. Newts were given intraperitoneal injections of human FSH throughout the periods of February–March, and December–January; periods in which newt FSH levels are normally very low. The effects of human FSH on adrenal gland activity were observed in the morphological features of the steroidogenic and chromaffin adrenal cells, and in the serum levels of aldosterone, corticosterone, norepinephrine and epinephrine. The effect of human FSH on the steroidogenic cells was significant during the February–March period; the quantity of cytoplasmic lipids decreased, and the corticosteroid serum levels increased. During the December–January period, the human FSH effects were negligible. The effect of human FSH on the chromaffin cells was significant during both the February–March, and the December–January periods. During February–March, the human FSH increased the numeric ratio of norepinephrine granules to epinephrine granules, and increased the epinephrine serum levels. During December–January, the human FSH decreased the numeric ratio of norepinephrine granules to epinephrine granules, and increased the norepinephrine serum levels. The results of the present study show that human follicle-stimulating hormone influences the activity of the newt adrenal gland, thus indicating a relationship between the annual sexual cycle and the annual adrenal cycle of the newt.

Non-mammalian vertebrate models and the endocannabinoid system: Relationships with gonadotropin-releasing hormone

Endocannabinoids, via cannabinoid receptors (CB1 and CB2), affect reproductive functions at both local and central level. Due to the high complexity of the endocannabinoid system, to the widespread distribution outside the nervous system and to the high degree of evolutionary conservation, a deep CB1 molecular characterization among species may be useful to elucidate the activity of endocannabinoids at multiple levels. In this review we report CB1 characterization in non-mammalian animal models and, in particular, in the anuran amphibian, the frog, Rana esculenta; we also describe its expression during the annual sexual cycle. Moreover, since reproductive functions are under control of gonadotropin releasing hormone (GnRH), cb1 mRNA and protein expression profile in the forebrain has been compared to those of GnRH-I, the mammalian form primarily involved in gonadotropin release.

Interplay between the Endocannabinoid System and GnRH-I in the Forebrain of the Anuran Amphibian Rana esculenta

The morphofunctional relationship between the endocannabinoid system and GnRH activity in the regulation of reproduction has poorly been investigated in vertebrates. Due to the anatomical features of lower vertebrate brain, in the present paper, we chose the frog Rana esculenta (anuran amphibian) as a suitable model to better investigate such aspects of the reproductive physiology. By using double-labeling immunofluorescence aided with a laser-scanning confocal microscope, we found a subpopulation of the frog hypothalamic GnRH neurons endowed with CB1 cannabinoid receptors. By means of semiquantitative RT-PCR assay, we have shown that, during the annual sexual cycle, GnRH-I mRNA (formerly known as mammalian GnRH) and CB1 mRNA have opposite expression profiles in the brain. In particular, this occurs in telencephalon and diencephalon, the areas mainly involved in GnRH release and control of the reproduction. Furthermore, we found that the endocannabinoid anandamide is able to inhibit GnRH-I mRNA synthesis; buserelin (a GnRH agonist), in turn, inhibits the synthesis of GnRH-I mRNA and induces an increase of CB1 transcription. Our observations point out the occurrence of a morphofunctional anatomical basis to explain a reciprocal relationship between the endocannabinoid system and GnRH neuronal activity.

UBPy/MSJ-1 system during male germ cell progression in the frog, Rana esculenta

mUBPy (mouse ubiquitin specific processing protease) is a de-ubiquitinating enzyme expressed in mouse testis and brain. In testis, it interacts with the DnaJ protein MSJ-1 (mouse sperm cell specific DnaJ first homologue), a molecular chaperone expressed in spermatids and spermatozoa. Since MSJ-1 is conserved among vertebrates, to demonstrate an evolutionarily conserved function of UBPy/MSJ-1 system, we assayed mUBPy presence in the anuran amphibian, the frog, Rana esculenta, during the annual sexual cycle. By Western blot we have detected a specific signal of 126 kDa in testis and isolated spermatozoa. During the annual sexual cycle, the signal gradually increases as soon as spermatogenesis resumes after the winter stasis. Using immunocytochemistry, we have localized the protein in spermatids and spermatozoa. In conclusion, UBPy/MSJ-1 system is available in R. esculenta testis suggesting a conserved fundamental function in spermatogenesis and sperm formation.

17β-Estradiol induces Akt-1 through estrogen receptor-β in the frog (Rana esculenta) male germ cells

Several lines of evidence support the key role of estrogens in male fertility. Here, we investigate the regulation of the serine/threonine kinase Akt-1 in the frog (Rana esculenta) testis during the annual sexual cycle and, whether 17beta-estradiol (E2) exerts a role in the Akt-1 activity. Akt-1 has been shown to be the mediator of growth factor-dependent cell proliferation, survival, and metabolism in a variety of cell types. First, we demonstrate by immunohistochemistry, the presence of estrogen receptor-beta (ERbeta), and Akt-1 in the spermatogonia (SPG), spermatocytes (SPC), and spermatids (SPT). Western-blot analysis revealed that ERbeta isoform (molecular weight 55 kDa) was highly expressed in May (reproductive period) with respect to January and November (winter stasis); in parallel, Akt-1 (molecular weight 60 kDa) is highly phosphorylated (Ser-473) during the period of active spermatogenesis (May) compared with the winter stasis (January and November). In addition, in vitro experiments demonstrate that E2 treatment induces the activation of Akt-1, and this effect is counteracted by the anti-estrogen ICI 182-780. In conclusion, our data show that E2 induces Akt-1 phosphorylation (Ser-473) possibly via ERbeta in frog (R. esculenta) male germ cells.

Type‐1 cannabinoid receptor expression in the frog, Rana esculenta, tissues: A possible involvement in the regulation of testicular activity

Endogenous cannabinoids and type-1 cannabinoid receptor (CB1) are widely produced and distributed in the central nervous system (CNS) and peripheral nerves in mammals. In addition, the detection of endocannabinoids and corresponding receptors in non nervous peripheral tissues indicates an involvement of the system in the control of a wide range of physiological activities, including reproduction. Recently, the existence of CB1 was also observed in lower vertebrates and in urochordate suggesting that the endocannabinoid system is phylogenetically conserved. Using RT-PCR, CB1 mRNA expression profiles were characterized in a wide range of tissues of the anuran amphibian, the frog, Rana esculenta. Besides a strong expression in the CNS, CB1 was also present in testis, kidney, liver, ovary, muscle, heart, spleen, and pituitary. The CB1 expression pattern has been characterized in both testis and CNS during the annual sexual cycle. In testis, CB1 is poorly expressed during the winter stasis of the spermatogenesis rising during the breeding season and resumption period. An expression profile mismatching to that observed in testis was detected in whole-brain preparations during the sexual cycle; in particular in the diencephalon, the encephalic area mainly involved in the control of reproductive functions. Furthermore, fluctuations inside isolated encephalic areas and spinal cord were observed all over the reproductive cycle. In conclusion, CB1 receptor is expressed in R. esculenta CNS and testis. As far as the gonad it concerns, our results suggest the involvement of the endocannabinoids in the control of reproductive function.