The Satellite DNA PcH-Sat, Isolated and Characterized in the Limpet Patella caerulea (Mollusca, Gastropoda), Suggests the Origin from a Nin-SINE Transposable Element.

Satellite DNA (sat-DNA) was previously described as junk and selfish DNA in the cellular economy, without a clear functional role. However, during the last two decades, evidence has been accumulated about the roles of sat-DNA in different cellular functions and its probable involvement in tumorigenesis and adaptation to environmental changes. In molluscs, studies on sat-DNAs have been performed mainly on bivalve species, especially those of economic interest. Conversely, in Gastropoda (which includes about 80% of the currently described molluscs species), studies on sat-DNA have been largely neglected. In this study, we isolated and characterized a sat-DNA, here named PcH-sat, in the limpet Patella caerulea using the restriction enzyme method, particularly HaeIII. Monomeric units of PcH-sat are 179 bp long, AT-rich (58.7%), and with an identity among monomers ranging from 91.6 to 99.8%. Southern blot showed that PcH-sat is conserved in P. depressa and P. ulyssiponensis, while a smeared signal of hybridization was present in the other three investigated limpets (P. ferruginea, P. rustica and P. vulgata). Dot blot showed that PcH-sat represents about 10% of the genome of P. caerulea, 5% of that of P. depressa, and 0.3% of that of P. ulyssiponensis. FISH showed that PcH-sat was mainly localized on pericentromeric regions of chromosome pairs 2 and 4-7 of P. caerulea (2n = 18). A database search showed that PcH-sat contains a large segment (of 118 bp) showing high identity with a homologous trait of the Nin-SINE transposable element (TE) of the patellogastropod Lottia gigantea, supporting the hypothesis that TEs are involved in the rising and tandemization processes of sat-DNAs.

Life History Traits and Longevity of the Invasive Asian Common Toad Duttaphrynus melanostictus (Schneider, 1799) in Madagascar.

We analyzed the body length, age structure, and age at sexual maturity of the invasive Asian common toad Duttaphrynus melanostictus from different sites in Toamasina, east Madagascar. We used skeletochronology as a proxy for age estimation, while gonads were histologically analyzed to determine the age of sexual maturity. The analysis of pooled age data from three sites investigated in 2016 showed that both sexes were larger, although not older, than those of native populations. For the individuals from Madagascar, the males were significantly smaller and younger (mean ± SD, SVL: 71.4 ± 1.6 mm; age: 1.8 ± 0.7 years) than the females (SVL: 78.42 ± 1.9 mm; age: 2.7 ± 1.3 years), when the data were pooled, but when the data were analyzed separately for each of the three sites, similar results were obtained only for one site. The oldest recorded male and female were 3 and 6 years old, respectively. Gonadal histology showed that the males and females reach sexual maturity after the first and second years of age, respectively. Further studies are needed to understand if the larger size and faster growth rates observed in the invasive population of D. melanostictus in Madagascar are a consequence of more favorable environmental conditions with respect to the native range (e.g., the availability of larger trophic niches, a lack of competitors, and lower predatory pressure), and we suggest to extend the monitoring of these life history traits to understand how they might influence the invasion.

Chromosome Diversity and Evolution of the Endemic Malagasy Velvet Geckos of the Genus Blaesodactylus (Reptilia, Gekkonidae).

We performed a molecular and phylogenetic analysis and a comparative cytogenetic study with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, + fluorochromes on several Blaesodactylus samples. The phylogenetic inference retrieved two main clades, the first comprises B. victori, B. microtuberculatus and B. boivini, while the second includes B. sakalava, B. antongilensis and B. ambonihazo. The available samples of B. sakalava form two different clades (here named B. sakalava clade A and clade B), which probably deserve a taxonomic re-evaluation. We found a karyological variability in Blaesodactylus in terms of chromosome number (2n = 40-42), morphology, location of NORs, and heterochromatin distribution pattern. Blaesodactylus antongilensis and B. sakalava clade A and B showed a karyotype of 2n = 40 mostly telocentric chromosomes. Pairs 1 and 6 were metacentric in B. sakalava clade A and B, while pair 1 was composed of subtelocentric/submetacentric elements in B. antongilensis. In contrast, B. boivini displayed a karyotype with 2n = 42 only telocentric chromosomes. NORs were on the first chromosome pair in B. boivini, and on the second pair in B. antongilensis. Adding our data to those available from the literature on evolutionarily related species, we highlight that the chromosome diversification in the genus probably proceeded towards a progressive reduction in the chromosome number and the formation of metacentric elements.

Evolutionary and Genomic Diversity of True Polyploidy in Tetrapods.

True polyploid organisms have more than two chromosome sets in their somatic and germline cells. Polyploidy is a major evolutionary force and has played a significant role in the early genomic evolution of plants, different invertebrate taxa, chordates, and teleosts. However, the contribution of polyploidy to the generation of new genomic, ecological, and species diversity in tetrapods has traditionally been underestimated. Indeed, polyploidy represents an important pathway of genomic evolution, occurring in most higher-taxa tetrapods and displaying a variety of different forms, genomic configurations, and biological implications. Herein, we report and discuss the available information on the different origins and evolutionary and ecological significance of true polyploidy in tetrapods. Among the main tetrapod lineages, modern amphibians have an unparalleled diversity of polyploids and, until recently, they were considered to be the only vertebrates with closely related diploid and polyploid bisexual species or populations. In reptiles, polyploidy was thought to be restricted to squamates and associated with parthenogenesis. In birds and mammals, true polyploidy has generally been considered absent (non-tolerated). These views are being changed due to an accumulation of new data, and the impact as well as the different evolutionary and ecological implications of polyploidy in tetrapods, deserve a broader evaluation.

First Insights on the Karyotype Diversification of the Endemic Malagasy Leaf-Toed Geckos (Squamata: Gekkonidae: Uroplatus).

We provide here the first karyotype description of eight Uroplatus species and a characterization of their chromosomal diversity. We performed a molecular taxonomic assessment of several Uroplatus samples using the mitochondrial 12S marker and a comparative cytogenetic analysis with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, +Chromomycin A3 (CMA3), +4',6-diamidino-2-phenylindole (DAPI). We found chromosomal variability in terms of chromosome number (2n = 34-38), heterochromatin composition and number and localization of loci or Nucleolar Organizer Regions (NORs) (alternatively on the 2nd, 6th, 10th or 16th pair). Chromosome morphology is almost constant, with karyotypes composed of acrocentric chromosomes, gradually decreasing in length. C-banding evidenced a general low content of heterochromatin, mostly localized on pericentromeric and telomeric regions. Centromeric bands varied among the species studied, resulting in CMA3 positive and DAPI negative or positive to both fluorochromes. We also provide evidence of a first putative heteromorphic sex chromosome system in the genus. In fact, in U. alluaudi the 10th pair was highly heteromorphic, with a metacentric, largely heterochromatic W chromosome, which was much bigger than the Z. We propose an evolutionary scenario of chromosome reduction from 2n = 38 to 2n = 34, by means of translocations of microchromosomes on larger chromosomes (often involving the NOR-bearing microchromosomes). Adding our data to those available from the literature, we show that similar processes characterized the evolutionary radiation of a larger gecko clade. Finally, we hypothesize that sex chromosome diversification occurred independently in different genera.

Comparative cytogenetics on eight Malagasy Mantellinae (Anura, Mantellidae) and a synthesis of the karyological data on the subfamily.

We performed a molecular and cytogenetic analysis on different Mantellinae species and revised the available chromosomal data on this group to provide an updated assessment of its karyological diversity and evolution. Using a fragment of the mitochondrial 16S rRNA, we performed a molecular taxonomic identification of the samples that were used for cytogenetic analyses. A comparative cytogenetic analysis, with Giemsa's staining, Ag-NOR staining and sequential C-banding + Giemsa + CMA + DAPI was performed on eight species: Gephyromantis sp. Ca19, G.striatus (Vences, Glaw, Andreone, Jesu et Schimmenti, 2002), Mantidactylus (Chonomantis) sp. Ca11, M. (Brygoomantis) alutus (Peracca, 1893), M. (Hylobatrachus) cowanii (Boulenger, 1882), Spinomantispropeaglavei "North" (Methuen et Hewitt, 1913), S.phantasticus (Glaw et Vences, 1997) and S. sp. Ca3. Gephyromantisstriatus, M. (Brygoomantis) alutus and Spinomantispropeaglavei "North" have a karyotype of 2n = 24 chromosomes while the other species show 2n = 26 chromosomes. Among the analysed species we detected differences in the number and position of telocentric elements, location of NOR loci (alternatively on the 6th, 7th or 10th pair) and in the distribution of heterochromatin, which shows species-specific patterns. Merging our data with those previously available, we propose a karyotype of 2n = 26 with all biarmed elements and loci of NORs on the 6th chromosome pair as the ancestral state in the whole family Mantellidae. From this putative ancestral condition, a reduction of chromosome number through similar tandem fusions (from 2n = 26 to 2n = 24) occurred independently in Mantidactylus Boulenger, 1895 (subgenus Brygoomantis Dubois, 1992), Spinomantis Dubois, 1992 and Gephyromantis Methuen, 1920. Similarly, a relocation of NORs, from the putative primitive configuration on the 6th chromosome, occurred independently in Gephyromantis, Blommersia Dubois, 1992, Guibemantis Dubois, 1992, Mantella Boulenger, 1882 and Spinomantis. Chromosome inversions of primitive biarmed elements likely generated a variable number of telocentric elements in Mantellanigricans Guibé, 1978 and a different number of taxa of Gephyromantis (subgenera Duboimantis Glaw et Vences, 2006 and Laurentomantis Dubois, 1980) and Mantidactylus (subgenera Brygoomantis, Chonomantis Glaw et Vences, 1994, Hylobatrachus Laurent, 1943 and Ochthomantis Glaw et Vences, 1994).

Life History Traits of Sperm Whales Physeter macrocephalus Linnaeus, 1758 Stranded along Italian Coasts (Cetartiodactyla: Physeteridae).

We investigated the relationship between age and body length, and age at sexual maturity of Physeter macrocephalus individuals stranded along the Italian coast. Our molecular analysis shows that all our samples belong to the C.001.002 haplotype, shared between Atlantic and Mediterranean populations. We show that males attain sexual maturity at 10 years, similar to those from other marine areas. However, considering the same body length class, Mediterranean males are older than Atlantic ones. Our finding of a Mediterranean pregnant female of only 6.5 m in length and an assessed age of 24-26 years is particularly noteworthy, considering that females reach sexual maturity at about 9 years and 9 m of total length in other regions. Comparing our results with the literature data, we highlight the positive correlation between lifespan, adult body length and weight of males from the Mediterranean and Atlantic Ocean. Regardless of whether the relatively small size of Mediterranean specimens is a consequence of an inbreeding depression or an adaptation to less favorable trophic conditions, we recommend to closely monitor this population from a conservation perspective. In fact, its low genetic diversity likely corresponds to a relatively limited ability to respond to environmental changes compared with other populations.

Chromosome Diversity and Evolution in Helicoide a (Gastropoda: Stylommatophora): A Synthesis from Original and Literature Data.

We performed a molecular and a comparative cytogenetic analysis on different Helicoidea species and a review of all the available chromosome data on the superfamily to provide an updated assessment of its karyological diversity. Standard karyotyping, banding techniques, and Fluorescence in situ hybridization of Nucleolus Organizer Region loci (NOR-FISH) were performed on fifteen species of three families: two Geomitridae, four Hygromiidae and nine Helicidae. The karyotypes of the studied species varied from 2n = 44 to 2n = 60, highlighting a high karyological diversity. NORs were on a single chromosome pair in Cernuella virgata and on multiple pairs in four Helicidae, representing ancestral and derived conditions, respectively. Heterochromatic C-bands were found on pericentromeric regions of few chromosomes, being Q- and 4',6-diamidino-2-phenylindole (DAPI) negative. NOR-associated heterochromatin was C-banding and chromomycin A3 (CMA3) positive. Considering the available karyological evidence on Helicoidea and superimposing the chromosome data gathered from different sources on available phylogenetic inferences, we describe a karyotype of 2n = 60 with all biarmed elements as the ancestral state in the superfamily. From this condition, an accumulation of chromosome translocations led to karyotypes with a lower chromosome number (2n = 50-44). This process occurred independently in different lineages, while an augment of the chromosome number was detectable in Polygyridae. Chromosome inversions were also relevant chromosome rearrangements in Helicoidea, leading to the formation of telocentric elements in karyotypes with a relatively low chromosome count.

Karyological Diversification in the Genus Lyciasalamandra (Urodela: Salamandridae).

We performed the first cytogenetic analysis on five out of the seven species of the genus Lyciasalamandra, including seven subspecies, and representatives of its sister genus Salamandra. All the studied species have a similar karyotype of 2n = 24, mostly composed of biarmed elements. C-bands were observed on all chromosomes, at centromeric, telomeric and interstitial position. We found a peculiar taxon-specific NOR configuration, including either heteromorphic and homomorphic NORs on distinct regions of different chromosomes. Lyciasalamandra a.antalyana and L. helverseni showed two homomorphic NORs (pairs 8 and 2, respectively), while heteromorphic NORs were found in L. billae (pairs 6, 12), L. flavimembris (pairs 2, 12), L. l. luschani (pairs 2, 12), L. l. basoglui (pairs 6, 12), L. l. finikensis (pairs 2, 6) and S. lanzai (pairs 8, 10). Homomorphic NORs with an additional supernumerary site were shown by S. s. salamandra (pairs 2, 8) and S. s. gigliolii (pairs 2, 10). This unexpected highly variable NOR configuration is probably derived from multiple independent NOR translocations and paracentric inversions and correlated to lineage divergence in Lyciasalamandra. These results support the taxonomic validity of the studied taxa and are consistent with a hypothesized scenario of synchronous evolution in the genus.

Dental fluorosis in the Vesuvius towns in AD 79: a multidisciplinary approach.

Background: Endemic fluorosis induced by high concentrations of fluoride in groundwater and soils is a major health problem in several countries, particularly in volcanic areas.Aim: To evaluate the occurrence of dental fluorosis resulting from exposure to high levels of environmental fluoride in 79 AD Herculaneum and close Vesuvius towns.Subjects and methods: The occurrence of dental fluorosis from teeth of the Herculaneum victims of the 79 AD eruption and some individuals from Pompeii (14-37 AD) and Nocera Inferiore (Salerno, IV sec. AD) was detected by means of Particle Induced Gamma-ray Emission technique (PIGE).Results: A clinical and analytical scenario of dental fluorosis resulted from the extreme high fluorine tooth content detected in teeth from Herculaneum and the Vesuvius area inhabitants. The adoption of PIGE technique has proved to be particularly effective in showing moderate as well as milder forms of dental fluorosis, otherwise not clearly detectable by clinical and histological analysis.Conclusions: Morphological, histological and elemental analysis of teeth of the 79 AD Herculaneum population show that in this area fluorosis occurred since Roman times.

Cold-blooded in the Ice Age: "refugia within refugia", inter-and intraspecific biogeographic diversification of European whipsnakes (Squamata, Colubridae, Hierophis).

In this work, we performed a biogeographic analysis with Bayesian binary MCMC (BBM) statistical dispersal-vicariance analysis (S-DIVA) and species distribution models (SDM) on three phylogenetically closely related Mediterranean whipsnakes (Hierophis gemonensis, H. carbonarius, H. viridiflavus), to investigate the pathways of their geographical diversification and locate putative refugial areas in the last glacial maximum (LGM). Our analysis suggests that the diversification processes between the studied species overall followed an east-west route, from eastern Greece to the Iberian Peninsula and continental France, highlighting a significant role of dispersal and vicariance processes at both inter- and intraspecific levels. In particular, the main lineage-splitting events between H. gemonensis, H. carbonarius and H. viridiflavus coincide with two events of vicariance, involving respectively eastern Greece and eastern Italy, and eastern Italy and western Italy, Iberian Peninsula and continental France. SDM analyses highlight the occurrence of multiple putative glacial refugia in the Balkans, Italy and southern France, which represent well the occurrence of distinct "refugia within refugia" in the main Mediterranean peninsulas. Furthermore, our results suggest how these refugia may have played an important role during the Quaternary climatic oscillations in shaping the current haplotype distribution of European whipsnakes.

To move or not to move: cranial joints in European gekkotans and lacertids, an osteological and histological perspective.

Lepidosaurs are frequently described as having highly kinetic skulls, and different forms of cranial kinesis have been described as being characteristic of their radiation. The model of amphikinesis proposed by Frazzetta, J Morphol 1962; 111:287-319, which was long considered a synapomorphy of the large suborder Sauria, is now much debated given its uncertain distribution among the various lizard taxa and the lack of data about its morphological correlates. In this article, we analyze the anatomical correlates of different forms of cranial kinesis, with particular regard to the putative saurian amphikinesis, describing the possible diverse skull movements of several species of European gekkotans (Hemidactylus turcicus, Mediodactylus kotschyi, and Tarentola mauritanica) and lacertids (Lacerta agilis, L. bilineata, Podarcis muralis, P. siculus, and Teira dugesii). Using serial and whole-mount histology, we found clear differences between gekkotans and lacertids in the structure of several cranial joints underlining the existence of two degrees of intracranial mobility. The lacertid species possess the anatomical features for streptostyly (quadrate joints) and metakinesis (parietal-supraoccipital and parabasisphenoid-pterygoid joints) and lack the anatomical correlates for mesokinesis (mobility of frontal-parietal and palatine-pterygoid joints) and amphikinesis (coupled mesokinesis, metakinesis, and streptostyly). In contrast, geckos present all the anatomical correlates for amphikinesis as described by the traditional quadratic crank model. Finally, we present a comprehensive summary of the different forms of squamate cranial kinesis, advancing two alternative hypotheses about the evolutionary origin of amphikinesis.

Karyological evidence for diversification of Italian slow worm populations (Squamata, Anguidae).

A karyological analysis on six Italian populations the slow worm (Anguis veronensis Pollini, 1818) was performed and their genetic differentiation at the mitochondrial 16S rRNA gene fragment from a Spanish sample has been assessed. The Italian populations were karyologically uniform, all showing 2n=44 elements, of which 20 were macrochromosomes and 24 microchromosomes. Comparison with literature data on Central European populations showed a difference on the morphology of the 10(th) chromosome pair: submetacentric in Italian populations and telocentric in the Central European ones. Our analysis showed the presence of a fragile site on chromosomes of this pair, suggesting its propensity for structural rearrangements. Analysis of the 16S rRNA gene fragment showed uniformity among Italian populations (uncorrected genetic distance of 0.4%), and their genetic distinctness from the Spanish individual (uncorrected genetic distance of 4.2%). Our results confirm the existence of two different Anguis fragilis Linnaeus, 1758 lineages, each one characterized by a different cytotype.

Replacement of sodium with choline in slow-cooling media improves human ovarian tissue cryopreservation.

Ovarian tissue cryopreservation is a promising technique for fertility preservation in young female cancer patients and efforts have been made to improve its effectiveness. During cooling and thawing, sodium ions significantly contribute to the 'solute effect' that plays a major role in disrupting cell membranes. Choline ions, which do not cross the cell membrane, should not contribute to the intracellular solute load. The present study assessed the effects of sodium substitution with choline in slow-cooling freezing media on human ovarian cortical strip cryopreservation. A total of 629 follicles (fresh control n=266; cryopreserved n=363), collected from ovarian biopsies of 11 women (22-40years) during laparoscopic surgery, were studied by light microscopy, immunohistochemistry and transmission electron microscopy to evaluate their morphology, apoptosis and ultrastructure. The results demonstrate that choline substitution leads to: (i) an improved preservation of oocytes and follicular cells; (ii) the recovery of a higher percentage of grade-1 follicles negative for p53, p21 and Apaf-1 apoptotic markers; (iii) a reduced mitochondrial damage as observed at an ultrastructural level; and (iv) a better preservation of ovarian tissue stroma. In conclusion, the use of choline-based media may represent a valuable tool to improve human ovarian tissue cryopreservation. Ovarian tissue cryopreservation is a promising fertility preservation approach for cancer patients before undergoing treatments that irreversibly reduce the ovarian reserve. Autotransplantation of ovarian cortical strips has resulted in viable offspring in animal models and human. Worldwide, 20 live births have been reported thus far following autotransplantation of frozen-thawed ovarian tissue. However, currently the success rate of this technology is far from being satisfactory. This could be due to inappropriate cryopreservation procedures that might impair the physiology of ovarian follicles. Sodium ions contained in freezing media significantly contribute to the 'solute effect' that plays a major role in disrupting cell membranes. Choline ions, which do not cross the cell membrane, would not be expected to contribute to the intracellular solute load. In the present study we assessed the effects of sodium substitution with choline in slow-cooling freezing media on human ovarian cortical strip cryopreservation. A total of 629 follicles, collected from ovarian biopsies of 11 women (aged 22-40years) during laparoscopic surgery, have been studied by light microscopy, immunohistochemistry and transmission electron microscopy to evaluate their morphology, apoptosis and ultrastructure. Results demonstrated that choline substitution allowed: (i) a better preservation of oocytes and follicular cells; (ii) the recovery of an higher percentage of healthy follicles negative for apoptotic markers; (iii) a lower mitochondria ultrastructural damage; and (iv) a better preservation of ovarian tissue stroma. In conclusion, the use of choline-based media could represent a valuable tool to cryopreserve human ovarian tissue for fertility preservation.

Seasonal variation in glycoconjugates of the pedal glandular system of the rayed Mediterranean limpet, Patella caerulea (Gastropoda: Patellidae).

Glycoconjugates secreted by the pedal system of the rayed limpet, Patella caerulea, were characterised in situ by histochemical and lectin-histochemical methods in individuals collected around the annual cycle, in November, March, and June. Stainings with periodic acid-Schiff (PAS), Alcian blue pH 2.5 (AB pH 2.5), Alcian blue pH 1.0 (AB pH 1.0), high-iron diamine-Alcian blue pH 2.5 and lectin binding assays with 9 lectins (Con A, WGA, succinylated-WGA, PNA, DBA, SBA, AAA, UEA-I, LTA) were performed. Four secreting cell types were observed in the sole, one in the peripheric region, and two in the sidewall. Glycoconjugate composition varied among cell types and also in one and the same cell type throughout the year. ß-Elimination followed by PAS and AB pH 2.5 stainings indicated that most saccharidic chains were O-linked to the protein backbone. Secretion by sole and peripheric region was acidic, carboxylated and/or sulfated, whereas that of the sidewall was neutral. Glucosaminylated and 1,4-fucosylated residuals were predominant in the cell types along the year, 1,2-fucosylated residuals being observed only in the sidewall cells in June. Mannosylated and/or glycosylated residuals were observed in all cells mostly in November. Galactosylated/galactosaminylated residuals were present mostly in the sidewall cells and in the sole subepidermal mucocytes in June. Mannosylated and/or glycosylated residuals in November are probably linked to gonad maturation or to higher locomotion and foraging activity, whereas galactosaminylation in the sole cells and 1,2-fucosylation and glucosaminylation in the sidewall cells in June are linked to a prolonged stationary state, increasing water adsorption to counteract dehydration and/or to modulate microbial interactions.

Molecular cytogenetic study of three common Mediterranean limpets, Patella caerulea, P. rustica and P. ulyssiponensis (Archaeogastropoda, Mollusca).

The present paper shows the results of chromosome banding and rDNA-FISH study performed on several specimens of different populations of Patella caerulea, Patella rustica and Patella ulyssiponensis. The taxonomic attribution of specimens was ascertained by the molecular phylogenetic analysis of the mitochondrial 16S rRNA gene. P. caerulea and P. rustica had 2n = 18 chromosomes with first seven of biarmed pairs and the remaining two uniarmed pairs. P. ulyssiponensis had 2n = 16 with all biarmed chromosomes. Ag-NOR loci were on the short arms of the first metacentric pair in the three studied limpets, whereas they showed a different pattern of heterochromatin distribution and composition. A chromosome mosaicism was observed in several P. caerulea specimens, which exhibited an unpaired metacentric element and loss of a telocentric pair. The obtained results suggest that in the genus Patella specific diversification was accompanied by variations in heterochromatin distribution and composition and reduction of chromosome number by Robertsonian centric fusion.

Estrogen-induced Akt-1 activity in the lizard (Podarcis s. sicula) testis.

There are always more evidences indicating that 17beta-estradiol (E2) is to be necessary for normal male fertility. Here we report the expression of the most ubiquitously expressed member of the akt family of genes, akt1, in the lizard (Podarcis s. sicula) testis. We have used a nonmammalian vertebrate model (the lizard P. s. sicula) to investigate the regulation of the serine/threonine kinase Akt activity, implicated in the control of cell proliferation, survival, and metabolism, in the testis during the annual sexual cycle and to study whether E2 exerts a role in the spermatogenesis through Akt-1 activity. Immunocytochemistry analysis show that Akt-1 proteins are present in the spermatogonia (SPG), and spermatocytes (SPC), and spermatids (SPT). The annual E2 profile shows a progressive increase during the active spermatogenesis (from April to June) and a peak in the month of August (spermatogonial mitosis). In parallel, Akt-1 (molecular weight 60 kDa) are highly phosphorylated during the period of active spermatogenesis and in post-refractory period (August) compared with the winter stasis (from November to March). Present results demonstrate that E2 treatment induces the activation of Akt-1, and this effect is counteracted by the anti-estrogen ICI 182-780.