Expression of Insl3 Protein in Adult Danio rerio.

Insulin-like peptide 3 (INSL3) is a biomarker for Leydig cells in the testes of vertebrates, and it is principally involved in spermatogenesis through specific binding with the RXFP2 receptor. This study reports the insl3 gene transcript and the Insl3 prepropeptide expression in both non-reproductive and reproductive tissues of Danio rerio. An immunohistochemistry analysis shows that the hormone is present at a low level in the Leydig cells and germ cells at all stages of Danio rerio testis differentiation. Considering that the insl3 gene is transcribed in Leydig cells, our results highlight an autocrine and paracrine function of this hormone in the Danio rerio testis, adding new information on the Insl3 mode of action in reproduction. We also show that Insl3 and Rxfp2 belonging to Danio rerio and other vertebrate species share most of the amino acid residues involved in the ligand-receptor interaction and activation, suggesting a conserved mechanism of action during vertebrate evolution.

Glyphosate Interference in Follicular Organization in the Wall Lizard Podarcis siculus.

Glyphosate (Gly) is a broad-spectrum herbicide widely used thanks to its high efficiency and low toxicity. However, evidence exists of its toxic effects on non-target organisms. Among these, the animals inhabiting agricultural fields are particularly threatened. Recent studies demonstrated that exposure to Gly markedly affected the morphophysiology of the liver and testis of the Italian field lizard Podarcis siculus. The present study aimed to investigate the effects of the herbicide on the female reproductive system of this lizard in order to have a full picture of Gly-induced reproductive impairment. The animals were exposed to 0.05 and 0.5 µg/kg of pure Gly by gavage for 3 weeks. The results demonstrated that Gly, at both doses tested, profoundly interfered with ovarian function. It induced germ cells' recruitment and altered follicular anatomy by anticipating apoptotic regression of the pyriform cells. It also induced thecal fibrosis and affected oocyte cytoplasm and zona pellucida organizations. At the functional levels, Gly stimulated the synthesis of estrogen receptors, suggesting a serious endocrine-disrupting effect. Overall, the follicular alterations, combined with those found at the level of the seminiferous tubules in males, suggest serious damage to the reproductive fitness of these non-target organisms, which over time could lead to a decline in survival.

Effects of Dibutylphthalate and Steroid Hormone Mixture on Human Prostate Cells.

Phthalates are a family of aromatic chemical compounds mainly used as plasticizers. Among phthalates, di-n-butyl phthalate (DBP) is a low-molecular-weight phthalate used as a component of many cosmetic products, such as nail polish, and other perfumed personal care products. DBP has toxic effects on reproductive health, inducing testicular damage and developmental malformations. Inside the male reproductive system, the prostate gland reacts to both male and female sex steroids. For this reason, it represents an important target of endocrine-disrupting chemicals (EDCs), compounds that are able to affect the estrogen and androgen signaling pathways, thus interfering with prostate homeostasis and inducing several prostate pathologies. The aim of this project was to investigate the effects of DBP, alone and in combination with testosterone (T), 17ß-estradiol (E2), and both, on the normal PNT1A human prostate cell-derived cell line, to mimic environmental contamination. We showed that DBP and all of the tested mixtures increase cell viability through activation of both estrogen receptor α (ERα) and androgen receptor (AR). DBP modulated steroid receptor levels in a nonmonotonic way, and differently to endogenous hormones. In addition, DBP translocated ERα to the nucleus over different durations and for a more prolonged time than E2, altering the normal responsiveness of prostate cells. However, DBP alone seemed not to influence AR localization, but AR was continuously and persistently activated when DBP was used in combination. Our results show that DBP alone, and in mixture, alters redox homeostasis in prostate cells, leading to a greater increase in cell oxidative susceptibility. In addition, we also demonstrate that DBP increases the migratory potential of PNT1A cells. In conclusion, our findings demonstrate that DBP, alone and in mixtures with endogenous steroid hormones, acts as an EDC, resulting in an altered prostate cell physiology and making these cells more prone to cancer transformation.

Molecular and Histological Effects of Glyphosate on Testicular Tissue of the Lizard Podarcis siculus.

The expansion of agriculture produces a steady increase in habitat fragmentation and degradation due to the increased use of pesticides and herbicides. Habitat loss and alteration associated with crop production play an important role in reptile decline, among which lizards are particularly endangered. In this study, we evaluated testicular structure, steroidogenesis, and estrogen receptor expression/localization after three weeks of oral exposure to glyphosate at 0.05 and 0.5 µg/kg body weight every other day in the field lizard Podarcis siculus. Our results show that glyphosate affected testicular morphology, reduced spermatogenesis, altered gap junctions and changed the localization of estrogen receptors in germ cells, increasing their expression; the effects were mostly dose-dependent. The result also demonstrates that glyphosate, at least at these concentrations, did not influence steroidogenesis. Overall, the data indicate that this herbicide can disturb the morphophysiology of the male lizard's reproductive system, with obviously detrimental effects on their reproductive fitness. The effects of glyphosate must be considered biologically relevant and could endanger the reproductive capacity not only of lizards but also of other vertebrates, including humans; a more controlled and less intensive use of glyphosate in areas devoted to crop production would therefore be advisable.

The Italian Wall Lizard Podarcis siculus as a Biological Model for Research in Male Reproductive Toxicology.

Spermatogenesis is a genetically driven differentiation process that occurs in the testis and leads to the formation of spermatozoa. This process is extensively studied in several experimental models, particularly in vertebrates that share the morphological structure and functionality of the mammalian testis. Although reptiles are not generally considered biological models, the lizard Podarcis siculus has represented a suitable organism for the study of spermatogenesis over the years. In this lizard, the process of spermatogenesis is regulated by the interaction between systemic factors such as gonadotropins and local factors, i.e., molecules produced by the somatic and germinal cells of the testis. Many exogenous substances are able to alter the production of these regulative factors, thus altering the course of spermatogenesis, and P. siculus has proven to be an excellent model for studying the effects of various endogenous or exogenous substances on mechanisms underlying spermatogenesis. This review summarizes the available data on the effects of different substances on the control of spermatogenesis, highlighting the induced morphological and molecular alterations. Overall, the data show that sex hormone levels as well as the final stages of spermatogenesis are most affected by an imbalance of endogenous compounds or contamination by environmental pollutants. This is helpful for the male individual, since the damage, not affecting the spermatogonial stem cells, can be considered transient and not irreversible.

Effects of environmental cocaine concentrations on COX and caspase-3 activity, GRP-78, ALT, CRP and blood glucose levels in the liver and kidney of the European eel (Anguilla anguilla).

Cocaine is one of the most widely used illicit drugs in the world, and as a result of incomplete removal by sewage treatment plants it is found in surface waters, where it represents a new potential risk for aquatic organisms. In this study we evaluated the influence of environmental concentrations of cocaine on the liver and the kidney of the European eel (Anguilla anguilla). The eels were exposed to 20 ng L-1 of cocaine for fifty days, after which, three and ten days after the interruption of cocaine exposure their livers and kidneys were compared to controls. The general morphology of the two organs was evaluated, as well as the following parameters: cytochrome oxidase (COX) and caspase-3 activities, as markers of oxidative metabolism and apoptosis activation, respectively; glucose-regulated protein (GRP)78 levels, as a marker of endoplasmic reticulum (ER)-stress; blood glucose level, as stress marker; serum levels of alanine aminotransferase (ALT), as a marker of liver injury and serum levels of C-reactive protein (CRP), as a marker of the inflammatory process. The liver showed morphologic alterations such as necrotic areas, karyolysis and pyknotic nuclei, while the kidneys had dilated glomeruli and the renal tubules showed pyknotic nuclei and karyolysis. In the kidney, the alterations persisted after the interruption of cocaine exposure. In the liver, COX and caspase-3 activities increased (COX: P = 0.01; caspase-3: P = 0.032); ten days after the interruption of cocaine exposure, COX activity returned to control levels (P = 0.06) whereas caspase-3 activity decreased further (P = 0.012); GRP78 expression increased only in post-exposure recovery specimens (three days: P = 0.007 and ten days: P = 0.008 after the interruption of cocaine exposure, respectively). In the kidney, COX and caspase-3 activities increased (COX: P = 0.02; caspase-3: P = 0.019); after the interruption of cocaine exposure, COX activity remained high (three days: P = 0.02 and ten days: P = 0.029 after the interruption of cocaine exposure, respectively) whereas caspase-3 activity returned to control values (three days: P = 0.69 and ten days: P = 0.67 after the interruption of cocaine exposure, respectively). Blood glucose and serum ALT and CRP levels increased (blood glucose: P = 0.01; ALT: P = 0.001; CRP: 0.015) and remained high also ten days after the interruption of cocaine exposure (blood glucose: P = 0.009; ALT: P = 0.0031; CRP: 0.036). These results suggest that environmental cocaine concentrations adversely affected liver and kidney of this species.

OctylPhenol (OP) Alone and in Combination with NonylPhenol (NP) Alters the Structure and the Function of Thyroid Gland of the Lizard Podarcis siculus.

Different environmental contaminants disturb the thyroid system at many levels. AlkylPhenols (APs), by-products of microbial degradation of AlkylPhenol Polyethoxylates (APEOs), constitute an important class of Endocrine Disrupting Chemicals (EDCs), the two most often used environmental APs being 4-nonylphenol (4-NP) and 4-tert-octylphenol (4-t-OP). The purpose of the present study was to investigate the effects on the thyroid gland of the bioindicator Podarcis siculus of OP alone and in combination with NP. We used radioimmunoassay to determine their effects on plasma 3,3',5-triiodo-L-thyronine (T3), 3,3',5,5'-L-thyroxine (T4), thyroid-stimulating hormone (TSH), and thyrotropin-releasing hormone (TRH) levels in adult male lizards. We also investigated the impacts of AP treatments on hepatic 5'ORD (type II) deiodinase and hepatic content of T3 and T4. After OP and OP + NP administration, TRH levels increased, whereas TSH, T3, and T4 levels decreased. Lizards treated with OP and OP + NP had a higher concentration of T3 in the liver and 5'ORD (type II) activity, whereas T4 concentrations were lower than that observed in the control group. Moreover, histological examination showed that the volume of the thyroid follicles became smaller in treated lizards suggesting that that thyroid follicular epithelial cells were not functionally active following treatment. This data collectively suggest a severe interference with hypothalamus-pituitary-thyroid axis and a systemic imbalance of thyroid hormones.

Spermatogenesis and regulatory factors in the wall lizard Podarcis sicula.

Spermatogenesis is an extraordinarily complex process, regulated by several factors, which leads to the differentiation of spermatogonia into spermatozoa. Among vertebrates, several reports have been focused on the lizard Podarcis sicula, a seasonal breeder and a good model for the study of reproductive processes. The goal of this review is to resume all the available data about systemic and above all local control factors involved in the control of P. sicula testicular activity. During the seasonal reproductive cycle, the variation of the expression levels of these factors determines significant variations that induce the activation or blocking of spermatogenesis. The data supplied in this review, in addition to analyze the current literature regarding the main actors of Podarcis sicula spermatogenesis, will hopefully provide a basic model that can be used for further studies on the intratesticular interaction between molecular factors that control spermatogenesis.

Adrenal gland response to endocrine disrupting chemicals in fishes, amphibians and reptiles: A comparative overview.

The adrenal gland is an essential component of the body stress response; it is formed by two portions: a steroidogenic and a chromaffin tissue. Despite the anatomy of adrenal gland is different among classes of vertebrates, the hormones produced are almost the same. During stress, these hormones contribute to body homeostasis and maintenance of ion balance. The adrenal gland is very sensitive to toxic compounds, many of which behave like endocrine-disruptor chemicals (EDCs). They contribute to alter the endocrine system in wildlife and humans and are considered as possible responsible of the decline of several vertebrate ectotherms. Considering that EDCs regularly can be found in all environmental matrices, the aim of this review is to collect information about the impact of these chemical compounds on the adrenal gland of fishes, amphibians and reptiles. In particular, this review shows the different behavior of these "sentinel species" when they are exposed to stress condition. The data supplied in this review can help to further elucidate the role of EDCs and their harmful impact on the survival of these vertebrates.

Pituitary adenylate cyclase-activating peptide (PACAP) and its receptors in Mus musculus testis.

To enlighten the involvement of PACAP/receptors system in the control of mammal testis, we investigated the expression of PACAP and the localization of PACAP and its receptors PAC1, VPAC1, and VPAC2 in the testis of Mus musculus. By molecular and immunohistochemical investigations, we highlighted that PACAP and its receptors are widely represented in germ cells of Mus testis, particularly in spermatocytes I, spermatids, and spermatozoa, strongly suggesting their involvement in spermatogenesis process. Moreover, for the first time in the adult mouse testis we highlighted that PACAP is present within Leydig cells, as PACAP receptors, confirming its involvement in the control of steroidogenesis in mouse.

Pituitary adenylate cyclase-activating polypeptide in the testis of the quail Coturnix coturnix: Expression, localization, and phylogenetic analysis.

To evaluate the involvement of pituitary adenylate cyclase-activating polypeptide (PACAP)/receptors system in the control of testis activity, we have investigated the expression and localization of PACAP and the distribution of its receptors in the testis of mature samples of quail Coturnix coturnix, and we have performed a phylogenetic analysis of PACAP in birds. Using histological, molecular, and bioinformatics tools, we demonstrated that (a) PACAP messenger RNA shows a high sequence identity with that reported in other birds studied so far and in other vertebrates. Furthermore, we showed that purifying selection acts on PACAP; (b) the PACAP peptide is present only in Leydig cells, whereas its receptors are localized within both Leydig and germ cells; (c) the synthesis of PACAP does not take place in seminiferous tubules. The role of PACAP in the control of spermatogenesis and steroidogenesis in birds is discussed. Finally, we talk about the phylogenetic and evolutionary relationships between PACAP in birds and in other vertebrates.

Seasonal expression and cellular distribution of star and steroidogenic enzymes in quail testis.

The quail Coturnix coturnix is a seasonal breeder with a physiological switch on/off of gonadic activity. Photoperiod and temperature are the major environmental factors regulating the spermatogenesis. To more thoroughly comprehend the steroidogenic pathways that govern the seasonal reproductive cycle, we have investigated the localization of StAR protein and steroidogenic enzymes (3ß-HSD, 17ß-HSD, P450 aromatase, and 5α-Red) as well as androgen and estrogen levels, in the testis of reproductive and nonreproductive quails. We demonstrated that StAR, 3ß-HSD, 17ß-HSD, P450 aromatase, and 5α-Red were always present in the somatic (Leydig and Sertoli cells) and germ cells (spermatogonia, spermatocytes I and II, spermatids, and spermatozoa). In addition, by western blot analysis, we demonstrated that 17ß-HSD, P450 aromatase, and 5α-Red showed the highest expression levels during the reproductive testis compared with nonreproductive one. Accordingly, we also found that during the reproductive phase the highest titres of testosterone, 17ß-estradiol, and 5α-dihydrotestosterone are recorded. In conclusion, our findings demonstrated that in C. coturnix: (a) both somatic and germ cells are involved in the local synthesis of sex hormones; (b) 17ß-HSD, P450 aromatase, and 5α-Red expressions, as well as testicular androgens and estrogens, increased in reproductive quail testis. This study strongly indicates that the steroidogenic process in quail testis exhibits seasonal changes with the promotion of both androgenic and estrogenic pathways in the reproductive period, suggesting their synergic mechanism in the spermatogenesis regulation.

D-Asp upregulates PREP and GluA2/3 expressions and induces p-ERK1/2 and p-Akt in rat testis.

D-Aspartate (D-Asp) is an endogenous amino acid that plays a central role in the development of the central nervous system (CNS) and functioning of the neuroendocrine system. In line with its functions, it is abundantly present in the CNS and reproductive systems of vertebrates and invertebrates. It has been implicated in the biosynthesis and/or secretion of hormones and factors that are involved in various reproductive functions, such as GnRH from the hypothalamus and testosterone from the testis. We conducted an in vivo study consisting of acute (i.p. injection of 2 µmol/g body weight) and chronic (15 days drinking solution) administration of D-Asp to adult rats to understand the signaling pathways elicited by D-Asp in the rat testis. We found that D-Asp upregulated the expression of prolyl endopeptidase (PREP), a serine protease having a pivotal role in the regulation of mammalian spermatogenesis and spermiogenesis. Immunofluorescence analysis revealed its overexpression in Leydig cells, Sertoli cells and spermatogonia. Moreover, PREP was found to co-localize with GluA2/3, an AMPA receptor subunit, whose protein expression also increased after D-Asp treatments. Finally, we found a significant increase in ERK and Akt activities in the testis of rats treated with D-Asp. Since PREP is known to be involved in regulating GnRH levels and in germ cell differentiation, we hypothesize D-Asp to play a pivotal role in regulating hormone homeostasis and spermatogenesis through activation of PREP, AMPAR, ERK and Akt.

P450 aromatase: a key enzyme in the spermatogenesis of the Italian wall lizard, Podarcis sicula.

P450 aromatase is a key enzyme in steroidogenesis involved in the conversion of testosterone into 17ß-estradiol. We investigated the localization and the expression of P450 aromatase in Podarcis sicula testes during the different phases of the reproductive cycle: summer stasis (July-August), early autumnal resumption (September), middle autumnal resumption (October-November), winter stasis (December-February), spring resumption (March-April) and the reproductive period (May-June). Using immunohistochemistry, we demonstrated that the P450 aromatase is always present in somatic and germ cells of P. sicula testis, particularly in spermatids and spermatozoa, except in early autumnal resumption, when P450 aromatase is evident only within Leydig cells. Using real-time PCR and semi-quantitative blot investigations, we also demonstrated that both mRNA and protein were expressed in all phases, with two peaks of expression occurring in summer and in winter stasis. These highest levels of P450 aromatase are in line with the increase of 17ß-estradiol, responsible for the spermatogenesis block typical of this species. Differently, in autumnal resumption, the level of P450 aromatase dramatically decreased, along with 17ß-estradiol levels, and testosterone titres increased, responsible for the subsequent renewal of spermatogenesis not followed by spermiation. In spring resumption and in the reproductive period we found intermediate P450 aromatase amounts, low levels of 17ß-estradiol and the highest testosterone levels determining the resumption of spermatogenesis needed for reproduction. Our results, the first collected in a non-mammalian vertebrate, indicate a role of P450 aromatase in the control of steroidogenesis and spermatogenesis, particularly in spermiogenesis.

Role of PACAP on testosterone and 17ß-estradiol production in the testis of wall lizard Podarcis sicula.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide that in mammalian testis is involved in the control of testosterone and 17ß-estradiol synthesis. A similar involvement was recently postulated in the testis of a nonmammalian vertebrate, the wall lizard Podarcis sicula. Indeed, we reported the presence of PACAP and its receptors throughout the reproductive cycle within both germ and somatic cells. Now, we investigated the effects of PACAP on steroidogenesis in significant periods of Podarcis reproductive cycle: winter stasis, reproductive period and summer stasis. Using different in vitro treatments, in the absence or presence of receptor antagonists, we demonstrated that in P. sicula testis PACAP is involved in the control of testosterone and 17ß-estradiol production. In particular we demonstrated that treatment with PACAP induced a testosterone increase only in stasis periods (winter and summer stasis); differently they induced a 17ß-estradiol production in all periods analyzed (summer stasis, winter stasis and reproductive period).

PACAP and PAC1 receptor in the reproductive cycle of male lizard Podarcis sicula.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide involved in multiple functions, including vertebrate reproduction. Recently, we reported the presence of PACAP in the testis of Italian wall lizard Podarcis sicula during reproductive period (May-June). Herein we investigated the PACAP mRNA expression and the localization of PACAP/PACAP receptor system, in the other periods of the Podarcis reproductive cycle, namely in summer stasis, early autumnal resumption, mid-autumnal resumption, winter stasis, and spring resumption. Using biomolecular and immunohistochemical investigations, we demonstrated that PACAP mRNA was widely expressed in all germ and somatic cells; in summer stasis (July-August) and early autumnal resumption (September) in particular, the mRNA was always found in Sertoli cells while was transiently expressed in germ and in Leydig cells. Differently from the mRNA, the protein was always present in germ and somatic cells independently from the reproductive cycle phase. As PACAP, the PAC1 receptor was always present in the testis, except for the summer stasis (July-August) and the early autumnal resumption (September), when PACAP was lacking in germ and somatic cells (Leydig and Sertoli cells). The present results strongly suggest that PACAP/PAC1 receptor system is widely represented during the reproductive cycle of male lizard. The possible involvement of PACAP/PACAP receptor system in the control of spermatogenesis is discussed.

The VIP/VPACR system in the reproductive cycle of male lizard Podarcis sicula.

Starting from the knowledge that in the reproductive period the Vasoactive Intestinal Peptide (VIP) is widely distributed in Podarcis sicula testis, we studied VIP expression and the localization of the neuropeptide and its receptors in the testis of the Italian wall lizard P. sicula in the other phases of its reproductive cycle (summer stasis, autumnal resumption, winter stasis, spring resumption). By Real Time-PCR, we demonstrated that testicular VIP mRNA levels change during the reproductive cycle, showing a cyclic trend with two peaks, one in the mid-autumnal resumption and the other in the reproductive period. By in situ hybridization and immunohistochemistry, we demonstrated that both VIP mRNA and protein were widely distributed in the testis in almost all the phases of the cycle, except in the early autumnal resumption. As regards the receptors, the VPAC1R was localized mainly in Leydig cells, while the VPAC2R showed the same distribution of VIP. Our results demonstrate that, differently from mammals, where VIP is present only in nerve fibres innerving the testis, an endotesticular synthesis takes place in the lizard and the VIP synthesis changes throughout the reproductive cycle. Moreover, the VIP/VPAC receptor system distribution observed in germ and somatic cells in various phases of the cycle, and particularly in the autumnal resumption and the reproductive period, strongly suggests its involvement in both spermatogenesis and steroidogenesis. Finally, the wider distribution of VIP in lizards with respect to mammals leads us to hypothesize that during the evolution the synthesis sites have been transferred from the testis to other districts, such as the brain.

Histopathological effects of long-term exposure to realistic concentrations of cadmium in the hepatopancreas of Sparus aurata juveniles.

In recent decades, cadmium has emerged as an environmental stressor in aquatic ecosystems due to its persistence and toxicity. It can enter water bodies from various natural and anthropogenic sources and, once introduced into aquatic systems, can accumulate in sediments and biota, leading to bioaccumulation and biomagnification in the food chain. For this reason, the effects of cadmium on aquatic life remain an area of ongoing research and concern. In this paper, a multidisciplinary approach was used to assess the effects of long-term exposure to an environmental concentration on the hepatopancreas of farmed juveniles of sea bream, Sparus aurata. After determining metal uptake, metallothionein production was assessed to gain insight into the organism's defence response. The effects were also assessed by histological and ultrastructural analyses. The results indicate that cadmium accumulates in the hepatopancreas at significant concentrations, inducing structural and functional damage. Despite the parallel increase in metallothioneins, fibrosis, alterations in carbohydrate distribution and endocrine disruption were also observed. These effects would decrease animal fitness although it did not translate into high mortality or reduced growth. This could depend on the fact that the animals were farmed, protected from the pressure deriving from having to search for food or escape from predators. Not to be underestimated is the return to humans, as this species is edible. Understanding the behaviour of cadmium in aquatic systems, its effects at different trophic levels and the potential risks to human health from the consumption of contaminated seafood would therefore be essential for informed environmental management and policy decisions.

Unravelling the ecotoxicological impacts of gadolinium (Gd) on Mytilus galloprovincialis embryos and sperm in seawater: A preliminary study.

As the demand for rare earth elements (REEs) continues to surge in diverse industrial and medical domains, the ecological consequences of their ubiquitous presence have garnered heightened attention. Among the REEs, gadolinium (Gd), commonly used in medical imaging contrast agents, has emerged as a pivotal concern due to its inadvertent introduction into marine ecosystems via wastewater release. This study delves into the complex ecotoxicological implications of Gd contamination, focusing on its impact on the embryonic development and sperm functionality of Mytilus galloprovincialis. The findings from this study underscore the potential hazards posed by this rare element, offering a critical perspective on the ecological risks associated with Gd. Notably, this exploratory work reveals that Gd exerts a significant embryotoxic effect at elevated concentrations, with an observed half maximal effective concentration (EC50) value of 0.026 mg/L. Additionally, Gd exposure leads to a considerable reduction in sperm motility and alters sperm morfo-kinetic parameters, especially at a concentration of 5.6 mg/L. The results highlight a dose-dependent relationship between Gd exposure and the prevalence of specific malformation types in Mytilus embryos, further providing crucial insights into the potential risks imposed by this rare earth element.

A network system for vitellogenin synthesis in the mussel Mytilus galloprovincialis (L.).

The aim of this study is to assess, by RT-PCR, in situ hybridization, electron microscopy, and immunohistochemistry, the site/s of vitellogenin (VTG) synthesis in the mussel Mytilus galloprovincialis. Our investigations demonstrate that, among the analyzed tissues, the synthesis of VTG occurs only in the female gonad, that is, within the oocyte and follicle and connective cells. Such a synthesis is just evident in early vitellogenic oocytes, whose cytoplasm is characterized by numerous RER cisternae and an extended Golgi complex surrounded by nascent yolk platelets. The synthesis of VTG goes on in vitellogenic oocytes assuming a pear form, and progressively reduces once the oocyte shows the pear or polygonal form, typical of those oocytes that have concluded the growth. The expression of VTG occurs also within follicle (auxiliary) and connective cells. In particular, it is noteworthy that follicle cells are characterized by numerous RER cisternae and an active Golgi complex surrounded by numerous vesicles and vacuoles containing electron dense material. The same material is also present along their plasma membrane, within the intercellular space between oocyte and follicle cells, and finally within invaginations of the oocyte surface, thus suggesting a VTG transfer to the oocyte via endocytosis. Differently, no VTG synthesis was observed within digestive gland. All together the findings here reported strongly suggest that in M. galloprovincialis, inside the gonad, the VTG synthesis occurs in the oocyte (autosynthesis) and in the follicle and adipogranular cells (heterosynthesis).