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.

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.

Molecular Alterations in Spermatozoa of a Family Case Living in the Land of Fires. A First Look at Possible Transgenerational Effects of Pollutants.

In our previous work, we reported alterations in protamines/histones ratio, in DNA binding of these proteins and their involvement in DNA oxidative damage in 84% of the young men living in the Land of Fires. In the present work, we extended our findings, evaluating any alterations in spermatozoa of a family case, a father and son, living in this area, to also give a first look at the possibility of transgenerational inherited effects of environmental contaminants on the molecular alterations of sperm nuclear basic proteins (SNBP), DNA and semen parameters. In the father and son, we found a diverse excess of copper and chromium in the semen, different alterations in SNBP content and low DNA binding affinity of these proteins. In addition, DNA damage, in the presence of CuCl2 and H2O2, increased by adding both the father and son SNBP. Interestingly, son SNBP, unlike his father, showed an unstable DNA binding and were able to produce DNA damage even without external addition of CuCl2, in line with a lower seminal antioxidant activity than the father. The peculiarity of some characteristics of son semen could be a basis for possible future studies on transgenerational effects of pollutants on fertility.

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.

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).

Chronic pro-oxidative state and mitochondrial dysfunctions are more pronounced in fibroblasts from Down syndrome foeti with congenital heart defects.

Trisomy of chromosome 21 is associated to congenital heart defects in ∼50% of affected newborns. Transcriptome analysis of hearts from trisomic human foeti demonstrated that genes involved in mitochondrial function are globally downregulated with respect to controls, suggesting an impairment of mitochondrial function. We investigated here the properties of mitochondria in fibroblasts from trisomic foeti with and without cardiac defects. Together with the upregulation of Hsa21 genes and the downregulation of nuclear encoded mitochondrial genes, an abnormal mitochondrial cristae morphology was observed in trisomic samples. Furthermore, impairment of mitochondrial respiratory activity, specific inhibition of complex I, enhanced reactive oxygen species production and increased levels of intra-mitochondrial calcium were demonstrated. Seemingly, mitochondrial dysfunction was more severe in fibroblasts from cardiopathic trisomic foeti that presented a more pronounced pro-oxidative state. The data suggest that an altered bioenergetic background in trisomy 21 foeti might be among the factors responsible for a more severe phenotype. Since the mitochondrial functional alterations might be rescued following pharmacological treatments, these results are of interest in the light of potential therapeutic interventions.

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.

Who Is the Intermediate Host of RNA Viruses? A Study Focusing on SARS-CoV-2 and Poliovirus.

The COVID-19 pandemic has sparked a surge in research on microbiology and virology, shedding light on overlooked aspects such as the infection of bacteria by RNA virions in the animal microbiome. Studies reveal a decrease in beneficial gut bacteria during COVID-19, indicating a significant interaction between SARS-CoV-2 and the human microbiome. However, determining the origins of the virus remains complex, with observed phenomena such as species jumps adding layers to the narrative. Prokaryotic cells play a crucial role in the disease's pathogenesis and transmission. Analyzing previous studies highlights intricate interactions from clinical manifestations to the use of the nitrogen isotope test. Drawing parallels with the history of the Poliovirus underscores the need to prioritize investigations into prokaryotic cells hosting RNA viruses.

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).

Morphological, Gene, and Hormonal Changes in Gonads and In-Creased Micrococcal Nuclease Accessibility of Sperm Chromatin Induced by Mercury.

Mercury is one of the most dangerous environmental pollutants. In this work, we analysed the effects of exposure of Mytilus galloprovincialis to 1, 10 and 100 pM HgCl2 for 24 h on the gonadal morphology and on the expression level of three stress genes: mt10, hsp70 and πgst. In this tissue we also evaluated the level of steroidogenic enzymes 3ß-HSD and 17ß-HSD and the expression of PL protein genes. Finally, we determined difference in sperm chromatin accessibility to micrococcal nuclease. We found alterations in gonadal morphology especially after exposure to 10 and 100 pM HgCl2 and hypo-expression of the three stress genes, particularly for hsp70. Furthermore, decreased labelling with both 3ß-HSD and 17ß-HSD antibodies was observed following exposure to 1 and 10 pM HgCl2 and complete absence at 100 pM HgCl2 exposure. Gonads of mussels exposed to all HgCl2 doses showed decreased expression of PL protein genes especially for PLIII. Finally, micrococcal nuclease digestions showed that all doses of HgCl2 exposure resulted in increased sperm chromatin accessibility to this enzyme, indicative of improper sperm chromatin structure. All of these changes provide preliminary data of the potential toxicity of mercury on the reproductive health of this mussel.

gH625-liposomes deliver PACAP through a dynamic in vitro model of the blood-brain barrier.

The blood-brain barrier (BBB) selectively protects the central nervous system (CNS) from external insults, but its function can represent a limit for the passage of therapeutic molecules. Numerous in vitro models of the BBB have been realized in order to study the passage of drugs for neurodegenerative diseases, but these in vitro models are not very representative of the physiological conditions because of a limited supply of oxygen and nutrients due to static conditions. To avoid this phenomenon, we used a millifluidic bioreactor model that ensures a circulation of the medium and, therefore, of the nutrients, thanks to the continuous laminar flow. This dynamic model consists of a double-culture chamber separated by a membrane on which brain endothelial cells are cultured in order to evaluate the passage of the drug. Furthermore, in the lower chamber, SH-SY5Y were seeded as 3D spheroids to evaluate the drug passage through these cells. As nanodelivery system, we used liposomes functionalized with viral fusion peptide to evaluate the passage of a neuroprotective agent, pituitary adenylate cyclase-activating polypeptide (PACAP), through the dynamic in vitro model of the BBB. We showed that our nanodelivery system, made of functionalized liposomes and loaded with specific molecules, efficiently crosses the in vitro fluid-dynamic model of the BBB. Our findings represent an important step for further experimental investigations on PACAP administration as a therapeutic agent by an enhanced drug delivery system. Our results can improve the diffusion of good practice in neuroscience laboratories, helping to spread the 3R rules.

Neuroprotective Effects of gH625-lipoPACAP in an In Vitro Fluid Dynamic Model of Parkinson's Disease.

Parkinson's disease (PD) is an aggressive and devastating age-related disorder. Although the causes are still unclear, several factors, including genetic and environmental, are involved. Except for symptomatic drugs, there are not, to date, any real cures for PD. For this purpose, it is necessary develop a model to better study this disease. Neuroblastoma cell line, SH-SY5Y, differentiated with retinoic acid represents a good in vitro model to explore PD, since it maintains growth cells to differentiated neurons. In the present study, SH-SY5Y cells were treated with 1-methyl-4-phenylpyridinium (MPP+), a neurotoxin that induces Parkinsonism, and the neuroprotective effects of pituitary adenylate cyclase-activating polypeptide (PACAP), delivered by functionalized liposomes in a blood-brain barrier fluid dynamic model, were evaluated. We demonstrated PACAP neuroprotective effects when delivered by gH625-liposome on MPP+-damaged SH-SY5Y spheroids.

Could SARS-CoV-2 Have Bacteriophage Behavior or Induce the Activity of Other Bacteriophages?

SARS-CoV-2 has become one of the most studied viruses of the last century. It was assumed that the only possible host for these types of viruses was mammalian eukaryotic cells. Our recent studies show that microorganisms in the human gastrointestinal tract affect the severity of COVID-19 and for the first time provide indications that the virus might replicate in gut bacteria. In order to further support these findings, in the present work, cultures of bacteria from the human microbiome and SARS-CoV-2 were analyzed by electron and fluorescence microscopy. The images presented in this article, in association with the nitrogen (15N) isotope-labeled culture medium experiment, suggest that SARS-CoV-2 could also infect bacteria in the gut microbiota, indicating that SARS-CoV-2 could act as a bacteriophage. Our results add new knowledge to the understanding of the mechanisms of SARS-CoV-2 infection and fill gaps in the study of the interactions between SARS-CoV-2 and non-mammalian cells. These findings could be useful in suggesting specific new pharmacological solutions to support the vaccination campaign.

Expression of vitellogenin in the testis and kidney of the spotted ray Torpedo marmorata exposed to 17ß-estradiol.

In vertebrates, the liver was long thought to be the only site of vitellogenin (Vtg) production, but recent studies demonstrated that Vtg is also expressed in extrahepatic districts. The aim of this paper is to assess, by in situ hybridization and immunohistochemistry, the expression of Vtg in the testis and kidney of Torpedo marmorata exposed to 17ß-estradiol (E(2)). In treated samples vtg mRNA and Vtg were detected contemporaneously only in the testis; differently the kidney cells were positive to Vtg antibody, but negative to vtg mRNA. This is the first study to assess that male germ cells, after an exposure to E(2), synthesize Vtg in a stage-dependent manner. The presence of Vtg and the modifications observed in the kidney after E(2) treatment are discussed.

Pituitary adenylate cyclase-activating polypeptide and its receptor PAC1 in the testis of Triturus carnifex and Podarcis sicula.

The pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon-related family that occurs in two amidated forms with 38 (PACAP38) and 27 (PACAP27) amino acids. First discovered in the brain, it was then localized in several peripheral tissues of mammals, including the testis. However, current knowledge of the expression and function of PACAP and its receptor PAC(1) in the reproductive system of non-mammalian vertebrates, and particularly in the testis, is still limited. The aim of this work was to study the presence of PACAP and its receptor PAC(1) in the testis of two non-mammalian vertebrates during the breeding season: the crested newt Triturus carnifex and the wall lizard Podarcis sicula. The expression and distribution of this neuropeptide and its receptor PAC(1) were investigated by using in situ hybridization and immunohistochemistry techniques. Our results demonstrated that PACAP and its receptor PAC(1) were highly represented in the testis of these two species. In particular, we showed that they are present within some germ cells and that PACAP, unlike in mammals, is expressed also in the somatic cells (Sertoli and Leydig cells) of the testis of these two non-mammalian vertebrates, suggesting that this neuropeptide is involved in the hormonal control of spermatogenesis and steroidogenesis.

Immunolocalization of aromatase P450 in the epididymis of Podarcis sicula and Rattus rattus.

The goal of this study was to evaluate P450 aromatase localization in the epididymis of two different vertebrates: the lizard Podarcis sicula, a seasonal breeder, and Rattus rattus, a continuous breeder. P450 aromatase is a key enzyme involved in the local control of spermatogenesis and steroidogenesis and we proved for the first time that this enzyme is represented in the epididymis of both P. sicula and R. rattus. In details, P450 aromatase was well represented in epithelial and myoid cells and in the connective tissue of P. sicula epididymis during the reproductive period; instead, during autumnal resumption this enzyme was absent in the connective tissue. During the non-reproductive period, P450 aromatase was localized only in myoid cells of P. sicula epididymis, whereas in R. rattus it was localized both in myoid cells and connective tissue. Our findings, the first on the epididymis aromatase localization in the vertebrates, suggest a possible role of P450 aromatase in the control of male genital tract function, particularly in sperm maturation.

Organization of the vitelline envelope in ovarian follicles of Torpedo marmorata Risso, 1810 (Elasmobranchii: Torpediniformes).

In Torpedo marmorata, the vitelline envelope (VE), an extracellular envelope surrounding the growing oocyte, consists of fibrils and amorphous materials that are deposited in the perivitelline space starting from the initial steps of oocyte growth. SDS-PAGE analysis of the isolated and purified VE reveals that it consists of different glycoproteins. Furthermore, our investigations showed that the 120 and 66 kDa glycoproteins are positive to an antibody directed against gp69/64 of the Xenopus laevis VE and are synthesized under the control of 17beta-estradiol in the liver, that, together follicle cells and the oocyte, is the biosynthetic site of VE components.