LA RIPRODUZIONE DEI PESCI: ASPETTI TEORICI ED APPLICATIVI

1. LA RIPRODUZIONE DEI PESCI: ASPETTI TEORICI ED APPLICATIVI

2. Reproductivecycle of teleosts

3. VARIABILITY OF THE REPRODUCTIVE CYCLE A majority of teleost fishes are seasonal breeders, while a few breed continuously. Among the seasonal breeders, there is wide variation in the time of the year when breeding occurs. Fresh water temperate zone fishes spawn in spring and early summer, while others such as the salmonids do so in autumn. The time of breeding of each species is so precisely timed that fry are produced in an environment in which the chances of survival are maximal. Natural selection possibly favors genomes of individuals that produce their young ones at a time most suitable for survival.

4. REPRODUCTIVE CYCLE PHASES In both males and females, the reproductive cycle involves two major phases: • the phase of gonadal growth and development (gametogenesis) • the phase of maturation, which culminates in ovulation/spermiation and spawning. The release of mature gametes to the external environment (spawning) is a highly synchronized event, leading to fertilization of the egg and development of the embryo. The success of reproduction depends on the successful progression through every process of the reproductive cycle, which leads to the production of good quality gametes.

5. Ovarian development: oogenesis, maturation and ovulation The ovaries are compartmentalized folds (the ovigerous lamellae) of the germinal epithelium that project transversally to the ovarian lumen. In the lamellae, the oocytes undergo the various phases of gametogenesis, until mature ova (i.e., eggs) are released into the ovarian cavity (cystovarian) or abdominal cavity (gymnovarian) (e.g., salmonids) at ovulation and then to the external environment during spawning. Ovulated ova may remain in the ovarian/abdominal cavity for a period of time before spawning. There, they maintain their maturational competence (fertilizing capacity) for a certain period of time, but if not spawned, the ova become “over-ripe” through a process of degeneration.

7. This is an important consideration in cultured fish whose reproduction is based on manual egg stripping and artificial insemination, because stripping should be performed before over-ripening occurs. The lapse of time between ovulation and over-ripening varies greatly among fish, from minutes (e.g., striped bass, Morone saxatilis) to days (e.g., salmonids) and depends greatly on water temperature. In salmonids, which do not have a complete mesovarium and the oocytes are ovulated directly into the abdominal cavity, the ovulated ova can remain for several days with no evident over-riping.

8. It initiates with the mitotic proliferation of the oogonia that undergo primary oocytes when entering to meiosis. The primary oocytes go through a primary growth phase (pre-vitellogenesis), which involves the appearance of pale material in the cytoplasm and formation of the two layers of surrounding granulosa and theca cells (i.e., follicular wall). The secondary growth phase (vitellogenesis) involves the synthesis and incorporation into the oocyte of vitellogenin (VTG), and is associated with a drastic increase in size. During vitellogenesis, new inclusions appear in the cytoplasm, such as the cortical alveoli (white circles), lipid globules (light grey circles) and yolk granules (dark grey circles) and the oocyte wall (i.e., zona radiata) and follicular wall become increasingly thick. At the end of vitellogenesis, the cytoplasm is filled completely with lipid globules and yolk granules at the onset of coalescence, the nucleus (germinal vesicle, GV) (black circle) is centrally located and a thick zona radiata surrounds the oocyte. At early maturation, lipid globules and yolk granules continue coalescence and the nucleus migrates to the animal pole (GV migration, GVM). As maturation advances, there is a massive coalescence of yolk granules and localization of the nucleus at a peripheral position. Hydration: relevant in fishes producing pelagic eggs Final oocyte maturation (FOM) is characterized by the dissolution of the GV membrane (GV break down, GVBD) and hydration of the oocyte. Oocytes are finally ovulated into the ovarian or abdominal cavity, and are released in the water during spawning.

9. Assessment of the Stage of gonadal development The determination of the stage of gonadal development in female breeders is an important tool in aquaculture. This can be determined by examination of biopsies of oocyte samples. The biopsy is performed in anesthetized females, by insertion of a cannula through the gonoduct and gentle aspiration of intra ovarian oocytes. The collected oocytes are observed under the binocular and classified according to their size, position of the GV (central, migrating or peripheral), degree of yolk granule coalescence, etc.; these classifications give a relative indication of the stage of gonad development of the females.

10. Progression of oocyte development from primary growth oocytes (PG), to secondary growth, which begins with the cortical alveolar (CA) stage and then proceeds through vitellogenesis (Vtg), which can be broken into substages associated with the extent of yolk globules or platelets in the ooplasm (primary [Vtg1], secondary [Vtg2], and tertiary [Vtg3]). Oocyte maturation (OM) occurs after the appropriate trigger and can include germinal vesicle migration (GVM), yolk coalescence (YC), germinal vesicle breakdown (GVBD), and in pelagic spawners, hydration (H). At ovulation, the follicle ruptures and the oocyte is released. Postovulatory follicles (POF) remain in the ovary, where they are resorbed.  

12. An oocyte surrounded by two layers of follicular cells that offer structural and functional support to the developing oocyte, mediating the internalization of external molecules, synthesizing hormones and factors necessary for the differentiation, growth and survival of the oocyte. Each oocyte is surrounded by an inner mono-layer of granulosa cells and an outer mono-layer of theca cells. Between the two follicular layers there is a thin basal membrane. Also, a thick acellular envelop surrounds the oocyte (zona pellucida). Granulosa cells microvilli and oocyte microvilli cross the zona pellucida, changing its name in zona radiata.The zona radiata develops progressively during gametogenesis, becoming increasingly thick and compact to constitute the egg chorion or egg shell.

13. Testicular development in males: spermatogenesis, maturation and spermiation In Teleosts, testes can be either tubular or lobular. In tubular testes the ends of the tubules hooks the efferent ducts, forming loops, while the lobular testes have blind ends. Within the wall of the tubules there are germinal cells at different stages of maturity. In the lobular testis there are cysts with germ cells at the same stage of maturation. Spermatozoa containing cysts can either open up and release spermatozoa in the lumen of the lobule (unrestricted testis), or can move up to the end of the lobule and release the spermatozoa near its end (restricted testis). After Mari Carmen Uribe, Harry J Grier and Víctor Mejía-Roa (2014) Comparative testicular structure and spermatogenesis in bony fishes. Spermatogenesis. 4(3): e983400.

14. Tubular testis This testis type is present in lower fishes, as salmonids, cyprinids and lepisosteids

15. Unrestricted lobular testis This testis type is found in neoteleosts, except for the atherinomorphs.

16. Restricted lobular testis of the poeciliid Poecilia latipinna This testis type is found in all Atherinomorpha (Atheriniformes, Beloniformes and Cyprinodontiformes). The restriction of spermatogonia to the termini of the lobules supports the monophyly of that group.

17. Sertoli stem cells? Spermiation Sperm stem cells Elongating spermatogonia Spermatogonia Round spermatids Schematic drawing of cystic spermatogenesis observed in fish and amphibians. In anamniote testes, a cyst of Sertoli cells surrounds each germ cell syncytium. Sertoli cells share their fate with the developing germ cell syncytium that they nourish, and eventually degrade when germ cells mature and spermiate. Tight junctions are established between Sertoli cells that cover haploid spermatids and more advanced germ cells. The spatial organization of cysts within the testis varies highly between species. They are aligned in the order of development in some fish, while others do not have such a polarized organization. Spermatocytes

18. Siti web su cui trovare info relative al teleosteo e diffuso modello animale zebrafish https://en.wikibooks.org/wiki/The_Zebrafish_in_Toxicology/Spermatogenesis http://zfishtoxpat.comoj.com/tesdet.html#

20. Types of gonadal development The ovarian development can be classified in three major types: • synchronousexhibited by those species spawning only once in their life (freshwater eel (Anguilla spp), and Pacific salmons (Oncorhynchus spp). In this type of ovary, all oocytes advance in synchrony through all phases of gametogenesis. Thus, only one type of developing oocyte class is present in the ovary. • group-synchronous exhibited by the seasonal spawners, those species that spawn one or more times during the annual reproductive season. In this type, a cluster of vitellogenic oocytes is recruited and advance synchronously through further stages of development, whereas the rest of the oocyte population remains arrested. The cluster of recruited oocytes will undergo maturation, ovulation and spawning. This type of ovarian development can be divided in two subgroups: single-batch and multiple-batch spawners. In the single-batch group synchronous species, only one batch of oocytes undergoes maturation every season and thus, they produce one single spawn per year (e.g., rainbow trout, Oncorhynchus mykiss). The multiple-batch group synchronous species are able to repeat this process several times during the spawning season, with the recruitment of successive batches of oocytes and thus the production of several spawns per year. The number of spawning depends on the number of recruitments, e.g., the European sea bass (Dicentrarchuslabrax) producing 2-4 spawns per season. • Asynchronous is exhibited by those species that produce multiple spawns through an expanded period of time (several months), normally on a daily basis. This is typical of some tropical species and in the Mediterranean sea the Sparusauratus(orata)

21. The testis development The development of the testes is more homogenous and could be described as an asynchronous type of development for all species. Male fish used to being fluent on a daily basis through a long period of time, normally overlapping the spawning period of the females. At every moment, several classes of cell development, from immature spermatogonia to spermatozoa, can be found in the testes. At the full spermiation period, the testes are mostly occupied by mature spermatozoa, ready for spermiation, while early in the season, a high percentage of less mature spermatocytes is present.

22. ENVIRONMENTAL REGULATION OF FISH REPRODUCTION Factors that determine survival and reproduction: Food availability and environmental conditions Fish have the ability, through a genetically determined bio-chemical threshold, to ascertain what size and/or age conditions are optimal to complete maturation both during the first and subsequent maturation episodes. Food availability for offspring and hence off-spring survival determines the timing of reproduction

23. Photoperiod, temperature, lunar cycles, weather cycles and ocean currents, control the seasonality of food availability and entrain maturational development of fish. Food availability and the ability to store energy determine when a fish attains a genetical threshold and proceeds to the completion of maturation. Maturational development of the fish is entrained by environmental factors to ensure that critical off-spring feeding periods coincide with peaks in food availability which are months or years after maturation is initiated.

24. Perhaps the most important proximate factor is photoperiod. photoperiod controls all aspects of maturational development, i.e., the entire brain-pituitary-gonad axis. In the rainbow trout: 1. the increasing spring photoperiod entrains the start of vitellogenesis/spermatogenesis, 2. the passage of photoperiod from spring to summer to autumn entrains the progress of vitellogenesis/spermatogenesis, 3. the decreasing autumn photoperiod entrains final maturation, ovulation and spermiation. Photoperiod plays an important role in the timing of reproduction of most temperate fish species such as the Atlantic salmon (Salmo salar, family: Salmonidae), European seabass (Dicentrarchus labrax, Percichthyidae), gilthead bream, (Sparus aurata, Sparidae), Atlantic cod (Gadus morhua, Gadidae), Atlantic halibut, (Hippoglossus hippoglossus, Pleuronectidae), sole (Solea solea, Soleidae), and tropical latitudes, such as the Nile tilapia (Oreochromus niloticus), grey mullet (Mugil cephalus, Mugilidae), catfish (Heteropneustes fossilis, Heteropneustidae)and common carp (Cyprinus carpio, Cyprinidae)

25. HORMONAL REGULATION OF FISH REPRODUCTION

26. The reproductive cycle is regulated by a cascade of hormones along the brain-pituitary-gonad (BPG) axis, the so-called reproductive axis

27. The pituitary gland of teleosts

29. At initial stages GTH stimulation (mainly FSH) induces the secretion of androgens (testosterone and 11-ketotestosterone ) in males and estrogens (estradiol ) in females, which act concomitantly with FSH in the control of gametogenesis. The E2 plays an additional important role in female gametogenesis, with the stimulation of VTG synthesis from the liver. 2. At the end of gametogenesis Secretion of LH from the pituitary induces a shift in the steroidogenic pathway of the gonad, stimulating the synthesis and secretion of progestin-like steroids, the maturation-inducing steroids (MIS). The concomitant action of LH with the MIS stimulates the process of gonadal maturation. This period is characterized by decreasing blood levels of FSH and androgens/estrogens and increasing blood levels of LH and MIS. Once gonadal maturation is completed, the brain GnRH system stimulates a high surge of LH secretion from the pituitary, which induces ovulation in the females, whereas in the males, relatively stable but elevated levels of LH induce spermiation. The GnRH-induced pre-ovulatory LH surge in the plasma is essential for successful ovulation. In fact, the demonstration that this characteristic LH surge was absent in captive fish that failed to ovulate, but not in wild fish ovulating spontaneously, set up the basis for the development of hormone based spawning induction therapies in aquaculture. The stress associated with captivity or the absence of appropriate environmental conditions in culture facilities may act on the brain-inhibiting neuroendocrine secretions.

30. At initial stages, pituitary FSH stimulation induces gonadal secretion of E2 in females and androgens in males (11KT)that regulate gonad development. In females, E2 plays an additional role on the liver, stimulating VTG synthesis. The period of gametogenesis is characterized by high blood levels of FSH and increasing levels of androgens in males, and E2 and VTG in females. At the end of gametogenesis, pituitary LH secretion induces the synthesis of maturation-inducing steroids (MIS), which regulate the process of gonadal maturation; this period is characterized by decreasing blood levels of FSH and androgens/estrogens and increasing blood levels of LH and MIS. At completion of maturation, a GnRH induced LH surge stimulates ovulation, spermiation and spawning.

31. Brain Gonadotropin-Releasing Hormone (GnRH)

33. The stimulatory action of GnRH on GTH secretion is dependent on the presence of GnRH receptors (GnRH-R) in the membrane of the pituitary gonadotrops. In fish, multiple GnRH-Rs have been identified and, in contrast to mammals, they do not show ligand specificity. Expression levels of the GnRH-R genes in the pituitary show a seasonal pattern, which is an important factor influencing the seasonal responsiveness of the pituitary to GnRH stimulation. Highest levels of GnRH-R and thus highest responsiveness of the pituitary occur at the pre-spawning period, whereas lowest GnRH-R levels are found during the resting period and early stages of gonadal development. This is critical not only for the natural development of the reproductive cycle, but also when applying hormonal therapies, as this affects greatly the efficiency of GnRHa-based hormonal treatments, depending on the moment of the cycle when the treatments are applied.

34. In addition to the primary GnRH stimulatory system, GTH secretion is under the influence of a brain inhibitory tone, the dopaminergic system • Neuronssecretingdopamine (DA) exert an inhibitory action on both the brain and pituitary. • DA effects: • Inhibits GnRH synthesis and GnRH release in the brain; • Down-regulates GnRH-R in the pituitary; • Interferes with the GnRH signal-transduction pathways, inhibiting GnRH-stimulated LH secretion from the pituitary. In many freshwater species, DA inhibits strongly the pre-ovulatory GnRH-stimulated LH surge and thus, ovulation and spawning. In contrast, an active DA inhibitory system seems to be very weak or absent in most marine fishes.

35. La dopammina (o dopamina) è un neurotrasmettitore endogeno della famiglia delle catecolammine. All'interno del cervello questa feniletilammina funziona da neurotrasmettitore, tramite l'attivazione dei recettori dopamminicispecifici e subrecettori. La dopammina è prodotta in diverse aree del cervello, tra cui la substantia nigra e l'area tegmentale ventrale (ATV). Grandi quantità si trovano nei gangli della base, soprattutto nel telencefalo, nell'accumbens, nel tubercolo olfattorio, nel nucleo centrale dell'amigdala, nell'eminenza mediana e in alcune zone della corteccia frontale. Nessun altro sistema neuronale ha ricevuto tanta attenzione negli ultimi 20 anni quanto quello dopamminergico. La dopammina è anche un neuro ormone rilasciato dall'ipotalamo. La sua principale funzione come ormone è quella di inibire il rilascio di prolattina da parte del lobo anteriore dell'ipofisi. A livello gastrointestinale il suo effetto principale è l'emesi. La dopammina può essere fornita come un farmaco che agisce sul sistema nervoso simpatico, producendo effetti come aumento della frequenza cardiaca e pressione del sangue. Ha formula chimica C6H3(OH)2-CH2-CH2-NH2. Il suo nome chimico è 4-(2-amminoetil)benzene-1,2-diolo e la sua sigla è "DA". Fa parte della famiglia catecolammine (un anello benzenico con due gruppi ossidrilici), al quale poi è legato un gruppo etilamminico. La dopammina è un precursore della noradrenalina e dell'adrenalina.

36. Although GnRH is the primary regulator of reproduction, the brain synthesizes other neurohormones and neurotransmitters that have been shown to stimulate LH secretion and participate in the regulation of fish reproduction The NPY is involved in the regulation of the nutritional status of the fish. NPY neurons exert stimulatory actions on both GnRH and GTH and seem to play an important role in mediating interrelationships between nutrition and reproduction. The neurotransmitter γ-amino-butiric acid (GABA). The GABA is the most relevant neurotransmitter of the brain; in mammals and in fish it exerts a stimulatory action over LH secretion.

37. Il neuropeptide Y (NPY) è un polipeptide molto diffuso nel sistema nervoso centrale e nel sistema nervoso autonomo; svolge diverse azioni, tra cui l'aumento dell'appetito e la modulazione della risposta vasocostrittrice innescata dai neuroninoradrenergici. Il neuropeptide Y fa parte delle famiglia che comprende il peptide YY e il polipeptide pancreatico. È formato da 36 aminoacidi: -OOC-Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gli-Glu-Asp-Ala-Glu-Asp--Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2 Funzioni nel sistema nervoso centrale Il neuropeptide Y è un potente stimolatore dell'appetito ed ha uno spiccato effetto oressizzante. Nel Sistema Nervoso Centrale, sia degli uomini che degli animali, è stato dimostrato il ruolo del NPY sia come ansiolitico (composto in grado di combattere i sintomi dell'ansia al pari dei classici farmaci) che anti depressivo. Questo neuropeptide è inoltre coinvolto nel consumo e abuso di alcolici, infatti le persone che soffrono di depressione e gli alcolisti hanno livelli alterati di questo neuropeptide nell'organismo. La localizzazione di NPY nell'ippocampo lo rende importante nei processi di apprendimento e memoria; in questa regione del cervello è inoltre capace di stimolare la proliferazione neuronale e ciò è in accordo con le sue proprietà antidepressive.

38. GABA (sigla di GammaAmminoButirrico Acido) Neurotrasmettitore di tipo inibitorio, il più importante del sistema nervoso centrale; è prodotto a partire dall’acido glutammico, per decarbossilazione catalizzata dell’enzima glutammicodecarbossilasi. Il GABA è ampiamente diffuso nel cervello e nel midollo spinale. Sono stati identificati diversi recettori postsinaptici per l’acido γ-amminobutirrico: i recettori GABAA e GABAC (questi ultimi presenti solo nella retina) sono di tipo ionotropico e controllano un canale di membrana specifico per il cloro; il recettore GABAB è, invece, di tipo metabotropico accoppiato a proteine G e controlla un canale per il potassio. I recettori GABAA sono il bersaglio di farmaci ansiolitici e ipnotici (le benzodiazepine e i barbiturici) o antiepilettici (l’acido valproico), che agiscono sul sistema nervoso potenziando il sistema GABAergico (cioè il sistema costituito dai neuroni che utilizzano il GABA). 

39. Pituitary Gonadotropins (GTH)

40. The two pituitary GTHs, FSH and LH, together with the placental chorionic gonadotropin (CG) are glycoprotein hormones. They are heterodimeric proteins, constituted by a common α subunit, noncovalently linked to a hormone-specific β subunit, which confers the biological specificity to the hormone The half-life of the GTHs in the bloodstream is determined mainly by its degree of glycosilation. Human CG (hCG) is used in the spawning induction protocols in fish since it is the highest glycosilated GTH and thus, it presents higher resistance to degradation than any other glycoprotein. The general view is that: FSH controls mainly early stages of gametogenesis, LH regulates FOM, ovulation and spermiation. There are important differences between fish species, most probably related to different patterns of gonadal development and different reproductive strategies.

41. The initiation of the reproductive cycle is characterized by increased FSH levels, which are maintained high during gametogenesis, whereas LH levels remain undetectable. During gonadal maturation, FSH levels decline and LH increase, showing a sharp LH peak prior to ovulation. In salmonid species, showing single-batch group synchronous ovarian development, FSH increase during early gametogenesis while LH predominates during FOM. Nonsalmonid species show a slightly different picture. In the gilthead seabream (Sparus aurata), with asynchronous ovarian development, both FSH and LH are expressed throughout the year, increasing both during the reproductive season . In other nonsalmonid species, exhibiting multiple-batch group synchronous or asynchronous ovarian development, such as the blue gourami (Trichogaster trichopterus), red seabream (Pagrus major), European seabass (Dicentrarchus labrax) and stickleback (Gasterosteus aculeatus), FSH and LH levels are found throughout the reproductive cycle, although in most cases FSH synthesis is advanced with respect to that of LH.

42. Gonad steroids

43. Steroidogenesis takes place in the somatic cells of the gonad, the granulosa and theca cells in the ovary and the interstitial Leydig cells and Sertoli cells in the testes. The major steroid hormones in the regulation of fish gametogenesis are the estrogen E2 in females and the androgen 11KT in males and, to a lower extent, dihydrotestosterone (DHT). The fish ovary also synthesizes T, which plays other reproductive related functions. Similarly, males also synthesize E2, but this is found in much lower levels than in females. The testes of male fish produce other androgens than 11KT (DHT, T), which exert complementary functions during testicular development.

44. Gonadal steroids effects • Regulation of gonadal development. • Both positive and negative feedback on the brain-pituitary axis and thus, • Regulation of GTH release. • A major positive action of the steroids is to enhance pituitary responsiveness to GnRH, probably by stimulating GnRH-R. • A major negative action of these steroid hormones is exerted through the dopaminergic system, increasing DA turnover and thus enhancing the DA inhibitory tone over GTH secretion. • In this way, the brain is constantly informed about the evolution of gonad development, through the action of the fluctuating circulating levels of steroids during the reproductive cycle.

45. Steroids regulate female oogenesis and maturation

46. At the end of the vitellogenic stage… Pituitary LH secretion induces a shift in the steroid biosynthetic activity of the ovary with a reduction in T and E2 production and enhancement of the synthesis of MIS. This is caused by reduction of aromatase activity and increased activity of enzymes of the MIS pathway. There are two major MIS identified in fish: 1. 17α,20β,dihydroxy-4-pregnen-3-one (17,20β-P) 2. 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S). They both probably act as MIS in most fishes, but normally one of them is the predominant MIS for a given species. The 17,20β-P is the major MIS in several salmonid and nonsalmonid species, while 20β-S is the major MIS in Atlantic croaker, spotted sea trout, striped bass and black porgy. The synthesis of MIS is also a two-cell process, by which the precursor 17β-hydroxyprogesterone is synthesized in theca cells and converted into 17,20β-P in the granulosa cells, by the enzyme 20β-hydosysteroid dehydrogenase.

47. Steroids regulating male spermatogenesis and maturation The LH is mainly involved in the stimulation of androgen production in Leydig cells. FSHstimulates 11KT production in Leydig cells through activation of specific enzymes (11α-hydroxylase and 11α-hydroxysteroid dehydrogenase) and regulates Sertoli cell activity during spermatogenesis. 11KT regulates the full process of spermatogenesis. FSH levels are high at early spermatogenesis, increase to maximum levels during the rapid testicular growth phase and then decline after spawning. On the other hand, LH is very low during early spermatogenesis, start increasing during the rapid testicular growth phase and peaks during spawning. As spermatogenesis advances, LH becomes important in supporting 11KT production. After completion of spermatogenesis, secretion of LH from the pituitary induces a shift in the steroidogenic pathway of the testes leading to the production of MIS, which in turn regulate sperm maturation.