Group of fish reproductive physiology
The hormonal control of the gonadal development: Gametogenesis and its hormonal control S.Zanuy Institute of Aquaculture of Torre de la Sal (IATS); Spanish Council for Scientific Research (CSIC), Ribera de Cabanes. 12595 Torre de la Sal, Castellón, Spain
E-mail:
[email protected]
1
HORMONAL CONTROL OF FISH REPRODUCTION: GAMETOGENESIS ENVIRONMENTAL FACTORS (Ligh, temperature, others)
www.amustard.com www.amustard.com
Sertoli cells FSH-R
GnRH
Cyst
Gonadotropins
Oocyte and sperm maturation
Basal Membrane
gonadal steroids
17α hydroxyprogesterone
Perilobular cells
Theca
Theca cells
Vitelogenesis Spermatogenesis
MIS 17α, 20ß-dihidroxi-4-pregnen-3 one
MPF
(cdc2 kinasa, ciclinaB)
Germinal vesicle
Receptor
Oocyte Granulosa
(Vitelogenin)
Germinal vesicle Yolk granules
Granulosa cells
The various environmental signals, mainly photoperiod and temperature but also visual-olfactory stimulus, social factors, water characteristic and quality, food availability, presence of substrate, etc., are perceived by the sensorial system of the fish and sent to the brain which reacts throughout the activation of multiple neuronal circuits which finally will act on the pituitary and stimulate gonadotropin (GtHs) synthesis, secretion and release to the blood. These are responsible for steroideogenesis which in turn are cause of the events leading to gamete production. In summary reproductive activity is controlled by the sequential participation of hormones and a complex cascade of events along the brainpituitary-gonadal axis.
2
Gametogenesis
The Ovary
Gametogenesis is the creation of gametes by meiotic division of gametocytes into various gametes. Males and females of a species that reproduce sexually have two different forms of gametogenesis: spermatogenesis (male) and oogenesis (female). Ovaries appear as paired elongated organs oriented longitudinally whithin the abdominal cavity. They are surrounded by a conjunctive tunica, the mesovarioum, adherent to the dorsal lining of the abdominal cavity under the swim bladder. Ovaries are compartimentalized by numerous septa formed by folds of the germinal epithelium called ovigerous lamella projecting into the ovarian lumen. These lamellae contain nests of oogonia and oocytes at early stages of entry into the meiotic prophase, and follicles at various stage of growth. In contrast to mammals oogonia keep on proliferating in adult females and thus renewing the stocks of young oocytes each sexual cycle.
3
MAJOR STEPS OF OOGENESIS 1.- Formation of primordial germ cells PGCs 2.- Transformation of PGCs into oogonnnia (sex differentiation) 3.- Transformation of oogonnnia into oocytes (onset of meiosis) 4.- Growth of oocytes (mainly vitelogenesis; meiotic arrest ) 5.- Maturation (resumption of meiosis) 6.- Ovulation
Pictures after Zanuy & Carrillo, unpublished
Growth
MaturationOvulation
In broad sense, oogenesis is the process by which primordial germinal cells become oocytes ready to be fertilized. Primordial germ cells (PGCs) are the embryonic precursors of the gametes. In most species, PGCs spend much of early development as nomadic residents within other developing tissues. Once they reach the gonad, germ cells undergo a sexually dimorphic process of differentiation that eventually (after days, weeks or years, depending on the organism) culminates in reductive divisions (meiosis) giving rise to the haploid sperm or egg. Once PGCs are differentiated into oogonia these transform into oocytes (onset of meiosis). In most teleost fish, after gonadal sex differentiation or at the beginning of each reproductive cycle a fraction of oogonia, present in the ovary, undergo a series of mitotic divisions followed by their entry into meiosis (transformation into oocytes). The mechanisms controlling oogonial selection, proliferation and meiotic commitment are largely unknown. We are going to focus our attention on the last steps of oogenesis namely growth, maturation and ovulation. Picture: Different stages of development of sea bass oocytes (Zanuy & Carrillo, unpublished)
4
THE PREVITELLOGENIC GROWTH Occurs at the beguining of the first meiotic arrest at the diplotene stage of chromosomal development
ca and mRNA Production of large amounts of ribosomal
Vitellogenin receptor and vitellogenin procesing enzymes
Production of large amounts of glicoproteins Incorporated ninto alveoli newly formed at the oocytes’s perifery
Oocyte lipid deposition lp Storage Earlyin the ooplasma as lipid droplets
ve
Structural changes take place at the ovarian follicle Vitellin envelope between the oocyte and the granulosa Late Pictures after Zanuy & Carrillo, unpublished
During this step occurring at the beginning of the first meiotic arrest the diameter of the ovarian follicle clearly increases. The nucleoli produce large amounts of ribosomal RNA as well as mRNA that encode proteins required for subsequent oocyte growth such as vitellogenin receptors and yolk processing enzymes (cathepsines). In addition large amounts of glycoproteins are synthesized by the oocyte during mid to late previtellogenic growth. These are incorporated into alveoli in the periphery of the oocyte. Cortical alveoli are important structures because their participation in the cortical reaction to fertilization. Oocyte lipid deposition is generally initiated during this step of oocyte growth. Finally, structural changes take place at the periphery of the ovarian follicle. The chorion or vitelline envelope (VE) starts to be deposited at the periphery of the oocyte and with its thickening, the microvilli ( intimate connection between the granulosa and the oocyte) elongate to maintain contact with granulosa cells via channels traversing the chorion (zona radiata). Early previtellogenic growth is non gonadotropine regulated. Late previtellogenic growth probably is gonadotropine regulated, at least the production of VE that is controlled by ovarian estrogens which in turn, are controlled by pituitary gonadotropins mainly FSH.
5
THE VITELLOGENIC GROWTH Accumulation of yolk coming from its hepatic precursor vitellogenin (Vtg)
VITELLOGENIN a glicolipophosphoprotein synthesized in the female liver under stimulation of estrogen -Lipovitelin (AAs and lipids
embryonic development)
-Phosvitin (delivers minerals Ca++
skeletal development)
-β´-component (no lipids, no phosphorus
unknown function)
Male and juvenile fish capable on synthesize Vtg after E2 or estrogen mimic contaminants (biomarker)
6
THE VITELLOGENIC GROWTH Pralichthys olivaceus Vtg purification and characterization M
Size exclusion chromatography
E
Native-PAGE
P
M
E
P
Western blot
A.J. Palumbo et al., 2007, Comp. Biochem. Physiol. Part A 146: 200–207
Two distinct Vtg transcripts/proteins with different functions and properties: 1. In teleost with pelagic eggs AAs source for hydration and buoyancy 2. Different responsiveness to estrogens relevant for endocrine disrupting studies
7
THE VITELLOGENIC GROWTH CA Yg Yv
cv
cp ZR
ZR
Gc TC -Vtg passes via extracellular space among theca cells, basal lamina and granulosa cells and across the canals in the chorion -Vtg bind to specific receptors at the oolema. The Vtg-receptor complex is internalized through coated pits. Those pinch off the oolema and forma coated vesicles which are the precursor of yolk globules
Vitellogenins are sequestered by the follicles to be incorporated into the oocyte as yolk. Ultraestructural studies demonstrate a possible route for the Vtg passage from the blood capillaries, via the extracellular space among the theca cells, across the basal lamina, through the spaces between adjacent granulosa layer, around the microvillar projections of the granulosa and those of the oocyte and across the channels of the zona radiata to make contact wit the oolema. On the oolema the Vtg binds to specific receptors. The Vtg-receptor complex is internalized by the formation of coated pits. These pinch off the oolema to form coated vesicles in the ooplasma that fuse with lysosome-like multivesicular bodies, were the first cleavage of Vtg occurs to form the yolk proteins, giving rise to the yolk granules or globules. The degradation of Vtgs to form yolk proteins is carried out by the lysosomal enzymes the cathepsins. Pictures of sea bass oocytes by Zanuy & le Menn (Unpublished results)
8
ENDOCRINE CONTROL OF VITELLOGENESIS Pituitary Gonadotropin (FSH) Testosterone Theca cell
17 β estradiol Ooplasma Germinal vesicle Liver
Granulosa cell
Follicle Yolk granules Vitellogenin
According to Nagahama, 1995, Curr. Top. Dev. Biol., 30:103-45
In salmonids vitellogenine uptake is stimulated by FSH but not by LH. The action of the gonadotropin is mediated by the follicular production of 17 beta Estradiol biosynthesized by ovarian follicles via an interaction of two cell layers, the theca and granulosa (two-cell-type model). The granulosa cell layer provides these steroid mediator but its production depends on the provision of the precursor steroid (Testosterone) by the thecal cell layer. Thus, both theca and granulosa cell layers cooperate in the production of steroidal mediator of oocyte growth. Gonadotropin (FSH) acts on the thecal and granulosa cell layers to stimulate testosterone and its aromatization to estradiol, respectively, through a receptor mediated adenilate cyclase cyclic-AMP system. In non salmonid teleost fish, the few existing evidences indicate that the process would be similar. Nevertheless, in other teleost such as Fundulus or medaka, steroideogenic thecal cells are not evident and the granulosa cells are the major sites for steroid synthesis. Thus, it is suggested that in this cases the involvement of two cell types is not required. The choriogenesis (fromation of the viteline enveloppe) proceeds during this step regulated as well by estradiol. This process is more rapid and sensitive to steroid than the formation of Vtg.
9
Endocrine control of vitellogenesis in sea bass A
8
E2 (ng/ml)
6
Group A
B
Group B
4 2
(mm) Oocyte diameter
0 4 VTG (mg/ml)
3 2 1 0
1500 1200 900 600 300 0
S
O
N D
J
F
M
A MY J
JL AG S
S
O
N
D
J
F
M
MONTHS
MONTHS Asturiano et al., J.Fish.Biol., 2000, 56 (5): 1155-1172
A) Estradiol and vitellogenin plasma levels increase as vitellogensis progresses and decreased just prior maturation-ovulation of oocytes starts. Maximum plasma levels of E2 (>5 ng/ ml)] are obtained during late vitellogenesis. VTG plasma levels increase throughout vitellogenesis peaking (c. 2.5 mg/ ml) at postvitelogenesis. B) yellow bars vitellogenic or postvitellogenic oocytes; red bars ovulated oocytes
10
ENDOCRINE CONTROL OF MATURATION Reinitiation and completion of the first meiotic division
GV
Maturation occurs before ovulation and the most visible event is the migration of the germinal vesicle towards the animal pole Pictures after Zanuy & Carrillo, unpublished
After migration towards the animal pole, Germinal Vesicle (GV) breaks down (GVBD) indicating that the end of prophase I has occurred. At the same time other processes such as condensation of the chromosomes, formation of the spindle and extrusion of the first polar body, take place. This late event marks the completion of meiosis I. The first meiotic division is immediately followed by the preparation of the second meiotic division that will remain blocked at metaphase II until fertilization. Other more or less obvious morphological features of oocyte maturation, depending of the species, are yolk clarification and increase of oocyte volume. Maturation is followed by ovulation that is the rupture of the follicle and the release of the eggs into the ovarian lumen. The figure shows the micropyle a small canal or aperture produced by a single specialized granulosa cell that phagocytes a funnel traversing the entire width of the chorion. Lather on at fertilization, this aperture will allow the entrance of the acrosomeless teleost spermatozoa.
11
ENDOCRINE CONTROL OF MATURATION Pituitary Gonadotropin (LH) 17α hidroxyprogesterone Theca cell MIH 17α, 20ß-Dihidroxy-4-pregnen-3one (DHP)
cdc2 kinase
MPF (cdc2 kinase, Cicline B)
P Receptors
MPF Cicline B
Germina Vesicle
Granulosa cell
Follicle
P
According to Nagahama, 1995, Curr. Top. Dev. Biol., 30:103-45
In teleosts oocyte maturation is regulated by three major mediators: gonadotropin, maturation inducing hormone (MIH) and maturation-promoting factorr (MPF). Recent evidences indicate that LH and not FSH is responsible for induction of follicular maturation in teleost. Thus, in response to a LH surge promoted by environmental, social or pheromonal stimuli, the oocyte will acquire maturational competence. That is, the follicle enclosed oocyte resumes meiosis when stimulated with MIH. To summarize, the term ovarian follicle maturation is defined as the suite of LH-induced changes that are necessary for resumption of meiosis. Similar to the situation during vitellogenesis a two cell model has been described for the maturing follicle. According to this model 17 alfahydroxyprogesterone formation occurs in the theca cells. These steroid diffuses into granulosa cells to be converted into 17alfa-20 beta-dihydroxy-4-pregnen-3one (DHP). The two cell type model production of DHP does not apply to all teleost as occurred for the production of E2, in certain teleos species the presence of the theca layer is not required. In other fishes 17alfa-20beta21trihydroxy-4-pregnen-3-one (20-beta S) acts as well as a MIH. The MIH binds to membrane receptors (MIHR) which number increases during final maturation. There are evidences suggesting that binding of MIH to MIHR inhibits adenylatecyclase activity, as a consequence the decline of c-AMP levles appears to activate cytosolic maturaton promoting factor (MPF) which directly is responsible for triggering resumption of meiosis. MPF is a complex consisting in the cell cycling regulator cdc2-kinase and cykiln B. MIH induces oocytes to de novo synthesize cyclin B which in turn activates the preexisting cd2-kinase through its threonine phosporilation, producing the active cd2-kinase
12
Endocrine control of sea bass oocyte maturation A
1,50 1,00
b (32) ab (7)
0,75
ab (4)
0,50
a (27)
a (11)
ab (6)
ab (3) a (42)
B
0,25 0,00 1,8
1,5 20β S (ng/ml)
1,2
(5)e d (7)
0,9
bcd bd (6) (11) bd (31)
0,6
ac abc (42) (3)
a (27)
0,3
Oocyte diámetre (mm)
17α 20β P (ng/ml) (DHP)
1,25
1500 1200 900 600 300 0
0,0 ATRE
OVUL
f MAT
e MAT
POSTVTG
a VTG
e VTG
PREVTG
Occyte developmental stage
S
O
N
D
E
F
M
MONTHS
Asturiano et al., J. Fish. Biol., 2000, 56 (5): 1155-1172
High plasma levels of DHP were observed during postvitellogenesis whereas those of 20 beta S raised during final maturation. These oocyte stage of development were maximum from mid December to end of February when ovulation occurred.
13
Pathway of steroid biosynthesis in the ovarian follicle of salmonids according to Nagahama et al. 1995
Cholesterol Pregnenolone
3ß-HSD Progesterone P-450 17α
CH3
CH2
C21
HO
H OH
20 ß-HSD O=
O=
17α, 20ß-dihydroxy-4pregnen-3-one
17a-hydroxyprogesterone P-450 17,20 liase Androstenedione
OH
17ß-HSD
C
C= O OH
C18
OH
P-450arom O=
Testosterone
OH
17 ß-estradiol
Oocyte growth
P-450scc
Ocyte maturation
Granulosa Cell
Thecal Cell
14
ng/ml
20 10 0 15
ng/ml
3
1
5
Testosterone
3
2
10
Hormonal profile at Oocyte Growth and Ovulation in sea bass
T
2
15
E2
17β Estradiol
1
5
ng/ml
0
1,00 0,75
2
1
3
17,20 β P
17α, 20β-dihydroxy -4pregnen-3one (DHP)
0,50 0,25
ng/ml
0,8
Diameter µ
0,00 1,2
1600
1
20 β S
3 2
17α, 20β-21-trihydroxy -4pregnen-3one (20βS)
0,0
Ov1 Ov2 Ov3 VTG
1200 800 400 0
S
O
N
D
E
F
M
A
3 2 1 0
VTG (mg/ml
0,4
Maturation Ovulation Asturiano et al., 2002; Sci. Mar. 66(3): 273-282
Successive elevations of plasma T and E were observed prior peaks of progestagens, which resulted from the shift in gonadal steroidogenesis and coincided with the maturation-ovulation of the different clutches of oocytes. This hormonal pattern was repeated several times depending on the number of ovulations per female. These results suggest a mechanism, based on shifts in gonadal steroidogenesis, which may be responsible for regulation of groupsynchronous ovarian development in sea bass.
15
Gametogenesis
The Testicle
Gametogenesis is the creation of gametes by meiotic division of gametocytes into various gametes. Males and females of a species that reproduce sexually have two different forms of gametogenesis: spermatogenesis (male) and oogenesis (female). Testicles appear as paired elongated organs oriented longitudinally within the abdominal cavity and consisting on anastomosing tubules that contain spermatocysts encircled by Sertoli cells in which spermatogensis occurs.
16
THE TELEOST TESTICLE Interstitial
Sertoli cells Spermatocyst
Basement membrane Perilobular cells
Sc
Sc
SC SA
SB SA
LC Sc
Fb SA
SA
Two compartments can be observed in the testes of teleost: the tubular and the interstitial. The interstitial consists on Leyding cells, fibroblast and blood and lymph cells. Electron microscopic and enzyme histochemical studies have demonstrated that Leyding cells are the main source of steroids in the testis. The tubular compartment contains germ cells and somatic cells or Sertoli cells. These cells described by Enrico Sertoli in 1865 fill a crucial nursing function in connection with spermatogenesis. Thus a well functioning Sertoli cell provides the developing germ cells with appropriated mitogens, differentiation factors and sources of energy. In consequence, the survival and development of germinal cells during spermatogenesis depends on their intimate association with Sertoli cells. Sertoli cells and germinal cells form a unit referred as spermatogenic cysts or spermatocyst and several spermatocysts give rise to a testicular tubule. The simplest spermatocyst is a single spermatogonia enveloped by few Sertoli cells which in turn are in contact with the basement membrane, which separates the germinal compartment from the interstitial compartment. During the mitotic phase the germ cell number of an spermatocyst increase in number and it is reasonable to assume that the number of Sertoli cells associated with a developing germinal cell clone increase as well. In Teleost fish, the cystic mode of spermatogenesis implies that the germ cells within the same spermatocyst (surrounded by the Sertoli cells), are at the same stage of differentiation. In amniotic vertebrates a given Sertoli cell concomitantly contacts members of several germ cell clones at any given time. Pictures O. Yilmaz (Master degree Thesis) and Carrillo & Zanuy (1977), Inv. Pesq. 41(1): 121-146
17
THE SPERMATOGENESIS IN THE SEA BASS Different types of spermatogonia
Spermatogonial renewal
Spermatogonial proliferation
I & II Spermatocytes
Meiosis
Spermatids
Spermiogenesis
Spermatozoa
Sperm maturation
Schema depicted after Miura and Miura, 2003; Fish. Physiol. Biochem. 28: 181-186/Pictures after O.Yilmaz’s Master of Aquaculture, 2005
Pre-spermatogonial steam cells divide mitotically to produce two types of cells: the self-renewing spermatogonial stem cells and the spermatogonial cells committed to further proliferation that will lead to meiosis. Spermatogonia divide mitotically in order to increase the germ cell population and provide a large number of cells to ensure fertility. The number of divisions before to enter in meiosis is species specific. Germ cells passes through three major phases: mitotic proliferation (spermatogonia renewal and proliferation), meiosis (spermatocytes I and II) and spermiogenesis (spermatids transform into spermatozoa). After the final mitotic proliferation spermatogonia B enter in meiosis giving rise to the preleptotenic primary spermatocites. These spermatocytes undergo a long prophase (subdivided in leptotene, zygotene, pachytene and dipoltene), during which DNA is duplicated and the chromosomes pair up with their corresponding chromosome. While paired enzymes cut sequences of DNA which are exchanged between the chromosomes (exchange of genes or recombination of genetic information). Following, chromosomes of maternal and paternal origin separate at subsequent meta,-ana and telophase and short living secondary spermatocytes are produced. These divide again without DNA replication giving rise to the haploid spermatids. These accomplish a cellular restructuring generating the flagellated spermatozoa (spermiogenesis). The acrosoma do not develop in most fishes since, as mentioned previously, the postovulatory oocyte has a mycropile which allow the sperm to entry. After completion of spermiogenesis the cyst wall opens to release the sperm (spermiation). In many fishes intratesticular spermatozoa has reduced fertilizing capacity. The accomplishment of the fertilizing capacity takes place in the sperm duct and is referred as sperm maturation or capacitation
18
Steroideogenic mediators of spermatogenesis and sperm maturation in salmonids Cholesterol CH3
Nagahama 1994; Int. J. Dev. Biol. 38: 217-229
Pregnenolone Progesterone 17a-hydroxyprogesterone
HOCH …..OH
O= Spermatogenesis
17α, 20ß-dihydroxy-4pregnen-3-one
Androstenedione
OH
Sperm maturation Testosterone
O
11-Ketotestosterone O
Two main steroid mediate spermatogenesis and sperm maturation in salmonids and most teleost a non-aromatizable 11 oxygenated androgen and a progestagen, respectively.
19
HORMONAL REGULATION OF SPERMATOGENESIS Pituitary
11-Ketotestosterona
BasementMembrane BasementMembrane
Sertoli Sertoli Cell Cell
Spermatogonia Spermatogonia Activin B proliferation of spermatogonia
Activin B proliferation of spermatogonia IGF I continuation of spermatogenesis IGF I continuation of spermatogenesis SRS21~MIS prevents spermatogensis SRS21~MIS prevents spermatogensis
R
FSH
-R
Activine B IGF I SRS21,
Receptor Activine B vs SRS21,
Spermatogenesis Spermatogenesis
Germinal cells
Leydig Leydig Cell Cell
Somatic cells
LH-R
According Nagahama, 1994, Int, J. Dev. Biol. 38:217-229 and Miura & Miura , 2003, Fish Physiol. Biochem. 28: 181-186
Gonadotropins
Testicle
There are not many studies regarding hormonal control of the renewal of spermatogonial steam cells, nevertheless, in the Japanese eel it has been demonstrated that it is stimulated by E2. Sertoli cells seem to be implicated in this action since they poses estradiol receptors. The shift from renewal of spermatogonial germ cells to spermatogonial proliferation leading to meiosis is gonadotropin dependent. Gonadotropins cause a surge in the secretion in the androgen 11-Ketotestosterone (11KT) in the Leydig cells. 11KT stimulate Sertoli cells to produce several mediators such as Activin B and Insuline like growthfactor I (IGF-I) . In the japanese eel 11KT initiates spermatogenesis whereas it seems that IGF is necessary to continue it. Activine B induces spermatogonial proliferation but not meiosis. An additional member regulating spermatogonial proliferation is the so-called Spermatogenesis Related Substance SRS21, which amino acid sequence shows a great similarity with the Mullerian Inhibiting Substance (MIS). Recombinant eel SRS21 suppress spermatogonial proliferation induced by 11KT. Thus it seems that spermatogonial proliferation is regulated by rivalry between Activin B and SRS21. FSH receptors at least in mammals are expressed in the Sertoli cells and thus FSH directly regulates its function. Although in fish the situation is not clear and it is claimed that an important role of FSH would be to modulate the release of growth factors by Sertoli cells. In addition FSH could participate in the stimulation of Sertoli cell proliferation
20
HORMONAL LEVELS DURING THE REPRODUCTIVE CYCLE OF SEA BASS
pg mRNA/ng 28s rRNA
6 5 4
c
3 2 1
bc a
a a
a
a
a
a
6
c
FSH-β
c
b
3 a
a
c
0
O
b
N
D
J
F
M
A
M
MONTHS 6
a a
100 80
LH (ng/ml)
11-Ketotestosterone (ng/ml)
7
0
% Spermiation
Cerdá et al., 1997; Aquaculture International, 5: 473-477
Mateos et al., 2003, Gen. Comp. Endocrinol 133: 216-232
b
8
2
b
bd
4 a
c
b d
a
a
a
J
Jl
0
60 40
O
20 0
A S O N D E F M A M J J A S O N D E F M A M
N
D
J
F
M
A
MONTHS
MY
J.M. Navas, et al., 2004, Ciencia Marinas 30: 527-536
MONTHS Pictures O. Yilmaz’s Master’s degree, 2005
21
ENDOCRINE REGULATION OF SPERM MATURATION
Gonadotropine
Testicle
LH-R
Leydig Leydig Cell Cell
17α -hidroxiprogesterone 20ß-HSD
Espermatozoa Espermatozoa
DHP and 20βS CA II/SRS22
Esperm duct Increase in seminal plasma PH Increase the level of AMPc interespermatic Sperm Motility
According Nagahama, 1994, Int, J. Dev. Biol. 38:217-229 and Miura & Miura , 2003, Fish Physiol. Biochem. 28: 181-186
Hipofisis
The accomplishment of the fertilizing capacity takes place in the sperm duct and is referred as sperm maturation or capacitation which is accompanied by fluid production (hydration). After two meiotic divisions the germ cells develop into spermatids. The spermatids transform into spermatozoa through spermiogenesis. This process is characterized by the occurrence of important morphological changes associated to the formation of the sperm head with condensed nucleus, a mid piece and a flagellum. In teleosts it is not yet clear whether a regulation mechanism exist in spermiogenesis. That is if spermiogenesis take place without hormones. Once spermiogenesis is completed sperm is not yet capable of fertilizing the eggs. In salmonids spermatozoa adquire the ability of motility during their passage through the sperm duct. Sperm maturation the passage form non functional gametes to mature spermatozoid involves physiological but not morphological changes. In salmonids and the japanese eel sperm maturation is induced by increasing the seminal pH which results in elevation of sperm cAMP levels.Sperm maturation is regulated by the endocrine system, in some teleost it has been suggested that 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) is related with the regulation of sperm maturation. These progestagen does not act directly in the sperm; Its action is mediated through an increase in the seminal plasma pH, which in turn increases the sperm content in cAMP allowing the acquisition of sperm motility. Nevertheless the mechanism by which DHP increases the seminal plasma pH remin unclear. Recently a factor related to the regulation of the increase in pH has been cloned from the testis of japanese eel. This factor called SRS22 is a homologue of a Carbonic anhydrase (CA) involved in the regulation of ion acidbase balance in various fluids an tissues. The following mechanism has been suggested the presence of DHP induces the activation of CAII/SRS22, this enzymatic activation causes an increase in the seminal plasma pH, and spermatozoa subsequently acquire the motile ability.
22
ng/ml
4 3 2 1 0
ng/ml
4 3
ng/ml
1 3
11KT
1
Espermatogenesis
3
1
1,2
0,0
ng/ml
17α, 20β-dihydroxy -4pregnen-3one (DHP)
3
1
0,4
1,2
2
20 β S 1
0,8
17α, 20β-21-trihydroxy -4pregnen-3one (20βS)
3
0,4 0,0
2
8
1
6 4
Esperm production
3
2 0
11Ketotestosterone
2
17,20 β P
0,8
1,6
Sperm Volume ml
Testosterone
2
2 0 1,6
Hormonal profile at spermatogenesis and Spermiation in sea bass
2
T
D
E
F
M
Asturiano et al., 2002; Sci. Mar. 66(3): 273-282
Successive elevations of plasma T and 11KT levels were observed prior to peaks of progestagens, which resulted from the shift in gonadal steroidogenesis and coincided with increases of sperm production. This suggest a mechanism, based on shifts in gonadal steroidogenesis, which may be responsible for regulation of waves of spermiation in sea bass.
23
Is it important to have an adequate knowledge of the endocrine control of fish reproductive process? In the nature the fish live in a changing environment and its reproduction is intimately related to the fluctuations of this environment. Thus they assure the survival of the offspring The synchronization with the environment is carried out by means of complex interactions between the components of the brain-pituitary-gonad axis The captivity modifies the environment and in addition it simplifies it. It can seriously affect diverse aspects of the reproduction So that the culture of a species is successful, this must reproduce in captivity Some important factors are fotoperiod, temperature, social interactions, rainfall, vegetation, tides, etc. When one or several of these factors is not adjusted the reproduction interrupts or undergoes some alteration. In captivity the presence of one or several of these factors or the interaction among them not always is possible. In this case the reproduction is impaired or altered.
24
Which reproductive alterations can be found? The species does not reproduce in captivity, total absence of gonadal recrudescencia in both or one of sexes There is gonadal recrudescencia but not maturation and/or spawning (absence of espermiation or ovulation) In protandric or protogynous hermaphrodite fish species, one of sexes reaches reproduction after some years Asynchrony between sexes during gonad recrudescence The rearing conditions can alter the proportion of sexes and cause the presence of the less suitable sex for the culture The intensive culture can cause premature gonadal maturation that affects the rate of growth and favors the appearance of diseases The emission of the gametos takes place but they are not viable and the fertilization does not take place Production of abnormal embryos and weak larvae that cannot overcome incubation, weaning and fingerling grow out
and etc., etc,etc.………………………….........................
25
Environment Sensorial receptors Melatonine
Breeders
Hipothalamus- Preoptic Region
& %
GnRHs +
Production of eggs and larvae
Othres - Dopamine
Hipophysis LH
FSH
+
Gonads Sexual Steroids
Intensive Culture In many cases captivity influence fish reproductive process by affecting hormonal events responsible for gamete production. When this occurs it is necessary to induce gonad recrudescence or/and spawning synchronization by means of intervention at different levels
26
How is it possible to control these hormonal events of the reproductive process in captivity?
27
Using GnRHs analogues
Cabrilla LHRHa: Dorada Japonesa Mugil pGlu-His-Trp-Ser-Tyr-Ala-Leu-Arg-Pro-NEt Corbina
rosácea
Salmón del Atlantico
European seabass Carpa india Trucha arcoiris
Mero Babre africano Carpa
Rodaballo
28
Important to determine the intraovarian oocyte developmental stage
Picture after Carrillo & Zanuy
Any hormonal therapy must be applied when the intraovarian oocytes are at the appropriated stage of development. Position of the germinal vesicle is determined either after transparency (upper pictures) or in fresh (lower pictures) after cannulation
29
Novel biotechnological applications outcome of advances in molecular biology
30
SINGLE CHAIN GONADOTROPIN Produced and released to the culture media by CHO cells scLH
scFSH N
LHβ
OOOO
CTP
N
N
α
N
N
FSHβ
OOOO
CTP
N
N
α
CTP of hCG as link between subunits: . 31 amino acids . Not secondary structure . Flexible and hydrophilic . Binds to the receptor . Longest half life Gómez et al., 2004; Aquaculture Europe 04: 372-373
The use of recombinant single chain gonadotropins are an interesting alternative
31
In conclusion…….
Fish culture benefits from the knowledge generated through the study of the mechanisms controlling the reproductive process of the fish, and to a great extent, its economic bonanza and the generation of important benefits is consequence of it.
32
Acknowledgements
Histology Service of the IATS
Thanks your attention Animalfor Husbandry service of the IATS
33