Turk. J. Vet. Anim. Sci.

30 (2006) 427-434
© TÜB‹TAK Research Article

Levels of Some Ovarian Hormones in the Pre- and Post Spawning

Periods of Chalcalburnus tarichi Pallas, 1811, and the Postovulatory
Structure of Follicles

Güler ÜNAL1,*, Hatice KARAK‹fi‹2, Mahmut ELP3

1
Department of Biology, Faculty of Arts and Science, Yüzüncü Y›l University, 65080 Van - TURKEY
2
Department of Biology, Faculty of Science, Ege University, 35040 Bornova, ‹zmir - TURKEY
3
Department of Fishery, Faculty of Agriculture, Yüzüncü Y›l University, 65080 Van - TURKEY

Received: 22.12.2003

Abstract: The structure of postovulatory follicles and the levels of ovarian 11-dehydrocorticosterone (11-DHC), estradiol-17β (E2),
17α-hydroxyprogesterone (17α-OH-P), and progesterone (P), before and after spawning, were studied in Chalcalburnus tarichi. It
was observed that postovulatory follicles are characterized by a highly vascular thecal layer and hypertrophied granulosa cells
containing dense smooth endoplasmic reticulum, many large mitochondria with tubular cristae, and lipid droplets. Ovary 11-DHC
level was low in the pre- and post-spawning periods (38.3 ± 4.9-22.3 ± 2.6 ng/ml). The level of E2 was 216.8 ± 31.1 ng/ml before
spawning. This hormone declined on the 1st and 5th day after spawning and increased again to the level of 216.8 ± 6.7 ng/ml on
the 10th day; however, the E2 level decreased significantly on the 15th day (61.5 ± 17.8 ng/ml) (P < 0.01). The 17α-OH-P level was
th
low (84.5 ± 49.4 ng/ml) before spawning and reached a peak (295.7 ± 52.5 ng/ml) on the 10 postspawning day, but a slight
th
decline was observed on the 15 day (190 ± 21.5 ng/ml) (P > 0.01). The P level was low (23.4 ± 18.3 ng/ml) before spawning.
Its level began to increase after spawning and reached a peak on the 10th postspawning day (200.7 ± 29.1 ng/ml) (P < 0.01), but
th
declined significantly on the 15 day (63.2 ± 19.5 ng/ml) (P < 0.01).
The results obtained from this study indicate that: a) In the postovulatory follicles, the granulosa cells produce steroids; b) 11-DHC
has no effect before or after spawning; c) E2 induces spawning; d) 17α-OH-P and P have an effect after spawning. It can be
concluded that the apoptotic cells in postovulatory follicles increased on the 15th day, at which time E2 and P hormones were at
their lowest levels after spawning in C. tarichi.

Key Words: Chalcalburnus tarichi, postovulatory follicle, ovarian steroids

‹nci kefali’nin (Chalcalburnus tarichi Pallas, 1811) Yumurta B›rakmadan Önce ve Sonraki
Periyotta Baz› Ovaryum Hormon Seviyeleri ve Foliküllerin Ovulasyondan Sonraki Yap›s›

Özet: Bu çal›flmada, inci kefalinde yumurta b›rakt›ktan sonra foliküllerin yap›s› ve yumurta b›rakmadan önce ve yumurta b›rakt›ktan
sonra ovaryum 11-dehydrocorticosteroit (11-DHC), östradiol-17β (E2), 17α-hydroxyprogesteron (17α-OH-P) ve progesteron (P)
seviyeleri araflt›r›ld›. Ovulasyondan sonra foliküllerin bol damarl› bir teka tabakas› ve bol miktarda düz endoplazmik retikulum,
tubüler kristal› çok say›da mitokondri ve lipit damlalar› içeren büyük granüloza hücreleri ile karakterize edildi¤i gösterilmifltir.
Ovaryum 11-DHC seviyesi yumurta b›rakmadan önce ve sonra düflüktür (38,3 ± 4,9-22,3 ± 2,6 ng/ml). E2 seviyesi yumurta
b›rakmadan önce 216,8 ± 31,1 ng/ml olarak belirlendi. Bu hormon yumurta b›rakt›ktan sonra 1. ve 5. günde azald› ve 10. günde
tekrar 216,8 ± 6,7 ng/ml’ye yükseldi. Bununla birlikte, E2 seviyesi 15. günde belirgin olarak düfltü (61,5 ± 17,8 ng/ml) (P < 0,01).
17α-OH-P seviyesi yumurta b›rakmadan önce düflüktü (84,5 ± 49,4 ng/ml) ve yumurta b›rakt›ktan sonra artarak 10. günde en
yüksek seviyeye ulaflt› (295,7 ± 52,5 ng/ml) fakat 15. günde hafif bir düflüfl gözlendi (190 ± 21,5 ng/ml) (P > 0,01). P seviyesi
yumurta b›rakmadan önce düflüktü (23,4 ± 18,3 ng/ml). Onun seviyesi yumurta b›rakt›ktan sonra artmaya bafllad› ve 10. günde
maksimum seviyeye ulaflt› (200,7 ± 29,1 ng/ml) (P < 0,01) fakat 15. günde belirgin olarak düfltü (63,2 ± 19,5 ng/ml) (P < 0,01).
Bu çal›flmadan elde edilen sonuçlar: a) Ovulasyondan sonraki foliküllerde granuloza hücrelerinin steroit sentezledi¤ini, b) 11-DHC’nin
yumurta b›rakmadan önce ve sonra etkili olmad›¤›n›, c) E2’nin yumurta b›rakmay› uyard›¤›n›, d) 17α-OH-P ve P’un yumurta
b›rakt›ktan sonra etkili oldu¤unu gösterir. ‹nci kefalinde, ovulasyondan sonraki foliküllerde apoptotik hücrelerin, E2 and P’nin
yumurta b›rakt›ktan sonra en düflük seviyede oldu¤u, 15. günde artt›¤› sonucu ç›kar›labilir.

Anahtar Sözcükler: Chalcalburnus tarichi, postovulasyon folikülleri, ovaryum steroidleri

* E-mail: [email protected]

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Levels of Some Ovarian Hormones in the Pre- and Post Spawning Periods of Chalcalburnus tarichi Pallas,
1811, and the Postovulatory Structure of Follicles

Introduction Ovaries from 5 different groups were studied: a) before

st th
Chalcalburnus tarichi is an endemic cyprinid species of spawning; b) 1 day after spawning; c) 5 day after
th th
the Lake Van Basin in Turkey. It spawns once a year and spawning; d) 10 day after spawning; e) 15 day after
has an ovary of the synchronous type. It is generally spawning. The fish were anesthetized with 100 ppm 3-
accepted that the fish’s ovarian follicles, as in other aminobenzoic acid ethyl ester (MS-222, Sigma). The
vertebrates, produce steroid hormones. In many fish, it ovaries of disected fish were removed immediately and
has been shown that the ovarian follicles synthesize stored in a deepfreeze at –80 ºC for later steroid analysis.
estrogens, such as estradiol-17β, estrone and estriol, The ovary follicles of the fish were fixed on the first day
progestogens, androgens, and corticosteroids (1,2). after the spawning for histological study. Fish 4-6 years
Ultrastructural and histochemical studies suggest that in old, which were 18-20 cm in size, were kept for both
some teleosts the special thecal cells (ST cells) and hormone analysis and histological study.
granulosa cells of ovarian follicles are the major cellular Electron microscopy
sites of ovarian steroidogenesis (3-7). It has been
Ovarian follicles were fixed in Karnovsky’s fluid for 2
accepted that the induction of final oocyte maturation by
h at 4 ºC. After washing in 0.2 M Cacodylate buffer (pH
gonadotropin in fish ovarian follicules is mediated by
7.4) they were postfixed in 1% OsO4 for 1 h at 4 ºC, and
maturation-inducing steroids produced by follicle cells
after dehydration were embedded in Epon 812. Thin
(8). In most teleosts, oocyte final maturation and
sections were placed on 1000 mesh copper grids and
ovulation is mediated by either 17,20β-dihydroxy-4
stained with saturated uranyl acetate followed by lead
pregnen-3-one (17,20β-P) (9,10) or 17,20β,21-
citrate, and were then examined under a JEOL 100 C
trihydroxy-4 pregnen-3-one (20β-S) (11,12); however,
electron microscope. For light microscopy, 1 µm thick
in Pagrus major (13) and Acipenser transmontanus (14),
sections were cut with a Reichert ultramicrotome and
it has been reported that both steroids have an effect
stained with methylene blue.
during final oocyte maturation. Moreover, in some fish,
endocrine gland changes during the ovulatory cycle have High performance liquid chromatography (HPLC)
been observed (15-17). It is reported that the special Three fish samples from each group and 1-g ovarian
thecal cells are the sites of steroid synthesis during tissue samples from each fish were used for hormone
vitellogenesis and that E2 induces vitellogenesis and analysis. The HPLC analysis was adapted from Shimizu et
spawning in C. tarichi (18). al. (19), Venkatesh et al. (20), and Battal and Ünal (21).
The aim of the present study was to clarify the Frozen tissue was powdered in liquid nitrogen and
histological features of the postovulatory follicles of C. methanol was added. The samples were homogenized in
tarichi and to determine ovarian estradiol 11- an Ultra Tissue Lysis (Ultrasonic Processor Jenway) and
dehydrocorticosterone (11-DHC), estradiol-17b (E2), centrifuged at 8500 xg for 30 min at 4 ºC. The extract
17β-hydroxyprogesterone (17β-OH-P), and was evaporated at 35 ºC and then steroids were
progesterone (P) levels in the periods before and after redissolved in 0.05 M phosphate buffer (pH 7.6). Next,
spawning. the samples were passed through Sep-Pak C18 cartridges
(Waters) that were preconditioned with 5 ml of distilled
water and 5 ml of methanol. Cartridge-adsorbing
Materials and Methods hormones were eluted with 5 ml of 80% methanol and
Mature C. tarichi samples were caught in May and the eluate was injected into HPLC (Shimadzu, LC-10 AD).
June 2000, during their spawning period, from the Steroids were separated with a chromatographic system
Karasu River where it enters Lake Van. The fish, which under isocratic conditions at a flow rate of 1.5 ml/min in
were ready to spawn, were stripped by gently touching a µBondapak column with acetonitrile:water (53:47, v/v)
their ventral region. These fish were kept in fiberglass as the mobile phase. The pressure and wavelength were
tanks (90 x 90 x 250 cm) with a continuous flow rate adjusted 2.8 psi and 254 nm, respectively. The elution
(1 l/s) at a temperature of 16-18 ºC, with a natural pattern of each steroid (11-DHC, E2, 17α-OHP and P) is
photoperiod. They were fed live zooplankton twice daily. shown in Figure 1.

428
 G. ÜNAL, H. KARAK‹fi‹, M. ELP

11

P
Absorbance (254 nm)

17 α-OHP
E2

0 2 4 6 8
Relation Time (min)
Figure 1. Elution patterns of 11-dehydrocoticosterone (11-DHC),
estradiol-17β (E2), 17β-hydroxyprogesterone (17β-OHP), Figure 2. Epon section (1 µm) of a postovulatory follicle 1 day after
and progesterone (P) separated using HPLC. Stationary ovulation. BV: blood vesicle; TL: thecal layer; GC: granulose
phase: µBondapak column. mobile phase: acetonitrile:water cells; methylene blue: bar = 60 µm.
(53:47). flow rate: 1.5 ml/min, detection: uv (254 nm).

in the cytoplasm of these cells and in the follicular lumen

All concentrations were expressed as mean ± SE. The (Figure 3b). Ultrastructurally, these cells consist of
data were analyzed by analysis of variance (ANOVA). The almost smooth endoplasmic reticulum distributed
significance level for differences was set at P = 0.01. throughout the cytoplasm, with tubules of varying
lengths (Figure 4) and mitochondria. The mitochondria
Results were oval or elongated, with tubular cristae. A Golgi
apparatus, which was poorly developed, appeared in the
Histology basal cytoplasm near the nucleus. Some of the granulosa
The most important feature of postovulatory follicles cells contained lysosome-like bodies.
is the thickness of the thecal layer, which contains many Hormone Levels
enlarged blood vessels. Postovulatory follicles are also
characterized by hypertrophied granulosa cells dispersed Ovarian steroid levels according to sampling date are
in a cavity formerly occupied by oocytes (Figures 2 and shown in Figure 5. Ovarian 11-DHC level was significantly
3a). In epoxy sections, the granulosa cells are cuboidal or lower than the other steroids (P > 0.01). Its level was
columnar with cytoplasmic processes projecting into the 35.8 ± 4.9 ng/ml before spawning and remained low
follicular lumen (Figure 3a). The nucleus, with one after spawning (P > 0.01).
prominent nucleolus, is located basally. Some droplets, Ovarian E2 level was 216.8 ± 31.1 ng/ml before
which stained dark with methylene blue, were observed spawning, and slightly decreased to 184.6 ± 25.6 ng/ml

429
Levels of Some Ovarian Hormones in the Pre- and Post Spawning Periods of Chalcalburnus tarichi Pallas,
1811, and the Postovulatory Structure of Follicles

Figure 3. Epon section (1 µm) of a postovulatory follicle 1 day after ovulation. a) Large vacuoles in the thecal layer (TL) and granulosa cells with
cytoplasmic processes. FL: follicular lumen. methylene blue: bar = 30 µm. b) Well vascularized thecal layer and stained dark lipid droplets
(L). FL: follicular lumen. methylene blue: bar = 30 µm.

17α-hydroxyprogesterone 11-dehydrocorticosteroid
350 Progesterone Estradiol-17β
300
Hormone Levels

250
200
150
100
50
0
Before s. 1 5 10 15
Days

Figure 5. Change in ovary steroid levels before and after spawning (s).

(295.7 ± 52.5 ng/ml). 17α-OH-P level slightly decreased

15 days after spawning (190 ± 21.5 ng/ml) (P > 0.01).
The ovarian P level was also low (23.4 ± 18.3 ng/ml)
before spawning (P < 0.01). After spawning, it gradually
increased to a prominent peak (200.7 ± 29.1 ng/ml) on
Figure 4. Electron micrograph of granulosa cells 1 day after ovulation.
the 10th day (P < 0.01). The high value was followed by
Smooth endoplasmic reticulum and lipid droplet (L). Bar =
0.237 µm. a sharp decline on the 15th day (63.2 ± 19.5 ng/ml) (P <
0.01).

on the 10th day after spawning, but again increased to

216.8 ± 6.7 ng/ml, and decreased dramatically (61.5 ± Discussion
17.8 ng/ml) on the 15th day (P < 0.01). Postovulatory follicles in C. tarichi the first day after
Before the spawning, ovarian 17α-OH-P level was the spawning were characterized by a highly vascularized
significantly lower (84.5 ± 49.4 ng/ml) than after thecal layer and hypertrophied granulosa cells. The
spawning. After spawning, this level increased rapidly to follicular layer, which was shown to synthesize the
242 ± 1.6 ng/ml (P < 0.01) and peaked on the 10th day steroid hormone in postovulatory follicles, varies among

430
 G. ÜNAL, H. KARAK‹fi‹, M. ELP

teleost fish (4,22). In the white-spotted salmon level peaked during vitellogenesis and then decreased
(Salvelinus leucomaenis), pink salmon (Oncorhynchus slightly, and this high level was maintained during oocyte
gorbuscha), and coho salmon (Oncorhynchus kisutch), the maturation. In salmonids, plasma E2, which was observed
special thecal cells in the thecal layer possess organelles at high levels during vitellogenesis, decreased to very low
characteristic of steroid production (4,5). In tilapia, levels during oocyte maturation and ovulation (15,25),
Oreochromis mossambicus, although the granulosa cells whereas, in bitterling, Acheilognathus rhombea, high
in postovulatory follicles increase in size and organelle levels of E2 observed during vitellogenesis were
content, the primary site of steroidogenesis seems to be maintained during oocyte maturation and ovulation (19).
the special thecal cells, which show an intense 3β-HSD The high level of E2 is explained by the requirement of
reaction (6). Matsuyama et al. (7) found that the cells continuous vitellogenesis during the short reproductive
that possess organelles characteristic of steroid- cycle of this species. High levels of E2 are known to
producing cells show negative histochemical reactions for prevent apoptosis in fish (26). According to our results,
3β-HSD activity in Pagrus major. However, in goldfish, in C. tarichi, apoptosis in postovulatory follicles is dense
Carassius auratus, 3β-HSD activity is positive, not only in on the 15th day after ovulation.
the granulose cells, which possess organelles In many teleosts, it has been reported that progestins,
characteristic of steroid-producing cells, but also in especially 17α-OH-P, 17α,20β-diOH-P, and 17,20β-21-
special thecal cells (3). In C. tarichi, the granulosa cells in P, have a major effect on oocyte maturation and ovulation
postovulatory follicles 1 day after spawning showed an (27-29). Each one of these may have an effect in
ultrastructure essentially similar to those in Carassius different species. In goldfish, Carassius auratus, both
auratus (3), such as smooth endoplasmic reticulum, large 17α-OH-P and 17α,20β-diOH-P increase significantly at
mitochondria with tubular cristae, and lipid droplets. the time of oocyte maturation and ovulation (30). In the
According to ultrastructural studies, granulosa cells same fish, however, plasma 17α,20β-diOH-P levels show
(steroid-producing cells) are where steroids are produced a peak before ovulation, but plasma levels of 17α-OH-P
in C. tarichi. Nevertheless, histochemical experiments are were not determined (16). Hence, in the same fish, Kime
necessary to determine the exact role of granulose cells. et al. (31) reported that 17α,20β-diOH-P, 11-
Goswami and Sundararaj (23) reported that deoxycortisol, and 17,20β-21-P may have an effect,
corticosteroids are terminal hormones, which act to depending on the maturation state of the ovaries or the
induce oocyte maturation and ovulation in catfish, method used to stimulate oocyte maturation.
Heteropneustes fossilis. In orange roughy, 11-DOC has Nonetheless, 11-deoxycortisol and the 20β-reduced
also been measured in the plasma of female and male derivative (17, 20β-21-P) may be significant in spawning
fish, but no change in relation to reproductive goldfish since 17,20β-diOH-P has been found in only low
development has been reported (24). Corticosteroids in levels (as sulfate). It is generally accepted that, in
brook trout, Salvelinus fontinalis, and yellow perch, Perca salmonids, 17,20β-diOH-P has an effect on oocyte
flavescens, are ineffective in germinal vesicle breakdown maturation and it is called a maturation-inducing steroid
and ovulation. Similarly, in the present study, 11-DHC (MIS) (15,11). Yet, in trout, Salmo gairdneri, both
was detected in the ovaries of C. tarichi, but its level did 17α,20β-diOH-P and 17α-OH-P were measured at the
not change after spawning. time of ovulation (32). Goetz and Bergman (33) showed
In C. tarichi, ovarian E2 level showed high values that in brook trout, Salvelinus fontinalis, and yellow
before and after spawning, but it decreased dramatically perch, Perca flavescens, 17α,20β-diOH-P induced
on the 15th day after spawning. E2 is produced primarily ovulation at a lower concentration than both 17α-OH-P
during vitellogenesis. In many teleosts, it has been shown and 11-deoxycortisol, and had a strong influence during
that E2 induces the synthesis and secretion of vitellogenic oocyte maturation. In the white sucker, Catostomus
protein by the liver (5,17,19). E2 levels and commersoni, 17α-P and 17,20-P levels are low in
gonadosomatic index values are correlated during prespawning fish and their levels are the highest in fish
vitellogenesis (25). In our previous study (18), the that have ovulated (34). The peak values of 17α,20β-
production of ovarian steroid hormones during diOH-P are higher than those of 17α-OH-P during
vitellogenesis and oocyte maturation was observed. E2 ovulation in bitterling, Acheilognathus rhombea (19). The

431
Levels of Some Ovarian Hormones in the Pre- and Post Spawning Periods of Chalcalburnus tarichi Pallas,
1811, and the Postovulatory Structure of Follicles

major role of 17α-OH-P is considered to be as a progesterone has no effect on ovulation in C. tarichi, but
precursor of 17α,20β-diOH-P rather than a direct according to the above-mentioned studies its metabolites
inducer of oocyte maturation and ovulation (35-37). The may have an influence. The progesterone level reached a
effects of steroids in oocyte maturation and ovulation peak on the 10th day after ovulation and then decreased
have been determined by in vitro experiments (36). 17α- dramatically to basal levels just on the 15th day. To the
OH-P is the immediate precursor to 17α,20β-diOH-P in best of our knowledge, no study about the effect of
ovaries, testes, and head kidneys of the Atlantic salmon, progesterone on apoptosis in fish has been published. In
Salmo salar (37). Sakai et al. (35) have also shown that one study carried out in cow luteal cells,
17,20β-P levels before spawning increased, depending aminoglutethimide added to culture media was shown to
on the exogenous addition of 17α-OH-P in vitro. In C. block progesterone synthesis, and an increase in
tarichi, the level of 17α-OH-P was low before spawning. apoptosis was observed during this block. In another
According to the results mentioned above, it may be study, adding progesterone to the culture media
concluded that 17α-OH-P has an indirect effect on this prevented apoptosis (39). Similar to this, it can be noted
species and it may be a precursor of 17α,20β-diOH-P. that in the present study, in C. tarichi, apoptosis increased
In our previous study (18), ovarian progesterone was on the 15th day after ovulation when E2 and P levels were
low level during the cortical alveoli, vitellogenesis, and low; however, the effect of these hormones should be
oocyte maturation phases. In the present study, these low studied further in vitro and in vivo.
levels were also maintained relatively before spawning. In conclusion, this study investigated steroid secretory
Moreover, in Fundulus heteroclitus, steroid levels in cells after ovulation and some ovary hormones levels
media were measured after incubation with before and after spawning in C. tarichi. Granulosa cells
progesterone, pregnenolone, and 25-hydroxycholesterol are where steroid synthesis in postovulatory follicles
of the isolated follicles and the substrates added to follicle occurs. 11-DHC has no effect during spawning or
cultures caused an elevation even above the basal levels of postspawning. E2 induces ovulation. 17β-OH-P and P
E2, 17α-OH,20β-DHP, and testosterone secretion. In all have an effect after spawning. It may be suggested that
cases, the incidence of oocyte maturation and the the decline in the levels of E2 and P increases apoptosis in
accumulation of steroids in the medium are dependent postovulatory follicles.
upon the concentration of added precursor (38).
Matsuyama et al. (7) have shown that progesterone has
no effect on germinal vesicle breakdown (oocyte Acknowledgments
maturation). When ayu, Plecoglossus altivelis, both This study was supported by Yüzüncü Yıl University
treated and untreated with a salmon gonadotropin, were Research Fund (Project 97/FED-044). The authors wish
incubated with 14C-labeled progesterone and 17α-OH-P to thank Dr. Peyami Battal, who analyzed the samples by
in the presence of NADH, metabolites of progesterone HPLC.
were observed in the ovaries (36). We can conclude that

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