F. Bardakci*, ▄. Ozansoyć and E. Koptagelç
*Department of Biology, Faculty of Science and Literature, Cumhuriyet University, 58140 Sivas, Turkey.
ćDepartment of Biology, Faculty of Education, Gazi University, Kirsehir, Turkey.
çDepartment of Histology and Embryology, Faculty of Medicine, 58140 Sivas, Turkey.
The process of gamet development has been studied in many fish species in detail owing to advanced histochemical techniques and electron microscopy ( 1). Most recent studies on oogenesis have dealt with sex differentiation (2, 3, 4, 5), oocyte growth and development (6, 7, 8, 9, 10) and specialised aspects of formation of primary envelope ( 11, 12, 13), cortikal alveoli formation and vitellogenezis ( 14, 15) and oocyte maturation (16). Nonetheless, there are limited numbers of studies on environmental factors affecting ovarium development (16), especially in its natural environment. In nature, these environmental factors largely determine the timing of reproduction and thus, the reproductive strategy of a species ( 17). Therefore, knowing the effect of environmental factors on the reproductive biology of a species is essential basis for adapting it to a fish culture ( 18).
So called "doctor fish" ( 19) Garra rufa Heckel, 1843 (Teleostei: Cyprinidae) lives in the pools of a hot-spring, which have a constant water temperature (approximately 35 oC) over the year, along with Cyprinion macrostomus Heckel, 1843 (Teleostei: Cyprinidae). As these two species feed on plaques of skin diseases and softened human skin tissue by water, it has believed to have treupatic effect on skin diseases, especially psoriasis ( 19). Since many patients with skin diseases and tourists visit the hot-spring for treatment and pleasure, there may be a requirement for the culture of this species as an aquarium fish and/or for conservation purposes. The present study compares oogenesis in doctor fish living in the pools of the hot-spring under constant water temperature, and in the Topardiš stream having seasonally fluctuating water temperature.
The study was undertaken during consecutive months between May 1991 and December 1991 from pools of the hot-spring and Topardic stream (Samples could not be collected for the remaining months because of winter). Both the hot-spring and Topardic stream are closely located in the province of Sivas in Central Anatolia region, Turkey. Adult females were collected from the pool of the hot-spring and Topardiš stream by employing scoop net and gill nets, respectively. Total length, body weight and ovarian weight were recorded. In the calculation of gonadosomatic index (GSI), the following formula was employed:
GSI (%) = Gonad Weight (g)/Body weight (g) x 100
HISTOMORPHOLOGY OF THE OVARY AND OOGONIA
The paired of ovaries are located in the dorsal region of the body cavity. Externally, ovary morphology of females from the hot-spring and Topardiš stream was similar except relatively reduced gonadal weight and size of ovaries of females from the hot-spring. Ovaries are surrounded by a single-layer germinal epithelium, which enspheres a thick tunica albuginea. The tunica albuginea consisting of muscle and connective tissues projects into lumen of ovary known ovigerous lamella in which oogonia and follicule cells are present. Oogonia are slightly oval and are apparent by their smaller size and larger nuclear-to-cytoplasmic ratio (Figure 1a). Older germ cells move toward distal parts of lamella and newly formed ones take place as oogenesis proceeds.
STAGES OF OOCYTE DEVELOPMENT
The process of oogenesis was classified according to size, appearance of nucleus and nucleoli and distribution of cytoplasmic inclusions in G. rufa females from both localities studied.
1. Chromatin-nucleolus stage (CNS)
The size of the oocytes at this stage is approximately 70 Ám in diameter in females from both localities. Chromatin-nucleolus stage is characterised by a large nucleus in central position and little ooplasm. The nucleus contains an eccentric primary nucleus, as well as a few smaller nucleoli and chromatin threads, many of which are associated with nucleoli (Figure 1b).
2. Peri-nucleolar stage (PNS)
Mean oocyte diameter for females from the hot-spring and Topardiš stream was 135 ▒ 21.21 Ám and 142 ▒ 35.35 Ám, respectively. Nucleolus divides to multiple nucleoli and resides in the periphery of nucleoplasm. At this stage, nucleus increases in size and nucleolus in number. In addition, "yolk nucleus" or "Balbiani bodies" appears in the ooplasm (Figure 1c). Oocyte is surrounded by a single layer of follicle cells.
3. Cortical-alveolar stage (CAS)
Mean oocyte diameter was 260 ▒ 14.12 Ám for females from the hot-spring and 370 ▒ 70.71 Ám for females from Topardiš stream. The major event in this stage is the appearance of cortical alveoli on the periphery of the oocyte. It has also noticed that a few nuclei pushed into the ooplasm. The number of nucleoli located around the nuclear membrane increase and nuclear membrane take an irregular structure. As this stage proceeds, cortical alveoli continue to accrue and increase in size (Figure 1d).
4. Vitellogenetic stage (VS)
Mean oocyte diameter for the females from the hot-spring and Topardiš stream was 525 ▒ 35.35 Ám and Topardiš stream 775 ▒ 35.36 Ám, respectively. The main event occurring at this stage is the accumulation of a hepatically derived yolk precursor protein, vitellogenin that is responsible for majority of oocyte growth. In the beginning of this stage, yolk spheres appear around the nucleus and as the stage proceeds all oocyte filled with them. (Figure 1e). Oocyte surrounded with a distinct teka layer, granulosa layer and zona radiata, which divides zona radiata interna and zona radiata extarna (Figure 1f).
5. Maturation stage (MS)
This is a final stage of oocyte development. Oocyte reaches its maximal size that is 700 ▒ 70.71 Ám and 1125 ▒ 48.49 Ám for the hot-spring females and for Topardiš females, respectively. Massive accumulation of yolk protein occupies the central portion of the oocyte (Figure 1g). The germinal vesicle is located near the animal pole just beneath the oocyte surface. The vitellin envelope becomes more compact and during its transformation into the chorion and the entire follicular epitelium appears to be pulled away from the vitelline envelope. After the germinal vesicle breakdown the oocyte ovulated into the ovarian lumen and becomes a mature egg.
Average lengths of females studied from the hot-spring and Topardiš stream were 73.5▒ 10.61 mm and 97▒ 18.38 mm, respectively. Similarly, average body weights of females from the hot-spring and Topardiš stream were 2.75▒ 0.35 g and 11.15▒ 4.45 g, respectively.
As shown in Figure 2, GSI(%) values of hot-spring females is lower than females from Topardiš stream but there is a concordance in its values between two populations over the period studied. Ovaries are in resting stage from September to December according to GSI(%) values and proportion of oocytes at the different stages both for the hot-spring and Topardiš population. At this period, ovaries of individuals from both populations are mainly filled with oocytes at peri-nucleolar stage and cortical alveolar stage (Figure 3, Figure 4)). At the pre-spawning stage, the size and weight of ovaries increases from May and gradually reaches to their maximal weight in July. The process of vitellogenesis starts at this stage. As seen from Figure 3, proportion of vitellogenic oocytes is the highest in the ovaries of females from Topardiš stream in June and gradually transforms to mature oocytes until July. On the other hand, proportion of mature oocytes of females from the hot-spring at the pre-spawning stage were found lower than those in Topardiš Stream (Figure 4). This striking difference indicates that transformation of vitellogenic oocytes to mature oocytes is hindered.In addition, while atretic oocytes (AO) are present only at the post-spawning stage in the ovaries of females from Topardiš stream (Figure 3), atresia at various stages of development has been seen in the ovaries of females from the hot-spring (Figure 1h and Figure 4).
The overall pattern of oocyte development in G. rufa is basically the same in all teleost species ( 7, 21, 15). The process of oogenesis has been divided to various stages according to size and various cell inclusions. For example, development of oocyte in the rainbow trout, Oncorhyncus mykiss ( 22), piefish Syngnathus scovelli ( 9) and mudfish, Labeo capensis ( 10) were divided to eight, six and six stages, respectively. Here we have established five developmental stages for the process of oocyte development of G. rufa species. There was no difference found between two populations of G. rufa studied with respect to the pattern of oocyte development, especially at the early stages of oocyte development. On the other hand, the size of oocytes from females in Topardiš stream was found higher than those in the hot-spring from peri-nucleolar stage on. The increase at the early stages of oocyte development is due mainly to accumulation of nonyolky cytoplasm and the major growth of oocytes at the later stages is attributable mainly to accumulation of a hepatically-derived, female specific, yolk precursor protein, vitellogenin ( 1, 8). While oocytes reach their maximal size of about 1125 m m in the population of Topardiš stream, oocytes of the hot-spring population remain only about 700 m m. These results indicate that there is a limited yolk accumulation in the oocytes of the hot-spring females. Wallace and Selman ( 16) have found that nutritional state of the fish directly effects the vitellogenic and maturational enlargement of oocyte within the ovary of Fundulus heteroclitus. They stated that starving fish for a certain period ceased the follicule size before it reached its maximal size and refeeding animals restored the oocyte growth. De Vlaming ( 23) have established that photoperiod, appropriate temperature and adequate food supply are important environmental factors for the regulations of reproductive patterns in most teleosts. It has been shown that high temperature (25-30 oC) suppressed gonadal activity in a cyprinid fish, Gnathopogen cacrulescens ( 24). Ízer et al. ( 25) and Bardakci ( 26) have found that there is a malnutrition in the pools of the hot-spring, by analysing gut content of Doctor fishes, G. rufa and Cyprinion macrostomus and water of hot-spring. It can be concluded that high temperature and lack of food has suppressed the gonadal activity. Although this study indicates that temperature and malnutrition have depressed the oocyte development, certain amount of oocytes in the ovaries of females from the hot-spring still reach to maturity. This is presumably a result of non-fluctuating temperature and a food supply from debris of human skin since people visiting period of year to the pools of the hot-spring coincides with the pre-spawning and spawning time of this species. Temperature is actually one of the environmental factors that accelerate the vitellogenesis, only if daily fluctuation in water temperature is not high ( 27) and sufficient amount of nutrition is available.
As seen from Figure 2, the GSI% values of the hot-spring females show an accord with those of Topardic stream, indicating that the process of seasonal gonad development is under the genetical factors although it can be affected by environmental factors. Lower GSI(%) values of the hot-spring females are presumably a consequence of limited food supply. GSI(%) values were found to be the highest in July for both populations, showing the starting period of spawning for both populations.
The occurrence of oocytes at the different developmental stages per microscopic field has showed that there was no difference between two populations at the post-spawning stage. From September to December, oocytes at peri-nucleolar and cortical alveolar stages are dominant in the ovaries of females from both populations. The most striking difference between two populations is that frequency of oocytes at the vitellogenic stage in the ovaries of females from the Topardiš stream were found higher than the hot-spring females. In addition, it is concluded that transformation of vitellogenic oocytes to mature oocytes is suppressed in females from the hot-spring since the proportion of mature oocytes did not increase gradually from May to July. This data confirms the suppression of vitellogenin accumulation in the oocytes of females in the hot-spring.
Follicular atresia is a very common phenomenon of the teleost ovary that is usually seen at the period of post-spawning in oocytes at any stage. On the other hand, it has been considered that environmental stress is the main cause of follicular atresia ( 1). Wallace and Selman ( 16) have seen follicular atresia after several weeks of starvation in Fundulus heteroclitus and suggested that a mobilisation of material from yolk-containing oocytes is for the physiological needs of the starving female under the experimental conditions. In the case of this study, constant high temperature and starvation are probably the main environmental stresses that causes the formation of atretic oocytes for the females in the pools of the hot-spring. It is clear from Figure 4 that atretic follicular are even present at the pre-spawning stage of the hot-spring females ranging from May to September.
Consequently, the process of oogenesis in both populations studied is similar in general but is under the stress of the high constant temperature and starvation in the hot-spring population. Therefore, vitellogenezis and maturation process is in part suppressed in the females of the hot-spring; thus, it has lower GSI(%) values but the same spawning period with the population of Topardiš stream. The data also confirms that environmental stress is a cause of occurrence of atretic oocytes in natural environments.
Authors are grateful to colleagues in the Department of Biology of Faculty of Science and literature and in the Histology and Embryology of Medical Faculty for their help during laboratory work of this study.