| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Testis; |
Research Group Endocrinology & Metabolism,3 Department of Biology, Faculty of Sciences, Utrecht University, 3508 TB Utrecht, The Netherlands
Institute of Marine Research,4 5817 Bergen, Norway
ABSTRACT
Precocious male puberty significantly compromises sustainability aspects of aquaculture in a number of finfish species. As part of a program aiming to understand and eventually control testis maturation in farmed Atlantic cod, we studied the first reproductive cycle. The gonadosomatic index shows a 41-fold increase from immature (August) to mature (March) stages, reaching almost 10% of the total body weight. The paired cod testes are composed of several lobes arranged around a central collecting duct. In each individual lobe, spermatogenesis occurs in a marked gradient of development, with undifferentiated spermatogonia in the periphery of the lobe and the most advanced germ cells in the vicinity of the collecting duct, suggesting a tight spatiotemporal organization of spermatogenesis in the testis lobes of this species. Spermatogonial proliferation starts in August and continues for about 6 mo. Meiosis and spermiogenesis are first observed in October and are completed in all cysts by February, when a 2-mo-long spawning season starts. Spermatogonia go through 11 mitotic divisions before differentiating to primary spermatocytes. Apoptosis is rare, but when observed it occurs mainly during the last spermatogonial generations. Our observations suggest a model in which a maturational wave progresses through each growing lobe that is first driven by appositional growth from the lobe's periphery, reflecting spermatogonial proliferation and cyst formation which, when ceasing, is terminated by completing spermiogenesis and spermiation that progress toward the lobe's periphery.
apoptosis, proliferation, seasonal reproduction, spermatogenesis, testis
Spermatogonial stem cells can either self-renew or produce spermatogonia committed to the developmental steps constituting spermatogenesis [1]. Via mitotic proliferation of spermatogonia, meiotic recombination in spermatocytes, and cellular differentiation of spermatids, mature haploid spermatozoa are formed [1, 2]. During spermatogenesis, the germ cells descending from a given stem cell stay interconnected via cytoplasmatic bridges, thereby constituting a synchronously developing germ cell clone. This sequence of developmental steps requires a specific microenvironment that is created by somatic cells, particularly the Sertoli cells. Germ cell survival and development critically depend on their continuous and close contact to Sertoli cells that provide structural and physiologic support, including paracrine interaction between these two cell types. In anamniote vertebrates (fish and amphibians), cytoplasmic extensions of Sertoli cells envelop the individual germ cell clones, forming spermatogenic cysts, which together constitute the germinal epithelium in the seminiferous tubules of the testis [3–5]. Hence, the main difference to spermatogenesis in higher vertebrates is that in cystic spermatogenesis a given Sertoli cell usually is in contact only with a single germ cell clone.
A crucial phase during spermatogenesis is the period of spermatogonial proliferation. Precise knowledge about the number of mitotic divisions that spermatogonia undergo is essential for analysing the regulatory mechanisms targeting spermatogenesis [6, 7]. Usually, a spermatogonium completes a species-specific, predictable number of mitotic cell cycles, before differentiating into spermatocytes, and hence entering meiosis; this number can vary between 2 and 14 in vertebrates [8].
During spermatogenesis, loss of a certain percentage of germ cells via apoptosis is normal in all species investigated, and it plays a critical role in determining spermatogenic efficiency [2, 9]. Germ cell death occurs exclusively or preferentially in certain developmental stages, also varying in a species-specific manner in quality and quantity [10]. For example, in rodents, apoptosis occurs mainly during the spermatogonial phase, referred to as density-dependent regulation, but can also occur during meiosis, related to irreparable chromosomal damage [6]. In teleosts, apoptotic germ cells have been observed mainly during the spermiogenic phase [11, 12]. Moreover, in seasonally breeding fish, germ cells not released during the reproductive season are phagocytized during the tubules' reorganization for the next season [13]. Sertoli cells are involved in both types of germ cell elimination in teleosts [14, 15].
The Atlantic cod (Gadus morhua, L.) is an economically important marine fish of the northern hemisphere. In recent years, natural stocks of Atlantic cod have been declining, and cod aquaculture has become increasingly relevant [16]. However, under farming conditions, male cod in particular commence and complete puberty much earlier than in the wild, and sexual maturation not only compromises flesh quality and growth performance [17] but also leads to unwanted introduction of genetic traits in wild populations via escaped mature fish or reproduction in aquaculture facilities, with the fry leaving the facilities. Attempts to prevent the initiation of testis maturation are an effective manner of controlling all aspects of unwanted reproduction, but they require understanding the regulation of this developmental process. Moreover, the cod belongs to an order of fish (gadiformes) in which spermatogenesis has not been studied in detail. We investigated (morphology, proliferation, apoptosis) the first maturation of cod testis over a period of 1.5 yr and found a novel pattern of development of the spermatogenic process that is presented in this paper.
A mixed population of male and female Atlantic cod of Norwegian Coastal Cod origin was reared in 7-m3 seawater tanks under ambient light conditions at Austevoll Aquaculture Research Station, Norway (60°N). The larvae were first fed on natural zooplankton in a semienclosed seawater pond at the Institute of Marine Research according to the method of Blom et al. [18] before transfer to the experimental tanks, where they were fed a commercial dry pellet diet ad libitum. All fish were treated and killed according to Norwegian National Legislation for laboratory animals. The water temperature ranged from 7.4°C to 9.4°C (mean ± SD = 8.1°C ± 0.3°C) during the experimental period.
Testis samples from 5–14 fish were analyzed (morphology, proliferation, apoptosis) monthly for 16 mo starting in July 2004, when the fish were 18 mo old (prepubertal). Body and testis weight was recorded to calculate gonadosomatic index (GSI = testis x 100 / body weight). Testis tissue was fixed in Bouin fluid by immersion, dehydrated, and embedded in paraffin according to conventional techniques. For all further studies, testis sections of 4 µm thickness were used.
The GSI values were log10-transformed and tested by one-way ANOVA, followed by Student-Newman-Keuls multiple comparisons test. A significance level of 0.05 was applied in the test.
Immunohistochemical Detection of Proliferation
Proliferation of spermatogonia and Sertoli cells was assessed by phosphorylated histone H3 (pH3) immunodetection. Histone H3 is a chromosomal protein component involved in the condensation of mitotic and meiotic chromosomes and becomes phosphorylated during late G2 phase, being present until metaphase in the cell cycle [19, 20] (i.e., it is detectable in cells preparing to divide). For this purpose, the sections were mounted on glass slides coated with 3-aminopropyl triethoxysilane (TESPA; Sigma, St. Louis, MO), and dried overnight at 37°C. The sections were deparaffinized and hydrated before incubation in a plastic chamber filled with 1 mM EDTA solution containing 0.05% Tween 20, pH 8.0 (Merck-Schuchardt, Hohenbrunn, Germany). For epitope retrieval, the glass chamber was transferred to a boiling water bath for 20 min and then left to cool to room temperature. Nonspecific protein binding sites were blocked with 5% goat serum (Vector Laboratories, Burlingame, CA) + 1% BSA (Sigma) in PBS for 30 min, followed by an incubation with a polyclonal rabbit anti-human phosphohistone H3 IgG preparation (Upstate, Charlottesville, VA; 1:200 dilution in 1% BSA in PBS, 1 h, room temperature). After being rinsed in PBS, sections were immersed in 0.35% hydrogen peroxide in PBS for 10 min to quench endogenous peroxidase activity. The subsequent incubation with biotinylated goat anti-rabbit IgG (1:100; Vector Laboratories) in 1% BSA in PBS lasted 30 min at room temperature, after which slides were incubated with avidin-biotin complex (ABC; Vector Laboratories) during 1 h, according to the manufacturer's protocol. DAB (3,3'-diaminobenzidine tetrahydrochloride, Dako, Glostrup, Denmark) substrate development was done for 30 sec. Nuclei were counterstained with 5% Mayer hematoxylin for 45 sec, and slides were mounted with Pertex (Cellpath Ltd., Hemel Hempstead, UK) after dehydration. For negative control, the primary antibody was replaced by the same concentration of normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
In Situ Detection of DNA Fragmentation
The in situ terminal deoxynucleotidyl transferase (TdT) mediated by deoxy-UTP-digoxigenin nick-end labeling (TUNEL) method was used to localize apoptotic cells, as described by van Bragt et al. [21]. Sections were counterstained and mounted as described above. For control purposes, the TdT enzyme was excluded from the TUNEL reaction mixture.
To determine whether apoptotic germ cell loss occurs during a specific phase of spermatogenesis, 100 cysts containing apoptotic cells were examined in each of seven males sampled in November that presented complete spermatogenesis (i.e., all germ cell stages present). These 700 cysts (100%) then were examined for the germ cell type they contained, and the incidence of apoptosis was expressed as percentage of cysts containing apoptotic spermatogonia, spermatocytes, or spermatids.
Morphometric Determination of the Number of Spermatogonial Generations
To assess the number of spermatogonial generations, five testes were fixed in November (GSI 5.98 ± 3.1) in 5% glutaraldehyde (Merck), and 5 mM phosphate buffer and were embedded in resin (Technovit 7100; Heraeus Kulzer, Wehrheim, Germany) according to conventional techniques. Serial sections of 3 µm were prepared and stained with 1% toluidine blue containing 1% borax.
To estimate the number of pachytene spermatocytes per cyst, and thereby to conclude how many mitotic divisions the spermatogonia completed before entering meiosis, it was necessary to determine the average volume of spermatogenic cysts containing pachytene spermatocytes and the average volume of a pachytene spermatocyte. The volume of pachytene spermatocytes was calculated from the nuclear volume and the proportion between nucleus and cytoplasm. To assess the volume of the nucleus, its diameter was measured (30 pachytene spermatocytes per animal; n = 5). Since the pachytene nucleus is round, its volume was estimated using the formula 4 / 3
R3 (R = diameter / 2), expressed in µm3. To calculate the proportion between nucleus and cytoplasm, a grid with 121 intersections was placed over the sectioned material at 400x magnification. For each animal, 1000 points over pachytene spermatocytes were counted.
The cyst volume was estimated using the Cavalieri method of reference volume [22] using serial sections. Only cysts comprised completely in the serial sections were used for evaluation. From the first to the last section of each cyst, the cyst area was measured and multiplied by the thickness of the section, providing the volume of the cyst per section, which when summed up provided the volume of the entire cyst. To measure the cyst area in each section, the Image J analysis program was used (http//:rsb.info.nih.gov/ij/features.html). We analyzed six to eight cysts per animal (n = 5).
Changes in Gonadosomatic Index and Testicular Lobe Composition
Cod testes are paired, longitudinal organs stretching dorsally through the length of the body cavity, connected to the dorsal body wall via the mesorchium. The spermatogenic parenchyma of the cod testis is composed of several lobes of similar morphology that are arranged around and drained by one central efferent duct per testis (Fig. 1). It is a highly dynamic tissue/organ as indicated by the dramatic (41-fold) changes in the gonadosomatic index (GSI; Fig. 2), which ranged from 0.2% (August) to 8.2% (March) on average.
|
|
Histologic analysis showed that the small, thin, rose-colored lobes of the immature testis (June/July; Fig. 1a) predominantly contained spermatogenic cysts with a single spermatogonium or small groups of spermatogonia; tubules did not yet show a continuous lumen (Fig. 3a). In fully mature males (February to April; Fig. 1c), the large, thick, white lobes were composed of spermatogenic tubules with a large lumen filled with spermatozoa (Fig. 3b). These two stages were connected by a developmental process that established in each lobe a marked gradient of spermatogenic cysts containing germ cells at different stages of maturation. Spermatogenic cysts with more advanced germ cells were situated closer to the central collecting duct, whereas cysts with germ cells at early stages of spermatogenesis were found in the distal, peripheral part of the lobe. This gradient was particularly evident in samples collected during the rapid growth phase (September to December) and was visible macroscopically (Fig. 1b) and microscopically (Fig. 4a). During this rapid growth phase, the periphery of the lobes was thin, rose colored, and contained mainly different spermatogonial generations (Fig. 4b). The off-white-colored, somewhat thicker central area of growing lobes contained predominantly spermatocytes and early (round) spermatids (Fig. 4c), whereas the white area close to the collecting duct contained late (elongated) spermatids and spermatozoa (Fig. 4d). With further increase of testis weight and GSI, the white area made up an increasing proportion of the growing lobe, eventually occupying the complete lobe in a whitening wave that emanated from the central collecting duct. Histologically, this was reflected in an increasing proportion of the tubules being filled with mature spermatozoa, whereas the more peripheral zone of spermatocytes and spermatogonia became progressively smaller, until all spermatogenic cysts had reached the spermiation stage (cysts open and Sertoli cells releasing spermatozoa into the tubular lumen), and the entire testis lobe was filled with sperm.
|
|
With the spawning-associated release of sperm, the GSI started to decrease (April). Low values were attained already in June in spent testes, when early spermatogonia were the only germ cells present next to residual sperm being progressively removed by Sertoli cells via phagocytosis (Fig. 3, c and d). Cysts were no longer observed in the testis lobes, and Sertoli cells formed an epithelial layer lining the tubular lumen (Fig. 3d). Figure 5 schematizes the developmental changes in a testicular lobe, highlighting its marked gradient of maturation.
|
Spermatogonial Generations and Analysis of Spermatogenesis
The average volume of a pachytene spermatocyte was 70 ± 2 µm3 per cell. Analyzing the volume of a total of 37 pachytene spermatocyte cysts, we found that two thirds of the cysts showed a volume indicating that the spermatogonia went through 11 mitotic cell cycles, whereas the volume of the remaining one third indicated that 12 mitotic cell cycles were completed. These data resulted in an average volume of pachytene spermatocyte cysts of 136 ± 25 mm3, suggesting that approximately 1900 pachytene spermatocytes were present per cyst. We therefore conclude that at least 11 mitotic cell cycles are completed before entering meiosis (n.b. 211 = 2048).
To evaluate further the mitotic phase of spermatogenesis, we analyzed sections after immunocytochemical detection of phosphorylated histone H3, a mitosis marker. Generally, the massive spermatogonial proliferation started in August when, besides single spermatogonia, the first cysts of late spermatogonia were observed proliferating through the lobe (Fig. 4e). Sertoli cells proliferated as well (Fig. 3a, inset), and their mitotic activity was observed mainly while they were associated with early spermatogonia. In October, the end of spermiogenesis was attained in the furthest progressed cysts in some of the males, and spermiation started (i.e., the cysts opened and spermatozoa were released in the tubular lumen). Free sperm was present in tubules near the collecting duct, but cysts with meiotic and postmeiotic germ cells as well as cysts still containing proliferating spermatogonia were found in other areas of the lobes (Fig. 4, e–g). Strong spermatogonial proliferation activity and formation of new spermatogonial cysts in the periphery of the lobes were observed until January. From February onward, however, formation of cysts with proliferating spermatogonia had stopped, and most cysts had entered meiosis so that proliferating spermatogonia were found only rarely in the testis and moreover did not form developing cysts, but were single or paired spermatogonia.
TUNEL analysis indicated that germ cell death did not occur frequently in cod spermatogenesis, since cysts presenting positive germ cells were rare. The highest incidence was observed during the last stages of spermatogonial proliferation, possibly just before entering meiosis (Fig. 4, i and j), involving 72% of the apoptotic germ cells, followed by spermatocytes and spermatids, which represented 12% and 11%, respectively; 5% remained nonidentified due to the lack of clear cytologic features when cells were far progressed into apoptosis. Germ cells in apoptosis showed the expected nuclear staining, reflecting the DNA fragmentation, but in some cysts the Sertoli cell cytoplasm was also labeled with the TUNEL technique (Fig. 4i).
The initiation of spermatogenesis marks the onset of puberty, which in cod is associated with an impressive growth of the testes. The GSI started to increase in September, reaching more than 8% of the total body weight in fully mature fish. Similar GSI values were observed in cod either captured in Canada (9%) [23, 24] or reared in Norway (10%) [25]. Higher maximum GSI values of up to 17% were found in a previous study with cod that were 1 yr older [26].
Testicular weight gain is attributable mainly to germ cell proliferation during spermatogenesis. Indeed, the variation in GSI values in cod testes correlated with the observed changes in testis histology. During periods with low GSI values (prepubertal-immature and postspawning-spent testes), the spermatogenic parenchyma showed only early spermatogonia and Sertoli cells in the germinal epithelium, while a lumen had not formed yet. The first elevation in GSI reflected the initiation of the mitotic activity of these two cell types. Hence, starting in August, proliferating spermatogonia were observed regularly until January and continued their development toward meiosis and spermiogenesis, so that spermatogenic cysts increased progressively both in number and size along with the GSI. Decreasing GSI values were observed after the end of spermatogenic activity and during the course of the spawning period, reflecting the release of sperm. The lowest GSI values were attained in June/July, reflecting completion of the removal of residual sperm via Sertoli cell phagocytosis, whereas the new spermatogenic wave had not started yet.
A very interesting feature of cod spermatogenesis is the maturational gradient in which spermatogenic cysts become organized during testis growth. This gradient of progressively mature germ cells is established in each lobe, suggesting that a spatiotemporal regulation of the lobe's histo-architecture is implemented throughout the testis. The origin and driving force of the gradient seems to be the peripheral rim of the lobe, where new cysts with proliferating spermatogonia are formed and which is therefore referred to as the germinative zone. The addition of new spermatogonial cysts results in appositional growth of the lobes and displacement of the germinative zone from the collecting duct. Further growth is achieved by the increasing cell number of the developing cysts while procession through spermatogenesis. The newly formed cysts do not appear to move during their growth/maturation while new cysts are being added from the lobe's peripheral germinative zone. The latter ceases in late January, as reflected by the disappearance of large cysts with spermatogonial cells positive for pH3. Consequently, since spermatogenesis continues at a predictable speed [27], all cysts reach the spermiation stage, and the tubules' lumina become filled with spermatozoa in a maturational wave proceeding from the central collecting duct area towards the periphery of the lobes. An akin pattern of spermiation (spermatozoa being released first from cysts near the collecting duct) was observed in the common snook (Centropomus undecimalis) [28]. The marked spatiotemporal organization of cod spermatogenesis in conjunction with the lobular composition of the spermatogenic parenchyma has not been described in other teleost fish, and it may represent a good model to study the activity of locally active factors controlling the spermatogenic process. For instance, it will be very interesting to study 1) what factors are responsible for the start of cyst formation in the germinative zone in August and its cessation in January, 2) how these hypothetical factors are regulated by the brain-pituitary system, and 3) how external factors (e.g., photoperiod) that are known to affect pubertal maturation [25] would modulate the regulation via the brain-pituitary system.
The present study is the first one describing in detail testis morphology and the histologic organization of spermatogenesis in a species from the order gadiformes, and introduces the concept of a maturational gradient in testicular lobes. The impressive overall growth of the testis represents the combined growth of several individual lobes that seem to function as independent units. Since cod are fish showing continuous growth during adult life, we anticipate that further growth of the testes in subsequent years would be realized via a caudal extension of the collecting ducts and the outgrowth of additional lobes. In salmonid fish, testis tissue also undergoes several-fold weight changes during spermatogenesis, but the spermatogenic parenchyma is organized in two compact organs and there is no predictable distribution of spermatogonial cell types along a maturational gradient [29]. Flatfish of the genus Solea do show testes where spermatogonia are located preferentially in the testicular periphery, but the testes are small and the increase in GSI during maturation of the sole testis is always very low [30].
In cod testis, all spermatids present in one cyst developed synchronously and presented the same shape and nuclear condensation. This is different from the situation in rainbow trout as described by Billard [31], who recorded certain heterogeneity during spermiogenesis within one cyst. Following spermiation, free spermatozoa were present in the gonad for a long period, from January to May, as previously described [16, 24]. After the spawning season, that is, from June, spermatogenic cysts in development were absent, and Sertoli cells phagocytized residual spermatozoa, then becoming more vesiculated. In some animals, spermatozoa were found in the testis up until the beginning of the second spermatogenic cycle, and they seemed to be eliminated later by Sertoli cells rather then being stored for the next season.
Although early spermatogonia were found preferentially in the peripheral rim of the testis lobes (germinative zone), some were also found dispersed throughout the epithelium of the seminiferous tubules, located as single cells between cysts of further advanced stages of spermatogenesis, a feature cod share with other teleosts [26, 28, 32, 33]. In our studies on proliferation, we observed that these cells were only rarely active, suggesting that these cells are quiescent and represent a reserve population.
Generally, the number of mitotic divisions of spermatogonia preceding meiosis is species specific in vertebrates [7, 34, 35]. In different teleosts, it has been found that spermatogonia divide 5 to 14 times before differentiating in spermatocytes [7, 11, 12, 33, 36–38]. The two known methods used for estimating the number of spermatogonial generations, counting the number of germ cells or measuring cyst diameter [7, 12], could not be used in cod testis. The former because the number of germ cells per cyst was too high, decreasing the confidence of the methodology, and the latter because the cysts in cod were irregularly shaped. Hence, an alternative method was developed for this study. It consisted of determining the average volume occupied by a cyst with pachytene spermatocytes, which was then divided by the average volume of a pachytene spermatocyte. With this approach, the number of mitotic divisions that spermatogonia undergo in cod was determined to be 11, which is among the highest found in vertebrates. The small difference between the theoretical (2048) and measured (1942) number of primary spermatocytes is in line with the very low incidence of apoptosis, implying that cod spermatogenesis, similar to the guppy Poecilia reticulata [11] or the zebrafish Danio rerio [39], is rather efficient among teleost fish, which in general seems more efficient than mammalian spermatogenesis [40, 41].
During the spawning season, female cod spawn 10–20 batches of eggs, with a new batch produced every 2–3 days [42]. This requires repeated mating and, therefore, a large number of spermatozoa for the external mode of fertilization. One reason that may contribute to the high spermatogenic efficiency in cod (and other fishes) is Sertoli cell proliferation, which typically accompanies the spermatogonial phase of cystic [5], in contrast with avian or mammalian, spermatogenesis. Hence, the number of Sertoli cells is adjusted to the optimum required during cystic spermatogenesis. In cod testis, Sertoli cell proliferation was observed by histone H3 phosphorylation immunohistochemistry. Although not studied systematically here, Sertoli cell proliferation was recorded mainly when these cells were associated with the first spermatogonial generations (i.e., in the periphery of the lobe, rather than when associated with meiotic or postmeiotic stages). This suggests that the germinative zone is an environment stimulating or allowing Sertoli cell proliferation.
As already mentioned, germ cell loss in cod spermatogenesis was relatively rare. However, when it occurred, it was more common during the late spermatogonial phase, particularly during the last mitotic divisions prior to spermatocyte formation. The degeneration of spermatogenic cells at the transition stage from spermatogonia to spermatocytes is frequently observed in seasonally breeding animals [43], although in some teleost fish the apoptotic incidence is highest during spermiogenesis [5, 11, 12]. Also, in the phylogenetically older dogfish (Squalus acanhtias), germ cell degeneration occurs during the differentiation to spermatocytes [44, 45], whereas this occurs during the meiotic phase in the spotted ray [46]. Interestingly, not all germ cells of a given cyst went into apoptosis, but only one to five spermatogonia in a given cyst. This situation is normally described in the literature, despite the intercellular bridges between cells originated from the same clone [47], and it indicates that it is rather a developmental problem of a specific germ cell than a problem of the complete clone or of the cyst-forming Sertoli cells. In the present study, besides the expected nuclear labeling of germ cells, Sertoli cells near apoptotic germ cells sometimes also presented TUNEL staining that was, however, a cytoplasmic staining. The latter might reflect an important and well-known task of Sertoli cells, the removal of apoptotic germ cells by phagocytosis.
In summary, cyst development occurs in a specific spatiotemporal organization during cod spermatogenesis, leading to an appositional growth of testis lobes. With 11 spermatogonial mitotic divisions and a high number of germ cells per cyst, combined with a low incidence of apoptosis, spermatogenesis in cod is one of the most efficient among teleost.
ACKNOWLEDGMENTS
The help from Fritz Kindt with composing the histology plates is highly appreciated.
FOOTNOTES
1Supported by Norwegian Research Council grant 159662/S40. 
Correspondence: 2Rüdiger W. Schulz, Department of Biology, Research Group Endocrinology & Metabolism, Faculty of Sciences, Utrecht University, Kruyt Building Room Z-203, Padualaan 8, 3584 CH Utrecht, The Netherlands. FAX: 31 30 2532837; e-mail: r.w.schulz{at}uu.nl
Received: 6 July 2007.
First decision: 6 August 2007.
Accepted: 4 September 2007.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  M. C. Leal, E. R. Cardoso, R. H. Nobrega, S. R. Batlouni, J. Bogerd, L. R. Franca, and R. W. Schulz Histological and Stereological Evaluation of Zebrafish (Danio rerio) Spermatogenesis with an Emphasis on Spermatogonial Generations Biol Reprod, July 1, 2009; 81(1): 177 - 187. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. F.L. Almeida, G. L. Taranger, B. Norberg, O. Karlsen, J. Bogerd, and R. W. Schulz Photoperiod-Modulated Testis Maturation in Atlantic Cod (Gadus morhua, L.) Biol Reprod, April 1, 2009; 80(4): 631 - 640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. d. Alvarenga and L. R. d. Franca Effects of Different Temperatures on Testis Structure and Function, with Emphasis on Somatic Cells, in Sexually Mature Nile Tilapias (Oreochromis niloticus) Biol Reprod, March 1, 2009; 80(3): 537 - 544. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
