The Female Reproductive System of Zearaja chilensis (Guichenot, 1848) (Chondrichthyes, Rajidae). Gametogenesis and Microscopic Validation of Maturity Criteria/ El Sistema Reproductor Femenino de Zearaja chilensis (Guichenot, 1848) (Chondrichthyes, Rajidae). Gametogenesis y Validacion Microscopica de los Criterios de Madurez.
Chondrichthyans are considered one of the most successful groups of animals from the evolutionary standpoint. These fish have adapted effectively over eons, resulting in a great diversity of species of rays, skates, sharks and chimeras. Their reproductive strategies, among other aspects of their life history, have made them highly susceptible to exploitation (Musick & Ellis, 2005). In the last years, the interest of fisheries worldwide has focused increasingly in elasmobranchs. This combination of factors has put these fish in a complicated situation, either from the conservational point of view as the economic (Dulvy et al, 2008). In Argentina, batoids are the most representative group of Chondrichthyans within the catches (Massa & Hozbor, 2011). This is an important topic, if we take into account that skates and rays are the most vulnerable group among cartilaginous fish, to overexploitation (Duvly & Reynolds, 2002).
The "red skate" Zearaja chilensis is an oviparous skate attaining about 120 cm in total length. Because of its large size, is highly appreciated by the fishing industry. Z. chilensis has been historically captured as by-catch (Rivera Gomez & Pettovelo, 2000) but, since 1999 it has become the target of directed fishery. Massa & Hozbor, have reported a decrease in the abundance of this species in the period between 1993 and 2005. As a result of this, Z. chilensis is considered by the IUCN Red List as "vulnerable, A4bd" (Kyne & Simpfendorfer, 2007).
Worldwide, most countries have implemented programs with a multidisciplinary approach, designed to address studies of the life history of commercially important Chondrichthyan species. Indeed, the importance of morphologic analysis on reproductive cycles has been recognized (Alonso Fernandez et al, 2011) and it has proven to be a useful tool as it provides accurate information of reproductive parameters, needed for drawing up conservation polices.
The aforementioned being considered the aim of this paper is to analyze the microscopic anatomy of the female's reproductive system of Z chilensis and to verify the accuracy of the macroscopic maturity criteria employed in the species.
MATERIAL AND METHOD
Females were collected from landings of the commercial fleet operating in the San Matias Gulf (41[degrees] 42'-42[degrees] 41' S; 63[degrees] 45'-65[degrees] 09' W). Measurements of the total length, disc width and weight of each specimen were recorded. Sexual maturity was determined by direct observation of ovaries according to Serra-Pereira et al. (2011). Small pieces of the ovaries, oviductal glands and uteri were fixed in Bouin mixture in seawater. The material was embedded in Paraplast[R], cut at 5 mm and stained with hematoxylin-eosin and Masson's trichromic stain for general observations. The periodic acid Schiff reaction and the Alcian blue technique at 2.5 pH level were employed to analyze the glycoproteins distribution. The Sudan Black technique was used to dye vitelline precursors.
The genital system of Z chilensis comprises two similarly sized ovaries, one ostium, two oviducts, two oviductal glands, two uteri and one urogenital sinus.
Ovary: Ovaries are similarly sized, elongated, dorsoventrally compressed organs, associated to the lymphomyeloid tissue known as the"epigonal organ" (Fig. 1). As ovarian maturation proceeds, the epigonal organ retracts to the caudal zone. This maturity wave occurs from cranial to caudal and from ventral to dorsal. The organ is surrounded by a simple columnar and ciliated epithelium that lies on a thin and vascularized connective tissue. The surface is folded and the epithelium acquires a pseudostratified aspect (Fig. 1). As follicles increase in number and size, the ovarian epithelium stretches. Follicles internalize according maturation progresses.
The ovarian parenchyma comprises oogonia and follicles in different stages of development (primordial, primary, developing, vitellated and atretic), all of them immersed in a loose connective tissue network. Diameter of different structures is shown in Figure 2.
Oogonia are characterized by their high nucleus: cytoplasm ratio. The nucleus is typically centric and euchromatic and the cytoplasm is slightly basophilic (Fig. 3). This cell type was found only in one sexually immature animal.
Follicles form when the oogonium associates with follicular cells. In the primordial one, the oocyte is surrounded by a monolayer of follicular squamous cells (Fig. 3). The germinal nucleus is euchromatic with heterochromatin clumps ("lampbrush chromosomes", Fig. 3). PAS reaction foregrounds the early deposit of glycoprotein patches between the oolema and follicular cells. Primary follicles characterize by a simple stratum of highly basophilic follicular cells, both cubic and columnar (Fig. 4). Interspersed between them, there are few globed-shaped cells with a slightly basophilic cytoplasm and a round euchromatic nucleus (Fig. 4). The oolema is densely folded and looks as a refractive line (Fig. 4). The zona pellucida is well defined and surrounds the entire oocyte (Fig. 4). This zone stains positively with PAS but there is no reaction to the AB technique.
According maturation progress, the follicle increases in size, the zona pellucida thickens and the thecas differentiate into an inner one, formed by eosinophilic squamous cells and an outer, comprising one or two layer of squamous basophilic cells (Fig. 5A).
The progression of the follicular maturation process implies the increase in thickness of the zona pellucida, the stratification of the follicular envelope and the formation of a vascular network between thecas (Fig. 5A). The follicular epithelium depicts two cellular types: the columnar eosinophilic cells and the globed-shaped ones. The latter show a basophilic cytoplasm and granules that stain with Sudan B (Fig. 5A). Granulosa cells and oolema interact through thin projections running through the zona pellucida (Fig. 5B).
Yolk accumulation occurs in a centripetal pattern (Fig. 6). Initially, small acidophilic granules appear underneath the plasmalema. Then, granules coalesce to form elliptic plates (Fig. 6). Microscopically, the yolk input begins when follicle attain about 1400 micrometer. Nevertheless, the macroscopic evidence of yolk accumulation (yellowish coloration) appears when follicles are up to 1.2 cm. The largest follicular diameter recorded was of 5.2 cm.
Atretic bodies were present in all studied fish. As the oocyte retracts, the follicular cells collapse. At the beginning of the process, follicular cells vacuolated and show clear evidences of apoptosis (Fig. 7). Finally, the connective tissue infiltrates the area, forming a scare-like structure (Fig. 7).
No evidence of corpora lutea was found.
Oviductal gland: The oviductal gland comprises a set of tubular glands, arranged in different zones according their morphology and dye affinity (Figs. 8-10). The lumen is coated by a simple columnar ciliated epithelium. Adenomers contain columnar ciliated supporting cells and glandular cells. Based on the nature of the secretion, it is possible to differentiate three zones (Figs. 8-10): a cranial mucous zone, a medium protein-producer zone and a mixed caudal zone. The dye affinity of each region is shown in Table I.
Uterus: The uterus of Z. chilensis comprises a folded mucosa lined by a simple, basophil, ciliated epithelium and with a highly vascularized chorion (Figs. 11-13). The complexity of the folds increases towards the urogenital sinus with the formation of branched villi whose epithelium flattens (Fig. 13). Epithelial cells show apical granules that react positively to PAS and AB 2.5. The muscular layer arranges in orthogonal sheets. The serosa is formed by a simple squamous or cubic epithelium (Fig. 11).
The plan of organization of cartilaginous fish is a highly conserved model throughout more than 400 million years (McEachran & Dunn, 1998). Within this scheme, the female reproductive tract is relatively similar throughout the group (McMillan, 2007). Zearaja chilensis is not an exception, presenting symmetrical functional ovaries and related organs.
The organization of the germinative epithelium of Z. chilensis seems to be an adaptation that allows distention and has also been recorded in other species (Galindez et al, 2014). During embryonic development, primordial germ cells originate in the vitelline endoderm and migrate to the germinal ridge, where they begin to multiply and differentiate into oogonia. The presence of oogonia has also been recorded in other Chondrichthyans. In some cases, the presence of these cells has been observed even in exemplars that were close to hatching size (Diaz-Andrade et al. 2009). Likewise, this cell type may be present in maturing females (Diaz-Andrade et al., 2011; Galindez et al., 2014). The reproductive potential of species rests, among other things, in the number of primordial germ cells that can be recruited as gametes. So, the presence of oogonia only in the first stages of sexual maturation could be associated to a low reproductive capacity. However, the data here exposed regarding this issue are in a preliminary stage and are presented only for informational purposes. Further studies are being performed to define more accurately the reproductive potential of this species.
The process of development and maturation of the follicles is called foliculogenesis. In Z. chilensis, the follicular epithelium stratifies during this process and once it does, it remains unchanged. This pattern is also observed in other batoids (Prisco et al. 2002). The presence of different follicular cells has also been reported in other Rajids (Diaz Andrade et al., 2009, 2011; Hamlett et al., 1999; Prisco et al., 2002). Moreover, Prisco et al. (2002) have reported at least three different types of follicular cells in Torpedo marmorata, distinguishable by their ultraestructural features. However, a more profound analysis, at an ultrastructural and cytochemical study, is needed for a more accurate typing of follicular cells in the red skate.
The zona pellucida of most vertebrates is an acellular, thin layer, composed by glycoproteins and produced by both, the follicular cells and the oocyte. In cartilaginous fish, this structure has an atypical dynamic, showing a drastically width increases even just at the beginning of yolk input (Davenport et al. 2011). The onset of the zona pellucida" s deposition in Z. chilensis starts at the beginning of folliculogenesis, in the primordial follicle. This is not the common pattern, so that in other species this acelular coat deposits at the primary follicle step (Hamlett et al., 1999). The chemical nature of the zona pellucida indicates the presence of neutral glycoproteins and the absence of acid mucins. These behaviors are common to most studied Chondrichthyan and differ from those exhibits for the genus Sympterygia (Diaz-Andrade et al., 2009, 2011).
The relationship between the oocyte and the follicular cells is not only crucial for yolk input but also for their modulation (Hutt & Albertini, 2007). In Z. chilensis the presence of cellular interactions between both cell types seems clear. However, the material employed for this work was stored in cold chambers for several days, so that structural details may have not been correctly preserved, making difficult an accurate conclusion.
Like other Chondrichthyans (Prisco et al., 2007), Z. chilensis exhibits a double theca. The connective nature of the inner one and the high vascularization at the beginning of vitellogenesis leads to assume that is involved in the transport of yolk precursors. The outer theca depicts cuboidal secreting cells, probably with a steroidogenic capacity, as it has been recorded in other species (Prisco et al., 2002).
According follicles mature there is an increase in size and structural complexity. The yolk input and accumulation provides sustenance for subsequent embryonic development. In Z. chilensis there is a clear evidence of precursor's translocations by means of granulosa cells, both columnar and globed-shaped cells. This has also been recorded in other species (Prisco et al., 2002; Diaz-Andrade et al., 2009).
Atretic follicles result from the involution of the oocyte and its resorption by phagocytosis, and can be triggered at any stage of follicular development (Hisaw & Hisaw, 1959). In the red skate, atretic follicles were observed in animals of all maturational stages. The atretia rate is an important parameter for the evaluation of ovarian fecundity in fish, so that more studies are needed in commercially important species, such as Z. chilensis.
Different methods are used to determine the sexual maturity stage in cartilaginous fish. Usually, the criteria are, essentially, macroscopic. In females, the diameter and color of "eggs", the overall appearance of the uterus and oviducal glands and the presence of eggs or embryos are used as indicators (Serra-Pereira et al.; Stehmann, 2002). In Z. chilensis there is a significant difference between the onset of vitellogenesis noticeable at a microscopic level and that seen at naked eye. If we take this into account, it seems clear that considering only macroscopic criteria to evaluate the maturity stage in a fish population could lead to overestimate the size at first sexual maturity and therefore, underestimate the potential of the resource. Although the histological work may seem cumbersome and expensivein a fishery management context, the quality of information provided and the accuracy in the estimation of sexual maturity is invaluable and may contribute to fine-tuning of policies to apply regarding the catches.
Once the oocyte is released from the ovary it is collected by the ostium, passes through the oviduct and arrives to the oviductal gland, which is a conglomerate of tubular glands that secrete the third egg envelopes. The three zones referred in this work correspond with the four fundamental zones reported in the oviductal gland of most elasmobranchs. In the red skate, the mucous-secreting zone corresponds to the club and papillary and the protein-producing zone is homologous to the baffle zone. On the other hand, the terminal zone, with a mixed secretion, matches the same-named zone of other species. In general, the oviductal gland is similar in all studied elasmobranchs, with some few exceptions (Hamlett et al., 1998). Considering that the function of the oviductal gland is to secrete the wrappers that protect the egg, it is clear that yolk sac species are those which exhibit the most complex organization and Z. chilensis is not an exception to this.
The general microscopic organization of the uterus of Z. chilensis agrees with that recorded in other elasmobranchs (Diaz-Andrade et al, 2013; Galindez et al, 2010). In oviparous species, the uterus houses the egg capsule during its sclerotization and thereafter, until oviposition. The uterus structure in these species is quite simple and leads to the assumption of a secondary role. However, the vascular net located below the lining epithelium indicates that this organ does not serve only as a container, but it fulfils an active role in the process of metabolites exchange between the embryo and the mother, the supply of oxygen necessary for the capsule's sclerotization and in the transport of the capsule through the uterus. This fact has also been recorded in other oviparous species (Diaz-Andrade et al., 2013).
Elasmobranchs possess a life history completely different from bony fish. These features make them more susceptible to environmental disturbances and fishing pressure (Field et al., 2009). Z. chilensis is an important economic resource in Argentina and their fins are highly prized and demanded in the Asian market (Paesch & Oddone, 2008). The directed fishing that this species has undergone in the last years has exerted an important pressure over the resource, evidenced mainly by the decrease in abundance and in the size at first maturity recorded in the last 20 years (Paesch & Oddone). All these factors make necessary an integral analysis of the life history of the red skate, before population's decline becomes irreversible.
In the last decades, Elasmobranchs have become the center of discussion in the fisheries community worldwide, so that Argentina, such as other fishing countries, has implemented a National Plan of Action for the Conservation and Management of sharks, rays and chimaeras (PAN--Sharks, Federal Fisheries Council, Res No. 6/2009). This work contributes to its guidelines, increasing the knowledge of a valuable species. In this context, the histological analysis of the reproductive tract, not only allows clarifying the mechanisms governing the reproductive cycles, but also provides significant information for sustainable fisheries management.
ACKNOWLEDGEMENTS. We thank the Fish Plant "Rio Salado" for providing us the specimens and the CONDROS Group of IMPAS for your cooperation. Special thanks go to Lic. Carolina Moya for collaborating in the sampling and Dr. Maria Constanza Diaz Andrade for your collaboration.
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Laboratorio de Histologia Animal
Departamento de Biologia
Bioquimica y Farmacia
Universidad Nacional del Sur
San Juan 670
Bahia Blanca, 8000
Anahi Wehitt *; Edgardo E. Di Giacomo **,*** & Elena J. Galindez *,**
* Laboratorio de Histologia Animal, Departamento de Biologia, Bioquimica y Farmacia, Universidad Nacional del Sur, Buenos Aires, Argentina.
** Instituto de Biologia Marina y Pesquera "Alte. Storni" (CONDROS).
*** Universidad Nacional del Comahue, Buenos Aires, Argentina.
This work was supported by the SGCyT-UNS, PGI 24/B173.
Caption: Fig. 1. A. General view of the ovary of a sexually immature female. F= follicle; eo= epigonal organ. Hematoxiline-eosine. Scale bar: 160 [micro]m. B. Ovarian wall. Arrow indicates the folded germinative epithelium; cf= follicular cells; o= oocyte. Masson's Trichromic stain. Scale bar: 20 [micro]m.
Caption: Fig. 2. Diameter of oogonia and follicles. Note that, since the beginning of meiosis, growth seems to be more widespread and appears to be slightly lower speed. However, there is insufficient information to statistically compare the slopes. Ordinate is in logarithmic scale.
Caption: Fig. 3. Oogonia and primordial follicle formation. Note the flat simple follicular epithelium (large arrows) and the incipient zona pellucida (short arrow). Asterisk indicates the "lampbrush chromosomes"; n= nucleus; oo= oogonia. Masson's Trichromic stain. Scale bar: 10 [micro]m.
Caption: Fig. 4. Primary follicle. Arrow head depicts the folded oolema abutting against the zona pellucida (asterisk); arrow depicts the theca; cu= cubic cells; gc= globed-shaped cells. Masson's Trichromic stain. Scale bar: 30 [micro]m.
Caption: Fig. 5. A. High magnification image of a stratified follicular epithelium. Note the cytoplasmic stain affinity of globed-shaped cells (asterisk); co= columnar cells; bv= blood vessel; it= inner theca; ot= outer theca. Sudan Black stain. Scale bar: 15 [micro]m. B. Arrow indicates the thin projections of the follicular cells. Masson's Trichromic stain. Scale bar: 15 [micro]m.
Caption: Fig. 6. Vitellating follicle; bv= blood vessel; co: columnar cells; ep= elliptic plate; gc= globed-shaped cells; it= inner theca; ot= outer theca; yg= yolk granules. Masson's Trichromic stain. Scale bar: 30 [micro]m.
Caption: Fig. 7. A. General view of a recent forming atretic body; ct= connective tissue; eo= epigonal organ; vc= vacuolated cells. Masson's Trichromic stain. Scale bar: 35 mm. B. Vacuolated follicular cells (asterisk). Masson's Trichromic stain. Bar 25 [micro]m.
Caption: Fig. 8. Oviductal gland. Mucous cranial zone (M) and protein zone (P). Note the cilia of the support cells (arrow) and nuclei of glandular cells (arrow heads); asterisk: protein granulation. Masson's Trichromic stain. Scalebar: 10 [micro]m.
Caption: Fig. 9. Oviductal gland. Glands of the mixed zone. Note the secretion in the lumen of the glandular ducts (asterisks) and the cilia (arrow); m: mucous adenomer; s: serous adenomer. Masson's Trichromic stain. Scale bar: 10 [micro]m.
Caption: Fig. 10. Oviductal gland. High magnification image of the mixed zone; d= duct; m= mucous adenomer; s= serous adenomer. Masson's Trichromic stain. Scalebar: 10 [micro]m.
Caption: Fig. 11.General view of the longitudinal section of the uterus. Arrow indicates the lining epithelium and arrow head shows the serosa; bv= blood vessel; ch= chorion; im= inner muscular sheet; om= outer muscular sheet. Scale bar: 150 [micro]m.
Caption: Fig. 12. Uterus. High magnification image of the folded mucosa showing the mucous epithelial surface (arrows); bv= blood vessel. Masson's Trichromic stain. Scale bar: 20 [micro]m.
Caption: Fig. 13. Uterus. General view of the mucosa showing the branched villi. Note the high vascularization of the chorion; bv= blood vessel; ch= chorion. Masson's Trichromic stain. Scale bar: 100 [micro]m.
Table I. Glycosaminoglycans reactivity of the oviductal gland. Zone Characteristics AB 2.5 PAS/AB 2.5 Mucous Simple tubular glands + Light fuchsia cranial zone Ramified tubular glands. + Fuchsia Simple tubular glands. ++ Violet and Fuchsia Protein Simple tubular glands. - -- zone With spinnerets Mixed Ramified tubular glands. +++ (dc) Light blue (mc) zone Duct with mucous cells. Adenomers with a mixture ++ (mc) of mucous and serous cells. - (sc) dc= duct cells; mc= mucous cells; sc= serous cells.