Ángel García-López

Sex steroids and metabolic parameter levels in a seasonal breeding fish (Sparus aurata L.)

The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the two first reproductive cycles (RCs), in which the specimens develops as males. During the first RC (RC1), 11-ketotestosterone (11-KT) and testosterone (T), the main androgens in fish, play different and specific roles in the testicular physiology as they peak at different stages of RC1. Moreover, the profiles of T serum levels during the second RC (RC2) demonstrated that this androgen is not essential in the testicular regression process that occurs during this cycle. However, changes in serum levels suggest that 17beta-estradiol (E2) orchestrates this process during both RCs. Moreover, the E2 serum levels recorded during RC2 indicate that there is a threshold level that determines the initiation of ovarian development during this cycle without promoting complete feminization. We analysed triglyceride, protein, glucose and lactate serum levels in order to establish a relationship between the mobilization and transfer of nutrients, the hormonal changes that take place during the RC and body composition, finding that in vivo serum levels of metabolites change significantly throughout the first two RCs, although the physiological relevance of such changes is still unknown. Triglyceride levels seem to be affected by the beginning of ovary development during RC2 but not by testicular recrudescence during RC1. Moreover, glucose and lactate might be important sources of energy during the resting and testicular involution stages.

Targeted gene expression profiling in European sea bass (Dicentrarchus labrax, L.) follicles from primary growth to late vitellogenesis

A real-time PCR-based gene expression survey was performed on isolated European sea bass follicles from primary growth to late vitellogenesis. Expression levels of 18 transcripts with demonstrated relevance during oogenesis, encoding gonadotropin, thyrotropin, estrogen, androgen, and vitellogenin receptors, steroidogenesis-related as well as growth and transcription factors were measured. Primary oocytes showed high mRNA levels of insulin-like growth factors 1 and 2, bone morphogenetic protein 4, estrogen receptor 2b, androgen receptor b, and SRY-box containing gene 17 together with low transcript amounts of gonadotropin receptors. Follicles at the lipid vesicles stage (i.e., the beginning of the secondary growth phase) showed elevated mRNA amounts of follicle stimulating hormone receptor (fshr) and anti-Mullerian hormone. Early-to-mid vitellogenic follicles showed high mRNA levels of fshr and cytochrome P450, family 19, subfamily A, polypeptide 1a while mid-to-late vitellogenic follicles expressed increasing transcript amounts of luteinizing hormone/choriogonadotropin receptor, steroidogenic acute regulatory protein, and estrogen receptors 1 and 2a. The molecular data presented here may serve as a solid base for future studies focused on unraveling the specific mechanisms orchestrating follicular development in teleost fish.

Endocrine and milt response of Senegalese sole, Solea senegalensis, males maintained in captivity

Improving fertilization success in captive Senegalese sole broodstocks has been a challenge in the last years. Recent reports suggest that low sperm volume and quality could be one of the reasons leading to poor fertilization rates, although further studies are needed to reach a conclusive explanation. Here, we report on several experiments focused on this issue. Seasonal profiles of plasma androgen levels (testosterone and 11-ketotestosterone) and sperm production and quality parameters were assessed, although no statistical correlations among them were identified. The response of males to female presence/absence was also analyzed. Long-term isolation from females decreased male androgen levels at the peak of the reproductive period, suggesting some kind of disrupting effects on the endocrine system. On the other hand, short-term exposure of previously isolated males to ripe females decreased androgen levels, possibly reflecting a rapid steroidogenic shift promoting final maturation of spermatozoa, and increased sperm viability, motility and velocity, thus, supporting the concept of positive effects of female contact on male sole performance. Further evidence sustaining the relevant female-to-male communication in sole reproduction was obtained after treating the females with progestagen 17α,20β-dihydroxy-4-pregnen-3-one (regarded as pre-ovulatory pheromone in fish) and registering a significant increase in sperm viability, velocity and motility in surrounding males. Finally, we found that a single administration of a 20 μg/kg GnRH analogue in males was effective in stimulating androgen release and sperm quality, although the effects were transient and thus, the use of sustained hormone delivery methods were suggested for improving efficiency. Our results point to velocity, viability, and motility as the most sensitive parameters in sole sperm, although further studies will have to evaluate whether these parameters have any relation with fertilization success in captive broodstocks of this important aquaculture species.

Bone morphogenetic protein 15 and growth differentiation factor 9 expression in the ovary of European sea bass (Dicentrarchus labrax): Cellular localization, developmental profiles, and response to unilateral ovariectomy

Vertebrate oocytes actively contribute to follicle development by secreting a variety of growth factors, among which bone morphogenetic protein 15 (BMP15/Bmp15) and growth differentiation factor 9 (GDF9/Gdf9) have been paid particular attention. In the present study, we describe the cellular localization, the developmental profiles, and the response to unilateral ovariectomy (a procedure implying the surgical removal of one of the ovaries) of protein and mRNA steady-state levels of Bmp15 and Gdf9 in the ovary of European sea bass, an important fish species for marine aquaculture industry. In situ hybridization and immunohistochemistry demonstrated that the oocyte is the main production site of Bmp15 and Gdf9 in European sea bass ovary. During oocyte development, Bmp15 protein expression started to be detected only from the lipid vesicle stage onwards but not in primary pre-vitellogenic (i.e. perinucleolar) oocytes as the bmp15 mRNA already did. Gdf9 protein and gdf9 mRNA expression were both detected in primary perinucleolar oocytes and followed similar decreasing patterns thereafter. Unilateral ovariectomy induced a full compensatory growth of the remaining ovary in the 2-month period following surgery (Á. García-López, M.I. Sánchez-Amaya, C.R. Tyler, F. Prat 2011). The compensatory growth elicited different changes in the expression levels of mRNA and protein of both factors, although the involvement of Bmp15 and Gdf9 in the regulatory network orchestrating such process remains unclear at present. Altogether, our results establish a solid base for further studies focused on elucidating the specific functions of Bmp15 and Gdf9 during primary and secondary oocyte growth in European sea bass.

Mechanisms of oocyte development in European sea bass (Dicentrarchus labrax L.): investigations via application of unilateral ovariectomy

Unilateral ovariectomy (ULO) was performed in European sea bass (Dicentrarchus labrax L.) during late pre-vitellogenesis/early vitellogenesis. Plasma steroid levels and the expression of a suite of potential oogenesis-relevant genes in the ovary, brain, and pituitary were evaluated with the aim of understanding their involvement in the compensatory oocyte development occurring within the remaining ovarian lobe. After 69 days of surgery the remaining ovarian lobe in ULO fish was gravimetrically equivalent to an intact-paired ovary of sham operated, control fish. This compensatory ovarian growth was based on an increased number of early perinucleolar oocytes and mid-late stage vitellogenic follicles without an apparent recruitment of primary oocytes into the secondary growth phase. Plasma steroid levels were similar in ULO and control females at all time points analyzed, suggesting an increased steroid production of the remaining ovarian lobe in hemi-castrated females. Results of the gene expression survey conducted indicate that the signaling pathways mediated by Fsh and Gnrh1 constitute the central axes orchestrating the observed ovarian compensatory growth. In addition, steroid receptors, Star protein, Igfs, and members of the transforming growth factor beta superfamily including anti-Mullerian hormone and bone morphogenetic protein 4 were identified as potentially relevant players within this process, although their specific actions and interactions remain to be established. Our results demonstrate that ULO provides an excellent in vivo model for elucidating the interconnected endocrine and molecular mechanisms controlling oocyte development in European sea bass.

Lectin-binding pattern of the senegalese sole Solea senegalensis oogenesis

The glycoconjugate pattern of developing ovarian follicles in wild and cultured Senegalese sole Solea senegalensis was investigated by means of lectin histochemistry. Ovaries from cultured fish contained oocytes up to the late vitellogenic stage, whereas they reached the hydration stage in wild specimens. The follicular cells bound MAL II, SBA, HPA, DBA, Con A, KOH‐sialidase (K‐s)‐WGA, GSA I‐B4 in the late vitellogenic stage, and in wild fish also SNA and K‐s‐PNA, whereas in the hydration stage SBA, HPA, DBA, and GSA I‐B4 only. The zona radiata reacted with SBA, HPA, DBA, Con A, and GSA I B4 in the late vitellogenic stage and in cultured fish also with UEA I, whereas in the hydration stage it stained with SBA only. The cortical alveoli bound SBA, HPA, RCA120 during the late vitellogenic stage, also SNA, PNA, K‐s‐PNA, GSA I‐B4 in cultured fish, DBA, and K‐s‐WGA in wild ones which stained with SBA, HPA, and GSA I‐B4 in the hydrated stage. The yolk reacted with Con A in the late vitellogenic oocytes, and also with MAL II, SNA, K‐s‐PNA, SBA, HPA, K‐s‐WGA, GSA I‐B4, UEA I in the hydrated ones. From perinucleolus to late vitellogenic stages, the oocyte nucleoplasm bound Con A, GSA I‐B4, GSA II, UEA I, and in wild fish also MAL II, SNA, LTA but only GSA I‐B4 reactivity in the early maturation stage. These findings demonstrate that the glycan pattern of fish ovarian follicles changes during the maturative stages and that it is affected by culture‐rearing conditions. Microsc. Res. Tech. 75:1124–1135, 2012.