Reynaldo Patiño

Ovarian follicle growth, maturation, and ovulation in teleost fish

Knowledge of ovarian follicle growth, maturation, and ovulation in teleosts is important to many aspects of basic and biomedical research, fisheries management, aquaculture, and environmental science. Follicle growth includes previtellogenic and vitellogenic stages. Although little progress has been made in our understanding of previtellogenic development, information on the regulation and mechanisms of vitellogenic growth has increased substantially in recent years. This new information pertains to the multiplicity of vitellogenin (Vg) genes, the oocyte Vg receptor, Vg processing enzymes (cathepsins) and their specific yolk products, and synthesis of various vitelline envelope precursor proteins in liver and ovary. Oocyte acquisition of lipids from sources other than Vg dominates follicle growth in many teleosts, especially perciformes, yet our understanding of the basic processes involved remains rudimentary. Considerable advances also have been made in our understanding of the endocrine regulation and mechanisms of ovarian follicle maturation. We have learned that ovarian follicle maturation involves a number of events including luteinizing hormone (LH)-dependent acquisition of oocyte maturational competence, LH induction of maturation-inducing hormone (MIH) synthesis, and MIH-dependent meiotic resumption (nuclear maturation) and cytoplasmic maturation. While much has been learned about mechanisms of maturational competence, MIH synthesis and meiotic resumption, our knowledge of cytoplasmic maturation is limited to descriptions of MIH-dependent yolk protein hydrolysis and associated oocyte hydration in a few species. It has become apparent that ovulation requires genomic activation regulated by nuclear MIH receptors, but the transduction pathways for the ovulatory MIH signal appear to be complex and are poorly understood.

SEX DIFFERENTIATION OF CHANNEL CATFISH GONADS : NORMAL DEVELOPMENT AND EFFECTS OF TEMPERATURE

Channel catfish have an XX female-XY male sex determination system. Although genetic males of this species are readily feminized with exogenous steroid, the pattern of gonadal sex differentiation and the effect of temperature on genetic sex determination are unknown. To document the pattern of gonadal sex differentiation, catfish from a mixed-sex progeny (XX female-XY made cross), known XY males (XX female-YY male cross), and steroid sex-reversed (XY) females were reared at 28°C and collected for histological analysis from Day 7 to Day 90 postfertilization. No signs of gonadal sex differentiation were detected from Day 7 to Day 16. On Day 19, about half the fish from the mixed-sex population as well as the sex-reversed females had gonads with proximal and distal tissue outgrowths. These outgrowths eventually fused to form an ovarian cavity. Germ cell meiosis in these gonads was first detected on Day 22 and growing ovarian follicles were seen at later stages. Thus, ovarian differentiation began about Day 19. Although presumptive testes lose their sensitivity to feminizing steroids about Day 19, the gonads of known males and of putative males from the mixed-sex population showed no clear signs of sex differentiation up to Day 90. Thus, the prolonged temporal dissociation between testicular commitment and differentiation indicates that these two events may be mediated by separate stimuli. To determine the effects of temperature on sex determination, fish of a mixed-sex population were reared at 27°C until Day 10; at 20°, 27°, or 34°C from Day 10 to Day 24; and then at 27°C until Day 102 when sex ratios and gonadal histology were determined. The sex ratio was significantly skewed toward females in fish treated at 34°C (1 male:1.68 female; P < 0.01) but no effects were seen at 20° or 27°C. This observation suggests that genetic and temperature-dependent mechanisms of sex determination are both functional in channel catfish and that this phenomenon is more widespread in fishes than previously believed. The testes of the Day 102 males examined had developed positive histological signs of sex differentiation, suggesting that testicular formation in channel catfish normally starts between Day 90 and 102.

The hypothalamus–pituitary–thyroid axis in teleosts and amphibians: Endocrine disruption and its consequences to natural populations

Teleosts and pond-breeding amphibians may be exposed to a wide variety of anthropogenic, waterborne contaminants that affect the hypothalamus-pituitary-thyroid (HPT) axis. Because thyroid hormone is required for their normal development and reproduction, the potential impact of HPT-disrupting contaminants on natural teleost and amphibian populations raises special concern. There is laboratory evidence indicating that persistent organic pollutants, heavy metals, pharmaceutical and personal care products, agricultural chemicals, and aerospace products may alter HPT activity, development, and reproduction in teleosts and amphibians. However, at present there is no evidence to clearly link contaminant-induced HPT alterations to impairments in teleost or amphibian population health in the field. Also, with the exception of perchlorate for which laboratory studies have shown a direct link between HPT disruption and adverse impacts on development and reproductive physiology, little is known about if or how other HPT-disrupting contaminants affect organismal performance. Future field studies should focus on establishing temporal associations between the presence of HPT-disrupting chemicals, the occurrence of HPT alterations, and adverse effects on development and reproduction in natural populations; as well as determining how complex mixtures of HPT contaminants affect organismal and population health.

Manipulations of the Reproductive System of Fishes by Means of Exogenous Chemicals

Abstract Environmental control of reproductive activity of captive fish is feasible (or potentially feasible) but, with few exceptions, is currently impractical for most species. Therefore, chemical methods of manipulating reproductive activity continue to be widely used in fish production operations worldwide. However, the control of fish reproduction in captivity cannot be exercised without regard to adequate environmental conditions, which can differ markedly for different species. This review provides a synopsis of relevant aspects of fish reproductive physiology and addresses current and promising future chemical methods of sex control, gonadal recrudescence, and spawning. Most research on the control of reproduction in fishes has focused on female physiology because ovarian development and maturation are easily disturbed by environmental stressors. Control of sex ratios by steroid treatment has become a well-established technique for several fish species, but the technique continues to be problemati...

Role of Arachidonic Acid and Protein Kinase C During Maturation-Inducing Hormone-Dependent Meiotic Resumption and Ovulation in Ovarian Follicles of Atlantic Croaker

Abstract The roles of arachidonic acid (AA) and protein kinase C (PKC) during in vitro maturation-inducing hormone (MIH)-dependent meiotic resumption (maturation) and ovulation were studied in ovarian follicles of Atlantic croaker (Micropogonias undulatus). The requirement for cyclooxygenase (COX) metabolites of AA was examined using a nonspecific COX inhibitor, indomethacin (IM), as well as two COX products, prostaglandin (PG) F2α and PGE2, whereas the role of lipoxygenase (LOX) was investigated using a specific LOX inhibitor, nordihydroguaiaretic acid (NDGA). The involvement of PKC was examined using phorbol 12-myristate 13-acetate (PMA), a PKC activator, as well as GF109203X (GF), a specific inhibitor of PKC and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), nonspecific inhibitor of protein kinases. Genomic mechanisms were examined with the transcription-inhibitor actinomycin D (ActD) and the functionality of heterologous (oocyte-granulosa) gap junctions (GJ) with a dye transfer assay. The AA (100 μM) and PGF2α (5 μM) did not induce maturation, and NDGA (10 μM) did not affect MIH-dependent maturation. However, IM (100 μM) partially inhibited MIH-dependent maturation. Conversely, AA and both PGs induced, and IM and NDGA inhibited, MIH-dependent ovulation in matured follicles. The PMA (1 μg/ml) did not induce maturation but caused ovulation in matured follicles, whereas PKC inhibitors (GF, 5 μM; H7, 50 μM) did not affect MIH-dependent maturation but inhibited MIH- and PMA-dependent ovulation. The PMA-dependent ovulation was inhibited by IM but not by NDGA. In addition, ActD (5 μM) blocked MIH-dependent, but not PMA-dependent, ovulation, and PGF2α restored MIH-dependent ovulation in ActD-blocked follicles. The AA and PGs did not induce, and GF did not inhibit, MIH-dependent heterologous GJ uncoupling. In conclusion, AA and PKC mediate MIH-dependent ovulation but not meiotic resumption or heterologous GJ uncoupling in croaker follicles, but a permissive role of COX products of AA during maturation is possible. A novel model of MIH-dependent ovulation is proposed in which 1) LOX and COX metabolites of AA are both required for ovulation, but at upstream and downstream sites of the pathway, respectively, relative to PKC, and 2) PKC is downstream of genomic activation.

Morphometric and Histopathological Parameters of Gonadal Development in Adult Common Carp from Contaminated and Reference Sites in Lake Mead, Nevada

Abstract This study examined the hypothesis that exposure to sublethal concentrations of contaminants alters the gonadal condition of feral common carp Cyprinus carpio. Adult common carp in Lake Mead, Nevada, were collected from a contaminated site (Las Vegas Bay) that receives municipal and industrial effluent and from a reference site (Overton Arm) with a relatively low level of contamination. Fish were sampled seven times over a 1-year period extending over two separate spawning seasons. Morphometric and histopathological parameters of gonadal and germ cell development were determined. In males, the pattern of seasonal changes in the gonadosomatic index (GSI) was similar between the sites and showed no clear association with site-specific seasonal temperature profiles. However, Las Vegas Bay males had consistently lower GSI values and, on one of the sampling dates, a lower proportion of sperm relative to other germ cell stages (determined histologically). Further, Las Vegas Bay males had a higher incid...

NOVEL BIOMARKERS OF PERCHLORATE EXPOSURE IN ZEBRAFISH

Perchlorate inhibits iodide uptake by thyroid follicles and lowers thyroid hormone production. Although several effects of perchlorate on the thyroid system have been reported, the utility of these pathologies as markers of environmental perchlorate exposures has not been adequately assessed. The present study examined time-course and concentration-dependent effects of perchlorate on thyroid follicle hypertrophy, colloid depletion, and angiogenesis; alterations in whole-body thyroxine (T4) levels; and somatic growth and condition factor of subadult and adult zebrafish. Changes in the intensity of the colloidal T4 ring previously observed in zebrafish also were examined immunohistochemically. Three-month-old zebrafish were exposed to ammonium perchlorate at measured perchlorate concentrations of 0, 11, 90, 1,131, and 11,480 ppb for 12 weeks and allowed to recover in clean water for 12 weeks. At two weeks of exposure, the lowest-observed-effective concentrations (LOECs) of perchlorate that induced angiogenesis and depressed the intensity of colloidal T4 ring were 90 and 1,131 ppb, respectively; other parameters were not affected (whole-body T4 was not determined at this time). At 12 weeks of exposure, LOECs for colloid depletion, hypertrophy, angiogenesis, and colloidal T4 ring were 11,480, 1,131, 90, and 11 ppb, respectively. All changes were reversible, but residual effects on angiogenesis and colloidal T4 ring intensity were still present after 12 weeks of recovery (LOEC, 11,480 ppb). Whole-body T4 concentration, body growth (length and weight), and condition factor were not affected by perchlorate. The sensitivity and longevity of changes in colloidal T4 ring intensity and angiogenesis suggest their usefulness as novel markers of perchlorate exposure. The 12-week LOEC for colloidal T4 ring is the lowest reported for any perchlorate biomarker in aquatic vertebrates.

Regulation of gonadal sex ratios and pubertal development by the thyroid endocrine system in zebrafish (Danio rerio).

We examined associations between thyroid condition, gonadal sex and pubertal development in zebrafish. Seventy-two-hour postfertilization larvae were reared in untreated medium or in the presence of goitrogens (sodium perchlorate, 0.82 mM; methimazole, 0.15 and 0.3 mM) or thyroxine (1 and 10 nM) for 30 days. Thyrocyte height, gonadal sex and gonadal development were histologically determined at 45 and 60 days postfertilization (dpf). Thyrocyte hypertrophy, an index of hypothyroidism, was observed at 45 and 60 dpf in perchlorate-treated but only at 45 dpf in methimazole-treated fish. Similarly, gonadal sex ratios were biased toward ovaries relative to control animals at 45 and 60 dpf in perchlorate-treated fish but only at 45 dpf in methimazole-treated fish. Gonadal sex ratios were biased toward testes at 45 and 60 dpf in thyroxine-treated fish. Spermatogenesis was delayed in testes from goitrogen-treated fish at 60 dpf relative to control values, but was unaffected in testes from thyroxine-treated individuals. Oogenesis seemed to be nonspecifically delayed in all treatments relative to control at 60 dpf. This study confirmed the previously reported association between hypothyroid condition and ovarian-skewed ratios, and hyperthyroid condition and testicular-skewed ratios, and also showed that male pubertal development is specifically delayed by experimental hypothyroidism. The simultaneous recovery from the hypothyroid and ovary-inducing effects of methimazole by 60 dpf (27 days post-treatment) suggests that the ovary-skewing effect of goitrogens is reversible when thyroid conditions return to basal levels before developmental commitment of gonadal sex. Conversely, the masculinizing effect of hyperthyroidism seems to be stable and perhaps permanent.

Ovarian follicle maturation and ovulation: An integrated perspective

Numerous studies with teleosts have addressed the regulation and mechanisms of oocyte maturation, but largely at the exclusion of ovulation. A smaller but still considerable number of studies have focused on ovulation, and ignored maturation. Consequently, little is known about the mechanistic linkages between these two events. New information is presented here indicating that luteinizing hormone regulates the acquisition not only of oocyte maturational competence, but also ovulatory competence. The thesis is presented that maturation and ovulation are closely integrated and overlapping events that are best viewed conceptually and experimentally as parts of a functional whole.

Spatial and temporal patterns of surface water quality and ichthyotoxicity in urban and rural river basins in Texas

The Double Mountain Fork Brazos River (Texas, USA) consists of North (NF) and South Forks (SF). The NF receives urban runoff and twice-reclaimed wastewater effluent, whereas the SF flows through primarily rural areas. The objective of this study was to determine and compare associations between standard water quality variables and ichthyotoxicity at a landscape scale that included urban (NF) and rural (SF) sites. Five NF and three SF sites were sampled quarterly from March 2008 to March 2009 for specific conductance, salinity, hardness, pH, temperature, and turbidity; and a zebrafish (Danio rerio) embryo bioassay was used to determine ichthyotoxicity. Metal and nutrient concentrations at all sites were also measured in addition to standard water quality variables in spring 2009. Principal component analyses identified hardness, specific conductance, and salinity as the water variables that best differentiate the urban NF (higher levels) from rural SF habitat. Nutrient levels were also higher in the NF, but no landscape scale patterns in metal concentrations were observed. Ichthyotoxicity was generally higher in NF water especially in winter, and multiple regression analyses suggested a positive association between water hardness and ichthyotoxicity. To test for the potential influence of the toxic golden alga (Prymnesium parvum) on overall ichthyotoxicity, a cofactor known to enhance golden alga toxin activity was used in the bioassays. Golden alga ichthyotoxicity was detected in the NF but not the SF, suggesting golden alga may have contributed to overall ichthyotoxicity in the urban but not in the rural system. In conclusion, the physicochemistry of the urban-influenced NF water was conducive to the expression of ichthyotoxicity and also point to water hardness as a novel factor influencing golden alga ichthyotoxicity in surface waters.