Oogenesis in teleosts: How fish eggs are formed

Gene Expression Profile Induced by 17α-Ethynyl Estradiol, Bisphenol A, and Genistein in the Developing Female Reproductive System of the Rat

Vitamins and minerals serve essential roles in cellular metabolism, maintenance and growth throughout life. They are also central components of many enzymes and transcription factors. However, the need for optimum amounts of key micronutrients at critical stages during the periovulatory period and subsequent embryonic and fetal life has become the focus of sustained research activity only recently. In addition to folic acid, the minerals zinc, iron and copper and the antioxidant vitamins A and E are of particular importance during pregnancy. Both excesses and deficiencies of these micronutrients can have profound and sometimes persistent effects on many fetal tissues and organs in the absence of clinical signs of deficiency in the mother. The consequences of micronutrient imbalance on the developing conceptus may not be apparent at the time of the nutritional insult, but may be manifest later in development. However, supplementary micronutrients provided later in gestation or during postnatal life cannot completely reverse the detrimental effects of earlier micronutrient imbalance. Importantly, deficiency of a specific micronutrient, such as zinc, during pregnancy can result in a greater incidence of fetal malformation and resorptions than general undernutrition. Given the range of micronutrients that affect development, the number of developmental stages susceptible to inappropriate micronutrient status and the diverse biochemical systems and types of tissue affected, it is challenging to propose a unifying hypothesis that could explain the effects of micronutrient imbalance on programming throughout gestation. Micronutrient imbalance can affect pregnancy outcome through alterations in maternal and conceptus metabolism, as a consequence of their essential role in enzymes and transcription factors and through their involvement in signal transduction pathways that regulate development. Micronutrient-induced disturbances in the balance between the generation of free oxygen radicals and the production of antioxidants that scavenge free radicals may provide an additional mechanistic explanation. The detrimental effects of many micronutrient deficiencies, particularly zinc and copper, can be alleviated by supplementary antioxidants, whereas deficiencies of antioxidant vitamins A and E are likely to reduce defence against free radical damage.

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Micronutrient programming of development throughout gestation

The notion of a gamete recognition system that alerts females to the presence of gametes in their reproductive tract profoundly influences our understanding of the physiology of events leading to conception and the bearing of offspring. Here, we show that the female responds to gametes within her tract by modulating the environment in which pregnancy is initially established. We found distinct alterations in oviductal gene expression as a result of sperm and oocyte arrival in the oviduct, which led directly to distinct alterations to the composition of oviductal fluid in vivo. This suggests that either gamete activates a cell-type-specific signal transduction pathway within the oviduct. This gamete recognition system presents a mechanism for immediate and local control of the oviductal microenvironment in which sperm transport, sperm binding and release, capacitation, transport of oocytes, fertilization, and early cleavage-stage embryonic development occur. This may explain the mechanisms involved in postcopulatory sexual selection, where there is evidence suggesting that the female reproductive tract can bias spermatozoa from different males in the favour of the more biologically attractive male. In addition, the presence of a gamete recognition system explains the oviduct's ability to tolerate spermatozoa while remaining intolerant to pathogens.

Modulation of the oviductal environment by gametes.

Understanding gene expression patterns in response to altered environmental conditions at different time points of the preimplantation period would improve our knowledge on regulation of embryonic development. Here we aimed to examine the effect of alternative in vivo and in vitro culture conditions at the time of major embryonic genome activation (EGA) on the development and transcriptome profile of bovine blastocysts. Four different blastocyst groups were produced under alternative in vivo and in vitro culture conditions before or after major EGA. Completely in vitro- and in vivo-produced blastocysts were used as controls. We compared gene expression patterns between each blastocyst group and in vivo blastocyst control group using EmbryoGENE's bovine microarray. The data showed that changing culture conditions from in vivo to in vitro or vice versa, either before or after the time of major EGA, had no effect on the developmental rates; however, in vitro conditions during that time critically influenced the transcriptome of the blastocysts produced. The source of oocyte had a critical effect on developmental rates and the ability of the embryo to react to changing culture conditions. Ontological classification highlighted a marked contrast in expression patterns for lipid metabolism and oxidative stress response between blastocysts generated in vivo versus in vitro, with opposite trends. Molecular mechanisms and pathways that are influenced by altered culture conditions during EGA were defined. These results will help in the development of new strategies to modify culture conditions at this critical stage to enhance the development of competent blastocysts.

Molecular Mechanisms and Pathways Involved in Bovine Embryonic Genome Activation and Their Regulation by Alternative In Vivo and In Vitro Culture Conditions

Retinol (vitamin A) is essential for reproduction, and retinoids have been suggested to play a role in ovarian steroidogenesis, oocyte maturation, and early embryonic development. Retinol is transported systemically and intercellularly by retinol-binding protein (RBP). Within the cell, cellular retinol-binding protein (CRBP) functions in retinol accumulation and metabolism. Since the actions of retinoids are mediated, in part, by retinoid-binding proteins, the objective of this study was to investigate cell-specific expression of RBP and CRBP in the bovine ovary. Immunocytochemical analysis (ICC) localized RBP to the thecal and granulosa cell layers of antral and preantral follicles with the most intense staining in the cells of large, healthy follicles. The tunica adventitia of arterial blood vessels also exhibited RBP staining. Immunostaining of CRBP was most intense in the granulosa cells of preantral follicles and present, but diminished, in thecal and granulosa cells of antral follicles. Within the corpus luteum, both proteins were observed in large luteal cells, but only RBP was observed in small luteal cells. Northern blot analysis demonstrated that thecal and granulosa cells from antral follicles and luteal tissue expressed RBP and CRBP mRNA. Synthesis and secretion of RBP by thecal cells, granulosa cells, and luteal cells were demonstrated by immune-complex precipitation of radiolabeled RBP from the medium of cultured cells or explants, followed by SDS-PAGE and fluorography. Follicular fluid was collected from small (<5 mm) and large (8-14 mm) follicles, pooled according to follicular size, and analyzed for retinol, RBP, estradiol-17beta, and progesterone. Concentrations of retinol, RBP, and estradiol were greater in the fluid of large follicles. Results demonstrate retinoid-binding protein expression by bovine ovaries and provide physical evidence that supports the concept that retinoids play a role in ovarian function.

Expression of retinol‐binding protein and cellular retinol‐binding protein in the bovine ovary

Retinoids are recognized as important regulators of vertebrate development, cell differentiation, and tissue function. Previous studies, performed both in vivo and in vitro, indicate that retinoids influence several reproductive events, including follicular development, oocyte maturation and early embryonic development. The present study evaluated in vitro effects of retinol addition to media containing maturing bovine oocytes and developing embryos in both a low oxygen atmosphere (7%) and under atmospheric oxygen conditions (20%). In the first experiment, abbatoir collected bovine oocytes were matured in the presence or absence of varying concentrations of retinol. After a 22–24 hour maturation period the oocytes were fertilized, denuded 18 hours later and cultured in a modified synthetic oviductal fluid (mSOF) in a humidified atmosphere at 38.5 degrees C, 5% CO2, 7% O2 and 88% N2. Cleavage rates did not differ among control and retinol-treated oocytes in all three experiments. Addition of 5 micromolar retinol to the maturation medium (IVM) tended (p < 0.07) to increase blastocyst formation (blastocyst/putative zygote; 26.1% +/- 2.2%) compared to the controls (21.9% +/- 1.9%). Further analysis revealed when blastocyst development rates fell below 20% in the control groups, 5 micromolar retinol treatment dramatically improved embryonic development, measured by blastocyst/putative zygote rate (14.4 +/- 2.1 vs 23.7 +/- 2.5; p < 0.02). The 5 micomolar retinol treatment also enhanced the blastocyst/cleaved rate by nearly 10% (23.7% vs 34.6%; p < 0.02). In the second and third experiments addition of 5 micromolar retinol to the embryo culture medium (IVC) under low oxygen conditions did not significantly improve cleavage or blastocyst rates, but 5 micromolar retinol significantly increased blastocyst development under 20% O2 conditions (p < 0.001). These studies demonstrate that supplementation of 5 micromolar retinol to the maturation medium may improve embryonic development of bovine oocytes indicated by their increased blastocyst rate. A significant improvement in the blastocyst development with the 5 micromolar retinol treatment under atmospheric conditions suggests a beneficial antioxidant effect during embryo culture.

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Retinol improves bovine embryonic development in vitro

Retinol and its metabolites, all-trans retinoic acid and 9-cis retinoid acid, are regulators of cellular growth, differentiation, and development and have been implicated in reproductive processes including folliculogenesis and embryonic survival. Three experiments were conducted to identify effects of retinoid treatment of superovulated ewes upon subsequent in vitro embryonic development. Ewes were treated with all-trans retinol (ROH), all-trans retinoic acid (RA), 9-cis retinoic acid (CIS), or vehicle (Control) on the first and last day of FSH treatment. Embryos were recovered at the morula stage, cultured in vitro for 96 h, and observed for blastocyst formation. Embryos from ROH-treated animals had a higher (p < 0.01) incidence of blastocyst formation than RA-, CIS-, or vehicle-treated animals (72% vs. 27%, 33% and 32%, respectively). In experiment 2, ewes were given ROH or vehicle and treated as above. ROH treatment resulted in an increased percentage of embryos forming blastocysts (70% vs. 22%, p < 0.05). In experiment 3, ewes were treated with ROH or vehicle, and embryos were collected at the 1- to 4-cell stage and cultured for 7 days. ROH treatment resulted in increased blastocyst formation (79% vs. 5%, p < 0.05). The majority of embryos (60% vs. 6%; p < 0.01)) from vehicle-treated animals failed to develop beyond the 8-cell stage in comparison with those from ROH animals. ROH treatment of superovulated ewes increased embryonic viability and positively impacted embryonic development.

Retinol Administration to Superovulated Ewes Improves In Vitro Embryonic Viability

Two studies were conducted to identify retinol-binding protein (RBP) expression in the ovine oviduct and to determine the role of ovarian steroids in its regulation. Ewes were salpingectomized on Days 1, 5, or 10 of their respective estrous cycles, and oviducts were homogenized for RNA analysis, fixed for immunocytochemistry (ICC), or cultured for 24 h for protein analysis. ICC localized RBP to the epithelium of all oviducts. RBP synthesis was demonstrated by immunoprecipitation of radiolabeled RBP from the medium of oviductal explant cultures. Explant culture medium from oviducts harvested on Day 1 contained significantly more RBP than medium from oviducts collected on Days 5 or 10. Slot-blot analysis demonstrated that steady-state RBP mRNA levels were significantly higher on Day 1 than Day 5 or 10. In the second experiment, ovariectomized ewes were treated with estradiol-17β (E 2 ), progesterone (P 4 ), E 2 +P 4 (E2+P4), or vehicle control, and oviducts were analyzed as above. P 4 alone or in combination with E 2 significantly reduced steady-state RBP mRNA levels compared to those in E 2 -treated animals. Oviductal explants from E 2 - and E2+P4-treated animals released 3- to 5-fold more RBP into the medium than control and P 4 treatments as determined by ELISA. RBP synthesis of metabolically labeled RBP was increased by E 2 and E2+P4 treatments. This study demonstrates that P 4 applied on an estradiol background negatively regulates RBP gene expression in the oviduct whereas estradiol appears to stimulate RBP synthesis and secretion.

Steroid Regulation of Retinol-Binding Protein in the Ovine Oviduct

Disclosed is the use of retinoids such as retinol, all trans retinoic acid, and 9-cis retinoic acid to enhance the success of assisted-reproduction. Administration of retinol to superovulated animals dramatically improved embryo viability and development as well as the pregnancy rates of animals implanted with embryos derived from such animals. Culturing presumptive embryos in vitro in the presence of retinol enhanced development of embryos from the presumptive zygotes compared to presumptive embryos not treated with retinol.

Dawn M. Eberhardt

Papers

Expression of retinol‐binding protein and cellular retinol‐binding protein in the bovine ovary

Retinol improves bovine embryonic development in vitro

Retinol Administration to Superovulated Ewes Improves In Vitro Embryonic Viability

Steroid Regulation of Retinol-Binding Protein in the Ovine Oviduct

Use of retinol in assisted-reproduction protocols