Assessment of Histiotrophic Nutrition Using Fluorescent Probes

Craig Harris


Histiotrophic nutrition is a process whereby the rodent visceral yolk sac (VYS) internalizes exogenous macromolecules, degrades them, and sends the degradation products to the embryo. Quantification and visualization of histiotrophic nutrition can be accomplished using fluorescent tracer molecules such as fluorescein isothiocyanate-conjugated albumin (FITC-albumin). The methods are simple and can provide complimentary functional and structural information in studies of the effects of embryotoxicants on visceral yolk sac function.

Key words : Rat visceral yolk sac, Proteolysis, Endocytosis, Fluorescence microscopy, Histiotrophic nutrition, Spectrofluorimetry

1 Introduction

During the critical period of organogenesis, the interstitially implanted mammalian conceptus spends a significant amount of time in a relatively hypoxic endometrial environment with little or no direct access to micronutrients and oxygen. Initially removed from micronutrient exchange during a period when the placenta develops and matures, the developing conceptus relies on the cap- ture of maternal proteins, carbohydrates, and lipids secreted from endometrial glands and related local sources to supply the amino acids, fuel, and other substrates required to support growth and differentiation of the embryo [1–5]. In vitro and in vivo studies estimate that during embryogenesis, up to 99% of all amino acids utilized by the embryo for new protein synthesis are derived from whole maternal proteins [6, 7]. The route of conceptal protein cap- ture is through the brush border endothelium of the visceral yolk sac (VYS), (1) an inverted absorptive interface in the rodent that encloses the entire embryo and remains structurally and function- ally active until parturition and (2) the inward-facing absorptive surface of the human VYS as a vestigial pouch that disappears toward the end of the first trimester [8, 9]. Despite their obvious anatomical differences, it is believed that both visceral yolk sac morphologies provide similar nutritive functions during organogenesis [1, 2]. The complete uptake of maternal nutrients relies on a combination of pinocytosis and the receptor-mediated endocytosis (RME) of proteins as outlined in Fig. 1. The majority of captured maternal proteins are selectively recognized by the membrane receptors megalin (S) and cubilin (Y) (Fig. 2), located at the base of microvilli in the VYS brush border endothelium [10–12]. Once bound, they activate endocytosis and become the contents of a pri- mary vesicle, the apical endocytotic vesicle (AEV) which will soon fuse with a lysosome (L) to access the proteolytic enzymes required for degradation of the protein to its constituent amino acids. These amino acids and all other vitamins, lipids, cofactors, and cargoes are then made available to the conceptus for its nutritional needs. A partial list of the wide variety of bulk serum proteins, specialized vitamin and cofactor carriers, and growth factors can be seen in Fig. 2 for each of the receptor/transporter subtypes. The collective actions of maternal protein endocytosis, lysosomal proteolysis, and cofactor release are referred to as histiotrophic nutrition pathways (HNP) as opposed to the placental micronutrient exchange mech- anisms of hemotrophic nutrition. The activities of HNP are the nearly exclusive mode of nutrient capture during mammalian organogenesis.

Perturbation of HNP activities can produce developmental consequences that range across a broad spectrum from minimal decreases in overall embryonic growth to complete death and destruction of the conceptus. Experiments conducted in rodent whole embryo cul- ture (rWEC) showed that the selective inhibition of lysosomal cathepsins (cysteine proteases) produced dose-dependent effects on embryonic growth and morphogenesis [3, 5, 13, 14]. Disruption of HNP activities can elicit a series of deleterious metabolic and regula- tory changes in the conceptus that are not only confined to the VYS. Substrates and cofactors of the one-carbon metabolism pathway (C1) such as methionine, folate, betaine, and choline are supplied primarily through the RME as cargoes bound to their respective car- rier proteins (Fig. 1; [4, 5]). Deficiency of these components affects nucleotide and S-adenosylmethionine (SAM) biosynthesis which are essential for DNA synthesis, cellular metabolic methylation, and epigenetic regulation of growth and development [15]. Limitations in amino acid availability have the obvious consequences of reduced protein biosynthesis in embryo and VYS, deleterious changes associ- ated with abnormal autophagy, amino acid starvation, and inade- quate glutathione biosynthesis [16]. Most HNP inhibitors tested to date also produce a net oxidation of embryo and VYS tissues initially related to the decreased ability to synthesize new GSH and maintain proper redox balance [3, 5, 16].

Fig. 1 Histiotrophic nutrition pathways (HNP) are responsible for the selective uptake of maternal proteins by the conceptal visceral yolk sac (VYS) brush border as the major supply route of embryonic nutrients. Maternal proteins, including bulk proteins from endometrial secretions, serum proteins, and carrier proteins with their lipid, vitamin, cofactor, and growth cargoes are presented at the VYS brush border where they bind to membrane receptors. Binding of maternal proteins to megalin and cubilin receptors (Fig. 2) initiates the process of receptor-mediated endocytosis (RME) where proteins and their cargoes are incorporated into an apical endocytotic vesicle (AEV). The AEV will then fuse with a lysosome (L) where cathepsins (cysteine proteases) degrade AEV contents producing free amino acids and releasing protein cargoes. Inhibition of proteolytic activity by agents such as leupeptin results in the continued significant accumulation of maternal proteins in the secondary vesicles. Free amino acids generated through the proteolysis of proteins are utilized in the VYS and embryo proper as substrates for protein biosynthesis and metabolic biosynthesis (C1 pathways, glutathione biosynthesis, etc.). Over 95% of all amino acids incorporated into embryonic and VYS proteins synthesized during organogenesis are derived from HNP activity. Methionine and other C1 vitamins, cofactors, and substrates are obtained from HNP activity. Disruption of their supply can result in metabolic disruption and altered epigenetic programming. Glutathione biosynthesis, critical for cellular protection and maintenance of the intracellular redox environment, depends on amino acids provided by HNP. Disruption of these pathways can lead to oxidative damage and altered differentiation during organogen- esis. Although not generally considered to be part of the HNP activities, megalin- and cubilin-mediated endocytoses are known to have additional critical developmental roles in embryonic tissues throughout embryogenesis, including the processes of gastrulation and neurulation [10].

Multiple steps in the sequence of HNP activity can be inter- rupted directly by developmental toxicants at the RME level or as a result of lysosomal protease inhibition in the later stages. Most known teratogens have not been assessed for their abilities to disrupt RME, although a high proportion of those that have been examined show significant inhibitory activity [5, 17]. A possible causative link between HNP function and structural abnormalities has not yet been examined in detail. Known developmental toxicants such as ethanol and the phthalate, MEHP, are direct inhibitors of RME, induce conditions of intracellular oxidation, and elicit growth deficits and structural malformations. On the other hand, agents such as the GSH synthesis inhibitor buthionine-S,R- sulfoximine (BSO) produce similar or greater oxidation yet pro- duce no observable negative effects on growth or development [5, 16, 17]. Recent and ongoing studies suggest that successful devel- opment may correlate more closely with the state of histiotrophic function than traditional measures such as cellular oxidation.

Fig. 2 The cell surface receptors megalin (S), cubilin (Y), and amnionless make up a typical recognition com- plex used for facilitating RME. Megalin and amnionless are anchored to the membrane by transmembrane domains, while cubilin is attached only to the outer leaf of the plasma membrane. CUB, EGF, complement type, and other binding domains are found in the receptors to aid in the recognition of selected proteins and carriers. A selected number of preferred proteins are listed for each receptor type, some of which are bound exclusively by megalin or cubilin while others can be bound by either or shared. Once bound, signals are transmitted to the intracellular carboxyl terminal NPXY motifs to initiate membrane invagination and endocytosis.

The rodent inverted visceral yolk sac (VYS) is essential for normal embryonic development in rodents and provides the primary functional interface between the embryo proper and the external (maternal) environment (Fig. 3a). With its outward-facing brush border endothelium (Fig. 3b), the rodent VYS is able to capture exogenous macromolecules via RME, degrade them to metabolic precursors, and release vitamins, cofactors, and nutrient precursors for support of conceptal growth and differentiation [6, 18, 19]. The latter process has been designated “histiotrophic nutrition” because of the near exclusive transport of whole proteins and their cargoes as opposed to “hemotrophic nutrition” which refers to the exchange of micronutrients via placental blood [20–23]. A num- ber of in vitro studies have utilized radiolabeled tracer molecules to quantify VYS-mediated histiotrophic nutrition and to monitor alterations in this pathway following chemical exposure. The fluo- rescent methods described here were developed as alternatives to radiolabeled techniques that have been used previously. Besides providing a means to quantify histiotrophic nutrition without the use of radioactivity, this approach also allows visualization of the process through the fluorescent microscope. Confocal microscopic and functional characterization of the effects of lysosomal cathep- sin inhibition (Fig. 3c), shows the apical orientation and relative density of AEVs (arrow) along the brush border epithelium with relatively little FITC-Ab accumulation in controls (left col- umn) due to efficient degradation to amino acids and their export. The top two panels of Fig. 3c show FITC-Ab confocal imaging of the surface view of an intact section of control VYS compared to a similar section of a VYS from a conceptus exposed to the selective cathepsin inhibitor Z-Phe-Ala-CHN2 [14]. The significant accu- mulation of non-degraded (acid-soluble) FITC-Ab (green) in api- cal vesicles of the VYS was significant and was verified with dual-labeled confocal imaging in the cross-sectional plane where the green FITC-Ab label accumulates in apical vesicles of VYS endothelium which were nuclear stained with propidium iodide (Fig. 3c lower panels). Studies to determine the effects of lysosome protease inhibition in the intact viable conceptus in rWEC on redox status and structural malformations also employed the thiol proteomic evaluation of altered protein levels following a 6 h GD 11 exposure to the protease inhibitor leupeptin (LEU). Significant protein accumulations of 2.22- to 21.08-fold for 14 of the most abundant proteins confirmed the identity of the specific maternal rat serum proteins captured and sequestered in the VYS following leupeptin exposure (Fig. 3d). Note that these selected proteins belong to the same general families of proteins selected for trans- port by megalin and cubilin (Fig. 2) [16].

Fig. 3 The use of fluorescent-labeled proteins such as FITC-albumin for assessment of RME and HNP activi- ties in viable rodent conceptuses can be justified and validated through imaging and biochemical assess- ment. The methods described in this chapter utilize the intact, viable rodent conceptus (embryo and associated extraembryonic membranes) grown in whole embryo culture (WEC). Panel a shows a light micro- graph of a typical gestation day 11 rat conceptus in WEC. The apical brush border endothelium is outward facing (maternal interface), and the active VYS vasculature is prominent. A cross-sectional transmission electron micrograph (Panel b) through the VYS shows the prominent brush border (BB) at the apex of the columnar endothelial cells, as well as the abundant apical endocytotic vesicles (AEV), characteristic of active endocytosis. Under normal conditions, the FITC-albumin is actively transported and rapidly degraded in the secondary vesicle of the VYS (control). Confocal micrographs (Panel c) show very little accumulation of acid-insoluble fluorescence, indicative of non-degraded proteins. When proteases are inhibited, as with the selective cathepsin inhibitor Z-Phe-Ala- CHN2, non-degraded whole proteins accumulate rapidly in the vesi- cles as indicated by increased green fluorescence. The extent of acid-soluble and acid-insoluble fluores- cence can be quantified based on its clearance from the culture media [14]. Confirmation that the accumulated proteins are of maternal serum (culture medium) origin (Panel d) was confirmed using ICAT thiol proteomic analysis and mass spectroscopy showing that the 14 most abundant proteins found in the VYS after leupeptin treatment were all maternal rat serum proteins. Numbers in brackets show treated/control concentration ratios after 6 h of leupeptin treatment on GD11 [16].

Although quantification of VYS fluorescence is possible with image analysis of data obtained using the confocal microscope, dis- tinctions between degradation products and whole proteins are not possible, and assessment of non-tissue-associated fluorescence is also not possible. On the other hand, the biochemical data alone cannot determine the location of the tracer molecules within the VYS or within individual cells and cannot identify heterogeneity within a population of cells. Thus, this work is a good example of how biochemical and histological approaches can complement each other to provide more robust information.

The selective inhibition of lysosomal proteases has been shown for natural proteases such as leupeptin, synthetic protease inhibitors like Z-Phe-Ala-CHN2, and therapeutic agents such as chloroquine (an ionotropic inhibitor) [13, 14]. Several known rodent embryo- toxins have been shown to significantly inhibit the RME and/or proteolytic degradation functions of histiotrophic nutrition.

2 Materials

2.1 Embryo Culture

1. Fine watchmaker’s forceps.
2. Disposable sterile Petri dishes.
3. Hanks’ balanced salt solution (HBSS, Gibco BRL, Gaithersburg, MD).
4. Heat-inactivated rat serum.

2.2 Fluorescent Labeling

1. FITC-albumin (11.2 mol FITC/mol albumin) Sigma Chemical Company (St Louis, MO) light sensitive, store in dark.
2. Bovine serum albumin (BSA).
3. 50 mM sodium phosphate buffer, pH = 6.0.
4. 500 mM Tris in H2O (without HCl).
5. 6% w/v trichloroacetic acid.
6. 6% trichloroacetic acid containing 1% SDS.
7. 1 N NaOH.
8. 0.1% Triton X-100.
9. 1.5 mL microcentrifuge tubes.
10. 10. Blue 50–1000 pipet tips (some with tips cut to a 3 mm opening with a razor blade).
11. 12 × 75 mm glass test tubes.
12. Black, round-bottom polypropylene 96-well plates (non- sterile) (Costar).

2.3 Fluorescence Confocal Microscopy

1. Microscope slides.
2. #1½ microscope cover slips (glass).
3. Permount™ microscope mounting media (Fisher Scientific, Pittsburgh, PA).
4. Methanol.
5. Acetone.

2.4 Equipment and Instrumentation

1. Ultrasonic cell disruptor (Misonix Sonicator 3000 with microtip).
2. UV/VIS 96-well plate reader for DCF protein assay (SpectraMax 384 Plus; SoftMax Pro Software; Molecular Devices).
3. Molecular Devices SpectraMax Gemini XS—multiwavelength fluorescence plate reader.
4. Vortex mixer.
5. Microtube centrifuge. Biofuge A (American Scientific Products).
6. Stereodissecting microscopes (Nikon Model AFX-DX and Wild Heerbrugg TYP 355110 with discussion bridge).
7. Fiber-optic illumination systems (Intralux 5000 or Fiber-Lite Model 190—Dolan-Jenner Industries).
8. Refrigerated centrifuge with rotors suited to accept 50 and
15 mL conical plastic centrifuge tubes. Allegra X-22R Centrifuge (Beckman Coulter).
9. Incubator (Fisher Isotemp-constant 37 °C; cabinet size must accommodate benchtop roller apparatus).
10. Roller apparatus (Wheaton Benchtop Roller with two roller decks).
11. Digital camera (Scion Corporation Model CFW-1301C Color Digital Camera) and NIH image software (free download from NIH) loaded on a compatible PC.
12. Leitz Aristoplan fluorescence microscope with differential interference contrast (DIC) optics, mercury arc lamp, and narrow bandpass filters for FITC and rhodamine fluorescence.
13. BioRad MRC600 laser scanning confocal microscope, FITC and rhodamine filters, BioRad COMOS software (BioRad).
14. Bottle gassing manifold (fabricated from glass tubing, blunt tip 18 Ga syringe needles, and latex tubing for use with a standard slide warmer).
15. PerkinElmer LS-5 fluorescence spectrophotometer (PerkinElmer).

3 Methods
3.1 Animals and Embryo Culture

Time-mated primigravida rats are the source of appropriately aged conceptuses (embryo plus extraembryonic membranes) that are explanted for use in whole embryo culture. A sperm-positive vagi- nal smear on the morning after copulation indicates pregnancy and is designated day 0 of gestation. Time-mate pregnant rats can be obtained from any reputable vendor.

On the morning of gestation day (GD) 10, pregnant dams are euthanized and their uteri removed. Conceptuses are dissected from the uterus and cultured as previously described (see Chapter 14). Conceptuses of an appropriate developmental stage (8–12 somite stage embryos with open neural tubes and dorsal flexure) are cultured in roller bottles containing medium consisting of 50–75% heat-inactivated rat serum in HBSS plus penicillin G (100 IU/mL) and streptomycin (50 IU/mL), allowing at least 1 mL of medium per conceptus. The medium is warmed to 37 °C and gassed for 30 min with 20% O2/5% CO2 prior to culture. Conceptuses are allowed to equilibrate to culture conditions in untreated medium in a roller incubator (37 °C) for 1–2 h before being exposed to tracer molecules on GD 10. GD 11 conceptuses can also be used for the assay either following culture of GD 10 conceptuses for 24 h or directly from explants from pregnant dams on GD11 (see Notes 1 and 2). The GD 11 cultures are gassed with 95% O2. Treatments can be added to the embryo culture media before or during the labeling procedure described below (see Note 3).

3.2 Labeling VYSs with Fluorescent Tracer Molecules In Vitro
3.2.1 General Information
3.2.2 Detailed Directions

FITC-labeled albumin or casein can be used to measure adsorptive endocytosis and proteolysis, which are the main processes of histio- trophic nutrition (see Note 4). Proteins conjugated with other, more photostable fluorophores can also be used (see Note 5). The tracer molecule is added directly to the culture medium at a nontoxic concentration, and then conceptuses are added to the medium containing the tracer molecule. After exposure to the tracer mole- cule for various times (between 1 and 6 h works well), uptake and degradation of the tracer molecule can be assessed using spectro- fluorometry and fluorescence microscopy (see Note 6). Pulse-chase experiments can be used to isolate the proteolytic step and high- light differences between control and treated conceptuses when using fluorescence microscopy (see Note 7).

1. Set up culture bottles (6 mL of culture medium in a 50 mL roller bottle) for each control and treated group for each time point. Using one bottle for each time point allows you to cal- culate the amount of degradation products released into the medium per unit time without correction for removal of a por- tion of the conceptuses at different times. You should also set up a “blank” which is one bottle containing unlabeled BSA instead of FITC-albumin. The blank is used to check/correct for background fluorescence of serum and any treatment.
2. Warm and gas the culture bottles prior to the start of the assay (20% oxygen for GD 10 culture, 95% oxygen for GD11 cul- ture). Add 100 μg FITC-albumin/mL to the medium of each control and treatment bottle just before adding the conceptuses (see Note 8). Allow the labeled protein to dissolve and swirl to disperse the tracer (this should occur rapidly in warm media).
3. Begin the assay by transferring conceptuses (~5 conceptuses/ bottle) from the acclimation culture bottles into the assay bot- tles (containing tracer) using a wide-mouth transfer pipette (see Note 9). Immediately remove triplicate samples of 20 and 250 μL of medium, and place in microcentrifuge tubes on ice. These will determine the media fluorescence at the start of the assay (total fluorescence from the 20 μL sample, acid-soluble fluorescence from the 250 μL samples). The media fluores- cence is important for calculation of clearance rate, which is calculated as the μL of media cleared/mg protein/hour (sam- ple calculation below).
4. Place the assay bottles containing conceptuses and FITC-albumin in a 37 °C roller incubator for the desired time course. At comple- tion of each time point, remove one roller bottle for each group (see Note 10). Remove triplicate samples of 250 μL of medium, and place in microcentrifuge tubes on ice. These will provide a measure of the acid-soluble fluorescence at this time point.

3.2.3 Collection and Processing of Tissues and Samples

Two methods have been used to measure tissue and fluid fluores- cence in conceptal samples with this protocol. One involves the manual determination of fluorescence intensity in individual sam- ples using a standard spectrofluorometer (PerkinElmer LS-5 fluo- rescence spectrophotometer). This procedure is somewhat time-consuming and has a higher propensity for sample-to-sample variability. The alternative method for measuring fluorescence intensity in conceptal samples uses identical extraction and sample preparation methods, but samples are transferred to a 96-well plate and read in a fluorescence plate scanner. The latter approach is more desirable because of reduced variability and much shorter analysis times. A description of the microplate method is included at the end of the general method below.
1. Using a wide-mouth transfer pipette, remove conceptuses from the labeling medium, and immediately rinse 3× in room temperature HBSS. Place conceptuses individually into a drop of 250 μL of 50 mM sodium phosphate buffer (pH = 6.0) in a dry 10 mm Petri dish, being careful to transfer a minimum volume of HBSS with it (a pipette tip with an opening about the same size of one intact conceptus (~3 mm) will help mini- mize adding too much extra volume).
2. Remove the ectoplacental cone from the conceptus using watchmaker’s forceps and discard it. Dissect the VYS and amnion away from the embryo by making small incisions using the watchmaker’s forceps (easier done with older conceptuses, but with experience GD10 conceptuses will work equally well). Exteriorize the embryo and release the EEF by gently agitating the VYS and amnion in the buffer drop (see Note 11).
3. Carefully remove the VYS from the buffer with the forceps, rinse 3× in HBSS, and place the VYS into 250 μL of 0.1% Triton X-100 in a microcentrifuge tube on ice to stop contin- ued proteolysis. The embryo proper and amnion do not pro- cess FITC-albumin, so it is not necessary to collect these unless other measurements are desired (e.g., protein content of embryo).
4. The buffer drop remaining in the Petri dish contains the EEF and is collected in a microcentrifuge tube and placed on ice. The volume should be approximately 250 μL.
5. After collecting samples, sonicate all VYS tissue samples to pre- pare a tissue homogenate. Be careful because the Triton X-100 has a tendency to foam during sonication. Use lower than nor- mal power settings on the sonicator to alleviate this. Once homogenized, collect a 20 μL sample of the homogenate, and save it in order to analyze protein content.
6. Next add 750 μL of 6% TCA to all tubes containing 250 μL, including the media tubes, to precipitate proteins. Do not add TCA to the tubes used to determine total media fluorescence (those with 20 μL of media). Instead add 230 μL of Triton X-100 and then 750 mL of 6% TCA with 1% SDS. The SDS will solubilize the tracer protein and not allow precipitation; thus the total media fluorescence reading will be obtained from the samples containing SDS.
7. Vortex the acidified samples, and place the tubes in the refrig- erator for 1–24 h to precipitate proteins. This is a good stop- ping point. At this point you should have triplicate samples of 20 μL of medium in acidified SDS from the beginning of the experiment and triplicate samples of 250 μL of medium in TCA from the beginning and each time point of the culture period. You should also have acidified samples of homogenized VYS tissue from each conceptus for determination of acid-solu- ble and acid-insoluble fluorescence in the VYS, as well as 20 μL of VYS homogenate for each conceptus for protein determination.
8. Centrifuge all tubes for fluorescent measurement at 10,000 × g
for 10 min.
9. Remove the supernatant to a clean 12 × 75 test tube. It should be a volume of ~900 μL. Respin the pellet (5 min), and remove any additional supernatant left with a small pipette tip. Add this to the rest of the supernatant. SDS samples should not have a pellet after centrifugation.
10. Add 1 mL of 500 mM Tris buffer and 150 μL of 1 N NaOH to the supernatant to bring the pH up to ~8.7 (optimal pH for FITC fluorescence). These samples can now be transferred into cuvettes or a microtiter plate and their FITC content determined spectrofluorometrically.
11. Set up the spectrofluorometer to use 495 nm light for excita- tion and 520 nm wavelength emission (use a narrow bandpass of ~10 nm). Zero the spectrofluorometer with the blank sam- ples taken from the media with unlabeled albumin. Since the fluorescence in tissue samples is compared to the total fluores- cence per μL media, no standard curve is necessary although one can be used (see below). Scale the fluorometer to give a large reading for the total media fluorescence (tubes containing 20 μL of media and SDS). The other samples will contain less fluorescence, but it should be consistently readable.
12. The pellets (acid-insoluble material left after precipitation and centrifugation of VYS homogenate) can be saved overnight (refrigerated and protected from light) if you want to read them later. To dissolve and resuspend the pellets, add 150 μL of 1 N NaOH to each pellet, vortex, and leave sit at room tem- perature for ~1 h to dissolve. Vortex several times; then add 250 μL of Triton X-100 once the pellet is soluble. Mix and give a quick centrifuge (~10 s at 10,000 × g) to bring the liq- uid to the bottom of the tube.
13. Remove each resuspended pellet sample to a clean, labeled 10 × 75 mm test tube. Add 1 mL of 500 mM Tris buffer and 750 μL of 6% TCA to the tubes, vortex and read the fluores- cence with same setting as the supernatant.

3.2.4 Measuring Fluorescence Intensity with Spectrofluorometry
3.2.5 Calculations

1. To measure FITC fluorescence, spectrofluorometer settings are 495 nm excitation and 520 nm emission with 10 nm slit widths (as described above).
2. For measurement of fluorescence using the microplate technique, a standard curve is included on each plate run to correct for plate-to-plate variability (see Note 12).
Standard curve for microtiter plate: From a 10 mg/mL FITC- albumin stock, make a 1:10,000 dilution by adding 1 μL of the stock to 9999 μL of diH2O, which provides 1 μg/μL FITC- albumin. Construct a standard curve as seen in Tables 1 and 2.
Samples are run in duplicate or triplicate and averaged for final quantitation. Set instrument wavelengths to 495 nm excitation and 520 nm emission, and run according to instrument instructions.

Raw fluorescence units can be converted into amount of media cleared of fluorescence (clearance) by the following procedure:
1. Determine the total amount of fluorescence units per μL of media for each bottle from the SDS samples (i.e., the reading of 20 μL media samples diluted with SDS/20 μL = fluores- cence units/μL).
2. Determine the total amount of media taken up by the tissue. Total uptake is the sum of the acid-soluble fluorescence in the medium, the acid-soluble fluorescence in the VYS and EEF (all conceptuses in that bottle), and the acid-insoluble fluorescence in the VYS and EEF (all conceptuses in that bottle). Divide this sum by the amount of fluorescence units/μL in the medium (number 1 above) and the total micrograms of protein from the VYS tissue in the culture bottle to determine the total μL of culture medium cleared per mg protein. Divide the volume (μL) of culture medium cleared per mg protein by the time of incubation to get the clearance rate in μL/mg protein/hour.
The total clearance includes the TCA-soluble fluorescence accumulated in the culture medium over time + EEF acid-soluble and acid-insoluble fluorescence + VYS acid-soluble and acid- insoluble fluorescence for each bottle. Both TCA-soluble and TCA-insoluble fluorescence in the tissue are added with the acid- soluble fluorescence in the media for total uptake; only TCA soluble.

1. Expose conceptuses to the tracer molecule in whole embryo culture as described above (see Note 13).
2. Remove conceptuses from the culture medium containing the tracer, and rinse 3× in HBSS using a wide-mouth transfer pipette. At this point the VYS tissue can be processed for fluo- rescent microscopy or placed in “chase” medium which does not contain tracer (see Note 13). The pulse-chase technique allows you to isolate the proteolytic phase of histiotrophic nutrition from the endocytotic phase.
3. To prepare the VYS tissue for fluorescent microscopy, remove and discard the ectoplacental cone with watchmaker’s forceps while the conceptus is in the HBSS. Dissect the VYS free from the embryo using watchmaker’s forceps by making a tear across the VYS and exteriorizing the embryo. The VYS can then be separated from the embryo with one incision at the attachment site.
4. Once the VYS is removed from the embryo, rinse it 3× in HBSS using a transfer pipette, and place it in a drop of HBSS on a clean cover slip.
5. Carefully open and flatten the VYS with watchmaker’s forceps on the cover slip, remove excess buffer with a transfer pipette, and blot the remaining buffer with tissue paper. The VYS should lie relatively flat on the cover slip (see Note 14).
6. Immediately fix the tissue by immersing the cover slip in −20 °C methanol for 5 min followed by −20 °C acetone for 5 min. The VYS tends to float off the cover slip during fixation but can be transferred by gently holding one edge with a watchmaker’s forceps. If a nuclear counterstain is desired, it can be added to the cold methanol. For example, instead of placing the VYS in pure −20 °C methanol for 5 min, it can be placed in −20 °C methanol containing a low concentration of propidium iodide (1 uM) for 3 min and then rinsed in pure −20 °C methanol for 2 min, before fixing in acetone.
7. Because of the thickness of the fixed VYS, welled microscope slides are needed for mounting (see Note 15). Add a small amount (1–2 drops) of Permount™ mounting media to mount the VYS with a coverslip on top.
8. Protect from light and let dry ~1 h at room temperature; then refrigerate to preserve fluorescence until you are ready to image (see Note 16). The FITC fluorescence is stable several weeks if kept refrigerated and protected from light.

3.3.2 Fluorescence Microscopy of Labeled VYSs

Whole mount VYSs can be viewed with a conventional microscope with phase contrast or differential interference contrast (DIC) microscopy to identify the tissue structure. DIC works well for unstained thick sections, and the structure of the VYS is readily apparent, including all cell types with surface and transverse views (Fig. 4). Once the tissue is visualized with DIC or phase contrast, the VYS fluorescence should be readily visualized by switching to fluorescent imaging. Conventional fluorescent microscopy (mer- cury arc lamp and FITC filter set) is used to locate the tracer mol- ecule. For dual-labeled samples, a rhodamine filter set will allow visualization of nuclear staining using propidium iodide. Photography will be suboptimal when using a conventional fluo- rescent microscope because of background fluorescence emanating from out-of-focus planes in the sample (due to the thickness of the preparation). For improved fluorescent images, a confocal micro- scope is ideal because it removes the glare associated with out-of- focus light emanating from the sample. The confocal microscope also allows three-dimensional visualization of the tissue and visual- ization of dual-labeled samples using computerized image overlay techniques (Fig. 4).

4 Notes

1. When first conducting the assay, gestational day (GD) 11 rat conceptuses are easier to use than GD10 conceptuses, because of their larger size and relative ease of dissecting the VYS from the embryo proper. Explanting GD11 conceptuses is more dif- ficult than GD10 conceptuses, however, so it may be easier to culture GD10 conceptuses for 24 h prior to setting up the assay than to explant GD11 conceptuses. Since conditioned media (culture media that was used to grow conceptuses over 24 h) has been found to possess proteolytic activity once the conceptuses are removed (J. Ambroso, unpublished observations), if you are culturing conceptuses on GD10 for treatment and evaluation on GD 11, fresh media should be used when exposing concep- tuses to labeled proteins, and conceptuses should be rinsed prior to placing them in medium containing a tracer molecule.
2. To get good results in quantitative studies with GD 10 con- ceptuses, it may be best to combine two VYS tissues in one sample rather than use individual samples (as for GD11).
3. Irreversible enzyme inhibitors can be added to the embryo culture prior to removing conceptuses to media containing the fluorescent tracer. If the treatment causes a reversible effect (e.g., competitive inhibition of endocytosis), then co-incubating the treatment with the fluorescent tracer is required.
4. A nondigestible tracer such as FITC-dextran can be used to measure fluid-phase endocytosis [25]. When a nondigestible tracer is used, all the fluorescence should remain acid insoluble during the incubation; in other words proteolysis is not a factor.
5. One traditional problem with fluorescent probes is photo- bleaching or decreased fluorescence following exposure to light. This requires protecting samples from light as much as possible, in particular during preparation for fluorescent microscopy work. However, newer fluorescent dyes such as BODIPY are less sensitive to photobleaching, and processing of these samples can be performed in ambient light.
6. Because unlabeled protein in the culture medium competes for membrane binding sites with the tracer protein, lower rates of uptake will be observed in cultures with higher concentrations of serum or unlabeled albumin. The minimal concentration of serum needed to support normal embryonic growth is ~33% serum (C. Harris unpublished data); however most studies of whole embryo culture use ≥50% serum/50% HBSS.
7. In a pulse-chase experiment, conceptuses are incubated in medium containing tracer molecule for a period of time to allow uptake of the tracer (pulse). Next, the labeled concep- tuses are placed in media without tracer and allowed to digest the tracer. If a treatment inhibits proteolysis in the VYS, the tracer will not be cleared from the lysosomes of the VYS epi- thelium during the chase period.
8. FITC-albumin concentrations of 500 μg/mL have been used for short periods of time and produce a large fluorescent sig- nal. However, this concentration in the medium is embryo- toxic over a 24 h culture period. FITC-albumin concentrations of 100 μg/mL do not affect embryonic development after 24 h in culture and provide an adequate fluorescent signal. It would be wise to test the toxicity of tracer protein conjugated to other fluorophores before their use in whole embryo culture.
9. Wide-mouth transfer pipettes can be made from glass pasteur pipettes by scoring and breaking the tapered portion of the pipette at an appropriate diameter and dulling the sharp edge where the pipette is broken by heating it with a Bunsen burner.
10. Start a timer and stagger the start times of each bottle to allow yourself time to dissect the conceptuses at the end of the assay (usually 15 min is sufficient for 3–5 conceptuses/time point).
11. During development of this method, it was noted that the exocoelomic and amniotic fluid contains concentrated amounts of fluorescence, nearly all acid-insoluble degradation products. In order to measure these products, the VYS and amnion need to be opened and agitated in the buffer drop to release the EEF.
12. The selection of appropriate 96-well plates is important to limit intra-plate variability when reading fluorescence. We pre- screened a number of blank plates under the exact reading conditions of the assay and found that black, round-bottom polypropylene plates (Costar) gave the most consistent results with the least well-to-well variability. When using the plate reader method, new batches of plates should always be pre- screened to avoid artifacts.
13. Exposure to fluorescent tracer molecule can be continuous or as a pulse exposure followed by a chase culture period when conceptuses are cultured in normal medium without tracer. During continuous exposure to the tracer, the VYS epithelium will attain high levels of fluorescence in the vacuolar system. During the chase period, fluorescence is normally cleared from the VYS epithelium by degradation of the tracer and diffusion out of the tissue, resulting in return to background levels of fluorescence over time. The pulse-chase method provides a good way to highlight inhibition of lysosomal proteolysis using fluorescence microscopy.
14. Because the intact VYS is round, flattening on a cover slip is sometimes difficult. Flattening the VYS can be aided by mak- ing small incisions with the watchmaker’s forceps and carefully removing the excess HBSS after placing the VYS on a slide prior to fixation. As the excess buffer is removed, the VYS naturally flattens onto the cover slip.
15. Welled microscope slides can be made by scoring and breaking cover slips into small pieces and cementing the pieces to a slide with Permount™ mounting medium. The pieces should be close enough together so that a coverslip will span the distance of the “well.” The sample can then be placed on the slide between the pieces of attached cover slip and one or two drops of Permount™ placed on top, followed by a coverslip.
16. Because the Permount™ does not completely solidify if only given 1 h to dry, it can be easy to contaminate the microscope lens with Permount™ while imaging the slides under oil immersion lenses. Thus care should be taken not to apply too much pressure on the cover slip with the microscope lens as this can cause the mounting media to extrude from beneath the coverslip. If the microscope lens does get contaminated with Permount™, clean up the lens with a cotton swab with a small amount of methanol or xylene on it.

Fig. 4 Typical results found for the FITC-albumin assessment of histiotrophic nutrition activity in cultured rat conceptuses using the methods described in this chapter. All values shown are calculated as clearances (μL cleared/min/mg protein in 3 h) as described in the Subheading 3. Values for visceral yolk sac (VYS), extra embryonic fluids (EEF), and embryo proper (EMB) are shown for acid-soluble relative fluorescence [FITC fluorescence associated with protease degraded amino acids and small peptides that will no longer precipitate in acid and insoluble relative fluorescence associated with whole proteins and large peptides that readily precipitate in acid]. Note that the control maintains a good balance between degraded and un-degraded spe- cies of fluorescence and that increasing concentrations of the protease inhibitor, leupeptin, dramatically reduces the soluble fraction in favor of increased acid-insoluble forms. Increasing concentrations result in a dose-dependent decrease in total clearance as shown [24].


1. Burton GJ (2001) Nutrition of the human fetus during the first trimester—a review. Placenta 22(Suppl A):S70–S77
2. Burton GJ (2002) Uterine glands provide his- tiotrophic nutrition for the human fetus during the first trimester of pregnancy. J Clin Endocrinol Metab 87(6):2954–2959
3. Sant KE, Dolinoy DC, Nahar MS et al (2013) Inhibition of proteolysis in histiotrophic nutri- tion pathways alters DNA methylation and one-carbon metabolism in the organogenesis- stage rat conceptus. J Nutr Biochem 24(8): 1479–1487
4. Sant KS, Dolinoy DC, Nahar MS, Harris C (2013) Histiotrophic nutrition informs DNA methylation in the rat conceptus. J Nutr Biochem In Press
5. Sant KE, Dolinoy DC, Jilek JL et al (2016) Mono-2-ethylhexyl phthalate (MEHP) alters histiotrophic nutrition pathways and epigenetic processes in the developing conceptus. J Nutr Biochem 27:211–218
6. Beckman DA, Pugarelli JE, Koszalka TR et al (1991) Sources of amino acids for protein syn- thesis during early organogenesis in the rat. 2. Exchange with amino acid and protein pools in embryo and yolk sac. Placenta 12(1):37–46
7. Beckman DA (1990) Sources of amino acids for protein synthesis during early organogenesis in the rat. I. Relative contributions of free amino acids and of proteins. Placenta 11(2):109–121
8. Padykula HA, Deren JJ, Wilson TH (1966) Development of structure and function in the mammalian yolk sac: I. Developmental mor- phology and vitamin B12 uptake of the rat yolk sac. Dev Biol 13(3):311–348
9. Zohn IE, Sarkar AA (2010) The visceral yolk sac endoderm provides for absorption of nutrients to the embryo during neurulation. Birth Defects Res A Clin Mol Teratol 88(8):593–600
10. Kozyraki R (2007) Multiligand endocytosis and congenital defects: roles of cubilin, mega- lin and amnionless. Curr Pharm Des 13(29): 3038–3046
11. Moestrup SK (2001) Megalin- and cubilin- mediated endocytosis of protein-bound vita- mins, lipids, and hormones in polarized epithelia. Annu Rev Nutr 21:407–428
12. Assémat E, Vinot S, Gofflot F et al (2005) Expression and role of cubilin in the internal- ization of nutrients during the peri-implanta- tion development of the rodent embryo. Biol Reprod 72(5):1079–1086
13. Ambroso JL (1993) Chloroquine embryotox- icity in the postimplantation rat conceptus in vitro. Teratology 48(3):213–226
14. Ambroso JL, Harris C (1994) In vitro embryo- toxicity of the cysteine proteinase inhibitors benzyloxycarbonyl-phenylalanine-alanine- diazomethane (Z-Phe-Ala-CHN2) and benzy- loxycarbony1-phenylalanine-phenylalanine- diazomethane (Z-Phe-Phe-CHN2). Teratology 50(3):214–228
15. Anderson OS, Sant KE, Dolinoy DC (2012) Nutrition and epigenetics: an interplay of dietary methyl donors, one-carbon metabolism and DNA methylation. J Nutr Biochem 23(8):853–859
16. Harris C, Jilek JL, Sant KE et al (2015) Amino acid starvation induced by protease inhibition produces differential alterations in redox status and the thiol proteome in organogenesis-stage rat embryos and visceral yolk sacs. J Nutr Biochem 26(12):1589–1598
17. Jilek JL, Sant KE, Cho KH et al (2015) Ethanol attenuates histiotrophic nutrition pathways and alters the intracellular redox environment and thiol proteome during rat organogenesis. Toxicol Sci 147(2):475–489
18. Freeman SJ, Beck F, Lloyd JB (1981) The role of the visceral yolk sac in mediating protein uti- lization by rat embryos cultured in vitro. J Embryol Exp Morphol 66:223–234
19. Freeman SJ, Lloyd JB (1983) Evidence that protein ingested by the rat visceral yolk sac yields amino acids for synthesis of embryonic protein. J Embryol Exp Morphol 73:307–315
20. Freeman SJ, Lloyd JB (1982) The effect of teratogenic antiserum on yolk sac function in rat embryos cultured in vitro. J Embryol Exp Morphol 71:63–74
21. Freeman SJ, Lloyd JB (1983) Inhibition of proteolysis in rat yolk sac as a cause of terato- genesis. Effects of leupeptin in vitro and in vivo. J Embryol Exp Morphol 78:183–193
22. Freeman SJ, Lloyd JB (1986) Evidence that suramin and aurothiomalate are teratogenic in rat by disturbing yolk sacmediated embryonic protein nutrition. Chem Biol Interact 58: 149–160
23. Hunter ES III, Phillips LS, Goldstein S, Sadler TW (1991) Altered visceral yolk sac function produced by a low molecular weight somato- medin inhibitor. Teratology 43:331–340
24. Harris C, Dixon M, Hansen JM (2004) Glutathione depletion modulates methanol, formaldehyde and formate toxicity in cultured rat conceptuses. Cell Biol Toxicol 20(3):133–145
25. Ambroso JL, Harris C (1994) Chloroquine accumulation and alterations Fluorescein-5-isothiocyanate in pinocytosis and proteolysis in the rat conceptus in vivo. Biochem Pharmacol 47:679–688.