In Vitro Culture of Rhesus Macaque (Macaca mulatta) Embryos

i. Summary

Production of nonhuman primate (NHP) embryos in vitro begins with recovery of gametes. Females undergo a controlled ovary stimulation to produce multiple preovulatory follicles from which oocytes may be recovered. Mature ova are subjected to in vitro fertilization (IVF) and presumptive zygotes are cultured to the intended stage of development. Essential to this practice is the culture medium unique to each step in the process. Here we describe medium preparation, oocyte recovery, in vitro fertilization and in vitro culture (IVC) of embryos in the Rhesus macaque model.

1. Introduction

Rhesus macaques represent a valuable resource in biomedical programs and conservation initiatives. Their close phylogenetic relationship to humans makes them an ideal species for studies investigating infectious disease and chronic degenerative disorders (1,2), as well as reproductive function – gametogenesis, tract biology, pregnancy, and assisted reproductive technologies (ART) (3–7). The advent of gene editing technology has made possible the generation of precise macaque disease models by injecting zygote stage embryos with editing material (e.g. CRISPR-Cas9 or TALENs) followed by transfer of cultured embryos into recipient dams (8,9). Additionally, rhesus monkeys provide a foundation for conservation strategies of endangered macaque species, such as the Celebes Crested or Pagai Island macaques, through development of macaque specific in vitro ART and banking of gametes and embryos (10–17).

The use of Rhesus macaques for all of these techniques requires, at some level, the ability to produce and culture embryos in vitro so that optimized protocols and validated medium become critical for success.

In this chapter, methods are sequentially provided to walk through the steps required to obtain the biological materials necessary for creation and culture of Rhesus macaque embryos. Protocols include 1) media preparation for gamete collection and culture, 2) ovarian stimulation with recombinant gonadotropins and a GnRH inhibitor for production of multiple preovulatory follicles, 3) oocyte recovery from follicle aspirates and grading of meiosis, and 4) IVF followed by IVC and the stages of embryo development. At the beginning of the methods section is a graphical timeline indicating the sequence of events beginning with initiation of the ovarian stimulation protocol and ending with embryo production. As with all United States Department of Agriculture (USDA) regulated laboratory species, appropriate Institutional Animal Care and Use Committee (IACUC) approval must be acquired prior to initiating any NHP studies.

2. Materials

Wear gloves during medium preparation and if necessary, spray with 70% ethanol (EtOH) to remove latex powder (toxic to embryos) before handling materials. Use ultra-pure water (≥18 MOhm) unless otherwise specified and prepare medium in clean glassware free of residue and debris (see Note 1).

2.1. Preparation of Culture Media

1. TALP-HEPES: 114 mM Sodium Chloride, 3.2 mM Potassium Chloride, 0.34 mM Sodium Phosphate Dibasic, 5.0 mM D-(+)-Glucose, 10.0 mM Sodium DL-lactate solution 60% (w/w), 28.2 mM Phenol Red, 2.0 mM Sodium Bicarbonate, 0.2 mM Gentamicin Sulfate, 11.0 mM HEPES, 0.5 mM Sodium Pyruvate, 0.5 mM Magnesium Chloride Hexahydrate, and 2.0 mM Calcium Chloride Dihydrate. On a magnetic stir plate with an appropriately sized stir bar, place a 1-liter glass beaker filled with ~700 mL ultra-pure water and adjust rotational speed to a “medium” setting. In the order listed in Table 1, weigh out and add chemicals allowing each to dissolve into solution before adding the next (see Note 2).

   Once all chemicals are thoroughly mixed and in solution, adjust the pH to 7.3–7.5 with either NaOH or HCl. Add sufficient ultra-pure water to bring the final volume to 1,000 mL. Measure and confirm that the osmolality is between 275–290 mOsm/kg. If the osmolality is greater than 290 mOsm/kg, an adjustment can be made by gradually adding more water and measuring again. If the osmolality is below the target range, a new batch of medium must be prepared. Filter medium through a 0.2 µm filter unit and store at 4°C for up to one month. Prior to use add 3 mg/mL of BSA, ensuring all crystals are completely dissolved and pass through a 0.2 µm filter. TALP-HEPES can be stored at 4°C for up to 7 days. Warm medium to 37°C prior to use.

2. TALP Stock Solutions: 157 mM Sodium Chloride stock solution, 166 mM Potassium Chloride stock solution, 120 mM Calcium Chloride Dihydrate stock solution, 120 mM Magnesium Chloride Hexahydrate stock solution, 295 mM D-(+)-Glucose stock solution, 167 mM Sodium Bicarbonate stock solution, and 1.7% Sodium DL-Lactate stock solution. Prepare stock solutions by weighing and adding the indicated chemicals to the specified volume of ultra-pure water listed in Table 2.

3. TALP Sodium Phosphate Stock (20 mM): Add 140 mg of sodium phosphate monobasic monohydrate to 50 mL of glucose stock solution.

4. TALP Medium: On a magnetic stir plate with an appropriately sized stir bar, place a 1000 mL glass beaker filled with ~300 mL of sodium chloride stock solution and adjust rotational speed to a “medium” setting. In the order listed in Table 3, weigh out or measure the indicated chemicals allowing each to dissolve into solution before adding the next. Adjust the pH to 7.3 – 7.5 and bring the final volume to 500 mL with sodium chloride stock solution. The osmolality should be between 285–295 mOsm/kg. Filter medium through a 0.2 µm filter unit and store at 4°C for up to one month. Prior to use add 3 mg/mL of BSA and 0.006g/100 mL sodium pyruvate (0.5 mM), ensuring all crystals are completely dissolved before passing through a 0.2 µm filter. TALP can be stored at 4°C for up to 7 days. Medium intended for culture should be equilibrated at 37°C in 5% CO2 in humidified air for at least 2 hours before use.

5. Sperm Activation Medium: 2 mg (10 mM) caffeine, 5 mg (10 mM) dibutyryl cAMP and 1 mL of sterile saline. Mix well. Make fresh prior to use for in vitro fertilization and maintain at 37°C.

6. Embryo Culture Medium: Commercially available single step, or single phase, culture medium better supports development of rhesus embryos from zygote to the blastocyst stage than sequential culture medium (18). Examples of medium include those formulated for human embryo culture (e.g. Global® by the LifeGlobal® Group) or similar (e.g. BO-IVC from IVF Bioscience). All media should contain 4 to 5 mg/mL protein (see Note 3). Culture medium should be equilibrated for at least 2 hours before use in an atomosphere of 6% CO₂, 5% O₂, and 89% N₂ at 37°C in humidified air to achieve a pH of approximately 7.3 (range 7.2–7.4). Altitude and characteristics of individual incubators may alter the concentration of CO₂ required to maintain the desired pH of the culture medium.

7. Blastocyst Cryopreservation: Commercial cryopreservation kits marketed for use with human blastocysts are suitable for rhesus macaque embryos.

Table 1:

Composition of HEPES-buffered Tyrode’s Albumin¹, Lactate, and Pyruvate Medium (TALP-HEPES).

Compound	MW (g/mol)	Amount per L	Concentration (mM)
Sodium Chloride	58.44	6.660 g	114.0
Potassium Chloride	74.55	0.240 g	3.2
Sodium Phosphate	141.96	0.048 g	0.3
D-(+)-Glucose	180.16	0.900 g	5.0
Sodium D,L-Lactate solution 60% (w/w)	112.06	1.870 mL	10.0
Phenol Red	354.38	0.010 g	28.2
Sodium Bicarbonate	84.01	0.168 g	2.0
Gentamicin Sulfate	202.27	0.050 g	0.2
HEPES	238.30	2.600 g	11.0
Sodium Pyruvate	110.04	0.060 g	0.5
Magnesium Chloride Hexahydrate	203.30	0.102 g	0.5
Calcium Chloride Dihydrate	147.01	0.294 g	2.0

¹3.0 mg/mL bovine serum albumin added prior to use.

Table 2:

Composition of stock solutions used to make Tyrode’s Albumin, Lactate, and Pyruvate (TALP) Medium.

Compound	Amount	MW (g/mol)	Ultra-Pure Water	Stock Concentration
Sodium Chloride	9.2 g	58.44	1000 mL	157 mM
Potassium Chloride	2.48 g	74.55	200 mL	166 mM
Calcium Chloride Dihydrate	3.52 g	147.01	200 mL	120 mM
Magnesium Chloride hexahydrate	4.88 g	203.30	200 mL	120 mM
D-(+)-Glucose	26.55 g	180.16	500 mL	295 mM
Sodium Bicarbonate	2.8 g	84.01	200 mL	167 mM
Sodium DL-lactate solution, 60% (w/w)	2.0 mL	112.06	70 mL	1.7% (149 mM)

Table 3:

Preparation of Tyrodes’s Albumin¹, Lactate, and Pyruvate¹ (TALP) Medium.

Component	Amount per 500 mL	Concentration (mM)
Sodium Chloride Stock	300 mL	114.0
Phenol Red	0.005 g	28.2
Sodium D,L-Lactate Stock	33.5 mL	10.0
Gentamicin Sulfate	25 mg	0.2
Potassium Chloride Stock	9.5 mL	3.2
Calcium Chloride Dihydrate Stock	8.5 mL	2.0
Magnesium Chloride Hexahydrate Stock	2.05 mL	0.5
Sodium Bicarbonate	75 mL	25.0
Sodium Phosphate Stock2	8.5 mL	0.3

¹Add 3 mg/mL BSA and 0.006g/100mL (0.5 mM) sodium pyruvate prior to use

²Prepared in glucose stock solution for a final concentration of 5.0 mM glucose.

2.2. Controlled Ovarian Stimulation (COS)

1. Recombinant human follicle stimulation hormone (r-hFSH).

2. Recombinant human luteinizing hormone (r-hLH).

3. Human chorionic gonadotropin (hCG): available commercially either as a recombinant product or purified from the urine of pregnant women.

4. Gonadotropin Releasing Hormone (GnRH) antagonist.

3. Methods

Both indoor and outdoor housed rhesus macaques undergo a seasonal breeding cycle where ovulation typically initiates in September and continues until May. The months of June-August constitute the off-season, where animals may still experience menses but ovulation of mature ova may not occur (19). When selecting animals to use for in vitro embryo production, it is best to avoid the off-season months as oocyte quality and embryo development can be affected. Additionally, using regularly cycling females between the ages of 6 and 12 years is ideal for providing developmentally competent ova. Characterization of the ovarian cycle can be determined by measuring estradiol-17β (E₂) and progesterone (P₄) in blood serum and has been well described (20–23). The following methods describe the steps necessary to produce and culture rhesus macaque embryos in vitro, including ovarian stimulation to recover multiple mature ova, sperm preparation, IVF, IVC, and embryo cryopreservation (Figure 1).

Figure 1.

Timeline of events for in vitro production of rhesus macaque embryos.

3.1. Controlled Ovarian Stimulation (COS)

For this 10-day protocol, r-hFSH and r-hLH are used to stimulate development of multiple ovarian follicles. GnRH antagonist is used to suppress the endogenous LH surge. The timed bolus of hCG initiates the ovulatory process inducing the signaling cascade for meiotic resumption resulting in mature ova (Figure 2).

Figure 2.

Schematic representation of the Controlled Ovarian Stimulation (COS) protocol used to promote development of multiple antral follicles in rhesus macaques. Protocol should be initiated within the first 4 days after the onset of menses.

1. Begin the COS protocol within the first 4 days after the onset of menses (beginning of the follicular phase). Blood samples to measure serum estradiol (E₂) and progesterone (P4) should be taken daily prior to morning administration of gonadotropins. On day 1 (first day of injections) of the protocol, E₂ concentrations should be 30–60 pg/mL and P₄ concentrations should be approximately 0 (<0.05 ng/mL) (see Note 4).

2. Administer 30 IU of r-hFSH intramuscularly (IM), in a.m. and p.m., with at least 8 hours between doses on days 1–8.

3. Similar to r-hFSH, administer 30 IU of r-hLH IM, a.m. and p.m., with at least 8 hours between doses on days 7 and 8 (See Note 5).

4. When serum E₂ concentrations are >200 pg/mL, administer a single dose of 1 mg/kg GnRH antagonist subcutaneously in the morning of the following day. If P₄ begins to rise prior to the hCG, a second dose of 1 mg/kg GnRH antagonist may be given.

5. On day 8, administer 1100 IU of hCG IM in the p.m., timed so follicle aspiration occurs 36 hours later. For example, an 8:00 p.m. dose on day 8 accommodates an 8:00 a.m. follicle aspiration on day 10. Serum P₄ should measure approximately 1ng/mL or higher the morning following hCG administration. If P₄ remains low following hCG, it is presumed the animal did not respond to the bolus and oocyte maturation may not have been initiated. In this case, the COS protocol is considered unsuccessful and retrieval for ova should not be attempted.

6. Aspirate follicles 36 hours following hCG to ensure collection of mature ova. Follicular aspirates should be collected into warmed TALP-HEPES with 5 IU/mL heparin added to prevent blood clot formation. If collection time is delayed beyond 37 hours, follicles may undergo ovulation diminishing the number of recoverable ova.

7. Isolate oocytes into warmed TALP-HEPES and wash free of any residual blood and follicle cells. Remove cumulus granulosa cells surrounding the oocyte by gentle pipetting to visualize the cytoplasm and assess meiotic stage (Figure 3). Metaphase II (MII) oocytes with an extruded polar body are considered mature and able to undergo fertilization. Oocytes that have not initiated meiotic resumption and have a visible germinal vesicle (GV) or those undergoing meiosis (GVBD or metaphase I, MI) are considered immature and may require additional culture time in TALP medium.

8. Wash ova selected for fertilization into fresh, equilibrated TALP. Add 5 ova per 100 µL drop (covered with oil and equilibrated) and maintain at 37°C in 5% CO₂ humidified air until insemination – this will later increase the total volume of each drop to 111 µL.

Figure 3.

Stages of meiosis in recovered rhesus macaque oocytes. A) Germinal vesicle (GV) intact (arrow), oocyte has not yet resumed meiosis and is considered immature; B) Metaphase I (MI), oocyte has resumed meiosis but not yet considered mature; C) Metaphase II (MII), ovum has completed meiosis, extruded the first polar body (arrow), and is now considered mature.

3.2. Collection and Processing of Spermatozoa for In Vitro Fertilization (IVF)

When performing IVF, fresh semen samples are preferred. They can be easily washed by centrifugation and typical samples maintain motility throughout culture. This protocol describes the process to wash the sperm from the seminal plasma, assess motility, and determine optimal concentration for IVF. Appropriate personal protection equipment (PPE) should always be worn when handling biological samples from nonhuman primates.

1. Semen collected by electro-ejaculation with penile stimulation will form a seminal coagulum that would otherwise function as a post-coital plug during natural breeding (24). The plug forms quickly at the time of semen collection, trapping spermatozoa in the plug. To recover the spermatozoa, samples should be maintained undisturbed for 30 minutes at ambient temperature to allow liquefaction of the outer layer of the plug and release of motile spermatozoa.

2. Transfer the liquefied portion of the sample to a 15 mL conical tube and record the volume.

3. Use 1 mL of warm (37°C) TALP-HEPES to rinse the semen plug and collection container. Add the wash to the same conical tube containing the liquefied sample and repeat the wash twice more. Add enough warmed TALP-HEPES into the conical tube to bring the final volume to 10 mL.

4. Centrifuge the sample for 8 minutes at 1400 rpm (no more than 400 x g) at room temperature.

5. Discard the supernatant, being careful not to disturb the medium close to the pellet that may contain live sperm. Suspend the sperm pellet with 10 mL of warmed TALP-HEPES.

6. Centrifuge the sample for another 8 minutes at room temperature.

7. Discard enough supernatant to leave a final volume of 1 mL. Mix gently, avoiding rapid pipetting as the shear force may damage the sperm.

8. Add 10 µL of the washed sperm to 190 µL of warmed TALP-HEPES in a 0.5 mL microcentrifuge tube. Mix gently then place 10 µL on a glass microscope slide and place a coverslip over the sample.

9. Using an upright compound phase contrast microscope, focus on the center of the sample to score a group of approximately 100 sperm for motility. Motile sperm are those that exhibit active, forward progression. Slow moving and twitching sperm should be scored as non-motile. Record the percentage of motile sperm.

10. Determine the concentration of the washed sperm sample and adjust the final concentration to 20 million sperm/mL in 1 m of TALP. Maintain the sperm sample at 37°C in 5% CO₂ humidified air until ready to perform IVF.

3.3. In Vitro Fertilization (IVF)

Once oocytes have been recovered and confirmed to have completed meiosis, insemination should occur within 4 hrs. This allows time to obtain and evaluate the semen sample for suitability and acquire additional samples if necessary. Sperm cells must undergo hyperactivation to initiate sperm capacitation, observed as increased motility characterized by pronounced flagellar movements, a non-linear trajectory, and acquisition of the ability to undergo the acrosome reaction upon zona binding before introduction to the ova (insemination) (25). This protocol describes the use of gametes intended to be used on the same day as they were collected, although oocytes that have undergone in vitro maturation (IVM) would be inseminated similarly but require a source of fetuin during IVM to prevent zona hardening (see Note 6). All incubations are performed at 37°C in 5% CO₂ humidified air.

1. To activate the sperm, combine 900 µL of the 20 million/mL sperm sample in TALP with 100 µL of the sperm activation solution and maintain in the incubator for 15 minutes. Check for activation by observing a small drop of sperm under the microscope for increased, rapid movement.

2. Add 10 µL of warmed sperm activation solution to each fertilization drop containing oocytes, then follow by adding 1 µL of activated sperm (see Note 7). It is best to observe the addition of sperm under the microscope to ensure the full microliter is delivered. Note the insemination time and incubate the oocytes with spermatozoa for 14–16 hours.

3. Upon completion of gamete co-incubation, remove the ova from the TALP drops and wash in TALP-HEPES. Use a micropipettor with a 125 µm diameter tip to gently remove remaining granulosa cells and attached sperm, which should dislodge easily from the zona without enzymatic treatment. Transfer the oocytes to a fresh well containing 500 µL of TALP-HEPES and note the presence of polar bodies (PB) and/or pronuclei (PN) (Figure 4, A-B).

4. When evaluations are complete, transfer presumptive zygotes to equilibrated medium for culture.

Figure 4.

Stages of embryo development. A) Zygote with 2 polar bodies (PB) in the perivitelline space (arrows); B) Zygote with 2 pronuclei (PN) present (arrows); C) 2-cell; D) 4-cell; E) 12-cell; F) compacted morula; G) blastocyst with clearly forming blastocoel (asterisk); H) expanded blastocyst; I) collapsed blastocyst.

3.4. In Vitro Embryo Culture (IVC)

While performing embryo culture, the amount of time the embryos are out of the incubator should always be minimized to avoid excessive changes in temperature and pH. When observing embryos for developmental progress, also assess the culture medium on multiple focal planes for signs of bacterial or fungal contamination.

1. Day 0 – On the day of IVF, prepare 2–60 mm culture plates each containing 4 drops (100 µL/drop) of embryo culture medium layered with enough mineral oil to completely cover the medium – approximately 7 mL. One plate will serve to wash embryos out of TALP-HEPES from step 4 above and the other will be used to culture embryos. Allow the plates to equilibrate at 37°C in an atmosphere of 6% CO₂, 5% O2, and 89% N₂ with humidified air overnight.

2. Day 1 - Wash presumptive zygotes recovered from the IVF culture through at least 3 drops of embryo culture medium in the wash plate before transferring to a fresh drop of culture medium, maintaining no more than 5 embryos/drop.

3. Day 2 - Most embryos will begin to cleave by the second day in culture. Observe individual embryos for developmental quality characteristics. The nuclei should be clearly visible and the blastomeres should have homogenous cytoplasm with little to no retraction (clearing) around the periphery. There should be no vacuole formation or fragmentation of blastomeres. Any zygotes that have not cleaved or are obviously degenerating, should be removed.

4. Day 3 - Developing embryos should be actively cleaving and have reached the 8–12 cell stage. Wash embryos through at least 3 drops of fresh, pre-equilibrated (18– 24 hours), culture medium and transfer to a new plate containing 100 µL drops culture medium covered in oil for continued culture.

5. Day 4 – All embryos in culture should have developed to the 12–16 cell stage.

6. Day 5 - Healthy embryos should begin to show signs of compaction, where individual blastomeres become indistinguishable (Figure 4F). All viable embryos should be washed and moved to fresh culture medium as before – 100 µL drops, pre-equilibrated for 18–24 hours, and covered in oil.

7. Day 6 – Early stage blastocysts should begin developing cavities that will form the blastocoel.

8. Day 7 – Blastocysts should have a well-formed blastocoel with minimal cellular fragmentation (Figure 4G). Embryos with continue development are washed and moved to fresh, pre-equilibrated culture medium – 100 µL drops, pre-equilibrated for 18–24 hours, and covered in oil.

9. Day 8 – Blastocysts may undergo expansion with a noticeable increase in size and a thinning of the zona pellucida (Figure 4H). Some embryos may collapse after expansion and have a similar appearance to a compacted morula (Figure 4I).

10. Day 9 – Expanded blastocysts of good quality may hatch and either be partially or completely extruded from the zona pellucida.

3.5. Blastocyst Cryopreservation

Blastocysts intended for embryo transfer can be cryopreserved until a recipient is properly synchronized (20). Protocols developed for use with human blastocysts are effective when followed as directed by manufacturer instructions. While the longevity of embryos stored in liquid nitrogen has yet to be determined, they can be frozen and subsequently thawed at the time of embryo transfer to produce viable offspring (23).