Embryo production and possible species preservation by nuclear transfer of somatic cells isolated from bovine semen

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Theriogenology 74 (2010) 1629 –1635 www.theriojournal.com

Embryo production and possible species preservation by nuclear transfer of somatic cells isolated from bovine semen Jie Liua, Mark Westhusina, Charles Longa, Gregory Johnsonb, Robert Burghardtb, Duane Kraemera,* a

Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA b Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843, USA Received 12 January 2010; received in revised form 21 June 2010; accepted 27 June 2010

Abstract Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals; this is especially important when an animal has died and the only viable genetic material available is frozen semen. Usefulness of somatic cells obtained from fresh (cultured) and frozen (isolated, not cultured) bovine semen for nuclear transfer was evaluated. Twelve ejaculates were collected from nine bulls representing three breeds: Charolais, Brahman, and crossbred Rodeo bull. All samples were processed immediately and cell growth was obtained from seven of the twelve ejaculates (58.3%). Cells from three bulls (with the best growth rates) were evaluated by optical microscopy and used in cloning experiments. In culture, these cells exhibited classic epithelial morphology and expressed cytokeratin and vimentin, indicating they were of epithelial origin. When cells from the three bulls were used as donor cells, 15.9% (18/113), 34.5% (29/84), and 14.4% (13/90) of the fused embryos developed into blastocysts, respectively. Of the blastocyst stage embryos, 38.9% (7/18), 72.4% (21/29), and 61.5% (8/13) hatched, respectively. Somatic cells isolated (not cultured) from frozen bovine semen were also used in the cloning experiments. Although cleavage occurred, no compact morulae or blastocysts were obtained. In conclusion, epithelial cell growth was obtained from fresh bovine ejaculates with relatively high efficiency. Somatic cells from semen can be used as nucleus donors to produce cloned blastocyst-stage embryos. © 2010 Elsevier Inc. All rights reserved. Keywords: Nuclear transfer; Somatic cell; Bovine; Semen; Species preservation

1. Introduction The birth of Dolly in 1997 [1] first demonstrated that fully differentiated adult somatic cells can be reprogrammed to produce live clones. Although an inefficient process, nuclear transfer has been widely used to produce genetically identical animals for research and commercial purposes. Endangered [2–5], exotic [6],

* Corresponding author. Tel.: ⫹ 1 979 845 4220; fax: ⫹ 1 979 458 3635. E-mail address: [email protected] (D. Kraemer). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.06.035

extinct, and transgenic animals (when combined with molecular biology techniques) [7–11] have also been produced by nuclear transfer techniques. Semen contains somatic cells, a potential source of nuclei for nuclear transfer to produce genetically identical animals. Many animals of genetic value have died and the only viable genetic material available is frozen semen. If somatic cells in semen could be used for nuclear transfer, this would represent an extremely valuable resource for preservation or restoration of these animals. Therefore, the objectives of this investigation were to evaluate: 1) the potential for isolating

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and propagating somatic cells from bovine semen for use in nuclear transfer; and 2) cleavage and blastocyst development rates of cloned embryos produced using somatic cells from fresh and frozen bovine semen. 2. Materials and methods 2.1. Reagents Dulbecco’s Modified Eagle Medium/ Nutrient Mixture F-12 (DMEM/F-12), M199 (Hanks’ salts), and M199 (Earle’s salts) were purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Bovine pituitary extract was obtained from BD Biosciences (Bedford, MA, USA). Fluorescein-conjugated goat-anti-mouse IgG was ordered from Zymed (San Francisco, CA, USA). Prolong antifade was ordered from Molecular Probes (Eugene, OR, USA). Bovine follicle stimulating hormone (bFSH) was obtained from Sioux Biochemical (Sioux Center, IO, USA). G1/G2 medium was purchased from Vitrolife (Englewood, CO, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise indicated. 2.2. Semen collection and somatic cell culture Twelve ejaculates were collected from nine bulls by electro-ejaculation [12,13], with one or two samples being collected from each animal. These nine bulls included three 1 y old Charolais, two 1 y old crossbred Rodeo bulls, one 5 y old Brahman, two 6 y old Brahmans, and one 8 y old Brahman. All bulls were in good health at the time of collection. Somatic cells were isolated and cultured as described [14,15], with minor modifications. Briefly, each semen sample was evenly distributed on top of four 15 mL conical tubes, each of which contained a column of 2.5 mL each of 20, 50, and 90% Percoll. The tubes were centrifuged at 400 ⫻ g for 20 min. Cells in the 20% and top half of the 50% Percoll layers were washed two or three times in DMEM/F12 (supplemented with 100 ␮g/mL gentamycin) by centrifugation at 400 ⫻ g for 10 min. After the last wash, cell pellets were re-suspended in DMEM/ F-12 supplemented with 10% FBS, 10 ng/mL epidermal growth factor, 30 ␮g/mL bovine pituitary extract, 5 ␮g/mL insulin, 10 ng/mL cholera toxin, 200 ␮g/mL gentamycin, 100 units/mL penicillin, 100 ␮g/mL streptomycin, and 0.25 ␮g/mL amphotericin B, and cultured in T-75 flasks with a seeding density of (0.8 –2.5) ⫻ 104 cells/cm2 at 37 °C with 5% CO2 in humidified air. Medium was changed 24 h after plating. Culture dishes

were checked for contamination once daily for 1 wk. Medium containing bacteria and/or fungi was replaced with fresh medium until contamination was eliminated. The concentration of gentamycin in the medium was stepwise decreased with medium changes to 150, 100, and finally 50 ␮g/mL. Penicillin, streptomycin, and amphotericin B were likewise gradually excluded from the culture medium (within 1–2 wk of culture). Cells were fed twice a week with fresh medium until 50 – 80% confluency was reached in 2–3 wk. These cultures were then trypsinized using 0.5 mg/mL trypsin to remove cells from culture surfaces. Cells were suspended in DMEM/F12 supplemented with 10% FBS, collected by centrifugation, frozen as suspension in DMEM/F-12 supplemented with 20% FBS and 10% dimethyl sulfoxide, and stored in liquid nitrogen to be used for immunofluorescence analysis or nuclear transfer experiments. Some of these cells were kept in culture to determine their life span. 2.3. Immunofluorescence analysis Cells cultured from semen of three different bulls were characterized by immunofluorescence microscopy, as previously described [14,16]. Cells from each bull were trypsinized, re-suspended, and the cells were counted using a hemocytometer. Cells from each bull were then transferred to a four-well chamber slide (Nunc, Naperville, IL, USA) at a concentration of 4 ⫻ 104 cells/well, cultured in DMEM/F-12 supplemented with 10% FBS, 10 ng/mL epidermal growth factor, 30 ␮g/mL bovine pituitary extract, 5 ␮g/mL insulin, 10 ng/mL cholera toxin, and 50 ␮g/mL gentamycin, and incubated at 37 °C with 5% CO2 in humidified air until the cells reached 60 to 70% confluence. The slides were then fixed in ⫺20 °C methanol for 10 min, permeabilized with 0.3% Tween 20 in 0.02 M PBS (PBS/Tween), and blocked in 10% (v/v) goat serum in antibody dilution buffer (AbDB; two parts 0.02 M PBS, pH 8.0, 0.3% Tween 20, 1% bovine serum albumin, and one part glycerol). After a quick rinse in PBS/Tween, cells in each of the four wells were incubated with 2 ␮g/mL mouse anti-IgG (negative control), anti-cytokeratin (Hybridoma 8.13), anti-vimentin (Hybridoma V9), or anti-␣-smooth muscle actin (F-3777) in AbDB at 4 °C overnight. After three rinses in PBS/Tween for 10 min each, the slides were incubated with fluorescein-conjugated goat-anti-mouse IgG in AbDB at room temperature for 1 h. Two drops of Prolong anti-fade containing DAPI were added to each slide, which was then gently overlaid with a cover glass. The slides were evaluated with an appropriate filter set using an Axioplan 2 mi-

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croscope (Carl Zeiss, Thornwood, NY, USA) interfaced with an Axioplan HR digital camera and Axiovision 4.3 software. Photographic plates were assembled using Adobe Photoshop (version 6.0, Adobe Systems Inc., San Jose, CA, USA). 2.4. Preparation of donor cells Cells obtained from semen of three bulls were used in cloning experiments. The cells were thawed and cultured (without serum starvation) to 90 –100% confluency. Immediately before nuclear transfer, the cells were trypsinized and re-suspended in M199 (Hanks’ salts) supplemented with 10% FBS. Passage number of these cells was between 3 and 14. Somatic cells isolated from frozen bovine semen, but not cultured, were also used in the cloning experiments. Fifteen to twenty straws (0.25 mL/straw) of semen were thawed each time to obtain dozens to several hundred somatic cells. The same isolation procedures used for fresh semen were applied. After isolation, somatic cells were held at 4 °C in DMEM/ F-12 supplemented with 10% FBS and 100 ␮g/mL gentamycin for 3 h before being used in cloning experiments. 2.5. Preparation of oocytes Cow ovaries were collected from a local abattoir and transported to the laboratory in warm saline solution within 2– 4 h. Oocytes were released from antral follicles (⬎3 mm in diameter) by mincing using a scalpel blade in a Petri dish filled with pre-warmed M199 (Hanks’ salts) supplemented with 5% FBS and 50 ␮g/mL gentamycin. Compact cumulus-oocyte complexes (COCs) with evenly distributed cytoplasm and at least two or three layers of cumulus cells were selected for maturation. After washing twice in maturation medium, 50 COCs were transferred to each well of a four-well dish (Nunc, Naperville, IL, USA) containing 500 ␮L of maturation medium per well. The COCs were incubated at 38.5 °C with 5% CO2 in humidified air for 18 h. Maturation medium was composed of M199 (Earle’s salts), 10% FBS, 0.045 unit/mL bFSH, 1 mg/mL estrogen, and 50 ␮g/mL gentamycin. These oocytes served as recipient cytoplasts for cloning by electrical fusion. Alternatively, oocytes were purchased from Ovitra Biotechnology (Hereford, TX, USA) and transported to the laboratory overnight in maturation medium in a portable incubator (temperature, 38 °C). These oocytes served as recipient cytoplasts for cloning by piezo-assisted direct injection.

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2.6. Enucleation After in vitro maturation, cumulus cells were removed by repeated pipetting in M199 (Hanks’ salts) supplemented with 0.3% hyaluronidase for 2–3 min. Oocytes with good morphology [17] and the presence of a first polar body were selected and stained in M199 (Earle’s salts) supplemented with 10% FBS, 5 ␮g/mL Hoechst 33342, and 5 ␮g/mL cytochalasin B for 10 –15 min at 38.5 °C. A drop (200 ␮L) of M199 (Hanks’ salts) supplemented with 10% FBS was placed on the cover of a 35 ⫻ 10 mm tissue culture dish (BD Falcon, Franklin Lakes, NJ, USA) and covered under mineral oil. The cover was then placed onto heated (38 °C) stage of an inverted microscope (Nikon Instruments Inc., Melville, NY, USA) equipped with Narishige micromanipulators (Narishige International USA, Inc., East Meadow, NY, USA). A group of 15 to 20 oocytes was transferred to the drop each time. Each oocyte was exposed to ultraviolet (UV) light briefly to locate the metaphase plate and sometimes the first polar body. The metaphase plate and polar body were then gently removed with a micropipette. Successful enucleation was confirmed by brief exposure of the oocyte to UV light. On average, each oocyte was exposed to UV light for 3 s or less. 2.7. Injection and fusion For cells cultured from fresh bovine ejaculates, a single cell was inserted into the perivitelline space of an enucleated oocyte by an injection pipette. After equilibration in fusion medium (0.28 M mannitol, 0.1 mM CaCl2, and 0.1 mM MgSO4), reconstructed oocytes were transferred to a drop of fusion medium placed on a 1 mm electrofusion chamber. After manual alignment, fusion was induced by applying two consecutive 2.4 KV/cm, 20 ␮s direct-current pulses delivered by an Eppendorf Multiporator (Eppendorf, North America). For somatic cells isolated from frozen semen samples, piezo-driven pulses were applied to penetrate through the zona pellucida and the oolemma. A single piezo pulse was attempted to break the somatic cell membrane before depositing it into the ooplasm [18]. Size of the injection pipette was ⬃15 ␮m in outer diameter. Cells cultured from fresh bovine semen were used as positive controls. 2.8. Activation From 1.5 to 2.0 h after fusion or direct injection, reconstructed couplets were equilibrated in fusion medium, then transferred to the electrofusion chamber containing a drop of fresh fusion medium. Activation was induced by applying

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Table 1 Number of semen collections from each bull and number of collections from which cell growth was obtained. Animal code

Breed

Age (y)

No. collections

No. collections with cell growth

Cell life span (passage no.)

1 2 3 4 5 6 7 8 9

Charolais Charolais Charolais Crossbred Rodeo Crossbred Rodeo Brahman Brahman Brahman Brahman

1 1 1 1 1 5 6 6 8

1 1 1 1 2 1 2 1 2

1 0 0 1 Contaminated Contaminated 2 1 2

⬎2 mo (⬎15) 0 0 ⬃1 mo (0) 0 0 ⬎2 mo (⬎15) ⬃2 mo (8) ⬎2 mo (⬎15)

two consecutive 0.4 KV/cm, 55 ␮s direct-current pulses, followed by incubation in M199 (Earle’s salts) supplemented with 10% FBS, 10 ␮g/mL cycloheximide and 5 ␮g/mL cytochalasin B for 5 h in a humidified 5% CO2, 5% O2, and 90% N2 gas mixture at 38.5 °C.

3. Results 3.1. Semen collection and somatic cell culture

Nuclear transfer embryos were washed three times in M199 (Earle’s salts) and once in commercially available G1 medium [19] and cultured in G1 medium in a humidified 5% CO2, 5% O2, and 90% N2 gas mixture at 38.5 °C. Three days later, embryos were transferred to G2 medium [19] and cultured for an additional 7 to 8 d. Cleavage and blastocyst development rates were examined on Days 3, 7 and 10 (Day 0 was the day of nuclear transfer) of in vitro culture.

Total number of cells obtained from each ejaculate varied, ranging from (0.8 – 4) ⫻ 106. Either no cell attachment was found or a few cells per ejaculate attached and started to divide. Cell growth was obtained from seven of the twelve ejaculates (58.3%; Table 1). Culture dishes containing cells from three ejaculates (from Bulls 5 and 6) had severe bacterial and/or fungi contamination and were discarded within 1 wk of culture. Cells obtained from two bulls (4 and 8) stopped dividing in 1 or 2 mo. Cells from the other three bulls (1, 7, and 9) proliferated for more than 2 mo and were used in subsequent experiments (Fig. 1A).

2.10. Statistical analysis

3.2. Immunofluorescence analysis

One-way ANOVA was used to compare blastocyst development rates of embryos produced using cells cultured from semen of three bulls. The same statistical method was used to compare cleavage rates of embryos produced by piezo injection of somatic cells obtained from frozen and fresh (positive control) semen. Differences were considered significant at P ⬍ 0.05.

A single cell type was successfully cultured from semen of the three bulls (Fig. 2). These cells (maybe partially urine derived) [20] had classic cobblestone morphology and expressed cytokeratin, indicating that they were of epithelial origin. These cells also expressed vimentin, indicating that they had undergone or were undergoing an epithelial-to-mesenchymal trans-

2.9. Embryo culture

Fig. 1. Cells cultured from bovine ejaculates and cloned blastocysts produced using these cells. (A) Cells cultured from bovine ejaculates (primary culture). These cells had classic cobblestone morphology and a discrete edge to the colony. (B) Blastocysts produced using epithelial cells cultured from bovine ejaculates. Scale bar: 50 ␮m.

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Fig. 2. Immunofluorescence analysis of cells (passages 2– 6) cultured from bovine semen. These cells were both cytokeratin and vimentin positive. (A) anti-cytokeratin. (B) anti-vimentin. Scale bar: 20 ␮m.

formation that is often observed in primary cultures of epithelial cells [16]. Little to no ␣-smooth muscle actin expression was observed; therefore, it was unlikely that they were of myofibroblast origin. 3.3. Somatic cell nuclear transfer When cells from three of the bulls, 1 y old Charolais, 6 y old Brahman, and 8 y old Brahman were used as donor cells, 15.9% (18/113), 34.5% (29/84), and 14.4% (13/90) of the fused embryos developed into blastocysts, respectively (Fig. 1B and Table 2). Of these blastocyst-stage embryos, 38.9% (7/18), 72.4% (21/ 29), and 61.5% (8/13) hatched, respectively. There was no difference (P ⬎ 0.05) among these three bulls for blastocyst development rates of embryos produced using cells from these bulls (Table 2). In the frozen semen group, 27.4% (82/299) of recombined couplets were either cleaved or fragmented. Of the 82 embryos, 48 were examined and 42 were determined to be fragmented (contained intact donor cells). All of the six cleaved embryos arrested at or before the eight-cell stage. No compact morulae or blastocysts were obtained. Based on staining with propidium iodide (20 ␮g/mL), ⬍10% of somatic cells isolated from frozen bovine semen were viable. In the positive control group, cleavage rate was 98.6% (73/74), which was greater than that of the frozen semen group (P ⬍ 0.05). Three blastocysts (4%) were obtained (Table 3).

4. Discussion Extensive work has been done to identify the types of somatic cells in human semen. Cells found are epithelial cells, and various major leukocyte subpopulations including granulocytes, monocytes/macrophages, B lymphocytes, helper and suppressor/cytotoxic T lymphocytes [21–23]. However, very limited similar work has been done on livestock, and none on companion animals. Phillips et al. [21] reported that epithelial cultures, uncontaminated by fibroblasts, were obtained from human semen samples. Epithelial cultures have also been obtained from sheep [14] and eland [24] semen. In the present study, cell growth was obtained from fresh bovine ejaculates with relatively high efficiency. A supplemented medium we previously used in culturing somatic cells from ram semen showed beneficial effects in growing these cells from bovine semen [14; unpublished results]. Although the genital area was cleaned before each semen collection, contamination remained a problem when growing these cells in culture. We found that 24 h after plating, gentle washing of the culture surface to which cells might have attached using culture medium helped remove bacteria and fungi, thus decreasing the concentrations of antibiotics required in the culture medium and increasing the

Table 2 In vitro development of cloned embryos produced by epithelial cells cultured from semen of three bulls. Donor (age)

Reconstructed oocytes

Fused couplets (%)

Cleaved (%)

Compact Morulae

Blastocysts (%)a

Hatched blastocysts (%)b

Charolais 1 y Brahman 6 y Brahman 8 y

182 118 140

113 (62.1) 84 (71.2) 90 (64.3)

98 (86.7) 72 (85.7) 79 (87.8)

26 33 13

18 (15.9) 29 (34.5) 13 (14.4)

7 (38.9) 21 (72.4) 8 (61.5)

a b

Percentage of fused couplets developing to blastocysts. Blastocyst development rates did not differ among bulls (P ⬎ 0.05). Percentage of blastocysts hatched.

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Table 3 In vitro development of cloned embryos produced by piezo-injection of somatic cells obtained from frozen and fresh (positive control) semen. Donor cell source Frozen semen Fresh semen (Brahman, 6 y)

No. oocytes injected 488 108

Survived injections

Cleaved (%) b

299 (61.3) 74 (68.5)

10 (4.4)* 73 (98.6)c

Compact morulae

Blastocyts (%a)

0 5

0 3 (4)

* Estimated number. a Percentage of recombined couplets developing to blastocysts. b,c Within a column, values without a common superscript differ (P ⬍ 0.05).

chances of obtaining cell growth. Frequent medium changes during the first 24 –72 h post plating also helped prevent contamination. The cells cultured from bovine ejaculates exhibited an epithelial morphology and expressed cytokeratin and vimentin intermediate filaments. The same expression pattern was observed in cells cultured from ovine semen [14]. Epithelial-to-mesenchymal transformation is often observed in primary cultures of epithelial cells; this likely accounted for vimentin expression in epithelial cells cultured from semen [16]. Although blastocysts were obtained when the cells derived from eland semen were used as karyoplast donors for nuclear transfer, the experimental number was small [24]. Our findings agreed with these results and confirmed that somatic cells in bovine semen may serve as nucleus donors for nuclear transfer. Intergeneric nuclear transfer of semen derived eland epithelial cells into enucleated bovine oocytes has been reported [25]. However, a high percentage of the cloned embryos had developmental arrest before the eight-cell stage and no blastocyst formation occurred. Intrinsic developmental potency of the donor cell may play a role in the cloning outcome [26]. Passage number of donor cells ranging from 3 to 14 were used, but no difference in cloning efficiency was found (unpublished results). Cloning by direct injection resulted in lower blastocyst rates than those by fusion (4% vs. 14.4 –34.5%). This may be partially due to the large size of the injection pipette used for injection. Although similar results have been reported for cattle [27], cloning by direct injection and fusion had similar blastocyst rates in pigs [28]. Isolating and propagating somatic cells from frozen semen can be difficult, especially with a limited number of semen straws/pellets. However, nuclei of the somatic cells in frozen semen may be used for nuclear transfer, even though the cell membrane and cytoplasm are damaged during the semen freezing process. In the present study, somatic cells isolated (not cultured) from frozen semen were also used in the cloning experiments. Electrical fusion (unpublished results) and piezo assisted

direct injection were applied to introduce nuclei of these cells into enucleated bovine oocytes. Although cleavage occurred and some embryos reached six- to eight-cell stages, no compact morulae or blastocysts were obtained. The majority of somatic cells in frozen semen were flat and their membranes were rigid. Several piezo pulses and drawing in and out of the injection pipette were attempted, with no change detected in the somatic cell membrane. It was likely that a whole cell with minor to no membrane damage was introduced into the oocyte cytoplasm. It may be possible to use enzymatic treatment or sonication to release or expose the nucleus for direct injection. Since some of the cloned embryos developed beyond two-cell stage, perhaps using nuclei of these blastomeres for a second round of cloning would be more successful. In conclusion, epithelial cells were cultured from fresh bovine ejaculates with relatively high efficiency and cloned blastocysts were produced from cells of three donor bulls. With optimized nuclear transfer protocol and improved cryopreservation procedures for semen, somatic cells in frozen semen could be a valuable resource for recovering lost genetics. Acknowledgments We thank Kimberly Green and Dr. Gabriela Foxworth at Global Genetics and Biologicals (Bryan, Texas) for kindly providing fresh bovine semen. Frozen bovine semen was donated by Dr. Brad Stroud at BioTech Productions (Weatherford, TX, USA). Special thanks to Nilay Chakraborty for helping with images and Gloria Y. Lee for assistance in preparing the manuscript. This work was supported by NIH Grant HL 073737-02. References [1] Wilmut L, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. Viable offspring derived from fetal and adult mammalian cells. Nature 1997;385:810 –3. [2] Lanza RP, Cibelli JB, Diaz F, Moraes CT, Farin PW, Farin CE, Hammer CJ, West MD, Damiani P. Cloning of an endangered

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