Production of diabetic offspring using cryopreserved epididymal sperm by in vitro fertilization and intrafallopian insemination techniques in transgenic pigs.

Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs.

The physiology and anatomy of pigs closely resemble those of humans; for this reason, pigs are regarded as a large animal model that can produce experimental data that are easily extrapolated to humans [1]. In recent years, genetic modification techniques have been used to develop pig strains that model several human diseases [2, 3].

We created genetically modified (GM) pigs that exhibit the symptoms of diabetes similar to maturity onset diabetes of the young 3 (MODY3). The diabetic symptoms of the GM pigs were induced by the dominant-negative effect of a transgene, human mutant hepatocyte nuclear factor 1 alpha (HNF-1α) [4]. In applying GM pigs to translational research, it must be ensured that enough animals can be supplied for experiments. Therefore, we aimed to establish an efficient procedure for proliferating our diabetes model pig by means of artificial reproductive technologies. In this study, the founder transgenic (Tg) cloned pig was generated by somatic cell nuclear transfer (SCNT) and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation.

Here, we report that in vitro fertilization (IVF) and intrafallopian insemination using cryopreserved epididymal sperm are useful options for reproducing the diabetes model pig. This reproductive system can theoretically be applied to other GM pigs, thereby enhancing the utility of disease model GM pigs.

Porcine fetal fibroblast cells carrying mutant human HNF-1α [4] were used to generate the founder Tg pigs by SCNT. SCNT was performed as described elsewhere using in vitro matured (IVM) oocytes as recipient cytoplasts [5]. In total, 333 SCNT embryos were generated and transferred to three estrus-synchronized gilts on day 1 or day 2; each recipient received 116, 109 or 108 embryos. The gilt that had received 116 embryos became pregnant and farrowed four Tg-cloned pigs. Three of the four piglets died within two days of birth for unidentified reasons or from being crushed by the recipient sow. The surviving piglet developed hyperglycemia (non-fasting blood glucose level: 374 mg/dl) at two weeks of age.

Administration of insulin started at three weeks of age, and the pig was raised for 15 months as the founder Tg-cloned boar. The non-fasting blood glucose level of the founder Tg-cloned boar was maintained at approximately 200 mg/dl by administration of a combination of regular insulin (Novolin R, Novo Nordisk, Bagsvaerd, Denmark) and long-acting insulin glargine (Lantus, Sanofi, Paris, France) (Table 1). The target blood glucose level was set higher than the normal blood glucose level for pigs (85–150 mg/dl) to avoid the risk of hypoglycemic coma.

Table 1.

Biochemical parameters in the plasma of the diabetes model founder transgenic-cloned pig

		Control (n = 10)	Founder
		Mean ± SD	4 monthsa	8 monthsa	12 monthsa	13 monthsb
ALT	(U/l)	28.2 ± 8.1	40.0	37.0	32.0	32.0
AST	(U/l)	24.8 ± 10.4	26.0	27.0	24.0	16.0
TP	(g/dl)	7.4 ± 0.7	6.7	6.5	6.1	6.5
TG	(mg/dl)	32.7 ± 27.1	48.0	28.0	41.0	74.0
T-CHO	(mg/dl)	70.7 ± 17.9	124.0	59.0	54.0	75.0
GLU	(mg/dl)	109.6 ± 26.1	246.0	230.0	195.0	536.0
1,5-AG	(mg/dl)	5.1 ± 1.8	-	1.2	1.1	1.0
BUN	(mg/dl)	9.9 ± 1.8	15.6	10.1	12.8	27.1
CRE	(mg/dl)	1.6 ± 0.5	0.8	1.5	1.4	1.5
Na	(mEq/l)	140.8 ± 2.9	136.0	134.0	143.0	135.0
K	(mEq/l)	4.9 ± 1.3	3.8	4.4	3.4	4.3
Cl	(mEq/l)	101.6 ± 2.4	99.0	94.0	97.0	94.0

a Treated with insulin. b Not treated with insulin. 1,5-AG, 1,5-anhydroglucitol; AST, aspirate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; Cl, chloride; CRE, creatinine; GLU, glucose; K, potassium; Na, sodium; T-CHO, total cholesterol; TG, triglyceride; TP, total protein.

The level of 1,5-anhydroglucitol (1,5-AG) in the founder Tg-cloned boar was lower than that in healthy pigs (Table 1), indicating that the animal developed hyperglycemia. By contrast, the levels of blood urea nitrogen (BUN) and of triglyceride (TG) in the blood during the insulin administration period were similar to those of healthy pigs (Table 1).

The founder Tg-cloned boar grew steadily and reached a peak weight of 169 kg at 12 months. The growth rate during the period between 3.5 months and 11 months was 3.7 kg/week, which was consistent with that of healthy wild-type (WT) pigs (3.6 kg/week). When insulin administration was temporarily stopped at 13 months, the founder pig started to lose weight, non-fasting blood glucose levels reached 536 mg/dl and BUN and TG levels worsened (Table 1). Therefore, these analyses demonstrated that the founder Tg-cloned boar exhibited typical diabetic symptoms that could be controlled by insulin administration.

Following the successful rearing of this founder Tg-cloned boar, we removed one testis at 8 months and the other at 12 months. Epididymal sperm was collected from each testis and cryopreserved using the method described by Kikuchi et al. [6] with a slight modification. After thawing, sperm samples were used for IVF and intrafallopian insemination.

The fertilization capacities of the Tg-cloned boar sperm at 8 months (DM-8m) and 12 months (DM-12m) of age were compared with those of WT boar sperm via IVF using IVM oocytes. IVF was performed using previously reported methods [7, 8] with slight modifications. A preliminary experiment revealed the optimal insemination concentration was 1.0×106 cells/ml for IVF with the WT boar sperm. Therefore, we adopted this condition for IVF with the Tg-cloned boar sperm.

The fertilization rate with the Tg-cloned boar sperm was significantly (P<0.05) lower than that with WT boar sperm: WT, 54.6%; DM-8m, 26.7%; DM-12m, 24.0%. IVF with the DM-8m sperm yielded rates of normal cleavage and blastocyst formation equivalent to IVF with WT sperm (Table 2). However, use of DM-12m sperm for IVF significantly reduced the rate of embryonic development (Table 2).

Table 2.

Efficiency of fertilization and development of porcine oocytes fertilized in vitro with cryopreserved boar sperm

Type of sperm	Inseminated sperm concentration (×106 cells/ml)	No. of oocytes			No. of oocytes
		Examined after insemination	Fertilized (%)	Normallyfertilized (%)	Cultured afterinsemination	Normally cleaved (%)	Developed to the blastocyst stage (%)
WT	1.0	97	53 (54.6)a	25 (47.2)a	69	42 (60.9)a	32 (46.4)a
DM-8m	1.0	45	12 (26.7)b	10 (83.3)	37	19 (51.4)a	14 (37.8)a
DM-12m	1.0	125	30 (24.0)b	28 (93.3)b	83	23 (27.7)b	14 (16.9)b

ab Significant differences (P<0.05).

The blood glucose level of the founder Tg-cloned boar was maintained at slightly higher levels than normal to avoid hypoglycemic coma. The fact that the Tg boar was maintained under suboptimal conditions for more than 6 months may have had a negative influence on sperm quality. Reduced sperm quality in male diabetic patients has been reported by several groups [9, 10]. Paasch et al. (2011) [11] reported increased accumulation of eppin (epididymal proteinase inhibitor) protein complex components, such as clusterin, lactotransferrin and semenogelin-1, in the sperm plasma membranes of diabetic patients. They also noted a possible influence of this eppin accumulation on the fertilization capacity of the sperm. A morphological study by Baccetti et al. (2002) [12] of diabetic patients’ sperm indicated an increased incidence of acrosomal abnormalities and aberrations of the sperm nuclei and tails. Together, these results suggest that the sperm quality of the Tg-cloned boar was adversely affected by diabetic symptoms.

The fertilization rate of porcine IVF has been reported to increase with sperm concentration [13, 14]. Thus, we examined whether elevated Tg-cloned boar sperm concentrations, above the level optimal for WT boar sperm (1.0×106), could improve the outcome of IVF. We found that embryo cleavage rates and blastocyst formation rates were highest at 2.5×106 cells/ml and 5×106 cells/ml for the DM-8m and DM-12m sperm, respectively (Table 3). Both of these concentrations are far above the optimal sperm concentration for WT boars in our experimental settings. These results may be coincident with the deteriorated fertility of the DM-12m sperm.

Table 3.

Determination of optimal sperm concentration for in vitro fertilization of cryopreserved diabetic boar sperm

Type of sperm	Inseminated spermconcentration (×106 cells/ml)	No. of oocytes			No. of oocytes
		Examined after insemination	Fertilized(%)	Normally fertilized (%)	Cultured after insemination	Normally cleaved (%)	Developed to the blastocyst stage (%)
DM-8m	1.0	45	12 (26.7)a	10 (83.3)	37	19 (51.4)	14 (37.8)
	2.5	125	86 (68.8)b	45 (52.3)	71	48 (67.6)a	41 (57.7)
	5.0	129	71 (55.0)b	45 (63.4)	66	31 (47.0)b	30 (45.5)
DM-12m	1.0	125	30 (24.0)a	28 (93.3)	83	23 (27.7)a	14 (16.9)a
	2.5	144	60 (41.7)b	51 (85.0)	95	43 (45.3)b	33 (34.7)b
	5.0	112	70 (62.5)c	58 (82.9)	67	38 (56.7)b	34 (50.7)b

ab, ac, bc Significant differences (P<0.05).

Next, we examined whether live piglets could be produced by IVF using cryopreserved Tg-cloned boar sperm. DM-12m sperm was used for IVF to verify the utility of the reduced-quality cryopreserved sperm. The insemination concentration was set at 5×106 cells/ml, and the resultant embryos at either the 1–8-cell stage (days 1–3) or the blastocyst stage (days 5–6) were transferred to estrus-synchronized recipients. In total, 319 embryos at the 1–8-cell stage were transferred into four recipient gilts; three became pregnant and gave birth to 22 live piglets. Seven of the live piglets (31.8%) were transgenic, carrying the human mutant HNF-1α transgene (Table 4). All of the Tg piglets exhibited hyperglycemia similar to the founder Tg-cloned boar, except for three of the Tg piglets that died before one week of age. Four of the Tg piglets developed to the weaning stage.

Table 4.

Production of offspring by in vitro fertilization and intrafallopian insemination of cryopreserved diabetic boar sperm

Methods	Recipients	No. of embryostransferred	No. of live piglets(tg piglets included)	No. of stillborn piglets(tg piglets included)	Proportion of tgpiglets/litter (%)	Average (mean ± SEM) bodyweight of piglets at birth (kg)
IVF-1a	P57	70	11 (4)	3 (0)	4/14 (28.6)	0.56 ± 0.05
	P84	112	7 (2)	0 (0)	2/7 (28.6)	1.09 ± 0.06
	P162	93	4 (1)	4 (3)	4/8 (50.0)	0.70 ± 0.13
	P58c	44	-	-	-	-
IVF-2b	P99	20	4 (1)	3 (1)	2/7 (28.6)	0.75 ± 0.10
	P135	15	5 (2)	0 (0)	2/5 (40.0)	0.84 ± 0.09
Intrafallopian insemination	B47	10d	6 (4)	2 (2)	6/8 (75.0)	1.07 ± 0.05
	B45c	10d	-	-	-	-
	B46c	8d	-	-	-	-
	B51c	14d	-	-	-	-

a Embryos were transferred at the 1–8-cell stage. b Embryos were transferred at the blastocyst stage. c Returned to estrus. d No. of ovulations.

A total of 35 blastocysts produced by IVF were transferred into two recipients, both of which became pregnant. The gilts produced 9 live piglets, of which three (33.3%) were transgenic (Table 4). One of those developed to the weaning stage and exhibited hyperglycemia.

These results demonstrated that DM-12m sperm could be used to produce viable piglets through IVF despite reduced fertility.

Piglets were also produced by intrafallopian insemination using cryopreserved Tg-cloned boar sperm. DM-8m sperm, which exhibited better fertilization capacity in IVF, was used to minimize the risk of infertility in this in vivo experiment. Insemination of four ovulation-induced gilts resulted in one pregnancy, which produced four live (66.7%) Tg piglets out of six farrowed (Table 4). Three of the four Tg piglets developed to the weaning stage and exhibited hyperglycemia.

In this study, we demonstrated that IVF and intrafallopian insemination using cryopreserved epididymal sperm were effective in reproducing the diabetes model Tg pig. Production of offspring through artificial reproductive technology was possible even using reduced-quality sperm from the founder Tg boar; therefore, this approach should be applicable for other pig strains that model human diseases.

Materials and Methods

Animal care

The Institutional Animal Care and Use Committee of Meiji University approved all animal experiments in this study (IACUC 09-0006).

In vitro maturation of porcine oocytes

SCNT and IVF were performed using IVM oocytes. Porcine ovaries were collected at a local abattoir and transported to the laboratory in Dulbecco’s phosphate-buffered saline (DPBS; Nissui Pharmaceutical, Tokyo, Japan) containing 75 µg/ml potassium penicillin G, 50 µg/ml streptomycin sulfate, 2.5 µg/ml amphotericin B and 0.1% (w/v) polyvinyl alcohol (PVA). Cumulus-oocyte complexes (COCs) were collected by aspiration from ovarian antral follicles with a diameter of 3.0–6.0 mm. COCs with at least three layers of compacted cumulus cells were selected and cultured in NCSU23 medium [15] supplemented with 0.6 mM cysteine, 10 ng/ml epidermal growth factor (EGF), 10% (v/v) porcine follicular fluid, 75 µg/ml potassium penicillin G, 50 µg/ml streptomycin sulfate, 10 IU/ml equine chorionic gonadotropin (eCG; ASKA Pharmaceutical, Tokyo, Japan) and 10 IU/ml human chorionic gonadotropin (hCG; ASKA Pharmaceutical). The COCs were cultured for 22 h with hormones in a humidified atmosphere of 5% CO2 and 95% air at 38.5 C, followed by additional culture for 18–20 h without hormones in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 38.5 C.

Production of Tg embryos by somatic cell cloning

Porcine fetal fibroblast cells (#111-4) carrying an expression vector for the mutant human HNF-1α cDNA (HNF-1αP291fsinsC), which had been established in our previous study [4], were used as nuclear donor cells. SCNT was performed using IVM oocytes as recipient cytoplasts as described elsewhere [5]. SCNT embryos were cultured in porcine zygote medium-5 (PZM-5; Research Institute for the Functional Peptides, Yamagata, Japan) in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 38.5 C for 1 or 2 days, and then they were transferred into an estrus-synchronized recipient.

Cryopreservation of boar spermatozoa from epididymides

The cauda epididymides of the founder Tg-cloned boar and a WT boar (17 months old) were removed under general anesthesia and transferred to the laboratory at ambient temperature. Luminal fluid containing spermatozoa was extruded from the distal portion of each cauda epididymis by air pressure from a syringe, followed by perfusion with approximately 10 ml of collecting solution [6]. The recovered sperm suspension was diluted to a concentration of 2.0×109 cells/ml by adding collecting solution. The sperm suspension was then cooled down from 25 C to 15 C over 60 min (average cooling rate: 0.17 C/min). After incubation for 2 h at 15 C, the sperm suspension was centrifuged at 800 × g for 10 min at 15 C and the supernatant was discarded. Precipitated spermatozoa were resuspended in equal amounts of Niwa and Sasaki Freezing (NSF)-I extender [6]. The sperm suspension was again cooled down from 15 C to 5 C over 120 min (average cooling rate: 0.08 C/min) and was then diluted to a concentration of 2.0×109 cells/ml with NSF-I extender. It was finally mixed with an equal volume of NSF-II extender containing 6% (v/v) glycerol [6]. The sperm suspension (0.2–0.3 ml) was loaded in 0.5-ml plastic straws (Cryo Bio System, Paris, France). The straws were the placed in liquid nitrogen vapor (–150 C) approximately 4 cm above the surface of liquid nitrogen for 20 min and were finally plunged into liquid nitrogen.

IVF

IVF was performed using IVM oocytes with expanded cumulus cells [16]. A straw containing frozen sperm was thawed by immersion in warm water (37 C) for 30 seconds. The sperm were then suspended in 5 ml DPBS supplemented with 0.1% bovine serum albumin (Wako Pure Chemical Industries, Osaka, Japan) and washed three times by centrifugation at 1,000 × g for 4 min. After washing, the sperm pellets were resuspended in porcine fertilization medium (PFM; Research Institute for the Functional Peptides) at a concentration of 1×107 cells/ml.

For insemination, 20 COCs that had been matured in vitro were placed in a 100-µl drop of PFM containing spermatozoa (1.0–5.0×106 cells/ml); the oocytes and sperm were incubated for 8 h at 38.5 C in a humidified atmosphere containing 5% CO2, 5% O2 and 90% N2. After insemination, the eggs were transferred to Tyrode lactose medium containing 10 mM Hepes and 0.3% (w/v) polyvinylpyrrolidone (Hepes-TL-PVP); cumulus cells and excess sperm were removed by gentle pipetting. Eggs that showed release of one or more polar bodies with normal cytoplasmic morphology were cultured (20–30 eggs/20–30 µl drop) in PZM-5 under paraffin oil (Kanto Chemical, Tokyo, Japan) in a plastic petri dish maintained in an atmosphere of 5% CO2, 5% O2 and 90% N2 at 38.5 C until transfer. Eggs were cultured beyond the morula stage by adding 10% (v/v) fetal calf serum to the medium.

Some eggs were fixed in 1:3 acetic acid:methanol after 12 h of culture and then stained with 1% aceto-orcein. Eggs with two pronuclei and two polar bodies were considered to have undergone normal fertilization. Other categories of fertilized eggs were identified as polyspermic: eggs with two pronuclei, two polar bodies and a swollen sperm head(s) and eggs with three or more pronuclei.

Embryo transfer

Crossbred (Large White / Landrace X Duroc) prepubertal gilts weighing 100–105 kg were used as recipients. To induce estrus in the gilts, 1000 IU of eCG was injected intramuscularly, followed by an injection of 1500 IU hCG (Kyoritsu Seiyaku Corporation, Tokyo, Japan) 66 h later.

SCNT embryos were transferred at the 1-cell (day 1) or 2–8-cell (day 2) stages into the oviducts of estrus-synchronized recipient gilts by midventral laparotomy under general anesthesia [5]. Transfer of in vitro-fertilized embryos was conducted at 1–8-cell (days 1–3) or blastocyst (days 5–6) stage. Embryos on days 1–2, day 3 and days 5–6 were respectively transferred to recipients at 51–54 h, 75–78 h and 147–150 h after the hCG injection. Pregnant recipients were allowed to farrow, and piglets obtained were raised with the standard diet and water ad libitum.

Intrafallopian insemination

The gilts used for the intrafallopian insemination experiment were induced to ovulate by an injection of 1000 IU eCG, followed 72 h later by administration of 1500 IU hCG. Intrafallopian insemination was performed by midventral laparotomy approximately 46 h after hCG injection. The ovaries of estrus-induced recipients were examined to confirm that ovulation had occurred. Frozen-thawed epididymal sperm suspended in Beltsville thawing solution [17] at a concentration of 2.7×107 cells/0.1 ml were injected into both ampullae of the fallopian tubes using a 3.5 Fr catheter (Tom Cat Catheter, Tyco Healthcare Group, Mansfield, MA, USA) with a syringe. The proportion of sperm with progressive motility was 30%.

Control of blood glucose levels in the diabetic founder boar

Blood glucose levels were measured daily (morning and evening) with a glucose test meter (GLUCOCARD GT-1820, Arkray, Kyoto, Japan) using blood samples from the ear vein. The diabetic founder pig received a subcutaneous injection of insulin glargine (0.5–1.1 U/kg) at the time of feeding in the morning. Blood glucose levels were measured again 60–90 min after feeding, and a subcutaneous injection of regular insulin (0–0.2 U/kg) was given to maintain a blood glucose level of approximately 200 mg/dl.

Biochemical analysis of blood components

Venous blood samples were collected in tubes containing heparin to determine the concentrations of aspirate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), creatinine (CRE), glucose (GLU), total cholesterol (T-CHO), triglyceride (TG), total protein (TP), sodium (Na), potassium (K) and chloride (Cl) using a dry-chemistry analyzer (FUJI DRI-CHEM 7000, FUJIFILM Corporation, Tokyo, Japan). The concentration of 1,5-anhydroglucitol (1,5-AG) was determined using the enzymatic method (SRL, Tokyo, Japan).

Determination of transgenic offspring

Determination of transgenic offspring was performed using PCR. Genomic DNA was extracted from offspring tail tissue, purified with a DNeasy Blood & Tissue Kit (QIAGEN, Tokyo, Japan) and amplified using TaKaRa Ex Taq (Takara Bio, Shiga, Japan) with the following set of primers: 5’-AGGACCTGAGCCTGCCGAGCAAC-3’ and 5’-AGGGCTCTCCATAGGCCCAGGCT-3’. The PCR conditions were as follows: 94 C for 3 min, followed by 25 cycles of 94 C for 30 s, 60 C for 15 s and 72 C for 20 s.

Statistics

Statistical analyses were performed using the SPSS 16.0 software (SPSS, Chicago, IL, USA). Differences in the rates of fertilization and embryo development between the DM sperm and control WT sperm were analyzed using a χ2 test.