The effects of maternal nutrition during the first 50 d of gestation on the location and abundance of hexose and cationic amino acid transporters in beef heifer uteroplacental tissues

Abstract

We hypothesized that maternal nutrition during the first 50 d of gestation would influence the abundance of hexose transporters, SLC2A1, SLC2A3, and SLC2A5, and cationic amino acid transporters, SLC7A1 and SLC7A2, in heifer uteroplacental tissues. Angus-cross heifers (n = 43) were estrus synchronized, bred via artificial insemination, and assigned at breeding to 1 of 2 dietary intake groups (CON = 100% of requirements to achieve 0.45 kg/d of BW gain or RES = 60% of CON intake) and ovariohysterectomized on day 16, 34, or 50 of gestation (n = 6 to 9/d) in a completely randomized design with a 2 × 3 factorial arrangement of treatments. Uterine cross-sections were collected from the horn ipsilateral to the corpus luteum, fixed in 10% neutral buffered formalin, sectioned at 5 µm, and stained via immunofluorescence for transporters. For each image, areas of fetal membrane (FM; chorioallantois), luminal epithelium (ENDO), superficial glands (SG), deep glands (DG), and myometrium (MYO) were analyzed separately for relative intensity of fluorescence as an indicator of transporter abundance. Analysis of FM was only conducted for days 34 and 50. No transporters in target areas were influenced by a day × treatment interaction (P ≥ 0.06). In ENDO, all transporters were differentially abundant from days 16 to 50 of gestation (P ≤ 0.04), and SLC7A2 was greater (P = 0.05) for RES vs. CON. In SG, SLC7A1 and SLC7A2 were greater (P ≤ 0.04) at day 34 vs. day 16. In DG, SLC2A3 and SLC7A1 were greater (P ≤ 0.05) for CON vs. RES heifers; furthermore, SLC7A1 was greater (P < 0.01) at day 50 vs. days 16 and 34 of gestation. In MYO, SLC7A1 was greater (P < 0.01) for CON vs. RES and was greater (P = 0.02) at days 34 and 50 vs. day 16. There were no differences in FM (P ≥ 0.06). Analysis of all uterine tissues at day 16 determined that SLC2A1, SLC2A3, and SLC7A2 were all differentially abundant across uterine tissue type (P < 0.01), and SLC7A1 was greater (P = 0.02) for CON vs. RES. Analysis of all uteroplacental tissues at days 34 and 50 demonstrated that all transporters differed (P < 0.01) across uteroplacental tissues, and SLC7A1 was greater (P < 0.01) for CON vs. RES. These data are interpreted to imply that transporters are differentially affected by day of gestation, and that hexose and cationic amino acid transporters are differentially abundant across utero-placental tissue types, and that SLC7A1 is responsive to maternal nutritional treatment.

Introduction

During early gestation in ruminants, the maternal and embryonic systems do not share a blood supply. Prior to the establishment of hemotrophic nutrition, the embryo must receive nutrients from uterine secretions called histotroph, which is secreted via transporters in the luminal and glandular epithelium to support the growth and development of the embryo (Gray et al., 2001; Bazer et al., 2011; Spencer et al., 2019). Hexoses and cationic amino acids have critical roles as energy substrates, vasoactive factors, and activators of the mammalian target of rapamycin (mTOR), all of which assist in conceptus growth, elongation, and successful maintenance of pregnancy (Kwun et al., 2003; Kim et al., 2011; Krause et al., 2011; Wang et al., 2015).

Expression of hexose and cationic amino acid transporters in uteroplacental tissues has been previously investigated in both the bovine (Lucy et al., 2012; Crouse et al., 2016a, b, 2017) and ovine models (Gao et al., 2009a, b). Furthermore, recently published data reported difference concentrations of hexoses and cationic amino acids in response to temporal and hormonal treatments (Forde et al., 2014; Crouse et al., 2019a; Simintiras et al., 2019) in Crossbred and Charolais-Limousin heifers, respectively. Recently, published data from our group utilizing the same animals and tissues as presented in this manuscript have elucidated effects of day of gestation and maternal nutritional treatment on the concentrations of glucose and fructose in fetal fluids and temporal effects on the concentrations of cationic amino acids and metabolites in fetal fluids (Crouse et al., 2019a). Furthermore, animals and tissues from the same studies demonstrated temporal and maternal nutritional effects on mRNA expression of the same transporters investigated within this manuscript (Crouse et al., 2016a, 2017).

The transporters analyzed in this manuscript were selected due to their roles in hexose and cationic amino acid transport, and previous analysis via rt-qPCR by our lab group (Crouse et al., 2016a, 2017). Glucose transporter SLC2A1 has a high affinity for glucose ranging from 3 to 5 mM depending on the tissue, with a uterine transport K_m of 3 mM (Mueckler, 1994; Augustin, 2010). In uterine tissue, SLC2A1 has been localized in the stroma, luminal epithelium, and glandular epithelium (Gao et al., 2009a; Frolova and Moley, 2011). Glucose transporter SLC2A3 has the highest known transport capacity of any glucose transporter at 1.5 mM (Thorens and Mueckler, 2010). Fructose is transported by the low affinity high-capacity fructose transporter, SLC2A5. In the bovine, high concentrations of SLC2A5 are found on fast-cleaving embryos which are more likely to develop to the blastocyst stage due to their enhanced nucleotide synthesis (Gutiérrez-Adán et al., 2004). Transporter SLC7A1 exhibits K_m values for l-arginine and l-lysine of 100 to 150 µM, and in sheep, knockout of SLC7A1 resulted in decreased arginine transport by 73%. Additionally, arginine catabolites, citrulline and ornithine, decreased by 76% and 40%, respectively (Wang et al., 2014). Transporter SLC7A2 has a 10-fold lower substrate affinity compared with SLC7A1 (Closs et al., 1993; Kavanaugh et al., 1994; Closs et al., 1997), and in ewes, SLC7A2 mRNA expression is greater in pregnant vs. cyclic ewes (Gao et al., 2009b).

Although mRNA expression data have been presented for both hexose and cationic amino acid transporters in bovine and ovine uteroplacental tissues under various experimental conditions, limited data are available on the effects of maternal nutrition and day of gestation on the location and abundance of hexose and cationic amino acid transporter proteins in beef heifer uteroplacental tissues during early pregnancy. Therefore, we tested the hypothesis that maternal nutrition during the first 50 d of gestation would influence the abundance and location of the hexose transporters, SLC2A1, SLC2A3, SLC2A5, and cationic amino acid transporter proteins, SLC7A1, and SLC7A2, in heifer uteroplacental tissues.

Materials and Methods

All animal procedures were approved by the North Dakota State University Institutional Animal Care and Use Committee.

Animals, housing, and diet

Animals, housing, and diet were previously described by Crouse et al. (2020). Briefly, crossbred Angus heifers (n = 43) were housed at the North Dakota State University Animal Nutrition and Physiology Center and individually fed (American Calan, Northwood, NH). All heifers were estrus synchronized (Bridges et al., 2008), artificially inseminated to a common sire, and randomly assigned to 2 intake groups: control (CON) heifers received 100% of NRC (2000) requirements for 0.45 kg/d gain in BW (actual average daily gain = 0.51 kg), and restricted heifers (RES) received 60% of CON delivery (actual ADG = –0.08 kg/d). Diets were formulated based on initial body weight at breeding to contain 2.25 Mcal/kg metabolizable energy, 9.75% crude protein, and 58.6% neutral detergent fiber on a dry matter basis and delivered as a total mixed ration consisting of grass hay, corn silage, alfalfa haylage, as well as a grain and mineral mixture. Dried distillers grains with solubles were top dressed and initially provided at a rate of 0.23 kg/d. The combined total mixed ration and top dress were fed individually to heifers daily in the Calan gate system, and the TMR and distillers grains offerings were adjusted weekly based on heifer average daily gain to meet targeted body weight gains.

Heifers were ovariohysterectomized at either day 16 (CON, n = 7; RES, n = 7), 34 (CON, n = 6; RES, n = 9), or 50 (CON, n = 7; RES, n = 7) of gestation. Thus, the experimental design was a completely randomized design with a 2 × 3 factorial arrangement of treatments. Heifers whose uterus did not contain a conceptus after surgery on day 16 were removed from the study. To be included for hysterectomy on day 34 or 50, heifers were ultrasounded on day 28 of gestation and again prior to hysterectomy to confirm the presence of a single embryo. Heifers were ovariohysterectomized via a standing surgical procedure with a left flank incision (McLean et al., 2016). Uterine and ovarian arteries as well as the cervix were ligated, and the uterus was clamped caudal to the internal bifurcation and cranial to the cervix. The uterus was incised along the clamp, and the entire uterine body and ovaries were collected and transported to the lab for tissue collection.

Sample collection and analysis

Sample collection followed the same protocol previously described by Crouse et al. (2020) and was completed within 30 min after ovariohysterectomy. Briefly, 3 stainless steel specimen pins (Ento Sphinx, Czech Republic) were passed through the gravid horn of the uterus at intervals of 1 cm with the center pin placed at the external bifurcation to prevent detachment of the fetal membranes. Cross-sections (0.5-cm wide) were collected from the horn ipsilateral to the corpus luteum (Grazul-Bilska et al., 2010, 2011, Crouse et al., 2020), fixed in 10% neutral buffered formalin, and embedded in paraffin blocks. Tissue sections (5 μm) were mounted onto glass slides, deparaffinized with xylene, and rehydrated with a series of graded ethanol washes followed by distilled water. Antigen retrieval was performed in 10 mM sodium citrate buffer, pH 6, with 0.05% Tween 20 in a 2100 retriever (Electron Microscopy Sciences, Hatfield, PA). Slides were rinsed twice with Tris-buffered saline with Triton X-100 (TBST; 0.05 M Tris, 0.15 M NaCl, 0.1% Triton X-100). To block nonspecific binding, slides were treated for 1 hr with 5% normal goat serum (Vector Laboratories, Burlingame, CA). Optimal antibody dilution tests were conducted for each transporter, and slides with no primary but secondary antibody only were included as negative controls to ensure no non-specific binding had occurred. Separate tissue sections were incubated with rabbit primary antibodies for SLC2A1 (sodium independent facilitative diffusion glucose transporter; Abcam, Cambridge, UK; ab625; rabbit polyclonal; 1:250; 97% homology), SLC2A3 (sodium independent facilitative diffusion high-capacity glucose transporter; Abcam; ab15311; rabbit polyclonal; 1:50; 86% homology), SLC2A5 (sodium independent facilitative diffusion fructose transporter; Abcam ab87847; rabbit polyclonal; 1:500; 80% homology), SLC7A1 (facilitative diffusion cationic amino acid transporter of arginine, lysine, and ornithine; Abcam; ab37588; rabbit polyclonal; 1:100; 91% homology), and SLC7A2 (facilitative diffusion cationic amino acid transporter of arginine, lysine, and ornithine; Abcam; ab140831; rabbit polyclonal; 1:100; 92% homology) followed by incubation with goat anti-rabbit secondary antibody Alexa 633 (1:250 dilution, Invitrogen A21235, Grand Island, NY) overnight at 4 °C on a rocking platform followed by incubation with 4,6-diamidino-2-phenylindole (DAPI; Life Technologies, Grand Island, NY) staining solution for 5 min, after which slides were rinsed with distilled water. Slides were mounted using EverBrite Mounting Medium (Biotium Inc., Fremont, CA). Large-area photomicrographs of complete cross-sections of the uterine wall along with fetal membranes were taken using the same exposure time and energy level for each slide with a Zeiss Imager M2 epifluorescence microscope using a 10× objective and AxioCam HRm camera, as well as a Zeiss piezo automated stage controlled by the MosaiX module of Zeiss AxioVision software (Carl Zeiss Microscopy, LLC; 1 Zeiss Dr., Thornwood, NY). For each image, the areas of fetal membrane (FM; chorioallantois), luminal epithelium (ENDO; uterine endometrium), superficial endometrial glands (SG), deep endometrial glands (DG), and myometrium (MYO) were included in MosaiX (Figure 1). Tissue areas with the highest and lowest abundance of antibody of interest were used for setting the threshold for the Confocal Laser Scanning Microscope (Zeiss LSM 700 Confocal Laser Scanning Microscope; ZeissThornwood, NY). Three areas of interest for each tissue were imaged with a Plan-Apochromat 40x/1.3 oil immersion lens. Images were analyzed using ImagePro Premier software (Media Cybernetics, Silver Spring, MD). Briefly, the areas of interest for each tissue were carefully outlined and analyzed for intensity using an automated macro that allowed objective comparisons of the samples. Data are presented as relative fluorescence units within the areas of interest as an indicator of transporter abundance. Due to FM not being attached at day 16 and FM being flushed at day 16 of gestation for mRNA expression (Crouse et al., 2016a, 2017), FM data were only available for days 34 and 50.

Figure 1.

MosaiX images of SLC2A1. FM (chorioallantois), uterine ENDO (luminal epithelium), SG, DG, and MYO. (A and D) MosaiX with DAPI. (B and E) MosaiX with AlexaFluor633. (C and F) Mosaics with AlexaFluor633 and DAPI. The white scale bar on each image is 50 μm.

Statistical analysis

Statistical analysis was conducted as previously described by Crouse et al. (2020) and is briefly described below. Fetal membranes were not available for analysis on day 16. Therefore, a complete analysis including all days and tissues (FM, ENDO, SG, DG, and MYO) across days of gestation was not conducted, but rather, days were analyzed separately as a day 16 only or as days 34 and 50 and are presented as described below.

Day × maternal nutrition. Data for individual transporters were analyzed using the GLM procedure of SAS 9.4 (SAS, 2013), with day, maternal nutrition treatment, and the day × maternal nutrition interaction included in the model, with individual heifer serving as the experimental unit and P-values ≤ 0.05 considered significant. Tendencies were discussed when P-values were ≤ 0.10.

Complete tissue analysis. Models were developed to compare the abundance of transporters among the respective maternal and fetal tissues evaluated. For samples collected on day 16 of gestation, all maternal tissues (ENDO, SG, DG, and MYO) were analyzed with the GLM procedure of SAS 9.4 with main effects of tissue and nutrition treatment in the model. The maternal tissue × maternal nutrition interaction was not significant (P ≥ 0.30) for any transporter investigated and was therefore removed from the model and not reported. For samples collected on days 34 and 50 of gestation, all uteroplacental tissues and maternal nutrition treatments were analyzed with the GLM procedure of SAS 9.4, with main effects of tissue and maternal nutrition in the model. Two-way interactions for treatment × maternal nutrition or 3-way interactions with day were not significant (P ≥ 0.61) for any transporter and were therefore removed from the model and not reported. Individual heifer served as the experimental unit and P-values ≤ 0.05 considered significant and tendencies were discussed when P-values were ≤ 0.10.

Results

Day × maternal nutrition

ENDO . No transporters were influenced by a day × maternal nutrition interaction (P ≥ 0.21; Table 1); however, all transporters were differentially abundant across day of pregnancy. Glucose transporter SLC2A1 protein abundance was greater (P < 0.01) at day 50 compared with days 16 and 34 of gestation (Table 1). Abundance of SLC2A3 protein was greater (P = 0.04) at day 50 compared with 16, with day 34 being intermediate and not different from days 16 and 50 of gestation. Fructose transporter SLC2A5 was more abundant (P = 0.04) for days 16 and 34 compared with day 50 (Table 1). Furthermore, abundance of both cationic amino acid transporters was greater at days 34 and 50 compared with day 16 of gestation (P ≤ 0.01). Finally, SLC7A2 was influenced by maternal nutrition, being more abundant (P = 0.05) for RES compared with CON heifers.

Table 1.

Protein abundance of hexose and cationic amino acid transporters in luminal epithelium (ENDO) as influenced by maternal nutrition from days 16 to 50 of gestation¹

			Day of gestation2					P-values3
Transporter4	Nutr. Trt5		16	34	50	Nutr. Avg6	SEM7	Day	Nutr.	Day × Nutr.
SLC2A1	CON		10.09	21.54	31.78	21.14	5.49	<0.01	0.68	0.71
	RES		11.85	23.38	31.56	19.28
		Day8	10.97b	17.98b	31.67a
SLC2A3	CON		5.56	5.60	5.57	5.57	0.02	0.04	0.80	0.42
	RES		5.52	5.60	5.59	5.57
		Day	5.54b	5.60a	5.58ab
SLC2A5	CON		14.32	14.52	12.40	13.75	1.75	0.04	0.26	0.43
	RES		13.67	14.53	8.10	12.10
		Day	14.00a	14.52a	10.25b
SLC7A1	CON		2.70	4.34	3.15	3.40	0.36	<0.01	0.81	0.21
	RES		2.64	3.86	3.91	3.47
		Day	2.67b	4.10a	3.53a
SLC7A2	CON		1.35	1.96	1.86	1.72d	0.26	0.01	0.05	0.84
	RES		1.62	2.48	2.40	2.16c
		Day	1.48b	2.22a	2.13a

¹Data are presented as relative fluorescence units.

²Day of gestation, days after insemination.

³Probability values for the effects of day, maternal nutrition, and day × maternal nutrition.

⁴SLC2A1, facilitated diffusion glucose uniporter; SLC2A3, facilitated diffusion glucose transporter; SLC2A5, fructose transporter; SLC7A1, arginine, lysine, and ornithine transporter; and SLC7A2, arginine, lysine, and ornithine transporter.

⁵CON, heifers fed a total mixed ration that met 100 % of NRC requirements to gain 0.45 kg daily; RES, heifers restricted to 60 % of the CON diet.

⁶Mean fluorescence of treatment groups across days 16, 34, and 50 of gestation.

⁷Average SEM for the day × maternal nutrition interaction (day 16 CON n = 7, day 16 RES n = 7, day 34 CON n = 6, day 34 RES n = 9, day 50 CON n = 7, day 50 RES n = 7).

⁸Mean fluorescence across maternal nutrition within day of gestation.

^a,bMeans within row without common superscript differ (P ≤ 0.05).

^c,dMeans within column without common superscript differ (P ≤ 0.05).

SG . No transporters were influenced by a day × maternal nutrition interaction (P ≥ 0.36). Transporters SLC7A1 and SLC7A2 were influenced by day of gestation, where SLC7A1 was more abundant (P < 0.01) at days 34 and 50 compared with day 16, and SLC7A2 was more abundant (P = 0.04) at day 34 compared with 16, with day 50 being intermediate and not different from days 16 and 34 (Table 2). No other transporters were affected by day of gestation or maternal nutrition (P ≥ 0.16; Table 2).

Table 2.

Abundance of hexose and cationic amino acid transporters in SG as influenced by maternal nutrition from days 16 to 50 of gestation¹

			Day of gestation2					P-values3
Transporter4	Nutr. Trt5		16	34	50	Nutr. Avg6	SEM7	Day	Nutr.	Day × Nutr.
SLC2A1	CON		3.17	2.32	3.00	2.83	0.52	0.16	0.28	0.89
	RES		3.91	2.74	3.23	3.29
		Day8	3.54	2.53	3.11
SLC2A3	CON		5.45	7.35	4.98	5.92	0.80	0.26	0.92	0.36
	RES		5.72	5.99	5.88	5.86
		Day	5.58	6.67	5.45
SLC2A5	CON		14.94	17.07	13.51	15.18	2.13	0.20	0.53	0.83
	RES		15.35	15.43	11.42	14.07
		Day	15.15	16.25	12.47
SLC7A1	CON		4.02	6.71	5.92	5.55	0.69	<0.01	0.20	0.58
	RES		3.28	5.39	5.92	4.87
		Day	3.65b	6.05a	5.92a
SLC7A2	CON		2.62	5.72	3.42	3.92	0.92	0.04	0.67	0.49
	RES		3.06	4.88	4.79	4.24
		Day	2.84b	5.3a	4.11ab

¹Data are presented as relative fluorescence units.

²Day of gestation = days after insemination.

³Probability values for the effects of day, maternal nutrition, and day × maternal nutrition.

⁴SLC2A1, facilitated diffusion glucose uniporter; SLC2A3, facilitated diffusion glucose transporter; SLC2A5, fructose transporter; SLC7A1, arginine, lysine, and ornithine transporter; and SLC7A2, arginine, lysine, and ornithine transporter.

⁵CON, heifers fed a TMR that met 100% of NRC requirements to gain 0.45 kg daily; RES, heifers restricted to 60% of the CON diet.

⁶Mean fluorescence of treatment groups across days 16, 34, and 50 of gestation.

⁷Average SEM for the day × maternal nutrition interaction (day 16 CON n = 7, day 16 RES n = 7, day 34 CON n = 6, day 34 RES n = 9, day 50 CON n = 7, day 50 RES n = 7).

⁸Mean fluorescence across maternal nutritional treatments within day of gestation.

^a,bMeans within row without common superscript differ (P ≤ 0.05).

DG. No transporters were influenced by a day × maternal nutrition interaction (P ≥ 0.35). Glucose transporter SLC2A1 tended (P = 0.09) to be greater at day 16 compared with days 34 and 50 (Table 3). Additionally, SLC7A1 was greater (P < 0.01) at day 50 compared with days 16 and 34 of gestation. Maternal nutrition affected the abundance of SLC2A3 such that SLC2A3 was greater (P = 0.05) for CON compared with RES heifers (Table 3).

Table 3.

Abundance of hexose and cationic amino acid transporters in DG as influenced by dietary maternal nutrition from days 16 to 50 of gestation¹

			Day of gestation2					P-values3
Transporter4	Nutr. Trt5		16	34	50	Nutr. Avg6	SEM7	Day	Nutr.	Day × Nutr.
SLC2A1	CON		9.93	6.51	8.63	8.35	1.61	0.09	0.18	0.68
	RES		12.69	9.01	8.80	10.16
		Day8	11.31	7.76	8.71
SLC2A3	CON		3.79	4.00	3.69	3.82a	0.42	0.90	0.05	0.50
	RES		3.22	2.76	3.44	3.14b
		Day	3.51	3.38	3.56
SLC2A5	CON		15.40	14.04	15.30	14.91	2.30	0.91	0.60	0.75
	RES		14.49	14.69	12.54	13.91
		Day	14.94	14.36	13.92
SLC7A1	CON		2.75	3.96	4.67	3.79a	0.59	<0.01	<0.01	0.35
	RES		2.25	2.41	3.75	3.15b
		Day	2.50d	3.19d	4.21c
SLC7A2	CON		2.84	3.32	2.23	2.79	0.48	0.23	0.65	0.71
	RES		2.92	2.69	2.24	2.62
		Day	2.88	3.01	2.23

¹Data are presented as relative fluorescence units.

²Day of gestation = days after insemination.

³Probability values for the effects of day, maternal nutrition, and day × maternal nutrition.

⁴SLC2A1, facilitated diffusion glucose uniporter; SLC2A3, facilitated diffusion glucose transporter; SLC2A5, fructose transporter; SLC7A1, arginine, lysine, and ornithine transporter; and SLC7A2, arginine, lysine, and ornithine transporter.

⁵CON, heifers fed a TMR that met 100% of NRC requirements to gain 0.45 kg daily; RES, heifers restricted to 60% of the CON diet.

⁶Mean fluorescence of treatment groups across days 16, 34, and 50 of gestation.

⁷Average SEM for the day × maternal nutrition interaction (day 16 CON n = 7, day 16 RES n = 7, day 34 CON n = 6, day 34 RES n = 9, day 50 CON n = 7, day 50 RES n = 7).

⁸Mean fluorescence across maternal nutritional treatments within day of gestation.

^a-bMeans within column without common superscript differ (P ≤ 0.05).

^c-dMeans within row without common superscript differ (P ≤ 0.05).

MYO . Fructose transporter SLC2A5 tended (P = 0.06) to be influenced by a day × maternal nutrition interaction such that at day 16, RES heifers were greater than RES heifers at day 50, with all other treatments and days being intermediate (Table 4). The abundance of SLC7A1 was greater (P = 0.02) at days 34 and 50 compared with 16 of gestation (Table 4). Finally, SLC7A1 protein was more abundant (P < 0.01) for CON vs. RES heifers.

Table 4.

Abundance of hexose and cationic amino acid transporters in MYO as influenced by maternal nutrition from days 16 to 50 of gestation¹

			Day of gestation2					P-values3
Transporter4	Nutr. Trt5		16	34	50	Nutr. Avg6	SEM7	Day	Nutr.	Day × Nutr.
SLC2A1	CON		2.29	1.81	2.48	2.19	0.35	0.47	0.50	0.24
	RES		2.77	2.41	1.99	2.39
		Day8	2.53	2.11	2.23
SLC2A3	CON		4.58	5.22	5.47	5.09	0.75	0.76	0.76	0.72
	RES		5.44	4.87	5.53	5.28
		Day	5.01	5.04	5.50
SLC2A5	CON		7.94	8.74	8.19	8.29	1.36	0.09	0.58	0.06
	RES		10.99	7.46	4.53	7.66
		Day	9.47	8.10	6.36
SLC7A1	CON		2.88	4.51	4.94	4.11a	0.53	0.02	<0.01	0.37
	RES		2.50	3.05	3.25	2.93b
		Day	2.69d	3.77c	4.09c
SLC7A2	CON		1.22	1.86	1.45	1.51	0.19	0.42	0.97	0.14
	RES		1.54	1.42	1.53	1.50
		Day	1.38	1.64	1.49

¹Data are presented as relative fluorescence units.

²Day of gestation = days after insemination.

³Probability values for the effects of day, maternal nutrition, and day × maternal nutrition.

⁴SLC2A1, facilitated diffusion glucose uniporter; SLC2A3, facilitated diffusion glucose transporter; SLC2A5, fructose transporter; SLC7A1, arginine, lysine, and ornithine transporter; and SLC7A2, arginine, lysine, and ornithine transporter.

⁵CON, heifers fed a TMR that met 100% of NRC requirements to gain 0.45 kg daily; RES, heifers restricted to 60% of the CON diet.

⁶Mean fluorescence of maternal nutrition groups across days 16, 34, and 50 of gestation.

⁷Average SEM for the day × maternal nutrition interaction (day 16 CON n = 7, day 16 RES n = 7, day 34 CON n = 6, day 34 RES n = 9, day 50 CON n = 7, day 50 RES n = 7).

⁸Mean fluorescence across maternal nutritional treatments within day of gestation.

^a,bMeans within column without common superscript differ (P ≤ 0.05).

^c,dMeans within row without common superscript differ (P ≤ 0.05).

FM . No transporters were affected by a day × maternal nutrition interaction (P ≥ 0.29) or the main effect of maternal nutrition (P ≥ 0.41; Table 5). Protein abundance of both SLC2A3 and SLC7A1 tended (P = 0.06 and P = 0.10, respectively) to be greater at day 34 compared with day 50 of gestation (Table 5).

Table 5.

Abundance of hexose and cationic amino acid transporters in FM as influenced by maternal nutrition from days 34 and 50 of gestation¹

			Day of gestation2				P-values3
Transporter4	Nutr. Trt5		34	50	Nutr. Avg6	SEM7	Day	Nutr.	Day × Nutr.
SLC2A1	CON		8.01	14.70	11.36	2.71	0.19	0.41	0.29
	RES		8.69	9.40	9.04
		Day8	8.35	12.05
SLC2A3	CON		4.74	3.31	4.02	0.60	0.06	0.70	0.41
	RES		4.11	3.54	3.82
		Day	4.24	3.42
SLC2A5	CON		13.96	14.59	14.28	2.03	0.49	0.52	0.70
	RES		14.49	16.74	15.61
		Day	14.23	15.66
SLC7A1	CON		3.15	2.09	2.63	0.61	0.10	0.82	0.99
	RES		3.30	2.23	2.76
		Day	3.23	2.16
SLC7A2	CON		1.51	1.8	1.65	0.42	0.12	0.84	0.75
	RES		1.48	1.92	1.70
		Day	1.49	1.86

¹Data are presented as relative fluorescence units.

²Day of gestation = days after insemination.

³Probability values for the effects of day, maternal nutrition, and day × maternal nutrition.

⁴ SLC2A1, facilitated diffusion glucose uniporter; SLC2A3, facilitated diffusion glucose transporter; SLC2A5, fructose transporter; SLC7A1, arginine, lysine, and ornithine transporter; and SLC7A2, arginine, lysine, and ornithine transporter.

⁵CON, heifers fed a TMR that met 100% of NRC requirements to gain 0.45 kg daily; RES, heifers restricted to 60% of the CON diet.

⁶Mean fluorescence of maternal nutritional treatment groups across days 16, 34, and 50 of gestation.

⁷Average SEM for the day × maternal nutrition interaction (day 34 CON n = 6, day 34 RES n = 9, day 50 CON n = 7, day 50 RES n = 7).

⁸Mean fluorescence across maternal nutritional treatments within day of gestation.

Maternal tissue analysis on day 16

In maternal tissues, SLC2A1 was greater (P < 0.01) in DG and ENDO compared with SG and MYO (Table 6). High-capacity glucose transporter SLC2A3 was greater (P < 0.01) in the ENDO, SG, and MYO compared with DG. Fructose transporter SLC2A5 tended (P = 0.07) to be greater in the ENDO, SG, and DG compared with MYO (Table 6). Cationic amino acid transporter SLC7A2 was greater (P < 0.01) in both glandular epithelia (SG and DG) compared with ENDO and MYO. In maternal tissues, SLC7A1 was not differentially abundant across tissue type; however, SLC7A1 was more abundant (P = 0.02) in CON vs. RES heifers (Table 6).

Table 6.

Abundance of hexose and cationic amino acid transporters in luminal epithelium (ENDO), SG, DG, and MYO as influenced by maternal nutrition at day 16 of gestation¹

	Tissue2					Maternal nutrition3		P-values4
Transporter5	ENDO	SG	DG	MYO	SEM6	CON ± SEM	RES ± SEM	Tissue	Mat. Nutr.
SLC2A1	10.97a	3.54b	11.31a	2.53b	1.63	6.37 ± 1.15	7.80 ± 1.15	<0.01	0.38
SLC2A3	5.54a	5.58a	3.51b	5.01a	0.29	4.84 ± 0.19	4.97 ± 0.21	<0.01	0.64
SLC2A5	14.00	15.15	14.94	9.47	1.66	13.15 ± 1.21	13.63 ± 1.12	0.07	0.78
SLC7A1	2.67	3.27	2.51	2.70	0.25	3.09 ± 0.18	2.48 ± 0.18	0.17	0.02
SLC7A2	1.48b	2.84a	2.88a	1.39b	0.32	2.01 ± 0.21	2.29 ± 0.23	<0.01	0.38

¹Data are presented as relative fluorescence units as an indicator of transport abundance.

²Tissue, ENDO, SG, DG, and MYO.

³CON, heifers fed a total mixed ration that meets 100% of NRC requirements to gain 0.45 kg daily; RES = heifers restricted to 60% of CON diet.

⁴Probability values for the effect of tissue and maternal nutrition (Mat. Nutr.) on the fluorescence of hexose and cationic amino acid transporter abundance.

⁵SLC2A1, facilitated diffusion glucose uniporter; SLC2A3, facilitated diffusion glucose transporter; SLC2A5, fructose transporter; SLC7A1, arginine, lysine, and ornithine transporter; and SLC7A2, arginine, lysine, and ornithine transporter.

⁶Average SEM for the main effect of maternal tissue. Day 16 CON n = 7, day 16 RES n = 7.

^a–cMeans within row without common superscript differ (P ≤ 0.05).

Complete tissue analysis

Protein abundance of glucose transporter SLC2A1 was affected by a day × tissue interaction (P < 0.01), being greater in ENDO at day 50 and next most abundant in ENDO at day 34 compared with all other days and tissues (Table 7). Furthermore, SLC2A1 was more abundant in FM at days 34 and 50 compared with MYO at days 34 and 50.

Table 7.

Abundance of hexose and cationic amino acid transporters in FM, luminal epithelium (ENDO), SG, DG, and MYO as influenced by maternal nutrition at days 34 and 50 of gestation¹

			Tissue2						Nutr. Trt				P-values3
Protein4	Day5		FM	ENDO	SG	DG	MYO	SEM7	CON	RES	SEM8	Tissue	Nutr.	Day	Day × Nutr.	Day × Tissue
SLC2A1	34		8.35cde	17.98b	2.53ef	7.76cdef	2.11f	2.12	8.04	7.45	1.69	<0.01	0.53	<0.01	0.84	<0.01
	50		12.05c	31.67a	3.11def	8.71cd	2.23f		12.12	10.99
		Tiss. Avg9	10.20	24.83	2.82	8.23	2.17
SLC2A3	34		4.34	5.60	6.70	3.50	5.04	0.43	5.41	4.66	0.31	<0.01	0.31	0.22	0.09	0.23
	50		3.42	5.58	5.43	3.46	5.49		4.60	4.79
		Tiss. Avg	3.88h	5.59g	6.07g	3.53h	5.27g
SLC2A5	34		14.23	14.52	16.25	14.36	8.10	1.28	13.66	13.32	0.79	<0.01	0.12	0.03	0.26	0.16
	50		15.66	10.25	12.47	13.92	6.36		12.80	10.67
		Tiss. Avg	14.95g	12.39g	14.36g	14.14g	7.23h
SLC7A1	34		3.23bc	4.10bc	6.05a	3.19c	3.78bc	0.37	4.34	3.60	0.23	<0.01	<0.01	0.71	0.20	0.05
	50		2.16d	3.53bc	5.92a	4.21b	4.10bc		4.15	3.81
		Tiss. Avg	2.69	3.81	5.98	3.70	3.94
SLC7A2	34		1.49	2.22	5.30	3.01	1.64	0.39	2.87	2.59	0.24	<0.01	0.77	0.14	0.16	0.30
	50		1.86	2.13	4.11	2.23	1.49		2.15	2.58
		Tiss. Avg	1.68i	2.18hi	4.71g	2.62h	1.56i

¹Data are presented as relative fluorescence units as an indicator of transport abundance.

²Tissue, fetal membranes (FM), endometrium (ENDO), superficial glands (SG), deep glands (DG), and myometrium (MYO).

³Probability values for the effect of tissue, maternal nutrition, day of gestation, day × maternal nutrition, and day × tissue on the fluorescence of hexose and cationic amino acid transporter abundance.

⁴SLC2A1, facilitated diffusion glucose uniporter; SLC2A3, facilitated diffusion glucose transporter; SLC2A5, fructose transporter; SLC7A1, arginine, lysine, and ornithine transporter; and SLC7A2, arginine, lysine, and ornithine transporter.

⁵CON, Heifers fed a TMR that meets 100% of NRC requirements to gain 0.45 kg daily; RES, heifers restricted to 60% of CON diet.

⁶Mean fluorescence of maternal nutritional treatment groups across uterine tissue type.

⁷Average SEM for day × tissue interaction. Day 34 CON n = 6, day 34 RES n = 9, day 50 CON n = 7, day 50 RES n = 7.

⁸Average SEM for the day × maternal nutrition interaction.

⁹Mean fluorescence across maternal nutrition within tissue type.

^a–cMeans within row without common superscript differ (P ≤ 0.05).

^d,eMeans within column without common superscript differ (P ≤ 0.05).

Protein abundance of the high-capacity glucose transporter SLC2A3 tended (P = 0.09) to be affected by the interaction of maternal nutrition × day of gestation interaction where CON heifers at day 34 of gestation had greater SLC2A3 compared with day 50 CON heifers and days 34 and 50 RES heifers (Table 7). Furthermore, there was a main effect of tissue (P < 0.01) where SLC2A3 was differentially abundant across uteroplacental tissue type, being more abundant in the ENDO, SG, and MYO compared with FM and SG (Table 7).

Protein abundance of the fructose transporter SLC2A5 was not affected by any 2- or 3-way interactions (P ≥ 0.16); however, SLC2A5 was more abundant at day 34 compared with 50 (P = 0.03). Further, there was a main effect (P < 0.01) of tissue type for SLC2A5 where SLC2A5 was more abundant in FM, ENDO, SG, and DG compared with MYO (Table 7).

Cationic amino acid transporter SLC7A1 was differently affected by the interaction (P = 0.05) for day × tissue type. In SG, SLC7A1 was more abundant (P = 0.05) at days 34 and 50 compared with all other tissues and days of gestation. Furthermore, SLC7A1 was least abundant at day 50 in FM with all other tissues and days being intermediate (Table 7). Additionally, at days 34 and 50 of gestation, SLC7A1 was more abundant (P < 0.01) for CON compared with RES heifers across all uteroplacental tissue types.

Finally, protein abundance of SLC7A2 exhibited a main effect of tissue, being most abundant (P < 0.01) in SG compared with all other uteroplacental tissue types. Furthermore, SLC7A2 was intermediate and more abundant in DG compared with FM and MYO, which were least abundant (Table 7).

Discussion

These data report the combined temporal and maternal nutrition impacts on the location and abundance of hexose and cationic amino acid transporters in bovine uteroplacental tissues throughout the first 50 d of gestation in beef heifers. These data are interpreted to imply that within individual tissue type, hexose and cationic amino acid transporters are most often differentially affected by day of gestation compared with maternal nutrition. Furthermore, these data are interpreted to imply that hexose and cationic amino acid transporters are differentially abundant across uteroplacental tissue type. Finally, these data are interpreted to imply that SLC7A1, when measured across all uteroplacental tissue types, is responsive to maternal nutritional treatment, which is similar to neutral amino acid transporters recently reported by Crouse et al. (2020).

Hexoses, such as glucose and fructose, are instrumental metabolites in fetal development. Both glucose and fructose activate mTOR via the hexosamine biosynthesis pathway (Kim et al., 2012; Wang et al., 2016). Furthermore, glucose is a principle energy substrate for fetal development and without sufficient glucose can lead to apoptosis (Moley et al., 1998). Fructose, while being the main hexose in fetal fluids (Kim et al., 2012; Crouse et al., 2019a) is not a main energy source for the conceptus but in swine, can be used for the biosynthesis of nucleic acids (White et al., 1982), which during the time of increased cell proliferation during early gestation is critical for embryonic development.

During early pregnancy, glucose is transported by the SLC2A (GLUT family) and SLC5A (SGLT family). The mRNA expression of these transporters in uteroplacental tissues has been evaluated in both the ovine (Gao et al., 2009a) and bovine model (Lucy et al., 2012; Crouse et al., 2016a, b, 2017; Leane et al., 2018) under multiple experimental conditions. Previously, Crouse et al. (2016b,2017) reported no difference in the mRNA expression of SLC2A1, SLC2A3, or SLC2A14 in uteroplacental tissues from the same study due to maternal restriction. Furthermore, intravenous glucose infusion from days 7 to 14 postestrus in lactating dairy cows did not alter the mRNA expression of glucose transporters SLC2A1, SLC2A3, SLC2A8, or SLC5A1 of Holstein cow endometrium at day 14 of gestation (Leane et al., 2018).

Glucose transporters SLC2A1 and SLC2A3 are the main glucose transporters utilized for transport of glucose to the fetus (Wooding et al., 2005), and both transporters either increased or maintained similar levels of abundance across all uterine tissues from days 16 to 50 of gestation. This pattern was similar to the mRNA expression of SLC2A1 and SLC2A3 from the same tissues, which increased or were similar in the intercaruncular endometrium from days 16 to 50 of gestation (Crouse et al., 2016b, 2017). Previously published data from this same study presented concentrations of nutrients in fetal fluids and reported no change in glucose concentrations of uterine flushes from days 16 to 50 of gestation (Crouse et al., 2019a) and no change in glucose concentrations of uterine flushes in response to maternal nutrition treatments. Furthermore, in response to venous infusion of glucose for 7 d before uterine flushing, Leane et al. (2018) reported no change in glucose concentrations of uterine flushes and similarly no change in mRNA expression of glucose transporters. These data may suggest a mechanism by which glucose transport is regulated to prevent excess transport of glucose from the maternal system to the fetal system during early gestation. Glucose transporter SLC2A1 is responsive to the activation of the mTOR pathway, suggesting that glucose transport and uptake may match substrate utilization in cellular growth and proliferation (Buller et al., 2008), thus matching glucose transport to the embryonic growth curve. The maintenance of glucose levels in uterine luminal fluid, as previously reported by Crouse et al. (2019a), and the maintenance in protein abundance of SLC2A1 and SLC2A3 in SG, DG, and FM further support this hypothesis while similar increases in concentrations of fructose occur (Crouse et al., 2019a; Simintiras et al., 2019) that may prevent oxidative stress and damage to early embryos, which will be discussed later.

Glucose transporter SLC2A3 is a high capacity glucose transporter in the placenta, with a k_m of 1.5 mM (Thorens and Mueckler, 2010) compared with SLC2A1 with a k_m of 6.9 mM (Burant and Bell, 1992). Interestingly, SLC2A3 decreased in mRNA expression (Crouse et al., 2016b) as well as protein expression as reported in the current data from days 16 to 50 of gestation. In contrast, both mRNA expression, as well as protein expression of SLC2A1 increased in FM from days 16 to 50 of gestation. Fetal fluids collected from this same study had decreased glucose concentrations in allantoic fluid from days 34 to 50 of gestation, which follows the same pattern as SLC2A3 (the main placental glucose transporter) protein abundance in FM, supporting that glucose transport across the placenta is driven by SLC2A3. Although neither SLC2A1 nor SLC2A3 transporter abundance was altered by maternal nutrition in FM, glucose concentrations of the allantoic and amniotic fluids were decreased in offspring of RES compared with CON heifers (Crouse et al., 2019a). The RNA-sequencing of fetal liver from offspring collected from this study had ten differentially expressed genes related to carbohydrate metabolism (Crouse et al., 2019b). Most notable was glucose-6-phosphatase, which is the final enzyme in gluconeogenesis, that was upregulated in fetuses from RES compared with CON heifers, and UDP-glucose phosphorylase 2, which produces UDP-glucose, the direct precursor to glycogen, that was downregulated in fetuses of RES compared with CON heifers. Although abundance of SLC2A1 and SLC2A3 was not different due to maternal nutrition treatments, as previously discussed, concentrations of glucose in allantoic and amniotic fluids were decreased in RES compared with CON heifers (Crouse et al., 2019a) and accompanied by alterations to expression of enzymes involved in carbohydrate metabolism (Crouse et al., 2019b) that may suggest altered fetal metabolism and programming of nutrient utilization by the fetus.

The abundance of fructose transporter SLC2A5 remained consistent from days 16 to 50 of gestation in most uteroplacental tissues, which was similar to SLC2A5 mRNA expression in the intercarauncular endometrium, which was also not altered by day of gestation in the same tissues (Crouse et al., 2017); however, mRNA expression of SLC2A5 in the FM decreased dramatically (80.17-fold to 27.39-fold greater than nonbred nonpregnant controls) from days 16 to 50, respectively, but there was no difference in mRNA expression from days 34 to 50 of gestation (Crouse et al., 2017). Similarly, we report no difference in the abundance of SLC2A5 in the chorioallantois (fetal membrane) from days 34 to 50 of gestation; however, we do not report SLC2A5 abundance in FM for day 16, and the similarity of SLC2A5 protein abundance to mRNA expression level on days 34 and 50 may suggest the importance of investigating SLC2A5 at day 16 for future studies, especially if the protein abundance is similar to mRNA expression at this time point. Previous data in bovine have demonstrated that fructose concentrations in fetal fluids are 5.5 mM (Holstein cows from days 28 to 42 of pregnancy; Lucy et al., 2012) and 5.0 mM (Angus-cross heifers day 50 of pregnancy; Crouse et al., 2019a), which is much greater than the concentration of glucose in the same fluids 1.39 mM (Lucy et al., 2012) and 1.45 mM (Crouse et al., 2019a). Furthermore, Simintiras et al. (2019) reported fructose and mannitol/sorbitol (the intermediate in the conversion of glucose to fructose) to be the metabolites with the greatest mean fold increase in uterine luminal fluid (18.39- and 28.53-fold, respectively, for fructose and mannitol/sorbitol) for high vs. normal progesterone cows with a 10.70- and 14.86-fold increase, respectively, from days 12 to 14 of the estrus cycle. Crouse et al. (2019a) reported a 7.44-fold increase in fructose from days 16 to 50 of gestation in uterine luminal fluid of heifers. These data suggest an important role of fructose in early pregnancy in a low oxygen environment and further support the need for investigation of SLC2A5 during the peri-implantation period of pregnancy. Finally, Crouse et al. (2019a) reported an interaction of fructose in allantoic fluid such that day 34 CON and RES heifers were greater than day 50 CON heifers, which were all greater than day 50 RES heifers. Although not significant, the interaction of maternal nutrition and day for whole uteroplacental tissue analysis of SLC2A5 on days 34 and 50 as reported herein was similar where the day 34 CON and RES heifers were greater than day 50 CON heifers, which were all greater than day 50 RES heifers, which reflects similarities between substrate concentration and transporter abundance.

Arginine is an important metabolite for the synthesis of nitric oxide via nitric oxide synthase and the metabolism of arginine to citrulline (Gao et al., 2009c). Furthermore, arginine may be metabolized to ornithine, which is a direct precursor of polyamines (Gao et al., 2009c). Arginine, nitric oxide, and polyamines are important for embryonic development via stimulation of migration and elongation of the trophectoderm, increased vascularity, and activation of the mTOR signaling pathway (Wang et al., 2016). Arginine and ornithine share the same cationic amino acid transporters SLC7A1, SLC7A2, and SLC7A3 (mRNA reported for SLC7A3; Crouse et al. 2016b, 2017], but protein not conducted for this study). Knockout of SLC7A1 resulted in decreased arginine transport by 73%, a decrease in arginine metabolism proteins including ornithine decarboxylase and nitric oxide synthase, as well as a 76% decrease in citrulline and 40% decrease in ornithine (Wang et al., 2014). These data demonstrated that SLC7A1 is a key transporter of arginine and is essential for conceptus survival. Building upon this, data presented herein demonstrate that SLC7A1 is also affected by maternal nutrition, with abundance decreased in uteroplacental tissues for RES compared with CON when whole uterine tissues were analyzed at day 16 of gestation and all uteroplacental tissues were analyzed on days 34 and 50 of gestation. However, neither the concentrations of arginine nor ornithine were decreased in RES compared with CON uterine luminal flushes, allantoic fluid, nor amniotic fluids collected from the same heifers as the current study (Crouse et al., 2019a). Analysis of vascularity and angiogenic factors in uteroplacental tissues from the same study (McLean et al., 2017) reported decreased vascular volume and vascular endothelial growth factor mRNA expression in the contralateral uterine horn of RES compared with CON heifers but no difference in the mRNA expression of endothelial nitric oxide synthase. Furthermore, RNA-sequencing on fetal liver collected from this same study at d 50 of gestation determined 45 genes that were differentially expressed were related to metabolism, and 4 of those genes were classified with roles in arginine metabolism or arginine biosynthesis. These included genes that were upregulated in fetuses of RES compared with CON heifers (aminoacylase 1 and guanidinoacetate N-methyltransferase) and genes that were downregulated in fetuses of RES compared with CON heifers (arginase 2 and glutamate dehydrogenase 1; Crouse et al., 2019b). The decrease in SLC7A1 abundance in RES compared with CON heifers is similar to recently published data in the same heifers (Crouse et al., 2020) where neutral amino acid transporters SLC1A1, SLC7A5, SLC38A2, and SLC38A7 abundance were decreased for RES compared with CON heifers. These data suggest that although nutrient concentrations of arginine or its metabolites were not different by maternal nutrition treatment, the main arginine transporter (SLC7A1; Wang et al., 2014) is responsive to moderate maternal nutrient restriction, and the observed alterations in transporter abundance during early gestation could be manifested as altered concentrations of arginine available to the fetus as gestation progresses.

These novel data presented herein build upon previously published data by multiple lab groups to elucidate the complex supply and demand relationship of maternal and embryonic metabolism during early gestation. These data are interpreted to imply that transporters are differentially abundant across uteroplacental tissue types during early gestation. Furthermore, these data are interpreted to imply that hexose transporters are not differentially affected by maternal nutritional treatment; however, concentrations of hexoses in fetal fluids are affected by maternal nutrition, which may suggest altered fetal metabolism in response to maternal nutrition. Additionally, these and other recently published data investigating fructose and the role of fructose in gestation necessitate the investigation of fructose transport and metabolism surrounding the peri-implantation period of gestation. Finally, these data suggest that the main arginine transporter in the uteroplacenta, SLC7A1, is responsive to maternal nutrition, which is similar to previously investigated neutral amino acid transporters in the same tissues presented within this manuscript.

Glossary

Abbreviations

DG

deep endometrial glands

ENDO

endometrium (luminal epithelium)

FM

fetal membrane (chorioallantois)

mTOR

mammalian target of rapamycin

MYO

myometrium

SG

superficial endometrial glands

SLC2A1

solute carrier family 2 member 1

SLC2A3

solute carrier family 2 member 3

SLC2A5

solute carrier family 2 member 5. GLUT5, fructose transporter

SLC7A1

solute carrier family 7 member 1. CAT-1, arginine, lysine, and ornithine transporter

SLC7A2

solute carrier family 7 member 2. CAT-2, arginine, lysine, and ornithine transporter

Conflict of Interest Statement

The authors declare no real or perceived conflicts of interest.