Journal Pre-proof

Regulation of proliferation, apoptosis, hormone secretion and gene expression by

acetyl-L-carnitine in yak (Bos grunniens) granulosa cells

Xu-dong Jiang, Yu Liu, Jian-fei Wu, San-ni Gong, Yao Ma, Xiang-dong Zi

PII: S0093-691X(23)00099-7
DOI: https://doi.org/10.1016/j.theriogenology.2023.03.016
Reference: THE 16629

To appear in: Theriogenology

Received Date: 24 October 2022

Revised Date: 13 March 2023
Accepted Date: 14 March 2023

Please cite this article as: Jiang X-d, Liu Y, Wu J-f, Gong S-n, Ma Y, Zi X-d, Regulation of proliferation,
apoptosis, hormone secretion and gene expression by acetyl-L-carnitine in yak (Bos grunniens)
granulosa cells, Theriogenology (2023), doi: https://doi.org/10.1016/j.theriogenology.2023.03.016.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.

© 2023 Published by Elsevier Inc.

CRediT authorship contribution statement

Xu-dong Jiang: Data curation, Formal analysis, Investigation,

Conceptualization, Writing-original draft. Yu Liu: Methodology,
Conceptualization. Jian-fei Wu: Conceptualization, Methodology. San-ni Gong:
Methodology. Yao Ma: Investigation. Xiang-dong Zi: Writing-review & editing,
Conceptualization, Funding acquisition.

f
r oo
-p
re
lP
na
ur
Jo
 1 Revised
2

3 Regulation of proliferation, apoptosis, hormone secretion and gene expression by

4 acetyl-L-carnitine in yak (Bos grunniens) granulosa cells

5 Xu-dong Jianga, Yu Liua, Jian-fei Wub, San-ni Gonga, Yao Maa, Xiang-dong Zia, *

f
oo
a
7 The Key Laboratory for Animal Science of National Ethnic Affairs Commission,

8 Southwest Minzu University, Chengdu 610041, PR China

r
9 b
-p
Zigong Psychiatric Research Center, Zigong 643020, PR China
re
10
lP

11
na

12
ur

13
Jo

14

15

16 * Corresponding author. Address: College of Animal and Veterinary Science,

17 Southwest Minzu University, Chengdu 610041, PR China. Fax: +86 28 85522310.

18 Email address: zixd@sina.com (X.-D. Zi).

19

1
20 Abstract

21 Supplementation with acetyl-L-carnitine (ALC) during in vitro maturation

22 significantly improves the rates of oocyte cleavage and morula and blastocyst

23 formation in sheep and buffalo; however, the mode of action of ALC in improving

24 oocyte competence is not completely understood. Therefore, the aim of this study was

25 to investigate the effects of ALC on proliferation, antioxidant properties, lipid droplet

26 accumulation and steroid hormone secretion in yak (Bos grunniens) granulosa cells

f
oo
27 (GCs). Yak GCs were identified using FSHR immunofluorescence. The cells were

r
28 -p
treated with different concentrations of ALC, cell proliferation was detected by cell
re
29 counting kit-8, and the optimal concentration and treatment time were determined for
lP

30 subsequent experiments. Then, reactive oxygen species (ROS) were detected by a

DCFH-DA probe, and lipid droplet accumulation was observed by oil red O staining.
na

31

32 Estradiol (E2) and progesterone (P4) in the medium were detected by ELISA, and the
ur

33 expression of genes related to cell proliferation, apoptosis, the cell cycle, antioxidants
Jo

34 and steroid synthesis was determined by RT‒qPCR. The results showed that 1 mM

35 ALC treatment for 48 h was the optimum treatment. It significantly increased cell

36 viability (P < 0.05), significantly decreased the amount of ROS and lipid droplet

37 content, and promoted P4 and E2 secretion (P < 0.05) of yak GCs. RT‒qPCR results

38 verified that GCs treated with 1 mM ALC for 48 h significantly increased the

39 expression of genes related to anti-apoptosis and the cell cycle (BCL-2, PCNA,

40 CCND1 and CCNB1), antioxidants (CAT, SOD2 and GPX1), and E2 and P4 secretion

41 (StAR, CYP19A1 and HSD3B1) (P < 0.05), but it significantly decreased the

2
42 expression of apoptosis genes (BAX and P53) (P < 0.05). In conclusion, ALC

43 increased the viability of yak GCs, reduced the amount of ROS and lipid droplets,

44 increased P4 and E2 synthesis and affected the expression of related genes in yak

45 GCs.

46 Keywords: Acetyl-L-carnitine; Yak; Granulosa cells; Proliferation; Gene expression

f
r oo
-p
re
lP
na
ur
Jo

3
47 1. Introduction

48 As the basis of female reproduction, the main functions of the ovaries are follicle

49 development and ovulation as well as hormone secretion. The growth and

50 development of follicles is an important component of fertility in female animals.

51 Follicles contain oocytes and granulosa cells (GCs), and the essence of follicular

52 growth and development is the proliferation and differentiation of GCs [1]. GCs are

53 linked to oocytes through oocyte-derived factors, and GCs are important because they

f
oo
54 provide steroid hormones and gonadotropin receptors for ovarian and follicle growth

r
55 -p
and development [2] and they provide nutrition to oocytes through gap junctions,
re
56 which in turn affects the reproductive capacity of females [3]. GC apoptosis is
lP

57 involved in the induction of follicular atresia [4-6].

The organism produces reactive oxygen species (ROS) during aerobic processes,
na

58

59 and a large accumulation of ROS causes oxidative stress, leading to inflammation

ur

60 development, senescence and even apoptosis of cells [7]. ROS are one of the factors
Jo

61 that trigger follicular atresia [8]. Lipid droplets, as important organelles of cells, are

62 involved in the lipid metabolism of cells [9]. The metabolism of lipids has an

63 important role in animal reproduction. Oocytes are rich in lipid droplets, which play a

64 crucial role in subsequent maturation as well as in the development of the embryo

65 [10]. Triglycerides gradually decrease with cell maturation, and lipid metabolism

66 serves as an important source of energy for oocyte development during the maturation

67 of porcine oocytes in vitro [11].

4
68 Acetyl-L-carnitine (ALC) is the acetyl ester of the trimethylated amino acid

69 L-carnitine (LC), and as a natural derivative of LC, it can be easily converted to LC

70 when needed [12]. ALC plays an important role in the oxidation of fatty acid

71 metabolic pathways as an effective substrate for mitochondrial energy metabolism [13]

72 and is also a strong antioxidant with a wide range of pharmacological properties, such

73 as antioxidant, antiapoptotic [14], anti-inflammatory [15] and lipid

74 metabolism-promoting properties [16]. Xu et al. [17] reported that ALC affects

f
oo
75 mitochondrial function, regulates oocyte-derived paracrine factors, and increases

r
76 -p
steroid hormone production, thereby improving the quality of mature oocytes and
re
77 improving embryonic development in vitro in buffalo. Supplementation with ALC
lP

78 during in vitro maturation (IVM) improves buffalo oocyte quality after vitrification by

enhancing mitochondrial function and altering the phospholipid composition of

na

79

80 vitrified oocyte membranes [18]. Treatment of oocytes from prepubertal lambs with
ur

81 ALC during IVM increased the blastocyst rate and altered the number, volume, and
Jo

82 distribution of mitochondria, vesicles, and lipid droplets [12].

83 Cell proliferation is regulated by cell cycle-related genes and apoptosis-related

84 genes, and CCND, CCNE and CCNB are all cycle-critical proteins [19]. PCNA acts as

85 a marker of cell proliferation and positively regulates the level of cell proliferation

86 [20]. Compared with healthy follicles, GCs in atretic follicles had reduced expression

87 of NRF-1, increased BAX expression and an increased ratio of BAX to BCL-2

88 expression [21], indicating that changes in the mitochondria-associated gene

89 expression patterns in GCs may lead to follicular atresia during follicle development.

5
 90 Antioxidant enzymes such as manganese superoxide dismutase (SOD2), catalase

91 (CAT) and glutathione peroxidase (GPX) ameliorate apoptosis due to ROS

92 accumulation [22]. The biosynthesis of P4 and E2 is a complex process regulated by

93 multiple proteins and key enzymes (StAR, CYP19A1 and HSD3B1), and the

94 biosynthesis of P4 and E2 is positively and linearly related to the expression of these

95 genes [23].

96 Yak (Bos grunniens) is an irreplaceable livestock species that lives mainly on the

f
oo
97 Tibetan Plateau at altitudes ranging from 2500 to 5000 m. Yaks provide meat and

r
98 -p
milk where few other farm animals will survive because of extreme climatic
re
99 conditions such as low oxygen content in air and cold temperature [24,25]. However,
lP

100 reproduction in yaks is low as a result of seasonal breeding, delayed puberty and a

lower frequency of estrus [26]. The in vitro oocyte maturation rate and embryo
na

101

102 development to morula and blastocyst rates are also low [27]. Currently, the effects of
ur

103 ALC on oocyte competence have been studied in sheep and buffalo [12,17]. GCs are
Jo

104 important somatic cells in follicles that are involved in follicular development, but the

105 role of ALC in GCs is not clear. Therefore, in this experiment, different

106 concentrations of ALC were added to the culture medium to investigate its effects on

107 the proliferation, ROS, lipids, hormone secretion and related genes of yak GCs and to

108 provide a theoretical basis for the use of ALC to improve oocyte IVM in yaks.

109 2. Materials and methods

110 2.1. Reagents and preparation

6
111 ALC, bovine serum albumin (BSA) and Triton X-100 were purchased from

112 Sigma‒Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), 0.25% trypsin,

113 penicillin‒streptomycin (pen-strep) solution, DMEM/F12 medium and PBS were

114 purchased from Gibco (Grand Island, NY, USA). Rabbit anti-bovine FSHR antibody

115 (AF5242) and goat anti-rabbit IgG (H+L) HRP (S0001) were purchased from Affinity

116 (Changzhou, China). Insulin-like Growth Factor 1 (IGF1) was purchased from Sino

117 Biological (Beijing, China). Testosterone (T102170) was purchased from Aladdin

f
oo
118 (Beijing, China). Follicle-stimulating hormone (FSH) was purchased from Vetoquinol

r
119 -p
(Paris, France). Cell Counting Kit-8 (CCK-8) was purchased from Meilunbio (Dalian,
re
120 China). The Oil Red O kit (G1262), DCFH-DA (CA1410), DAPI (C0065) and
lP

121 reactive oxygen species assay kit (R1200) were purchased from Solarbio (Beijing,

China). TRIzol (No. 15596026), an RNA Reverse Transcription kit (M631) and
na

122

123 PowerUpTM SYBRTM Green Master Mix (A25742) were purchased from Thermo
ur

124 Fisher Scientific, Invitrogen (USA). The Bovine Progesterone (P4) ELISA Kit and
Jo

125 Bovine Estrogen (E2) ELISA Kit were purchased from Jianglai Biology (Shanghai,

126 China).

127 2.2. Isolation and identification of GCs

128 Animal care and use were approved by the Southwest Minzu University Animal

129 Care and Use Committee. All female yaks were slaughtered under exactly the same

130 conditions. The ovaries of 18 healthy female yaks were collected in this experiment.

131 GCs were isolated, cultured and evaluated with some modifications as described

132 previously [28]. Briefly, the collected ovaries were placed in 75% alcohol for 30 s.

7
133 Yak ovaries were then washed three times with saline containing 1% pen-strep

134 solution preheated to 34‒35 °C. Only cumulus-oocyte complexes (COCs) aspirated

135 from follicles with diameters of 3‒8 mm were selected for in vitro fertilization (IVF)

136 of the yak [29]; therefore, GCs were isolated from such follicles [30]. Follicles were

137 extracted by aspiration and centrifuged at 1,000 × g for 5 min, and the precipitate was

138 washed 3 times with PBS containing 1% pen-strep prewarmed at 37 °C. Cells were

139 adjusted to 106 cells/mL using complete medium (DMEM-F12 containing 10% FCS

f
oo
140 and 1% pen-strep) and incubated in an incubator at 37 °C with 5% CO2. The culture

r
141 -p
medium was replaced with fresh complete medium 24 h later. Cells were cultured for
re
142 two to three days and then passaged, and second generation cells were used for the
lP

143 assay (within one week). The isolated GCs were identified using immunofluorescence

staining as described by Yousefi et al. [31], with minor modifications. Briefly, yak
na

144

145 GCs were fixed for 20 min with 4% paraformaldehyde and then washed with PBS.
ur

146 The GCs were permeabilised in 0.5% Triton X-100 in PBS for 20 min and then
Jo

147 incubated with 5% FBS for 30 min. After incubation with rabbit anti-FSHR antibody

148 (1:200) overnight at 4 °C, the GCs were incubated with goat anti-rabbit IgG (1:500)

149 for 40 min and DAPI for 15 min. After blocking the slices, they were observed and

150 photographed using laser confocal microscopy (ZISS, LSM880, Germany). The

151 subsequent experiments were performed using second-generation cells for the assay

152 (within one week).

153 2.3. Effects of ALC on the survival rate of GCs

8
154 GCs were seeded in 96-well plates at a 5×103 concentration per well. ALC

155 concentrations were based on previous reports of the concentrations for ALC

156 supplementation in lamb and buffalo oocytes [12,17]. After 24 h of cell adhesion, the

157 cells were treated with different concentrations (0, 0.1, 0.5, 1, 2.5, 5, 10 and 20 mM)

158 of ALC complete medium for 36 h, washed twice with prewarmed PBS at 37 °C,

159 added to prepared complete medium containing 10% CCK-8 complete medium, and

160 incubated for 4 h in the incubator at 37 °C, and the absorbance at 450 nm was

f
oo
161 measured using an enzyme marker (Thermo Scientific, MULTISKAN Sky 51119670,

r
162 -p
Finland). The CCK-8 results were analyzed as described previously by Lin et al. [32].
re
163 The control group and the 36 h treatment optimal concentration group were set
lP

164 up for five time periods of 24, 36, 48, 60 and 72 h. The CCK-8 method was used to

determine the cell survival rate of each group and to screen the optimal culture time.
na

165

166 The optimal concentration at the best treatment time was the ALC treatment group
ur

167 (ALC), and the complete medium was the control group (CON) for subsequent
Jo

168 experiments.

169 2.4. ROS staining assay

170 The GCs were seeded in 12-well plates at the density of 5×105 cells/mL. The

171 ROS content of GC was analyzed as previously described by Wang et al [33], with

172 minor modifications. Briefly, DCFH-DA was diluted to a 10 μmol/L working solution

173 with PBS and stored at -20 °C. After treatment, the cells were washed three times

174 with PBS, 1 mL of working solution was added, and the dishes were gently shaken

175 and incubated in the incubator for another 30 min. Wash three times with PBS and

9
176 stain with DAPI for 15 min. The fluorescence intensity was observed by confocal

177 microscopy (ZISS, LSM880, Germany) at excitation 525 nm and emission 460 nm.

178 Image analysis was performed using ImageJ software.

179 2.5. Oil Red O staining

180 The GCs were seeded in 12-well plates at the density of 5×105 cells/mL. The control

181 and ALC treatment groups were cultured in 12-well plates and stained with Oil Red O

182 according to the manufacturer's instructions. Then, the cells were rinsed twice with

f
oo
183 PBS, and images were taken by microscopy (Olympus, CKX53, Japan). GC lipid

r
184 -p
droplet content was analyzed as previously described by Maoduo et al. [34]. Briefly,
re
185 300 µl of pure isopropanol was added to the wells, and the optical density was
lP

186 measured at 510 nm on an enzyme calibrator (Thermo Scientific, MULTISKAN Sky

51119670, Finland) after shaking for 30 min at 37 °C.

na

187

188 2.6. Effect of ALC on steroidogenesis of yak GCs

ur

189 The GCs were seeded in 12-well plates at the density of 5×105 cells/mL. The
Jo

190 effect of ALC on steroidogenesis of yak GCs was evaluated as previously described

191 [35], with minor modifications. Briefly, yak GCs were incubated for 48 h in 10% FCS

192 at 38.5 °C and then cultured for an additional 48 h in the presence of FSH (30 ng/mL),

193 IGF1 (30 ng/mL), and testosterone (500 ng/mL, as an estrogen precursor) in FCS-free

194 medium with 1 mM ALC in the treatment group and 0 mM ALC in the control group.

195 The medium was collected for determination of E2 and P4 concentrations via ELISA

196 kits, and the experiment was repeated three times. The sensitivities of the E2 and P4

10
197 assays were 7.6 pg/mL and 38.87 pg/mL, respectively. The intrasample coefficient of

198 variation was <10%, and the intersample coefficient of variation was <10%.

199 2.7. Real-time PCR analysis

200 The GCs were seeded in 12-well plates at the density of 5×105 cells/mL. For the

201 yak GCs in the control and ALC treatment groups, the supernatant was withdrawn and

202 discarded, washed three times with PBS, and total RNA was extracted using the Total

203 RNA Extraction Kit. cDNA was synthesized according to the instructions of reverse

f
oo
204 transcription kits and stored in a -20 °C freezer. All primers (Table 1) were designed

r
205 -p
using Primer Premier 5.0 software according to the bovine gene sequences in
re
206 GenBank. The relative mRNA level was normalized to that of β-actin [36]. The
lP

207 synthesis was performed by Chengdu Qingke Yuxi Biotechnology Co, Ltd, China.

Table 1
na

208

209 Primer sequences for quantitative real-time PCR.

ur

Gene Primer sequences（5’-3’） Product length (bp)

Jo

BCL-2 F: TTCGCCGAGATGTCCAGT 132

R: CCCTCCGAACTCAAAGAAGG

BAX F: CGAGTGGCGGCTGAAATGT 93

R: GGCCTTGAGCACCAGTTTG

P53 F: CCCATCCTCACCATCATCAC 80

R: GCACAAACACGCACCTCAA

PCNA F: AGAGCTGAAGATAACGCGG 181

R: GATCTCGGCATATACGTGCAA

11
CASP3 F: GACCATAGCAAAAGGAGCAGT 130

R: TTCTGCAATAGTCCCCTCTG

CCNE1 F: CACCGATGTCTCTGTTCGCT 111

R: CCACACTGGCTTCTCACAGT

CCND1 F:GCCGAGGAGAACAAGCAG 180

R:CGTCAGGCGGTGATAGGA

CCNB1 F:GAAGACGGAGCGGATCCAAA 122

f
oo
R:CCAGTGACTTCACGACCCAT

r
SOD2 -p
F: ACCTCAACGTCGCCGAGG 260
re
R: CCAACCGGAGCCTTGGAC
lP

GPX1 F:CCTGAAGTACGTCCGACCAG 289

R:GTCAGGCTCGATGTCGATGG
na

CAT R:GTCAGGCTCGATGTCGATGG 162

ur

R:GTCAGGCTCGATGTCGATGG
Jo

STAR F: TGGCATGGCCACACTCTATG 111

R: GTCCTTGAGGGACTTCCAGC

HSD3B1 F: TCCGGGTGCTAGACAAAGTC 171

R: TGACGTCAATGACAGAGGCG

CYP19A1 F:TGACCGTCTGTGCCGATTC 120

R:TGTTAGAGGTGTCCAGCATGATG

β-actin F: CCATCGGCAATGAGCGGTTCC 146

R: CGTGTTGGCGTAGAGGTCCTTG

12
210

211 The RT‒qPCR system was 15 µl: cDNA 1 µl, PowerUp™ SYBR™ Green

212 Master Mix 7.5 µl, 0.5 µl each of upstream and downstream primers, and ddH2O 5.5

213 µl. Reaction conditions: UDG enzyme activation 50 °C; predenaturation at 95 °C for

214 2 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, 39 cycles; final

215 reading of the dissolution curve. Three replicates per group were calculated using the

216 2-ΔΔct method for RT‒qPCR to obtain the results of three replicate experiments.

f
oo
217 2.8. Statistical analysis

218 Data are expressed as the means ± standard errors of the means (SEM) of three

r
219
-p
yaks (n = 3) with three technical replicates per animal in each experiment. Statistical
re
220 comparisons were performed using Student’s unpaired t test or one-way ANOVA
lP

221 (with Tukey’s multiple comparisons test as the post hoc test). P < 0.05 was
na

222 considered statistically significant.

ur

223 3. Results
Jo

224 3.1. Identification of yak GCs

225 FSHR is a GC-specific expressed protein, and immunofluorescence staining was

226 used to stain and identify yak GCs. The results showed that more than 80% of the

227 cells in the field of view were positive for FSHR expression, indicating that the

228 isolated cultured cells were GCs (Fig. 1).

229 3.2. Effects of ALC concentration and treatment time

230 As shown in Fig. 2, compared with the control group, the cell viability of yak

231 GCs was increased in the 0.1, 0.5, 1, 2.5 and 5 mM ALC groups and decreased in the

232 10 and 20 mM ALC groups at 36 h of in vitro culture (IVC). The highest viability
13
233 observed in the 1 mM ALC group was significantly greater than that in the other

234 groups (P<0.05).

235 As shown in Fig. 3, the addition of 1 mM ALC increased the cell viability of yak

236 GCs at 24, 36, 48, 60 and 72 h. The cell viability increased gradually from 24 h to 48

237 h. After 48 h, the growth of cell viability slowed down gradually. The highest cell

238 viability increase was observed at 48 h, with significant differences (P < 0.05)

239 compared to 24, 36, 60 and 72 h. Therefore, the medium (DMEM-F12 containing 10%

f
oo
240 FBS and 1% pen-strep solution) was set as the CON group, and 1 mM ALC + 48 h

r
241 was set as the ALC group. -p
re
242 3.3. Effect of ALC on ROS in GCs
lP

243 The ROS levels of yak GCs in the control group and the 1.0 mM ALC-treated

group were examined after IVC for 48 h using the fluorescent probe DCFH-DA. The
na

244

245 control group served as a positive control (Fig. 4A) for the 1 mM ALC-treated GCs
ur

246 (Fig. 4B). The mean fluorescence density of ROS in the two experimental groups was
Jo

247 analyzed (Fig. 4C), and it was found that the ROS level in the 1 mM ALC-treated

248 group was significantly lower than that in the control group (P < 0.01). The results

249 indicated that 1 mM ALC treatment of GCs reduced ROS levels.

250 3.4. Effect of ALC on lipid droplets

251 The content of lipid droplets in GCs was identified using Oil Red O. The results

252 showed that the control group had more lipid droplets (Fig. 5A), while for GCs treated

253 with ALC for 48 h, there was an obvious decrease in the number of lipid droplets (Fig.

254 5B). The results of lipid droplet quantification showed that the content of lipid

14
255 droplets within the ALC-treated group was significantly reduced compared to the

256 control group (P < 0.01) (Fig. 5C).

257 3.5. Effect of ALC on the secretion of E2 and P4 from GCs

258 As shown in Fig. 6, the E2 level in the culture medium of the ALC treatment was

259 significantly greater than that of the control group (P < 0.05), and P4 was extremely

260 significantly greater than that of the control group (P < 0.01) after 48 h of culture.

261 3.6. Effects of ALC on proliferation, apoptosis and cycle-related genes in yak GCs

f
oo
262 The expression of a proliferation-related gene (PCNA) and apoptosis-inhibiting

r
263 -p
gene (BCL-2) was upregulated (P < 0.01) in the ALC treatment group compared with
re
264 the CON group after 48 h of culture, while the expression levels of proapoptotic genes
lP

265 (BAX, P53 and CASP3) were decreased (P < 0.01) (Fig. 7A). The expression levels of

cell cycle-related genes (CCND1 and CCNB1) were upregulated (P < 0.01) in
na

266

267 response to ALC treatment (Fig. 7B).

ur

268 3.7. Effect of ALC on E2 and P4 secretion- and antioxidant-related gene expression
Jo

269 There was a significant upregulation (P < 0.05) of StAR, CYP19A1 and HSD3B1,

270 which might be associated with P4 and E2 secretion in yak GCs (P < 0.01) (Fig. 7C).

271 There was a significant upregulation of antioxidant-related genes (CAT, SOD2 and

272 GPX1) (P < 0.01) in the GCs in the 1 mM ALC group compared with the control

273 group after 48 h of culture (Fig. 7D).

274 4. Discussion

275 GCs, an important component of ovarian follicles, play an important role in

276 follicular development and ovulation or atresia [2]. The proliferation and

15
277 differentiation of GCs as well as the signaling linkage with oocytes determine the fate

278 of oocytes. LC is a naturally occurring antioxidant in mammals, and ALC, as an

279 acetyl ester of LC, is present in plasma as free carnitine and acetyl esters of various

280 lengths, which promote β-oxidation of fatty acids and have good antioxidant,

281 anti-apoptotic and other effects. In the present study, we found that the addition of a

282 low concentration of ALC to the culture medium has a promoting effect on the

283 proliferation of yak GCs.

f
oo
284 To further investigate the effect of different concentrations of ALC at different

r
285 -p
treatment times on the proliferation of yak GCs, we found that different
re
286 concentrations of ALC treatment for 36 h had some effects on the proliferation
lP

287 viability of yak GCs. The 0.1, 0.5, 1, 2.5 and 5 mM treatment groups promoted cell

proliferation, while the 10 and 20 mM treatment groups inhibited cell proliferation,

na

288

289 among which the 1 mM ALC treatment group had the best effect on cell proliferation
ur

290 and the 20 mM treatment group almost lost viability. This is similar to the findings of
Jo

291 Pan et al. [37]. They reported that treatment with 1 mM ALC improved proliferation

292 and alleviated senescence of starved adipose-derived stem cells (ADSCs),

293 accompanied by reduced ROS and increased protein expression of SOD1 and CAT,

294 but ALC at a high concentration (10 mM) markedly aggravated cell apoptosis and

295 senescence. In the present study, the proliferation of GCs was promoted within 48 h

296 of culture, and thereafter, the proliferation ability of cells gradually decreased.

297 Therefore, 1 mM treatment for 48 h was selected as the best treatment group for

298 subsequent experiments. In a study by Xu et al. [17], the addition of 2.5 mM ALC to

16
299 buffalo cumulus cells treated for 24 h was found to have a greater proliferative effect

300 on the cells, but the treatment was not continued for subsequent time periods.

301 PCNA is a key molecule in cell replication, and it was found that the addition of

302 ALC to mouse fibroblast medium significantly increased PCNA expression, stabilized

303 mitochondrial membranes, increased energy supply to organelles, and protected cell

304 proliferation [38]. ALC can reduce cell proliferation due to serum and glucose by

305 downregulating CASP3-deficient mouse adipose-derived mesenchymal stem cell

f
oo
306 (AD-MSC) apoptosis [39]. It was shown that CCND and CCNE promote cell

r
307 -p
transition from G1 to S phase, while CCNB promotes cell transition from G2 to M
re
308 phase and is a key protein for mitotic entry [19]. In this study, we observed that in yak
lP

309 GCs cultured in 1 mM ALC, the expression levels of BCL-2, PCNA, CCNB1 and

CCND1 were upregulated, but the expression levels of BAX, CASP3 and P53 were
na

310

311 downregulated. This indicates that ALC inhibits apoptosis and promotes cell
ur

312 proliferation by promoting the normal progression of the cell cycle in yak GCs.
Jo

313 ROS are chemically active molecules produced during aerobic metabolism that

314 can cause oxidative stress and lead to cellular DNA damage and apoptosis [40].

315 Antioxidants, a class of free radical scavengers, protect cells from ROS and free

316 radical damage under normal physiological conditions in the presence of SOD, GPX

317 and CAT. For example, CAT can reduce ROS production in cells by breaking down

318 intracellular hydrogen peroxide into water and oxygen [41]; SOD2, one of the

319 important members of the superoxide dismutase family, is the first line of defense of

320 the antioxidant system, mainly located in mitochondria, and can scavenge superoxide

17
321 anion radicals, thus protecting cells from ROS damage [42]. Several studies have

322 shown that ALC reduces ROS and alleviates oxidative stress in vivo. For example,

323 when ADSCs were cultured using medium containing 1 mM ALC, senescence was

324 alleviated while reducing ROS and increasing the protein expression of SOD1 and

325 CAT, improving cell proliferation [37]. Feed supplementation with ALC significantly

326 reduced arsenic-induced oxidative stress in the rat hippocampus, modulated

327 antioxidant defenses, and improved mitochondrial function [43]. The addition of ALC

f
oo
328 to buffalo oocyte cultures significantly reduced ROS, thereby improving oocyte

r
329 -p
quality and embryo development [17]. In this study, we found that ALC significantly
re
330 scavenged ROS from yak GCs and upregulated the expression levels of antioxidant
lP

331 genes (CAT, SOD2 and GPX1), and we hypothesized that ALC scavenged

intracellular ROS by promoting the expression of antioxidant genes.

na

332

333 Lipid droplets, as important organelles of cells, are involved in lipid metabolism,
ur

334 transmembrane transport and signaling [9]. ALC, as a precursor of acetylcholine, not
Jo

335 only regulates the release of energy from fatty acid β-oxidation but also participates in

336 the tricarboxylic acid cycle itself to generate ATP [44]. LC can increase the

337 development of oocytes and embryos by improving mitochondrial viability, and

338 increasing β-oxidation can enhance oocyte and embryo development. Porcine oocytes

339 matured in vitro in medium containing 1.25 mg/mL LC IVM were found to have

340 more active mitochondria, fewer lipid droplets and promote lipid metabolism [45]. It

341 has been shown that the addition of LC to embryo medium can enhance the morula

342 embryo stage of bovine embryo development by significantly reducing lipid content

18
343 and promoting lipid utilization [46]. Under the action of carnitine lipid acyltransferase

344 Ι, the 3-hydroxyl group of LC combines with the acyl group of lipid acyl-CoA to form

345 lipid ACL, which later enters the mitochondria from the cytoplasm via translocase

346 and is then separated by the catalytic action of carnitine lipid acyltransferase II to

347 reform lipid acyl-CoA for β-oxidation, which in turn promotes lipid metabolism. It

348 has been found that inhibition of lipid metabolism in bovine GCs decreases cell

349 proliferation [47]. In this study, the results of oil red O staining and lipid droplet

f
oo
350 quantification revealed that the addition of 1 mM ALC to the culture medium could

r
351 -p
significantly reduce lipid droplet volume and promote lipid metabolism in yak GCs,
re
352 thus providing more energy to the cells and increasing the proliferation of yak GCs.
lP

353 GCs are important hormone-like secretory cells in female animals that can

secrete cytokines such as E2 and P4 [2] and provide nutrition to oocytes through gap
na

354

355 junctions [3]. The addition of E2 and P4 during IVM of oocytes significantly
ur

356 increased the maturation rate of oocytes, and the addition of E2 and P4 during the
Jo

357 IVM of Egyptian buffalo oocytes also promoted GC proliferation [48]. StAR is

358 present in mitochondria and facilitates the transport of cholesterol from the outer

359 mitochondrial membrane to the inner membrane; this transport of cholesterol is the

360 rate-limiting step in the synthesis of steroid hormones, and after entering the

361 mitochondria, cholesterol undergoes a series of changes in the tissues to synthesize

362 steroid hormones such as P4, testosterone, E2 and aldosterone [49]. CYP19A1, a key

363 enzyme in estrogen synthesis, catalyzes the conversion of androgens to estrogens in

364 GCs [50]. HSD3B1 is the enzyme required for the final step in the synthesis of P4,

19
365 turning pregnenolone into P4 [51]. Some studies have reported that inhibition of lipid

366 metabolism in bovine GCs reduces P4 secretion and may affect follicular growth [47].

367 It has been shown that the addition of 2.5 mM ALC to the IVM medium of buffalo

368 oocytes can upregulate the expression of StAR, P450scc and GDF9 and significantly

369 increase E2 secretion and slightly increase P4 secretion [17]. The present study found

370 that yak GCs cultured in vitro in medium containing 1 mM ALC for 48 h could

371 promote lipid metabolism, upregulate the steroid-related genes STAR, CYP19A1 and

f
oo
372 HSD3B1, and promote the secretion of E2 and P4 in yak GCs.

r
373 5. Conclusion -p
re
374 We found that yak GCs cultured in medium containing 1 mM ALC for 48 h
lP

375 improved the cell viability of yak GCs, accompanied by increased expression of

PCNA, BCL-2, CCNB1 and CCND1 and decreased expression of BAX, CASP3 and
na

376

377 P53. ALC reduced ROS and alleviated oxidative stress by increasing SOD2, GPX1
ur

378 and CAT levels. The secretion of E2 and P4 in yak GCs was increased by upregulation
Jo

379 of STAR, CYP19A1 and HSD3B1. In addition, ALC reduced the amount of GC lipid

380 droplets and promoted fat metabolism in yak GCs. These findings indicate that ALC

381 may play a positive role in the IVM of yak oocytes.

382 Acknowledgments

383 This work was supported by the National Major Science and Technology

384 Projects of China (No. 2018YFD0502303) and the Fundamental Research Funds for

385 the Central Universities, Southwest Minzu University (2021PTJS26).

386 CRediT authorship contribution statement

20
387 Xu-dong Jiang: Data curation, Formal analysis, Investigation,
388 Conceptualization, Writing-original draft. Yu Liu: Methodology,
389 Conceptualization. Jian-fei Wu: Conceptualization, Methodology. San-ni Gong:
390 Methodology. Yao Ma: Investigation. Xiang-dong Zi: Writing-review & editing,
391 Conceptualization, Funding acquisition.

392 Declaration of Competing Interest

393 The authors declare that there are no conflicts of interest.

394 References

f
oo
395 [1] Maalouf SW, Liu WS, Pate JL. MicroRNA in ovarian function. Cell Tissue Res

r
396 2016;363:7‒18. https://doi.org/10.1007/s00441-015-2307-4

397
-p
[2] Eppig JJ. Oocyte control of ovarian follicular development and function in
re
398 mammals. Reproduction 2001;122:829‒38.
lP

399 https://doi.org/10.1530/rep.0.1220829
na

400 [3] Alam MH, Miyano T. Interaction between growing oocytes and granulosa cells in
ur

401 vitro. Reprod Med Biol 2019;19:13‒23. https://doi.org/10.1002/rmb2.12292

Jo

402 [4] He Y, Deng H, Jiang Z, Li Q, Shi M, Chen H, et al. Effects of melatonin on

403 follicular atresia and granulosa cell apoptosis in the porcine. Mol Reprod Dev

404 2016;83:692‒700. https://doi.org/10.1002/mrd.22676

405 [5] Hughes FM Jr, Gorospe WC. Biochemical identification of apoptosis

406 (programmed cell death) in granulosa cells: evidence for a potential mechanism

407 underlying follicular atresia. Endocrinology 1991;129:2415‒22.

408 https://doi.org/10.1002/mrd.22676

21
409 [6] Murdoch WJ. Programmed cell death in preovulatory ovine follicles. Biol Reprod

410 1995;53:8‒12. https://doi.org/10.1210/endo-129-5-2415

411 [7] Mao X, Gu C, Chen D, Yu B, He J. Oxidative stress-induced diseases and tea

412 polyphenols. Oncotarget 2017;8:81649‒61.

413 https://doi.org/10.18632/oncotarget.20887

414 [8] Meng L, Wu Z, Zhao K, Tao J, Chit T, Zhang S, et al. Transcriptome Analysis of

415 porcine granulosa cells in healthy and atretic follicles: role of steroidogenesis

f
oo
416 and oxidative stress. Antioxidants (Basel) 2020;10:22.

r
417 -p
https://doi.org/10.3390/antiox10010022
re
418 [9] Olzmann JA, Carvalho P. Dynamics and functions of lipid droplets. Nat Rev Mol
lP

419 Cell Biol 2019;20:137‒55. https://doi.org/10.1038/s41580-018-0085-z

[10] Sturmey RG, Reis A, Leese HJ, McEvoy TG. Role of fatty acids in energy
na

420

421 provision during oocyte maturation and early embryo development. Reprod
ur

422 Domest Anim 2009;44:50‒8. https://doi.org/10.1111/j.1439-0531.2009.01402.x

Jo

423 [11] Sturmey RG, Leese HJ. Energy metabolism in pig oocytes and early embryos.

424 Reproduction 2003;126: 197‒204. https://doi.org/10.1530/rep.0.1260197

425 [12] Reader KL, Cox NR, Stanton JA, Juengel JL. Effects of acetyl-L-carnitine on

426 lamb oocyte blastocyst rate, ultrastructure, and mitochondrial DNA copy number.

427 Theriogenology 2015;83:1484‒92.

428 https://doi.org/10.1016/j.theriogenology.2015.01.028

429 [13] Qiao N, Chen H, Du P, Kang Z, Pang C, Liu B, et al. Acetyl-L-carnitine induces

430 autophagy to promote mouse spermatogonia cell recovery after heat stress

22
431 damage. Biomed Res Int 2021;2021:8871328.

432 https://doi.org/10.1155/2021/8871328

433 [14] Wang S, Xu J, Zheng J, Zhang X, Shao J, Zhao L, et al. Anti-Inflammatory and

434 Antioxidant effects of acetyl-L-carnitine on atherosclerotic rats. Med Sci Monit

435 2020;26:e920250. https://doi.org/10.12659/MSM.920250

436 [15] Zhang ZY, Fan ZK, Cao Y, Jia ZQ, Li G, Zhi XD, et al. Acetyl-L-carnitine

437 ameliorates mitochondrial damage and apoptosis following spinal cord injury in

f
oo
438 rats. Neurosci Lett 2015;604:18‒23. https://doi.org/10.1016/j.neulet.2015.06.021

r
439 -p
[16] Traina G. The neurobiology of acetyl-L-carnitine. Front Biosci (Landmark Ed)
re
440 2016;21:1314‒29. https://doi.org/10.2741/4459
lP

441 [17] Xu HY, Yang XG, Lu SS, Liang XW, Lu YQ, Zhang M, et al. Treatment with

acetyl-l-carnitine during in vitro maturation of buffalo oocytes improves oocyte

na

442

443 quality and subsequent embryonic development. Theriogenology 2018;118:80‒9.

ur

444 https://doi.10.1016/j.theriogenology.2018.05.033.
Jo

445 [18] Xu HY, Geng SS, Li TT, Fu Q, Lu SS, Liang XW, et al. Maturation of buffalo

446 oocytes in vitro with acetyl-L-carnitine improves cryotolerance due to changes in

447 mitochondrial function and the membrane lipid profile. Reprod Fertil Dev

448 2019;31:386‒94. https://doi.org/10.1071/RD18102

449 [19] Lim S, Kaldis P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation.

450 Development 2013;140:3079‒93. https://doi.org/10.1242/dev.091744

23
451 [20] Strzalka W, Ziemienowicz A. Proliferating cell nuclear antigen (PCNA): a key

452 factor in DNA replication and cell cycle regulation. Ann Bot 2011;107:1127‒40.

453 https://doi.org/10.1093/aob/mcq243

454 [21] Zhang G, Wan Y, Zhang Y, Lan S, Jia R, Wang Z, et al. Expression of

455 Mitochondria-Associated Genes (PPARGC1A, NRF-1, BCL-2 and BAX) in

456 Follicular Development and Atresia of Goat Ovaries. Reprod Domest Anim

457 2015;50:465-73. https://doi: 10.1111/rda.12514

f
oo
458 [22] Bastaki M, Huen K, Manzanillo P, Chande N, Chen C, Balmes JR, et al.

r
459 -p
Genotype-activity relationship for Mn-superoxide dismutase, glutathione
re
460 peroxidase 1 and catalase in humans. Pharmacogenet Genomics 2006;16:279-86.
lP

461 https://doi: 10.1097/01.fpc.0000199498.08725.9c

[23] Zheng X, Boerboom D, Carrière PD. Transforming growth factor-beta1 inhibits

na

462

463 luteinization and promotes apoptosis in bovine granulosa cells. Reproduction

ur

464 2009;137:969-77. https://doi: 10.1530/REP-08-0365

Jo

465 [24] Wiener G, Han JL, Long RJ. The Yak. 2nd ed. Bangkok: The Regional Office for

466 Asia and the Pacific of the Food and Agriculture Organization of the United

467 Nations; 2003.

468 [25] Qiu Q, Zhang G, Ma T, Qian W, Wang J, Ye Z, et al. The yak genome and

469 adaptation to life at high altitude. Nat Genet 2012;44:946‒9.

470 https://doi.org/10.1038/ng.2343

24
471 [26] Zi XD. Reproduction in female yaks (Bos grunniens) and opportunities for

472 improvement. Theriogenology 2003;59:1303‒12.

473 https://doi.org/10.1016/s0093-691x(02)01172-x

474 [27] Zi XD, Luo B, Xia W, Zheng YC, Xiong XR, Li J, et al. Characterization of

475 transcriptional complexity during pre-implantation development of the yak (Bos

476 grunniens) using RNA-Seq. Reprod Domest Anim 2018;53(3):759-68. https://doi:

477 10.1111/rda.13167

f
oo
478 [28] Wang Y, Yang C, Elsheikh N, Li C, Yang F, Wang G, et al. HO-1 reduces heat

r
479 -p
stress-induced apoptosis in bovine granulosa cells by suppressing oxidative
re
480 stress. Aging (Albany NY) 2019;11:5535–47.
lP

481 https://doi.org/10.18632/aging.102136

[29] Xiao X, Zi XD, Niu HR, Xiong XR, Zhong JC, Li J, et al. Effect of addition of
na

482

483 FSH, LH and proteasome inhibitor MG132 to in vitro maturation medium on the
ur

484 developmental competence of yak (Bos grunniens) oocytes. Reprod Biol

Jo

485 Endocrinol 2014;12:30. https://doi: 10.1186/1477-7827-12-30

486 [30] Zhao D, Fan Y, Xiong X, Yin S, Fu W, Ma Y, et al. Molecular characterization

487 of TRIB1 gene and its role in regulation of steroidogenesis in bos grunniens

488 granulosa cells. Theriogenology 2022;191:1‒9.

489 https://doi.10.1016/j.theriogenology.2022.07.012.

490 [31] Yousefi S, Soleimanirad J, Hamdi K, Farzadi L, Ghasemzadeh A, Kazemi M, et

491 al. Distinct effect of fetal bovine serum versus follicular fluid on

25
492 multipotentiality of human granulosa cells in in vitro condition. Biologicals

493 2018;52:44‒8. https://doi.org/10.1016/j.biologicals.2018.01.002

494 [32] Lin M, Hua R, Ma J, Zhou Y, Li P, Xu X, et al. Bisphenol A promotes autophagy

495 in ovarian granulosa cells by inducing AMPK/mTOR/ULK1 signalling pathway.

496 Environ Int 2021;147:106298. https://doi.org/10.1016/j.envint.2020.106298

497 [33] Wang Y, Yang C, Elsheikh NAH, Li C, Yang F, Wang G, et al. HO-1 reduces

498 heat stress-induced apoptosis in bovine granulosa cells by suppressing oxidative

f
oo
499 stress. Aging (Albany NY) 2019;11(15):5535-5547. https://doi:

r
500 10.18632/aging.102136. -p
re
501 [34] Maoduo Z, Hao Y, Wei W, Feng W, Dagan M. Effects of LPS on the
lP

502 accumulation of lipid droplets, proliferation, and steroidogenesis in goat

luteinized granulosa cells. J Biochem Mol Toxicol 2019;33:e22329.

na

503

504 https://doi.org/10.1002/jbt.22329
ur

505 [35] Chiminelli I, Spicer LJ, Maylem ERS, Caloni F. In vitro effects of Enniatin A on
Jo

506 steroidogenesis and proliferation of bovine granulosa cells. Toxins (Basel)

507 2022;14:714. https://doi: 10.3390/toxins14100714

508 [36] Wang L, Li J, Zhang L, Shi S, Zhou X, Hu Y, et al. NR1D1 targeting CYP19A1

509 inhibits estrogen synthesis in ovarian granulosa cells. Theriogenology

510 2022;180:17-29. https://doi: 10.1016/j.theriogenology.2021.12.009

511 [37] Pan T, Qian Y, Li T, Zhang Z, He Y, Wang J, et al. Acetyl l-carnitine protects

512 adipose-derived stem cells against serum-starvation: regulation on the network

26
513 composed of reactive oxygen species, autophagy, apoptosis and senescence.

514 Cytotechnology 2022;74:105‒21. https://doi.org/10.1007/s10616-021-00514-y

515 [38] Pillich RT, Scarsella G, Risuleo G. Reduction of apoptosis through the

516 mitochondrial pathway by the administration of acetyl-L-carnitine to mouse

517 fibroblasts in culture. Exp Cell Res 2005;306:1‒8.

518 https://doi.org/10.1007/s10616-021-00514-y

519 [39] Abdolmaleki A, Ghayour MB, Behnam-Rassouli M. Protective effects of

f
oo
520 acetyl-L-carnitine against serum and glucose deprivation-induced apoptosis in rat

r
521 -p
adipose-derived mesenchymal stem cells. Cell Tissue Bank 2020;21(4):655‒666.
re
522 https://doi.org/10.1007/s10561-020-09844-1
lP

523 [40] Luo X, Jia S, Ma Q, Zhong M, Gao P, Yu Z, et al. Suppressive effects of

subchronic aluminum overload on the splenic immune function may be related to

na

524

525 oxidative stress in mice. Biol Trace Elem Res 2014;157:249‒255.

ur

526 https://doi.10.1007/s12011-014-9888-8.
Jo

527 [41] Habashy WS, Milfort MC, Rekaya R, Aggrey SE. Cellular antioxidant enzyme

528 activity and biomarkers for oxidative stress are affected by heat stress. Int J

529 Biometeorol 2019;63:1569‒84. https://doi.org/10.1007/s00484-019-01769-z

530 [42] Villaverde AISB, Netherton J, Baker MA. From past to present: The link

531 between reactive oxygen species in sperm and male infertility. Antioxidants

532 (Basel) 2019;8:616. https://doi.org/10.3390/antiox8120616

533 [43] Keshavarz-Bahaghighat H, Sepand MR, Ghahremani MH, Aghsami M, Sanadgol

534 N, Omidi A, et al. Acetyl-L-carnitine attenuates arsenic-induced oxidative stress

27
535 and hippocampal mitochondrial dysfunction. Biol Trace Elem Res

536 2018;184:422‒35. https://doi.org/10.3390/antiox8120616

537 [44] Longo N, Frigeni M, Pasquali M. Carnitine transport and fatty acid oxidation.

538 Biochim Biophys Acta 2016;1863:2422‒35.

539 https://doi.org/10.1016/j.bbamcr.2016.01.023

540 [45] Somfai T, Kaneda M, Akagi S, Watanabe S, Haraguchi S, Mizutani E, et al.

541 Enhancement of lipid metabolism with L-carnitine during in vitro maturation

f
oo
542 improves nuclear maturation and cleavage ability of follicular porcine oocytes.

r
543 Reprod Fertil -p Dev 2011;23:912‒20.
re
544 https://doi.org/10.1016/j.bbamcr.2016.01.023
lP

545 [46] Sutton-McDowall ML, Feil D, Robker RL, Thompson JG, Dunning KR.

Utilization 6of endogenous fatty acid stores for energy production in bovine
na

546

547 preimplantation embryos. Theriogenology 2012;77:1632‒41.

ur

548 https://doi.org/10.1016/j.theriogenology.2011.12.008
Jo

549 [47] Elis S, Desmarchais A, Maillard V, Uzbekova S, Monget P, Dupont J. Cell

550 proliferation and progesterone synthesis depend on lipid metabolism in bovine

551 granulosa cells. Theriogenology 2015;83:840‒53.

552 https://doi.org/10.1016/j.theriogenology.2014.11.019

553 [48] Amer HA, Hegab AR, Zaabal SM. Some studies on the morphological aspects of

554 buffalo oocytes in relation to the ovarian morphology and culture condition. In

555 Vitro Cell Dev Biol Anim 2009. https://doi.org/10.1007/s11626-009-9224-3

28
556 [49] Khalil SR, Awad A, Ali SA. Melamine and/or formaldehyde exposures affect

557 steroidogenesis via alteration of StAR protein and testosterone synthetic enzyme

558 expression in male mice. Environ Toxicol Pharmacol 2017;50:136‒44.

559 https://doi.org/10.1016/j.etap.2017.01.018

560 [50] Stocco C. Aromatase expression in the ovary: hormonal and molecular regulation.

561 Steroids 2008;73:473‒87. https://doi.org/10.1016/j.steroids.2008.01.017

562 [51] Thomas JL, Duax WL, Addlagatta A, Brandt S, Fuller RR, Norris W.

f
oo
563 Structure/function relationships responsible for coenzyme specificity and the

r
564 -p
isomerase activity of human type 1 3 beta-hydroxysteroid dehydrogenase/
re
565 isomerase. J Biol Chem 2003;278:35483‒90.
lP

566 https://doi.org/10.1074/jbc.M304752200
na
ur
Jo

29
567 Figure legends
568 Fig. 1. Identification of yak follicular GCs by FSHR immunofluorescence.

569 Second-generation cells were used in the experiment for the identification of yak GCs.

570 GCs were incubated at 37 °C and 5% CO2 for 48 h using complete medium. Brownish

571 yellow fluorescence indicates the specific expression of FSHR, and blue fluorescence

572 indicates the nucleus. Bar = 20 μm.

573 Fig. 2. Effect of different concentrations of ALC in culture medium on the viability of

f
oo
574 yak GCs at 36 h. The data shown are the mean ± SEM of three yaks (n = 3) with three

r
575 -p
replicates per animal. The different lowercase letters indicate P < 0.05, one-way
re
576 ANOVA (Tukey’s multiple comparisons post hoc test).
lP

577 Fig. 3. Effect of 1 mM ALC in culture medium on the viability of yak GCs at

different treatment times. The data shown are expressed as the mean ± SEM of three
na

578

579 yaks (n = 3) with three replicates per animal. The different lowercase letters indicate
ur

580 P < 0.05, one-way ANOVA (Tukey’s multiple comparisons post hoc test).
Jo

581 Fig. 4. Comparison of the concentrations of ROS between yak GCs cultured in

582 untreated medium (CON) and in medium containing 1.0 mM ALC. (A). Control

583 group ROS. (B). ALC treatment group (n = 3). (C). Average fluorescence density of

584 ROS. **P < 0.01, t test. The data shown are the mean ± SEM of three yaks (n =3)

585 with three replicates per animal.

586 Fig. 5. Effects of ALC on lipid droplet content in GCs of yaks between yaks GCs

587 cultured in untreated medium (CON) and in medium containing 1.0 mM ALC. GCs

588 were incubated for 48 h and then treated to detect lipid droplet content. (A). Oil red O

30
589 staining in the control group (n = 3). (B). Oil red O staining in the ALC-treated group

590 (n = 3); Scale bars = 50 μm. (C). Oil Red O-stained intracellular lipids were extracted

591 with isopropanol and quantified by measuring the absorbance at 510 nm (n = 3). **P

592 < 0.01, t test. Data shown are the mean ± SEM from three independent experiments.

593 Fig. 6. Effect of ALC on the concentrations of estradiol (E2) and progesterone (P4) in

594 yak GCs between yak GCs cultured in untreated medium (CON) and in medium

595 containing 1.0 mM ALC. GCs were incubated for 48 h, the supernatants were

f
oo
596 harvested, and estradiol (A) and progesterone (B) production were measured by

r
597 -p
ELISA (n = 3). *P < 0.05, **P < 0.01, t test. Data shown are the mean ± SEM of
re
598 three yaks (n =3) with three replicates per animal.
lP

599 Fig. 7. Effect of ALC on gene expression levels in yak GCs. (A). Proliferation and

apoptosis-related genes (n = 3); (B). Cell cycle-related genes (n = 3); (C) Steroid
na

600

601 synthesis genes (n = 3); (D). Antioxidant-related genes (n = 3). Yak GCs were
ur

602 cultured in untreated medium (CON) and medium containing 1.0 mM ALC for 48 h.
Jo

603 **P < 0.01, t test. Data shown are the mean ± SEM of three yaks (n =3) with three

604 replicates per animal.

31
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Jo
ur
na
lP
re
-p
ro
of
Highlights

⚫ Treatment with 1 mM acetyl-L-carnitine (ALC) increased viability of granulosa

cells

⚫ ALC decreased the amount of reactive oxygen species and lipid droplet content

⚫ ALC promoted progesterone and estradiol secretion of yak granulosa cells

⚫ ALC increased the expression of antiapoptotic, cell cycle and antioxidant genes

f
⚫ ALC decreased the expression of apoptotic genes of yak granulosa cells

r oo
-p
re
lP
na
ur
Jo