Hypoglycemia is a neglected metabolic disorder. Thus, we evaluated the protective effect of hypoxia-preconditioned human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) on hypoglycemic testicular injury. We examined 56 testes from 28 animals: 7 rats with insulin-induced hypoglycemia (HG group), 7 hypoglycemic rats which received an intratesticular injection of hUCB-MSCs (HG-MSC group), and 14 untreated control rats. Testosterone level, testicular catalase (CAT) activity, and malondialdehyde (MDA) level were analyzed. Immunostaining for specific testicular germ and somatic cell markers was performed. Proliferating and apoptotic cells were detected by anti-PCNA and anti-caspase-3, respectively. Morphometrical data were statistically analyzed. The hypoglycemic rats showed a significant decrease in testosterone level and CAT activity and a significant increase in MDA production. Examination of histological structure and protein expression of diverse germ cell markers revealed collapsed tubules that were lined by degenerated germ cells, decreased lactate dehydrogenase type C immune expression, as well as decreased proliferating and increased apoptotic cells number in hypoglycemic testes. Injection of MSCs improved testicular biochemical parameters, preserved germ cells and somatic cells, and decreased apoptosis. In conclusion, hypoxia-preconditioned hUCB-MSCs attenuate rat testicular injury caused by insulin-induced hypoglycemia. Avoidance and rapid management of hypoglycemia are necessary to avoid significant testicular injury.

1.
Abd Allah
SH
,
Pasha
HF
,
Abdelrahman
AA
,
Mazen
NF
.
Molecular effect of human umbilical cord blood CD34-positive and CD34-negative stem cells and their conjugate in azoospermic mice
.
Mol Cell Biochem
.
2017
Apr
;
428
(
1-2
):
179
91
.
[PubMed]
0300-8177
2.
Aebi
H
.
Catalase in vitro
.
Methods Enzymol
.
1984
;
105
:
121
6
.
[PubMed]
0076-6879
3.
Ahn
SW
,
Gang
GT
,
Kim
YD
,
Ahn
RS
,
Harris
RA
,
Lee
CH
, et al.
Insulin directly regulates steroidogenesis via induction of the orphan nuclear receptor DAX-1 in testicular Leydig cells
.
J Biol Chem
.
2013
May
;
288
(
22
):
15937
46
.
[PubMed]
0021-9258
4.
Alves MG, Socorro S, Silva J, Barros A, Sousa M, Cavaco JE, et al. In vitro cultured human Sertoli cells secrete high amounts of acetate that is stimulated by 17β-estradiol and suppressed by insulin deprivation. Biochim Biophys Acta. 2012 Aug;1823(8):1389–94.
5.
Anand
S
,
Bhartiya
D
,
Sriraman
K
,
Patel
H
,
Manjramkar
D
.
Very Small Embryonic-Like Stem Cells Survive and Restore Spermatogenesis after Busulphan Treatment in Mouse Testis
.
J Stem Cell Res Ther
.
2014
;
216
(
4
):
2
16
.2157-7633
6.
Arikawe
AP
,
Oyerinde
A
,
Olatunji-Bello
II
,
Obika
LF
.
Streptozotocin diabetes and insulin resistance impairment of spermatogenesis in adult rat testis: central vs. local mechanism
.
Niger J Physiol Sci
.
2012
Dec
;
27
(
2
):
171
9
.
[PubMed]
0794-859X
7.
Athwal VS, Pritchett J, Llewellyn J, Martin K, Camacho E, Raza SM, et al. SOX9 predicts progression toward cirrhosis in patients while its loss protects against liver fibrosis. EMBO Mol Med. 2017 Dec;9(12):1696–1710.
8.
Banerjee
A
,
Anuradha
,
Mukherjee
K
,
Krishna
A
.
Testicular glucose and its transporter GLUT 8 as a marker of age-dependent variation and its role in steroidogenesis in mice
.
J Exp Zool A Ecol Genet Physiol
.
2014
Nov
;
321
(
9
):
490
502
.
[PubMed]
1932-5231
9.
Bernardino
RL
,
D’Souza
WN
,
Rato
L
,
Rothstein
JL
,
Dias
TR
,
Chui
D
, et al.
Knockout of MCT1 results in total absence of spermatozoa, sex hormones dysregulation, and morphological alterations in the testicular tissue
.
Cell Tissue Res
.
2019
Nov
;
378
(
2
):
333
9
.
[PubMed]
0302-766X
10.
Chen H, Stanley E, Jin S, Zirkin BR. Stem Leydig cells: from fetal to aged animals. Birth Defects Res C Embryo Today. 2010 Dec;90(4):272–83.
11.
Chen
B
,
Chen
D
,
Jiang
Z
,
Li
J
,
Liu
S
,
Dong
Y
, et al.
Effects of estradiol and methoxychlor on Leydig cell regeneration in the adult rat testis
.
Int J Mol Sci
.
2014
May
;
15
(
5
):
7812
26
.
[PubMed]
1661-6596
12.
Creasy D, Bube A, de Rijk E, Kandori H, Kuwahara M, Masson R, et al. Proliferative and nonproliferative lesions on the rat and mouse male reproductive system. Toxicol Pathol. 2012 Aug;40(6 Suppl):40–121S.
13.
Desimone ME, Weinstock RS. Non-diabetic hypoglycemia. In: Endotext [Internet]: MDText.com, Inc.; 2017.
14.
Doi
H
,
Kitajima
Y
,
Luo
L
,
Yan
C
,
Tateishi
S
,
Ono
Y
, et al.
Potency of umbilical cord blood- and Wharton’s jelly-derived mesenchymal stem cells for scarless wound healing
.
Sci Rep
.
2016
Jan
;
6
(
1
):
18844
.
[PubMed]
2045-2322
15.
Ehmcke
J
,
Schlatt
S
.
Identification and characterization of spermatogonial subtypes and their expansion in whole mounts and tissue sections from primate testes. Germline Stem Cells
.
Springer
;
2008
. pp.
109
18
.
16.
El-Behery EI, El-Naseery NI, El-Ghazali HM, Elewa YHA, Mahdy EAA, El-Hady E, et al. The efficacy of chronic zinc oxide nanoparticles using on testicular damage in the streptozotocin-induced diabetic rat model. Acta Histochem. 2019 Jan;121(1):84–93.
17.
Elewa
YH
,
Mohamed
AA
,
Galal
AA
,
El-Naseery
NI
,
Ichii
O
,
Kon
Y
.
Food Yellow4 reprotoxicity in relation to localization of DMC1 and apoptosis in rat testes: roles of royal jelly and cod liver oil
.
Ecotoxicol Environ Saf
.
2019
Mar
;
169
:
696
706
.
[PubMed]
0147-6513
18.
El Omar
R
,
Beroud
J
,
Stoltz
JF
,
Menu
P
,
Velot
E
,
Decot
V
.
Umbilical cord mesenchymal stem cells: the new gold standard for mesenchymal stem cell-based therapies?
Tissue Eng Part B Rev
.
2014
Oct
;
20
(
5
):
523
44
.
[PubMed]
1937-3368
19.
Errea
A
,
Cayet
D
,
Marchetti
P
,
Tang
C
,
Kluza
J
,
Offermanns
S
, et al.
Lactate inhibits the pro-inflammatory response and metabolic reprogramming in murine macrophages in a GPR81-independent manner
.
PLoS One
.
2016
Nov
;
11
(
11
):
e0163694
.
[PubMed]
1932-6203
20.
Galipeau
J
,
Sensébé
L
.
Mesenchymal stromal cells: clinical challenges and therapeutic opportunities
.
Cell Stem Cell
.
2018
Jun
;
22
(
6
):
824
33
.
[PubMed]
1934-5909
21.
Gouda
ZA
,
Khalifa
ME
,
Shalaby
SM
,
Hussein
S
.
Mechanistic effect of human umbilical cord blood derived mesenchymal stem cells on the submandibular salivary gland in ovariectomized rats
.
Biochem Cell Biol
.
2018
Feb
;
96
(
1
):
57
67
.
[PubMed]
0829-8211
22.
Gupta
G
.
Isozymes of glycolytic pathway in sperm: the unique sites for contraception
.
Int J Bioassays
.
2013
;
2
:
1354
74
.2278-778X
23.
Hashimoto T, Brooks GA. Mitochondrial lactate oxidation complex and an adaptive role for lactate production. Med Sci Sports Exerc. 2008 Mar;40(3):486–94.
24.
Hsiao
C
,
Ji
A
,
Chang
C
,
Cheng
C
,
Lee
L
,
Ho
J
.
Local Injection of Mesenchymal Stem Cells Protects Testicular Torsion-induced Germ Cell Injury
.
Cytotherapy
.
2016
;
18
(
6
):
116
26
. 1465-3249
25.
Jia
YF
,
Feng
Q
,
Ge
ZY
,
Guo
Y
,
Zhou
F
,
Zhang
KS
, et al.
Obesity impairs male fertility through long-term effects on spermatogenesis
.
BMC Urol
.
2018
May
;
18
(
1
):
42
.
[PubMed]
1471-2490
26.
Johnson
ES
,
Koepsell
TD
,
Reiber
G
,
Stergachis
A
,
Platt
R
.
Increasing incidence of serious hypoglycemia in insulin users
.
J Clin Epidemiol
.
2002
Mar
;
55
(
3
):
253
9
.
[PubMed]
0895-4356
27.
Kheirandish M, Gavgani SP, Samiee S. The effect of hypoxia preconditioning on the neural and stemness genes expression profiling in human umbilical cord blood mesenchymal stem cells. Transfus Apher Sci. 2017 Jun;56(3):392–9.
28.
Kobayashi
T
,
Namekawa
J
,
Shimomoto
T
,
Yasui
M
,
Iijima
T
,
Itano
Y
, et al.
The effects of long-lasting hypoglycemia on male reproductive organs in rats
.
J Toxicol Sci
.
2015
Dec
;
40
(
6
):
719
25
.
[PubMed]
0388-1350
29.
Kumar
,
G. L.
&
J.
Kiernan
(
2010
)
Special stains and H&E.
Connection, 14.
30.
Kyselová P, Zourek M, Rusavý Z, Trefil L, Racek J. Hyperinsulinemia and oxidative stress. Physiol Res. 2002;51(6):591–5.
31.
Lavrentieva
A
,
Majore
I
,
Kasper
C
,
Hass
R
.
Effects of hypoxic culture conditions on umbilical cord-derived human mesenchymal stem cells
.
Cell Commun Signal
.
2010
Jul
;
8
(
1
):
18
.
[PubMed]
1478-811X
32.
Liang J, Wang N, He J, Du J, Guo Y, Li L, et al. Induction of Sertoli-like cells from human fibroblasts by NR5A1 and GATA4. Elife. 2019 Nov;8:e48767.
33.
Lue
Y
,
Erkkila
K
,
Liu
PY
,
Ma
K
,
Wang
C
,
Hikim
AS
, et al.
Fate of bone marrow stem cells transplanted into the testis: potential implication for men with testicular failure
.
Am J Pathol
.
2007
Mar
;
170
(
3
):
899
908
.
[PubMed]
0002-9440
34.
Nayernia
K
,
Lee
JH
,
Drusenheimer
N
,
Nolte
J
,
Wulf
G
,
Dressel
R
, et al.
Derivation of male germ cells from bone marrow stem cells
.
Lab Invest
.
2006
Jul
;
86
(
7
):
654
63
.
[PubMed]
0023-6837
35.
Nematollahi LR, Kitabchi AE, Stentz FB, Wan JY, Larijani BA, Tehrani MM, et al. Proinflammatory cytokines in response to insulin-induced hypoglycemic stress in healthy subjects. Metabolism. 2009 Apr;58(4):443–8.
36.
Odet
F
,
Duan
C
,
Willis
WD
,
Goulding
EH
,
Kung
A
,
Eddy
EM
, et al.
Expression of the gene for mouse lactate dehydrogenase C (Ldhc) is required for male fertility
.
Biol Reprod
.
2008
Jul
;
79
(
1
):
26
34
.
[PubMed]
0006-3363
37.
Oliveira
PF
,
Martins
AD
,
Moreira
AC
,
Cheng
CY
,
Alves
MG
.
The Warburg effect revisited—lesson from the Sertoli cell
.
Med Res Rev
.
2015
Jan
;
35
(
1
):
126
51
.
[PubMed]
0198-6325
38.
Oltmanns
KM
,
Fruehwald-Schultes
B
,
Kern
W
,
Born
J
,
Fehm
HL
,
Peters
A
.
Hypoglycemia, but not insulin, acutely decreases LH and T secretion in men
.
J Clin Endocrinol Metab
.
2001
Oct
;
86
(
10
):
4913
9
.
[PubMed]
0021-972X
39.
Pattappa
G
,
Heywood
HK
,
de Bruijn
JD
,
Lee
DA
.
The metabolism of human mesenchymal stem cells during proliferation and differentiation
.
J Cell Physiol
.
2011
Oct
;
226
(
10
):
2562
70
.
[PubMed]
0021-9541
40.
Proia P, Di Liegro CM, Schiera G, Fricano A, Di Liegro I. Lactate as a metabolite and a regulator in the central nervous system. Int J Mol Sci. 2016 Sep;17(9):1450.
41.
Rato L, Alves MG, Socorro S, Duarte AI, Cavaco JE, Oliveira PF. Metabolic regulation is important for spermatogenesis. Nat Rev Urol. 2012 May;9(6):330–8.
42.
Roche KC, Gracz AD, Liu XF, Newton V, Akiyama H, Magness ST. SOX9 maintains reserve stem cells and preserves radioresistance in mouse small intestine. Gastroenterology. 2015 Nov;149(6):1553–63.e10.
43.
Sagaradze
G
,
Basalova
N
,
Kirpatovsky
V
,
Ohobotov
D
,
Nimiritsky
P
,
Grigorieva
O
, et al.
A magic kick for regeneration: role of mesenchymal stromal cell secretome in spermatogonial stem cell niche recovery
.
Stem Cell Res Ther
.
2019
Nov
;
10
(
1
):
342
.
[PubMed]
1757-6512
44.
Schneider
CC
,
Ateschrang
A
,
Königsrainer
I
,
Glatzle
J
,
Bühler
S
,
Schaefer
R
, et al.
Lactate influences the gene expression profile of human mesenchymal stem cells (hMSC) in a dose dependant manner
.
Cell Physiol Biochem
.
2012
;
30
(
6
):
1547
56
.
[PubMed]
1015-8987
45.
Selleri
S
,
Bifsha
P
,
Civini
S
,
Pacelli
C
,
Dieng
MM
,
Lemieux
W
, et al.
Human mesenchymal stromal cell-secreted lactate induces M2-macrophage differentiation by metabolic reprogramming
.
Oncotarget
.
2016
May
;
7
(
21
):
30193
210
.
[PubMed]
1949-2553
46.
Shen
C
,
Lie
P
,
Miao
T
,
Yu
M
,
Lu
Q
,
Feng
T
, et al.
Conditioned medium from umbilical cord mesenchymal stem cells induces migration and angiogenesis
.
Mol Med Rep
.
2015
Jul
;
12
(
1
):
20
30
.
[PubMed]
1791-2997
47.
Strober
W
.
Trypan blue exclusion test of cell viability
.
Curr Protoc Immunol
.
2015
Nov
;
111
(
1
):
1
.
[PubMed]
1934-3671
48.
Wei
X
,
Peng
G
,
Zheng
S
,
Wu
X
.
Differentiation of umbilical cord mesenchymal stem cells into steroidogenic cells in comparison to bone marrow mesenchymal stem cells
.
Cell Prolif
.
2012
Apr
;
45
(
2
):
101
10
.
[PubMed]
0960-7722
49.
Xing
X
,
Zhang
Z
,
Zhong
L
,
Ju
G
,
Zou
X
,
Zhu
Y
, et al.
Differentiation of human umbilical cord mesenchymal stem cells into steroidogenic cells in vitro
.
Exp Ther Med
.
2016
Dec
;
12
(
6
):
3527
34
.
[PubMed]
1792-0981
50.
Xiong
W
,
Wang
H
,
Wu
H
,
Chen
Y
,
Han
D
.
Apoptotic spermatogenic cells can be energy sources for Sertoli cells
.
Reproduction
.
2009
Mar
;
137
(
3
):
469
79
.
[PubMed]
1470-1626
51.
Yan
HH
,
Mruk
DD
,
Lee
WM
,
Cheng
CY
.
Blood-testis barrier dynamics are regulated by testosterone and cytokines via their differential effects on the kinetics of protein endocytosis and recycling in Sertoli cells
.
FASEB J
.
2008
Jun
;
22
(
6
):
1945
59
.
[PubMed]
0892-6638
52.
Yang
RF
,
Liu
TH
,
Zhao
K
,
Xiong
CL
.
Enhancement of mouse germ cell-associated genes expression by injection of human umbilical cord mesenchymal stem cells into the testis of chemical-induced azoospermic mice
.
Asian J Androl
.
2014
Sep-Oct
;
16
(
5
):
698
704
.
[PubMed]
1008-682X
53.
Yang
Y
,
Li
Z
,
Wu
X
,
Chen
H
,
Xu
W
,
Xiang
Q
, et al.
Direct reprogramming of mouse fibroblasts toward Leydig-like cells by defined factors
.
Stem Cell Reports
.
2017
Jan
;
8
(
1
):
39
53
.2213-6711
54.
Yazawa
T
,
Mizutani
T
,
Yamada
K
,
Kawata
H
,
Sekiguchi
T
,
Yoshino
M
, et al.
Differentiation of adult stem cells derived from bone marrow stroma into Leydig or adrenocortical cells
.
Endocrinology
.
2006
Sep
;
147
(
9
):
4104
11
.
[PubMed]
0013-7227
55.
Yousefzade G, Nakhaee A. Insulin-induced hypoglycemia and stress oxidative state in healthy people. Acta Diabetol. 2012 Dec;49(Suppl 1):S81–5.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.