Alonso Sedano

Theriogenology 161 (2021) 26e40
Contents lists available at ScienceDirect
Theriogenology
journal homepage: www.theriojournal.com
Changes in miRNA levels of sperm and small extracellular vesicles of

seminal plasma are associated with transient scrotal heat stress in
bulls
Maíra Bianchi Rodrigues Alves a, Rubens Paes de Arruda a, Leonardo Batissaco a,
Laura Nataly Garcia-Oliveros a, Vitor Hugo Guilger Gonzaga a,
Moreira Nogueira a, Fla
Vinícius Jose via dos Santos Almeida a,
^mara Cristine Costa Pinto , Gabriella Mamede Andrade b, Felipe Perecin b,
Sa a
Juliano Coelho da Silveira b, Eneiva Carla Carvalho Celeghini a, *

a ~o Paulo, Pirassununga, Sa
Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sa ~o Paulo, Brazil
b ~o Paulo, Pirassununga, Sa
Department of Veterinary Medicine, School of Animal Science and Food Engineering, University of Sa ~o Paulo, Brazil
a r t i c l e i n f o a b s t r a c t
Article history: Scrotal heat stress affects spermatogenesis and impairs male fertility by increasing sperm morphological
Received 26 November 2019 abnormalities, oxidative stress and DNA fragmentation. While sperm morpho-functional changes trig-
Received in revised form gered by scrotal heat stress are well described, sperm molecular alterations remain unknown. Recently,
21 November 2020
spermatozoa were described as accumulating miRNAs during the last steps of spermatogenesis and
Accepted 23 November 2020
through epididymis transit, mainly by communication with small extracellular vesicles (sEVs). Herein,
Available online 27 November 2020
the aim was to investigate the impact of scrotal heat stress in miRNAs profile of sperm, as well as, seminal
plasma sEVs. Six Nelore bulls (Bos indicus) were divided into two groups: Control (CON; n ¼ 3) and
Keywords:
Epididymis
Scrotal Heat Stress (SHS; n ¼ 3; scrotal heat stressed during 96 h by scrotal bags). The day that the scrotal
Epididymosomes bags were removed from SHS group was considered as D0 (Day zero). Seminal plasma sEVs were isolated
Exosomes from semen samples collected seven days after heat stress (Dþ7) to evaluate sEVs diameter, concen-
microRNAs tration, and 380 miRNA levels. Sperm morpho-functional features and profile of 380 miRNAs were
Semen evaluated from semen collected 21 days after heat stress (Dþ21). As a control, sEVs and sperm were
Testicular thermoregulation analyzed seven days before heat stress (D-7). Only semen parameters that were not significantly different
(P > 0.05) among bulls on D-7 were addressed on Dþ7 and Dþ21. While no alterations in diameter and
concentration were detected in sEVs on Dþ7 between CON and SHS groups, three sEVs-miRNAs (miR-
23b-5p, 489 and 1248) were down-regulated in SHS bulls compared to CON on Dþ7; other three
(miR-126-5p, 656 and 1307) displayed a tendency (0.05 < P < 0.10) to be altered. Sperm oxidative
stress was higher, and the level of 21 sperm miRNAs was altered (18 down-, 3 up-regulated) in SHS bulls
compared to CON on Dþ21. Functional analysis indicated that target genes involved in transcription
activation, as well as cell proliferation and differentiation were related to the 18 down-regulated sperm
miRNAs (miR-9-5p, 15a, 18a, 20b, 30a-5p, 30b-5p, 30d, 30e-5p 34b, 34c, 106b, -126-
5p, 146a, 191, 192, 200b, 335 and 449a). Thus, the scrotal heat stress probably impacted
testicular and epididymis functions by reducing the levels of a substantial proportion of sEVs and sperm
miRNAs. Our findings suggest that miR-126-5p was possibly trafficked between sEVs and sperm and
provide new insights on the mechanism by which sperm acquire miRNAs in the last stages of sper-
matogenesis and sperm maturation in cattle.
© 2020 Elsevier Inc. All rights reserved.
* Corresponding author.
E-mail addresses: maira.bianchi@gmail.com (M.B.R. Alves), arrudarp@usp.br (R.P. Arruda), leonardo.batissaco@usp.br (L. Batissaco), natalygarcia624@gmail.com
(L.N. Garcia-Oliveros), vitorhugomedvet@gmail.com (V.H.G. Gonzaga), vnogueira86@gmail.com (V.J.M. Nogueira), flavia.s.almeida@hotmail.com (F.S. Almeida),
scristinecostapinto@usp.br (S.C.C. Pinto), gabriellamamede@gmail.com (G.M. Andrade), fperecin@usp.br (F. Perecin), julianodasilveira@usp.br (J.C. da Silveira), celeghin@
usp.br (E.C.C. Celeghini).
https://doi.org/10.1016/j.theriogenology.2020.11.015
0093-691X/© 2020 Elsevier Inc. All rights reserved.
M.B.R. Alves, R.P. Arruda, L. Batissaco et al. Theriogenology 161 (2021) 26e40
1. Introduction proliferation as well as offspring healthy [22e24]. While these

molecules are accumulated in sperm during the last stages of
Heat stress negatively affects the reproductive processes and spermatogenesis (spermiogenesis) [25], injuries in spermatogen-
might decrease fertility potential in both males and females [1]. In esis might dysregulate the miRNAs content in sperm, which
male domestic mammalian, such as in men, heat stress could potentially impairs sperm molecular quality and harms, conse-
impair testicular thermoregulation triggering testicular hyper- quently, male fertility potential. In addition to spermiogenesis,
thermia [2]. Since the spermatogenesis in these species relies on miRNA molecules are acquired by spermatozoa during their transit
testicular thermoregulation capability, heat stress may generate a through the epididymis, mainly by the interaction with epididymal
testicular degeneration process [3,4]. Testicular degeneration is the extracellular vesicles, i.e. epididymosomes [26e28]. The epidid-
most prevalent factor involved in bull infertility in tropical climates ymal small extracellular vesicles (sEVs) are included in the epi-
[5]. In humans, testicular degeneration is associated with varico- didymosomes released in the lumen of the epididymis, and form
cele, a common abnormality that affects the male reproductive the seminal plasma in addition to prostasomes and other extra-
tract and may impair human fertility [6]. Indeed, testicular cellular vesicles [29]. Since the epididymal sEVs are small vesicles
degeneration disturbance is highly prevalent in many species since (100e200 nm) containing a molecular profile regulated and
several factors are involved in triggering testicular hyperthermia released by the epididymal epithelial cells [27,30,31], an injury in
and, thus, testicular degenerative processes, such as high environ- epididymis may alter the miRNA profile from these epididymal
mental temperature, scrotal traumas, and infectious/febrile pro- sEVs, changing the sperm miRNA levels in consequence. However,
cesses [7,8]. it remains unknown whether injuries to epididymis could impact
Although the paradigm referring to hypoxia as the mediator of sEV miRNAs content in bulls.
the testicular heat stress effects has been recently questioned [9], it Therefore, considering that (1) heat stress is an important factor
is postulated that testicular heat stress triggers an increase in cell related to male infertility, (2) molecular aspects related to sperm
metabolism associated with higher activity of the epithelial cells are poorly investigated, (3) sperm could accumulate miRNAs during
from seminiferous tubule [10]. From this perspective, the oxygen the last steps of spermatogenesis and through the epididymal
level arriving in testis is not enough to supply the increased de- passage by interacting with sEVs, and (4) seminal plasma sEVs
mand, which creates an environment of hypoxia that results in contain important miRNAs that can modulate sperm; on the pre-
oxidative stress, DNA fragmentation, and cell apoptosis [11,12]. As a sent study, we aimed to investigate the effect of scrotal heat stress
consequence, the germ cells from seminiferous tubule show higher on miRNA profile of seminal plasma sEVs and sperm. The results of
expression of hypoxia-inducible factor, antioxidant enzymes and the present study provide knowledge about the regulation of the
caspases during the experimental induction of the process [12]. sperm miRNAs during the last stages of spermatogenesis (mainly
Additionally, testicular heat stress is associated with sperm spermiogenesis) and sperm maturation, as well as shed light on the
morpho-functional quality impairment (e.g., decrease in sperm mechanisms involved in dysfunctions of the testis and epididymis.
motility and increase in sperm abnormal morphology, as well as
sperm DNA fragmentation), decreasing in serum testosterone
2. Material and methods
concentration, and male infertility issues [13e15]. However, to the
best of our knowledge, molecular alterations specifically in sperm
2.1. Animals
are not yet described during the process.
Hormones and a large repertoire of molecules drive the proper
Six healthy Nelore bulls (Bos indicus) with the age of 34 ± 0.40
progression of the complex spermatogenesis physiological process,
months and body weight of 651.70 ± 10.30 kg were used in this
in which an undifferentiated cell (i.e., spermatogonium) proliferates
study. Bulls were kept in paddocks during the experiment period
and differentiates into spermatocytes, which generate spermatids,
with access to water ad libitum and under grazing conditions sup-
that then differentiate into spermatozoa [16]. Among other mo-
plemented with concentrate and minerals. Environmental tem-
lecular factors, the small (~22 nt) non-coding-RNA microRNA
perature was measured every 5 min by weather station. The
molecules (miRNA) also participate in the regulation of this process
minimum and maximum environmental temperature measured
[17]. The miRNA molecules are very conserved among different
was 7.9 C and 34.6 C, respectively. The mean temperature during
species and modulate protein translation. The action of miRNA
the total period was 20.96 C. All experimental procedures per-
molecules involves the binding to an Argonaute protein forming
formed were in accordance with legal and ethical standards. The
the RNA-induced silencing complex (i.e., RISC complex), which
Ethics Committee on Animal Use of the School of Veterinary
identifies the miRNA target genes and inhibits their translation by ~o Paulo (CEUA/
Medicine and Animal Science of the University of Sa
decreasing the target-mRNAs-transcripts or by blocking the
FMVZ) approved the present experiment by the protocol numbers
translation process [18]. While Dicer is a key enzyme involved in
1912050516 and 5162170616.
miRNA biogenesis, the absence of Dicer in epithelial cells from
testicular seminiferous tubule, as well as in epididymal principal
cells, potentially impairs male fertility by triggering, respectively, 2.2. Experimental design
spermatogenesis disorders and miRNA changes in the epididymal
lumen [19,20]. In that regard, sperm related miRNAs have been Bulls were divided into Control (CON; n ¼ 3) and Scrotal Heat
recently recognized as key players during spermatogenesis and Stress (SHS; n ¼ 3) groups. SHS bulls were submitted to transient
sperm maturation processes. scrotal insulation for 96 h through insertion of scrotal bags made
Since spermatozoa are composed of a restricted cytoplasm and a especially for bull. The bags were designed according to Alves et al.
highly condensed chromatin, these cells are recognized as tran- [5]. The day that the scrotal bags were removed from SHS group
scriptionally inert and by having a limited quantity of RNA mole- was considered as D0 (Day zero); in this sense, semen samples were
cules [21]. However, spermatozoa were recently shown as key collected: 7 days before SHS (D-7), and 7 (Dþ7) and 21 (Dþ21) days
transporters of ribonucleic acid molecules (e.g., miRNAs) to the after SHS. Semen collections were performed by electroejaculation
oocyte, contributing embryonic first cleavage rates and cell (Autojac®; Neovet, Uberaba, Brazil) in a pre-warmed (37 C) 15 mL
tube protected from light.
27
Considering that the epididymal transit period in bulls is around was removed in order to evaluate sEVs characteristics regarding
10e15 days [32,33], the semen (n ¼ 6) was collected and processed diameter and concentration. The remaining volume was used for
seven days after (Dþ7) SHS induction, according to Fig. 1, for RNA extraction and evaluation of the miRNA relative expression.
evaluation of miRNAs altered by transient scrotal insulation in
small extracellular vesicles (sEVs) of the seminal plasma. These
sEVs were evaluated for quality (diameter, protein markers and 2.4. Assessment of small extracellular vesicles quality
concentration) and miRNA levels. For evaluation of the miRNAs
altered by transient SHS in sperm that underwent spermiogenesis 2.4.1. Quantification of diameter and concentration
and meiosis stages (last steps of spermatogenesis) during the heat For the evaluation of sEVs diameter size and concentration,
stress induction [33], semen (n ¼ 6) was collected 21 days (Dþ21) sEVs’ solution was diluted 1:500 (v:v) in PBS after isolation and
after heat stress induction as shown in Fig. 1. For both analyses analyzed using NanoSight® NS300 and NanoSight NTA software v
(sEVs and sperm), semen (n ¼ 6) was collected seven days before 3.1 (Malvern Panalytical, Malvern, United Kingdom). Five 30-sec
(D-7) scrotal heat stress to evaluate the characteristics previous to videos were recorded with camera at level 14, gain 2 and temper-
the scrotal heat stress induction in CON and SHS groups (Fig. 1). ature of 38.5 C. It was considered the sEVs concentration, as well as
After SHS, sperm concentration, sperm motility and sperm ab- diameter size mean and median. Beads of 50 nm, 100 nm and
normalities were, respectively, 201.66 ± 30.86 106 sperm/mL, 150 nm (Beads, Malvern, United Kingdom) were previously used for
71.66 ± 1.66% and 3.16 ± 0.92% in CON group and equipment standardization.
104.16 ± 25.34 106 sperm/mL, 28.33 ± 20.88% and 77.83 ± 3.08%
in SHS group. Sperm were then evaluated for sperm morpho-
2.4.2. Western Blot analysis
functional quality (integrity of sperm plasma and acrosome mem-
sEVs were evaluated for the presence or absence of the
branes, sperm mitochondrial membrane potential, sperm oxidative
following protein markers: ALIX (1:750, sc49267, Santa Cruz
stress, and sperm DNA fragmentation) and isolated to perform the
Biotechnology, Dallas, TX, USA), HSP70 (1:500, sc66048, Santa Cruz
investigation of the profile of sperm miRNAs.
Biotechnology, Dallas, TX, USA) and CD9 (1:500, sc13118, Santa Cruz
When the origin of the solution and reagents used during the
Biotechnology, Dallas, TX, USA). For that, two semen pool samples
study is not mentioned, they were purchased from Sigma-Aldrich
were submitted to centrifugation/ultracentrifugation protocol and
(St. Louis, MO, USA).
the isolated sEVs were lysed in 50 mL of RIPA buffer. Laemmli buffer
and 2-mercaptoethanol were added to the samples (1:4) that was
2.3. Isolation of small extracellular vesicles fraction from seminal then boiled at 95 C for 5 min. For each Western Blot analysis, 12 mL
plasma of each sample was loaded in a 12% SDS-polyacrylamide gels at
100V for 2 h. Proteins were transferred to a nitrocellulose mem-
After collection, a semen aliquot of 2.5 mL was separated and brane (#1620112 Bio-Rad) using Bio-Rad products and system for
submitted to a centrifugation/ultracentrifugation protocol adapted 2 h at 80V. These membranes were blocked with 5% BSA (Sigma-
from Frenette et al. [34] in order to obtain seminal plasma sEVs. For Aldrich) in Tris buffered saline (1X - TBST; 100 mM NaCl, 0.1%
that, semen was first centrifuged at 700g for 10 min to remove Tween 20 and 50 mm Tris, pH 7.4) for 1 h and incubated with the
sperm. Then, supernatant was centrifuged twice at 4,000g for primary antibodies in 1X-TBST containing 1% BSA overnight at 4 C.
20 min, followed by centrifugation of 16,500g for 30 min to remove Afterward, the blots were washed three times for 5 min with 1X-
any remaining cellular debris. The supernatant was centrifuged TBST, incubated with an HRP-conjugated secondary antibody
twice at 120,000 g at 4 C for 70 min. At the end, 50 mL of PBS diluted in 1X-TBST (1:2000) for 1 h at room temperature. Finally,
(10 mg/mL sodium chloride, 0.2 mg/mL potassium chloride, blots were washed another three times with 1X - TBST and treated
1.44 mg/mL dibasic sodium phosphate and 0.24 mg/mL potassium with Clarity™ Western ECL substrate (Bio-Rad) to visualize the
phosphate) was added to the pellet and homogenized. An aliquot proteins.
Fig. 1. Schematic figure of the experimental design. Nelore bulls (Bos indicus) were divided into Control (CON; n ¼ 3) and Scrotal Heat Stress (SHS; n ¼ 3; submitted to transient
scrotal heat stress for 96 h by using scrotal bags) groups. Semen was collected: seven days before scrotal heat stress induction (D-7); seven days after scrotal heat stress induction
(Dþ7); and 21 days after scrotal heat stress induction (Dþ21). Seminal plasma sEVs (small extracellular vesicles) were evaluated regarding characteristics (diameter and con-
centration) and miRNA levels at Dþ7 according to the epididymis transit period in bulls [33]. Spermatozoa were evaluated concerning sperm morpho-functional characteristics
(sperm motility, sperm abnormalities, sperm plasma and acrosome membranes integrity, sperm mitochondrial membrane potential, sperm oxidative stress and sperm DNA
fragmentation) and miRNA levels at Dþ21, to assess the effects of heat stress during last stages of spermatogenesis (spermiogenesis) according to Rahman et al. [33]. Seminal plasma
sEVs and spermatozoa were collected at D-7 to evaluate the characteristics and miRNA levels before stress.
28
2.4.3. Transmission electron microscopy iron sulfate at 37 C for 90 min). Then, MDA concentration was
Transmission electron microscopy was performed to visualize measured by a spectrophotometer (Ultrospec 2200 Pro Amersham
sEVs morphology. For such, sEVs from two semen pools were iso- Biosciences; Piscataway, NJ, USA) at a wavelength of 532 nm. The
lated by centrifugation/ultracentrifugation protocol and sEV pellets MDA concentration was adjusted by the sperm concentration,
were fixed with 2.5% glutaraldehyde, 0.1 M cacodylate and 4% measured using a Neubauer chamber and, thus, expressed in ng of
paraformaldehyde (pH 7.2 to 7.4) for 2 h at room temperature. Next, TBARS per 106 sperm for both spontaneous and induced TBARS.
ultracentrifugation was performed (120,000g/70 min at 4 C) and
the sEV pellets were resuspended in ultrapure water (Milli-Q; 2.5.3. Sperm DNA fragmentation
Millipore Corporation, Merk, Burlington, MA, USA) and ultra- Sperm DNA fragmentation was evaluated by assessing the per-
centrifuged (120,000g/70 min at 4 C) again. The isolated sEVs were centage of sperm with susceptibility to DNA acid-induced dena-
diluted in 100 mL of ultrapure water and placed in a copper grid turation by Acridine Orange Chromatin Structure assay adapted
coated with Pioloform® (Agar Scientific, Essex, United Kingdom) from Tejada et al. [38] using epifluorescence microscopy. For that,
for 5 min. The grid was immediately placed in a drop of 2% aqueous 500 mL of in natura (fresh) semen was centrifuged twice at 750g for
uranyl acetate for 3 min. The excess solution was removed, and the 10 min. The pellet was resuspended to a final concentration of
reading was performed in a transmission electron microscope (FEI 25 106 sperm/mL and 7 mL of the diluted semen was placed in a
of 200 kV model Tecnai20 emitter LAB6). slide and air-dried for 60 min. The slides were then placed in a
buffer solution (2.9% sodium citrate tribasic dehydrate and 9 mM
2.5. Assessment of sperm quality ethylenediaminetetraacetic acid) with 1 mM of 1% dithiothreitol
(DTT) for 30 min. Then, the slides were placed only in the buffer
Sperm quality was evaluated by the same technician and con- solution for 20 min. At the end, slides were fixed overnight in
sisted of the evaluation of plasma and acrosome membranes Carnoy’s solution (300 mL of 99.9% methanol and 100 mL of glacial
integrity, mitochondrial membrane potential, oxidative stress, and acetic acid) and, after fixation, the slides were air-dry for 10 min.
DNA fragmentation. Thus, 3 mL of acridine orange stain solution was placed in the slide
during for 5 min at room temperature. The solution was washed
2.5.1. Sperm plasma and acrosome membranes integrity and with sodium citrate solution. DNA fragmentation was quantified by
mitochondrial membrane potential counting 500 sperm distinguishing orange-stained (abnormal
The percentage of spermatozoa with the integrity of plasma and DNA) and green-stained (normal DNA) analyzed at 1000 magnifi
acrosome membranes and high potential of the mitochondrial cation by epifluorescence microscopy (model 80i, Nikon, Tokyo,
membrane was evaluated adjusting the technique proposed by Japan) using triple filter (D/F/R, C58420) with UV-2E/C (excitation
Celeghini et al. [35] using epifluorescence microscopy. Semen was 340e380 nm and emission 435e485 nm), B-2E/C (excitation
first diluted with TALP sperm [36] to a final sperm concentration of 465e495 nm and emission 515e555 nm) and G-2E/C (excitation
25 106 sperm/mL. Then, to 150 mL of diluted semen was added 540-525 nm and emission 605e655 nm). The percentage of DNA
2 mL of 0.5 mg/mL Hoechst 33342 (Thermo Fisher Scientific, Wal- fragmentation was calculated by the formula:

tham, MA, USA), 3 mL of 0.5 mg/mL propidium iodide (Sigma- % DNA fragmentation ¼ number of abnormal DNA sperm
100
total number of sperm
Aldrich), 2 mL of 153 mM JC-1 (5,5,6,6-tetrachloro-1,1,3,3-
tetraethylbenzimidazolylcarbocyanine iodide, Thermo Fisher Sci-
entific, Waltham, MA, USA) and 50 mL of 100 mg/mL FITC-PSA 2.6. Isolation of sperm
(fluorescein isothiocyanate-conjugated Pisum sativum agglutinin;
Sigma-Aldrich). Samples were incubated for 8 min at 37 C. Then, Spermatozoa were isolated performing a Percoll gradient in
4 mL of incubated sperm solution was placed in a slide covered with order to proceed with sperm miRNA profile evaluation. For that, an
a cover slip and 200 cells were evaluated by epifluorescence mi- aliquot of in natura semen that was previously evaluated by sperm
croscopy (model 80i, Nikon, Tokyo, Japan) using the triple filter (D/ quality was diluted in semen extender (Botubov® - Botupharma,
F/R, C58420) with UV-2E/C (excitation 340e380 nm and emission Botucatu, Sa ~o Paulo, Brazil) to sperm concentration of 100 106
435e485 nm), B-2E/C (excitation 465e495 nm and emission sperm/mL. Then, 200 mL of diluted semen was submitted to Percoll
515e555 nm) and G-2E/C (excitation 540-525 nm and emission gradient protocol in which semen was first centrifuged at 3600g for
605e655 nm). Sperm were classified according to the fluorescence 7 min. At the end, sperm pellet was suspended in 500 mL of PBS and
emitted in the percentage of sperm with plasma and acrosome centrifuged at 520g for 5 min. The supernatant was removed and
membrane integrity and high mitochondrial membrane potential 750 mL of TRIZOL® LS Reagent (Life Technologies, Carlsbad, CA, USA)
(IPIAH), percentage of cells with plasma membrane integrity, per- plus 8 mL of PolyAcryl Carrier® were added. The solution was stored
centage of cells with acrosome membrane integrity and percentage at 80 C.
of cells with high mitochondrial membrane potential.
2.7. Assessment of miRNA profile
2.5.2. Sperm oxidative stress
Oxidative stress was evaluated by the susceptibility to lipid For RNA extraction from isolated sEVs and sperm, miRNeasy
peroxidation using the technique of spontaneous and induced Mini Kit® (Qiagen, Hilden, Germany) was used following the
thiobarbituric acid reactive substances (TBARSs assay) according to manufacturer’s recommendation. Total RNA (including miRNAs)
Nichi et al. [37] and Alves et al. [15]. The spontaneous TBARS was measured by NanoDrop™ 1000 (Thermo Fisher Scientific,
technique evaluated the susceptibility to lipid peroxidation Waltham, MA, USA). After, 100 ng of total RNA was used to cDNA
measuring malondialdehyde (MDA) concentration that is already synthesis performed with the miScript RT kit (Qiagen, Hilden,
present on seminal plasma. Induced TBARS technique evaluated the Germany) following the manufacturer’s recommendation using
susceptibility to lipid peroxidation by incubating sperm with an 2 mL of 5x HiFlex buffer, 1 mL of 10x nucleic acid mix and 1 mL of the
oxidative stress induction system (20 mM ascorbic acid and 4 mM enzyme reverse transcriptase. The final volume reaction (10 mL) was
29
incubated for 1 h at 37 C and at 95 C for 5 min for the inactivation 3.2. Seminal plasma sEVs from Control and Scrotal Heat Stress
of the enzyme. Finally, for real-time PCR, 380 miRNAs groups were similar in size and concentration, but three sEVs-
(Supplementary Table S1) primers at 10 mM were mixed with PCR associated-miRNAs were down-regulated in Scrotal Heat Stress
mix that was prepared using miScript SYBR Green® kit (Qiagen, group
Hilden, Germany) according manufacturer’s recommendation. PCR
was performed in QuantStudio™ 6 Flex (Thermo Fisher Scientific, Seminal plasma sEVs from CON and SHS groups were similar
Waltham, MA, USA) with the following cycle conditions: an initial regarding size and concentration seven days after (Dþ7) scrotal
incubation of 95 C/15 min and 45 cycles of 94 C/15 s, 55 C/30 s heat stress induction (Table 1). sEVs size diameter mean and me-
and 70 C/30 s and using a 384-well plate. The melting-curve was dian were, respectively, 179.43 ± 9.60 nm and 121.77 ± 6.50 nm in
used to confirm the amplification of a single product. The miRNAs the CON group and 200.80 ± 20.96 nm and 146.27 ± 18.70 nm in the
were only considered as detected when the number of cycles SHS group. sEVs concentration was 1142.00 ± 134.00 109/mL and
required for the fluorescent signal to cross the threshold (Ct value) 1393.33 ± 364.00 109/mL in CON and SHS group, respectively,
was lower than 37. As endogenous references (housekeepings) we after scrotal heat stress. The sEVs diameter mean and median as
used bta-miR-99b and RNU43 snoRNA for sEVs and bta-miR-99b well as the concentration were also similar seven days before (D-7)
and bta-miR-425-5p for sperm. Endogenous references determi- transient scrotal heat stress between CON and SHS groups (see
nation was based on consistent detection among the groups ac- Supplementary Table S3).
cording to the NormFinder software [39e41]. Regarding the 380 miRNAs evaluated in sEVs, 353 were detected
in both groups. Seven miRNAs and five miRNAs were exclusively
2.8. Functional analyses detected in CON and SHS groups, respectively (Fig. 3A;
Supplementary Table S4). Out of the 353 detected in both groups,
For functional enrichment analysis, miRNAs that showed three miRNAs (miR-23b-5p, 489 and 1248) were down-
different relative expression between groups were investigated for regulated in sEVs from SHS group compared to CON group on
predicted target genes by the TargetScanHuman platform (http:// Dþ7 (Fig. 3B; Supplementary Table S5). The functional analysis
www.targetscan.org/vert_72/). For this, bovine and human showed that these three down-regulated sEVs-associated-miRNAs
mature miRNA homologous sequence was analyzed are involved in the modulation of target genes (see Supplementary
(Supplementary Table S2). The top 80 genes of the list were iden- Table S6) associated with negative regulation of processes
tified according to De Bem et al. [42] and all genes together and regarding cell biosynthesis, macromolecule biosynthesis, nucleic
simultaneously were analyzed by the David - Functional Annota- acid metabolism and transcription as shown in Fig. 3D. Three other
tion Tool (David Bioinformatics Resources 6.7, NIAID/NIH; https:// miRNAs (miR-126-5p, 656 and 1307) displayed a tendency to
david.ncifcrf.gov/). The signaling pathways were ranked according present different relative levels between CON and SHS groups
to the P value (-log10) and number of genes involved. (Fig. 3C). All these miRNAs (miR-23b-5p, 489, 1248, -126-
5p, 656 and 1307) were present at similar relative levels seven
2.9. Statistical analysis days before (D-7) scrotal heat stress induction between CON and
SHS (see Supplementary Table S7).
Statistical analysis was performed in Statistical Analysis System
software (SAS Institute Inc., 9.3). Significance level was considered 3.3. Sperm oxidative stress was present at a higher level in Scrotal
when P 0.05; statistical tendency was considered when 0.05 < Heat Stress group
P 0.10. Data were verified to normality using Shapiro-Wilk test.
When necessary, outliers were removed or data were transformed. After 21 days from scrotal heat stress (Dþ21), semen was
Analysis of variance was performed using the MIXED procedure of collected, and sperm characteristics were evaluated. Concerning
SAS. Data was compared first between CON and SHS groups 7 days evaluation of sperm membranes, oxidative stress and DNA frag-
before (D-7) heat stress. Only variables that were present at similar mentation, the sperm oxidative stress was present at a higher level
values (P > 0.05) on D-7 were addressed 7 days (Dþ7) and 21 days in SHS group regarding the induced lipid peroxidation compared do
(Dþ21) after scrotal heat stress induction for sEVs and sperm, CON on Dþ21, indicating increase of susceptibility to oxidative
respectively. For miRNAs, delta-Ct (DCt) values were calculated by stress in sperm (Table 2). The same sperm characteristics were also
subtracting each miRNA Ct value from the geometric mean of evaluated seven days before (D-7) scrotal heat stress and were
housekeeping’s small RNAs (bta-miR-99b and RNU43 e for sEVs; similar between CON and SHS groups as shown in Supplementary
bta-miR-99b and bta-miR-425-5p e for sperm) and was used to Table S8.
perform the statistical analysis. Then, relative expression was
calculated using 2-DCt method. The relative expression was shown 3.4. Scrotal heat stress altered sperm miRNA expression
as results in the tables and graphics as well as to generate the heat
map image using MetaboAnalyst 3.0 platform. Sperm samples miRNA relative expression was evaluated using
a panel of 380 miRNAs. Among these, 294 miRNAs were detected in
3. Results both CON and SHS groups 21 days after scrotal heat stress (Dþ21).
Additionally, 30 and six miRNAs were exclusively detected in sperm
3.1. Characterization of seminal plasma sEVs (small extracellular from CON and SHS groups on Dþ21, respectively (Fig. 4;
vesicles) Supplementary Table S9). After 21 days from scrotal heat stress
induction (Dþ21), 21 sperm miRNAs were present at a different
For characterization of the sEVs fraction obtained by centrifu- level (P 0.05) between CON and SHS (Figs. 4 and 5;
gation/ultracentrifugation protocol, the isolated vesicles were Supplementary Table S10) and were present at a similar level seven
evaluated regarding size, morphology and specific proteins. The days before (D-7) heat stress induction (see Supplementary
mean size of the sEVs isolated for both groups (CON þ SHS groups) Table S11). While only three of 21 miRNAs (bta-miR-378d, 454
for all samples was 190.10 ± 11.35 nm (Fig. 2A, B and 2C). Addi- and 1246) were up-regulated in SHS group, 18 of 21 miRNAs (bta-
tionally, the isolated sEVs presented cup-shape morphology miR-9-5p, 15a, 18a, 20b, 30a-5p, 30b-5p, 30d, 30e-
(Fig. 2D) as well as proteins such as ALIX, HSP70 and CD9 (Fig. 2E). 5p 34b, 34c, 106b, -126-5p, 146a, 191, 192, 200b, 335
30
Fig. 2. Characterization of small extracellular vesicles (sEVs) isolated from seminal plasma. In A and B, nanoparticle-tracking measurement of particle size and concentration in each
sample from Control (A) and Scrotal Heat Stress (B) groups. In C, the diameter of the seminal plasma sEVs in samples of Control (CON) and Scrotal Heat Stress (SHS). In D,
transmission electronic microscopy representative photomicrographs of sEVs isolated from bovine seminal plasma. Scale bar: 100 nm. In E, Western blot analysis of Alix, HSP70 and
CD9 proteins of sEVs fraction isolated from seminal plasma from CON and SHS groups. All quantitative data are presented as means and SEM.
and 449a) were down-regulated in this group (Fig. 5; in sEVs, six miRNAs exclusively in sperm and 324 miRNAs were
Supplementary Table S10). Enrichment analysis of the 18 down- detected in both sEVs and sperm (Fig. 8; Supplementary Fig. S1).
regulated sperm miRNAs in SHS group showed that these miR- Out of these common 324 miRNAs, 23 miRNAs were present at
NAs modulate genes (see Supplementary Table S12) involved in different relative levels in sEVs or sperm comparing CON and SHS
regulation of transcription, regionalization (process involved with groups. None of these 23 miRNAs were detected both altered in
cell differentiation in which the cell responds to a specific envi- sEVs and sperm. Thus, two (miR-489 and -1248) out of 23 miRNAs
ronment) and embryonic organ development as shown in Fig. 6. were detected altered in sEVs of CON and SHS and 21 (miR-9-
Also, signaling pathways important to cell signal transmission as 5p, 15a, 18a, 20b, 30a-5p, 30b-5p, 30d, 30e-
MAPK and cAMP signaling pathway, cell structure as regulation of 5p 34b, 34c, 106b, -126-5p, 146a, 191, 192, 200b,
actin cytoskeleton, cell proliferation as cancer pathways and cell 335, 378d, 449a, 454, -and 1246) out of 23 miRNAs were
cycle and development as Hippo and Wnt signaling pathways were detected altered in sperm of CON and SHS. The miR-23b-5p,
involved in the genes regulated by these miRNAs (Fig. 7A and B). differently detected in sEVs of CON and SHS groups, was exclu-
sively detected in sEVs (Fig. 8). Regarding the three miRNAs (miR-
126-5p, 656 and 1307) that tended to be altered in sEVs of CON
3.5. sEVs and sperm presented 324 common miRNAs, but only miR- and SHS groups (Fig. 3C), all of them were also detected in sperm of
126-5p seems to be altered in both sEVs and sperm of CON and SHS CON and SHS groups. Out of these three miRNAs, only miR-126-5p
was down-regulated in sperm from SHS group (Fig. 5) and tended
When evaluating miRNAs detected in sEVs on Dþ7 and also (P ¼ 0.06) to be down-regulated (Fig. 3C) in sEVs of SHS group.
detected in spermatozoa on Dþ21 independently of the experi-
mental group (CON and SHS), 41 miRNAs were detected exclusively
Table 1
Diameter and concentration (mean ± SEM) of seminal plasma small extracellular vesicles seven days after (Dþ7) scrotal heat stress.
Characteristics of seminal plasma small extracellular vesicles Experimental groups P value
Control (n ¼ 3) Scrotal Heat Stress (n ¼ 3)
Diameter mean (nm) 179.43 ± 9.60 200.80 ± 20.96 0.40

Diameter median (nm) 121.77 ± 6.50 146.27 ± 18.70 0.28
Concentration (x 109/mL) 1142.00 ± 134.00 1393.33 ± 364.00 0.55
31
Fig. 3. Analysis of the miRNAs’ relative levels in small extracellular vesicles (sEVs) from seminal plasma seven days after heat stress (Dþ7). In A, Venn diagram of the panel of 380
miRNAs detected in sEVs fraction isolated from seminal plasma from Control (CON) and Scrotal Heat Stress (SHS) groups. The miRNA was considered detected when its level was
identified in at least two different samples of the same group. The detailed list of the miRNA levels detected in sEVs is reported in Supplementary Table S4. In B, three miRNAs that
were present at different (P < 0.05) relative levels between CON and SHS groups. In C, three miRNAs that showed a tendency (0.05 < P 0.10) to present different relative levels
between CON and SHS groups. Only miRNAs that were detected in the three samples from each group were considered for analysis shown in B and C. In D, the top 10 gene ontology
functional annotation charts of the target genes regulated by the three down-regulated sEVs-associated-miRNAs (shown in B). The bars indicate the number of genes involved in
each process (axis Y left) and the line indicates the -log10 P value (axis Y right). The numbers indicate the functional annotation chart and gene ontology (GO) reference of each
biological process. One asterisk (*) indicates statistical difference (P < 0.05) and two asterisks (**) indicate statistical tendency (0.05 < P 0.10) between groups. Relative expression
is shown as 2-DCt. All quantitative data are presented as means and SEM.
4. Discussion [43] and early embryonic development [22,23], and are also asso-
ciated with distinct male fertility phenotypes [23,39], the impact of
Since spermatogenesis from domestic mammalian and men scrotal heat stress in sperm-borne miRNAs profile remains un-
relies on testicular thermoregulation capability, elevated scrotal known. Herein, scrotal heat stress was induced in bulls to promote
temperatures may increase testicular temperature and result in dysfunction in the testicular thermoregulation capability, with the
impairment of sperm quality, as well as fertility issues. Recently, aim to investigate the alterations in: (1) the sEVs-associated-
spermatozoa were described as accumulating RNA molecules, e.g. miRNAs; and (2) the sperm-associated-miRNAs (Fig. 1). These
miRNAs (microRNAs), during the last stages of spermatogenesis findings revealed that scrotal heat stress changes the levels of
and passage through the epididymis by communicating with small miRNAs in the sEVs, as well as in the spermatozoa, respectively,
extracellular vesicles (sEVs) released from epididymis epithelial seven and 21 days after scrotal heat stress induction, by predomi-
cells [27]. While spermatic miRNAs play key roles during repro- nantly driving to down-regulation. It has also been shown that
ductive events related to spermatogenesis [17], sperm maturation sperm-associated-miRNAs were larger impacted by transient
Table 2
Sperm morpho-functional features (mean ± SEM) evaluated 21 days after (Dþ21) scrotal heat stress.
Sperm morpho-functional features Experimental groups P value
Control (n ¼ 3) Scrotal Heat Stress (n ¼ 3)
Sperm plasma and acrosome membranes integrity and mitochondrial membrane potential
IPIAHa (%) 73.11 ± 7.20 41.66 ± 19.97 0.21
Plasma membrane integrity (%) 69.08 ± 1.21 47.33 ± 22.09 0.38
Acrosome membrane integrity (%) 76.58 ± 3.54 64.16 ± 16.99 0.51
High mitochondrial membrane potential (%) 73.75 ± 1.98 63.66 ± 6.61 0.21
Sperm oxidative stress and sperm DNA fragmentation
Spontaneous lipid peroxidation (ng/106 sperm) 100.36 ± 4.89 114.73 ± 11.55 0.31
Induced lipid peroxidation (ng x 106/106 sperm) 5.89 ± 0.63b 16.12 ± 6.43a 0.04
Sperm DNA fragmentation (%) 0.00 ± 0.00 20.66 ± 20.66 0.31
a,b
Different letters on the same line indicate statistical difference (P < 0.05) between groups.
a
IPIAH: sperm with intact plasma and acrosome membranes and high mitochondrial membrane potential.
32
Fig. 4. Venn diagram of 380 sperm miRNAs from Control (CON) and Scrotal Heat Stress (SHS) groups. The miRNA was considered detected when its relative levels was identified in
at least two different samples of the same group. Out of 294, 21 miRNAs were present at different relative levels (P 0.05) between CON and SHS groups. The detailed list of the
miRNA levels detected in spermatozoa is reported in Supplementary Table S9.
scrotal heat stress than sEVs-associated-miRNAs. Thus, herein a characterization revealed that the isolated sEVs had diameter mean
new factor (scrotal heat stress) has been shown to interfere on the of 190.10 ± 11.35 nm independently of the analyzed group (CON or
miRNAs profile from seminal plasma sEVs and spermatozoa. SHS), in accord to seminal plasma sEVs described in semen from
Furthermore, to the best of our knowledge, our study shows for the bulls, mice and men [44,46]. This finding also corroborated the
first time that transient scrotal heat stress changes the molecular diameter size of sEVs proposed by The ry et al. [30] in the nomen-
content of seminal plasma sEVs and spermatozoa in cattle. clature guidelines of EVs. The sEVs morphology and the positive
Epithelial cells in the epididymis release different populations of signal to the protein markers ALIX, HSP70 and CD9 also confirmed
extracellular vesicles (EVs), i.e. epididymosomes, that compose the the isolation of sEVs after the ultracentrifugation process [47,48].
seminal plasma with other different types of EVs. While the With the information in mind that sEVs from epididymis cauda are
diameter size of epididymosomes is between 50 and 250 nm [28], enriched in CD9 marker [47] and that we observed a positive signal
other seminal plasma EVs derived from prostate (i.e. prostasomes) of the isolated sEVs to this protein marker, we considered here that
display size between 40 and 500 nm [29,44]. Out of epi- the population of sEVs obtained from the seminal plasma were in
didymosomes, there are included the epididymal sEVs that own a part derived from the epididymis, that was under scrotal heat stress
regulated profile of molecules and minor size (between 100 and in the experimental conditions of this study.
200 nm) [30]. Even that free forms and bound forms of molecules, Since in the present study the sEVs obtained from seminal
such as proteins and miRNAs, are transferred to sperm in the plasma were in part derived from the epididymis, we expected that
epididymal lumen, the sEVs enclosed in the epididymis lumen are SHS induction impacted the sEVs molecular profile. For this pur-
key sperm maturation modulators [31,45]. At interest, sEVs traffic pose, we collected and isolated sEVs from seminal plasma seven
in a specific and highly regulated mode the main maturation- days after scrotal heat stress induction respecting bulls’ epididymis
associated-molecules from the epithelial cells to the sperm transit period according to Rahman et al. [33]. Then, we analyzed
[31,45]. Thus, in the present study, we investigated the miRNA the miRNA relative expression in seminal plasma sEVs from CON
molecules comprised in the sEVs instead of the free miRNAs. Since and SHS bulls; three miRNAs were down-regulated in sEVs from
we obtained sEVs from total seminal plasma, the isolated fraction the seminal plasma of SHS bulls. At this point, it is important to
was not exclusively composed of epididymal sEVs. From this, after emphasize that seminal plasma sEVs have been studied as potential
performing a modified protocol of ultracentrifugation, in which it non-invasive diagnostic tools. In that regard, the miR-23b was
was possible to obtain a smaller extracellular vesicles subpopula- portrayed as down-regulated in seminal plasma EVs from oligoas-
tion, we characterized the seminal plasma sEVs. The thenozoospermic subfertile men compared to normozoospermic
33
Fig. 5. Non-supervised heatmap and hierarchical clustering of differentially expressed miRNAs in sperm of Control (CON) and Scrotal Heat Stress (SHS) groups 21 days (Dþ21) after
scrotal heat stress. Samples are organized in columns. CON samples (n ¼ 3) are indicated by a light-gray bar above them and are labelled as CON_1, CON_2 and CON_3. SHS samples
(n ¼ 3) are indicated by a dark-gray bar above them and are labelled as SHS_1, SHS_2 and SHS_3. The 21 differentially expressed (P 0.05) miRNAs are organized in rows, and each
row represents one miRNA (labelled to the right). The scale represents the Z-scores of miRNAs’ relative level. The dendrogram on the top refers to the hierarchical clustering of
samples. The dendrogram to the left refers to the hierarchical clustering of miRNAs.
fertile men [49]. Conversely, herein, we found that miR-23b-5p potential biomarkers of spermatogenesis healthy [50] as well as
showed a lower relative level in seminal plasma sEVs from SHS reproductive infections [51]. Regarding the epididymis, the lack of
bulls. Indeed, these sEVs-associated-miRNAs have been used as one miRNA biogenesis component (Dicer enzyme) in epididymal
principal cells is associated with miRNA profile alteration in
epididymal EVs [20]. Here, our data shed light on a probable
mechanism by which sEVs-associated-miRNAs are modulated by
external epididymal disturbs.
In silico analysis revealed that the three down-regulated seminal
plasma sEVs-associated-miRNAs from SHS bulls regulate target
genes involved in the blockade and/or reduction of chemical re-
actions and formation of substances (impeding the biosynthetic
processes, e.g. biosynthesis of macromolecules and nitrogen com-
pounds), as well as transcription and gene expression. On the other
hand, the sEVs-associated-miRNAs target genes were related to the
activation of the phosphorylation of peptidyl-tyrosine. To under-
stand these results, it is necessary to comprehend the heat stress
effects on the male reproductive system. In that regard, while
testicular heat stress is well characterized by increasing the cellular
metabolism [52], the impact of the heat stress directly in the
epididymis is not well known; the hypothesis is that the epididymis
undergoes analogous modifications [53]. From this perspective, the
epididymis epithelial cells under heat stress conditions may in-
crease the metabolism, producing higher levels of metabolites as
well as ATP (energy) resulting in the generation of high amounts of
Fig. 6. Top 10 gene ontology functional annotation charts of the target genes related to reactive oxygen species (ROS), which are very deleterious to sperm,
the 18 down-regulated sperm-associated-miRNAs in Scrotal Heat Stress group. The since these cells are composed by oxidative stress susceptible
bars indicate the number of genes involved in each process (axis Y left) and the line
components (i.e. a restricted cytoplasmic that lacks important
indicates the -log10 P value (axis Y right). The numbers indicate the functional anno-
tation chart and gene ontology (GO) reference of each biological process. antioxidant enzymes as well as plasmatic membrane rich in
34
Fig. 7. Top 10 signaling pathways regulated by the target genes related to the 18 down-regulated sperm-associated-miRNAs in Scrotal Heat Stress group. In A, the signaling
pathways are arranged according the P value (-log10). In B, the signaling pathways are arranged according the number of involved genes.
polyunsaturated fatty acids) [54]. With all of these in mind, we scrotal heat stress is well known for modifying sperm quality
hypothesized here that lower relative levels of these three sEVs- concerning the sperm morpho-functional features, recently sperm
associated-miRNAs potentially drive the modulation of transcripts molecular features have been also shown as important to deter-
and proteins that regulate negatively the biosynthesis processes, to mine male fertility ability [22,23,55]. From this, the sperm quality
generate a smaller amount of ROS. Indeed, the modulation of these concept has been updated to be composed of sperm morpho-
negative-processes-transcripts probably balances the increase in functional features added to the molecular features of spermato-
cell metabolism and ATP production, which might act as a defen- zoa. In that regard, there is a lack of information regarding the ef-
sive mechanism to prevent high amounts of ROS. In parallel, in fects of scrotal heat stress on sperm molecular features. In order to
addition to the miRNAs and respective target genes’ potential verify sperm molecular changes associated with spermatogenesis
response to the excess of ATP production, this excess may activate impairment, we collected semen to isolate spermatozoa 21 days
the tyrosine phosphorylation pathway to self-regulate the levels of (Dþ21) after scrotal heat stress when the collected sperm were
ATP and, consequently, decrease ROS generation. However, tyrosine passing through spermatogenesis last stages during heat stress in
phosphorylation activation could promote premature sperm accord to Rahman et al. [33]. Considering that during the final
capacitation [54], which might is deleterious to in natura semen stages of spermatogenesis (i.e. spermiogenesis) occur key events in
quality. Another important point that must be addressed here is which spermatozoa store transcripts to initialize the shape of the
that the negative regulation of biosynthesis processes induced by sperm molecular features [25], we decided to collect and isolate
the target genes of sEVs-associated-miRNAs may decrease the sperm 21 days after scrotal heat stress to investigate the sperm
biosynthesis of key proteins involved on sperm maturation, which miRNAs altered by the testicular disturb. First, we investigated the
probably impairs sperm maturation and thus male fertility. How- sperm morpho-functional features in which the susceptibility to
ever, further studies are necessary to confirm these hypotheses. oxidative stress was the only parameter impacted by scrotal heat
The induction of scrotal heat stress using insulation bags is a stress at 21 days after stress. In that regard, the SHS group displayed
suitable practice for promoting a decrease in sperm quality and a high level of induced lipid peroxidation compared to the CON
subfertility/infertility in several species [8,13,15]. During the in- group. This means that spermatozoa from SHS bulls were less
duction period, the insulation bags block the scrotal surface capa- resistant to combat oxidative stress, displaying higher vulnerability
bility to exchange heat with the external environment, which to undergo lipid peroxidation and DNA fragmentation, which may
impairs the spermatogenesis by keeping the internal testicular impair the fertility rates [56]. Corroborating our data, Garcia-
temperature similar to body internal temperature [15]. While Oliveros et al. [57] revealed an increase in spermatozoa lipid
35
Fig. 8. Venn diagram of the panel of 380 miRNAs detected in seminal plasma small extracellular vesicles (sEVs) seven days after scrotal heat stress (Dþ7) and in spermatozoa 21
days after scrotal heat stress (Dþ21) independently of the experimental group (Control and Scrotal Heat Stress). The miRNA was considered detected when its level was identified in
at least two different samples of the same group. 1miRNAs detected in both sEVs and sperm that only presented alteration in relative level between Control and Scrotal Heat Stress
groups in sperm. 2miRNAs detected in both sEVs and sperm that only presented alteration in relative level between Control and Scrotal Heat Stress groups in sEVs. *miRNA detected
exclusively in sEVs that presented alteration in relative level between Control and Scrotal Heat Stress groups in sEVs.
peroxidation 21 days after scrotal heat stress in bulls followed by an well-characterized in spermatozoa, since its presence is associated
increase in sperm DNA fragmentation seven days afterward. On the with higher first cleavage rates in mice embryo [22]. Thus, taking
other hand, the spermatozoa vulnerability to undergo lipid perox- into account all of the findings previously mentioned, low levels of
idation was only detected as increased 49 days after scrotal heat miR-34b and 34c in spermatozoa are probably associated with a
stress in rams [15]. reduction in sperm quality and a decrease in male fertility ability.
Regarding the sperm molecular features, we found 21 miRNAs In addition to miR-34 family, other miRNAs (i.e., miR-200b
altered in the spermatozoa after scrotal heat stress induction. From and 449a) were also down-regulated in spermatozoa obtained
these results, the miR-34 family is very important to be emphasized from SHS bulls as well as in testicular tissues obtained from mon-
here, since it plays a key role in mice spermatogenesis [58,59]. Out keys induced to testicular heat stress [61]. The corroboration be-
of this miRNA family, the down-regulation of both miR-34b tween the findings regarding heat-stressed spermatozoa and
and 34c is involved with impairment of mitochondrial function testicular tissue suggest that miR-200b and 449a are probably
as well as oxidative stress in human pathological conditions [60]. In accumulated by sperm during spermatogenesis. However, addi-
the present study, since both miR-34b and 34c were found as tional studies are necessary to confirm this hypothesis. We also
down-regulated in the spermatozoa from SHS bulls, which were detected miR-146a down-regulated in the spermatozoa from SHS
also highly susceptible to oxidative stress, we hypothesize here that bulls. Since high levels of miR-146a are associated with lower levels
spermatozoa with lower levels of miR-34b and 34c are probably of inflammatory signaling [63], we considered here that the low
predisposed to undergo oxidative stress by displaying mitochon- levels of this miRNA in heat-stressed testicular may contribute to
drial dysfunction. However, further studies must be performed in activating pro-inflammatory pathways related to testicular heat
order to support this statement. Corroborating our data, lower stress response [64]. Interestingly, miR-15a displayed down-
levels of miR-34b and 34c were detected in monkeys’ heat- regulation in SHS bulls as well as in men affected by varicocele
stressed testicular tissue and men with low sperm motility and [65]; the low levels of miR-15a are associated with higher levels of
concentration [61,62]. Individually, the sperm-borne miR-34c is the heat shock protein, HSPA1B, which preserves the three-
36
dimensional conformation of the proteins and increases its own potentially accelerates the recovery of the testicular tissue to heat
expression in stresses situations [65]. Finally, miR-126-5p was stress effects. At interest, the spermatozoa produced during this
detected down-regulated in sperm and tended to be down- condition are able to accumulate these altered miRNAs reflecting
regulated also in seminal plasma sEVs from SHS group, which the spermatogenesis quality. However, further experiments are
suggests that miR-126-5p might be modulated by communication necessary to confirm these hypotheses.
between sEVs and sperm during epididymal transit. However, Finally, our data provide evidence that scrotal heat stress im-
further studies are necessary to confirm this hypothesis. pacts sperm quality regarding two different aspects: (1) sperm
Out of the total of 380 miRNAs evaluated in spermatozoa, 21 morpho-functional features, by increasing the spermatozoa sus-
sperm miRNAs (21/380, i.e. 5.52%) were altered by scrotal heat ceptibility to oxidative stress; and (2) sperm molecular features, by
stress induction. At interest, most of them (18/21 miRNAs, i.e. modifying the sperm miRNA profile. In that regard, the sperm-
85.5%) were down-regulated in sperm from bulls of SHS group associated-miRNAs miR-15a, 34b, 34c, 146a, 200b
when compared to CON. Similar miRNA profiles are found when and 449a show a potential role in modulating spermatogenesis
cellular hypoxia is induced. In this condition, the Argonaute protein injuries in bulls since these molecules were also involved in similar
becomes non-receptive to the Dicer enzyme blocking the RISC processes in other studies. Further studies are needed to test these
complex formation (RNA-induced silencing complex); conse- sperm miRNA molecules as non-invasive spermatogenesis quality
quently, the biogenesis of new miRNA molecules in the cell is biomarkers. In addition, our results showed that miR-126-5p was
blocked [66]. Since hypoxia condition is also described in testicular the only miRNA that assorted in a similar manner in sEVs and
heat stress [12], the inhibition of miRNA biosynthesis by RISC sperm. While miR-126-5p tended to be down-regulated in sEVs of
complex formation blockade might be the source of the large SHS group seven days after scrotal heat stress, it was down-
population of miRNAs altered with low levels after scrotal heat regulated in spermatozoa of SHS group 21 days after heat stress,
stress induction. which suggests traffic of this miRNA between sEVs and sperm
In silico analysis of the 18 down-regulated sperm miRNAs during epididymal transit. Though, additional studies are required
altered in spermatozoa from SHS bulls disclosed that their target to confirm these statements. Finally, since sperm quality is pivotal
genes activate transcription and gene expression, regulate cell to control male fertility success, the sperm morpho-functional and
differentiation processes as pattern specification, regionalization molecular changes might impair male fertility ability; the impact of
and embryo organ development. Since the low expression of the 18 these sperm miRNA molecules in fertilization and embryo devel-
miRNAs results in the target genes modulation, the transcription opment remains to be investigated in cattle.
and gene expression processes as well as cell differentiation are
probably activated. Additionally, modulation of the target genes of 5. Conclusion
the 18 down-regulated sperm miRNAs may activate the following
signaling pathways: p53, Hippo, Wnt, MAPK and cAMP. Interest- Our findings showed that miRNA levels from seminal plasma
ingly, p53 signaling is activated in the response to DNA damage, sEVs (miR-23b-5p, 489 and 1248) and sperm (miR-9-
that is triggered by scrotal heat stress in testicular tissue [11], which 5p, 15a, 18a, 20b, 30a-5p, 30b-5p, 30d, 30e-
may create mechanisms to respond to the down-regulation of 5p 34b, 34c, 106b, -126-5p, 146a, 191, 192, 2
specific miRNAs in order to modulate genes involved in DNA 00b, 335, 378d, 449a, 454, -and 1246) are changed by tran-
damage response. In parallel, while the Hippo and Wnt signaling sient scrotal heat stress, which triggers lower relative expression in
pathways stimulate cell proliferation, stem cell self-renewal and a substantial number of sEVs- and sperm-associated-miRNAs.
tissue regeneration [67,68], and the MAPK signaling pathway Moreover, our findings suggest that specific sperm-associated-
amplify cell proliferation, differentiation and inflammatory re- miRNAs (miR-15a, 34b, 34c, 146a, 200b and 449a) are po-
sponses signals [69], the modulation of these pathways may tential modulators of spermatogenesis injuries in bulls, since they
accelerate the recovery of the testicular tissue to heat stress effects. were previously associated with inflammatory and heat stress
Finally, the cAMP signaling pathway activation is related to sperm processes. Our data also suggest that the miR-126-5p might be
capacitation [70]; thus, the spermatozoa might be susceptible to delivered by sEVs to spermatozoa, since this miRNA displayed
premature analogous capacitation-associated events. Since previ- similar variation in relative expression in seminal plasma sEVs and
ously to the protamination process during spermiogenesis is one sperm. The results of the present study shed light on a probable
key point in which spermatozoa accumulate miRNA molecules, we mechanism by which sEVs- and sperm-miRNA potentially portray
considered here that these miRNAs were may generated in sper- the scrotal heat stress effects in the last stages of spermatogenesis,
matids in response to testicular heat stress and then accumulated mainly spermiogenesis, as well as sperm maturation in bulls. These
in spermatozoa, triggering molecular dysfunction in these sperm findings might open new avenues to understand the relationship
and potentially decreasing their molecular quality. between sEVs and sperm miRNAs as well as provide new insights
In summary, our study showed that relevant alterations in into the concept of sperm quality in cattle.
miRNA relative expression in seminal plasma sEVs and spermato-
zoa were induced by scrotal heat stress, by mainly triggering lower
levels of the miRNA molecules. The down-regulation of miRNAs Ethics approval and consent to participate
potentially implicates in the modulation of their target genes. In
~o Paulo,
All experiments were conducted at the University of Sa
that regard, our data showed that the seminal plasma sEVs-
associated-miRNAs target genes were mainly involved in the Brazil and all the experiments and procedures performed in the
blockade and/or reduction of cellular biosynthesis processes. Since study were approved by the Ethics Committee on Animal Use of the
seminal plasma sEVs are released by epididymis epithelial cells, the School of Veterinary Medicine and Animal Science of the University
~o Paulo (CEUA/FMVZ) and approved the present experiment by
of Sa
modulation of these target genes may balance the possible increase
in cell metabolism stimulated by scrotal heat stress. In addition, our the protocol numbers 1912050516 and 5162170616.
results revealed that 18/21 miRNAs (i.e., 85.5%) were down-
regulated in spermatozoa from SHS bulls; the sperm-associated- Consent for publication
miRNAs target genes were involved in cell proliferation, differen-
tiation and regeneration. The modulation of these processes Not applicable.
37
Availability of data and materials Dþ7 7 days after heat stress

Dþ21 21 days after heat stress
The datasets used during the current study are available from DNA deoxyribonucleic acid
the corresponding author on reasonable request unless presented EVs extracellular vesicles
in the manuscript and supplementary data. h hours
HP sperm with high mitochondrial membrane potential
Funding MDA malondialdehyde
min minutes
This work was supported by Saeo Paulo Research Foundation miRNAs microRNAs
(FAPESP). M.B.R.A. was supported by grant #2015/09154e6, n number of samples
FAPESP, J.C.S. was supported by grants #2014/22887e0, FAPESP and PI sperm with plasma membrane integrity
#2015/21829e9, FAPESP, and E.C.C.C. was supported by grant PIAIH sperm with plasma and acrosome membrane integrity
#2016/05395e1, FAPESP. and high mitochondrial membrane potential
pre-miRNA precursor miRNA transcripts
Authors’ contributions pri-miRNA primary miRNA transcripts
RISC RNA-induced silencing complex
M.B.R.A. designed the study, performed EVs and sperm isolation, RNAs ribonucleic acid
analyzed EVs in NanoSight, performed the molecular analysis SEM standard error of the mean
(mRNA extraction, cDNA synthesis and real-time PCR analysis), ran sEVs small extracellular vesicles
the statistical analysis and was a major contributor in writing the SHS scrotal heat stress
manuscript; R.P.A. and F.P. contributed to the study design and V Volts
manuscript writing; L.B., V.H.G.G. AND V.J.M.N. performed animal
management and semen collection; L.N.G.O performed sperm Appendix A. Supplementary data
quality analysis; F.S.A. and S.C.C.P. contributed to sperm quality
analysis; G.M.A. performed Western-Blot analysis; J.C.S. contrib- Supplementary data to this article can be found online at
uted to design the study, statistical analysis and write the manu- https://doi.org/10.1016/j.theriogenology.2020.11.015.
script; E.C.C.C. was the PI and contributed to the study design and
writing the manuscript. All authors reviewed the manuscript. References
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Leonardo Batissaco: performed animal management and semen temperatures. Anim Reprod Sci 1997;45:255e61.
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Theriogenology 161 (2021) 26e40

Contents lists available at ScienceDirect

Changes in miRNA levels of sperm and small extracellular vesicles of

Juliano Coelho da Silveira b, Eneiva Carla Carvalho Celeghini a, *

1. Introduction proliferation as well as offspring healthy [22e24]. While these

Characteristics of seminal plasma small extracellular vesicles Experimental groups P value

Control (n ¼ 3) Scrotal Heat Stress (n ¼ 3)

Diameter mean (nm) 179.43 ± 9.60 200.80 ± 20.96 0.40

Sperm morpho-functional features Experimental groups P value

Control (n ¼ 3) Scrotal Heat Stress (n ¼ 3)

Availability of data and materials Dþ7 7 days after heat stress

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