Theriogenology 195 (2023) 229e237

Contents lists available at ScienceDirect

Theriogenology
journal homepage: www.theriojournal.com

The effects of follicle stimulating hormone (FSH)-induced controlled

ovarian hyperstimulation and nutrition on implantation-related gene
expression in caruncular tissues of non-pregnant sheep
€
Ozlem Bedir a, Aykut Gram a, b, Anna T. Grazul-Bilska c, Mariusz P. Kowalewski a, d, *
a
Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, 8057, Zurich, Switzerland
b
Department of Histology and Embryology, Faculty of Veterinary Medicine, Erciyes University, 38280, Kayseri, Turkey
c
Department of Animal Sciences, North Dakota State University, Fargo, ND, 58108, USA
d
Center for Clinical Studies (ZKS), Vetsuisse Faculty, University of Zurich, 8057, Zurich, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history: Disturbances at the conceptus-maternal interface can have detrimental effects on pregnancy outcome.
Received 22 August 2022 Additionally, changes in body condition and exogenously administered gonadotropins could affect
Received in revised form ovarian and uterine function, including cell proliferation and ovulation rates, and alter endometrial
21 October 2022
receptivity. In ruminants, endometrial caruncles maintain placental function via interaction with fetal
Accepted 27 October 2022
Available online 1 November 2022
chorionic cotyledons. Here, the effects of feeding regimens on the expression of selected genes known to
be involved in uterine receptivity were investigated in the caruncles of control and FSH-superovulated
ewes. Sheep were grouped according to their diet: control fed (CF), overfed (OF) or underfed (UF), and
Keywords:
Sheep
were either superovulated with FSH (SOV) or untreated (CON, naturally cycling) (n ¼ 3e5/group). Car-
Feeding uncular samples for the assessment of the transcript levels of 11 target genes were collected at either the
Superovulation early (day 5) or mid-luteal (day 10) phases of the luteal lifespan, resulting in 12 groups of animals. The
FSH day of the estrous cycle affected the expression of ITGAV, ITGB3, FGF10 and IGFBP3 mRNA. There was
Endometrium lower expression of MUC1, and higher expression of FGF10, ITGB3 and FN1, on day 10 in CF_SOV animals.
mRNA expression Compared with CF, expression of integrins (ITGB3, ITGA5 and ITGA4) was higher in OF and UF, and higher
transcript levels of HGF and IGFBP3 in UF animals on day 10. Expression of ITGA5, ITGB1, -3, -5 and MUC1
was greater in OF_SOV than CF_SOV at day 10. In conclusion, it appears that imbalanced nutrition, by
altering the expression of genes responsible for intercellular communication, cell adhesion, and encoding
for growth factors, could affect the uterine responsiveness to exogenously applied hormonal stimulation
and, likely, uterine receptivity.
© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).

1. Introduction mechanisms, including sex steroids, and by local autocrine and

In mammalian species, successful implantation relies on the
coordinated and well-timed functional interaction between the
conceptus and the uterus. This well-orchestrated process starts
with the apposition, attachment, and adhesion of endometrial
surface epithelia and conceptus trophectoderm [1,2]. All of these
complex cellular and molecular events are controlled by endocrine
* Corresponding author. Institute of Veterinary Anatomy Vetsuisse Faculty Uni-
versity of Zürich Winterthurerstrasse 260 CH-8057 Zürich, Switzerland.
E-mail addresses: ozlem.bedir@uzh.ch (O. € Bedir), aykutgram@erciyes.edu.tr
(A. Gram), anna.grazulbilska@ndus.edu (A.T. Grazul-Bilska), kowalewski@vetanat.
uzh.ch (M.P. Kowalewski).
paracrine factors. In domestic ruminants, including the sheep, im-
plantation also relies on secretions from both the embryo, which
signals its presence through interferon tau, and the endometrial
tissues that are involved in the secretion of endometrial histotroph
to nourish the conceptus [3]. Furthermore, in contrast to humans
and rodents [2] or domestic carnivores [4,5], which have an inva-
sive type of implantation, sheep show superficial implantation [6].
Endometrial surface epithelium is a polarized cell layer that
mediates cell-cell and cell-extracellular matrix (ECM) interactions,
thereby playing an important role during implantation. At the time
of implantation, uterine epithelia lose the nonadhesive properties
of apical plasma membranes, allowing the attachment of the
embryonal trophectoderm to the uterine surface [6]. This

https://doi.org/10.1016/j.theriogenology.2022.10.033
0093-691X/© 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
€ Bedir, A. Gram, A.T. Grazul-Bilska et al.
O.
nonadhesive characteristic of the uterine epithelium is partially
acquired by their apical expression of mucins (MUCs) inhibiting
cell-cell and cell-ECM adhesion [6,7], e.g. MUC1 [8,9]. The expres-
sion and function of MUCs have been investigated in the uteri of
several species including humans [10], sheep [7], pigs [11] and ro-
dents [12], with immunohistochemical staining demonstrating a
decrease in MUC1 on the apical surface of the ovine luminal
epithelium prior to implantation [7]. Other cell adhesion molecules
are involved in the regulation of implantation in mammalian spe-
cies [13e16]. These include integrins as well as ECM proteins and
other factors inducing cellular differentiation, motility, and adhe-
sion [17e19]. Accordingly, increased endometrial expression of
selected integrins prior to implantation has been shown in dogs,
humans, sheep, and cows [7,17,20e22]. In the sheep, selected
integrin subunits, including ITGAV, ITGA4, ITGA5, ITGB1, ITGB3, and
ITGB5, have been confirmed to be dynamically expressed on both
the conceptus trophectoderm and the apical surface of the endo-
metrial luminal epithelium as a part of the utero-placental interface
during the peri-implantation and early placentation, and were
shown to be involved in forming integrin adhesion complexes,
which is a hallmark of activated integrins [7,13]. Their functionality
is thought to be related to controlling the assembly of matrix
components at the embryo-maternal interface. While the expres-
sion of ITGB3 was shown to decrease following implantation
[13,23], the others maintained their expression levels during
pregnancy. In mice and humans, altered expression of ITGAV and
ITGB3 has been demonstrated to interfere with fertility [24e26].
Furthermore, in species showing an invasive type of placentation,
including rats and humans, the expression of integrins and their
typical mechanosensory receptor, fibronectin (FN1), is associated
with the implantation, decidualization, and placentation processes
[27,28]. Moreover, in humans, several integrins of the b1 family that
are expressed in endometrial cells bind ECM proteins, including
FN1 [29].
Cross-talk between integrins and stromal-derived growth fac-
tors, e.g. hepatocyte growth factor (HGF), fibroblast growth factor-
10 (FGF10), and insulin-like growth factor binding protein 3
(IGFBP3), during ovine implantation has also been inferred
[30e32]. HGF and FGF10 are involved in the regulation of epithelial
proliferation and differentiation at the time of implantation in the
ovine uterus [33,34]. Furthermore, an investigation of the effect of
P4 on the endometrial expression of stromal-derived growth fac-
tors, their receptors and related IGFBPs, implicated FGF10, IGFBP1
and IGFBP3 in the P4-dependent modulation of endometrial
epithelial function in sheep [31,35]. In particular, P4 stimulates the
expression of IGFBP1 and IGFBP3 within the ovine uterine luminal
and superficial glandular epithelium during the peri-implantation
period [31].
Changes in dietary intake and body condition including
adiposity might also affect the endocrine system [36,37], ovarian
and uterine function including cell proliferation and ovulation rates
[38,39], and could alter endometrial receptivity in sheep [40,41]. A
positive correlation between body condition score, body weight,
and reproductive performance has been reported in seasonal-
calving dairy cattle [42]. In ewes, different feeding regimens, e.g.,
overfeeding or underfeeding, were associated with delayed puberty
and reduced conception and pregnancy rates [43], with under-
feeding also associated with poorer oocyte quality and lower rates
of zygote cleavage and embryo development to the morula and
blastocyst stages following IVF [44]. Additionally, imbalanced
nutrition e.g., overfeeding or underfeeding, also affected the
expression of IGF1 and IGF2 in ovine caruncular samples [45].
Together, these observations highlight the importance of an
optimal diet and body condition score for proper reproductive
performance, uterine receptivity and physiology in domestic
230
Theriogenology 195 (2023) 229e237
animal species.
Reproductive function can be affected by the use of exogenous
gonadotropins associated with assisted reproductive technologies
(ARTs), which have been widely used to treat infertility in humans
as well as in domestic mammalian species for several decades
[46e48]. Among the gonadotropins, FSH is most commonly
administered to induce follicular development or superovulation in
domestic animal species as well as in humans [49,50]. FSH treat-
ment results in the development of multiple follicles in domestic
mammalian species including cows and sheep [51,52], which in
turn cause an increase in circulating P4 levels [40,51], and therefore
affecting the uterine endocrine milieu. Accordingly, in humans,
ovarian stimulation with FSH reduces integrin expression, delays
the maturation of the glandular epithelium and formation of
pinopodes in the uterus, and seems to shift the window of im-
plantation [53]. In sheep, FSH-induced superovulation altered the
expression of IGF1, PGR, as well as ESR1 and ESR2 [39,45], and
caused accumulation of lipid droplets in the cytoplasm of endo-
metrial surface and glandular epithelial cells [38], showing the
potential adverse effects of hormonal manipulation on uterine
receptivity and physiology.
The expression of genes responsible for intercellular commu-
nication and cell adhesion (e.g., integrins and cadherins), or
encoding for growth factors, during the estrous cycle and preg-
nancy has been intensively investigated in ovine uterus [7], bovine
caruncular and intercaruncular endometrium [54], porcine
placenta [55], and human myometrium [28]. However, the effect of
ovarian hyperstimulation under different feeding regimes on the
expression of genes involved in uterine receptivity still needs to be
elucidated.
In the present study, we hypothesized that controlled ovarian
stimulation would alter the expression of genes involved in im-
plantation in the uterine caruncles area depending on the plane of
the diet and stage of the estrous cycle, implying possible effects on
pregnancy outcomes. Therefore, we investigated the effects of FSH-
induced ovarian hyperstimulation under different feeding regimes
(i.e., maintenance diet, overfeeding and underfeeding) on the
mRNA expression of integrin subunits (ITGA4, ITGA5, ITGVA, ITGB1,
ITGB3, ITGB5), FN1, MUC1 and selected growth factors (HGF, FGF10,
and IGFBP3) in ovine caruncular tissue on days 5 and 10 of the
estrous cycle.
2. Material and methods
2.1. Animals, experimental design, and sample collection
All experimental procedures were performed in accordance
with animal welfare legislation and approved by the North Dakota
State University's Institutional Animal Care and Use Committee (#
A12013), Fargo, USA. All samples were derived from healthy, non-
lactating, sexually mature non-pregnant Rambouillet ewes, and
were also used in our previous studies [45]. A brief overview of the
experimental design is presented here, as a detailed description has
already been presented elsewhere [45,56]. Ewes (total n ¼ 60, i.e.,
n ¼ 20 for each feeding group), 3e5 years old, were individually
penned in the regular breeding season between August and
December. They were randomly divided into three different feeding
groups: Maintenance (control fed, CF; 100% National Research
Council [NRC] requirements; 2.4 Mcal of metabolizable energy
[ME]/kg body weight (BW)); overfed (OF; 200% NRC requirements);
and underfed (UF; 60% NRC requirements). Feeding of animals in
each dietary group was initiated 60 days before the onset of the first
experimental estrus cycle and was performed twice a day (at 8 a.m.
and 3 p.m.). Ewes were weighed every week, and the diet of each
animal was adjusted to achieve the optimal body weight and body
€ Bedir, A. Gram, A.T. Grazul-Bilska et al.
O.
condition score on the first day of the estrous cycle. Initial and final
body weight and body condition score were reported elsewhere
[51]. Estrus synchronization of the animals was managed via
intravaginal insertion of controlled internal drug release (CIDR)
devices for 14 days. The first sign of estrus was seen 36 h after the
removal of the CIDR, with the day of estrus considered as day 0 of
the estrous cycle. For the superovulation group (SOV), animals from
different diet groups (n ¼ 30, n ¼ 10/each feeding group) were
injected twice-daily with: 5 mg/injection, 4 mg/injection, or 3 mg/
injection, lowering dosages of follicle stimulating hormone (FSH)
on days 13, 14 and 15, respectively (FSHeP; Sioux Biochemical,
Sioux Center, IA, USA) [38]. As a control (CONT), ewes from each
feeding group (n ¼ 30, n ¼ 10/feeding group) were injected with
saline (10 ml, 0.9% NaCl in water). Endometrial caruncle samples
were collected on day 5 (early-luteal phase) and day 10 (mid-luteal
phases) of the second estrous cycle (i.e., first estrous cycle after
superovulation). Finally, 12 groups of animals were involved in this
study, each of n ¼ 5, i.e., CF/OF/UF, FSH þ/, d 5 or 10 [45]. Tissue
samples were immediately frozen on dry ice and stored at 80  C
until mRNA extraction followed by evaluation of gene expression.
2.2. Total RNA extraction and semi-quantitative real-time
(TaqMan) PCR
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to
isolate total RNA, according to the manufacturer's instructions. The
quality and quantity of the isolated RNA was measured with a
nanodrop 2000C® spectrophotometer (Thermo Fisher Scientific
AG, Reinach, CH). RNA integrity numbers (RIN) were assessed with
an Agilent 2200 TapeStation System and ranged between 8.0 and
10.0. Then, reverse transcription (RT) was performed following the
previously published protocol [57]. Briefly, genomic DNA contam-
ination was eliminated by using RQ1 RNase-free DNase (Roche
Diagnostics, Basel, CH). RT was performed with random hexamers
used as primers in the presence of the other reagents from Applied
Biosystems by Thermo Fisher in an Eppendorf Mastercycler (Vau-
daux- Eppendorf AG, Basel, CH) and complementary DNA (cDNA)
corresponding to 100 ng RNA per sample was used for detection of
each target gene. An automated cycler ABI PRISM 7500 Sequence
Detection System (Applied Biosystems by Thermo Fisher) was used
to perform the semi-quantitative (TaqMan) PCR. A non-template
control was performed by using autoclaved water instead of
cDNA. As a RT-minus control, samples were run with all the com-
ponents of the amplification reaction except for reverse transcrip-
tase to detect any possible genomic DNA contamination. All
commercially available and self-designed TaqMan systems are
shown in Table 1. For self-designed primers Primer Express soft-
ware version 2.0 (Applied Biosystems by Thermo Fisher Scientific)
was used and 6-carboxyfluorescein (6-FAM)- and 6-
carboxytetramethylrhodamine (TAMRA)-labeled TaqMan probes
were ordered from Microsynth (Balgach, CH). Pre-designed,
commercially available TaqMan systems were purchased from
Applied Biosystems. To normalize the target gene expression,
GAPDH and ACTB were used as reference genes, based on the
available literature, and their stability was checked by using the
online tool https://www.heartcure.com.au/reffinder/?
type¼reference. The comparative CT (DDCT) method was used to
calculate relative gene expression and it was performed according
to the ABI 7500 manufacturer's protocols and as described previ-
ously [57,58]. The gene showing the lowest expression was chosen
as a calibrator for calculating relative gene expression. The effi-
ciency of TaqMan systems was validated by using the CT slope
model which was set at approximately 100%.
231
Theriogenology 195 (2023) 229e237
2.3. Statistical analysis
GraphPad 3.06 software (San Diego, CA, USA) was used for
statistical analysis. The statistical approach was initiated by
comparing samples with respect to the stage of the estrous cycle
(day 5 versus day 10), with the expression of target genes in un-
treated controls compared using an unpaired two-tailed Student's
t-test. In the next step, following interactions have been assessed:
the interaction between day of estrus cycle and response to diet, as
well as the effect of time on the interaction between FSH-induced
superovulation and nutrition (two-way ANOVA). The effects of
FSH-induced superovulation and feeding on the expression of all
target genes were then evaluated with respect to the day of cyclus;
due to high individual variations in the results, a Kruskal-Wallis test
(non-parametric ANOVA), followed by the Mann-Whitney test was
performed. The statistical analysis software SPSS version 24, IBM,
Armonk, NY, USA was used. For all tests, P < 0.05 was considered as
statistically significant. All numerical results for relative gene
expression are presented as means ± standard deviation (SD).
3. Results
3.1. Gene expression patterns of selected integrins in ovine
caruncular tissues
The mRNA expression of several integrin subunits: ITGA4, ITGA5,
ITGAV, ITGB1, ITGB3, and ITGB5 was detected in all the caruncular
tissue samples (Fig. 1). While the transcriptional levels of ITGA4,
ITGA5, ITGB1 and ITGB5 did not differ significantly between days 5
and 10 (P > 0.05; Fig. 1), both ITGAV and ITGB3 showed time-
dependent expression and lower abundance in caruncles from
CF_CONT at day 10 compared with day 5 (P < 0.005 and P < 0.05,
respectively) (Fig. 1C and E). The transcript levels of ITGA4, -5, and
ITGB5 were affected by the plane of nutrition and FSH treatment at
both days 5 and 10 of the estrous cycle, while the effects on the
other integrins were only detected on day 10.
Significant interactions (P < 0.05) were found between time
(day of estrus cycle) and maternal diet for ITGA5, ITGAV, ITGB1 and
ITGB5. Additionally, ITGA5, ITGB1 and ITGB5 mRNA showed time-
dependent (P < 0.05) interaction between FSH treatment and
plane of nutrition (Fig. 1) (for more details s. below).
As for the expression of ITGA4, it was greater in OF_CONT and
UF_CONT, compared with CF_CONT on day 10 (P < 0.05 and P < 0.01
respectively). FSH treatment caused upregulation of ITGA4 in
OF_SOV compared with the OF_CONT (P < 0.05). Additionally,
ITGA4 mRNA expression was greater on day 10 in UF_SOV compared
with CF_SOV (P < 0.05). On day 5, ITGA4 expression was greater in
OF_CONT compared with CF_CONT (Fig. 1A).
For ITGA5, its significant interaction between day of the estrus
cycle and maternal diet (P < 0.05) was reflected in the greater levels
in UF_CONT and OF_CONT compared with CF_CONT, on days 5 and
10 (P < 0.001e0.05; Fig. 1B). Expression of ITGA5 mRNA was also
greater in UF_SOV ewes on day 5 (P < 0.01), and in OF_SOV on day
10 (P < 0.001), compared with their respective CF_SOV. Moreover,
FSH increased the ITGA5 expression in OF_SOV animals compared
with OF_CONT on day 10 (P < 0.05) resulting in its significant, time-
dependent interaction between FSH treatment and plane of
nutrition for ITGA5 mRNA expression, found at day 10 (P < 0.04;
Fig. 1B).
The significant interaction between day of the estrus cycle and
nutrition observed for ITGAV and ITGB1 (P < 0.03 and P < 0.02
respectively) was reflected in lower ITGB1 mRNA levels on day 10 in
CF_CONT than in UF_CONT (P < 0.01), and lower ITGAV and ITGB1
expression in OF_CONT than UF_CONT (both P < 0.05). In the case of
ITGB1, the significant (P < 0.001) time-dependent interaction
€ Bedir, A. Gram, A.T. Grazul-Bilska et al.
O. Theriogenology 195 (2023) 229e237

Table 1
List of self-designed and commercially available primers used for Real-Time (TaqMan) PCR. F, forward primer; R, reverse primer; P, TaqMan probe.

Gene Name Accession number TaqMan System Product length (bp)

ITGA4 Integrin subunit alpha 4 XM_004004537.4 Commercially available: 97

Applied Biosystem, prod.
No. 0a04837603_m1
ITGA5 Integrin subunit alpha 5 XM_027967221.1 Commercially available: 95
Applied Biosystem, prod.
No. 0a04817753_g1
ITGAV Integrin subunit alpha V XM_012110914.3 Commercially available: 108
Applied Biosystem, prod.
No. 0a03213933_m1
ITGB1 Integrin subunit beta 1 XM_027976359.1 Commercially available: 96
Applied Biosystem, prod.
No. 0a03223518_m1
ITGB3 Integrin subunit beta 3 XM_012186282.3 Commercially available: 85
Applied Biosystem, prod.
No. 0a04294965_m1
ITGB5 Integrin subunit beta 5 XM_027956579.1 Commercially available: 95
Applied Biosystem, prod.
No. 0a04891121_m1
FN1 Fibronectin 1 XM_004004907.4 Commercially available: 56
Applied Biosystem, prod.
No. 0a04675746_m1
MUC1 Mucin 1, cell surface associated XM_027976040.1 Commercially available: 90
Applied Biosystem, prod.
No. 0a04914473_g1
HGF Hepatocyte growth factor XM_004007806.4 Commercially available: 69
Applied Biosystem, prod.
No. 0a04783932_m1
FGF10 Fibroblast growth factor 10 NM_001009230.1 Commercially available: 70
Applied Biosystem, prod.
No. 0a04658603_m1
IGFBP3 Insulin like growth factor binding protein 3 NM_001159276.1 Commercially available: 66
Applied Biosystem, prod.
No. 0a04654707_g1
GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase NM_001190390.1 F:50 -GGCACAGTCAAGGCAGAGAAC-30 114
R:50 -CACGTACTCAGCACCAGCATCA-30
P: 5 -AAGGCCATCACCATCTTCCAGGAGC-30
ACTB Actin Beta U39357.1 F:50 - AGAGGCATCCTGACCCTCAA-30 93
R:50 - GTTGTAGAAGGTGTGGTGCCAGAT-30
P:5‘ -TACCCCATTGAGCACGGCATTGTCA-30

between superovulation and nutrition was observed on day 10, and
was mirrored in higher ITGB1 levels in OF_SOV, compared to
CF_SOV (P < 0.05) (Fig. 1D). Similarly, ITGAV was higher in OF_SOV,
than in CF_SOV (P < 0.01) (Fig. 1C). Additionally, compared with
their controls, on day 10 superovulation resulted in higher ITGAV
levels (OF_CONT vs. OF_SOV; P < 0.05, Fig. 1C), but lower ITGB1
levels (UF_CONT vs. UF_SOV; P < 0.05, Fig. 1D).
Similar to the expression patterns for both ITGA4 and ITGA5,
ITGB3 mRNA levels were strongly potentiated by the plane of
nutrition and greater in OF_CONT and UF_CONT compared with
CF_CONT on day 10 (P < 0.05 and P < 0.001, respectively) (Fig. 1E).
Moreover, ITGB3 mRNA expression was greater in CF_SOV than
CF_CONT on day 10 (vs, P < 0.01). Furthermore, ITGB3 was greater in
OF_SOV than in CF_SOV (P < 0.05) (Fig. 1E).
Regarding ITGB5, its significant (P < 0.001) interaction between
day of estrus cycle and feeding, was mirrored in greater mRNA
levels in UF_CONT on days 5 and 10 compared with CF_CONT (both
P < 0.01), and in UF_CONT compared with OF_CONT at day 5
(P < 0.05) (Fig. 1F). Reflecting the significant (P < 0.001) time-
dependent interaction between FSH treatment and plane of
nutrition at day 10, ITGB5 was also greater in OF_SOV and UF_SOV,
compared with CF_SOV at that day (P < 0.01 and P < 0.01, respec-
tively). It was also greater in OF_SOV than OF_CONT (P < 0.01) at
day 10 (Fig. 1F).
3.2. Expression of FN1, MUC1 and growth factors (HGF, FGF10,
IGFBP3) in ovine caruncular tissues
Expression of mRNA encoding FN1, MUC1 (Fig. 2), as well as
growth factors (HGF, FGF10, IGFBP3) (Fig. 3), was detected in all
caruncular tissue samples examined. Whereas day of the estrous
cycle affected the mRNA expression of FGF10 and IGFBP3 resulting
in lower expression of both factors in CF_CONT on day 10 than day 5
(P < 0.001 and P < 0.05 respectively) (Fig. 3B and C), FN1, MUC1 and
HGF did not differ significantly (P > 0.05) (Figs. 2A and 3A).
Significant interactions (P < 0.01) were found between time
(day of estrus cycle) and maternal diet was found for FGF10.
Additionally, together with FN1, FGF10 showed time-dependent
(P < 0.05) interaction between FSH treatment and plane of nutri-
tion (Fig. 2).
In detail, the time-dependent interaction between FSH-induced
superovulation and nutrition for FN1 mRNA expression was found
at day 10 (P < 0.04) and was mirrored in its higher levels in CF_SOV
than CF_CONT (P < 0.05), and in OF_SOV than OF_CONT (P < 0.05)
on that day. However, FN1 transcripts were less abundant in
UF_SOV than UF_CONT on days 5 and 10 (both P < 0.05; Fig. 2A). The
expression of MUC1 mRNA was reduced in response to FSH treat-
ment in CF_SOV compared with CF_CONT on day 10 (P < 0.05)
(Fig. 2B). In SOV animals its expression was higher in OF_SOV and
UF_SOV compared to CF_SOV on day 10 (P < 0.01 for both; Fig. 2B).
Neither diet nor FSH treatment affected MUC1 levels on day 5
(Fig. 2B).

232
€ Bedir, A. Gram, A.T. Grazul-Bilska et al.
O. Theriogenology 195 (2023) 229e237

Fig. 1. Relative gene expression of integrin subunits; ITGA4 (A), ITGA5 (B), ITGAV (C), ITGB1 (D), ITGB3 (E), ITGB5 (F), as calculated by Real Time (TaqMan) PCR in ovine caruncular
endometrial tissue in the early luteal phase (day 5) and the mid-luteal phase (day 10), in control and FSH-superovulated ewes, control fed (CF), overfed (OF) and underfed (UF).
Time-dependent effects were assessed for control fed, untreated animals serving as experimental controls. Blue lines indicate effects of plane of nutrition and dashed lines represent
differences between superovulated groups. Numerical data are presented as the mean ± SD. Bars with asterisks differ at: * ¼ P < 0.05, ** ¼ P < 0.01, **** ¼ P < 0.001. CONT: non-
superovulated control, SOV: superovulated with FSH.

The expression of HGF mRNA was greater in UF_SOV than Regarding FGF10, the significant (P < 0.01) interaction between
UF_CONT on day 5 (P < 0.05). On day 10, HGF was greater in day of estrus cycle and feeding was reflected in lower mRNA levels
UF_CONT than CF_CONT (P < 0.05), with no effects of diet or FSH observed at day 10 in OF_CONT, but greater in UF_CONT, compared
treatment (Fig. 3A). with CF_CONT (both P < 0.05). The time-dependent interaction

Fig. 2. Relative gene expression of FN1 (A) and MUC1 (B) as calculated by Real Time (TaqMan) PCR in ovine caruncular endometrial tissue in the early luteal phase (day 5) and the
mid-luteal phase (day 10), in control and FSH-superovulated ewes, control fed (CF), overfed (OF) and underfed (UF). Time-dependent effects were assessed for control fed, untreated
animals serving as experimental controls. Dashed lines represent differences between superovulated groups. Numerical data are presented as the mean ± SD. Bars with asterisks
differ at: * ¼ P < 0.05, ** ¼ P < 0.01. CONT: non-superovulated control, SOV: superovulated with FSH.

233
€ Bedir, A. Gram, A.T. Grazul-Bilska et al.
O. Theriogenology 195 (2023) 229e237

Fig. 3. Relative gene expression of HGF (A), FGF10 (B) and IGFBP3 (C) as calculated by Real Time (TaqMan) PCR in ovine caruncular endometrial tissue in the early luteal phase (day
5) and the mid-luteal phase (day 10), in control and FSH-superovulated ewes, control fed (CF), overfed (OF) and underfed (UF). Time-dependent effects were assessed for control fed,
untreated animals serving as experimental controls. Blue lines indicate effects of plane of nutrition. Numerical data are presented as the mean ± SD. Bars with asterisks differ at:
* ¼ P < 0.05, ** ¼ P < 0.01, *** ¼ P < 0.005, **** ¼ P < 0.001. CONT: non-superovulated control, SOV: superovulated with FSH.

between superovulation and nutrition found for FGF10 on day 10 number of implantation sites in mice [63].
(P < 0.02) was mirrored in its increased mRNA levels in CF_SOV and In the present study, the combined effects of nutrition plane and
OF_SOV (both P < 0.005) but lowered in UF_SOV (P < 0.05) hormonal hyperstimulation on the expression of all investigated
compared to their respective controls (CF_CONT, OF_CONT and integrins were observed at both the early and mid-luteal phases.
UF_CONT) (Fig. 3B). The key functions of integrins at the time of implantation are to
IGFBP3 mRNA expression was greater in UF_CONT compared bind glycoproteins or other molecules responsible for adhesion in
with CF_CONT on day 10 (P < 0.01), and was greater in CF_CONT on ECM and involved in proliferation and differentiation of uterine
day 5 than day 10 (P < 0.05) (Fig. 3C). epithelial cells, and to stabilize adhesion of the conceptus to the
uterine epithelium [7]. Therefore, taking into account the biological
importance of integrins as cell adhesion molecules, our findings
4. Discussion
strongly suggest that alteration of their expression due to imbal-
anced nutrition could affect uterine responsiveness to the
Building on our previous results [38,39,45], this study deepens
implanting embryo, with hormonal stimulation having modulatory
our understanding of the effects of diet and hormonal manipulation
effects.
on ovine uterine caruncles during the early and mid-luteal phase,
Despite the common use of FSH during ARTs in animals as well
the latter being the day corresponding to uterine preparation for
as humans, supraphysiological FSH concentrations may compro-
implantation. Importantly, however, since our model only included
mise oocyte developmental competence and cumulus oocyte
non-pregnant animals, information regarding the effects of the
complex (COC) maturation [64]. Following this line, investigations
presence of embryos needs to be added in the future.
in mice revealed that FSH-induced ovarian hyperstimulation might
As observed here, and supported by previous studies [18,23], the
directly impact embryo genetic health, as embryos from FSH-
stage of the luteal phase affected ITGAV and ITGB3 but not ITGA4, -5,
stimulated mice had an increased incidence of chromosomal ab-
ITGB1 and -5 transcripts. However, in contrast to previous work [8],
normalities, fetal deformities and delayed fetal development [65].
MUC1 was similar in both stages of estrous cycle in the present
Similarly, the age-related excessive intrafollicular FSH levels may
study. Interestingly, all of the investigated integrins were affected
compromise oocyte quality by disrupting the transzonal delivery of
by diet. This was substantiated by a significant interaction found
cumulus-derived molecules required for energy production,
between the day of estrus cycle and nutrition for some of them, i.e.,
oxidative stress control, meiotic accuracy, DNA damage repair, and
ITGA5, ITGAV, ITGB1 and ITGB5. Together with MUC1 all integrins
gene expression as the ovarian reserve is gradually depleted [66].
were modified following induction of superovulation with FSH.
This is particularly relevant in humans, as hyperstimulation with
Integrins are the key mediators of matrix effects such as cell-cell
FSH appears to function better in patients under the age of 35,
and cell-ECM adhesion, having an impact on cell shape and regu-
resulting in higher oocyte quality rather than increased oocyte
lation of gene expression [9,59]. Their well-balanced expression is
availability [48]. In domestic animals, high FSH doses may
needed for implantation, trophoblast migration and proliferation
contribute to variability in superovulation response and outcomes
[17], with altered expression affecting fertility. Accordingly, in mice,
in cattle, with higher dosages of FSH resulting in an increased
knockout of selected integrins, like ITGA5 and ITGB1 [60,61], results
number of antral follicles, but with no effect on the number of
in embryonal lethality during the peri-implantation period [62].
embryos [67]. Age-related increases in FSH levels lead to increased
Similarly, functional blockade of ITGAV and ITGB3 reduces the
234
€ Bedir, A. Gram, A.T. Grazul-Bilska et al.
O.
female reproductive failure in mice [68], and the accelerated aging
was also proposed to be related to increased recruitment of folli-
cles, accelerating the depletion of reserves [69].
As shown in the present study, the expression of integrins, as
well as of MUC1 and FN1 was affected by superovulation during the
mid-luteal phase, which corresponds to the initiation of embryonal
recognition in pregnant animals. Thus, by altering expression of
endometrial genes critical for embryo attachment and adhesion,
FSH treatment may have effects on the window of implantation in
sheep.
Furthermore, the observed time-dependent interaction be-
tween superovulation and nutrition for FN1, ITGB5 and ITGA5 em-
phasizes that feeding and hormonal treatment jointly play a major
role in the mRNA expression of uterine caruncular adhesion mol-
ecules during critical stages in uterine preparation for implantation.
In this context, the changes in the expression of endometrial genes
critical for embryo attachment that occur after ovarian hyper-
stimulation have previously been associated with alterations in the
E2 to P4 ratio in humans and pigs [70,71]. In line with this, higher
P4 plasma levels in the early- and mid-luteal phases were observed
in FSH-treated ewes, compared with untreated controls [72].
Furthermore, different nutrition planes, e.g., overfeeding or un-
derfeeding, have been shown to increase uterine PGR expression in
sheep [39,45] with increased circulating P4 concentrations in
overfed animals [37]. It has also been suggested that altered PGR
expression might be of further functional importance [45]; in
humans and domestic animals, reduced endometrial PGR expres-
sion is considered necessary for the regulation of adhesion mole-
cules, mucins, and IFN-related genes required for implantation
[32,73,74]. This might also apply to the expression of MUC1, which
is regulated by E2 and P4 as well as by the presence of an embryo
[9,12,75], and possibly other adhesion molecules. Furthermore, as
indicated in our previous study, the ESR1:ESR2 ratio appeared
altered by different feeding regimes and superovulation in sheep
[45]. Thus, together with PGR, the expression of both E2 receptors
was upregulated in over- and underfed animals relative to their
normally-fed controls [45]. As for the ESR1:ESR2 ratio, whereas FSH
treatment increased transcriptional levels of ESR1, it suppressed
ESR2. If translated to the respective protein levels, leading to al-
terations of downstream cascades involving the expression of cell
adhesion molecules, the functional consequence could be in dis-
turbances at the embryo-maternal interface.
The expression of stromal-derived growth factors, like HGF,
FGF10 and IGFBP3, is regulated in uterine tissues by P4 in sheep [31].
Similarly, in the present study, expression of both FGF10 and IGFBP3
was time-dependent between the early and mid-luteal phases.
Moreover, besides showing a significant interaction between the
day of estrus cycle and nutrition, strong FSH treatment effects and
time-dependent interaction between superovulation and nutrition
were observed for FGF10. Interestingly, whereas lowered expres-
sion of FGF10 and IGFBP3 was expected in the mid-luteal phase
[31,34], their expression was upregulated in underfed animals at
day 10. FGF10 appears to diminish FSH responsiveness at the
transcriptional level, since intrafollicular treatment with FGF10
lowers aromatase (CYP19A1) expression, E2 production, and follicle
development in cows [76]. Consequently, the available evidence
implies that FGF10 plays a role during follicular development in
cows [76,77]. Additionally, in the mouse, FGF10 serves as a para-
crine factor for E2-dependent regulation of uterine epithelial cell
proliferation [78]. These findings further our previous observations
of modulatory effects of FSH in combination with diet upon other
growth factors in ovine caruncular endometrium, i.e., IGF1, IGF2 and
VEGFA [45].
Cumulatively, these results add valuable information and com-
plement our previously published data [38,39,45] on the possible
235
Theriogenology 195 (2023) 229e237
effects of nutritional plane and superovulation upon ovine uterine
functionality. This study has demonstrated that uterine expression
of genes responsible for intercellular communication, cell adhesion,
and encoding for growth factors is affected by imbalanced nutrition
as well as FSH-induced ovarian hyperstimulation. Plane of nutrition
impacts the uterine responsiveness to exogenously applied hor-
monal stimulation and, likely, implantation and pregnancy out-
comes in sheep. Concomitantly, the translational value of our
findings to other mammals is emphasized, highlighting the
possible causes for the disruption of uterine functionality, embryo
development, and embryo-maternal cross-talk during preparation
for implantation in domestic animal species, as well as in humans.
Funding
This project was partially supported by the Ministry of Higher
Education of the Republic of Turkey through the Scholarship Pro-
gramme YLSY and from the Vetsuisse Faculty of Zurich via the
Institute of Veterinary Anatomy, and by the USDA-AFRI grant 2011-
67016-30174.
CRediT authorship contribution statement
€
Ozlem Bedir: Conceptualization, Formal analysis, Data curation,
Writing e original draft, was involved in developing the concept of
the present study, experimental design, generating data, analysis
and interpretation of data and drafting the manuscript. Aykut
Gram: Project administration, Writing e original draft, was
involved in the laboratory part of the project, knowledge transfer,
drafting and revising the manuscript. Anna T. Grazul-Bilska:
Conceptualization, Data curation, was involved in developing the
concept of the study, experimental design, animal care and treat-
ment, tissue collection and processing, interpretation of data and
revising the manuscript. Mariusz P. Kowalewski: Conceptualiza-
tion, Formal analysis, Writing e original draft, was involved in
developing the concept of the present study, experimental design,
knowledge transfer, analyzing and interpreting the data and
writing and revising the manuscript, All authors read and approved
the final manuscript.
Declaration of competing interest
The authors declare that they have no conflicts of interest. All
authors read and approved the final version of the manuscript.
Acknowledgements
Authors are thankful to Dr. Sharon Mortimer (Oozoa Biomedical,
Inc., Vancouver, Canada) for careful editing of the manuscript. Part
of the laboratory work was performed using the logistics of the
Center for Clinical Studies, Vetsuisse Faculty, University of Zurich.
References
[1] Hamid HY, Abu Z, Zakaria B. Embryo implantation: shedding light on the roles
of ovarian hormones, cytokines and growth factors in the implantation pro-
cess. Afr J Biotechnol 2012;11:16297e304. https://doi.org/10.5897/
AJB12.1857.
[2] Hyttel P, Sinowatz F, Vejlsted M. Essentials of domestic animal embryology.
first ed. London: Elsevier Ltd; 2010.
[3] Bazer FW, Spencer TE, Johnson GA. Interferons and uterine receptivity. Semin
Reprod Med 2009;27:90e102. https://doi.org/10.1055/s-0028-1108013.
[4] Siemieniuch MJ, Jursza E, Szostek AZ, Zschockelt L, Boos A, Kowalewski MP.
Placental origin of prostaglandin Fin the domestic cat. Mediat Inflamm
2014;2014. https://doi.org/10.1155/2014/364787.
[5] Kowalewski MP, Kazemian A, Klisch K, Gysin T, Tavares Pereira M, Gram A.
Canine endotheliochorial placenta: morpho-functional aspects. Adv Anat
Embryol Cell Biol 2021;234:155e79. https://doi.org/10.1007/978-3-030-
€ Bedir, A. Gram, A.T. Grazul-Bilska et al.
O.
77360-1_8.
[6] Spencer TE, Johnson GA, Bazer FW, Burghardt RC. Implantation mechanisms:
insights from the sheep. Reproduction 2004;128:657e68. https://doi.org/
10.1530/rep.1.00398.
[7] Johnson GA, Bazer FW, Jaeger LA, Ka H, Garlow JE, Pfarrer C, et al. Muc-1,
integrin, and osteopontin expression during the implantation cascade in
sheep. Biol Reprod 2001;65:820e8. https://doi.org/10.1095/
biolreprod65.3.820.
[8] Raheem KA, Marei WFA, Campbell BK, Fouladi-Nashta AA. In vivo and in vitro
studies of MUC1 regulation in sheep endometrium. Theriogenology 2016;85:
1635e43. https://doi.org/10.1016/j.theriogenology.2016.01.018.
[9] Aplin JD. Adhesion molecules in implantation. Rev Reprod 1997;2:84e93.
https://doi.org/10.1530/ror.0.0020084.
[10] Horne AW, Lalani EN, Margara RA, Ryder TA, Mobberley MA, White JO. The
expression pattern of MUC1 glycoforms and other biomarkers of endometrial
receptivity in fertile and infertile women. Mol Reprod Dev 2005;72:216e29.
https://doi.org/10.1002/mrd.20307.
[11] Bowen JA, Bazer FW, Burghardt RC. Spatial and temporal analyses of integrin
and Muc-1 expression in porcine uterine epithelium and trophectoderm
in vitro. Biol Reprod 1997;56:409e15. https://doi.org/10.1095/
biolreprod56.2.409.
[12] Surveyor GA, Pemberton L, F Co, Julian J, Pimental RA, Wegner C, et al.
Expression and steroid hormonal control of Muc-1 in the mouse uterus.
Endocrinology 1995;136:3639e47.
[13] Seo H, Frank JW, Burghardt RC, Bazer FW, Johnson GA. Integrins and OPN
localize to adhesion complexes during placentation in sheep. Reproduction
2020;160:521e32. https://doi.org/10.1530/REP-20-0273.
[14] Fayazi M, Beigi Boroujeni M, Salehnia M, Khansarinejad B. Ovarian stimulation
by exogenous gonadotropin decreases the implantation rate and expression
of mouse blastocysts integrins. Iran Biomed J 2014;18:8e15. https://doi.org/
10.6091/ibj.1236.2013.
[15] Illera MJ, Lorenzo PL, ting Gui Y, Beyler SA, Apparao KBC, Lessey BA. A role for
avb3 integrin during implantation in the rabbit model. Biol Reprod 2003;68:
766e71. https://doi.org/10.1093/biolreprod/68.3.766.
[16] Lessey BA, Castelbaum AJ, Buck CA, Lei Y, Yowell CW, Sun J. Further charac-
terization of endometrial integrins during the menstrual cycle and in preg-
nancy. Fertil Steril 1994;62:497e506. https://doi.org/10.1016/s0015-
0282(16)56937-4.
[17] Burghardt RC, Johnson GA, Jaeger LA, Ka H, Garlow JE, Spencer TE, et al.
Integrins and extracellular matrix proteins at the maternal-fetal interface in
domestic animals. Cells Tissues Organs 2002;172:202e17. https://doi.org/
10.1159/000066969.
[18] Burghardt RC, Burghardt JR, Taylor JD, Reeder AT, Nguen BT, Spencer TE, et al.
Enhanced focal adhesion assembly reflects increased mechanosensation and
mechanotransduction at maternal-conceptus interface and uterine wall dur-
ing ovine pregnancy. Reproduction 2009;137:567e82. https://doi.org/
10.1530/REP-08-0304.
[19] García P, Nieto A, S anchez MA, Pizarro M, Flores JM. Expression of alphav,
alpha4, alpha5 and beta3 integrin subunits, fibronectin and vitronectin in goat
peri-implantation. Anim Reprod Sci 2004;80:91e100. https://doi.org/
10.1016/S0378-4320(03)00157-X.
[20] Apparao KBC, Murray MJ, Fritz MA, Meyer WR, Chambers AF, Truong PR, et al.
Osteopontin and its receptor avb3 integrin are coexpressed in the human
endometrium during the menstrual cycle but regulated differentially. J Clin
Endocrinol Metab 2001;86:4991e5000. https://doi.org/10.1210/
jc.86.10.4991.
[21] Kimmins S, Lim HC, MacLaren LA. Immunohistochemical localization of
integrin alpha V beta 3 and osteopontin suggests that they do not interact
during embryo implantation in ruminants. Reprod Biol Endocrinol 2004;2:
1e13. https://doi.org/10.1186/1477-7827-2-19.
[22] Bukowska D, Kempisty B, Jackowska M, Wo zna M, Antosik P, Piotrowska H,
et al. Analysis of integrins and vascular endothelial growth factor isoforms
mRNA expression in the canine uterus during perimplantation period. Pol J
Vet Sci 2011;14:253e8. https://doi.org/10.2478/v10181-011-0038-3.
[23] Frank JW, Steinhauser CB, Wang X, Burghardt RC, Bazer FW, Johnson GA. Loss
of ITGB3 in ovine conceptuses decreases conceptus expression of NOS3 and
SPP1: implications for the developing placental vasculature. Biol Reprod
2021;104:657e68. https://doi.org/10.1093/biolre/ioaa212.
[24] Lessey BA, Damjanovich L, Coutifaris C, Castelbaum A, Albeida SM, Buck CA.
Integrin adhesion molecules in the human endometrium. Correlation with the
normal and abnormal menstrual cycle. J Clin Invest 1992;90:188e95. https://
doi.org/10.1172/JCI115835.
[25] Thomas K, Thomson AJ, Sephton V, Cowan C, Wood S, Vince G, et al. The effect
of gonadotrophic stimulation on integrin expression in the endometrium.
Hum Reprod 2002;17:63e8. https://doi.org/10.1093/humrep/17.1.63.
[26] Hynes RO. Targeted mutations in cell adhesion genes: what have we learned
from them? Dev Biol 1996;180:402e12. https://doi.org/10.1006/
dbio.1996.0314.
[27] McLendon BA, Kramer AC, Seo H, Bazer FW, Burghardt RC, Johnson GA.
Integrin adhesion complex organization in sheep myometrium reflects
changing mechanical forces during pregnancy and postpartum. Biology
2021;10:508. https://doi.org/10.3390/biology10060508.
[28] Burkin HR, Rice M, Sarathy A, Thompson S, Singer CA, Buxton ILO. Integrin
upregulation and localization to focal adhesion sites in pregnant human
myometrium. Reprod Sci 2013;20:804e12. https://doi.org/10.1177/
236
Theriogenology 195 (2023) 229e237
1933719112466303.
[29] Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Molecular biology of
the cell. fifth ed. New York: Garland Science; 2008.
[30] Uehara Y, Minowa O, Mori C, Shiota K, Kuno J, Noda T, et al. Placental defect
and embryonic lethality in mice lacking hepatocyte growth factor/scatter
factor. Nature 1995;373:702e5. https://doi.org/10.1038/373702a0.
[31] Satterfield MC, Hayashi K, Song G, Black SG, Bazer FW, Spencer TE. Proges-
terone regulates FGF10, MET, IGFBP1, and IGFBP3 in the endometrium of the
ovine uterus. Biol Reprod 2008;79:1226e36. https://doi.org/10.1095/
biolreprod.108.071787.
[32] Bazer FW, Wu G, Spencer TE, Johnson GA, Burghardt RC, Bayless K. Novel
pathways for implantation and establishment and maintenance of pregnancy
in mammals. Mol Hum Reprod 2010;16:135e52. https://doi.org/10.1093/
molehr/gap095.
[33] Chen C, Spencer TE, Bazer FW. Expression of hepatocyte growth factor and its
receptor c-met in the ovine uterus. Biol Reprod 2000;62:1844e50. https://
doi.org/10.1095/biolreprod62.6.1844.
[34] Chen C, Spencer TE, Bazer FW. Fibroblast growth factor-10: a stromal medi-
ator of epithelial function in the ovine uterus. Biol Reprod 2000;63:959e66.
https://doi.org/10.1095/biolreprod63.3.959.
[35] Osgerby JC, Gadd TS, Wathes DC. Expression of insulin-like growth factor
binding protein-1 (IGFBP-1) mRNA in the ovine uterus throughout the oes-
trous cycle and early pregnancy. J Endocrinol 1999;162:279e87. https://
doi.org/10.1677/joe.0.1620279.
[36] Sosa C, Abecia JA, Carriquiry M, Forcada F, Martin GB, Palacín I, et al. Early
pregnancy alters the metabolic responses to restricted nutrition in sheep.
Domest Anim Endocrinol 2009;36:13e23. https://doi.org/10.1016/
j.domaniend.2008.08.003.
[37] Kaminski SL, Redmer DA, Bass CS, Keisler DH, Carlson LS, Vonnahme KA, et al.
The effects of diet and arginine treatment on serum metabolites and selected
hormones during the estrous cycle in sheep. Theriogenology 2015;83:
808e16. https://doi.org/10.1016/j.theriogenology.2014.11.017.
[38] Grazul-Bilska AT, Khanthusaeng V, Bass CS, Kaminski SL, Navanukraw C,
Redmer DA. Lipid droplets in the ovine uterus during the estrous cycle: effects
of nutrition, arginine, and FSH. Theriogenology 2017;87:212e20. https://
doi.org/10.1016/j.theriogenology.2016.08.023.
[39] Grazul-Bilska AT, Thammasiri J, Kraisoon A, Reyaz A, Bass CS, Kaminski SL,
et al. Expression of progesterone receptor protein in the ovine uterus during
the estrous cycle: effects of nutrition, arginine and FSH. Theriogenology
2018;108:7e15. https://doi.org/10.1016/j.theriogenology.2017.11.008.
[40] Bass CS, Redmer DA, Kaminski SL, Grazul-Bilska AT. Luteal function during the
estrous cycle in arginine-treated ewes fed different planes of nutrition.
Reproduction 2017;153:253e65. https://doi.org/10.1530/REP-16-0526.
[41] Foren-Fernandez A, Sosa C, Abecia J, Vazquez M, Forcada F, Meikle A. Dietary
restriction in sheep : uterine functionality in ewes with different body re-
serves during early gestation. Theriogenology 2019;135:189e97. https://
doi.org/10.1016/j.theriogenology.2019.06.023.
[42] Roche JR, Macdonald KA, Burke CR, Lee JM, Berry DP. Associations among body
condition score, body weight, and reproductive performance in seasonal-
calving dairy cattle. J Dairy Sci 2007;90:376e91. https://doi.org/10.3168/
jds.S0022-0302(07)72639-5.
[43] Grazul-Bilska AT, Borowczyk E, Bilski JJ, Reynolds LP, Redmer DA, Caton JS,
et al. Overfeeding and underfeeding have detrimental effects on oocyte
quality measured by in vitro fertilization and early embryonic development in
sheep. Domest Anim Endocrinol 2012;43:289e98. https://doi.org/10.1016/
j.domaniend.2012.05.001.
[44] Grazul-Bilska AT, Neville TL, Borowczyk E, Sharma A, Reynolds LP, Caton JS,
et al. Ovarian and uterine characteristics and onset of puberty in adolescent
offspring: effects of maternal diet and selenium supplementation in sheep.
Theriogenology 2014;81:887e95. https://doi.org/10.1016/
j.theriogenology.2013.12.024.
€
[45] O Bedir, Gram A, Dorsam ST, Grazul-Bilska AT, Kowalewski MP. Plane of
nutrition and FSH-induced superovulation affect the expression of steroid
hormone receptors and growth factors in caruncular tissue of non-pregnant
sheep. Domest Anim Endocrinol 2021;78. https://doi.org/10.1016/
j.domaniend.2021.106683.
[46] Leese HJ, Donnay I, Thompson JG. Human assisted conception: a cautionary
tale. Lessons from domestic animals. Hum Reprod 1998;13:184e202. https://
doi.org/10.1093/humrep/13.suppl_4.184.
[47] Santos MA, Kuijk EW, Macklon NS. The impact of ovarian stimulation for IVF
on the developing embryo. Reproduction 2010;139:23e34. https://doi.org/
10.1530/REP-09-0187.
[48] Buratini J, Dal Canto M, De Ponti E, Brambillasca F, Brigante C, Gippone S, et al.
Maternal age affects the relationship of basal FSH and anti-Müllerian hor-
mone concentrations with post-ICSI/IVF live birth. Reprod Biomed Online
2021;42:748e56. https://doi.org/10.1016/j.rbmo.2020.12.005.
[49] Menchaca A, Vilario M, Crispo M, De Castro T, Rubianes E. New approaches to
superovulation and embryo transfer in small ruminants. Reprod Fertil Dev
2010;22:113e8. https://doi.org/10.1071/RD09222.
[50] Marshall KL, Rivera RM. The effects of superovulation and reproductive aging
on the epigenome of the oocyte and embryo. Mol Reprod Dev 2018;85:
90e105. https://doi.org/10.1002/mrd.22951.
[51] Kraisoon A, Redmer DA, Bass CS, Navanukraw C, Dorsam ST, Valkov V, et al.
Corpora lutea in superovulated ewes fed different planes of nutrition. Domest
Anim Endocrinol 2018;62:16e23. https://doi.org/10.1016/
€ Bedir, A. Gram, A.T. Grazul-Bilska et al.
O.
j.domaniend.2017.08.002.
[52] Chu T, Dufort I, Sirard M. Effect of ovarian stimulation on oocyte gene
expression in cattle. Theriogenology 2012;77:1928e38. https://doi.org/
10.1016/j.theriogenology.2012.01.015.
[53] Nikas G, Develioglu OH, Toner JP, Jones HW. Endometrial pinopodes indicate a
shift in the window of receptivity in IVF cycles. Hum Reprod 1999;14:787e92.
https://doi.org/10.1093/humrep/14.3.787.
[54] Mansouri-Attia N, Aubert J, Reinaud P, Giraud-Delville C, Taghouti G, Galio L,
et al. Gene expression profiles of bovine caruncular and intercaruncular
endometrium at implantation. Physiol Genom 2009;39:14e27. https://
doi.org/10.1152/physiolgenomics.90404.2008.
[55] Garlow JE, Ka H, Johnson GA, Burghardt RC, Jaeger LA, Bazer FW. Analysis of
osteopontin at the maternal-placental interface in pigs. Biol Reprod 2002;66:
718e25. https://doi.org/10.1095/biolreprod66.3.718.
[56] Gram A, Redmer DA, Kowalewski MP, Dorsam ST, Valkov V, Warang P, et al.
Angiopoietin expression in ovine corpora lutea during the luteal phase: effects
of nutrition, arginine and follicle stimulating hormone. Gen Comp Endocrinol
2018;269:131e40. https://doi.org/10.1016/j.ygcen.2018.09.003.
[57] Kowalewski MP, Schuler G, Taubert A, Engel E, Hoffmann B. Expression of
cyclooxygenase 1 and 2 in the canine corpus luteum during diestrus. Ther-
iogenology 2006;66:1423e30. https://doi.org/10.1016/
j.theriogenology.2006.01.039.
[58] Kowalewski MP, Dyson MT, Manna PR, Stocco DM. Involvement of peroxi-
some proliferator-activated receptor gamma in gonadal steroidogenesis and
steroidogenic acute regulatory protein expression. Reprod Fertil Dev 2009;21:
909e22. https://doi.org/10.1071/RD09027.
[59] Bowen JA, Burghardt RC. Cellular mechanisms of implantation in domestic
farm animals. Semin Cell Dev Biol 2000;11:93e104. https://doi.org/10.1006/
scdb.2000.0155.
[60] Fa€ssler R, Meyer M. Consequences of lack of b1 integrin gene expression in
mice. Genes Dev 1995;9:1896e908. https://doi.org/10.1101/gad.9.15.1896.
[61] Stephens LE, Sutherland AE, Klimanskaya IV, Andrieux A, Meneses J,
Pedersen RA, et al. Deletion of b1 integrins in mice results in inner cell mass
failure and peri-implantation lethality. Genes Dev 1995;9:1883e95. https://
doi.org/10.1101/gad.9.15.1883.
[62] Van Der Flier A, Badu-Nkansah K, Whittaker CA, Crowley D, Bronson RT, Lacy-
Hulbert A, et al. Endothelial a5 and av integrins cooperate in remodeling of
the vasculature during development. Development 2010;137:2439e49.
https://doi.org/10.1242/dev.049551.
[63] Illera MJ, Cullinan E, Gui Y, Yuan L, Beyler SA, Lessey BA. Blockade of the a(v)
b3 integrin adversely affects implantation in the mouse. Biol Reprod 2000;62:
1285e90. https://doi.org/10.1095/biolreprod62.5.1285.
[64] Buratini J, Soares ACS, Barros RG, Dellaqua TT, Lodde V, Franciosi F, et al.
Physiological parameters related to oocyte nuclear differentiation for the
improvement of IVM/IVF outcomes in women and cattle. Reprod Fertil Dev
2021. https://doi.org/10.1071/rd21278.
[65] Van Der Auwera I, D'Hooghe T. Superovulation of female mice delays
237
Theriogenology 195 (2023) 229e237
embryonic and fetal development. Hum Reprod 2001;16:1237e43. https://
doi.org/10.1093/humrep/16.6.1237.
[66] Buratini J, Dellaqua TT, Dal Canto M, La Marca A, Carone D, Mignini Renzini M,
et al. The putative roles of FSH and AMH in the regulation of oocyte devel-
opmental competence: from fertility prognosis to mechanisms underlying
age-related subfertility. Hum Reprod Update 2021:1e23. https://doi.org/
10.1093/humupd/dmab044.
[67] Souza a H, Sartori R, Guenther JN, Caraviello D, Monson R, Wiltbank MC. Effect
of semen source and dose of FSH on superovulatory response and embryo
production in Holstein heifers. Anim Reprod 2007;4:70e6.
[68] McTavish KJ, Jimenez M, Walters KA, Spaliviero J, Groome NP, Themmen AP,
et al. Rising follicle-stimulating hormone levels with age accelerate female
reproductive failure. Endocrinology 2007;148:4432e9. https://doi.org/
10.1210/en.2007-0046.
[69] Vollenhoven B, Hunt S. Ovarian ageing and the impact on female fertility.
F1000Research 2018;7:1835. https://doi.org/10.12688/
f1000research.16509.1.
[70] Ka H, Seo H, Choi Y, Yoo I, Han J. Endometrial response to conceptus-derived
estrogen and interleukin-1b at the time of implantation in pigs. J Anim Sci
Biotechnol 2018;9:1e17. https://doi.org/10.1186/s40104-018-0259-8.
[71] Mirkin S, Nikas G, Hsiu JG, Díaz J, Oehninger S. Gene expression profiles and
structural/functional features of the peri-implantation endometrium in nat-
ural and gonadotropin-stimulated cycles. J Clin Endocrinol Metab 2004;89:
5742e52. https://doi.org/10.1210/jc.2004-0605.
[72] Grazul-Bilska AT, Kirsch JD, Bilski JJ, Kraft KC, Windorski EJ, Luther JS, et al.
Superovulation in Sheep : number and weight of the corpora lutea and serum
progesterone. Sheep Goat Res J 2007;22:26e31.
[73] Wetendorf M, Wu SP, Wang X, Creighton CJ, Wang T, Lanz RB, et al. Decreased
epithelial progesterone receptor A at the window of receptivity is required for
preparation of the endometrium for embryo attachment. Biol Reprod
2017;96:313e26. https://doi.org/10.1095/biolreprod.116.144410.
[74] Roberts RM, Cross JC, Leaman DW. Interferons as hormones of pregnancy.
Endocr Rev 1992;13:432e52. https://doi.org/10.1210/edrv-13-3-432.
[75] Brayman MJ, Julian JA, Mulac-Jericevic B, Conneely OM, Edwards DP,
Carson DD. Progesterone receptor isoforms A and B differentially regulate
MUC1 expression in uterine epithelial cells. Mol Endocrinol 2006;20:
2278e91. https://doi.org/10.1210/me.2005-0343.
[76] Gasperin BG, Ferreira R, Rovani MT, Santos JT, Buratini J, Price CA, et al. FGF10
inhibits dominant follicle growth and estradiol secretion in vivo in cattle.
Reproduction 2012;143:815e23. https://doi.org/10.1530/REP-11-0483.
[77] Buratini J, Pinto MGL, Castilho AC, Amorim RL, Giometti IC, Portela VM, et al.
Expression and function of fibroblast growth factor 10 and its receptor,
fibroblast growth factor receptor 2B, in bovine follicles. Biol Reprod 2007;77:
743e50. https://doi.org/10.1095/biolreprod.107.062273.
[78] Chung D, Gao F, Jegga AG, Das SK. Estrogen mediated epithelial proliferation
in the uterus is directed by stromal Fgf10 and Bmp8a. Mol Cell Endocrinol
2015;400:48e60. https://doi.org/10.1016/j.mce.2014.11.002.