Impaired seminiferous tubule development in neonatal boars after 

in utero heat stress
S.S. Faltas1, C. Nguyen1, R. Metcalf1, C. Bernhard2, D. Lagundžin3, K. Sharma3, K. Sharp2, T. Safranski2, M. Lucy2, A.T.
Desaulniers1
1
School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE. 2Division of Animal Sciences, University of Missouri,
Columbia, MO. 3Mass Spectrometry and Proteomics Core Facility, University of Nebraska Medical Center, Omaha, NE. 
Introduction Summary
Boar fertility is threatened by climate change. It is very costly to effectively cool 6 million • IUHS boars exhibit fewer pre-spermatogonia than their thermoneutral counterparts
pregnant sows that gestate during the summer in the United States. This leads to in utero heat • IUHS boars exhibit smaller seminiferous tubules than their thermoneutral
stress (IUHS) of 66 million offspring annually. The boar is a major driver of swine counterparts
reproductive success and genetic progress because a typical boar will produce over 8,000 • IUHS reduces boar semen quality, but the underlying mechanism behind this
progeny/year, while a sow will produce about 23 progeny/year. Previous research indicates impairment remains unknown
• This data provides evidence that proteins important for seminiferous tubule function
that IUHS reduces boar semen quality. Therefore, the negative effects of IUHS on boar
are impaired by IUHS
fertility is a large detriment to the swine industry. The overall objective of this project is to • Determining the mechanism of testicular impairment could lead to answers on how to
determine how IUHS affects seminiferous tubule development and the expression of proteins mitigate IUHS
important for Sertoli and germ cell function. Understanding how IUHS affects testis biology
will enable us to develop effective intervention strategies. Conclusion
Data analysis shows that IUHS inhibits seminiferous tubule area, which is positively correlated with
Acknowledgements Sertoli and germ cell number. The number of Sertoli and germ cells determine lifetime sperm
• University of Nebraska-Lincoln Undergraduate Creative Activities and Research production capacity. Thus, these data suggest that IUHS impairs adult sperm production via
disrupting proliferation of Sertoli and germ cells during fetal development. Our results also show that
Experience (UCARE) important testicular proteins are also affected by IUHS. IUHS-induced reproductive failure could
• UNL Institutional Seed Funds  Fig. 1. Representative histological images of testicular tissue from IUTN control (n = 8) and IUHS contribute to global food shortages, considering pork is the most consumed meat worldwide.
• USDA/ARD Hatch Multistate Enhanced Program  experimental (n = 8) boars at 400x magnification. Determining the mechanism of impairment in neonatal boars during gestation will allow for the
• USDA-NIFA  development of strategies to combat a looming food shortage crisis. In addition, pigs are also
• Mizzou Swine Farm  excellent biomedical models, so these results have implications for human fertility as well.

Methods and Results

*
Animal Trial
• Utilized swine model (white crossbred)
15

• Gilts synchronized, bred, and assigned to thermal treatment (n = 18/treatment)

• Fetal boars exposed to IUHS or IUTN (in utero thermal neutral) conditions:
• IUHS: 28 – 38 °C from d 27 – 57 of gestation
10

-Log10 P-value
• n = 54
• IUTN: 17 – 22 °C from d 27 – 57 of gestation
• n = 45
5

•Sows farrowed and reared progeny in thermal neutral conditions

•Offspring were castrated at post-natal day 3
0

Figure 1. Effect of IUHS on the development of seminiferous tubules (STs) within the testis of -6 -4 -2 0
Log2 Ratio
2 4 6

Figure 4. The testicular proteome is significantly affected by IUHS. Volcano plot

Histological Analysis
Nonsignificant and does not meet FC threshold Nonsignificant and greater than upper FC threshold

neonatal boars (3 days of age). IUHS boars (n = 54) had a reduction in average area of
Nonsignificant and less than lower FC threshold Significant and does not meet FC threshold
Significant and greater than upper FC threshold Significant and less than lower FC threshold

•
demonstrating 519 proteins that are upregulated (red) or downregulated (green) in testes
seminiferous tubules compared to IUTN controls (n = 45).*P < 0.05 Testes were fixed in 10% buffered formalin
• Tissue was paraffin embedded, sectioned, and stained with hematoxylin and eosin
from IUHS (n=8) compared with IUTN (n=8) boars.
• The following metrics were quantified via histological analysis using an Olympus Table 1. Proteins important for Sertoli and/or germ cell function were differentially
microscope/camera and CellSens software:
expressed in the testes of neonatal IUHS (n=8) compared with IUTN (n=8) boars.
• Average seminiferous tubule area: area of each seminiferous tubule recorded from
two 400x images/animal and divided by the number of tubules per image Fold Change in
• Pre-spermatonia were also quantified at the University of Missouri-Columbia Protein​ IUHS Compared  
P-value​ Associated Function ​
to IUTN​

E3 ubiquitin-protein ligase HUWE1 Ubiquitination

Proteomics
• Protein was extracted from fixed testicular tissue on a subset of pigs (n = 8/treatment) Immune
galectin 1
• Proteomics was performed at the University of Nebraska Medical Center Proteomics Core
Response
Facility (Omaha, NE) adiponectin Spermatogenesis
Figure 2. Effect of IUHS on the number of pre-spermatogonia within the testis of neonatal
boars (3 days of age). IUHS boars (n = 126) had a reduction in the number of pre- • Protein identification was performed by searching MS/MS data against the NCBI Germ Cell
database (selected for Sus scrofa, 2019_01, 63657 entries) in Proteome Discoverer
calpain 2
spermatogonia compared to IUTN controls (n = 127).*P < 0.05 Apoptosis
IUTN (n = 8) IUHS (n = 8)
• The relative abundance between groups was defined as significantly upregulated or Sperm
downregulated based on a fold change ≥ 2 and P-value < 0.05 casein kinase II subunit alpha
Morphology
• ShinyGO v0.76, a web-based resource (http://bioinformatics.sdstate.edu/go/) was used to Inositol monophosphatase 1 Unknown
conduct Gene Ontology analysis
• Database enrichment analysis was performed using the Uniport database heterogeneous nuclear ribonucleoprotein A1 Spermatogenesis

heterogeneous nuclear ribonucleoprotein D0 Spermatogenesis

histone H2A type 2-B Postfertilization

Figure 3. The testicular proteome is significantly affected by IUHS. Heat map demonstrating 519 Inhibits Estradiol
proteins that are upregulated (red) or downregulated (green) in testes from IUHS (n=8) compared mitogen-activated protein kinase 3
Production
with IUTN (n=8) boars.
Testis