The ratio of VEGF angiogenic to anti-angiogenic isoforms appears to determine different biological functions in reproduction.  Reduced amounts of angiogenic VEGF isoforms inhibit testis sex-specific vasculature and normal cord formation in organ cultures while reduction of inhibitory isoforms increased vasculature and perturbed cords.  In the female, using peri-natal ovarian cultures, inhibition of angiogenic VEGFisoforms reduced vascular development and inhibited follicle progression while conversely reductions in inhibitory isoforms or increases in angiogenic isoforms enhanced follicle development. Thus, regulation of the Vegfa gene to produce angiogenic or anti-angiogenic isoforms may be a mechanism to alter sex-specific vascular development, formation of seminiferous cords, and/or follicle progression within mammalian gonads

Bott, Rebecca C

Clopton, Debra T

Cupp, Andrea S.

Pohlmann, Renee M

Ten Broeck, Robin A

English

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Faculty Papers and Publications in Animal 
Science Animal Science Department 
2008 
Vascular Endothelial Growth Factor (VEGF) Isoforms may 
Regulate Sex-Specific Vascular Development, Cord Formation and 
Follicle Progression in Developing Gonads 
Andrea S. Cupp 
University of Nebraska-Lincoln, acupp2@unl.edu 
Rebecca C. Bott 
South Dakota State University, Rebecca.Bott@sdstate.edu 
Renee M. Pohlmann 
University of Nebraska-Lincoln, rmcfee3@unl.edu 
Robin A. Ten Broeck 
University of Nebraska-Lincoln, robs9227@yahoo.com 
Debra T. Clopton 
University of Nebraska-Lincoln, dclopton@unlnotes.unl.edu 
Follow this and additional works at: https://digitalcommons.unl.edu/animalscifacpub 
 Part of the Animal Sciences Commons 
Cupp, Andrea S.; Bott, Rebecca C.; Pohlmann, Renee M.; Ten Broeck, Robin A.; and Clopton, Debra T., 
"Vascular Endothelial Growth Factor (VEGF) Isoforms may Regulate Sex-Specific Vascular Development, 
Cord Formation and Follicle Progression in Developing Gonads" (2008). Faculty Papers and Publications 
in Animal Science. 595. 
https://digitalcommons.unl.edu/animalscifacpub/595 
This Article is brought to you for free and open access by the Animal Science Department at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Papers and 
Publications in Animal Science by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
1 
 
VASCULAR ENDOTHELIAL GROWTH 1 
FACTOR (VEGF) ISOFORMS MAY REGULATE 2 
SEX-SPECIFIC VASCULAR DEVELOPMENT, 3 
CORD FORMATION AND FOLLICLE 4 
PROGRESSION IN DEVELOPING GONADS 5 
AS Cupp, RC Bott, RM Pohlmann, RA Ten Broeck and DT Clopton 6 
Department of Animal Science 7 
University of Nebraska-Lincoln, Lincoln, NE, USA 8 
Summary 9 
  The ratio of VEGF angiogenic to anti-angiogenic isoforms appears 10 
to determine different biological functions in reproduction.  Reduced 11 
amounts of angiogenic VEGF isoforms inhibit testis sex-specific 12 
vasculature and normal cord formation in organ cultures while reduction 13 
of inhibitory isoforms increased vasculature and perturbed cords.  In the 14 
female, using peri-natal ovarian cultures, inhibition of angiogenic 15 
VEGFisoforms reduced vascular development and inhibited follicle 16 
progression while conversely reductions in inhibitory isoforms or 17 
increases in angiogenic isoforms enhanced follicle development. Thus, 18 
regulation of the Vegfa gene to produce angiogenic or anti-angiogenic 19 
isoforms may be a mechanism to alter sex-specific vascular development, 20 
formation of seminiferous cords, and/or follicle progression within 21 
mammalian gonads.   22 
Introduction 23 
Infertility affects 40-70 million couples; of those approximately 24 
50% of the infertility problems are attributed to male-related factors which 25 
include: low sperm count, abnormal spermatogenesis, and reduced 26 
androgen production [1, 2].  Formation of testicular cords, and sex-27 
specific vasculature, are the two morphological hallmarks that distinguish 28 
a testis from an ovary. Female-related infertility factors include: ovulatory 29 
disorders, anovulation due to polycystic ovarian disease and ovarian 30 
hyperstimulation syndrome, or premature ovarian failure. Some or all of 31 
these female infertility problems may be caused by improper prenatal 32 
development of the fetal gonad, reduction of number of primordial 33 
follicles or disruption of progression of folliculogenesis. 34 
Neovascularization of the ovary and continued formation of follicle 35 
vasculature are critical events in normal reproductive function.  36 
The VEGF family is composed of five ligands:  VEGF (VEGF-A), 37 
VEGF-B, VEGF-C, VEGF-D and Placenta Growth Factor.  VEGF 38 
(VEGF-A) is the best characterized and most potent VEGF molecule. 39 
VEGF works through both Fms-like tyrosine kinase 1 (FLT1) and Kinase 40 
2 
 
domain region receptor (KDR), to elicit its effects on endothelial cell 41 
migration, differentiation, proliferation and survival and apoptosis. VEGF 42 
is transcribed from a single gene that has 8 exons and is alternatively 43 
spliced into different isoforms each containing a different number of 44 
amino acids. The most common angiogenic isoforms are VEGF205, 188, 45 
164, 144, and 120 [3]. 46 
 In 2002, an additional isoform, VEGF164b, was identified which 47 
contained part of the 3’ UTR that is now determined to be exon 8b. 48 
Furthermore, recent studies have demonstrated that the human VEGF165b 49 
isoform is anti-angiogenic in function and inhibits signal transduction 50 
through KDR [4, 5].  Thus, this isoform is inhibitory to the actions of 51 
VEGF.  Therefore, it appears that for every angiogenic isoform there is a 52 
sister inhibitory isoform that is formed when exon 8a is replaced with 53 
exon 8b.  These inhibitory (anti-angiogenic) isoforms serves to modulate 54 
the functions of the angiogenic VEGF isoforms.  55 
Materials and Methods  56 
Rat Testis Organ cultures: E13 testes with attached mesonephros 57 
were placed on Millicell CM filters (Millipore, Bedford MA) in drops of 58 
medium floating on the surface of 0.4 ml of CMRL 1066 media (Gibco 59 
BRL, Gaithersburg, MD) at conditions reported [6, 7].  One organ from 60 
each animal was designated as a vehicle control, while its pair was 61 
subjected to a VEGF receptor signal transduction inhibitor, VEGFR-TKI 62 
(8 µM), or a VEGF antagonist, Je-11 (10 µg/ml) [8]. Whole-mount IHC 63 
and Confocal Microscopy: After culture, the organs were fixed in 4% 64 
paraformaldehyde.  Samples were washed, blocked and whole-mount IHC 65 
was conduted as reported [8]. Vascular Density Quantification was 66 
conducted as reported using the staining index in Scion Image [8]. 67 
Ovarian Organ Cultures: Ovaries were dissected from postnatal day 3 and 68 
4 (P3/4) rats (day of birth was considered to be P0).  One ovary from each 69 
animal was designated as a control, while its pair was subjected to 70 
Treatment with 8 µM VEGFR-TKI; Calbiochem, La Jolla, CA or KDR 71 
signal transduction inhibitor, V1, (30 µM; Calbiochem, La Jolla, CA), 72 
VEGFA164 (R & D Systems Inc., Minneapolis, MN) or VEGF165b 73 
antibody (5ng/ml or 50ng/ml) (Abcam, Cambridge, MA).  All of these 74 
treatments were added daily to the culture medium of the treated wells. 75 
Results 76 
Treatment of testis organ cultures with tyrosine kinase inhibitors to 77 
the VEGF receptor signal transduction pathway (VEGFR-TKI) or to 78 
VEGF antagonists (Je11) disrupted both sex-specific vascular 79 
development and seminiferous cord formation.  Vascular density was 80 
reduced by 90 and 46%, respectively (P < 0.01).  Conversely, treatment 81 
3 
 
with VEGF angiogenic isoforms: VEGF164, VEGF120 or an antibody to 82 
the exon 8b which binds inhibitory isoforms increased vascular density 83 
50-100% over controls and resulted in swollen and perturbed testis cord 84 
formation.  Thus angiogenic VEGF isoforms are important in establishing 85 
the sex-specific vascular development and too much inhibitory isoforms 86 
may alter the ability for this vasculature and subsequent testis cord 87 
formation to occur. 88 
In the female, treatment of perinatal rat ovaries signal transduction 89 
inhibitors (VEGFR-TKI), antagonists to KDR (V1) arrested follicle 90 
development to later secondary follicle stages (P < 0.05).  In contrast, 91 
treatment with angiogenic VEGF isoforms or an antibody to inhibitory 92 
isoforms increased vascular development and accelerated follicle 93 
progression to later secondary follicle stages (P < 0.05).  Thus, we propose 94 
that amount of angiogenic to inhibitory VEGF isoforms modulates follicle 95 
progression and may determine whether an ovarian follicles continues to 96 
progress or undergoes atresia. 97 
Conclusion 98 
 Approximately two million couples seek treatment for infertility 99 
every year and less than half find successful treatments [9, 10].  Infertility 100 
problems in at least half of these couples are a result of male-related 101 
factors that are created by testicular dysgenesis. Many of the problems 102 
associated with testicular dysgenesis are proposed to involve a disruption 103 
in embryonic differentiation of cells within the indifferent gonad resulting 104 
in altered testicular development.  Elucidating the factors involved in sex-105 
specific vascular development will allow for a better understanding of how 106 
transcription factors coordinate regulation of growth factors to result in a 107 
testis-specific vascular system.  Furthermore, delineating the interaction of 108 
VEGF angiogenic and inhibitory isoforms in ovarian follicle arrest and or 109 
progression may also  be an interesting piece in the puzzle of disorders 110 
that result in female infertility. 111 
 112 
References 113 
1. Skakkebaek, N.E., Testicular dysgenesis syndrome: new 114 
epidemiological evidence. Int J Androl, 2004. 27(4): p. 189-91. 115 
2. Skakkebaek, N.E., et al., Association between testicular dysgenesis 116 
syndrome (TDS) and testicular neoplasia: evidence from 20 adult 117 
patients with signs of maldevelopment of the testis. Apmis, 2003. 118 
111(1): p. 1-9; discussion 9-11. 119 
3. Veikkola, T. and K. Alitalo, VEGF's receptors and angiogenesis. 120 
Semin Cancer Biology, 1999. 9: p. 211-220. 121 
4 
 
4. Bates, D., et al., VEGF165b, an Inhibitory Splic Variant of Vascular 122 
Endothelial Growth Factor, Is Down-Regulated in Renal Cell 123 
Carcinoma. Cancer Research, 2002. 62: p. 4123-4131. 124 
5. Woolard, J., et al., VEGF165b, an Inhibitory Vascular Endothelial 125 
Growth Factor Splice Variant: Mechanism of Action, In vivo Effect 126 
on Angiogenesis and Endogenous Protein Expression. Cancer 127 
Research, 2004. 64: p. 7822-7835. 128 
6. Cupp, A., G. Kim, and M. Skinner, Expression and Action of 129 
Transforming Growth Factor Beta (TGFβ1, TGFβ2, and TGFβ3) 130 
during Embyonic Rat Testis Development. Biology of 131 
Reproduction, 1999. 60: p. 1304-1313. 132 
7. Cupp, A.S., et al., Effect of transient embryonic in vivo exposure to 133 
the endocrine disruptor methoxychlor on embryonic and postnatal 134 
testis development. J Androl, 2003. 24(5): p. 736-45. 135 
8. Bott, R.C., et al., Vascular endothelial growth factor and kinase 136 
domain region receptor are involved in both seminiferous cord 137 
formation and vascular development during testis morphogenesis 138 
in the rat. Biol Reprod, 2006. 75(1): p. 56-67. 139 
9. Carlsen, E., et al., Longitudinal changes in semen parameters in 140 
young Danish men from the Copenhagen area. Hum Reprod, 2005. 141 
20(4): p. 942-9. 142 
10. Shaban, S., Male Infertility Overview. 143 
http://www.ivf.com/shaban.html.,  2000. 144 
 145 
 146 
 147 
Corresponding address:  Andrea S Cupp, A224i Animal Science 148 
Department, 38th and Fair Street, University of Nebraska-Lincoln, 149 
Lincoln, NE 68583-0908.  acupp2@unl.edu 150 
Portions of this research were funded by grants to ASC from 151 
NIH/NICHD:  HD41546-01; HD045350-01. 152 


Vascular Endothelial Growth Factor (VEGF) Isoforms may Regulate Sex-Specific Vascular Development, Cord Formation and Follicle Progression in Developing Gonads

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Papers and Publications in Animal Science Animal Science Department 2008 Vascular Endothelial Growth Factor (VEGF) Isoforms may Regulate Sex-Specific Vascular Development, Cord Formation and Follicle Progression in Developing Gonads Andrea S. Cupp University of Nebraska-Lincoln, acupp2@unl.edu Rebecca C. Bott South Dakota State University, Rebecca.Bott@sdstate.edu Renee M. Pohlmann University of Nebraska-Lincoln, rmcfee3@unl.edu Robin A. Ten Broeck University of Nebraska-Lincoln, robs9227@yahoo.com Debra T. Clopton University of Nebraska-Lincoln, dclopton@unlnotes.unl.edu Follow this and additional works at: https://digitalcommons.unl.edu/animalscifacpub  Part of the Animal Sciences Commons Cupp, Andrea S.; Bott, Rebecca C.; Pohlmann, Renee M.; Ten Broeck, Robin A.; and Clopton, Debra T., "Vascular Endothelial Growth Factor (VEGF) Isoforms may Regulate Sex-Specific Vascular Development, Cord Formation and Follicle Progression in Developing Gonads" (2008). Faculty Papers and Publications in Animal Science. 595. https://digitalcommons.unl.edu/animalscifacpub/595 This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Papers and Publications in Animal Science by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 1  VASCULAR ENDOTHELIAL GROWTH 1 FACTOR (VEGF) ISOFORMS MAY REGULATE 2 SEX-SPECIFIC VASCULAR DEVELOPMENT, 3 CORD FORMATION AND FOLLICLE 4 PROGRESSION IN DEVELOPING GONADS 5 AS Cupp, RC Bott, RM Pohlmann, RA Ten Broeck and DT Clopton 6 Department of Animal Science 7 University of Nebraska-Lincoln, Lincoln, NE, USA 8 Summary 9   The ratio of VEGF angiogenic to anti-angiogenic isoforms appears 10 to determine different biological functions in reproduction.  Reduced 11 amounts of angiogenic VEGF isoforms inhibit testis sex-specific 12 vasculature and normal cord formation in organ cultures while reduction 13 of inhibitory isoforms increased vasculature and perturbed cords.  In the 14 female, using peri-natal ovarian cultures, inhibition of angiogenic 15 VEGFisoforms reduced vascular development and inhibited follicle 16 progression while conversely reductions in inhibitory isoforms or 17 increases in angiogenic isoforms enhanced follicle development. Thus, 18 regulation of the Vegfa gene to produce angiogenic or anti-angiogenic 19 isoforms may be a mechanism to alter sex-specific vascular development, 20 formation of seminiferous cords, and/or follicle progression within 21 mammalian gonads.   22 Introduction 23 Infertility affects 40-70 million couples; of those approximately 24 50% of the infertility problems are attributed to male-related factors which 25 include: low sperm count, abnormal spermatogenesis, and reduced 26 androgen production [1, 2].  Formation of testicular cords, and sex-27 specific vasculature, are the two morphological hallmarks that distinguish 28 a testis from an ovary. Female-related infertility factors include: ovulatory 29 disorders, anovulation due to polycystic ovarian disease and ovarian 30 hyperstimulation syndrome, or premature ovarian failure. Some or all of 31 these female infertility problems may be caused by improper prenatal 32 development of the fetal gonad, reduction of number of primordial 33 follicles or disruption of progression of folliculogenesis. 34 Neovascularization of the ovary and continued formation of follicle 35 vasculature are critical events in normal reproductive function.  36 The VEGF family is composed of five ligands:  VEGF (VEGF-A), 37 VEGF-B, VEGF-C, VEGF-D and Placenta Growth Factor.  VEGF 38 (VEGF-A) is the best characterized and most potent VEGF molecule. 39 VEGF works through both Fms-like tyrosine kinase 1 (FLT1) and Kinase 40 2  domain region receptor (KDR), to elicit its effects on endothelial cell 41 migration, differentiation, proliferation and survival and apoptosis. VEGF 42 is transcribed from a single gene that has 8 exons and is alternatively 43 spliced into different isoforms each containing a different number of 44 amino acids. The most common angiogenic isoforms are VEGF205, 188, 45 164, 144, and 120 [3]. 46  In 2002, an additional isoform, VEGF164b, was identified which 47 contained part of the 3’ UTR that is now determined to be exon 8b. 48 Furthermore, recent studies have demonstrated that the human VEGF165b 49 isoform is anti-angiogenic in function and inhibits signal transduction 50 through KDR [4, 5].  Thus, this isoform is inhibitory to the actions of 51 VEGF.  Therefore, it appears that for every angiogenic isoform there is a 52 sister inhibitory isoform that is formed when exon 8a is replaced with 53 exon 8b.  These inhibitory (anti-angiogenic) isoforms serves to modulate 54 the functions of the angiogenic VEGF isoforms.  55 Materials and Methods  56 Rat Testis Organ cultures: E13 testes with attached mesonephros 57 were placed on Millicell CM filters (Millipore, Bedford MA) in drops of 58 medium floating on the surface of 0.4 ml of CMRL 1066 media (Gibco 59 BRL, Gaithersburg, MD) at conditions reported [6, 7].  One organ from 60 each animal was designated as a vehicle control, while its pair was 61 subjected to a VEGF receptor signal transduction inhibitor, VEGFR-TKI 62 (8 µM), or a VEGF antagonist, Je-11 (10 µg/ml) [8]. Whole-mount IHC 63 and Confocal Microscopy: After culture, the organs were fixed in 4% 64 paraformaldehyde.  Samples were washed, blocked and whole-mount IHC 65 was conduted as reported [8]. Vascular Density Quantification was 66 conducted as reported using the staining index in Scion Image [8]. 67 Ovarian Organ Cultures: Ovaries were dissected from postnatal day 3 and 68 4 (P3/4) rats (day of birth was considered to be P0).  One ovary from each 69 animal was designated as a control, while its pair was subjected to 70 Treatment with 8 µM VEGFR-TKI; Calbiochem, La Jolla, CA or KDR 71 signal transduction inhibitor, V1, (30 µM; Calbiochem, La Jolla, CA), 72 VEGFA164 (R & D Systems Inc., Minneapolis, MN) or VEGF165b 73 antibody (5ng/ml or 50ng/ml) (Abcam, Cambridge, MA).  All of these 74 treatments were added daily to the culture medium of the treated wells. 75 Results 76 Treatment of testis organ cultures with tyrosine kinase inhibitors to 77 the VEGF receptor signal transduction pathway (VEGFR-TKI) or to 78 VEGF antagonists (Je11) disrupted both sex-specific vascular 79 development and seminiferous cord formation.  Vascular density was 80 reduced by 90 and 46%, respectively (P < 0.01).  Conversely, treatment 81 3  with VEGF angiogenic isoforms: VEGF164, VEGF120 or an antibody to 82 the exon 8b which binds inhibitory isoforms increased vascular density 83 50-100% over controls and resulted in swollen and perturbed testis cord 84 formation.  Thus angiogenic VEGF isoforms are important in establishing 85 the sex-specific vascular development and too much inhibitory isoforms 86 may alter the ability for this vasculature and subsequent testis cord 87 formation to occur. 88 In the female, treatment of perinatal rat ovaries signal transduction 89 inhibitors (VEGFR-TKI), antagonists to KDR (V1) arrested follicle 90 development to later secondary follicle stages (P < 0.05).  In contrast, 91 treatment with angiogenic VEGF isoforms or an antibody to inhibitory 92 isoforms increased vascular development and accelerated follicle 93 progression to later secondary follicle stages (P < 0.05).  Thus, we propose 94 that amount of angiogenic to inhibitory VEGF isoforms modulates follicle 95 progression and may determine whether an ovarian follicles continues to 96 progress or undergoes atresia. 97 Conclusion 98  Approximately two million couples seek treatment for infertility 99 every year and less than half find successful treatments [9, 10].  Infertility 100 problems in at least half of these couples are a result of male-related 101 factors that are created by testicular dysgenesis. Many of the problems 102 associated with testicular dysgenesis are proposed to involve a disruption 103 in embryonic differentiation of cells within the indifferent gonad resulting 104 in altered testicular development.  Elucidating the factors involved in sex-105 specific vascular development will allow for a better understanding of how 106 transcription factors coordinate regulation of growth factors to result in a 107 testis-specific vascular system.  Furthermore, delineating the interaction of 108 VEGF angiogenic and inhibitory isoforms in ovarian follicle arrest and or 109 progression may also  be an interesting piece in the puzzle of disorders 110 that result in female infertility. 111  112 References 113 1. Skakkebaek, N.E., Testicular dysgenesis syndrome: new 114 epidemiological evidence. Int J Androl, 2004. 27(4): p. 189-91. 115 2. Skakkebaek, N.E., et al., Association between testicular dysgenesis 116 syndrome (TDS) and testicular neoplasia: evidence from 20 adult 117 patients with signs of maldevelopment of the testis. Apmis, 2003. 118 111(1): p. 1-9; discussion 9-11. 119 3. Veikkola, T. and K. Alitalo, VEGF's receptors and angiogenesis. 120 Semin Cancer Biology, 1999. 9: p. 211-220. 121 4  4. Bates, D., et al., VEGF165b, an Inhibitory Splic Variant of Vascular 122 Endothelial Growth Factor, Is Down-Regulated in Renal Cell 123 Carcinoma. Cancer Research, 2002. 62: p. 4123-4131. 124 5. Woolard, J., et al., VEGF165b, an Inhibitory Vascular Endothelial 125 Growth Factor Splice Variant: Mechanism of Action, In vivo Effect 126 on Angiogenesis and Endogenous Protein Expression. Cancer 127 Research, 2004. 64: p. 7822-7835. 128 6. Cupp, A., G. Kim, and M. Skinner, Expression and Action of 129 Transforming Growth Factor Beta (TGFβ1, TGFβ2, and TGFβ3) 130 during Embyonic Rat Testis Development. Biology of 131 Reproduction, 1999. 60: p. 1304-1313. 132 7. Cupp, A.S., et al., Effect of transient embryonic in vivo exposure to 133 the endocrine disruptor methoxychlor on embryonic and postnatal 134 testis development. J Androl, 2003. 24(5): p. 736-45. 135 8. Bott, R.C., et al., Vascular endothelial growth factor and kinase 136 domain region receptor are involved in both seminiferous cord 137 formation and vascular development during testis morphogenesis 138 in the rat. Biol Reprod, 2006. 75(1): p. 56-67. 139 9. Carlsen, E., et al., Longitudinal changes in semen parameters in 140 young Danish men from the Copenhagen area. Hum Reprod, 2005. 141 20(4): p. 942-9. 142 10. Shaban, S., Male Infertility Overview. 143 http://www.ivf.com/shaban.html.,  2000. 144  145  146  147 Corresponding address:  Andrea S Cupp, A224i Animal Science 148 Department, 38th and Fair Street, University of Nebraska-Lincoln, 149 Lincoln, NE 68583-0908.  acupp2@unl.edu 150 Portions of this research were funded by grants to ASC from 151 NIH/NICHD:  HD41546-01; HD045350-01. 152 

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Vascular Endothelial Growth Factor (VEGF) Isoforms may Regulate Sex-Specific Vascular Development, Cord Formation and Follicle Progression in Developing Gonads

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