The goal of this project is to decipher the cellular signals that instigate leukocyte infiltration into the testis after exposure to mono-(2-ethylhexyl) phthalate (MEHP) and to determine the functional significance of these cells in the pathogenesis of MEHP-induced germ cell apoptosis. We have previously revealed a paracrine interaction between Sertoli cells (SCs) and germ cells (GCs) that initiates GCs to undergo apoptosis via FasL- Fas signaling. Central to this pathway is the inhibition of TIMP2 (tissue inhibitor of metalloproteinase 2) production by SCs after MEHP exposure. This allows for the consequent activation of matrix metalloproteinase 2 (MMP2) in the adluminal space that both generates a soluble form of tumor necrosis factor-? (sTNF?) as well as cause a disorganization of proteins that compose the blood-testis barrier (BTB) between adjacent SCs. A preliminary characterization of Fischer rat testis after MEHP (1 g/kg, p.o.) treatment at two peripubertal ages (PND 28 and 35) showed a robust infiltration of CD11b+ immunoreactive cells in the interstitial space prior to the peak induction of GC apoptosis. The CD11b antibody recognizes an antigen expressed on macrophages, neutrophils, monocytes, natural killer and dendritic cells. Intriguingly, we observed that the most pronounced infiltration of these cells occurs in peripubertal aged rats versus mature rats; and that C57BL/6J mice at both ages do not have a significant infiltration in response to MEHP exposure. The differential species and age- dependent sensitivity to MEHP-induced testicular injury is well recognized, although the mechanisms that account for these differences remain unresolved. Further, preliminary studies also show a dramatic increase in the expression of monocyte chemoattractant protein-1 (MCP-1) in peritubular myoid cells (PTMCs) in testis from PND 28 rats, but not in mature rats. MCP-1 is a prototypical chemokine for signaling the influx of leukocytes into tissues and is itself secreted in response to cytokines such as sTNF?, IL-1?, Il-1? and/or interferon-?. Taken together, our previous work and preliminary data, have led to the development of the hypothesis that MEHP-induced SC injury incites the production of cytokines, such as sTNF?, that trigger PTMCs to release chemokines that initiate the infiltration of leukocytes into the testis and act to enhance the extent of GC apoptosis. Moreover, a provocative underlying hypothesis of this proposal is that leukocytes account, in part, for the mechanism of the observed age- and species-dependent sensitivity to MEHP. To test these hypotheses, the first specific aim will use both acute and repeated MEHP treatments to characterize the leukocyte subtypes and timing of their influx into the testis following exposure. In the second aim, the chemokines and cytokines elicited, as well as their specific cellular source in the testis, will be examined. In the last ai, the functional significance of testis leukocyte infiltration in the pathogenesis of MEHP-induced injury will be directly tested. It is predicted that insights gained from this work will be useful or predicting susceptible individuals and preventing human reproductive health risks this class of chemicals.

Public Health Relevance

The focus of this research proposal is to distinguish the contribution of the innate immune system (macrophages, monocytes, neutrophils, natural killer and dendritic cells) in the loss of germ cells from the postnatal male testis after exposure to phthalates; a class of compounds used in consumer products and found ubiquitously in the environment. This work is relevant to human health as we hypothesize that immune cell infiltration into the testis underlies the susceptibility of individuals to phthalate-induced testiular toxicity. It is anticipated that the mechanistic insights provided from this research will be usefu for predicting and preventing human reproductive health risks to this class of chemicals found broadly dispersed in the environment.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES016591-10
Application #
9605031
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Schug, Thaddeus
Project Start
2008-04-01
Project End
2019-11-30
Budget Start
2018-12-01
Budget End
2019-11-30
Support Year
10
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Texas Austin
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
170230239
City
Austin
State
TX
Country
United States
Zip Code
78759
Bao, Jianqiang; Perez, Carlos J; Kim, Jeesun et al. (2018) Deficient LRRC8A-dependent volume-regulated anion channel activity is associated with male infertility in mice. JCI Insight 3:
Voss, Jorine J L P; Stermer, Angela R; Ghaffari, Rashin et al. (2018) MEHP-induced rat testicular inflammation does not exacerbate germ cell apoptosis. Reproduction 156:35-46
Stermer, Angela R; Murphy, Caitlin J; Ghaffari, Rashin et al. (2017) Mono-(2-ethylhexyl) phthalate-induced Sertoli cell injury stimulates the production of pro-inflammatory cytokines in Fischer 344 rats. Reprod Toxicol 69:150-158
Stermer, Angela R; Myers, Jessica L; Murphy, Caitlin J et al. (2016) Female mice with loss-of-function ITCH display an altered reproductive phenotype. Exp Biol Med (Maywood) 241:367-74
Murphy, Caitlin J; Richburg, John H (2014) Implications of Sertoli cell induced germ cell apoptosis to testicular pathology. Spermatogenesis 4:e979110
Richburg, John H; Myers, Jessica L; Bratton, Shawn B (2014) The role of E3 ligases in the ubiquitin-dependent regulation of spermatogenesis. Semin Cell Dev Biol 30:27-35
Harman, James G; Richburg, John H (2014) Cisplatin-induced alterations in the functional spermatogonial stem cell pool and niche in C57/BL/6J mice following a clinically relevant multi-cycle exposure. Toxicol Lett 227:99-112
Murphy, Caitlin J; Stermer, Angela R; Richburg, John H (2014) Age- and species-dependent infiltration of macrophages into the testis of rats and mice exposed to mono-(2-Ethylhexyl) phthalate (MEHP). Biol Reprod 91:18
Lin, Yi-Chen; Richburg, John H (2014) Characterization of the role of tumor necrosis factor apoptosis inducing ligand (TRAIL) in spermatogenesis through the evaluation of trail gene-deficient mice. PLoS One 9:e93926
Dwyer, Jessica L; Richburg, John H (2012) Age-dependent alterations in spermatogenesis in itchy mice. Spermatogenesis 2:104-116

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