Changes in testicular gene expression following reduced estradiol synthesis: A complex pathway to increased porcine Sertoli cell proliferation

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Highlights

  • Increase in Sertoli cells after reduced estradiol from longer proliferation interval.

  • Prolonged reduction in early juvenile estradiol required to increase Sertoli cells.

  • Increase in Sertoli cell numbers first detectable at 6.5 weeks of age.

  • Few and varying genes show dramatic up or down regulation at 2, 3, or 5 weeks of age.

  • Gene expression indicates stimulated pathways differ at 2, 3, and 5 weeks of age.

Abstract

Porcine Sertoli cell number including number present at puberty is increased if testicular estradiol synthesis is reduced during the neonatal interval. Evaluating the changes in gene expression during the crucial interval of suppressed estradiol that leads to the increased Sertoli cell population will increase our understanding of Sertoli cell biology but this evaluation first required a more precise determination of the critical interval for treatment and timing of a detectable response. Previously, reduced testicular estrogens from 1 week of age were accompanied by increased Sertoli cell number at 6.5 weeks of age but the age at which Sertoli cell numbers were initially increased was unknown, one of the current objectives. Additional experiments were designed to further delineate the essential timing of treatment for the Sertoli cell response. Finally, changes in gene expression induced by the reduced estradiol synthesis were evaluated to elucidate molecular mechanisms. Experimental design typically consisted of one member of littermate pairs of boars treated with the aromatase inhibitor, letrozole, beginning at 1 week of age and the remaining member treated with canola oil vehicle. Weekly treatments continued through 5 weeks of age or tissue collection, whichever came first. Increases in Sertoli cell numbers were not detectable prior to 6.5 weeks of age and persistent treatment through 5 weeks of age was required to induce the increase in Sertoli cell numbers. This increase resulted from prolonging the first interval of Sertoli cell proliferation in the treated animals. Few genes exhibited dramatically altered transcription and similarities in pathway analysis or principal modified genes were quite limited in 2, 3, and 5-week-old boars. The critical timing and prolonged treatment required and the sequential changes in gene expression suggest a complex mechanism is involved in this model of increased proliferation of Sertoli cells.

Introduction

Sertoli cells play key roles in the differentiation of the testis, from the initial organization of the developing gonad into primitive cords to the formation of the blood-testis barrier and initiation of spermatogenesis. Sertoli cells continue to have a crucial role in male reproductive function through adult life as they interact with developing germ cells to support spermatogenesis, explaining the relationship between the number of Sertoli cells and total sperm output in post-pubertal males (Berndtson and Thompson, 1990; Berndtson et al., 1987). Hence, proliferation of Sertoli cells prior to puberty has the potential to significantly influence sperm production capacity in post-pubertal life. Although Sertoli cell proliferation begins during fetal development, proliferation continues in early neonatal life and in species with an extended prepuberal interval, like the pig, during immediate pre-pubertal development. Understanding the regulation of Sertoli proliferation may uncover approaches to increase lifetime sperm production capacity or to restore sperm production capacity lost by perturbations or insults to normal testicular development before the onset of puberty.

Studies on the physiological regulation of Sertoli cell proliferation have utilized primarily rats and mice, with much less understood in other species. Clearly, fundamental differences exist among species and major determinants of Sertoli cell numbers in rodents do not translate well to livestock species, specifically the pig. Results from most studies support a significant stimulatory role for FSH in mice and rats (Migrenne et al., 2012; O'Shaughnessy et al., 2012) for instance, but evidence suggests that FSH has a lesser role in Sertoli cell proliferation in pigs (Ford et al., 1997; McCoard et al., 2003; Swanlund, N'Diaye, Loseth et al., 1995). Similarly, induced hypothyroidism stimulates Sertoli cell proliferation in rodents but does not appear to do so in the pig (Hess et al., 1993; Joyce et al., 1993; Klobucar et al., 2003; Lara et al., 2020; Palmero et al., 1989; Tarn et al., 1998). Compensatory testicular hypertrophy following removal of one testis (hemicastration) is a valuable experimental strategy to increase Sertoli cell proliferation in the remaining testis. Indeed, hemi-castration increases Sertoli cell proliferation of the remaining testis in multiple species including laboratory rodents, pigs and cattle (Kosco et al., 1987; Mirando et al., 1989; Putra and Blackshaw, 1985). Once again however, while this initial response may be mediated by inhibin, activin, and FSH in laboratory rodents (Brown et al., 1991; Pitetti et al., 2013), this does not appear to be the case in pigs (Clark et al., 1996; Kosco et al., 1987). Two waves of postnatal Sertoli cell proliferation exist in pigs and in primates, one initiated in early neonatal development and the second initiated pre-pubertally (Franca et al., 2000; Sharpe et al., 2003). Inhibition of testicular estrogen synthesis in neonatal pigs using letrozole, an inhibitor of the aromatase enzyme complex, conspicuously increases Sertoli cell numbers during development. Specifically, reducing endogenous estrogens during the first postnatal wave of proliferation can increase the Sertoli cell population, testicular size and sperm production after puberty by 25% (At-Taras et al., 2006). In contrast, the second, pre-pubertal wave of Sertoli cell proliferation does not appear to be estrogen-sensitive (Berger et al., 2012). This provides a novel experimental approach to elucidate regulation of proliferation of Sertoli cells in the pig and further our overall understanding of mechanisms regulating Sertoli cell numbers.

The restriction of the estrogen-sensitive wave of Sertoli cell proliferation to a brief window early in neonatal life provides an opportunity to define the molecular mechanisms that initiate or sustain this enhanced proliferation in pigs. However, adequately defining that window is crucial for such analysis. Prior work from our laboratory demonstrated that the stimulation of Sertoli cell numbers following continuous inhibition of testicular estrogen synthesis from 1 week of age was detectable at 6.5 weeks (Berger and Conley, 2014; Berger et al., 2012; Berger et al., 2013) and did not involve reduced apoptosis (Kao et al., 2012) but earlier time periods were not comprehensively evaluated in previous studies. Since more precise determination of timing will facilitate understanding of molecular mechanisms, one objective of the current studies was to determine if the initial increase in Sertoli cell numbers was detectable at an earlier time point (2,3, or 5 weeks). Precise determination of critical timing within the early window will also assist in determining critical molecular mechanisms. Treatment at 1, 3, and 5 weeks was sufficient to induce the response at 6.5 weeks of age and a single treatment reduced endogenous estrogens for at least 2 weeks in this interval but treatment at 1 or at 1 and 5 weeks was insufficient to increase Sertoli cell numbers at 6.5 weeks of age (Berger et al., 2012; Kao et al., 2012). To clarify critical time points, a second objective was to further evaluate the early treatment interval (a single treatment at 3 weeks of age and weekly treatment beginning at 1 day of age). With the critical timing for reduction in endogenous estrogens better defined, changes in gene expression were assessed by RNA sequencing (RNAseq) to increase understanding of the molecular mechanisms. Major differences in gene expression between control and treated intact boars at 5 weeks of age, just prior to the divergence of Sertoli cell numbers, were confirmed by qPCR analysis. These same genes were evaluated in an alternate model of enhanced Sertoli cell proliferation (6.5 week old, hemicastrated boars with and without reduced estrogen synthesis) since estrogen-induced divergence of Sertoli cell numbers is delayed beyond 6.5 weeks of age in the hemicastrated boars (Berger and Conley, 2014).

Section snippets

Design of animal experiments

All animal experimentation was conducted in accordance with the Guide for the Care and Use of Agricultural Animals in Research and Teaching and approved by the UC Davis Institutional Animal Care and Use Committee. Boars in the single time point sensitivity experiment were crossbred with a mixture of Yorkshire, Duroc, Hampshire and PIC genetics. All other boars were derived from breeding stock and semen obtained from PIC North America (Hendersonville, TN, USA). Animals were housed at the UC

Results

Weekly treatment with letrozole beginning at 1 week of age did not affect Sertoli cell numbers at 2, 3, or 5 weeks of age, indicating treatment induced Sertoli cell proliferation after 5 weeks of age. (Fig. 1). An increase in Sertoli cell numbers at 6.5 weeks of age was again observed in littermates receiving weekly treatment with letrozole compared with vehicle-treated littermates (Fig. 2). Treatment did not affect body weight (17.03 vs 17.17 kg, SEM = 1.01), nor was testis weight

Discussion

Reducing estrogen synthesis by inhibiting aromatase from 1 to 5 weeks prolongs the first wave of Sertoli cell proliferation, with increased numbers evident by 6.5 weeks of age. Similar numbers of Sertoli cells are present in vehicle-treated boars and in letrozole-treated littermates through 5 weeks of age. Previous data repeatedly demonstrated that Sertoli cell numbers are increased at 6.5 weeks of age following this treatment through 5 weeks of age and numbers remained increased at 20 weeks of

Acknowledgements

The research was partially supported by USDA NIFA NRICGP 2008-35203-19082. This work was also supported by MSP 3171 from USDA, a W.K. Kellogg Endowment, Henry A. Jastro Research Awards to ST and the infrastructure support of the Department of Animal Science, College of Agricultural and Environmental Sciences, and the California Agricultural Experiment Station of the University of California, Davis. Sequencing was completed at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley,

References (61)

  • V. Plaks et al.

    Adaptive immune regulation of mammary postnatal organogenesis

    Dev. Cell

    (2015)
  • C.Y. Tarn et al.

    Effects of 6-N-propyl-2-thiouracil on growth, hormonal profiles, carcass and reproductive traits of boars

    Anim. Reprod. Sci.

    (1998)
  • G.A. Traustadottir et al.

    The imprinted gene Delta like non-canonical Notch ligand 1 (Dlk1) is conserved in mammals, and serves a growth modulatory role during tissue development and regeneration through Notch dependent and independent mechanisms

    Cytokine Growth Factor Rev.

    (2019)
  • T.M. Williams et al.

    Macrophages in renal development, injury, and repair

    Semin. Nephrol.

    (2010)
  • P. Aliberti et al.

    Gonadotrophin-mediated miRNA expression in testis at onset of puberty in rhesus monkey: predictions on regulation of thyroid hormone activity and DLK1-DIO3 locus

    Mol. Hum. Reprod.

    (2019)
  • E.E. At-Taras et al.

    Reducing estrogen synthesis does not affect gonadotropin secretion in the developing boar

    Biol. Reprod.

    (2005)
  • E.E. At-Taras et al.

    Reducing estrogen synthesis in developing boars increases testis size and total sperm production

    J. Androl.

    (2006)
  • T. Berger et al.

    Reduced endogenous estrogen and hemicastration interact synergistically to increase porcine sertoli cell proliferation

    Biol. Reprod.

    (2014)
  • T. Berger et al.

    Alteration in Porcine Testicular Gene Expression in Response to Reduced Testicular Estradiol Synthesis

    (2020)
  • T. Berger et al.

    Stimulation of Sertoli cell proliferation: defining the response interval to an inhibitor of estrogen synthesis in the boar

    Reproduction

    (2012)
  • W.E. Berndtson et al.

    Changing relationships between testis size, Sertoli cell number and spermatogenesis in Sprague-Dawley rats

    J. Androl.

    (1990)
  • W.E. Berndtson et al.

    Relationship of absolute numbers of Sertoli cells to testicular size and spermatogenesis in young beef bulls

    J. Anim. Sci.

    (1987)
  • G.M. Boratyn et al.

    BLAST: a more efficient report with usability improvements

    Nucleic Acids Res.

    (2013)
  • J.L. Brown et al.

    Effects of follicular fluid administration on serum bioactive and immunoactive FSH concentrations and compensatory testicular hypertrophy in hemicastrated prepubertal rats

    J. Endocrinol.

    (1991)
  • D. de Rie et al.

    An integrated expression atlas of miRNAs and their promoters in human and mouse

    Nat. Biotechnol.

    (2017)
  • Ron Edgar et al.

    Gene expression omnibus: NCBI gene expression and hybridization array data repository

    Nucleic Acids Res.207-210

    (2002)
  • N. Ferrand et al.

    Glucocorticoids induce CCN5/WISP-2 expression and attenuate invasion in oestrogen receptor-negative human breast cancer cells

    Biochem. J.

    (2012)
  • J.J. Ford et al.

    Negative relationship between blood concentrations of follicle-stimulating hormone and testicular size in mature boars

    J. Anim. Sci.

    (1997)
  • L.R. Franca et al.

    Cell proliferation and hormonal changes during postnatal development of the testis in the pig

    Biol. Reprod.

    (2000)
  • R.A. Hess et al.

    Adult testicular enlargement induced by neonatal hypothyroidism is accompanied by increased Sertoli and germ cell numbers

    Endocrinology

    (1993)
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      Therefore, these findings indicate that TZD may inhibit the chicken SC proliferation through inducing excessive ROS and impaired mitochondrial function, as well as the alterations of cell proliferation-related pathways. Reducing testicular estradiol increased the SC number including number present at puberty during the neonatal interval (Berger et al., 2021). Our previous study found that low doses of exogenous 17β-estradiol (0.0001–0.1 μM) promoted the viability and cell cycle of SCs (Zhang et al., 2021).

    Data reported in this publication were deposited in the NCBI Gene Expression Omnibus (Edgar et al., 2002) and accessible as GEO Series accession number GSE154933 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE154933).

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