227 Lactocrine programming enhances testicular growth and alters the testicular proteome in the neonatal boar.

The boar has the greatest cumulative impact on swine reproductive performance and drives genetic progress in the herd. However, developing boars may be exposed to agrichemicals directly and/or indirectly via drinking water. Atrazine is an herbicide heavily used in U.S. corn production and an endocrine-disrupting chemical. Atrazine concentrations over 20 µg/L have been detected in groundwater of agricultural states, exceeding the Environmental Protection Agency limit (3 µg/L). This study aims to determine if maternal consumption of environmentally relevant atrazine levels in drinking water affects testicular development in exposed offspring. Pregnant littermate gilts drank water containing either atrazine (20 µg/L; n = 4) or vehicle control [0.002% (v/v) ethanol; n = 3)] ad libitum from 28 days of gestation through parturition (~127 days total). Atrazine-exposed (ATZ; n = 24) and control (n = 12) boars were necropsied on post-natal day 10. Body weight was recorded, and blood was collected for quantification of thyroxine and anti-müllerian hormone (AMH; product of Sertoli cells). Testes were trimmed, weighed, and fixed for histological analysis. Testicular samples stained with hematoxylin and eosin were utilized to quantify interstitial and tubular area, average area of individual seminiferous tubules and the number of seminiferous tubules per field. Data were analyzed via the MIXED procedure of the Statistical Analysis System with a model that included treatment as the fixed effect, boar as the experimental unit, and body weight as a covariate for testis weight. Results indicated an effect of treatment on testis development. For example, the testes of ATZ males were 29% smaller than control boars (2.7±0.2 versus 3.8±0.3 g; P = 0.0021), indicating a reduction in Sertoli cell number. Histological analysis revealed alterations in testis composition. ATZ exposed boars had larger seminiferous tubules than control boars (P = 0.0246), leading to a greater total tubular area (P = 0.0101). Consequently, total interstitial area – a region containing steroidogenic Leydig cells – was reduced in ATZ exposed males (P = 0.0116). Results also indicated an effect of atrazine on endocrine function. For example, serum concentrations of thyroxine were reduced by 9.5% in ATZ boars compared to controls (P = 0.0124). Thyroxine is a known modulator of neonatal Sertoli cell proliferation. In addition, serum AMH concentrations were 52.5% greater in ATZ boars compared with control animals (P = 0.0011). Previous work in other species has linked elevated serum AMH concentrations with poor spermatogenesis and testicular degeneration. These data collectively suggest that maternal ATZ exposure impairs testicular development and Sertoli cell function in offspring. Given that Sertoli cells determine sperm production capacity, these alterations may predispose a lifetime of reproductive failure. Therefore, maternal consumption of environmentally relevant ATZ levels may hinder the development of fertile offspring destined for the boar stud.

Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects

We previously demonstrated that androgens alone, in the complete absence of gonadotropins, initiated qualitatively complete spermatogenesis in hypogonadal (hpg) mice. Although germ cell to Sertoli cell ratios were normal in hpg mice with androgen-induced spermatogenesis, testicular size. Sertoli cell and germ cell numbers only reached 40% of those of non-hpg mice, and Sertoli cell numbers were unaffected by androgen treatment started at 21 days of age. We postulated that these observations were due to diminished gonadotropin-dependent. Sertoli cell proliferation during perinatal life while the Sertoli cells still exhibited normal carrying capacity for mature germ cells. In order to test this hypothesis, we examined the effects of administering androgens and gonadotropins to hpg mice during the first 2 weeks of postnatal life when Sertoli cells normally continue to proliferate. The study end-points were Sertoli and germ cell numbers in hpg mice following induction of spermatogenesis by 8 weeks treatment with 1 cm subdermal silastic testosterone implants. Newborn pups (postnatal day 0-1) were injected s.c. with recombinant human FSH (rhFSH) (0.5 IU/20 microliters) or saline once daily for 14 days, with or without a single dose of testosterone propionate (TP) (100 micrograms/20 microliters arachis oil) or human chorionic gonadotropin (hCG) (1 IU/20 microliters). Untreated hpg and phenotypically normal littermates were studied as concurrent controls. At 21 days of age, all treated weaning mice received a 1 cm silastic subdermal testosterone implant and, finally, 8 weeks after testosterone implantation, all mice were killed. As expected, qualitatively complete spermatogenesis was induced in all groups by testosterone despite undetectable circulating FSH levels. Exogenous rhFSH increased testis size by 43% (P < 0.002) but a single neonatal dose of either TP or hCG reduced the FSH effect although neither TP nor hCG had any effect alone. In contrast, a single neonatal dose of TP or hCG increased final seminal vesicle size whereas FSH had no effect. FSH and TP treatment significantly increased absolute numbers of testicular spermatids compared with saline treatment, whereas hCG and TP significantly increased testicular sperm when expressed relative to testis size. Stereological evaluation of Sertoli and germ cell numbers demonstrated a rise in the absolute numbers of Sertoli and all germ cell populations induced by neonatal administration of hormones. When expressed per Sertoli cells the numbers of germ cells in the treated mice were between 85 and 90% of non-hpg controls. We conclude that exogenous FSH treatment during the first 2 weeks of postnatal life, coinciding with the natural time of Sertoli cell proliferation, increases Sertoli cell numbers and thereby the ultimate size of the mature testis and its germ cell production. Thus neonatal gonadotropin secretion may be a critical determinant of the sperm-producing capacity of the mature testis. In addition, neonatal exposure to androgens could be important for the imprinting of sex accessory organs in hpg mice, with the long-term effects of altering the sensitivity of the accessory organs to exogenous testosterone later in life.

Androgens may be important regulators of Sertoli cell (SC) proliferation perinatally, with implications for the testicular dysgenesis syndrome (TDS) hypothesis. Fetal exposure of rats to 500 mg/kg . d di(n-butyl) phthalate (DBP) reduces fetal testosterone production and SC number at birth, but SC number recovers to normal by postnatal d (Pnd)25. It is unclear when and how SC proliferation is affected prenatally by DBP exposure or when and how postnatal compensation occurs. This study addressed these questions and investigated whether continued maternal exposure to DBP or to flutamide from Pnd1-Pnd15 could prevent SC number compensation, because this would have implications for how sperm counts might be lowered in TDS. DBP exposure attenuated SC proliferation by 7-18% throughout embryonic d (e)15.5-e21.5 (P < 0.05 at e21.5). After birth, SC proliferation increased significantly (>1.5-fold) between Pnd6 and Pnd10 in prenatally DBP-exposed animals, explaining the compensation. Continued maternal administration of DBP after birth attenuated (19% reduction) SC number compensation at Pnd25 and maternal administration of flutamide (100 mg/kg . d) to prenatally DBP-exposed animals was even more effective (42% reduction), suggesting the postnatal compensatory increase in SC proliferation after prenatal DBP exposure is androgen dependent. SC maturation (Pnd25) was unaffected, based on analysis of expression of key proteins, but lumen formation/expansion was attenuated in parallel with treatment-induced reduction in SC number. Our results provide further evidence that perinatal SC proliferation is androgen dependent and, importantly, show that similar exposure of mothers to antiandrogenic chemicals before birth and during lactation reduces final SC number, with implications for the origin of low sperm counts in TDS.

Are testicular cortisol and WISP2 involved in estrogen-regulated Sertoli cell proliferation?

Ontogeny of androgen and estrogen receptor expression in porcine testis: Effect of reducing testicular estrogen synthesis

The direct correlation between Sertoli cell number and sperm production capacity highlights the importance of deciphering external factors that modify Sertoli cell proliferation. A growing body of evidence in vitro suggests that metformin, the main pharmacological agent for type 2 diabetes treatment in children, exerts anti-proliferative effects on Sertoli cells. The aims of this study were to investigate the effect of metformin administration during postnatal period on Sertoli cell proliferation and on cell cycle regulators expression and to analyze the impact of this treatment on the sperm production capacity in adulthood. Sprague Dawley rat pups were randomly divided into two groups: MET (receiving daily 200mg/kg metformin, from Pnd3 to Pnd7 inclusive) and control (receiving vehicle). BrdU incorporation was measured to assess proliferation. Gene expression analyses were performed in Sertoli cells isolated from animals of both groups. Daily sperm production and sperm parameters were measured in adult male rats (Pnd90) that received neonatal treatment. MET group exhibited a significant decrease in BrdU incorporation in Sertoli cells. Concordantly, MET group showed a reduction in cyclin D1 and E2 expression and an increase in p21 expression in Sertoli cells. In addition, metformin-treated animals displayed lower values of daily sperm production on Pnd90. These results suggest that metformin treatment may lead to a decrease in Sertoli cell proliferation, a concomitant altered expression of cell cycle regulators and ultimately, a reduction in daily sperm production in adult animals.

Sertoli cell proliferation in juvenile boars and microRNA

Role of Sertoli cell number and function on regulation of spermatogenesis

Neonatal hypothyroidism increases adult Sertoli cell populations by extending Sertoli cell proliferation. Conversely, hyperthyroidism induces premature cessation of Sertoli cell proliferation and stimulates maturational events like seminiferous tubule canalization. Thyroid hormone receptors alpha1 and beta1, which are commonly referred to as TRalpha1 and TRbeta1, respectively, are expressed in neonatal Sertoli cells. We determined the relative roles of TRalpha1 and TRbeta1 in the thyroid hormone effect on testicular development and Sertoli cell proliferation using Thra knockout (TRalphaKO), Thrb knockout (TRbetaKO), and wild-type (WT) mice. Triiodothyronine (T3) treatment from birth until Postnatal Day 10 reduced Sertoli cell proliferation to minimal levels in WT and TRbetaKO mice versus that in their untreated controls, whereas T3 had a diminished effect on TRalphaKO Sertoli cell proliferation. Seminiferous tubule patency and luminal diameter were increased in T3-treated WT and TRbetaKO testes. In contrast, T3 had no effect on these parameters in TRalphaKO mice. In untreated adult TRalphaKO mice, Sertoli cell number, testis weight, and daily sperm production were increased or trended toward an increase, but the increase in magnitude was smaller than that seen in WT mice following neonatal hypothyroidism. Conversely, in TRbetaKO mice, Sertoli cell number, testis weight, and daily sperm production were similar to those in untreated WT mice. In addition, Sertoli cell number and testis weight in adult WT and TRbetaKO mice showed comparable increases following hypothyroidism. Our results show that TRalphaKO mice have testicular effects similar to those seen in WT mice following neonatal hypothyroidism and that TRbetaKO mice, but not TRalphaKO mice, have normal Sertoli cell responsiveness to T3. Thus, effects of exogenous manipulation of T3 on neonatal Sertoli cell development are predominately mediated through TRalpha1.

Changes in Sertoli cell numbers and testicular structure during normal development and compensatory hypertrophy were assessed in crossbred Meishan x White Composite males. Boars were assigned at birth to unilateral castration at 1, 10, 56, or 112 days or to remain as intact controls through 220 days. The first testes removed were compared to assess testicular development. At 220 days, testicular structure was evaluated in boars representing the 25% with the largest (Lg) testis and the 25% with the smallest (Sm) testis in each treatment group. The number of Sertoli cells per testis reached a maximum by Day 56 in Sm testis but not until Day 112 in Lg testis boars, indicating a longer duration of Sertoli cell proliferation in Lg testis boars. Unilateral castration of Lg testis boars on Days 1, 10, 56, and 112 caused the weight of the remaining testis to hypertrophy by 149%, 135%, 119%, and 120%, respectively, and total sperm production to increase to 127%, 128%, 97%, and 106%, respectively. However, Sertoli cell numbers changed little in hemicastrate boars. In Lg testis boars, compensatory hypertrophy primarily involved proliferation of Leydig cells and expansion of existing Sertoli cells with little increase in Sertoli cell numbers, but in Sm testis boars, it involved expansion of existing Leydig and Sertoli cells without increase in cell numbers. These results indicate that Lg and Sm testis boars display intriguing differences during both development and compensatory hypertrophy, and they identify a unique animal model for further studies of factors that program and control Sertoli cell proliferation.

Proliferation and maturation of Sertoli cells is a major determinant of sperm production. Understanding the function of androgens during Sertoli cell proliferation and maturation is the long term goal of this research. The current objective is to determine the effects of blocking androgen receptor activation during the first wave of Sertoli cell proliferation. In the pig, Sertoli cell proliferation occurs in two waves, which begin postnatally and continue until just before puberty. During Sertoli cell proliferation, androgens were thought to be relatively quiescent. Work in rodent models suggests that androgens are required for normal Sertoli cell maturation following the proliferative phase. We propose that blocking androgen binding to the androgen receptor during the first wave of Sertoli cell proliferation will stimulate Sertoli cell proliferation. To evaluate this, pairs of littermate male pigs were treated daily with flutamide, which is a non steroidal anti-androgen or with canola oil vehicle from 1 week of age until 6.5 weeks of age, covering the first interval of Sertoli cell proliferation. Blood was collected weekly from animals to analyze hormone levels. Tissues were recovered at 6.5 weeks (n=3) immediately following treatment or at 11 weeks (n=3) of age, 4.5 weeks after treatment. Testosterone was measured using radioimmunoassay and Sertoli cell numbers were counted using GATA4 immunohistochemistry. Circulating plasma testosterone was on average six times greater in the flutamide-treated animals compared with the vehicle treated littermates. Increased testosterone is an indicator that flutamide effectively blocked androgen receptor activation. After treatment with flutamide, there was a two-fold increase in testes size and a two-fold increase in Sertoli cell numbers at 6.5 weeks, suggesting a role for testosterone in Sertoli cell proliferation, contrary to previous thoughts. At 11 weeks of age, however, the animals treated with flutamide exhibited no significant difference in Sertoli cell numbers compared with the vehicle controls. Increased understanding of endogenous hormone action will provide a better understanding of the factors controlling Sertoli cell proliferation and ultimately the hormonal control of fertility. Research supported in part by NRI 2008-35203-19082 from USDA NIFA, Kellogg, Hatch, and W2171 funds. (poster)

17β-estradiol rescues the damage of thiazolidinedione on chicken Sertoli cell proliferation via adiponectin

Thyroid hormone inhibits neonatal Sertoli cell proliferation and recent results have shown that thyroid hormone upregulates cyclin-dependent kinase inhibitors (CDKIs) p27Kip1 and p21Cip1 (also known as CDKN1B and CDKN1A, respectively) in neonatal Sertoli cells. This suggests that these CDKIs, which negatively regulate the cell cycle, could be critical in Sertoli cell proliferation. Consistent with this hypothesis, mice lacking p27Kip1 develop testicular organomegaly, but Sertoli cell numbers have not been determined. Likewise, effects of loss of p21Cip1 or both p27 and p21 on Sertoli cell number and testicular development were unknown. To determine if p27 and/or p21 regulate Sertoli cell proliferation, we measured Sertoli cell proliferation at Postnatal Day 16 and testis weight, Sertoli cell number, and daily sperm production (DSP) in 4-mo-old wild-type (WT), p21 knockout (p21KO), p27 knockout (p27KO), and p27/p21 double-knockout (DBKO) mice. Testis weights were increased 27%, 42%, and 86% in adult p21KO, p27KO, and DBKO mice, respectively, compared with WT. Sertoli cell number also was increased 48%, 126%, and 126% in p21KO, p27KO, and DBKO mice, respectively, versus WT. DSP in p21KO, p27KO, and DBKO testes also showed significant increases compared with WT mice. Although DSP was increased, there were increased spermatogenic defects observed in both p27KO and DBKO mice compared with WT. These data indicate that both p27 and p21 play an inhibitory role in regulating adult Sertoli cell number such that loss of either CDKI produces primary increases in Sertoli cell number and secondary increases in DSP and testis weight. Furthermore, loss of both CDKIs causes additive effects on DSP and testis weight, suggesting a central role for these CDKIs in testis development.

Thyroid hormones participate in regulating growth and homeostatic processes in vertebrates, including development and adult functioning of the reproductive system. Here we report a new stimulatory role of thyroid hormone on the proliferation of Sertoli cells (SCs) and single, type A undifferentiated spermatogonia (A(und)) in adult zebrafish testes. A role for T3 in zebrafish testis is suggested by in situ hybridization studies, which localized thyroid receptor α (thrα) in SCs and the β (thrβ) mRNA in Sertoli and Leydig cells. Using a primary zebrafish testis tissue culture system, the effect of T3 on steroid release, spermatogenesis, and the expression of selected genes was evaluated. Basal steroid release and Leydig cell gene expression did not change in response to T3. However, in the presence of FSH, T3 potentiated gonadotropin-stimulated androgen release as well as androgen receptor (ar) and 17α-hydroxylase/17,20 lyase (cyp17a1) gene expression. Moreover, T3 alone stimulated the proliferation of both SCs and A(und), potentially resulting in newly formed spermatogonial cysts. Additional tissue culture studies demonstrated that Igf3, a new, gonad-specific member of the IGF family, mediated the stimulatory effect of T3 on the proliferation of A(und) and SCs. Finally, T3 induced changes in connexin 43 mRNA levels in the testis, a known T3-responsive gene. Taken together, our studies suggest that T3 expands the population of SCs and A(und) involving Igf signaling and potentiates gonadotropin-stimulated testicular androgen production as well as androgen sensitivity.