Ontogeny of TRα1 expression in the mouse testis and epididymis during postnatal development.

Thyroid hormone (T3 ) acts on the testis via thyroid hormone receptor alpha 1 (TRα1), though the cellular localization of TRα1 in testis remains controversial. Studies on the presence of TRα1 in the epididymis are also lacking. The present study, therefore, examined the cellular localization and expression pattern of TRα1 in testis and epididymis of Parkes mice during postnatal development. Immunohistochemical results showed localization of TRα1 in interstitial and tubular compartments of the testis all through the development. On postnatal day (PND) 14, only leptotene spermatocytes showed TRα1-immunoreactivity in the testis, while at PND 28, 42, and 90, a diverse staining pattern for TRα1 was seen in almost all the seminiferous tubules mainly in leptotene spermatocytes, round and elongating spermatids, and in Leydig cells. Further, qRT-PCR and immunoblot analyses showed that TRα1 was expressed in the testis at the transcript as well as protein level throughout the postnatal development. TRα1 was also seen in principal cells of the epididymis, with maximal expression at PND 90. TRα1 was also present in cauda epididymidal spermatozoa of adult mice at PND 90. The results suggest that TRα1 is expressed in the testis and epididymis and that it may help to regulate the spermatogenic process and male fertility.

Effect of Trigonella foenum-graecum L. seed extract on the reproductive system of male mice and possible mechanism of its action on spermatogenesis.

Fenugreek seed exhibits antidiabetic, antineoplastic, hepatoprotective, antidepressant and immunomodulatory properties. Fenugreek also causes antifertility effects in rodents. However, the impact of fenugreek seed on male reproduction and the possible mode of its action are not properly evaluated. Herein, we examined the effect of aqueous seed extract of fenugreek (FSE) and the possible mechanism of its action on male reproductive health in mice. Parkes mice were orally administered FSE (600 mg/kg body weight/day) or distilled water for 28 and 56 days, respectively. Various sperm parameters, histopathology, serum testosterone level and fertility indices were assessed. Furthermore, steroidogenic enzymes activities, oxidative status and germ cell dynamics in the testis were evaluated. Toxicological endpoints were also assessed. Treatment with FSE caused degenerative changes in the testis histoarchitecture. The treatment also affected various sperm parameters and concentrations of sialic acid and fructose in the epididymis and seminal vesicle, respectively. Fenugreek treatment also had negative impact on oxidative status and germ cell dynamics in the testis; fertility indices were also affected in female mice impregnated by the extract-treated male mice, though libido of the treated male mice remained unaffected. Results show that treatment with FSE caused adverse effects on the male reproductive health and pregnancy outcome in Parkes mice.

Decabromodiphenyl ether (BDE-209) exposure to lactating mice perturbs steroidogenesis and spermatogenesis in adult male offspring.

Decabromodiphenyl ether (BDE-209) is widely used as a flame retardant in many products like electronic equipments, plastics, furniture and textiles. BDE-209, a thyroid hormones (THs)-disrupting chemical, affects male reproductive health through altered THs status in mouse model. The present study was designed in continuation to our earlier work to elucidate whether early life exposure to BDE-209 has a long term potential risk to male reproductive health. This study, therefore, aimed to evaluate the effect of maternal BDE-209 exposure during lactation and to elucidate possible mechanism(s) of its action on male reproduction in adult Parkes mice offspring. Lactating female Parkes mice were orally gavaged with 500, and 700 mg/kg body weight of BDE-209 in corn oil from postnatal day (PND) 1 to PND 28 along with 6-propyl-2-thiouracil (PTU)-treated positive controls and vehicle-treated controls. Male pups of lactating dams were euthanized at PND 75. Maternal BDE-209 exposure during lactation markedly affected histoarchitecture of testis and testosterone production with concomitant down-regulation in the expression of various steroidogenic markers in adult offspring. Maternal exposure to BDE-209 during lactation also interfered with germ cell dynamics and oxidative status in testes of adult mice offspring. A decreased expression of connexin 43 and androgen receptor was also evident in testes of these mice offspring; further, number, motility and viability of spermatozoa were also adversely affected in these mice. The results thus provide evidences that maternal exposure to BDE-209 during lactation causes reproductive toxicity in adult mice offspring.

Decreased expression of orexin 1 receptor in adult mice testes during alloxan-induced diabetes mellitus perturbs testicular steroidogenesis and glucose homeostasis.

Diabetes mellitus (DM) affects male reproductive system and causes infertility. The male reproductive health is largely dependent upon uptake and proper utilization of glucose by testicular cells. Results show involvement of orexin A (OXA) and its receptor (OX1R) in regulation of steroidogenesis and glucose homeostasis in adult mice testis. However, the role of OX1R in regulation of testicular functions during hyperglycemia has not been investigated so far. The present study, therefore, examined the role of OX1R in regulation of steroidogenesis and glucose homeostasis in testis of adult mice during alloxan-induced type 1 DM. A significant decrease was noted in body weight and testis weight in alloxan-treated mice compared to controls. The blood glucose level, however, was markedly increased in treated animals than in controls. Further, serum and intratesticular level of testosterone, activities of testicular steroidogenic enzymes, and expressions of various steroidogenic markers, OX1R, glucose transporter 3 (GLUT3) and Wilms' tumor gene (WT1) were downregulated in treated mice. The level of glucose, activity of lactate dehydrogenase (LDH) and lactate concentration in the testes of diabetic mice were also decreased; a significant increase in the number of testicular apoptotic cells with concomitant increase in the expression of caspase-3 was noted in these mice. Furthermore, DM affected germ cell proliferation with decreased expression of proliferating cell nuclear antigen (PCNA). Results thus suggest that type 1 DM impairs testicular steroidogenesis and glucose homeostasis through inhibition of OXA/OX1R signaling cascade due to decreased OX1R expression in adult mice, thereby affecting germ cell survival and their proliferation in the testis.

Effect of neonatal hypothyroidism on prepubertal mouse testis in relation to thyroid hormone receptor alpha 1 (THRα1).

Thyroid hormones (THs) are important for growth and development of many tissues, and altered thyroid status affects various organs and systems. Testis also is considered as a thyroid hormone responsive organ. Though THs play an important role in regulation of testicular steroidogenesis and spermatogenesis, the exact mechanism of this regulation remains poorly understood. The present study, therefore, is designed to examine the effect of neonatal hypothyroidism on prepubertal Parkes (P) strain mice testis in relation to thyroid hormone receptor alpha 1 (THRα1). Hypothyroidism was induced by administration of 6-propyl-2-thiouracil (PTU) in mother's drinking water from birth to day 28; on postnatal day (PND) 21 only pups, and on PND 28, both pups and lactating dams were euthanized. Serum T3 and T4 were markedly reduced in pups at PND 28 and in lactating mothers, while serum and intra-testicular testosterone levels were considerably decreased in pups of both age groups. Further, serum and intra-testicular levels of estrogen were significantly increased in hypothyroid mice at PND 28 with concomitant increase in CYP19 expression. Histologically, marked changes were noticed in testes of PTU-treated mice; immunohistochemical and western blot analyses of testes in treated mice also revealed marked decrease in the expression of THRα1 at both age groups. Semiquantitative RT-PCR and western blot analyses also showed reductions in both testicular mRNA and protein levels of SF-1, StAR, CYP11A1 and 3ß-HSD in these mice. In conclusion, our results suggest that neonatal hypothyroidism alters localization and expression of THRα1 and impairs testicular steroidogenesis by down-regulating the expression SF-1, thereby affecting spermatogenesis in prepubertal mice.

Effect of polybrominated diphenyl ether (BDE-209) on testicular steroidogenesis and spermatogenesis through altered thyroid status in adult mice.

Polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants (BFRs), have been widely used in many products to minimize the risk of fire, mainly by mixing in polymer products. BDE-209, a congener of PBDEs having structural similarity with thyroid hormones, acts as an endocrine disruptor by interfering with thyroid homeostasis. However, little is known about the effect of BDE-209 exposure on testicular steroidogenesis and spermatogenesis. This study was therefore conducted in adult mice to examine the effect of BDE-209 on testicular steroidogenesis and spermatogenesis in relation to thyroid status, and to explore possible mechanism(s) of its action. Adult Parkes strain male mice were orally gavaged with 750 and 950mg/kg BW of BDE-209 in corn oil for 35days. Significant reductions were noted in the levels of serum total T3, T4 and testosterone in mice treated with 950mg/kg BW of BDE-209 compared to controls; histologically, testes showed nonuniform degenerative changes in the seminiferous tubules as both affected and normal tubules were observed in the same section; further, number and viability of spermatozoa were also adversely affected in cauda epididymidis of these mice. Semiquantitative RT-PCR and western blot analyses also showed significant reductions in both testicular mRNA and protein levels of steroidogenic factor 1 (SF-1), steroidogenic acute regulatory (StAR) protein, cytochrome P450scc (CYP11A1), 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) in 950mg dose treated-mice compared to controls. Immunohistochemical and immunoblot analyses further revealed a marked decrease in proliferating cell nuclear antigen (PCNA) positive cells in testes of 950mg dose of BDE-209-treated mice. However, 750mg dose of BDE-209 had no effect on the above parameters. In conclusion, our results suggest that exposure of BDE-209 to adult mice causes reduction in serum levels of thyroid hormones and altered thyroid status may partly result into impairment of testicular steroidogenesis because of down-regulated expression of SF-1, thereby causing suppression of spermatogenesis.

Knockdown of type 2 orexin receptor in adult mouse testis potentiates testosterone production and germ cell proliferation.

Orexins (OXs) are neuropeptides which regulate various physiological processes. OXs exist in two different forms, mainly orexin A (OXA) and orexin B (OXB) and their effects are mediated via OX1R and OX2R. Presence of OXB and OX2R in mouse testis is also reported. However, the role of OXB/OX2R in the male gonad remains unexplored. Herein we investigated the role of OXB/OX2R system in testicular physiology under in vivo and ex vivo conditions. Adult mice were given a single dose of bilateral intratesticular injection of siRNA targeting OX2R and were sacrificed 96 h post-injection. OX2R-knockdown potentiated serum and intratesticular testosterone levels with up-regulation in the expressions of major steroidogenic proteins. Germ cell proliferation also increased in siRNA-treated mice. Results of the ex vivo experiment also supported the findings of the in vivo study. In conclusion, OX2R may regulate testosterone production and thereby control the fine-tuning between steroidogenesis and germ cell dynamics.

Localization and expression of Orexin B (OXB) and its type 2 receptor (OX2R) in mouse testis during postnatal development.

The orexins (OXs) were first reported in hypothalamus of rat, and they play an important role in diverse physiological actions. The OXs consist of orexin A (OXA) and orexin B (OXB) peptides and their actions are mediated via two G-protein-coupled receptors, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R), respectively. Presence of OXA and OX1R has been also reported in peripheral organs like reproductive tissues. These findings, therefore, highlight a possible role of OXs and their receptors in male reproductive health. Though, expression and localization of OXB and OX2R in the testis and their role in spermatogenesis are not finally clarified. Herein, we elucidated the localization and the patterns of expression of OXB and OX2R in Parkes mice testes during postnatal development. Results suggest that the precursor prepro-orexin (PPO), OXB and OX2R are expressed at the transcript and protein levels in mouse testis throughout the postnatal development. Immunostaining further showed the localization of OXB and OX2R both in interstitium and tubular compartments of the testis. On 7 day postpartum (7 dpp), only spermatogonia showed immunoreactivity of OXB and OX2R, while at 14, 28, 42 and 90 dpp, immunolocalization of OXB and OX2R were noted in the seminiferous tubules, especially in leptotene, pachytene spermatocytes, round and elongating spermatids, and in Leydig cells and Sertoli cells. The immunoreactivity of OXB and OX2R appeared to be stage-specific in adult mouse testis. The results suggest the expression of OXB and OX2R in mouse testis and their possible regulatory role in spermatogenesis and steroidogenesis.

A comprehensive review on the decabromodiphenyl ether (BDE-209)-induced male reproductive toxicity: Evidences from rodent studies.

Polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants (BFRs), are employed in various manufactured products to prevent fires, slow down their spread and reduce the resulting damages. Decabromodiphenyl ether (BDE-209), an example of PBDEs, accounts for approximately 82 % of the total production of PBDEs. BDE-209 is a thyroid hormone (TH)-disrupting chemical owing to its structural similarity with TH. Currently, increase in the level of BDE-209 in biological samples has become a major issue because of its widespread use. BDE-209 causes male reproductive toxicity mainly via impairment of steroidogenesis, generation of oxidative stress (OS) and interference with germ cell dynamics. Further, exposure to this chemical can affect metabolic status, sperm concentration, epigenetic regulation of various developmental genes and integrity of blood-testis barrier in murine testis. However, the possible adverse effects of BDE-209 and its mechanism of action on the male reproductive health have not yet been critically evaluated. Hence, the present review article, with the help of available literature, aims to elucidate the reproductive toxicity of BDE-209 in relation to thyroid dysfunction in rodents. Further, several crucial pathways have been also highlighted in order to strengthen our knowledge on BDE-209-induced male reproductive toxicity. Data were extracted from scientific articles available in PubMed, Web of Science, and other databases. A thorough understanding of the risk assessment of BDE-209 exposure and mechanisms of its action is crucial for greater awareness of the potential threat of this BFR to preserve male fertility.

Maternal BDE-209 exposure during lactation causes testicular and epididymal toxicity through increased oxidative stress in peripubertal mice offspring.

Decabromodiphenyl ether (BDE-209), a flame retardant, interferes with thyroid homeostasis and androgen biosynthesis. BDE-209 evokes hyperglycemia through impaired glucose homeostasis in rat liver. This study is in continuation to our earlier work for a better understanding of whether or not BDE-209 affects testicular and epididymal physiology in relation to oxidative status in peripupertal mice offspring. Lactating female Parkes mice were orally gavaged with 500 and 700 mg/kg body weight of BDE-209 in corn oil from postnatal day (PND) 1 to PND 28. Male pups of lactating dams were sacrificed at PND 42. Maternal BDE-209 exposure during lactation increased apoptosis and oxidative status with altered expressions of various cell survival (Bcl-2), apoptotic (Bax and caspase-3) and oxidative stress (Nrf2 and HO-1) markers in testes and epididymis of peripubertal mice offspring. Testicular glucose and lactate concentrations were markedly reduced in these pups with down-regulation in GLUT3 and GLUT8 expressions and decreased LDH activity. Maternal BDE-209 exposure markedly affected fertility potential, epididymal histology, sialic acid concentration and sperm quality with decreased expression of epididymal Cx43 and AR in these mice offspring. Results thus suggest that maternal BDE-209 exposure during lactation causes reproductive toxicity in peripubertal mice offspring.

The hypothalamic neuropeptide orexin A- a possible regulator in glucose homeostasis and germ cell kinetics in adult mice testes.

Orexin A (OXA), a hypothalamic neuropeptide, regulates food intake, sleep-wake cycle and energy balance by binding to its receptor (OX1R). Apart from brain, OXA and OX1R are also present in peripheral organs including reproductive tissues. Mammalian reproduction depends on uptake and proper utilization of glucose in the testes. This study, therefore, examined role of OXA/OX1R system in regulation of glucose homeostasis in adult mouse testis under in vivo and ex vivo conditions. Binding of OXA to OX1R was blocked using an OX1R antagonist, SB-334867. Mice were given a single bilateral intratesticular injection of the antagonist at doses of 4 and 12µg/mouse and sacrificed 24 h post-injection. In order to understand the direct role of OXA in testes of adult mice, an ex vivo experiment was performed where binding of OXA to OX1R in the testis was blocked by using the same OX1R antagonist. The antagonist treatment affected testicular glucose and lactate concentration with concomitant down-regulation in the expression of glucose transporters 3 and 8. A decreased activity in lactate dehydrogenase enzyme and imbalance between germ cell survival and proliferation were also noted in testes in treated mice. The results of ex vivo study supported the results obtained from in vivo study. The findings thus suggest involvement of OXA/OX1R system in regulation of testicular glucose homeostasis and germ cell kinetics in adult mice.

Inhibition of testicular steroidogenesis and impaired differentiation of Sertoli cells in peripubertal mice offspring following maternal exposure to BDE-209 during lactation suppress germ cell proliferation.

Polybrominated diphenyl ethers (PBDEs) are used for fire prevention purpose. BDE-209, a congener of PBDEs, is thyroid hormones (THs)-disrupting chemical because of its structural similarity with THs. Testis is considered as THs-responsive organ and is more susceptible to chemical agents during peripubertal period. This study, therefore, evaluated the effect and possible mechanism(s) of action of maternal exposure to BDE-209 during lactation on germ cell proliferation, testicular steroidogenesis and on differentiation of Sertoli cells (SCs) in relation to altered THs status in peripubertal mice offspring. Lactating Parkes mice were gavaged with 500 and 700 mg/kg BW of BDE-209 in corn oil from postnatal day (PND) 1 to PND 28 along with 6-propyl-2-thiouracil (PTU)-treated positive controls and vehicle-treated controls. Male pups of lactating dams were sacrificed at PND 42. Maternal exposure to BDE-209 during lactation markedly affected testicular histopathology, germ cell proliferation and steroidogenesis with down-regulated expression of various steroidogenic markers in peripubertal mice offspring. Decreased expressions of maturational markers of SCs with a decline in serum THs levels were also evident in these offspring. Results thus suggest that maternal BDE-209 exposure during lactation impairs germ cell proliferation via inhibition of steroidogenic pathway and differentiation of SCs in peripubertal mice offspring.

Maternal BDE-209 exposure during lactation perturbs steroidogenesis, germ cell kinetics and THRα1 expression in testes of prepubertal mice offspring.

Decabromodiphenyl ether (BDE-209), a congener of polybrominated diphenyl ethers (PBDEs), is used as flame retardant and affects thyroid homeostasis. Thyroid hormones (THs) play crucial role in Leydig cell differentiation and steroidogenesis during early life. Present study examined the effect of maternal BDE-209 exposure during lactation on testicular steroidogenesis and spermatogenesis in relation to thyroid hormone receptor alpha 1 (THRα1) and possible mechanism(s) of its action in prepubertal Parkes mice offspring. Lactating female Parkes mice were orally gavaged with 500, and 700 mg/kg body weight of BDE-209 in corn oil from postnatal day (PND) 1 to PND 28. Lactating mothers and male pups were sacrificed on PND 28. Maternal BDE-209 exposure markedly affected testicular histopathology, steroidogenesis and germ cell dynamics with downregulated expressions of various steroidogenic markers in mice offspring. Serum THs levels were markedly reduced in both pups and lactating mothers compared to controls. Expression of proliferating cell nuclear antigen and THRα1 also deceased in testes of BDE-209-exposed mice offspring. In silico analysis by molecular docking was performed successfully for steroidogenic facor-1 (SF-1) and THRα1 with BDE-209 and T3. Maternal BDE-209 exposure during lactation affects testicular steroidogenesis, spermatogenesis and expression of THRα1 in prepubertal mice offspring through downregulation of SF-1.

Maternal exposure to polybrominated diphenyl ether (BDE-209) during lactation affects germ cell survival with altered testicular glucose homeostasis and oxidative status through down-regulation of Cx43 and p27Kip1 in prepubertal mice offspring.

BDE-209, a congener of polybrominated diphenyl ethers (PBDEs), is mainly used as flame retardant and accounts for over 82% of total PBDE usage. PBDEs have recently been detected in human milk and cord blood. BDE-209 possesses weak estrogenic/anti-estrogenic properties and evokes hyperglycemia through impaired glucose homeostasis in rat liver. However, little is known of the effect of lactational exposure to BDE-209 on germ cell survival in relation to testicular glucose homeostasis, estradiol and oxidative status during prepubertal period. The present study, therefore, evaluated the effect of maternal exposure to BDE-209 during lactation on above-mentioned parameters with reference to Cx43 and p27Kip1 in prepubertal Parkes (P) mice offspring. Lactating female P mice were orally gavaged with 500, and 700mg/kg body weight of BDE-209 in corn oil from postnatal day (PND) 0 to PND 28; male pups were euthanized at PND 21 and 28. Maternal exposure to BDE-209 during lactation increased germ cell apoptosis with altered expressions of various cell survival and apoptotic markers along with decreased expression of Cx43 and p27Kip1 in prepubertal mice offspring. Testicular glucose and lactate concentrations were markedly reduced in these pups with down-regulation in GLUT3 and GLUT8 expressions and decreased LDH activity. Rise in oxidative stress in testes with increased estrogen level in these pups was also noted. In conclusion, our results suggest that maternal BDE-209 exposure during lactation affects germ cell survival with altered testicular glucose homeostasis and oxidative status through down-regulation of Cx43 and p27Kip1 in prepubertal mice offspring.