Reducing endogenous estrogen during development alters hormone production by porcine Leydig cells and seminiferous tubules

High levels of estrogen produced by boar testes and the presence of estrogen receptors in both interstitial and tubular compartments are consistent with a direct role for estrogen in regulation of testicular cell function. This study investigated the importance of estrogen on hormone production by Leydig cells and seminiferous tubules in the developing boar. Thirty-six 1-week-old littermate pairs of boars were treated weekly with vehicle or 0.1 mg/kg BW Letrozole, an aromatase inhibitor, until castration at 2, 3, 4, 5, 6, 7, or 8 months. Tissue was collected and Leydig cells and seminiferous tubules were isolated. In a separate study, five untreated boars (ages 1.5-4 months) were castrated and Letrozole was added in vitro to Leydig cell and seminiferous tubule cultures. Leydig cells were cultured for 24h with and without porcine LH. Media were assayed for estradiol (E(2)) and testosterone (T) concentrations by RIA. Seminiferous tubules were cultured for 4h with and without porcine FSH; media were assayed for E(2) and immunoreactive inhibin (INH). In vivo aromatase inhibition decreased basal E(2) and increased basal T production by cultured Leydig cells. Basal seminiferous tubule production of E(2) but not INH was reduced. Decreasing estrogen synthesis in vivo did not alter LH-induced Leydig cell E(2) production or FSH-induced seminiferous tubule INH production. INH production decreased with advancing age regardless of treatment. In conclusion, in vivo aromatase inhibition altered baseline steroid production by cultured Leydig cells and seminiferous tubules but had little effect on response to gonadotropins.     

Expression of connexin 43 protein in testes, epididymides and prostates of stallions

REASONS FOR PERFORMING STUDY: Connexin 43 (Cx43) is a ubiquitously distributed gap junction protein in testes and other reproductive tissues. Adjacent cells share ions and small metabolites through intercellular channels, which are present in gap junctions. Previously, Cx43 has not been reported in testes, epididymides and prostates either in healthy stallions or cryptorchid horses. OBJECTIVES: To demonstrate the expression pattern of Cx43 in the reproductive tissues of stallions and examine whether naturally occurring bilateral cryptorchidism has any influence on distribution and expression of Cx43. METHODS: The expression and the presence of Cx43 protein were detected by means of immunohistochemistry and Western blot analysis using a polyclonal rabbit anti-Cx43 antibody. RESULTS: In stallions, gap junctions appeared as structures localised to cell-cell contacts between adjacent cells. In testes, Cx43 expression was detected in the interstitial tissue and seminiferous tubules, between Leydig and Sertoli, as well as Sertoli and germ cells. In epididymides, Cx43 was localised between epithelial cells, whereas in prostates, between secretory cells of the glandular epithelium. In the cryptorchid, a clear reduction of Cx43 signal was observed in all reproductive tissues. CONCLUSIONS: Coupling of Leydig cells via gap junctions may suggest that steroidogenic function of the testis is under the influence of these intercellular channels. Within seminiferous tubules, the expression was found to be stage-specific, pointing to its role in coordinating spermatogenesis. Differential distribution of Cx43 protein in the reproductive tract of normal and cryptorchid stallions indicates that expression is clearly dependent on the physiological status of the horse. POTENTIAL RELEVANCE: Detection of Cx43 expression in equine testicular, epididymal, and prostatic cells is important for a better understanding of the role of intercellular membrane channels in direct cell communication within the reproductive tract of stallions.     

High plasma estradiol-17beta levels in dogs with benign prostatic hyperplasia and azoospermia

The semen quality of 22 dogs (4 to 7 years old) with benign prostatic hyperplasia (BPH) was examined at the hospital of our university, and 4 of the 22 BPH dogs were diagnosed as azoospermic. The mean peripheral plasma estradiol-17beta (E2) level (17.3 pg/ml) of the 18 BPH dogs with spermatogenic function was higher than that of 5 normal male dogs and their mean T level (1.7 ng/ml) was lower. The mean E2 level (27.3 pg/ml) of the 4 BPH dogs with azoospermia was significantly higher than the value in the BPH dogs with spermatogenic function (P<0.01), and the mean T level (1.1 ng/ml) was significantly lower (P<0.05). Five normal male dogs were given 10 intramuscular injections of estradiol benzoate (E2B) 5 microg/kg, at 3-day intervals to investigate the relationship between high plasma E2 levels and the cause of the BPH and azoospermia. Their testes and prostates were measured and biopsied both before and 30 days after the start of E2B injections. At 30 days after the start of the E2B injections, the mean peripheral plasma T levels had decreased by half, and the mean testicular volume had decreased to 88% of original volume. The numbers of spermatocytes, spermatids, and spermatozoa in the seminiferous tubules of all of the dogs were significantly lower (P<0.05, 0.01). In addition, the mean prostatic volume increased to 130%, the mean height of the glandular epithelium decreased, and the glandular lumen became increased in diameter. These findings indicate that both BPH and serious spermatogenic dysfunction may be simultaneously induced by protracted high plasma E2 levels in dogs.     

Decreased sperm number and motile activity on the F1 offspring maternally exposed to butyl p-hydroxybenzoic acid (butyl paraben)

Butyl p-hydroxybenzoic acid (butyl paraben, BP) is widely used as a preservative in food and cosmetic products. Routledge et al showed that BP is weakly estrogenic in both in vitro and in vivo (rat uterotrophic) analyses. We investigated whether maternal exposures to BP during gestation and lactation periods affected the development of the reproductive organs of the F1 offspring. Pregnant Sprague-Dawley rats were injected subcutaneously with 100 or 200 mg/kg of BP from gestation day (GD) 6 to postnatal day (PND) 20. In the group exposed to 200 mg/kg of BP, the proportion of pups born alive and the proportion of pups surviving to weaning were decreased. The body weights of female offspring were significantly decreased at PND 49. The weights of testes, seminal vesicles and prostate glands were significantly decreased in rats exposed to 100 mg/kg of BP on PND 49. In contrast, the weights of female reproductive organs were not affected by BP. The sperm count and the sperm motile activity in the epididymis were significantly decreased at doses of 100 and 200 mg/kg of BP. In accordance with the sperm count in the epididymis, the number of round spermatids and elongated spermatids in the seminiferous tubule (stage VII) were significantly decreased by BP. Testicular expression of estrogen receptor (ER)-alpha and ER-beta mRNA was significantly increased in 200 mg/kg of BP treated group at PND 90. Taken together, these results indicated that maternal exposure of BP might have adverse effects on the F1 male offspring.     

Relationship between serum sex hormone concentrations and histology of seminiferous tubules of captured baleen whales in the Western North Pacific during the feeding season

The present study was conducted to obtain new information on relationships among serum testosterone (T), estradiol-17 beta (E(2)), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) concentrations and histology of seminiferous tubules in captured common minke and Bryde's whales during the feeding season. Blood samples and testes were collected from common minke (n=39 for blood samples, n=15 for testes) and Bryde's (n=14 for blood samples, n=7 for testes) whales captured from May 2001 to August 2001 in the Western North Pacific. Serum T concentrations, in 35.9% of the common minke and 57.1% of Bryde's whales, were below the detection limit (< 2.5 pg/ml). There were no significant differences in the serum concentrations of E(2), FSH, and LH among immature, mature common minke and Bryde's whales except that LH levels of immature Bryde's whales was higher than those of common minke whales. In most seminiferous tubules of mature whales, only a single-layer of spermatogonia was observed. However, spermatozoa were observed in seminiferous tubules in 2/13 of mature common minke and 4/4 of mature Bryde's whales with the low or undetectable T levels. These results indicate that the low serum T concentrations reflect the inactivity of spermatogenesis in both baleen whales, and that it is not possible to assess gonadal activity in either common minke or Bryde's whales using serum sex hormone concentrations during the feeding season.     

Rat Uterine Oxytocin Receptor and Estrogen Receptor α and β mRNA Levels are Regulated by Estrogen Through Multiple Estrogen Receptors

Estrogen action is mediated through several types of receptors (ERs), such as ERα, ERβ and putative membrane ERs. Oxytocin receptor (OTR) and ER expression levels in the rat uterus are regulated by estrogen; however, which types of ERs are involved has not been elucidated. This study examined OTR, ERα and ERβ levels in ovariectomized rats treated with 17β-estradiol (E2), an ERα agonist (PPT), an ERβ agonist (DPN) or estren (Es). E2 and PPT increased OTR mRNA levels and decreased ERα and ERβ mRNA levels 3 and 6 h posttreatment. DPN decreased ERα and ERβ mRNA levels at 3 and 6 h, while OTR mRNA levels increased at 3 h and decreased at 6 h. OTR mRNA levels increased 3 h after the Es treatment and then declined until 6 h. ERα and ERβ mRNA levels decreased by 3 h and remained low until 6 h posttreatment with Es. The ER antagonist ICI182,780 (ICI) suppressed the increases in OTR mRNA levels induced 3 h after the Es treatment. However, ICI and tamoxifen (Tam) had no significant effect on ERα and ERβ mRNA levels in the Es-treated or vehicle-treated group. In intact rats, proestrus-associated increases in OTR mRNA levels were antagonized by both ICI and Tam. However, decreases in ERα and ERβ mRNA levels were not antagonized by Tam and ICI, respectively. Therefore, uterine OTR gene expression is upregulated by estrogen through the classical nuclear (or non-nuclear) ERs, ERα and ERβ, while the levels of these ERs are downregulated by estrogen through multiple pathways including Es-sensitive nonclassical ERs.     

The Expression of Epidermal Growth Factor (EGF) and its Receptor (EGFR) During Post‐Natal Testes Development in the Yak

Growth factors play critical role in cell proliferation, regulate tissue differentiation and modulate organogenesis. Several growth factors have been identified in the testes of various mammalian species in last few years. In present investigation, the objective was to determine the expression of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in yak testicular tissue by relative quantitative real time polymerase chain reaction (RT‐PCR), Western blot (WB) and immunohistochemistry (IHC) from mRNA and protein levels. The testicular tissues were collected from male yak at 6 and 24 months old. Results of RT‐PCR and WB showed that the expression quantity of EGF and EGFR at 24 months of age was higher than at 6 months, and the increase rate of EGFR on mRNA and protein levels was higher than the increase rate EGF during post‐natal testes development. Positive staining for EGF and EGFR was very low and mainly localized to Leydig cells testes at 6 months of age with immunohistochemistry, and seminiferous tubules were not observed. At 24 month of age, both the EGF and EGFR could be detected in Leydig cells, peritubular myoid cells, sertoli cells and germ cells of the yak testes. However, EGF and EGFR were localized to preferential adluminal compartment and basal compartment in the seminiferous tubules, respectively. In conclusion, the findings in present studies suggest that EGF and EGFR as important paracrine and/or autocrine regulators in yak testes development and spermatogenesis.     

Leydig cell hyperplasia in an ENU-induced mutant mouse with germ cell depletion

Repro22 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing depletion of both male and female germ cells. In the present study, we investigated the male phenotypes of the mutant mouse at the adult stage. The repro22/repro22 homozygous mice showed reduced body weights as well as markedly reduced testis weights. Histological examination of the testes at 4 and 10 months of age showed no germ cells in the seminiferous tubules of the affected testis while a number of Sertoli cells were observed in the tubules. In addition to the germ cell depletion, the testes of the affected mouse contained expanded intertubular spaces that were filled by Leydig cell-like interstitial cells. These interstitial cells were confirmed to be Leydig cells by immunohistochmical staining using anti-3beta-HSD antibody. The estimated number of Leydig cells in the affected testes at 10 months of age increased approximately 2 fold compared with those of normal testes. Furthermore, the plasma testosterone levels of the affected mice at 10 months of age were significantly higher than those of the normal mice. These findings indicated that the repro22/repro22 mouse developed hyperplasia of Leydig cells that was presumably caused by the absence of germ cells in the seminiferous tubules.     

Effects of antiepileptic drugs carbamazepine and sodium valproate on fertility of male rats

The effects of carbamazepine and sodium valproate on fertility of male rats were studied. The tested drugs were given orally to male rats for 30 and 60 consecutive days. Mating performance, sex organs weights, semen quality, plasma concentrations of sexual hormones as well as histopathological findings were the criteria used to evaluate the reproductive efficiency of treated males. Oral administration of carbamazepine and sodium valproate for 30 and 60 consecutive days significantly decreased the testicular weight, sperm cell concentration, live sperms and percentage of progressively motile spermatozoa. Both drugs significantly increased the percentage of morphologically abnormal spermatozoa. A decrease in plasma testosterone, FSH and LH and an increase in prolactin levels were observed in the treated groups. Histopathological examination showed mild to moderate degenerative changes in the testes of the treated rats while the prostate glands and seminal vesicles appeared normal. A recovery period of 30 days was accompanied by marked changes in the tested parameters towards initial values.     

抑制芳香化酶活性对母鸡性腺分化和性行为的影响

为研究雌激素在鸡性腺和性行为分化过程中的作用,本试验在受精蛋孵化第3天气室注入100μL生理盐水或芳香化酶抑制剂(AI),出雏后常规饲养到8月龄性成熟,观察其性行为的表现,检测性腺结构和血清性激素水平.结果发现,AI处理获得的性反转母鸡出现雄性第二性征和雄性交配行为,性腺形态和组织学检查结果表明其性腺得到了不同程度的反转(1)完全反转母鸡右侧性腺发育成为睾丸,而左侧性腺出现不同程度的反转.两侧性腺均出现精细管结构,管径较正常窄,发育不完整,精细管内大量分布生殖细胞,右侧性腺的间质较左侧发达;(2)不完全反转母鸡的右侧性腺退化,左侧仍为完整卵巢.血清中雌二醇(E2)含量为不完全反转母鸡＞正常母鸡＞完全反转母鸡＞正常公鸡;睾酮(T)含量为完全反转母鸡＞不完全反转母鸡＞正常公鸡＞正常母鸡,雄性交配频率与血清T/E2的比值呈正相关.以上结果表明,在性分化前用AI阻断雌激素的合成可引起雌性性腺结构和激素分泌功能及性行为雄性化,而血清T和E2含量及其比值决定了性反转母鸡雄性交配行为的频率和强度.     

Estrogen inhibits interleukin-18 mRNA expression in the mouse uterus

Interleukin-18 (IL-18) is a proinflammatory cytokine expressed in female reproductive organs in humans, rats and mice. The physiological roles of uterine IL-18 and the regulatory mechanisms of IL-18 gene expression are unclear. The present study aimed to clarify the effects of estradiol-17beta (E2) and progesterone (P4) on IL-18 mRNA expression in the mouse uterus. Distribution and expression levels of IL-18 mRNA were studied using an RNase protection assay. Expression of IL-18 mRNA was observed in all organs studied, including testes, ovaries and uteri. The uterine IL-18 mRNA level of estrous mice was higher than that of diestrous mice. E2 treatment (1, 5, 25 or 250 ng/mouse) decreased uterine IL-18 mRNA levels in ovariectomized mice dose-dependently. E2 treatment acutely decreased IL-18 mRNA levels 3 h after injection, but these levels returned to the initial level after 48 h. P4 treatment (1 mg/mouse) decreased uterine IL-18 mRNA levels after 12 h, but levels returned to the initial level after 48 h. Both uterine IL-18 and IL-18Ralpha mRNAs were detected in cultured endometrial epithelial and stromal cells. These results suggest that uterine IL-18 expression is reduced by sex steroid hormones and that IL-18 acts on endometrial cells in a paracrine or autocrine manner.     

Curcumin mitigates fenthion-induced testicular toxicity in rats: histopathological and immunohistochemical study

Fenthion is a widely used organophosphorus pesticide in agriculture that induces different cytotoxic effects, including male reproductive toxicity. The present work aimed to study the ameliorative effects of curcumin, a potential therapeutic agent against several chronic diseases, on reproductive toxicity induced by the organophosphate insecticide fenthion. Forty adult male albino rats were divided into four groups. The control group received distilled water. The curcumin group was administered curcumin at a dose of 100 mg kg^?1 body weight. The fenthion group was administered fenthion at a dose of 0.001 mg kg^?1. The fenthion/curcumin group was administered fenthion and curcumin together at the same doses. After four weeks of daily oral administration, the animals were sacrificed and their testes were excised. Specimens were processed for histopatholological and immunohistochemical investigations. Light and electron microscopic analysis visualised dramatic degenerative changes in seminiferous tubules as indicated by atrophy, necrosis, vacuolation and decreased number of spermatogenic cells. Moreover, the fenthion group showed a significant reduction in the proliferating cell nuclear antigen (PCNA)-immunoreactivity. Decreased PCNA-immunoreactivity reflected the depletion in the proliferation rate of spermatogenic cells and suggesting arrested spermatogenesis. Curcumin administration to fenthion-treated rats revealed mild degenerative changes with partial improvement of active spermatogenesis. In conclusion, these data may confirm the cytoprotective potency of curcumin against fenthion-induced cyto-toxicity.     

雄性雏鸵鸟生殖器官的形态学研究

为了探明雄性雏鸵鸟生殖器官的形态学特点,采用石蜡切片和HE染色技术,对6羽45日龄雄性非洲雏鸵鸟的生殖器官进行了研究,并与家禽的相关结构进行了比较。在光镜下观察了雄雏鸟生殖器官的形态结构,结果表明:(1)鸵鸟睾丸没有睾丸纵隔和睾丸小叶,曲精小管中精原细胞排列紧密且整齐,睾丸间质较少;(2)附睾由睾丸网、输出小管和附睾管组成;(3)输精管管壁由黏膜、肌层和外膜3层组成;(4)阴茎由2个圆形的纤维性淋巴体、淋巴间隙和微血管共同构成。     

Testicular damage after exposure to carbendazim depends on the number of patent efferent ductules.

To study how long-term testicular damage depends on the patency of the efferent ductules (EFDs), rat testes and epididymides were examined after a single exposure to carbendazim (methyl 2-benzimidazole carbamate; MBC). The number of patent EFDs was determined in sections of the caput epididymides at 8, 16, 32 and 70 days post-treatment, and the testes were grouped into the following categories: those with intact EFDs, those with partially patent EFDs, or those with totally occluded EFDs. In each testis, 100 seminiferous tubules were examined for the presence of abnormalities. The mean weight of testes with partially patent EFDs was significantly higher compared with the control, whereas that of testes with totally occluded EFDs was significantly lower. Histologically, most seminiferous tubules of the testes with intact EFDs were normal. The testes with partially patent EFDs contained normal, degenerative and atrophic seminiferous tubules at various frequencies depending on the number of patent EFDs, and it was evident that as the number of patent EFDs increased, the number of normal seminiferous tubules also increased at any interval. In these testes, the number of normal seminiferous tubules increased progressively as the post-treatment interval increased, irrespective of patency of the EFDs. In the testes with totally occluded EFDs, atrophic seminiferous tubules were the most numerous. These results indicate that whether or not the testis is able to survive the long-term deleterious effects of MBC depends largely EFD patency.     

Seasonal Changes in the Immunolocalization of Cytoskeletal Proteins and Laminin in the Testis of the Black‐Backed Jackal (Canis mesomelas)

Manipulation of the reproductive activity of jackals is dependent on a thorough understanding of the reproductive biology of this species. This study describes seasonal morphological changes in the adult testis of the black‐backed jackal in relation to the immunoexpression of the basement membrane marker, laminin and the cytoskeletal proteins, cytokeratin, smooth muscle actin and vimentin. Laminin was immunolocalized in basement membranes surrounding seminiferous tubules, as well as in basement membranes associated with Leydig, peritubular myoid and vascular smooth muscle cells. Scalloped basement membranes enclosed seminiferous tubules in regressing testes. The seminiferous epithelium and interstitial tissue in all animals studied were cytokeratin immunonegative. Smooth muscle actin was demonstrated in vascular smooth muscle cells, as well as in peritubular myoid cells encircling seminiferous tubules. Vimentin immunoreactivity was exhibited in the cytoplasm of Sertoli cells, Leydig cells, vascular endothelial cells, vascular smooth muscle cells and fibrocytes. Vimentin immunostaining in Sertoli, Leydig and peritubular myoid cells varied depending on the functional state of the testis. The results of the study have shown that dramatic seasonal histological changes occur in the testes of the jackal. In addition, the use of immunohistochemistry accentuates these morphological changes.     

The morphological postnatal development of the testis of the Nubian bucks

This study was designed to monitor the morphological development of the reproductive tract of the Nubian bucks in relation to puberty. Thirty-two Nubain male kids were used in the study. The animals were slaughtered at intervals of 2 weeks starting from 1 day old up to 24 weeks of age. Tissue samples were obtained from the testes and processed for ultrastructural studies. The boundary tissue of the newly forming seminiferous tubule adhered closely to the basal lamina. It consisted of a single continuous layer of myoid cells, the outer surface of which was covered by scattered fibroblasts. The ultrastructural study of the boundary of the seminiferous tubule revealed that it consisted of three layers; inner fibrous, middle and outer cellular. The seminiferous tubules at week one were lined by two layers of epithelia; spermatogonia and Sertoli cells in the basal layer, and primary spermatocytes in the second layer. A gradual increase in the diameter of the tubules and epithelial height continued to increase with age. Furthermore, spermatocytes number showed an increase with age. In conclusion, based on the appearance of spermatozoa in the lumina of the seminiferous tubules, puberty age was achieved between weeks 18 and 20.     

Klinefelter syndrome (39 XXY) in an adult Siberian tiger (Panthera tigris altaica).

Fibroblast cultures of a skin biopsy from an adult intact male Siberian tiger (Panthera tigris altaica) revealed an abnormal standard and G-banded karyotype diploid chromosome number of 2n = 39 XXY due to an extra sex chromosome as opposed to the expected 2n = 38 XY. The tiger was euthanatized 1 yr later due to acute multifocal intervertebral disc disease. Histopathology of the reproductive tract demonstrated a paucity of seminiferous tubules and these were devoid of spermatagonia. An increase in fibrous connective tissue was noted in sections of the prostate and epididymis, and expansion of the fibrous interstitium was observed in the testes.     

Effects of chronic heat stress on the reproductive capacity of male Rex rabbits

The effects of chronic heat stress on the reproductive system of rabbits have not been thoroughly examined. Therefore, we investigated the effects of heat stress on the reproductive performance of male Rex rabbits and documented the recovery process after stress withdrawal. Thirty age-matched, male Rex rabbits were placed into artificial climate chambers and randomly divided into three groups: control, heat stress, and recovery. Our results indicate that both libido of rabbit and sperm density were decreased significantly during the 9 weeks exposure to heat stress. Semen volume was mildly affected. Histomorphological and TUNEL assay results showed that chronic heat treatment adversely affected microscopic structure of the testes, and elevated the apoptosis of germ cells in the seminiferous tubules distinctly. However, these adverse effects induced by heat were transient as all measured parameters returned to control levels following the recovery period.     

Administration of estradiol-3-benzoate down-regulates the expression of testicular steroidogenic enzyme genes for testosterone production in the adult rat

ABSTRACT. To study the role of estrogen in the testes, testosterone and testicular steroidogenic enzyme mRNA levels were investigated in male Sprague-Dawley rats 24 hr after intramuscular administration of a single dose of estradiol-3-benzoate (EB). EB administration resulted in a greater decrease in intra-testicular and serum testosterone in 10-week-old rats than in 3- or 5-week-old rats. A dose of 2 microg EB/kg had the lowest observed effect. The level of serum luteinizing hormone (LH) was unchanged at any dose. Semiquantitative RT-PCR analysis revealed that, of the four major testicular steroidogenic enzymes, mRNA levels of cytochrome P450 side-chain cleavage and 17beta-hydroxysteroid dehydrogenase type-III were significantly reduced, and mRNA levels of cytochrome P450 17alpha-hydroxylase/ C17-20 lyase (P450c17) were reduced severely and significantly, by EB administration. However, the level of 3beta-hydroxysteroid dehydrogenase type-I mRNA was not changed. In addition, the P450c17 mRNA level in EB-treated rats was much lower than that in the testes of hypophysectomized rats, with the level in the latter being equal to that in control rats. LH is secreted into blood periodically, the effects of estrogen on the LH secretion pattern of the pituitary gland, for example, in frequency and amplitude of LH pulse, were difficult to detect with the methods of the present study. The results indicated, at least, that EB administration down-regulates P450c17 gene expression predominantly, resulting in the inhibition of testosterone production. From the differences in the steroidogenic enzyme expressions between hypophysectomized and EB-treated rats, it was suggested that EB acts on the testis directly or indirectly though not via alteration of LH secretion and induces reduction of P450c17 mRNA level.