Role of estrogen in spermatogenesis in initial phase males of the three‐spot wrasse (Halichoeres trimaculatus): Effect of aromatase inhibitor on the testis

The three‐spot wrasse, Halichoeres trimaculatus, is a protogynous hermaphrodite. Under appropriate social conditions, female fish can become male. Previous studies indicated that estrogens are important regulators of sex change in this fish. However, the role of estrogen in the male is not known. To clarify the involvement of estrogen in spermatogenesis in hermaphrodite fish, we treated initial phase (IP) males for 10 weeks with exemestane, an aromatase inhibitor (AI), to block estrogen synthesis. Fish treated with AI exhibited decreases in gonadal weight, plasma estrogen levels, and spermatogonial proliferation in the testis, together with increases in androgen levels. Additionally, we confirmed that exogenous estrogen treatments stimulated the renewal and proliferation of spermatogonia in the testis of IP males. These results indicate that estrogens play an important role in regulating spermatogenesis in this fish. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Effect of intensive exercise‐induced testicular gametogenic and steroidogenic disorders in mature male Wistar strain rats: a correlative approach to oxidative stress

Aims: In order to investigate the effects of intensive exercise on reproductive dysfunctions in relation to oxidative stress, a total of 12 male rats (age: 3 months, weight: 127 ± 2.86 g) were randomly divided into: (1) control group (CG, n = 6) and (2) experimental group (Exp. G, n = 6). Methods: An exercise protocol of 3 h swimming day^?1, 5 days week^?1 was followed for 4 weeks in Exp. G, with no exercise in CG. All the animals were killed; blood, testes and the accessory sex organs were collected for estimation of different parameters. Results: A significant diminution (P < 0.001) was noted in testicular Δ⁵, 3β‐hydroxy‐steroid dehydrogenase (Δ⁵, 3β‐HSD), 17β‐hydroxy steroid dehydrogenase (17β‐HSD); plasma levels of testosterone, luteinizing hormone (LH); preleptotine spermatocytes (pLSc), midpachytene spermatocytes (mPSc) and stage 7 spermatids (7Sd); with no significant alteration in follicle stimulating hormone (FSH) and spermatogoia A (Asg) after intensive exercise. A significant elevation (P < 0.001) in malondialdehyde (MDA) and conjugated dienes (CD) along with significant reduction (P < 0.001) in glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione‐s‐transferase (GST) and peroxidase were found in testes of Exp. G. Moreover, the somatic index of testes and accessory sex organs were also decreased significantly (P < 0.001) after exercise. High correlations have been found in 17 β‐HSD with CAT (r = 0.90, P < 0.05) and peroxidase (r = 0.83, P < 0.05), epididymal somatic index with CD (r = ?0.91; P < 0.05) and GSH (r = 0.84, P < 0.05). Conclusion: The present study focused an chronic intensive exercise‐induced oxidative stress that may cause dysfunctions in male reproductive system including steroidogenesis and spermatogenesis.     

Characterization of histamine projections and their potential cellular targets in the mouse retina

The vertebrate retina receives histaminergic input from the brain via retinopetal axons that originate from perikarya in the posterior hypothalamus. In the nervous system, histamine acts on three G-protein-coupled receptors, histamine receptor (HR) 1, HR2 and HR3. In order to look for potential cellular targets of histamine in the mouse retina, we have examined the retina for the expression of histamine and the presence of these three receptors. Consistent with studies of retina from other vertebrates, histamine was only found in retinopetal axons, which coursed extensively through the ganglion cell and inner plexiform layers. mRNA for all three receptors was expressed in the mouse retina, and immunohistochemical studies further localized HR1 and HR2. HR1 immunoreactivity was observed on dopaminergic amacrine cells, calretinin-positive ganglion cells and axon bundles in the ganglion cell layer. Furthermore, a distinct group of processes in the inner plexiform layer was labeled, which most likely represents the processes of cholinergic amacrine cells. HR2 immunoreactivity was observed on the processes and cell bodies of the primary glial cells of the mammalian retina, the Müller cells. This distribution of histamine and its receptors is consistent with a brain-derived source of histamine acting on diverse populations of cells in the retina, including both neurons and glia.     

芳香化酶在成年小鼠脊髓的表达

目的:探讨芳香化酶在正常成年小鼠脊髓组织中的表达特征。方法:取成年C57SJL小鼠,应用免疫组织化学、免疫荧光、免疫印迹检测正常雄性与雌性成年小鼠脊髓组织芳香化酶的分布特征。结果:芳香化酶在正常成年小鼠脊髓组织中的颈、胸、腰段均有表达,主要在灰质神经元中表达,尤其是第Ⅳ、Ⅴ、Ⅶ、Ⅸ层。脊髓腰段前角芳香化酶阳性运动神经元数量在雌雄间无明显的差异。结论:正常成年小鼠脊髓组织表达芳香化酶,不仅在后角感觉神经元中,前角运动神经元也确有表达,且其分布特征在雌雄间无明显差异。     

Immunolocalization of Androgen Receptor and Estrogen Receptors in Skin Tags

Abstract

Skin tags (STs) are benign connective tissue tumors of the dermis. Several clinical observations suggested the involvement of sex steroids in their development. This study aimed at investigating the possible role of androgen receptor (AR) and estrogen receptors (ERs) in STs pathogenesis through their immunohistochemical (IHC) localization in skin biopsies of this disease and to correlate their expression with different clinical and histopathological parameters. Using IHC techniques, we examined 62 cases with STs and 30 gender- and age-matched, healthy subjects, representing the control group. ERα, ERβ, and AR were upregulated in STs compared to normal skin in epidermis and dermis (p?<?.001 for all). Higher AR H score was significantly associated with axillary STs (p?=?.02), skin colored tags (p?=?.03), acanthosis, and papillomatosis (p?=?.04 for both). Higher ERα H score was significantly associated with hyperpigmented tags (p?<?.001) and positive family history (p?=?.003). Higher ERβ H score was significantly associated with female gender and obesity (p?=?.004 for both). Higher ERα and AR H scores were significantly associated with loose collagen arrangement (p?=?.02, p?=?.004, respectively). Higher AR, ERα, and ERβ H scores were significantly associated with the presence of mast cells (p?=?.01, p?=?.04, p?=?.002, respectively) and dilated blood vessels (p?=?.006, p?=?.04, p?=?.04, respectively). In conclusion, AR and ERs may share in STs pathogenesis through their effect on keratinocytes, fibroblasts, and mast cells.     

Immunolocalization of aromatase in human minor salivary glands of the lower lip with primary Sjogren's syndrome

The enzyme aromatase Is Involved In the conversion of androgens to estrogens and in the modulation of various androgenlc and estrogenlc actions. Abnormalities of estrogen metabolism have been postulated to play roles in the development and/or pathophyslology of Sjdgren's syndrome. In the present study, aromatase was immunolocal-ized In 75 cases of Inflammatory disorders of human minor salivary glands of the lower lip. These included cases of primary Sjögren's syndrome (19 cases), of chronic slaladenitis (34 cases) and of mucous extravasation cysts (22 cases), in order to clarify the possible involvement of in situ estrogen production in primary Sjögren's syndrome. Aromatase Immunoreactlvlty was detected In myoepithelial cells of acini and in interstitial cells adjacent to acini and ducts In 13/19 (68%) cases of primary Sjögren's syndrome. In contrast, aromatase expression was detected In only six of 34 (18%) cases of chronic sialadenttis and in seven of 22 (32%) cases of mucous extravasation cyst. These results suggest that Increased aromatase expression in minor salivary glands with primary Sjogren's syndrome in premenopausal women may be involved in the biological features of primary Sjogren's syndrome through the production of estrogens in situ and possibly through the aggravation of the inflammatory reaction.     

Sex and androgenic steroid receptor expression in hepatic adenomas

Sex hormones and anabolic-androgenic steroids are implicated in the development and progression of hepatic adenomas (HA). We studied the expression of their receptors in HA and adjacent liver. Archival tissue sections of 27 HA (16 resections, four needle biopsies, seven aspirations) from 18 patients, and the adjacent liver, were immunostained with monoclonal antibody to estrogen receptor (ER, 1/80) (Dako, Carpinteria, CA), progesterone receptor (PR, 1/50) (BioGenex, San Ramon, CA), and androgen receptor (AR, 1/80) (BioGenex). An avidin-biotin complex technique was used with microwave antigen retrieval. Nuclear expression was assessed as 1 + to 3+ intensity, with semiquantitation of the percentage of nuclei immunopositive. Five percent or more nuclei immunopositive was regarded as positive. The 18 patients included 16 females of 34 years mean age (range, 16 to 49) with an available history of oral contraceptives in five; the two men were 24 and 30 years, with no history of androgenic steroids. ER, PR, and AR were present in seven (26%) (1 ± 2+ intensity, 5% to 10% of nuclei) of HA, seven (26%) (1 ± 2+ intensity, 5% to 30% of nuclei) and nine (33%) (1 ± 3+ intensity, 5% to 80% of nuclei), respectively. In the adjacent liver in 11 cases, there were one (9%) ER, (2+ intensity, 5% of nuclei), four (36%) PR (1 ± 2+ intensity, 5% to 20% of nuclei), and two (18%) AR (2 ± 3+ intensity, 10% of nuclei). Receptors are present and may mediate the action of sex hormones or androgenic steroids on HA and adjacent liver, but in less than one third of patients. This may have therapeutic implications.     

F4/80-positive cells rapidly accumulate around tubuli recti and rete testis between 3 and 4 weeks of age in the mouse: an immunohistochemical study

PROBLEM: Previous studies demonstrated that F4/80 antigen (murine macrophage-specific antigen)-positive cells in testes of normal adult mice accumulate particularly in the interstitium adjacent to the tubuli recti and rete testis (i.e., the central region). However, it remains unknown whether this accumulation is a congenital or acquired phenomenon. METHOD OF STUDY: The distribution of F4/80-positive cells on frozen sections of testes obtained from various aged mice was immunohistochemically examined to determine when the positive cells specifically accumulate in the central region. RESULTS: F4/80-positive cells were homogeneously distributed throughout the testicular interstitium with no specific accumulation until 2 weeks of age. However, at 3 weeks of age, the density of positive cells in the central region became slightly, but significantly, higher than that in the interstitium between the seminiferous tubules. Between 3 and 4 weeks of age, the cell density in the central region increased rapidly, the density at 4 weeks of age reaching the level of the mature testes of 8-week-old mice. CONCLUSION: These results demonstrate that the specific accumulation of F4/80-positive cells in the central region is an acquired phenomenon, which starts and ends before puberty.     

Testosterone-immunopositive primordial germ cells in the testis of the bullfrog, Rana catesbeiana

In amphibia, steroidogenesis remains quiescent in distinct seasonal periods, but the mechanism by which spermatogenesis is maintained under low steroidogenic conditions is not clear. In the present study, testosterone location in the testes of Rana catesbeiana was investigated immunohistochemically during breeding (summer) and nonbreeding (winter) periods. In winter, the scarce interstitial tissue exhibited occasional testosterone immunopositivity in the interstitial cells but the cytoplasm of primordial germ cells (PG cells) was clearly immunopositive. By contrast, in summer, PG cells contained little or no immunoreactivity whereas strong immunolabelling was present in the well-developed interstitial tissue. These results suggest that PG cells could retain testosterone during winter. This androgen reservoir could be involved in the control of early spermatogenesis in winter and/or to guarantee spermiogenesis and spermiation in the next spring/summer. The weak or negative immunoreaction in the summer PG cells might reflect consumption of androgen reservoir by the intense spermatogenic activity from spring to summer. Thus, besides acting as stem cells, PG cells of R. catesbeiana could exert an androgen regulatory role during seasonal spermatogenesis.     

Immunohistochemical Analysis of Histone H3 Modifications in Germ Cells during Mouse Spermatogenesis

Histone modification has been implicated in the regulation of mammalian spermatogenesis. However, the association of differently modified histone H3 with a specific stage of germ cells during spermatogenesis is not fully understood. In this study, we examined the localization of variously modified histone H3 in paraffin-embedded sections of adult mouse testis immunohistochemically, focusing on acetylation at lysine 9 (H3K9ac), lysine 18 (H3K18ac), and lysine 23 (H3K23ac); tri-methylation at lysine 4 (H3K4me3) and lysine 27 (H3K27me3); and phosphorylation at serine 10 (H3S10phos). As a result, we found that there was a significant fluctuation in the modifications; in spermatogonia, the stainings for H3K9ac, H3K18ac, and H3K23ac were strong while that for H3K4me3 was weak. In spermatocytes, the stainings for H3K9ac, H3K18ac, H3K23ac, and H3K4me3 were reduced in the preleptotene to pachytene stage, but in diplotene stage the stainings for H3K18ac, H3K23ac, and H3K4me3 seemed to become intense again. The staining for H3K27me3 was nearly constant throughout these stages. In the ensuing spermiogenesis, a dramatic acetylation and methylation of histone H3 was found in the early elongated spermatids and then almost all signals disappeared in the late elongated spermatids, in parallel with the replacement from histones to protamines. In addition, we confirmed that the staining of histone H3S10phos was exclusively associated with mitotic and meiotic cell division. Based upon the above results, we indicated that the modification pattern of histone H3 is subject to dynamic change and specific to a certain stage of germ cell differentiation during mouse spermatogenesis.     

The relationship between mouse thymocyte receptors for syngeneic and allogeneic erythrocytes and receptors for IgG

Mouse thymocytes lose the affinity for syngeneic erythrocytes in the rosette formation test after heating in the isotonic medium for 1 h at 45 degrees. The thymocyte receptors for syngeneic erythrocytes appear in the medium after heating. The rosetting of heated and washed thymocytes is restored after incubation with the supernatant obtained from heating in medium thymocytes. The receptors for allogeneic erythrocytes were not separated from thymocytes under the same conditions of heating and washing. The receptors for syngeneic erythrocytes separated by heating can be adsorbed on the column with IgG-Sepharose conjugate and probably are connected with the receptors for Fc portion of IgG.     

新生小鼠胃内组胺免疫反应细胞的形态及分布

目的：观察出生后小鼠胃内组胺免疫反应细胞的个体发生、分布、形态及数量变化。方法：免疫组织化学技术。结果：小鼠出生后第5天,胃体部粘膜上皮中出现组胺阳性细胞,此后随着胃体部粘膜的发育,组胺阳性细胞数量明显增多,密集分布于胃体部粘膜下1/3处的上皮内。胃粘膜下层中也可见少量组胺弱阳性细胞。上皮内的组胺阳性细胞多为闭合型,胞体较小,常聚集、环抱壁细胞。结论：小鼠胃体部粘膜中组胺阳性细胞出现的时间较G细胞、D细胞、EC细胞晚,随着小鼠的生长发育,其数量呈显著性增加。位于胃粘膜下1/3处上二皮内的组胺阳性细胞可能为肠嗜铬样细胞（ECL细胞）,ECL细胞释放的组胺,有可能通过旁分泌的方式作用于壁细胞。     

小鼠胃肠道胃泌素细胞的个体发生

目的：观察小鼠胃肠道内胃泌素细胞(G细胞)的个体发生、分布、形态及数量变化,为进一步揭示G细胞的功能及相关研究提供形态学基础。方法：免疫组织化学技术。结果：胎龄17d小鼠胃窦部出现G细胞,18d小肠中出现G细胞。小鼠胃肠道G细胞的数量胚胎期以十二指肠段最多,而出生后胃窦部最多。可见G细胞的突起伸抵上皮游离面,伸至其它细胞之间,或伸至基膜及毛细血管周围,并可见免疫反应阳性物质逸入腔内和逸出纽胞。结论：小鼠胃肠道G细胞的发生与数量变化有其种属特异性。G细胞可能同时具备内分泌、旁分泌和外分泌方式。     

增殖细胞核抗原在小鼠生后肾脏发育中的表达

目的：观察小鼠出生后肾脏发育过程中增殖细胞核抗原（PCNA）的表达特征,探讨出生后小鼠肾脏发育过程中细胞增殖的规律。方法：应用免疫组织化学技术检测小鼠出生后1、3、7、14、21、28和70d肾脏PCNA的表达。结果：小鼠出生后1～70d,皮质中的生肾区、肾小体、肾小管、髓放线以及髓质中的肾小管和集合管PCNA阳性细胞的表达具有一定的规律,早期PCNA阳性表达丰富,随着肾脏发育逐渐成熟而表达减弱直至消失。在70d成年小鼠肾脏中,没有检测到PCNA阳性细胞。结论：出生后小鼠肾脏皮质中的生肾区、肾小体、肾小管、髓放线以及髓质中小管的细胞增殖规律是由高逐渐降低的,直至成年完全停止。     

不同ER、PR状态乳腺癌中AR的表达及其意义

目的探讨AR在不同ER、PR状态乳腺癌中的表达及意义。方法采用免疫组化方法检测AR、ER、PR在173例乳腺癌中的表达,依据结果分组:(1)AR状态分组:AR阳性组和AR阴性组;(2)ER、PR状态分组:En组(ER、PR均阴性)、Ep组[ER和(或)PR阳性];(3)AR、ER、PR联合分组:En-AR+(En组且AR阳性)、En-AR-(En组且AR阴性)、Ep-AR+(Ep组且AR阳性)、Ep-AR-(Ep组且AR阴性),其中En-AR-又称为均阴性组,其他三组统称为部分或完全阳性组。不同分组方法比较与临床病理特征的关系。结果Ep组AR阳性率62.8%(54/86),En组AR阳性率37.9%(33/87),两组差异有显著性(P=0.001),AR阳性组体积小、核分裂少、组织学分级低(P0.05);En-AR-组表现为核分裂多、组织学分级高(P0.01),此外En组内AR阳性者核分裂少、组织学分级低(P0.05),Ep组内AR阳性者临床分期高(P=0.000),En-AR+、Ep-AR+、Ep-AR-比较均无差异。结论AR在不同激素状态乳腺癌中表达的意义不同,ER、PR均阴性乳腺癌表达AR者预后较好,ER、PR阳性乳腺癌表达AR者临床分期高。在选择针对性药物时应考虑到不同激素受体状态的组合。     

Atypical development of Sertoli cells and impairment of spermatogenesis in the hypogonadal (hpg) mouse

Testes of hypogonadal (hpg) mice show arrested postnatal development due to congenital deficiencies of gonadotrophin-releasing hormone (GnRH) and gonadotrophin synthesis and secretion. Follicle-stimulating hormone (FSH), androgen or oestrogen treatment restore qualitatively normal spermatogenesis in hpg testes. Understanding the cellular and molecular changes accompanying hormone-induced spermatogenesis in hpg mice requires detailed morphological analyses of the germ cells and Sertoli cells in the untreated hpg testis. We compared seminiferous epithelial cytology in adult hpg, immature and adult wild-type mice using unbiased optical disector-based stereology, immunolocalization of Sertoli cell microtubules (MT), espin (a component of the blood-testis barrier), markers of Sertoli cell maturity (p27(kip1) and WT-1), and electron microscopy. Hpg testes had marked reductions in weight, seminiferous cord volume and length, and severe spermatogenic impairment with germ cells per testis < 1% of adult wild-type testes. Sertoli cell nuclei expressed WT-1 in hpg testes, but often were centrally located, similar to 9-14-day-old wild-type testes, and they expressed p27(kip1), indicating that hpg Sertoli cells were post-mitotic. Hpg testes had significantly (P < 0.05) reduced Sertoli cells per testis (0.56 million) compared with 10-day wild-type (1.15 million) and adult wild-type testes (2.06 million). Immunofluorescence labelling of normal adult Sertoli cells showed supranuclear MT columns and basally located espin, but these features were absent in 10-day-old and hpg Sertoli cells. Hpg Sertoli cells showed pleomorphic nuclear ultrastructure with mature-type nucleoli, similar to normal adult-type Sertoli cells, but hpg Sertoli cells exhibited incomplete tight junctions that lacked ectoplasmic specializations. We conclude that in hpg mice, chronic gonadotrophin insufficiency restrains Sertoli cell proliferation and maturation, forming pseudo-adult-type Sertoli cells that are incapable of supporting germ cell proliferation and maturation.     

Gene suppression of mouse testis in vivo using small interfering RNA derived from plasmid vectors

We evaluated whether inhibiting gene expression by small interfering RNA (siRNA) can be used for an in vivo model using a germ cell-specific gene (Tex101) as a model target in mouse testis. We generated plasmid-based expression vectors of siRNA targeting the Tex101 gene and transfected them into postnatal day 10 mouse testes by in vivo electroporation. After optimizing the electroporation conditions using a vector transfected into the mouse testis, a combination of high- and low-voltage pulses showed excellent transfection efficiency for the vectors with minimal tissue damage, but gene suppression was transient. Gene suppression by in vivo electroporation may be helpful as an alternative approach when designing experiments to unravel the basic role of testicular molecules.     

An insight into insulin-like factor 3 regulate its receptor RXFP2 in mouse gubernaculum testis cells

The etiology of testicular dysgenesis syndrome is multifactorial and involves abnormalities in the anatomical structures and endocrine factors. Several studies have shown that the abnormal development of the gubernaculum may affect testicular descent, and the insulin-like factor 3 (INSL3) appears to play an important role in development of the gubernaculum have been proved. INSL3 binds its specific receptor (Relaxin family peptide 2, RXFP2), which was highly expressed in gubernaculum, to produce a crucial effect in the first transabdominal descent stage, but its mechanism still remain unclear. In this study, in order to explore how does INSL3 regulate its receptor RXFP2, we cultured mouse gubernaculum testis cells in vitro, which was treated by INSL3, and examined the expression of RXFP2 in mouse gubernaculum testis cells. The results displayed that INSL3 changed RXFP2 expression, and we found that low dose INSL3 can increase RXFP2 expression, the mechanism of above-mentioned might be related with the hormesis of INSL3.