An in vitro prototype of a porcine biomimetic testis-like cell culture system: a novel tool for the study of reassembled Sertoli and Leydig cells / 亚洲男科学杂志(英文版)

At present, there is no reliable in vitro assembled prepubertal testis-like biomimetic organ culture system designed to assess the functional effects of human gonadotropins on Sertoli and Leydig cells. Spermatogenesis is regulated by endocrine, paracrine, and juxtacrine factors (testicular cross-talk), mainly orchestrated by gonadotropins such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH) that play a pivotal role by stimulating Leydig and Sertoli cells, respectively. The aim of our study was to set up an in vitro prepubertal porcine bioengineered construct as a new model for experimental studies on reassembled Sertoli and Leydig cells. We have evaluated Sertoli and Leydig cells obtained from 15- to 20-day-old neonatal pig testes in terms of purity and function. Subsequently, purified Sertoli and enriched Leydig cells were subjected to coincubation to obtain an in vitro prepubertal porcine testis-like culture system. We performed enzyme-linked immunosorbent assay (ELISA) for anti-Müllerian hormone (AMH), inhibin B, and testosterone secretion in the medium, and Real-Time PCR analysis of AMH, inhibin B, FSH-r, aromatase, LHr, and 3β-HSD mRNA expression levels. This in vitro testis-like system was highly responsive to the effects of human gonadotropins and testosterone. AMH mRNA expression and secretion declined, and inhibin-B increased, while FSH-receptor expression was downregulated upon FSH/LH exposure/treatment. Finally, the production of testosterone was increased selectively upon LH treatment. In summary, our proposed model could help to better determine the action of human gonadotropins on Sertoli and Leydig cells. The potential usefulness of the system for shedding light into male infertility-related issues is evident.

Analysis of glucose-dependent insulinotropic peptide receptor (GIPR) and luteinizing hormone receptor (LHCGR) expression in human adrenocortical hyperplasia / Análise da expressão dos receptores do peptídeo insulinotrópico dependente de glicose (GIPR) e do hormônio luteinizante (LHCGR) nas hiperplasias adrenocorticais humanas

OBJECTIVE: To analyze the aberrant expression of the GIPR and LHCGR in different forms of adrenocortical hyperplasia: ACTH-independent macronodular adrenal hyperplasia (AIMAH), primary pigmented nodular adrenocortical disease (PPNAD) and diffuse adrenal hyperplasia secondary to Cushing's disease (DAHCD). METHODS: We quantified GIPR and LHCGR expressions using real time PCR in 20 patients with adrenocortical hyperplasia (seven with AIMAH, five with PPNAD, and eight with DAHCD). Normal adrenals tissues were used as control and the relative expression was compared with β-actin. RESULTS: GIPR and LHCGR expressions were demonstrated in all tissues studied. Median GIPR and LHCGR mRNA levels were 1.6; 0.4; 0.5 and 1.3; 0.9; 1.0 in adrenocortical tissues from AIMAH, PPNAD and DAHCD respectively. There were no differences between GIPR and LHCGR expressions in all tissues studied. CONCLUSIONS: GIPR and LHCGR overexpression were not identified in the studied cases, thus suggesting that this molecular mechanism is not involved in adrenocortical hyperplasia in our patients.

OBJETIVO: Analisar a expressão aberrante do GIPR e do LHCGR em diferentes formas de hiperplasias adrenocorticais: hiperplasia adrenal macronodular independente de ACTH (AIMAH), doença adrenocortical nodular pigmentada primária (PPNAD) e hiperplasia adrenal difusa secundária à doença de Cushing (DAHCD). MÉTODOS: Quantificou-se por PCR em tempo real a expressão desses receptores em 20 pacientes: sete com AIMAH, cinco com PPNAD e oito com DAHCD. Adrenais normais foram utilizadas como controle e a expressão relativa desses receptores foi comparada à expressão da β-actina. RESULTADOS: A expressão desses receptores foi demonstrada em todos os tecidos estudados. A mediana da expressão do GIPR e do LHCGR foi de 1,6; 0,4; 0,5 e de 1,3; 0,9; 1,0 nos tecidos dos pacientes com AIMAH, PPNAD e DAHCD, respectivamente. Não houve diferença significativa na expressão desses receptores nos tecidos estudados. CONCLUSÕES: Hiperexpressão do GIPR e do LHCGR não foi observada, sugerindo que esse mecanismo não está envolvido na patogênese molecular da hiperplasia adrenal nesses pacientes.

Modification of ovine luteinizing hormone subunits with SMPT and its effect on subunit recombination, immunological activity, receptor binding and steroidogenic activity.

The increasing use of heterobifunctional cross-linking agents in the design of defined conjugates for selective targeting and inducing immune response has prompted us to study the role of epsilon-NH2 group modification of oLH subunits, their recombination and effect on immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of alpha oLH and beta oLH subunits were separately modified by using SMPT. The alpha oLH-SMPT modified derivatives hybridize to beta oLH. Similarly, the beta oLH-SMPT derivatives recombined with alpha oLH. The recombination was judged by gel filtration chromatography and RP-HPLC analysis. The sequential modification of subunits led to progressive reduction in immunoreactivity and receptor binding activity. The modification of six or more epsilon-NH2 groups in alpha oLH although recombine fully with native beta oLH but failed to react to anti-oLH antibody. Moreover, the steroidogenic activity was also abolished. Introduction upto four SMPT groups in alpha oLH compromised immunological and biological activities but further addition of two or more SMPT groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two amino groups in the receptor binding and steroidogenic activity. The present investigation clearly demonstrate that only 1:2-3 molar ratio of oLH subunits:SMPT could generate the site(s) in the subunits of the oLH that retained reasonable immunological, receptor binding and biological activity of the hormone. Therefore, this molar ratio may be used in future for the design and synthesis of bioeffective hormonotoxins.

Testosterone productivity and histostructural changes of autotransplanted rat Leydig cells

To investigate the possibility of in vivo transplantation of Leydig cells as a new biologic androgen replacement therapy, the Leydig cells procured from 6 week-old male Sprague-Dawley rats were autotransplanted, and the level of testosterone secretion and histostructural changes were observed. The renal subcapsular and intraperitoneal transplant showed higher levels of testosterone compared to subcutaneous or scrotal counterparts, and the number of transplanted cells was correlated with the level of measured testosterone. Furthermore, if the Leydig cells were transplanted intraperitoneally after the uptake on synthetic collagen, testosterone levels were higher than the ones simply transplanted without synthetic collagen uptake, resulting in 27 fold increase at 3 months. The activity of 125I-hCG decreased 20 to 40% at each month after transplantation compared to the normal levels, but no statistical significance was noted among different periods. The histologic examination revealed neovascularized capillaries and well demarcated sheet-like group of eosinophilic Leydig cells were observed at 4 weeks. But the evidence of destructive changes such as a focal inflammation with central dystropic ossification could be noted after 3 month. On electron microscopy, the marked indentation of nucleus and presence of lipochrome pigment were seen, and the number and size of smooth endoplasmic reticulum and mitochondria were reduced after 3 month. In conclusion, testosterone output could be increased to the physiologic range by increasing the number of transplant cells or utilizing collagen uptake but further effort is necessary on delaying or preventing the structural and functional decrement of Leydig cells.

Binding of radiolabelled luteinizing hormone to intact and ovariectomised rat uterus.

Binding of ovine LH to uterine tissue preparation from intact and ovariectomised rat clearly indicates that uterus possesses specific binding sites for LH. Binding characteristics of LH to uterine tissue preparation from intact rat showed saturability with high affinity and low capacity. Scatchard plot analysis showed dissociation constant of the specific binding site to be 0.12 x 10(-9) mol/l and the number of binding sites was 2.31 +/- 0.05 f mol/mg protein. Ovariectomy did not change the binding affinity but effected a decrease in the number of binding sites (1.7 +/- 0.08 f mol/mg protein). LH treatment of ovariectomized (ovx) rat had no effect on binding affinity but significantly increased the number of binding sites (3.23 +/- 0.1 f mol/mg protein). Reduction of uterine weight due to ovariectomy and marked increase of ovx rat uterine weight by LH administration indicate a source of estrogen in ovx rat. An in vitro uterine tissue slice (from intact and ovx rat) incubation showed depletion of 17 beta-estradiol (E2) content in ovx rat which significantly elevated on LH addition. Data suggest that LH binding to rat uterine tissue has biological relevance.

Effect of lysine residue modification of ovine luteinizing hormone by heterobifunctional crosslinking reagent SPDP on subunit-subunit association, receptor binding and biological activity.

The increasing use of heterobifunctional crosslinking agent in the design of hormone-carrier conjugates for selective targeting or inducing immune response against the hormone has prompted us to study the effect of epsilon-NH2 group modification of oLH-subunit, their recombination, immunoreactivity, receptor binding and biological activity. The epsilon-NH2 groups of oLH alpha and oLH beta subunits were modified by using SPDP. The SPDP modified oLH alpha derivatives hybridize to native OLH beta as judged by RP-HPLC analysis. The sequential modification of alpha and beta subunits led to progressive reduction in immunoreactivity and receptor binding activities. The steroidogenic potential of oLH beta.SPDP.alpha oLH recombinant was relatively comparable. The modification of six or more epsilon-NH2 groups in oLH alpha although recombine fully with native oLH beta but failed to react to anti-oLH antibody. Moreover, steroidogenic activity was also abolished. Introduction up to four SPDP groups in oLH alpha compromised immunological and biological activities but further addition of two more SPDP groups completely abolished antibody reactivity, receptor binding and steroidogenic activity indicating the importance of later two -NH2 groups in the receptor recognition and steroidogenic potential.

Hormonotoxins: synthesis, characterization and bioefficacy of some defined disulfide linked conjugates of ovine LH with a ribosome inactivating protein, gelonin.

In order to synthesise a bioeffective hormonotoxin for selective targeting to specific cells in the gonads, gelonin, a single chain RIP obtained from an Indian plant, Gelonium multiflorum of Euphorbeaceae family was covalently linked to oLH with the use of N-succinimidyl-3-(2-pyridyldithio)propionate, generating a linkage containing a disulfide bond and a amide bond. The hormonotoxins were separated according to their molecular weight (indirectly according to oLH:gelonin molar ratio) and a complete biochemical analysis was performed. The linkage occurred through the epsilon-NH2 group of alpha oLH as judged from RP-HPLC analysis. The conjugates were devoid of ingredients as determined by SDS-PAGE and RP-HPLC analysis. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity of oLH and gelonin to their antibodies. Hormonotoxins bind to the Leydig tumour cells via oLH part leaving gelonin free as judged by competitive displacement analysis. The hormonotoxin was internalized to the sufficient degree to effectively inhibit protein synthesis. The cytotoxicity of 1:1 molar ratio conjugate was relatively higher than that of others. The cytotoxicity of presently described more defined hormonotoxins exhibited higher receptor binding and cytotoxicity than the hormonotoxins reported earlier [Singh, et al., J Biol Chem, 264 (1989) 3089].

Degradación de gonadotropina corioníca humana unida a receptores en células de leydig / Degradation of receptor-bound human chrionic gonadotrophin in leyding cells

Se determina la cinética de degradación de la 125-hCG unida a su receptor en células de Levding, aisladas de ratas maduras. Se analiza el efecto de la temperatura y de agentes lisosomotrópicos sobre la velocidad de degradación del complejo H-R. Se comparan estos resultados con los reportados en la literatura, los cuales se refieren a células de Leyding tumorales de ratón