Cultured Testis Fragments Research Articles

Age-related changes in human Leydig cell status.

What are the consequences of ageing on human Leydig cell number and hormonal function? Leydig cell number significantly decreases in parallel with INSL3 expression and Sertoli cell number in aged men, yet the in vitro Leydig cell androgenic potential does not appear to be compromised by advancing age. There is extensive evidence that ageing is accompanied by decline in serum testosterone levels, a general involution of testis morphology and reduced spermatogenic function. A few studies have previously addressed single features of the human aged testis phenotype one at a time, but mostly in tissue from patients with prostate cancer. This comprehensive study examined testis morphology, Leydig cell and Sertoli cell number, steroidogenic enzyme expression, INSL3 expression and androgen secretion by testicular fragments in vitro. The majority of these endpoints were concomitantly evaluated in the same individuals that all displayed complete spermatogenesis. Testis biopsies were obtained from 15 heart beating organ donors (age range: 19-85 years) and 24 patients (age range: 19-45 years) with complete spermatogenesis. Leydig cells and Sertoli cells were counted following identification by immunohistochemical staining of specific cell markers. Gene expression analysis of INSL3 and steroidogenic enzymes was carried out by qRT-PCR. Secretion of 17-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone by in vitro cultured testis fragments was measured by LC-MS/MS. All endpoints were analysed in relation to age. Increasing age was negatively associated with Leydig cell number (R = -0.49; P < 0.01) and concomitantly with the Sertoli cell population size (R= -0.55; P < 0.001). A positive correlation (R = 0.57; P < 0.001) between Sertoli cell and Leydig cell numbers was detected at all ages, indicating that somatic cell attrition is a relevant cellular manifestation of human testis status during ageing. INSL3 mRNA expression (R= -0.52; P < 0.05) changed in parallel with Leydig cell number and age. Importantly, steroidogenic capacity of Leydig cells in cultured testis tissue fragments from young and old donors did not differ. Consistently, age did not influence the mRNA expression of steroidogenic enzymes. The described changes in Leydig cell phenotype with ageing are strengthened by the fact that the different age-related effects were mostly evaluated in tissue from the same men. In vitro androgen production analysis could not be correlated with in vivo hormone values of the organ donors. In addition, the number of samples was relatively small and there was scarce information about the concomitant presence of potential confounding variables. This study provides a novel insight into the effects of ageing on human Leydig cell status. The correlation between Leydig cell number and Sertoli cell number at any age implies a connection between these two cell types, which may be of particular relevance in understanding male reproductive disorders in the elderly. However aged Leydig cells do not lose their in vitro ability to produce androgens. Our data have implications in the understanding of the physiological role and regulation of intratesticular sex steroid levels during the complex process of ageing in humans. This work was supported by grants from Prin 2010 and 2017. The authors have no conflicts of interest. N/A.

Testicular function in cultured postnatal mouse testis fragments is similar to that of animals during the first wave of spermatogenesis.

Previously, we evaluated optimal organ culture conditions to produce elongated spermatids in an in vitro mouse testis culture system. However, differences in testicular function between the cultured testis fragments and animal testis have not been determined. To examine how closely cultured testis fragments in vitro approximates what typically occurs during the first wave of spermatogenesis in vivo, C57BL/6J mouse testis fragments obtained on postnatal day (PND) 5 were cultured in AlbuMAX™ I/ α-Minimal Essential Medium for 15, 23, 30, 35, 42, and 49 days, and compared to mouse testes obtained at PND 5, 14, 20, 24, 28, 30, 35, and 40. At the specified days of culture or PND of mice, the following analyses were conducted: histology, flow cytometry for haploid cell detection, qPCR for spermatid markers, and liquid chromatography/mass spectrometry for testosterone levels. Round spermatids were initially observed at 23 days, and their percentage of the total number of cells continued to increase with culture time, as did gene expression of the spermatid markers and haploid cell percentage in the cultured testis fragments. These results were similar in temporal sequence to those in animals. Testosterone levels in the testis fragments reached a maximum at Day 49. These findings show this in vitro mouse testis organ culture model may be a useful and convenient tool for mechanistic studies. However, because germ cell differentiation in all seminiferous tubules was not observed, improvements in the system/methods are needed to more closely replicate spermatogenesis as observed in animals.

Gene expression profiling of cultured mouse testis fragments treated with ethinylestradiol.

The assessment of xenobiotic-induced testicular toxicity is important in drug development. Nonetheless, in vitro models to test drugs and chemicals that may cause testicular toxicity are lacking, requiring the continued use of animal models for those studies. We previously evaluated an in vitro mouse testis organ culture system using ethinylestradiol (EE), a well-studied testicular toxicant, and demonstrated a dose-dependent relationship between adverse effects to germ cell differentiation and increasing EE concentrations. However, we terminated that study after 20 days of culture due to oxygen deficiency during germ cell differentiation. Therefore, in the current study, we aimed to identify gene(s) with potential for supporting the histopathological evaluations of testicular toxicity using in vitro testis organ culture system. We cultured testis fragments obtained from mice at postnatal day (PND) 5 in α-Minimal Essential Medium containing 40 mg/mL AlbuMAX™ I and treated them with 0.01 or 1 nM EE on day 1 of culture. On day 20, we collected testis fragments for RNA sequencing analysis and quantitative polymerase chain reaction (qPCR). We found that phospholipase C, zeta 1 and testis-specific serine kinase 4 genes, that are involved in spermatogenesis and predominantly expressed in the testis, were significantly reduced in testis fragments treated with the highest concentration of EE. Also, cytochrome P450, family 26, subfamily b, polypeptide 1 (Cyp26b1) and interleukin 16 (Il16) were up-regulated in the highest EE-treated groups. Further studies are needed to confirm the variations of these gene expression using other testicular toxicants.

An in vitro system for the long-term tissue culture of juvenile rainbow trout (Oncorhynchus mykiss) testis

In vitro cultivation of fish gonad fragments continues to be an important experimental approach to answer both fundamental and applied scientific questions. The aim of the present investigation was to test whether juvenile rainbow trout (Oncorhynchus mykiss) testes cultured for a week or more were physiologically competent. Trout testis fragments (approximately 1 mm2) were placed on pieces of flat, culture plate insert filter, in a drop of liquid medium (modified Leibovitz's L-15), and floated on the medium surface in a multi-well culture plate. Culture plates were covered and incubated in air at 15 degrees C. Three different endpoints were used to test whether cultured testis fragments remained healthy and functional. First, a comparison of the histological appearance of testis fragments cultured in vitro for different periods up to 8 days with intact testes indicated that no differences were evident. Secondly, testis fragments incubated in medium supplemented with bovine calf serum (BCS) at concentrations of 2.5% and 25% BCS had significantly greater proliferation of interstitial and spermatocyst cells as measured by nuclear 5-bromo-2'-deoxyuridine incorporation. Finally, testis fragments cultured for 5 days and transplanted back into the donor fish underwent precocious sexual maturity in response to a regime of salmon pituitary extract injections and produced fertile sperm as determined by their ability to successfully fertilize eggs. These experiments demonstrate the utility of this method for in vitro culture of juvenile rainbow trout testis fragments.

Regulatory role of BMP2 and BMP7 in spermatogonia and Sertoli cell proliferation in the immature mouse.

The aim of this study was to determine the action of bone morphogenetic proteins (BMPs) on testicular cell proliferation during early postnatal life, a definite developmental time at which crucial changes in germ cell and Sertoli cell maturation occur. We investigated the effect of BMP2 and BMP7, two factors which belong to the relatively distant decapentaplegic (DPP) and 60 A classes of the large BMP family, upon spermatogonial and Sertoli cell proliferation, and we examined the expression of activin/BMP type II and type I receptors. We used in vitro cultured testis fragments from 7-day-old mice, highly purified populations of somatic and germ cells and total testes from mice of different ages. Cell proliferation was assessed by BrdU labelling and [3H]-thymidine incorporation. Ribonuclease protection assays and Northern blotting were performed to analyse receptor expression. We have demonstrated a stimulatory action of BMP2 and BMP7 in spermatogonia and Sertoli cell proliferation respectively. ActRIIB is the type II receptor expressed most in spermatogonia, whereas Sertoli cells specifically expressed BMPRIIB, in addition to ActRIIB. By contrast, the presence of ActRIIA was undetectable in either germ or somatic cells. The type I receptors ActRIA, ActRIB and BMPRIA were all found in both cell types, indicating that the observed effect of BMP2 and BMP7 on testicular cell proliferation may be mediated by a number of combinatorial interactions in the receptor complexes. These findings suggest that BMPs are involved in physiological paracrine signalling during the first wave of spermatogenesis.

245.Characterisation of prostaglandin production in the normal and inflamed rat testis

Prostaglandins E2 (PGE2) and F2α (PGF2α) play a role in Leydig cell function and in suppression of macrophage inflammatory functions. We predict that PGs also may play a role in interstitial fluid (IF) formation in the testis. Prostaglandin synthesis involves one of two distinct forms of cyclooxygenase (COX): constitutively expressed COX-1 and inducible COX-2. We recently demonstrated expression of both enzymes in macrophages, somatic and germ cells of the adult rat testis, and that COX-2 may the more important form in this organ. Adult male Sprague-Dawley rats were maintained on normal feed or 0.15% celebrex, a specific COX-2 inhibitor, for 5 weeks. Rats were subsequently treated with saline, or lipopolysaccharide (LPS; 0.1 mg/kg or 5 mg/kg), 6 h prior to collection of tissues. PGE2 was measured by RIA in medium of cultured testis fragments and testicular cells from normal rats (� 10 μg/mL LPS, 24 h, 37�C), and in testicular interstitial fluid. PGE2 was constitutively produced by whole testis, Sertoli cells, Leydig cells and round spermatids, but not by resting macrophages or pachytene spermatocytes in culture. Stimulation with LPS upregulated PGE2 in macrophage cultures, but not in other cells or whole testis. Normal PGE2 levels in IF were 16–20 ng/mL; levels were not altered by low-dose LPS, but were reduced by high-dose LPS. Celebrex caused a reduction in IF PGE2 levels in both the normal and low-dose groups, but not in the high-dose group. Celebrex elevated IF volume (25–50%) in all groups. Our experiments show cell-type specific regulation of PGE2 production in the rat testis, and predict a role for COX-2 elicited PGs in the IF regulation and in post-meiotic cell function. Paradoxically, low-level inflammation does not alter testicular PGE2 levels, as somatic and germ cells, which do not respond to LPS, appear to contribute most to the local levels of PGE2.