Davor Ježek

The expression of neurotrophins and their receptors in the prenatal and adult human testis: evidence for functions in Leydig cells

Previous studies have demonstrated local functions for neurotrophins in the developing and mature testis of rodents. To examine whether these signaling molecules are present and also potentially active in the human testis, we characterized immunohistochemically the expression and cellular localization of the known neurotrophins and their receptors during prenatal testicular development as well as in the adult human testis. Results obtained revealed the presence of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 and 4, as well as neurotrophin receptors p75NTR, TrkA, TrkB, and TrkC during testis morphogenesis. These proteins were also detectable in the adult human testis, and their local expression could be confirmed largely by immunoblot and RT-PCR analyses. Remarkably, the Leydig cells were found to represent the predominant neurotrophin/receptor expression sites within both fetal and adult human testes. Functional assays performed with a mouse tumor Leydig cell line revealed that NGF exposure increases cellular steroid production, indicating a role in differentiation processes. These findings support previously-recognized neuronal characteristics of Leydig cells, provide additional evidence for potential roles of neurotrophins during testis morphogenesis and in the mature testis, and demonstrate for the first time a neurotrophin-induced functional activity in Leydig cells.

Catecholamine-synthesizing enzymes in the adult and prenatal human testis

Catecholamines play functional roles in the mature and developing mammalian testis but the cell types responsible for their local synthesis are still controversially discussed. Here, we demonstrate that four enzymes involved in the biosynthesis of catecholamines, namely, tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine β-hydroxylase (DBH) and phenylethanolamine- N-methyltransferase (PNMT), are expressed in Leydig cells of the human testis. Tyrosine hydroxylase, the key enzyme of the biosynthesis of catecholamines, was localized to Leydig cells both at the transcript level (by RT-PCR analyses and by in situ hybridization assays) and at the protein level (by immunoblotting and by immunohistochemistry). The other enzymes were also demonstrated in Leydig cells by RT-PCR and immunohistochemical analyses. The presence of TH, AADC, DBH, and PNMT in human Leydig cells was found, in addition, by immunohistochemical approaches carried out on sections from prenatal human testes. Thus, the present study identifies the Leydig cells as the presumed sites of catecholamine production in both the mature and fetal human testes and further supports the previously recognized neuroendocrine characteristics of this cell type.

Effects of high doses of testosterone propionate and testosterone enanthate on rat seminiferous tubules — a stereological and cytological study

The effects of exogenous testosterone on various testicular variables has become of increasing significance because of its potential use in male contraception. For this reason, high doses of two testosterone esters [testosterone propionate (TP) and testosterone enanthate (TE)] were used in a study of their influence on the morphology, length and curvature of the seminiferous tubules of the rat testis, and on cytological smears of the seminiferous tubules epithelium. TP was given for 14 days (3 mg/100 g body weight, i. m.) to assess the acute effects of testosterone on the seminiferous tubules. TE was administered for 60 days (in the same manner as TP) to study possible chronic effects on the rat testis. After TP and TE treatment the seminiferous tubule epithelium showed disorganization and desquamation of spermatogenic cells. In the TP-treated testes the tubules lined with Sertoli cells only were observed. The values for the length and curvature of seminiferous tubules of the TP- and TE-treated rats were significantly reduced (p<0.001). All these changes were observed earlier in the TP-treated than in the TE-treated animals. In cytological smears of the testis of the TP- and TE-treated rats an increase of vacuoles and residual bodies in Sertoli cell cytoplasm was noted. In addition, a reduction of spermatogenic cells, particularly sperms, was manifest in the smears after treatment. Large groups of Sertoli cells were seen in the smears from these testes.

Thrombosis following ovarian hyperstimulation syndrome

Abstract The aim of this review is to analyse the pathophysiology and complications of thrombosis in conjuction with ovarian hyperstimulation syndrome (OHSS) following ovulation induction and to suggest practical guidelines usefull for the prevention and treatment. Although the incidence of thrombosis varies from 0.2% among in vitro fertilization (IVF) cycles and up to 10% for severe cases of the syndrome, it represents the most dangerous complication of OHSS. Different changes in haemostatic markers have been found to create a state of hypercoagulability, but no single standard test is available to estimate the state of thrombosis. The role of markers for thrombophilia is controversial. Thromboses are mostly venous (67–75%) involving upper limbs and neck, then arterial (25–33%) which are mainly intracerebral. The predominant sites of venous thromboembolism in the upper part of the body may be explained by higher concentrations of estrogens drained through lymphatic ducts from ascites and by compression of rudimentary branchyal cysts. Once early diagnosis is established, it is crucial to use an anticoagulant treatment with heparin proceeded with thromboprophylaxis. However, identification of patients at risk and preventive measures of OHSS are the best means in reducing the risk of thrombosis after ovarian stimulation. Chinese abstract 本综述的目的是分析促排卵导致的卵巢过度刺激综合征（OHSS）并发血栓形成的病理生理学特征与并发症，并针对其预防与治疗提出实用的临床指南。虽然体外受精治疗中血栓形成的发生率介于0.2∼10%之间，它却是OHSS最危险的并发症。现已发现了多种造成高凝状态的凝血标志物，但还没有单一的检验标准用于评估血栓形成的状态。关于标志物在血栓形成倾向中所起的的作用存在争议。血栓多为静脉血栓（67∼75%），包括上肢与颈部；而动脉血栓（25∼33%）多位于大脑内部。静脉血栓多位于身体上部，可能是由于腹水中较高浓度的雌激素经淋巴管排出，及残留囊肿的加压作用。一旦早期诊断确立，重要的是应用抗凝血剂治疗及肝素预防血栓。但是，鉴定高危患者与预防OHSS的发生是降低卵巢刺激后血栓风险的最好办法。

Investigation of Spermatozoa and Seminal Plasma by Fourier Transform Infrared Spectroscopy

Fourier transform infrared (FT-IR) spectra of human spermatozoa and seminal plasma were recorded and analyzed. The procedure that was established for sample preparation enabled acquisition of reproducible spectra. The parameter I1087/I966 for controlling spectra reproducibility was defined. The assignment of bands was carried out using an empirical approach and the origin of the “sperm specific doublet”, the bands at 968 cm−1 and 981 cm−1, was determined. The principal component regression (PCR) algorithm was used to define the specific spectral regions correlating to characteristics of spermatozoa, such as concentration, straight-line velocity (VSL), and beat cross frequency (BCF). Then, simple spectral parameters, such as band intensities and band ratios, were tested to determine which one best correlates to characteristics of spermatozoa. The region of the amide I band, between 1700 cm−1 and 1590 cm−1, was defined as a specific spectral region that correlates to the concentration of spermatozoa. The parameter that gave the linear dependence to the concentration of spermatozoa was the intensity of the amide I band. For VSL, the bands between 1119 cm−1 and 943 cm−1 were defined as the specific spectral region. The relative amount of nucleic acids with respect to proteins showed linear dependence on the straight-line velocity of spermatozoa. BCF showed the best correlation to the bands between 3678 cm−1 and 2749 cm−1, which largely represent lipids and proteins. These results suggest that FT-IR spectroscopy can serve as an adjunct to conventional histopathology studies.

Dopamine agonists in prevention of ovarian hyperstimulation syndrome

Abstract The aim of this review is to analyze the efficacy of different dopamine agonists in the prevention of ovarian hyperstimulation syndrome (OHSS). Cabergoline, quinagolide and bromocriptine are the most common dopamine agonists used. There are wide clinical variations among the trials in the starting time (from the day of human chorionic gonadotrophin (hCG) to the day following oocyte retrieval); the duration of the treatment (4–21 days), the dose of cabergoline (0.5 mg or 0.25 mg orally) and in the regimens used. At present, the best known effective regimen is 0.5 mg of cabergoline for 8 days or rectal bromocriptine at a daily dose of 2.5 mg for 16 days. Dopamine agonists have shown significant evidences of their efficacy in the prevention of moderate and early-onset OHSS (9.41%), compared with a placebo (21.45%), which cannot be confirmed for the treatment of late OHSS. It would be advisable to start with the treatment on the day of hCG injection or preferably a few hours earlier. The use of dopamine agonists should be indicated in patients at high risk of OHSS, as well as in patients with a history of previous OHSS even without evident signs of the syndrome. Chinese abstract 这篇综述的目的是分析不同的多巴胺受体激动剂在预防卵巢过度刺激综合征（OHSS）中的疗效。卡麦角林，喹高利特和溴隐亭是最常见的多巴胺受体激动剂。在本研究的开始阶段，卡麦角林的使用时间（从HCG日至取卵日4天-21天不等），使用剂量（0.5mg或0.25mg口服）和使用方案等临床因素变化很大。目前，最有效的方案是卡麦角林每天0.5mg口服8天，或者直肠给药0.25mg每天，使用16天。多巴胺受体激动剂有显著地预防中度和早发性卵巢过度刺激综合征(9.41%)的疗效，与对照组（21.45%）相比较，发病率显著降低，但是其治疗晚期OHSS的效果尚不确定。目前，多建议在HCG注射的当日开始使用，提前几个小时效果可能更好。多巴胺受体激动剂应当对具有高OHSS发生风险的患者以及之前有OHSS史,甚至没有发病迹象的患者也可以使用。

EFFECTS OF ORCHIECTOMY ON THE RAT PAROTID GLAND : AN ULTRASTRUCTURAL AND STEREOLOGICAL STUDY

The relation of the tests/testosterone with the structure and function of the mammalian parotid gland has so far been poorly investigated. The present study deals with the morphology of the rat parotid gland and its changes after orchiectomy and testosterone substitution. The glands of control and experimental animals (orchiectomized and orchiectomized with testosterone substitution) were analyzed by electron microscopy and stereology. In orchiectomized animals 30-60 days after castration, a significant reduction of the volume of the acini and the duct system as well as a significant increase of the connective tissue volume per cubic millimeter of the gland were noted. The volume and length of the intercalated and the striated ducts per cubic millimeter of parotid tissue are significantly reduced 45-60 days after orchiectomy. Excretory ducts seem to be unaffected by orchiectomy. The structure of the rat parotid acini is also changed by castration, indicating a reduction of acinar-cell activity. In controls, the volume of acinar cells with wide cisternae of rough endoplasmic reticulum is 3 times larger than the volume of acinar cells with regular and narrow cisternae of rough endoplasmic reticulum. After orchiectomy, the volume of acinar cells with wide cisternae of rough endoplasmic reticulum is significantly decreased, while the volume of acinar cells with regular and narrow cisternae of rough endoplasmic reticulum is significantly increased. Exogenously given testosterone can prevent or alleviate the mentioned effects of orchiectomy on the gland. It is concluded that orchiectomy affects the rat parotid gland, demonstrating the existence of an interaction between the testis and the mammalian parotid gland.

Macrophages and Leydig cells in testicular biopsies of azoospermic men.

A number of studies have indicated that testicular macrophages play an important role in regulating steroidogenesis of Leydig cells and maintain homeostasis within the testis. The current paper deals with macrophages (CD68 positive cells) and Leydig cells in patients with nonobstructive azoospermia (NOA). Methods employed included histological analysis on semi- and ultrathin sections, immunohistochemistry, morphometry, and hormone analysis in the blood serum. Histological analysis pointed out certain structural changes of macrophages and Leydig cells in NOA group of patients when compared to controls. In the testis interstitium, an increased presence of CD68 positive cells has been noted. Leydig cells in NOA patients displayed a kind of a mosaic picture across the same bioptic sample: both normal and damaged Leydig cells with pronounced vacuolisation and various intensity of expression of testosterone have been observed. Stereological analysis indicated a significant increase in volume density of both CD68 positive and vacuolated Leydig cells and a positive correlation between the volume densities of these cell types. The continuous gonadotropin overstimulation of Leydig cells, together with a negative paracrine action of macrophages, could result in the damage of steroidogenesis and deficit of testosterone in situ.

Chemically Defined Protein-Free in vitro Culture of Mammalian Embryo Does Not Restrict Its Developmental Potential for Differentiation of Skin Appendages

In a unique serum- and protein-free chemically defined in vitro culture model of postimplantation mammalian development the epidermis differentiates regularly, although the differentiation of other tissues is impaired due to the lack of the serum. The present study in that model was done to estimate more carefully the degree of epidermal differentiation in defined media supplemented with some growth- or differentiation-stimulating substances. The main objective was to discover by grafting in vivo to the richer environment whether simple protein-free culture conditions restrict an inherent embryonic potential for differentiation of skin appendages. Embryonic parts of E9.5 gastrulating Fischer rat embryos were cultivated for 2 weeks in the protein-free Eagle’s minimum essential medium supplemented with holotransferrin, apotransferrin, insulin and/or Na2SeO3 and in controls cultivated in protein-free medium or in serum-supplemented medium. In all experiments there was a high incidence of differentiation of the epidermis. A high level of epidermal differentiation was confirmed for the first time at the ultrastructural level. A well-differentiated cornified layer and cells connected with desmosomes containing keratohyaline masses and cytokeratin filaments were found. A strong immunohistochemical signal for the proliferating cell nuclear antigen was always detected in the basal layer of the epidermis showing that those cells were still able to proliferate. Finally, embryos precultivated for 1 or 2 weeks in the protein-free medium and media supplemented with apotransferrin or serum were grafted under the kidney capsule for an additional 2 weeks. It was discovered that even after spending 2 weeks in the simple protein-free medium in vitro, embryos retained their developmental potential for differentiation of skin appendages (hair and sebaceous glands).

Localisation of lanosterol 14α-demethylase in round and elongated spermatids of the mouse testis: an immunoelectron microscopic and stereological study

Abstract Lanosterol 14α-demethylase (CYP51) is a microsomal cytochrome P450 enzyme involved in the postsqualene cholesterol biosynthetic pathway. CYP51 removes 14α-methyl group from lanosterol], forming FF-MAS (folicular fluid meiosis activating sterol) which accumulates in gonads. The goal of our study is to determine the expression of CYP51 protein in the male gonad. Using electron microscopic immunogold techniques, CYP51 is localised on inner and outer acrosomal membranes of male germ cells, the round and elongated spermatids. Significance of CYP51 localization on the acrosome which is a Golgi-derived organelle is not known, but we propose that CYP51-formed FF-MAS can function as a signalling sterol during fertilisation.