Spermatogenetic Process Research Articles

Effect of chocolate and Propolfenol on rabbit spermatogenesis and sperm quality following bacterial lipopolysaccharide treatment

The aims of the study were to evaluate the effects of chocolate and propolis-enriched diets on rabbit spermatogenesis, sperm motility, and ultrastructure following bacterial lipopolysaccharide (LPS) treatment. Thirty-two New Zealand White rabbits were divided into four groups. The LPS-Propolfenol® group received propolis (500 mg/kg/day) in their diet for 15 days, while the LPS-chocolate group was fed 70% cacao chocolate (1 g/1 kg/day) for the same period. Following the diet treatments, rabbits in the LPS-Propolfenol® and LPS-chocolate groups, and an LPS group received a single intraperitoneal dose of 50μg/kg LPS, and the control group received only saline. Kinematic sperm traits were evaluated with a computer assisted sperm analyzer (CASA) system, and ultrastructural characteristics were examined by transmission electron microscopy (TEM). Testicular and epididymal tissues were observed by light microscopy and TEM and multiplex real time reverse transcriptase-polymerase chain reaction (RT-PCR) assay was used to detect and quantify toll-like receptor-4 (TLR-4) gene expression. The values of the analyzed semen parameters of rabbits treated with LPS-Propolfenol® and LPS-chocolate did not show any variations compared with the control group, but they were lower in rabbits treated only with LPS. Alterations observed in the testicular tissue of LPS treated-rabbits were not detected in specimens from the LPS-chocolate and LPS-Propolfenol® groups, which showed normal spermatogenesis. The TLR-4 mRNA expression was similar in controls, in LPS treated, and in LPS-chocolate groups, but it was significantly (p < 0.01) decreased in LPS-Propolfenol® rabbits. In conclusion, a chocolate and propolis-enriched diet showed a protective effect on the spermatogenetic process of buck rabbits following LPS treatment.

Molecular chaperones, cochaperones, and ubiquitination/deubiquitination system: involvement in the production of high quality spermatozoa.

Spermatogenesis is a complex process in which mitosis, meiosis, and cell differentiation events coexist. The need to guarantee the production of qualitatively functional spermatozoa has evolved into several control systems that check spermatogenesis progression/sperm maturation and tag aberrant gametes for degradation. In this review, we will focus on the importance of the evolutionarily conserved molecular pathways involving molecular chaperones belonging to the superfamily of heat shock proteins (HSPs), their cochaperones, and ubiquitination/deubiquitination system all over the spermatogenetic process. In this respect, we will discuss the conserved role played by the DNAJ protein Msj-1 (mouse sperm cell-specific DNAJ first homologue) and the deubiquitinating enzyme Ubpy (ubiquitin-specific processing protease-y) during the spermiogenesis in both mammals and nonmammalian vertebrates.

Uncommon formation of two antiparallel sperm bundles per cyst in tenebrionid beetles (Coleoptera)

Several species of Tenebrionidae are stored-grain pests. Since they belong to a specious family, the systematics of these beetles is still in doubt. In insects, spermatogenesis and the spermatozoa exhibit great diversity, and are therefore commonly used in phylogenetic and taxonomic analyses. During the spermatogenetic process in these organisms, the cells originating from a single spermatogonium develop synchronically in groups referred to as cysts. At the end of this process, there is usually only one sperm bundle per cyst, with all the cells in the same orientation. This paper details the spermiogenesis of the tenebrionid beetles Tenebrio molitor, Zophobas confusa, Tribolium castaneum and Palembus dermestoides using whole mount and histological sections of the cysts. In these species, spermatogenesis is similar to that which occurs in most insects. However, during spermiogenesis, the nuclei of the spermatids migrate to two opposite regions at the periphery of the cyst, leading to the uncommon formation of two bundles of spermatozoa per cyst. This feature is possibly an apomorphy for Tenebrionidae.

The apoptotic function analysis of p53, Apaf1, Caspase3 and Caspase7 during the spermatogenesis of the Chinese fire-bellied newt Cynops orientalis.

BackgroundSpontaneous and stress-induced germ cell apoptosis during spermatogenesis of multicellular organisms have been investigated broadly in mammals. Spermatogenetic process in urodele amphibians was essentially like that in mammals in spite of morphological differences; however, the mechanism of germ cell apoptosis in urodele amphibians remains unknown. The Chinese fire-belly newt, Cynops orientalis, was an excellent organism for studying germ cell apoptosis due to its sensitiveness to temperature, strong endurance of starvation, and sensitive skin to heavy metal exposure.Methodology/Principal FindingsTUNEL result showed that spontaneous germ cell apoptosis took place in normal newt, and severe stress-induced apoptosis occurred to spermatids and sperm in response to heat shock (40°C 2 h), cold exposure(4°C 12 h), cadmium exposure(Cd 36 h), and starvation stress. Quantitative reverse transcription polymerase chain reactions (qRT-PCR) showed that gene expression of Caspase3 or Caspase7 was obviously elevated after stress treatment. Apaf1 was not altered at its gene expression level, and p53 was significantly decreased after various stress treatment. Caspase assay demonstrated that Caspase-3, -8,-9 enzyme activities in newt testis were significantly elevated after heat shock (40°C 2 h), cold exposure(4°C 12 h), and cadmium exposure(Cd 36 h), while Caspase3 and Caspase8 activities were increased with Caspase9 significantly decreased after starvation treatment.Conclusions/SignificanceSevere germ cell apoptosis triggered by heat shock, cold exposure, and cadmium exposure was Caspase3 dependent, which probably involved both extrinsic and intrinsic pathways. Apaf1 may be involved in this process without elevating its gene expression. But starvation-induced germ cell apoptosis was likely mainly through extrinsic pathway. p53 was probably not responsible for stress-induced germ cell apoptosis in newt testis. The intriguing high occurrence of spermatid and sperm apoptosis probably resulted from the sperm morphology and unique reproduction policy of Chinese fire-belly newt, Cynops orientalis.

Complex chromosomal rearrangements in infertile males: complexity of rearrangement affects spermatogenesis

In this report, we describe 10 male cases of complex chromosome rearrangements (CCRs) with fertility problems: seven of them showed impairment of spermatogenesis, oligoasthenoteratozoospermia or azoospermia; in the other three cases, recurrent abortions were observed. The CCRs were characterized by conventional fluorescence in situ hybridization (FISH) and multicolor FISH methods as well as by the routine G-banding technique. CCRs found in three cases with recurrent abortions were double two-way exchanges, which were the simplest forms of CCRs; three oligoastenoteratozoospermic cases were double two-way exchanges or three-way exchanges. However, the CCRs in four azoospermic cases were much more complicated forms of CCRs. From our results and a review of the literature, we conclude that the complexity of CCRs might affect the severity of spermatogenetic impairment rather than the number of chromosomes involved or the location of breakpoints.

Local desynchronization of cellular development within mammalian male germ cell clones

Mammalian male germ cells go through the process of spermatogenesis as cell clones. The members of a clone are joined by intercellular bridges which are responsible for synchronizing the spermatogenetic process. The present ultrastructural study on pubertal and adult golden hamster and mouse testes, however, demonstrates that a strict synchronization of germ cell development is not attained by this clonal association in any single instance. Local desynchronization is either indicated by intraclonal divergence at the stage of the cell cycle of the respective germ cells or it is manifested in clones in which single cells are affected by degeneration while other members of the clone are not. The results are discussed with a view to a possible significance of intraclonal desynchronization of male germ cell development in local impairment of spermatogenesis and loss of germ cells.

Morphological appraisal of gametogenesis. Spermatogenetic process in mammals with particular reference to man.

The process of spermatogenesis in man can be subdivided into prenatal, postnatal and adult phases. Special attention is devoted to the cytomorphology of the germ cells in these phases, with a more detailed description of the proliferation, maturation and differentiation of the germ cells in the adult. Thus, spermatogonial renewal, meiotic division and the morphological transformations of the spermatids into mature spermatozoa are described. An outline of the structures characteristic of the latter is also given. The histological organization of the seminiferous epithelium is considered, whereby the concepts of the cycle, stage of the cycle and wave of the seminiferous epithelium are discussed. The duration of both the cycle and that of spermatogenesis of the adult are considered. The present review on mammalian, and chiefly human spermatogenic processes aims at: surveying of current concepts, and compiling of the more important facts and problems of spermatogenesis. The nomenclature and definitions advanced here arise from the concept of continuity of the processes of spermatogenesis from embryo to adult.

Effects of L-Acetylcarnitine (LAC) on the Post-Injury Recovery of Mouse Spermatogenesis Monitored by Flow Cytometry 1. Recovery after X-Irradiation/Über den Einfluß von L-Acetylcarnitin (LAC) auf die Wiederherstellung der Mäusespermatogenese mittels Überw

L-acetylcarnitine plays a key role in sperm metabolism and in the whole spermatogenetic process. In the present work, the influence of L-acetylcarnitine, administered i.p. (100 mg/kg body weight), on the recovery processes of mouse spermatogenesis after local acute irradiation with 10 Gy X-rays has been investigated. The effects were monitored 28, 35, 40, 45, 50, 55, and 60 days after irradiation by flow cytometric analysis of cellular DNA content. In the LAC-treated animals, the fraction of tetraploid cells is higher at 28 (p less than 0.05) and 45 days (p less than 0.02). Corresponding with the timing of the stages of murine spermatogenesis, the round spermatid fraction is higher at 45 days (p less than 0.1) and the elongated spermatid fraction is higher at 50 days (p less than 0.1) after irradiation. In addition, the LAC-treated animals show a faster recovery throughout the maturation process, from tetraploid to round and elongated spermatids. These results indicate that the presence of exogenous LAC could enhance the recovery of spermatogonial cells.

In vitro spermatogenesis as a method to bypass pre-meiotic or post-meiotic barriers blocking the spermatogenetic process: genetic and epigenetic implications in assisted reproductive technology

Pregnancies achieved by assisted reproduction technologies and particularly by ooplasmic injections of either in vivo or in vitro generated immature male germ cells are susceptible to genetic risks inherent to the male population treated with assisted reproduction and additional risks inherent to these innovative procedures. The documented, as well as the theoretical risks, are discussed in this review. These risks represent mainly the consequences of genetic abnormalities underlying male infertility and may become stimulators for the development of novel approaches and applications in the treatment of infertility. Recent data suggest that techniques employed for in vitro spermatogenesis, male somatic cell haploidization, stem cell differentiation in vitro and assisted reproductive technology may also affect the epigenetic characteristics of the male gamete, the female gamete, or may have an impact on early embryogenesis. They may be also associated with an increased risk for genomic imprinting abnormalities. Production of haploid male gametes in vitro may not allow the male gamete to undergo all the genetic and epigenetic alterations that the male gamete normally undergoes during in vivo spermatogenesis.

Sertoli cell index and spermatic reserves in adult captive African lions ( Panthera leo, Linnaeus, 1758)

The intrinsic yield of spermatogenesis and the supporting indexes of the Sertoli cells are the best indicators for the spermatic production capacity in a species. The aim of the present study was to quantify the intrinsic yield of the spermatogenetic process, as well as the Sertoli cell index and spermatic reserves. Testicular fragments of five adult African lions was fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 μm thickness. In the seminiferous epithelium of the African lions, 10.3 primary spermatocytes at pre-leptotene phase are produced by the type-A spermatogonia. During meiotic divisions, only 2.7 spermatids were produced from the primary spermatocytes. The general spermatogenesis production in the African lions was approximately 22.1 cells, and each Sertoli cell was able to sustain and maintain approximately 14.9 cells of the germinative line, from which 7.9 are round spermatids. A total of 103 × 10 6 spermatozoa are produced by each testis gram at each cycle of the seminiferous epithelium. The spermatic reserve of lion is below the amplitude observed in mammals.

Spermatozoa and Chronic Treatment with Finasteride: A TEM and FISH Study

Finasteride is a specific inhibitor of the 5alpha reductase enzyme originally approved for the treatment of benign prostatic hypertrophy and also for the treatment of androgenetic alopecia (AGA) in men at a dose of 1 mg/day. We report on three cases of young men recruited at our Centre for Male Infertility who had used finasteride for five years. Semen quality was investigated by light microscopy to evaluate sperm concentration and motility. Sperm morphology was performed by transmission electron microscope (TEM) and the data were analyzed. The presence of Y microdeletions was investigated by PCR. Meiotic segregation was explored by fluorescence in situ hybridization (FISH). Patient 1 was azoospermic, patients 2 and 3 showed a normal sperm concentration and severely reduced progressive motility. TEM analysis revealed altered sperm morphology consistent with necrosis and FISH data revealed elevated diploidy and sex chromosome disomy frequencies. This examination was repeated 1 year after the men had suspended the use of finasteride, without receiving any other treatment. A recovery of spermatogenetic process was observed. Motility and morphology improved whereas the meiotic pattern did not change presenting elevated diploidy and sex chromosome disomy frequency.

Response: Responsiveness to Tamoxifen Citrate and Testosterone Undecanoate Is Independent of the Severity of Idiopathic Oligozoospermia

To the Editor: I am sorry for the misunderstanding and thank the Editor because he gave me the opportunity to explain the mechanisms that provoked my mistake. My confusion originates from the following wording: “total sperm count × 106/mL” present in the abstract and in table 1, page 916, of the Adamopoulos article (Adamopoulos et al, 2003). In this regard, I have intended “sperm concentration/mL,” instead of total ejaculated sperm. The World Health Organization (WHO) handbook does not list any sperm pattern alike: “total sperm count × 106/mL,” and, most importantly, fractional values (ie,… × 106/mL) always indicate a concentration (WHO, 1999). Almost all articles regarding therapy for oligoasthenospermia take into consideration sperm concentration per milliliter instead of total ejaculated sperm (WHO, 1989, 1992; Rege et al, 1997; Kamischke et al, 1998; Rolf et al, 1999; Vicari and Calogero, 2001; Foresta et al, 2002; Wong et al, 2002; Cavallini et al, 2003; Lenzi et al, 2003, 2004; Zawackzi et al, 2003). It is uncommon to present data regarding number of total ejaculated sperm, even though an increase in sperm concentrations in OAT (oligo-astheno-teratospermia) infertile males has been thought to be associated with disproportionately higher fecundability (Adamopoulos et al, 2003), because the in vivo and in vitro fertilization proved to be more strictly linked to the quality of spermatogenesis than to the number of ejaculated sperm (Tomlinson et al, 1992; Patrizio et al, 1994; Parinaud et al, 1996a,b; Aboulghar et al, 1997; Verheyen et al, 1997; De Croo et al, 2000). In this regard, sperm concentration per milliliter is more closely linked to the spermatogenetic process than to the number of total ejaculated sperm (Biagiotti et al, 2002).

Estudo morfológico do ciclo reprodutivo e variabilidade nos níveis de glicose, de glicogênio e de lipídeo ao longo do ano em pato doméstico (Anas platyrhynchus)

Histologic studies on the seminiferous epithelium were performed to determine the reproductive annual cycle of domestic duck in mesothermic tropical region and to correlate it with metabolic parameters such as blood glucose, hepatic and muscular glycogen and lipids, which are basic metabolic substrates related to the reproductive process. The annual reproductive cycle of the duck could be divided in four successive phases along the year, which are named reproductive, regressive, quiescent and recrudescent phases. The reproductive phase began in winter (July) reaching its maximum peak in October. The regressive phase occurred in the end of spring and in the beginning of summer (December). The quiescent phase started in the summer between January and February. The recrudescent phase occurred in autumn and winter (June) when the spermatogenetic process resumed. Concerning the energetic metabolism only the blood glucose was related to the reproductive cycle, giving energetic support to this reproductive process

Rapid signal transduction in Sertoli cells.

The importance of non-genomic signaling as a complementary route for cell regulation has recently become evident. This rapid mechanism is utilized not just by peptide hormones, but also by steroids and other lipid-related substances, resulting in amplification and fine-tuning of the signals. The Sertoli cell is the principal target for hormone action in the seminiferous tubules. The involvement of FSH, testosterone and tri-iodothyronine (T(3)) in the spermatogenetic process is widely known. This paper discusses some rapid responses to FSH, retinol, testosterone and T(3) in the control of Sertoli cell function. Studies employing various methodologies and techniques are described and several hypotheses are considered in an attempt to explain the interactions of hormones with plasma membrane receptors. Recent knowledge about these new signaling mechanisms and cross-talk between them opens new fields of research on the communication and integration of the multiple hormonal signaling systems.

In vivo and in vitro effects of prochloraz and nonylphenol ethoxylates on trout spermatogenesis

We investigated the effects of in vivo exposure to non-lethal concentrations of two chemicals commonly discharged into the aquatic environment, prochloraz and nonylphenol diethoxylate (NP2EO – Igepal® 210), on the development of spermatogenesis in trout. The in vitro effects on basal and insulin-like growth factor-1 (IGF-I) stimulated DNA synthesis by early germ cells were also studied. In vivo, rainbow trout were exposed for 2 or 3 weeks to waterborne prochloraz (21 and 175 nmol/l) and/or NP2EO (68–970 nmol/l) renewed continuously, or periodically. Only the highest concentrations of NP2EO (225–970 nmol/l) induced a significant increase in blood plasma vitellogenin in juvenile or maturing male trout. When prepubertal fish were exposed for 15 days to prochloraz, the spermatogenetic process was significantly inhibited as shown by the stage of gonadal development reached 3 weeks after exposure. This effect was, to a great extent, reversible within 9 weeks post-exposure. When fish in the initial stage of spermatogenesis were exposed for 21–27 days to 580 nmol/l NP2EO, a 20–40% reduction of the gonadosomatic index was observed 4.5 weeks post-exposure, and the spermatogenetic process was partly inhibited. In vitro, testicular cells obtained at different stages of spermatogenesis were cultured for 4.5 days in the presence or not of the tested molecules and with IGF-I or not. 3H-thymidine ( 3H-Tdr) incorporation was measured according to Loir (Mol. Reprod. Dev. 53 (1999) 424) and 125I-IGF-I specific binding was determined according to Le Gac et al. (Mol. Reprod. Dev. 44 (1996) 35). Irrespective of the spermatogenetic stage, basal 3H-Tdr incorporation was decreased by prochloraz concentrations ⩾10 μmol/l. The presence of IGF-I (10–100 ng/ml) stimulated 3H-Tdr incorporation; this response to IGF-I began to decrease at 25–50 μmol/l prochloraz. In parallel, a dose-dependent increase of IGF-I specific binding was induced by prochloraz 1–100 μmol/l. Similarly, basal and IGF-I-stimulated 3H-Tdr incorporation was decreased by nonylphenol polyethoxylate (NpnEO; starting at 10 μmol/l), NP2EO and NP (30 μmol/l); a dose-dependent increase of IGF-I specific binding was also induced by NP and NPnEO. While 1–100 nmol/l 17β-estradiol had no effect in our in vitro system, Triton® X-100 acted as NPnEO on 3H-Tdr incorporation. Beside their known endocrine disrupting effects on sex steroid production or action, these lipophilic molecules could act on germ cells by disrupting cell membrane receptivity to peptide hormones like growth factors.

Notes on the presence of a single sperm duct in Procambarus clarkii (Girard, 1852) (Decapoda: Cambaridae) from Italian waters

The present work deals with the discovery of a distinctive morphology in the male reproductive apparatus of the red swamp crayfish, Procambarus clarkii (Girard, 1852), from two Italian lakes (where it was introduced a few years ago from different foreign countries). Anatomy and histology of the male reproductive apparatus were investigated in a large sample of the crayfish populations. Males were characterized by the presence of a tri-lobate testis and, as a rule, of a single sperm duct (on the right body side). Furthermore, a comparison of the same anatomical structures was made taking into consideration two other crayfish species: Astacus leptodactylus and Orconectes limosus. Observations on these crustaceans showed a bi-lobate testis and no morphological differences between the normally developed left and right vasa deferentia. The presence of a single sperm duct and of three testicular lobes in P. clarkii might be related to its burrowing-reproductive habit. One single vas deferens may be an adaptation to burrow environments where copulation is not affected by water flow, while three testicular lobes may act as a mechanism to make the spermatogenetic process active all year round, thus enhancing the probabilities of success in reproduction.

Residual Bodies Stimulate Rat Sertoli Cell Plasminogen Activator Activity

Previous studies suggested indirectly that residual bodies (RB) may play a role in the regulation of the proteolytic system within the testis and in the coordination of the spermatogenetic process. In the present study, we examined the effects of RB recovered from adult rat testes by centrifugal elutriation on Sertoli cell plasminogen activator (PA) levels, by zymography and ELISA procedures. Addition of RB to Sertoli cell cultures prepared from 20-day-old rat testes resulted in a dramatic stimulation of PA. Effects were dose- and time-dependent. Phagocytosis of RB by Sertoli cells leads to a rapid stimulation of Sertoli cell interleukin-1α (IL-1α), a cytokine potentially involved in the regulation of spermatogenesis; the effects of IL-1α were investigated. We found that IL-1α augmented PA levels and that immunodepletion of Sertoli cell–RB cocultures with anti-IL-1α antibodies abrogated the stimulatory effects of RB on PA. Together, the present findings indicate that RB enhance Sertoli cell PA and that IL-1α may be involved in that control.

Testicular Response to Exogenous Gonadotropins in Infant Cynomolgus Monkeys

The response of the testis to exogenous gonadotropins was examined by determining serum testosterone (T) levels and by observing histopathological changes of the testes using 5–7 month-old males. After a single combined injection of 100 IU plus 15 IU human menopausal gonadotropin (hMG) or 500 IU human chorionic gonadotropin (hCG) plus 15 IU hMG, the serum T levels reached peak values 72 h after administration. During repeated aministration of the combined injection of 100 IU hCG plus 15 IU hMG at five 3-day intervals (Day 0, 3, 6, 9, 12), the serum T levels reached peak values 3 days after the first injection, which were about 3 times higher than the mean serum T levels of adult monkeys. The serum T levels decreased gradually on subsequent days of treatment. Three days after the fifth injection (Day 15), the T levels were equal to the T levels of adult monkeys. The testicular volumes on Day 15 were significantly higher than those on the first day of injection. The histopathological examination of the testes (210, 238 mg) obtained from two treated males revealed hyperplasia and hypertrophy of the Leydig cells and spermatogonias, spermatocytes, and mitotic germinal cells were observed in the seminiferous tubule. Only spermatogonia were found in the one testis (150 mg) obtained from an untreated male. From the above results, it was confirmed that the testes of infant cynomolgus monkeys produced T levels in the same way as the testes of adult monkeys in response to the administration of exogenous gonadotropins. Additionally, the initiation of the spermatogenetic process in the testes of infant monkeys was stimulated by exogenous gonadotropins, therefore suggesting that this method could become a useful means to evaluate the testicular function of infant males.

Spermatocytes and round spermatids of rat testis: protein patterns.

Spermatogenesis is a process in the testis that involves meiotic cell division and spermiogenesis. The mechanisms of regulation and its associated proteins are mostly unknown. This publication shows the two-dimensional (2-D) gel electrophoresis protein map obtained from rat testis using nonlinear 3.5-10 immobilized pH gradients for the first-dimensional separation. Eighteen proteins were successfully identified in the SWISS-PROT protein database using amino acid analysis of proteins recovered from polyvinylidene difluoride (PVDF) membranes and verified for one of them by comparison with Anderson's rat liver reference map. Fourteen new polypeptides were identified and four were previously known. Two of these new proteins were closely related to the spermatogenetic process. T-complex protein 1 is expressed in large amounts in germ cells. Androgen-dependent sperm-coating glycoprotein is secreted by epididymal cells. In order to detect changes in protein expression during meiosis and spermiogenesis, spermatocytes and round spermatid cell populations were purified by centrifugal elutriation and compared. In this way several proteins not found in the spermatocyte 2-D images could be high-lighted. The sperm-coating glycoprotein was thus shown to be present in large amounts in round spermatids.

Ultrastructure of primary spermatocyte in fish (Tilapia: Oreochromis niloticus): The synaptonemal complex

The Primary Spermatocytes represent a stage in spermatogenesis when the first meiotic cell division occurs. They are derived from Spermatogonium or Stem cell through mitotic division. At the zygotene phase of meiotic prophase the Synaptonemal complex appears in these cells in the space between the paired homologous chromosomes. Spermatogenesis and sperm structure in fish have been studied at the electron microscope level in a few species? However, no work has yet been reported on ultrastructure of tilapia, O. niloticus, spermatozoa and spermatogenetic process. In this short communication we are reporting the Ultrastructure of Primary Spermatocytes in tilapia, O. niloticus, and the fine structure of synaptonemal complexes seen in the spermatocyte nuclei.