Sertoli cells

The extent of Sertoli cell proliferation during fetal and neonatal development determines the final adult testis size and potential for sperm output. To gain further knowledge of the factors that regulate Sertoli cell proliferation, the present study used a new approach to analyse changes in morphology and proliferation in the postnatal testis by combining organ culture with morphometric analysis. Fragments of rat testes from days 0 to 10 postpartum were cultured in contact with DMEM for 6 h or 72 h and fixed. The effects of ovine folliclestimulating hormone (FSH) and activin were studied in an additional 72-h organ culture experiment using day 9 testes. Bromodeoxyuridine (BrdU) was added for the last 6 h of culture to mark proliferating cells. Two-µm sections of the fragments were analysed for morphological changes of the seminiferous cords, and the proportion of BrdU-labelled Sertoli and germ cells was determined. Assessment of 6-h samples revealed growth characteristics consistent with those observed in vivo during days 1-10 of postnatal development. From day 2 onwards, the volume fraction of seminiferous cords began to increase, while significant growth in cross-sectional area of the cords occurred only after day 6. In these culture conditions, germ cell proliferation and testicular architecture was consistent with that expected for the age of the tissue at time of explant. The proportion of dividing Sertoli cells declined from 15-20% at days 0-4 postpartum to below 10% at day 10 postpartum in the 6-h culture, and it was low or abolished in the 3-day culture at all time points. Activin and FSH together, but not singly, stimulated Sertoli cell proliferation in the 72-h culture. This paper presents a new approach to analysis of in vitro testis development. The combination of fragment culture and stereological analysis permits rigorous and detailed assessment of developmental changes in the postnatal testis.

The objective was to characterize testicular development in Blackbelly sheep, focusing primarily on Sertoli cell number. Lambs (n = 43) were allotted into eight groups, and surgically castrated at 0, 3, 6, 9, 12, 15,18 or 21 weeks of age (n = 4-6 lambs per group). Testes were fixed and paraffin-embedded, cross-sections (5 mm) were stained and evaluated with quantitative morphometry techniques. Testis weight increased at a greater rate between 9 and 15 weeks of age, which was associated with remarkable changes in testicular histology, including increases in tubular tissue volume, and tubule diameter and length. Spermatogenesis started in a period between 9 and 12 weeks, lumen and elongated spermatids were observed for the first time at 12 weeks (78% of the tubules) and 15 weeks (37% of the tubules), respectively. Total number of Sertoli cells (mean AE S.E.M.) increased steadily from birth (531 AE 76 Â 10 6 ) to 15 weeks (12,008 AE 1722 Â 10 6 ), with no changes afterwards. Sertoli cell number per gram of testicular tissue decreased as lambs were older, with the most remarkable change occurring between Weeks 9 and 12. An early increase in serum LH was observed at 6 weeks of age, with testosterone (T) increasing at Weeks 12 and 21. In conclusion, Sertoli cells maintained the capacity of proliferating from birth to 15 weeks of age in Blackbelly sheep; furthermore, the period of accelerated testis growth was associated with increased serum T concentration and with important changes in testicular morphology, as a consequence of the beginning and establishment of spermatogenesis and Sertoli cell maturation. #

Molecular mechanisms responsible for diabetes-induced testicular dysfunction are not well understood. This study investigated oxidative stress, stage-dependent DNA base modification and expression of poly (ADP ribose) polymerase 1 (PARP1) in the testes of Streptozotocin-induced diabetic rats. Hyperglycemia led to testicular dysfunction characterized by impaired sperm parameters and testicular structure at the end of first (DM1) and third (DM3) month after the induction of diabetes. In the testis, total oxidant levels increased and total antioxidant levels decreased, which led to the induction of oxidative stress status. The oxidative stress up-regulated the levels of 8-oxo-7, 8-dihydro-2'-deoxyguanosine-an oxidized form of the DNA base, deoxyguanosine-in a stage-dependent manner. In DM1, stage VII-IX tubules showed more cytoplasmic expression of 8-oxo-7, 8-dihydro-2'-deoxyguanosine in all germ cell types and the Sertoli cells than did the other stage tubules, which suggested mitochondrial DNA damage. In DM3, mainly a stage-dependent nuclear expression of 8-oxo-7, 8-dihydro-2'-deoxyguanosine was observed in germ cells, but not in the Sertoli cells. Diabetes increased the cytoplasmic expression of 4-hydroxynonenal and concurrently inhibited the expression of both full length and 89 kDa large cleaved-fragment of PARP1 in DM1 and DM3. The germ and Sertoli cells showed the nuclear expression of the protein in a stage-dependent manner, which decreased from DM1 to DM3. The increase in oxidative DNA damage and a decrease in PARP1 led to a stagedependent induction of apoptosis of testicular cells. In conclusion, diabetes-induced oxidative stress, oxidative DNA damage and apoptosis occur in parallel with PARP1 inhibition in the testis.

Yq microdeletions are the leading genetic cause of male infertility and its detection in clinically relevant for appropriate genetic counseling. The objective of this study was to determine the frequency of Y microdeletion in a group of Tunisian infertile men and to compare the prevalence of these abnormalities with other countries and other Tunisian reported series. Totally, 105 Tunisian idiopathic infertile men (74 azoospermic and 31 severe oligozoospermic) were screened for the presence of Y chromosome microdeletions. The screening of Yq microdeletions was performed by two multiplex PCRs using six STS markers recommended by the EAA/EMQN. No microdeletions were detected in the men with severe oligozoospermia. In the azoospermic group, 2/74 (2.7%) patients showed Y chromosome microdeletions. Both had complete deletion of the AZFc region. No microdeletion was identified in the AZFa region or in the AZFb region. The estimated frequency of Y chromosome microdeletions in the present survey was similar to some other reports but lower than that of previous reports in Tunisian populations.

The major herbicide used worldwide, Roundup, is a glyphosate-based pesticide with adjuvants. Glyphosate, its active ingredient in plants and its main metabolite (AMPA) are among the first contaminants of surface waters. Roundup is being used increasingly in particular on genetically modified plants grown for food and feed that contain its residues. Here we tested glyphosate and its formulation on mature rat fresh testicular cells from 1 to 10000 ppm, thus from the range in some human urine and in environment to agricultural levels. We show that from 1 to 48 h of Roundup exposure Leydig cells are damaged. Within 24-48 h this formulation is also toxic on the other cells, mainly by necrosis, by contrast to glyphosate alone which is essentially toxic on Sertoli cells. Later, it also induces apoptosis at higher doses in germ cells and in Sertoli/germ cells co-cultures. At lower non toxic concentrations of Roundup and glyphosate (1 ppm), the main endocrine disruption is a testosterone decrease by 35%. The pesticide has thus an endocrine impact at very low environmental doses, but only a high contamination appears to provoke an acute rat testicular toxicity. This does not anticipate the chronic toxicity which is insufficiently tested, and only with glyphosate in regulatory tests.

Bioassays constitute a unique approach to determine the functional aspects of gonadotropins. Indeed, these highly complex glycoprotein hormones, including pituitary lutropin (LH) and follitropin (FSH), are heterogeneous in terms of both peptidic and carbohydrate moieties, and, as a consequence, the bioactivity of the different molecular forms often does not match their immunoreactivity. In this article, we review the different types of LH and FSH bioassays. Conventional methods for measuring FSH bioactivity are first described and include the in vivo Steelman and Pohley bioassay, the radioligand receptor assays (RRAs), the in vitro Sertoli cell bioassay, the in vitro granulosa cell bioassay, and the inhibin immunoassay. Recent methods based on cell lines transfected with cloned receptors, particularly the human FSH receptor, are then described. Methods for developing these assays are presented, and the advantages and disadvantages of the different bioassays are discussed.

The Sertoli cell received its family name in a paper published by von Ebner [1] in which he described the cells as “the cells of Sertoli.” It is amazing that the early scientists were able to deduce the cell’s basic structure so well, especially when consideration is given to the poor resolution of microscopes in the 1800s (see Chapter 1) and the lack of proper fixation, embedding, sectioning, and staining. In fact, Sertoli’s original observations were so intuitive that few scientists at that time bothered to study the cell in great depth, because most everyone accepted his descriptions and went on to other more important topics of the day.
Most scientists waited for the improved resolution provided by the electron microscope before returning to the study of the Sertoli cell. From 1865, when Sertoli published his famous observations, until 1950, approximately 85 manuscripts were published with Sertoli cell or other descriptive names for this cell in the title or as key words. Then in 1953 the first paper to observe the testis with the transmission electron microscope was published [2]. However, it was most likely the work of Don Fawcett at Harvard in the 1950s [3, 4] that firmly established the importance of study- ing Sertoli cell morphology, because from 1951 until 1973, the year that Lonnie Russell published his first manuscript on testicular morphology [5], the number of publications dealing with the Sertoli cell jumped to approximately 440. From 1973 to 1990, about 4000 Sertoli cell papers were published, which is an indica- tion of the recognition of the importance of Sertoli cells in testicular function. Such growth in Sertoli cell inter- est is a direct result of the tremendous impact that morphological studies, especially those of Dr. Russell, have had on the reproductive sciences.

The blood-testis barrier (BTB) is known for its ability to create an immune privilege site in the seminiferous epithelium, but less is known of the blood-epididymal barrier (BEB). It is already established that the fully functional BTB and BEB are much more complex and consist of anatomical/physical (tight junctions, basolateral and apical membranes), physiological and immunological components, which are all necessary to make a functioning barrier in the testis and epididymis. However, comparative data for metazoans suggest that an effective Sertoli cell barrier is not entirely necessary for the development of germ cells during spermatogenesis or that our knowledge about the barrier structure/function in metazoans is still immature. This chapter compares the unique barrier formed by the Sertoli cells of the testis to that formed by the apical junctional complexes of the epididymal epithelium.

S permatogenesis is a complex biological process of cellular transformation that produces male haploid germ cells from diploid spermatogonial stem cells. This process has been simplified morphologically by recognizing cellular associations or 'stages' and 'phases' of spermatogenesis, which progress through precisely timed and highly organized cycles. These cycles of spermatogenesis are essential for continuous sperm production, which is dependent upon numerous factors, both intrinsic (Sertoli and germ cells) and extrinsic (androgens, retinoic acids), as well as being species-specific.

Dimorphism between testis and ovary in germ cells proliferative behavior, shows remarkable differences in foetal and neonatal period [14.5 days post conception (dpc)--7 days post partum (dpp)]. Immunostaining of the foetal testis, with the PCNA and Ki-67 antibodies [estimation of Labeling Index (LI)], reveals increasing germ cells population until birth. Afterwards, a sharp decline in the first 3 days of postnatal life and a transient increase, between 3 and 5 dpp, is observed. Then, the mitotic activity of germ cells ceases. In the foetal ovary, germ cells proliferation reaches a peak value before birth, decreasing thereafter Somatic (Sertoli or follicular) cells behave similarly in both sexes. Increased mitotic activity is observed throughout the examined period. Thus, the gonadal dimorphism in proliferative behavior, concerns only germ cell lineage and is established during the foetal and neonatal period.

Lithium is frequently used as an effective drug for the treatment of several psychiatric disorders in human. This alkali element and its salt, at its higher doses, may lead to various side effects or has several toxic effects after prolonged therapeutic use. To test this hypothesis, the present study was designed to investigate the adverse effect of subchronic exposure of lithium carbonate on reproductive organs of male rat. Rats were exposed to lithium carbonate at doses of 500, 800, 1100 mg/kg of diet for 90 days. The weight of reproductive organs, histology of testis, epididymis, seminal vesicle, prostate, testicular interstitial fluid volume (IFV), testosterone level, sperm morphology and fertility index were analyzed. Treatment with higher doses of lithium carbonate (i.e. 800, 1100 mg/kg diet) significantly reduced testes, epididymis and accessory sex organs weights, whereas, lower dose (500 mg/kg diet) did not show any untoward effect. Similarly, the sperm number from cauda epididymis and daily sperm production was significantly decreased with higher doses of lithium carbonate. The serum testosterone levels and IFVs were also reduced significantly. Seminal vesicle and prostate secretions were completely blocked and spermatozoa were not seen in the lumen of epididymis and vas deference. Histological studies have revealed that lithium carbonate (1100 mg/kg) caused degeneration of spermatogenic cells and vacuolization of sertoli cells cytoplasm in the testis. The sperm transit rate and production of abnormal spermatozoa were significantly (P < 0.01) increased. When the lithium carbonate-treated males were mated with normal cyclic females, the fertility index declined to 50% even after 30 days of withdrawal of lithium carbonate treatment. These results clearly suggest that subchronic exposure of lithium carbonate promote reproductive system toxicity and reduces fertility of male rats.

In mammalian testes, Sertoli cells play a key physiological role in germ cell development. Previous research has implicated local ATP release as a potential mechanism of Sertoli cell stimulation. We show that, in mouse Sertoli cells, two different receptor proteins are activated by ATP. Receptor activation, in turn, causes elevation of calcium ion levels inside the cells. By using a novel method to visualize such calcium signals, we identify mitochondria as essential elements of calcium regulation in the testis.

Sertoli cells play a key role in the establishment of an adequate luminal environment in the seminiferous tubules of the male reproductive tract. Secretion of the seminiferous tubular fluid (STF) is vital for the normal occurrence of spermatogenesis and for providing a means of transport to the developing spermatozoa. However, several studies on this subject have not completely clarified the origin and composition of this fluid. Electrolyte and water are central components of STF. Sertoli cells secrete an iso-osmotic fluid with a higher content of K+ than the blood and express various membrane and water transporters (Na+/K+-ATPase; Ca2+-ATPase; V-type ATPase; Cl− channels; CFTR Cl− channels; K+ channels; L-, T- and N-type Ca2+ channels; Na+/H+ exchangers; Na+-driven HCO3−/Cl− exchangers (NDCBEs); Na+/HCO3− cotransporters (NBCes); Na+–K+–2Cl− cotransporter; Na+/Ca2+ exchanger; and aquaporins 0 and 8) involved in cellular and secretory functions. Studies with knockout mice for some of these transporters showed tubular fluid accumulation and associated infertility, revealing the relevance of these processes for the normal occurrence of spermatogenesis. Nevertheless, the role of the several membrane transporters in the establishment of STF electrolyte composition needs to be further elucidated. This review summarizes the available data on the ionic composition of STF and on the Sertoli cell membrane mechanisms responsible for ion and water movement. Deepening the knowledge on the mechanisms involved in the secretion, composition and regulation of SFT is essential and will be a major step in understanding the infertility associated with some pathological conditions.

Ultractructural study of seminiferous tubules in ageing men revealed a varying degree of spermatogenetic arrest associated with changes in the Sertoli cells. Approximately half of the Sertoli cells showed a normal mature nuclear appearance although the cytoplasm was altered morphologically. These cells were classified as containing abundant lipid droplets (30 %), containing large cytoplasmic vacuoles fdled with an amorphous material similar to that in the tubule lumen and surrounded by junctional specializations (8 %), multinucleated Sertoli cells (4 %), or Sertoli cells with numerous mitochondria displaying tubular cristae (2%). The remaining 7 % of Sertoli cells had an immature nuclear appearance and sparse development of the cytoplasmic organelles; these cells probably represent dedifferentiated Sertoli cells. Although individual differences were marked, a correlation between the increase in gonadotrophin levels and changes in both germ cell development and Sertoli cell structure was observed.

The aim of the study was to analyze molecular mechanisms involved in FSH and basic fibroblast growth factor (bFGF) regulation of lactate production in rat Sertoli cells. The regulation of the availability of pyruvate, which is converted to lactate, could be a mechanism utilized by hormones to ensure lactate supply to germ cells. On one hand, the regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) expression could result in increased glycolysis, while an increase in pyruvate availability may also result from a lower conversion to acetyl-CoA by negative regulation of pyruvate dehydrogenase complex (PDC) activity by phosphorylation. Sertoli cell cultures obtained from 20-day-old rats were used. Stimulation of the cultures with FSH or bFGF showed that FSH increases Pfkfb1 and Pfkfb3 expression while bFGF increases Pfkfb1 mRNA levels. Additionally, we observed that FSH-stimulated lactate production was inhibited in the presence of a PFKFB3 inhibitor, revealing the p...

Endogenous and environmental estrogens have been proved to affect male reproduction in vertebrates. Both positive and negative effects in the regulation of the reproductive tract have been described. Since it is well known that amphibians represent a useful model to study several aspects concerning reproductive activity, we have taken advantage of the frog, Rana esculenta, to study the involvement of estrogens in sperm release. We show here that pituitary hormones increased the number of peritubular myoid cells (PMCs) expressing Fra-1 and induced testicular morphological changes related to sperm release. The estrogen antagonist ICI182-780 counteracted the hypophysis driven effects. In vivo and in vitro experiments demonstrated that 17β-Estradiol acted directly on the testis to switch-on Fra-1 in PMCs. Furthermore, impairment of estrogen activity significantly reduced sperm release mainly affecting the detachment of spermatozoa from Sertoli cells (spermiation). Therefore, estrogens c...

Transgenic mouse models have contributed considerably to our understanding of the cellular and molecular mechanisms by which androgens control spermatogenesis. Cell-selective ablation of the androgen receptor (AR) in Sertoli cells (SC) results in a complete block in meiosis and unambiguously identifies the SC as the main cellular mediator of the effects of androgens on spermatogenesis. This conclusion is corroborated by similar knockouts in other potential testicular target cells. Mutations resulting in diminished expression of the AR or in alleles with increased length of the CAG repeat mimick specific human forms of disturbed fertility that are not accompanied by defects in male sexual development. Transcriptional profiling studies in mice with cell-selective and general knockouts of the AR, searching for androgen-regulated genes relevant to the control of spermatogenesis, have identified many candidate target genes. However, with the exception of Rhox5, the identified subsets of genes show little overlap. Genes related to tubular restructuring, cell junction dynamics, the cytoskeleton, solute transportation and vitamin A metabolism are prominently present. Further research will be needed to decide which of these genes are physiologically relevant and to identify genes that can be used as diagnostic tools or targets to modulate the effects of androgens in spermatogenesis.

Hormonal regulation is essential to spermatogenesis. Sertoli cells (SCs) have functions that reach far beyond the physical support of germ cells, as they are responsible for creating the adequate ionic and metabolic environment for germ cell development. Thus, much attention has been given to the metabolic functioning of SCs. During spermatogenesis, germ cells are provided with suitable metabolic substrates, in a set of events mediated by SCs. Multiple signaling cascades regulate SC function and several of these signaling pathways are hormone-dependent and cell-specific. Within the seminiferous tubules, only SCs possess receptors for some hormones rendering them major targets for the hormonal signaling that regulates spermatogenesis. Although the mechanisms by which SCs fulfill their own and germ cells metabolic needs are mostly studied in vitro, SC metabolism is unquestionably a regulation point for germ cell development and the hormonal control of these processes is required for a normal spermatogenesis.

The first edition of The Sertoli Cell was an appropri- ate vision of the late Professor Lonnie D. Russell, because he studied the Sertoli cell in more depth than most other modern-day scientists. He published more than 200 papers, of which nearly half were focused on the Sertoli cell, including the first book devoted to the cell, which he coedited with Michael D. Griswold [1]. Therefore, this chapter is written in honor of Lonnie because he was a fun-loving friend and visionary scientist who always used the microscope and his imagination to find new insights into complex scien- tific problems of the testis, and in particular the Sertoli cell. Lonnie’s devotion to this cell was exemplified by the license plate that he attached to his automo- bile, which read “Sertol 1,” and by his cat whose name was also “Sertoli.” Factual events surrounding Sertoli’s life were gathered from reading numerous reviews [2–9], particularly those of the distinguished scholar Brian P. Setchell [10,11], whose foreword in the first book provided a sincere and deserved admirable look at Enrico Sertoli [12].

Histochemical demonstration of alkaline phosphatase activity appears to be useful in identifying rat peritubular cells in primary testicular cell culture. In both frozen sections of rat testis and Mirsky&#39;s fixed, methacrylate-embedded rat testis, the reaction product localized primarily in peritubular cells, vascular endothelium and occasionally in interstitial cells, with much smaller amounts of reaction product associated with elongating spermatids in the germinal epithelium. Occasional late-stage tubules (X-XIV) showed weak reactivity in the epithelium, associated with spermatocytes or Sertoli cells. Ultrastructurally, Gomori-method reaction product was localized to peritubular cells, lymphatics, and spermatogonia in stage VII; no staining was found consistently in Sertoli cells. In isolated cell preparations enriched for Sertoli and germ cells, 1 to 8% of the cells demonstrated alkaline phosphatase activity, while greater than 50% of the cells stained positive for alkaline p...

Benzene-1,3-diamidoethanethiol (BDETH 2 ) is an exceptional precipitant for removing soft heavy metals from water. The present work will detail the bonding arrangement of BDETH 2 to the metals Cd, Hg, and Pb, along with the full characterization data of the BDET−M compounds. It was found that the Hg compound has a linear S−M−S geometry. The characterization data consisted of Mp, EA, IR, Raman, MS, XANES, EXAFS, and solid-state multinuclear NMR. Krepps, M. K.; Atwood, D. A. Water Res. 2000, 34, 3005. (2) Matlock, M. M.; Howerton, B. S.; van Aelstyn, M. A.; Henke, K. R.; Atwood, D. A. Water Res. 2003, 37, 579. (3) Matlock, M. M.; Howerton, B. S.; van Aelstyn, M. A.; Nordstrom, F. L.; Atwood, D. A. EnViron. Sci. Technol. 2002, 36, 1636. (4) Matlock, M. M.; Howerton, B. S.; Atwood, D. A. Ind. Eng. Chem. Res. 2002, 41, 1579. (5) Matlock, M. M.; Howerton, B. S.; Robertson, J. D.; Atwood, D. A.

The aims of the present study were to analyze the gonadal structure of Pseudoplatystoma fasciatum males during their annual cycle to enhance understanding of their reproductive biology and to improve the hormonally induced reproduction and culture of this species in hatcheries. We adopted the recently proposed method that establishes reproductive classes that are based on variations of the germinal epithelium within the year. Five reproductive classes were established: maturation (early, middle, and late), regression and recrudescence. Our observations revealed that in the spawning season P. fasciatum testes display two main functions: sperm production and sperm storage. We also concluded that the analysis of the variation of germinal epithelium was satisfactory when applied to this freshwater catfish and should be adopted for other fish species.

Outer dense fibers are a major constituent of the sperm tail and outer dense fiber 2 (ODF2) protein is one of their major components. ODF2 shares partial homology with cenexin 1 and cenexin 2, regarded as centriolar proteins. We show that ODF2 and cenexin 2 transcripts are the product of differential splicing of a single gene, designated Cenexin/ODF2 and that cenexin 1 is an incomplete clone of ODF2. ODF2 terminates in exon 20b whereas in cenexin 2 this exon is spliced out and translation terminates in exon 24. We demonstrate a transcriptional switch during rat testicular development, from somatic-type to testis-type ODF2 and cenexin transcripts during the onset of meiosis. The switch is completed when spermiogenesis is established. ODF2 immunoreactive sites were visualized in the acroplaxome, along the sperm tail and the centrosome-derived sperm head-to-tail coupling apparatus. An unexpected finding was the presence of ODF2 antigenic sites, but not cenexin antigenic sites, in the dense fibrillar component of the nucleolus of Sertoli cells, spermatogonia and primary spermatocytes. The characterization of the genomic origin, processing and developmental expression of ODF2 transcript isoforms and their protein products can help reconcile differences in the literature on the role of ODF2 and cenexin in the centrosome. Furthermore, the finding of ODF2 in the dense fibrillar component of the nucleolus suggests that this protein, in addition to its presence in sperm outer dense fibers and centrosome, highlights and adds to the nucleolar function during spermatogenesis and early embryogenesis. Mol. Reprod. Dev. 75: 1591-

The development of the fetal testis requires complex hormonal regulation and thus forms a highly sensitive detector for hormonal disruption (Sharpe 2006). Phthalate esters are known to be endocrine disruptors, chemi cals that alter normal endocrine function and regulation, in particular, during male sex determination and differentiation (Foster 2005; Henley and Korach 2006; Waring and Harris 2005). Di-(2-ethylhexyl) phthalate (DEHP) is the most common chemical additive used in the manufacture of plastics and is consequently distributed widely, for instance, in infant toys, cosmetics, food packaging, and medical devices [Agency for Toxic Substances and Disease Registry (ATSDR) 2002]. Because DEHP is not covalently bound to the polymers, it leaches from the matrix and is endemic to the human environment (Hashizume et al. 2002; Rudel et al. 2003), so humans are constantly exposed to phthalates through oral, dermal, and inhalation routes (Koch et al. 2006; Wormuth et al. 2006). After exposure, DEHP is rapidly hydrolyzed by esterases in the gut, liver, and blood into mono-(2-ethylhexyl) phthalate (MEHP), which is believed to be the active molecule (Gray 1986). MEHP is then hydrolyzed to mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP) and metabolized into mono-(2-ethyl-5-oxo hexyl) phthalate

Cyclophosphamide (CP) is extensively used as an antineoplastic agent for the treatment of various cancers, as well as an immunosuppressive agent. However, despite its wide spectrum of clinical uses, CP is known to cause several adverse effects including reproductive toxicity. Crataegus monogyna is one of the oldest pharmaceutical plants that have been shown to be cytoprotective by scavenging free radicals. The present study was conducted to assess whether Crataegus monogyna fruits aqueous extract with anti-oxidant properties, could serve as a protective agent against reproductive toxicity during CP treatment in a rat model. Male Wistar rats were categorized into four groups. Two groups of rats were administered CP at a dose of 5 mg in 5 ml saline/kg/day for 28 days by oral gavages. One of these groups received Crataegus monogyna aqueous extract at a dose of 20 mg/kg/day orally four hours after cyclophosphamide administration. A vehicle treated control group and a Crataegus monogyna ...

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 10day 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-dayold 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. Seminiferous epithelium of the hypogonadal mouse, M. Myers et al. All studies were carried out in C3H hpg mice from a breeding colony at the School of Biomedical Sciences, University of Nottingham, originally derived from a stock purchased from Jackson ImmunoResearch Laboratories, Inc. (Bar Harbor, ME, USA). Testes from hpg adults 170-190 days of age ( n = 15) were compared with those from wild-type littermates of hpg mice from the same colony: 140-190 days of age ( n = 9), 9 days old ( n = 3), 10 days old ( n = 10) or 14 days old ( n = 3). All animal procedures were approved by the University of Nottingham Local Ethical Review Committee and Seminiferous epithelium of the hypogonadal mouse, M. Myers et al.

Huntingtin interacting protein 1 (HIP1) is an endocytic adaptor protein with clathrin assembly activity that binds to cytoplasmic proteins, such as F-actin, tubulin, and huntingtin (htt). To gain insight into diverse functions of HIP1, we characterized the male reproductive defect of HIP1 À/À mice from 7 to 30 weeks of age. High levels of HIP1 protein were expressed in the testis of wild-type mice as seen by Western blots and as a reaction over Sertoli cells and elongating spermatids as visualized by immunocytochemistry. Accordingly, major structural abnormalities were evident in HIP1 À/À mice with vacuolation of seminiferous tubules caused by an apparent loss of postmeiotic spermatids and a significant reduction in mean profile area. Remaining spermatids revealed deformations of their heads, flagella, and/or acrosomes. In some Sertoli cells, ectoplasmic specializations (ES) were absent or altered in appearance accounting for the presence of spherical germ cells in the epididymal lumen. Quantitative analyses of sperm counts from the cauda epididymidis demonstrated a significant decrease in HIP1 À/À mice compared to wild-type littermates. In addition, computer-assisted sperm analyses indicated that velocities, amplitude of lateral head displacements (ALH), and numbers and percentages of sperm in the motile, rapid, and progressive categories were all significantly reduced in HIP1 À/À mice, while the numbers and percentages of sperm in the static category were greatly increased. Taken together, these various abnormalities corroborate reduced fertility levels in HIP1 À/À mice and suggest a role for HIP1 in stabilizing actin and microtubules, which are important cytoskeletal elements enabling normal spermatid and Sertoli cell morphology and function. Mol. Reprod. Dev. 74: 341-359, 2007. ß

The aim of this study was to investigate the protective effects of taurine against histomorphological and ultrastructural changes in the testes of Swiss albino mice caused by acute in vivo exposure to AlCl 3 . Light microscopy revealed that a single intraperitoneal (i.p.) dose of AlCl 3 (25 mg kg -1 Al 3+ ) was associated with sloughing, tubular atrophy, germ-cell degeneration, and foci of Leydig cell hyperplasia. In addition, transmission electron microscopy showed a destruction of inter-Sertoli cell tight junctions, apoptotic cell death of spermatogonia and primary spermatocytes, various types of abnormalities in spermatid morphology, accumulation of lipid droplets, reduction of the smooth endoplasmic reticulum (sER), and mitochondrial damage in Leydig cells. Taurine post-treatment at i.p. dose of 1 g kg -1 diminished these changes and signifi cantly reduced the number of affected tubules compared to Al-poisoned mice. This is the fi rst study to evidence that taurine protects against pathological changes in the testicular tissue of Al-treated mice.

Sertoli cells (SCs) glucose metabolism is crucial for spermatogenesis since developing germ cells consume lactate produced by SCs as their main energy source. Recently, androgens and estrogens have been implicated in SCs energy metabolism modulation, although the molecular mechanisms remained undisclosed. Here, we report the effect of sex steroid hormones on key points of cultured rat SCs glycolytic pathway. We used primary cultures of immature rat SCs treated with 17β-estradiol (E2) or 5α-dihydrotestosterone (DHT). The transcript levels of glucose transporters (GLUTs), phosphofructokinase 1 (PFK-1) and lactate dehydrogenase C (LDH C) were analyzed after 25 and 50 h of culture by qPCR. Protein levels of GLUTs, PFK-1, LDH and monocarboxylate transporter 4 (MCT4) after 25 and 50 h were determined by western blot and LDH activity was also assessed. Our results show that both E2 and DHT downregulated the transcript levels of PFK-1, GLUT1 and GLUT3 after 50 h. However, only DHT-treated cells presented a downregulation of LDH C transcript levels. Interestingly, the protein levels of these enzymes and transporters remained unaltered except in DHT-treated cells that presented a significant decrease on GLUT1 protein levels evidencing a possible site for the regulation of SCs glucose metabolism by androgens. Taken together, our results provide evidence that sex steroid hormones action in SCs energy metabolism is mediated through modulation in glycolysis-related transporters and enzymes, particularly at the transcriptional level. DHT decreased GLUT1 protein levels and increased LDH activity after 25 h, evidencing key points for this hormone action in the regulation of SCs metabolism.

The aims of the present study were to analyze the gonadal structure of Pseudoplatystoma fasciatum males during their annual cycle to enhance understanding of their reproductive biology and to improve the hormonally induced reproduction and culture of this species in hatcheries. We adopted the recently proposed method that establishes reproductive classes that are based on variations of the germinal epithelium within the year. Five reproductive classes were established: maturation (early, middle, and late), regression and recrudescence. Our observations revealed that in the spawning season P. fasciatum testes display two main functions: sperm production and sperm storage. We also concluded that the analysis of the variation of germinal epithelium was satisfactory when applied to this freshwater catfish and should be adopted for other fish species.

Sertoli cells (SCs) glucose metabolism is crucial for spermatogenesis since developing germ cells consume lactate produced by SCs as their main energy source. Recently, androgens and estrogens have been implicated in SCs energy metabolism modulation, although the molecular mechanisms remained undisclosed. Here, we report the effect of sex steroid hormones on key points of cultured rat SCs glycolytic pathway. We used primary cultures of immature rat SCs treated with 17β-estradiol (E2) or 5α-dihydrotestosterone (DHT). The transcript levels of glucose transporters (GLUTs), phosphofructokinase 1 (PFK-1) and lactate dehydrogenase C (LDH C) were analyzed after 25 and 50 h of culture by qPCR. Protein levels of GLUTs, PFK-1, LDH and monocarboxylate transporter 4 (MCT4) after 25 and 50 h were determined by western blot and LDH activity was also assessed. Our results show that both E2 and DHT downregulated the transcript levels of PFK-1, GLUT1 and GLUT3 after 50 h. However, only DHT-treated cells presented a downregulation of LDH C transcript levels. Interestingly, the protein levels of these enzymes and transporters remained unaltered except in DHT-treated cells that presented a significant decrease on GLUT1 protein levels evidencing a possible site for the regulation of SCs glucose metabolism by androgens. Taken together, our results provide evidence that sex steroid hormones action in SCs energy metabolism is mediated through modulation in glycolysis-related transporters and enzymes, particularly at the transcriptional level. DHT decreased GLUT1 protein levels and increased LDH activity after 25 h, evidencing key points for this hormone action in the regulation of SCs metabolism.

a b s t r a c t 25 Inflammatory reactions that result from microbial infections, both localized and systemic, are reported to 26 cause transient or permanent male infertility. The cellular mechanisms underlying the inhibitory effect of 27 microbial infection on spermatogenesis is not fully understood. However, there is evidence that sper-28 matogenesis is affected by bacterial lipopolysaccharides (LPS) that induce acute inflammatory responses. 29 The aim here was to use LPS treatments to investigate the potential oxidative stress and toxicity in pri-30 mary cultures of adult rat Sertoli cells. The Sertoli cells were established and incubated with different 31 concentrations of LPS (5, 10 or 20 lg/ml) for 6, 12 and 24 h. Lipid peroxidation (LPO) and hydrogen per-32 oxide (H 2 O 2 ) production, along with superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase 33 (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), reduced glutathione (GSH), lactate, 34 lactic acid dehydrogenase (LDH), c-glutamyl transpeptidase (c-GT) and b-glucuronidase were measured 35 in these cells. LPO as well as H 2 O 2 production were significantly increased while antioxidant enzyme 36 activities and GSH concentration were significantly depressed. Effects were dose and time-dependent 37 at all incubation periods with 10 and 20 lg/ml LPS. Moreover, markers of Sertoli cell function such as lac-38 tate production, LDH, c-GT and b-glucuronidase activities were decreased in a time and dose-dependent 39 manner. Incubation of Sertoli cells with 5 lg/ml LPS for 12 and 24 h significantly increased oxidative sta-40 tus but significantly decreased the antioxidant enzyme activities, GSH concentration and Sertoli cell 41 markers. In contrast, the oxidative and antioxidant status and markers of Sertoli cell function did not 42

In mammals, the pre-Sertoli cell of the male genital ridge is the first cell type to display sex specific differentiation and differential gene expression. The genetic cascade driving the differentiationof pre-Sertoli cells and ultimately testis formationis beginning to be unravelled, but many questions remain. A better understanding of the transcriptome of pre-Sertoli cells immediately after sex determination is essential in order to further understand this differentiationprocess. A mouse model expressing Red Fluorescent Protein (RFP) under the control of a hybrid mouse/pig SRY promoter (HybSRYp-RFP) was used to purify cells from embryonic day 12.0 (e12.0) male genital ridges. To compare the transcriptomes of HybSRYp-RFP cell populations versus age matched whole female genital ridges, RNA was extracted and used to generate molecular probes that were hybridized onto Affymetrix Mouse Genome 430 2.0 micro-arrays. The expression of genes considered markers for pre-Sertoli cells, including Sox9, Mis, Dhh and Fgf9 were identified within the HybSRYp-RFP expressing cell population, while markers for germ cells (Oct4, SSEA-1) and endothelial cells (Ntrk3) were not identified. In contrast, markers for ovarian somatic cell expression, including Fst and Bmp2, were identified as overexpressed within the ovarian cell population. In a general fashion, genes identified as 2.5-fold over expressed in HybSRYp-RFP expressing cells coded notably for cell signalling and extra cellular proteins. The expression of Sox10, Stc2, Fgf18, Fgf13 and Wnt6 were further characterized via whole mount in situ hybridization (WISH) on male and female genital ridges between e11.5 and e14.5. Sox10, Fgf18, Fgf13 and Stc2 gene expression was detected within the male genital ridges while Wnt6 was found diffusely within both the male and female genital ridges. These data represent the earliest comprehensive microarray expression analysis of purified presumptive pre-Sertoli cells available to date. Mol. Reprod. Dev. 74: 1491–1504, 2007. © 2007 Wiley-Liss, Inc.

aO 2 -Macroglobulin and clusterin are two putative Sertoli cell secretory products; however, the regulator(s) modulating their secretion by Sertoli cells is not known. Recent studies from this laboratory have shown that the testicular a 2 -macroglobulin, unlike its liver homologue, is not an acute-phase reactant and its concentration is not affected by acute inflammation. We sought to determine whether FSH, testosterone, and other biomolecules would affect the secretion of oi,-macroglobulin and clusterin by Sertoli cells as well as whether peritubular myoid cells would affect the secretion of these proteins by Sertoli cells. It was noted that Sertoli cells cultured in vitro secreted increasing amounts of oa 2 -macroglobulin and clusterin as a function of time. FSH (50-1000 ng/ml) and testosterone (10-'-10 -5 M) had no apparent effect on the secretion of a 2 -macroglobulin and clusterin by Sertoli cells. Addition of interleukin-6 to Sertoli cell-enriched cultures, in doses known to stimulate u,-macroglobulin secretion by hepatocytes, did not affect the ot,-macroglobulin secretion. However, dexamethasone at 10-7-10 -5 M stimulated t,-macroglobulin secretion by Sertoli cells dose-dependently while the addition of interleukin-6 had no synergistic effect on dexamethasone-stimulated at 2 -macroglobulin secretion. These findings suggest that the synthesis and/or secretion of ot2-macroglobulin by Sertoli cells is regulated by a mechanism distinct from that of the liver. Peritubular cells cultured alone did not produce any measurable amounts of either aot-macroglobulin or clusterin; however, co-cultures of peritubular cells with Sertoli cells induced a significant increase in i2,-macroglobulin and clusterin secretion by Sertoli cells. Addition of testosterone to the Sertoli cell-peritubular cell co-culture system did not affect the levels of 2,-macroglobulin and clusterin. In summary, FSH and testosterone, at doses known to affect Sertoli cell function, did not affect the secretion of aot-macroglobulin and clusterin; however, peritubular cells modulate the secretion of both a 2 -macroglobulin and clusterin by Sertoli cells.