Number Of Spermatogenic Cells Research Articles

Effects of nitrate and atrazine on larval development and sexual differentiation in the northern leopard frog Rana pipiens

Pollution from agrochemicals may be contributing to the global decline in amphibian populations. Environmentally relevant concentrations of nitrate and/or atrazine on anuran development and gonadal differentiation were tested. Four replicates of 20 tadpoles per tank (80/treatment) were exposed from Taylor-Kollros stage 2 to 3 to stage 23 to 34 to either 10 mg/L nitrate, 10 microg/L atrazine, a combined exposure of 10 mg/L nitrate plus 10 microg/L atrazine, or untreated controls. No treatment-dependent effects on weight, snout-vent or hind limb length, or time to forelimb emergence were observed. The proportions of females increased in all treatments compared to the controls, especially in the combined treatment (chi2 = 17.90, df = 6, p = 0.0065, combined = 66.4% female, control = 41% female). The frequency of intersex was low in all treatments. No treatment-related effects on the total number of spermatogenic cells were observed, but the ratio of cell types differed in that testes from animals in the treated groups exhibited more spermatogonia, fewer spermatocytes, and more spermatids than the control (significantly different, Kruskal-Wallis, p < 0.05). Ovaries from animals treated with nitrate or atrazine exhibited larger immature (previtellogenic) and mature (vitellogenic) follicles, but ovaries from the combined treatment had larger immature follicles only. Testicular oocytes were observed in the nitrate-only and atrazine-only treatments, and the control treatment, but not the combined treatment. Overall, this study has demonstrated changes in sex ratios that are more marked in response to combined nitrate/atrazine exposure than with these chemicals alone. Histological evidence suggests that premature maturation of gonad may occur as a result of nitrate and/or atrazine exposure during larval development.

Efficacy of Cryopreservation of Testicular Sperm in Hypo Spermatogenesis

Testicular sperm using ICSI could achieve optimal fertilization and pregnancy. Hypospermatogenesis is defined as a decrease in the number of spermatogenic cells, so that few mature spermatozoa are formed. This study was performed to assess the fertilizing ability and its embryonic developmental capacity using fresh- and frozen-thawed testicular sperm in hypospermatogenesis patients.

Temporal effects of mancozeb on testes, accessory reproductive organs and biochemical constituents in albino mice

Mancozeb, a fungicide of ethylenebisdithiocarbamate group was orally administered at 800 mg/kg body weight to male Swiss albino mice for 5, 10, 20 and 30 days. Daily body weight of the mice were recorded. The mice were sacrificed by cervical dislocation after 24 h of terminal exposure of mancozeb. Testes weight decreased significantly in 20 and 30 days mancozeb treated mice. However, there was a significant decrease in the number of spermatogonia, diameter of spermatocytes and spermatids in 20 days and number of spermatids in 10 days mancozeb treated mice. Histologic studies of the testis of the mice treated with mancozeb for long duration revealed spermatogenesis inhibition reflected by significant decrease in the number of spermatogenic cells and sperms, when compared with that of controls. In the mice treated with mancozeb for 20 and 30 days showed significant decrease in the weight of the prostate gland. However weight of Cowper's glands decreased significantly in 30 days mancozeb treated mice. There was significant decrease in the kidney, spleen and liver weight, where as thyroid weight increased significantly in mice treated with mancozeb for 30 days. However, thymus weight increased significantly only in the mice treated with mancozeb for 10, 20 and 30 days. In mice treated with mancozeb for 20 days caused significant decrease in the level of protein and a significant increase in the level of total lipids in the testis. However, there was significant decrease in the level of glycogen in the kidney. In mice treated with mancozeb for 30 days caused significant decrease in the levels of protein and glycogen and significant increase in the level of total lipids in the testis and liver and a significant decrease in the protein, glycogen and total lipids in the kidney. These observed effects of mancozeb on testis and biochemical constituents may be due to hormonal imbalance in any of the stages in the hypothalamo–hypophysial–testicular axis.

Seasonal changes in spermatogenesis and testicular steroidogenesis in wild male raccoon dogs (Nyctereutes procynoides).

Testes of 15 wild adult male raccoon dogs (Nyctereutes procynoides) obtained from September 2000 to April 2001 were studied to clarify seasonal changes in spermatogenesis and testicular steroidogenesis. There were marked seasonal variations in the testis weight and size with values relatively low in September and highest in March. Spermatogonia and primary spermatocytes were observed in September, while spermatogonia, spermatocytes and round spermatids were present in January, and all types of spermatogenic cells including mature spermatozoa were found in the mating season (February and March). The number of spermatogenic cells reached their peak values in February and March. In addition, steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3 betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). P450scc and P450c17 were identified in Leydig cells and spermatids in February, whereas these enzymes were present only in Leydig cells in September. 3betaHSD was found in Leydig cells in September and February with more intense staining in February. The localization of P450arom changed seasonally: no immunostaining in September; more extensive immunostaining in Leydig cells, Sertoli cells, and elongating spermatids in February. These results suggest that seasonal changes in the testis weight and size of wild male raccoon dogs are correlated with changes in spermatogenesis. Seasonal changes in testicular steroidogenesis suggest that the synthesis of androgen and estrogen reaches its peak in the mating season.

Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility.

Many differentiating spermatogenic cells die by apoptosis during the process of mammalian spermatogenesis. However, very few apoptotic spermatogenic cells are detected by histological examination of the testis, probably due to the rapid elimination of dying cells by phagocytosis. Previous in vitro studies showed that Sertoli cells selectively phagocytose dying spermatogenic cells by recognizing the membrane phospholipid phosphatidylserine (PS), which is exposed to the surface of spermatogenic cells during apoptosis. We examined here whether PS-mediated phagocytosis of apoptotic spermatogenic cells occurs in vivo. For this purpose, the PS-binding protein annexin V was microinjected into the seminiferous tubules of normal live mice, and their testes were examined. The injection of annexin V caused no histological changes in the testis, but significantly increased the number of apoptotic spermatogenic cells as assessed by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. The number of Sertoli cells did not change in the annexin V-injected testes, and annexin V itself did not induce apoptosis in primary cultured spermatogenic cells. These results indicate that annexin V inhibited the phagocytic clearance of apoptotic spermatogenic cells and suggest that PS-mediated phagocytosis of those cells occurs in vivo. Furthermore, the injection of annexin V into the seminiferous tubules brought about a significant reduction in the number of spermatogenic cells and epididymal sperm in anticancer drug-treated mice. This suggests that the elimination of apoptotic spermatogenic cells is required for the production of sperm.

Effects of an Endocrine Disruptor on Male Reproductive Organs.

A 5-day Hershberger assay, a 20-day pubertal male assay, a one-month oral administration study and a reproductive toxicity study were carried out using an antiandrogenic chemical, flutamide (FL). The effects of FL were as follows: weight decrease in male reproductive organs such as the prostate and seminal vesicles were observed at ≥0.6 mg/kg/day as the most sensitive parameter in the Hershberger assay and the one-month oral administration study. Reduction in number of spermatogenic cells was found at ≥0.6 mg/kg/day as the most sensitive parameter in the 20-day pubertal male assay. A decrease in the anogenital distance was observed at ≥2.5 mg/kg/day as the most sensitive parameter in the reproductive toxicity study. These effects were compared with the reported results of one-month oral administration study of an estrogenic chemical, ethinylestradiol (EE), and an androgenic chemical, methyltestosterone (MT). Differences in the results were as follows: serum hormone levels of LH and testosterone were increased by the FL-treatment, but decreased with EE despite testicular atrophy in all cases. While EE and MT produced atrophic changes in Leydig cells, FL induced their hyperplasia. Thus, different results were obtained regarding serum hormone levels and histopathological changes in Leydig cells with the endocrine disruptor. Attention should be concentrated on these two parameters to clarify and characterize the mechanisms of action of so called endocrine disruptors.

Biological effects observed in postnatal ontogeny of male rats subjected to acute and chronic prenatal γ-irradiation

Changes in the number of spermatogenic cells and spermatozoa, RNA and DNA contents, and weights of the thymus, spleen, and testes of 7- and 19-week-old rats subjected to acute and chronic prenatal irradiation in a dose of 1 Gy attest to long-lasting disturbances in the body.

Recognition of Phosphatidylserine on the Surface of Apoptotic Spermatogenic Cells and Subsequent Phagocytosis by Sertoli Cells of the Rat

In a primary co-culture of spermatogenic and Sertoli cells of the rat, many spermatogenic cells die by apoptosis and are subsequently engulfed by Sertoli cells. We investigated the mechanism of this phagocytosis reaction. Testicular cells from 20-day-old rats were cultured, and spermatogenic cells and Sertoli cells were separated. When the recovered spermatogenic cells were maintained without Sertoli cells, the viability of the cells decreased and they became more susceptible to phagocytosis by Sertoli cells. Phagocytosis was severely impaired when liposomes containing acidic phospholipids, such as phosphatidylserine, phosphatidylinositol, and cardiolipin, were included in the reaction, whereas those consisting of neutral phospholipids showed little effect. Such anionic liposomes were more efficiently engulfed by Sertoli cells than were the other neutral liposomes. Also, the number of spermatogenic cells that exposed phosphatidylserine to the surface increased when cells were maintained in single culture. The results indicate that upon induction of spermatogenic cell apoptosis, phosphatidylserine and probably other acidic phospholipids, which are normally localized in the inner leaflet of the plasma membrane, translocate to the outer leaflet and serve as a signal for phagocytosis by Sertoli cells.

Evaluation of Recovery from Cyclophosphamide Testicular Toxicity in Rats.

The testicular toxicity of cyclophosphamide (CP) in rats and recovery of normal spermatogenesis over an 8-week period were assessed. Male rats were administered 40mg/kg CP by gavage daily for 1 week and sacrificed at 1 day, 3 weeks, and 8 weeks after the cessation of treatment. The numbers of seminiferous epithelial cells were counted in the seminiferous tubules in stages II-III, V, VII, and XII of the spermatogenic cycle. Spermatogonia were decreased in number at 1 day after the last treatment, but most of the surviving spermatogonia including type A had proliferative activity, as demonstrated by PCNA staining. After 3-week recovery, the numbers of spermatogonia were improved, but the numbers of spermatocytes and round spermatids were reduced in some stages. Furthermore, focal substantial changes such as atrophy of seminiferous tubules were present in the testis. After 8-week recovery, the numbers of spermatogenic cells up to round spermatids were comparable to those in control specimens, and histopathologically no remarkable changes were observed in the testis. However, lower weights and sperm counts for testis and epididymis were still found at this time. These findings suggested that histopathological changes as subtle as decreased number of spermatogonia should be detected in evaluations of male chemical reproductive toxicity. Our findings also suggested that PCNA staining may be useful for predicting reversibility of testicular toxicity.

Characteristic features of preleptotene spermatocytes in Xenopus laevis: increase in the nuclear volume and first appearance of flattened vesicles in these cells.

In Xenopus laevis, the spermatogenic cells derived from a primary spermatogonium divide synchronously and form a cyst surrounded by somatic Sertoli cells. To clarify how many times the spermatogonia divide mitotically before differentiating to primary spermatocytes, the number of spermatogenic cells in the cysts was counted. Each cyst which consisted of cells morphologically identical to secondary spermatogonia contained discrete numbers of cells such as 2(6), 2(7), or 2(8) cells. On the other hand, the number of the germ cells in a cyst of zygotene/pachytene primary spermatocytes was approximately 2(8). These results indicate that spermatogenic cells which had finished eight mitotic divisions and showed a similar morphology to secondary spermatogonia were preleptotene spermatocytes. The nuclei of the preleptotene spermatocytes increased in volume concomitantly with premeiotic DNA replication and differentiation into zygotene/pachytene primary spermatocytes. Therefore the cells morphologically indistinguishable from secondary spermatogonia but with larger nuclei than typical secondary spermatogonia were identified as preleptotene spermatocytes. Electron microscopic observations of the preleptotene spermatocytes identified as above showed that flattened vesicles, 0.1-0.5 micron in length, about 0.05 micron in width, and with more electron-dense membrane than typical endoplasmic reticulum, first appeared in the cytoplasmic area of preleptotene spermatocytes.

Morphological evaluation of cyclophosphamide testicular toxicity in rats using quantitative morphometry of spermatogenic cycle stages.

The testicular toxicity of cyclophosphamide (Cp) in rats was evaluated by quantitative morphometry of spermatogenic cycle stages. Nine-week-old male Sprague-Dawley rats in Group 1 were given a single oral administration of 100 mg/kg of Cp, and were sacrificed at 1, 7, 14 and 21 days thereafter. Rats in Group 2 were orally given 100 mg/kg/day of Cp for 2 days, followed by 50 mg/kg/day for the next 3 days, and were sacrificed at 1 and 4 days after the last administration. The numbers of seminiferous epithelia were counted in the seminiferous tubules of stages II, V, VII and XII of the spermatogenic cycle. The data were expressed as numbers of spermatogenic cells per Sertoli cells per seminiferous tubule cross section. Animals in Group 1 showed decreased preleptotene spermatocytes at Day 7, decreased zygotene spermatocytes at Day 14, and decreased pachytene spermatocytes at Day 21. In group 2, testicular toxicity could also be clearly detected by this morphometric approach. The present morphometric study thus indicates that testicular toxicity can be detected from Day 7 even after a single administration of Cp.

Preliminary Report on Histological Characteristics of Male Reproductive Organs in Senile Japanese Macaques

Histological characteristics of male reproductive organs were observed macrographically and microscopically in Japanese macaques aged 20 years old or more. As the senility proceeded, testicular weight and the diameter of the seminiferous tubules decreased. Likewise, the weights and epithelial heights of the seminal vesicle and prostate gland decreased in old males. Histologically, a proliferation of interstitial tissue was notable in every organ examined. Seminiferous epithelium decreased in height as the age of animal approached thirty years. Similarly, disorder of cellular association and decrease in the number of spermatogenic cells were observed in the seminiferous tubules.

The Expression Pattern of the c- kit Ligand in Gonads of Mice Supports a Role for the c- kit Receptor in Oocyte Growth and in Proliferation of Spermatogonia

The tyrosine kinase receptor c- kit and its ligand KL are required for postnatal development of germ cells, in addition to their role in primordial germ cells. To clarify their function, a detailed examination of the pattern of expression of KL in postnatal gonads was undertaken. In ovaries, the expression of KL as seen by RNA blot analysis and by RNase protection assays is relatively high at birth (P0), low from P5 to P8, and high from P12 onward. KL expression is relatively high in testes of all ages. The forms of KL RNA present in the testes suggest that from P5 onward the membrane-bound form of KL predominates, while in the ovary significant amounts of both forms are present. As observed by in situ hybridization and immunohistochemistry, in the newborn ovary KL is highly expressed in central cords whose cells contribute to the formation of central growing follicles. Expression is low in follicle cells of small growing follicles and increases to high levels in three-layered follicles during late oocyte growth. Large amounts of the ligand are found within growing oocytes, After oocyte growth ceases, expression continues only in the outer layers of multilayered follicles. In the testis, from P0 through P9, KL expression is distinct in Sertoli cells, but not in germ cells. Thereafter, the intensity of KL expression declines as the number of spermatogenic cells increases within the tubules. KL in Sertoli cells appears to be concentrated basally at the stage of the cycle of the seminiferous epithelium when it is known to interact with differentiating type A spermatogonia. These data are consistent with a role for KL in oocyte growth and in facilitating proliferation and/or differentiation of type A spermatogonia.

Seasonal changes in testicular structure and localization of a sperm surface glycoprotein during spermatogenesis in sea urchins.

Testes of the sea urchin, Strongylocentrotus purpuratus, undergo morphological changes consistent with a reproductive season that includes the winter months in the northern hemisphere (c. November to March). These changes can be observed in the basal germinal epithelia (BGE) of testicular acini as alterations in the gradients of meiosis and spermatogenesis from the peripheral (basal) to the inner (luminal) regions. Early in the season, large numbers of spermatogonia and primary spermatocytes along with large, spherical nutritive phagocytes (NPs) occupy most of the BGE. Relatively few secondary spermatocytes, spermatids, and spermatozoa are present. In the middle of the season more spermatocytes, spermatids, and spermatozoa are observed within well-defined spermatogenic columns that run between elongated NPs. Late in the season the BGE diminishes as the number of spermatogenic cells within it decreases dramatically and the lumen becomes distended with mature spermatozoa. To trace the origin and location of a 210 KD sperm surface antigen during spermatogenesis, a monoclonal antibody that recognizes this antigen, MAb J18/2, was used to probe testis sections. Indirect immunofluorescence microscopy detected the antigen throughout the BGE. Immunogold electron microscopy further localized it to the intracellular vesicles within spermatogenic cells of all stages. In mature spermatozoa the 210 KD surface antigen was detected not only on the sperm surfaces overlying the acrosomes and tails but also within the acrosome, suggesting the presence of an intracellular store of this antigen. Other than the occasional detection of antigen within the residual bodies of NPs, which contain phagocytosed spermatozoa, no antigen was detected in somatic cells of the testis, indicating that it is generated within the spermatogenic cells themselves.

Flow cytometric quantification of rat spermatogenic cells after hypophysectomy and gonadotropin treatment.

DNA flow cytometry was evaluated as a tool to analyze stage-specific changes that occur in absolute cell numbers in the testes. Hypophysectomy was selected as a model system for perturbing testicular cell types, since the cytological sequelae of this treatment post-hypophysectomy in the rat are well documented in the literature. Rat spermatogenic cells in stages II-V, VII, and IX-XIII of the seminiferous epithelial cycle (as defined by Leblond and Clermont, 1952) were quantified in numbers per standard length of seminiferous tubule by DNA flow cytometry after hypophysectomy and subsequent gonadotropin treatment. In agreement with previous histological studies, we found that acrosome- and maturation-phase spermatids disappeared from the seminiferous epithelium after 17 days post-hypophysectomy, whereas meiosis and early spermiogenesis continued at least 164 days. The number of meiotic cells and round spermatids gradually decreased after hypophysectomy. Changes were observed as early as Day 6 post-hypophysectomy. Treatment with human chorionic gonadotropin (hCG) alone maintained most cell numbers within normal limits, and follicle-stimulating hormone (FSH) was needed in addition to hCG to maintain the normal number of cells with the amount of DNA contained in primary spermatocytes and spermatogonia in G2/M-phase (4C) in stages IX-XIII and elongated spermatids (1C') in stages II-V of the epithelial cycle. The absolute numbers of spermatogenic cells at different phases of maturation provide a useful reference for quantitative studies of spermatogenesis. Pathological changes in the seminiferous epithelium can be detected and quantified by DNA flow cytometry.

Culture patterns and sorting of rat Sertoli cell secretory proteins.

A cocultivation chamber and two types of permeable substrates have been used to study: (1) the culture patterns of rat Sertoli and peritubular cells, and Sertoli cells cocultured with spermatogenic cells or peritubular cells; and (2) the polarized secretion of Sertoli cell-specific proteins transferrin, S70 and S45-S35 heterodimeric protein. Substrates included a nylon mesh (with openings of 100 micron) coated with extracellular matrix (ECM) material and an uncoated microporous filter (with pores of 0.45 micron). Sertoli cells cultured on ECM-coated nylon mesh organized a continuous sheet of multilayered epithelial cells essentially devoid of spermatogenic cells while peritubular cells formed a layer of squamous cells. Sertoli cells cultured on uncoated microporous substrate formed a continuous sheet of cuboidal epithelial cells with numerous basal cytoplasmic processes projecting into the substrate and abundant apically located spermatogenic cells, while peritubular cells organized one or two layers of loose squamous cells. [35S]methionine-labelled secretory proteins resolved by two-dimensional polyacrylamide gel electrophoresis and autoradiography displayed cell-specific patterns that were slightly influenced by the type of substrate. Sertoli cells cocultured with peritubular cells on uncoated microporous substrate under conditions that enabled separation of apical and basal surfaces, secreted proteins in a polarized fashion. While transferrin was released bidirectionally, S45-S35 heterodimeric protein was released apically. S70 was detected in both apical and basal compartments. We conclude from these studies that: (1) the number of spermatogenic cells decreases when Sertoli-spermatogenic cell cocultures are prepared on ECM-coated nylon substrate; and (2) Sertoli cells in coculture with spermatogenic or peritubular cells on uncoated microporous substrate, organize continuous sheets displaying polarized protein secretion.

Testicular aspiration biopsy in evaluation of fertility of mink (Mustela vison).

A 19-gauge needle biopsy was taken of the testis of mink in late January. When scores from 1 to 10 were given according to the developmental stage and number of spermatogenic cells, males scoring 8-10 returned significantly better breeding results than did males having scores less than 7. The biopsy did not affect libido or induce other disturbances of fertility. Fine-needle aspiration biopsy of the testis is possibly the most convenient and accurate infertility assay in mink breeding.

A QUANTITATIVE ANALYSIS OF THE ANNUAL TESTICULAR CYCLE OF THE BRITTLE-STARAMPHIPHOLIS KOCHIIBY MEANS OF AUTORADIOGRAPHIC INVESTIGATION

This paper concerns the annual testicular cycle of the brittle-star Amphipholis kochii (Echinodermata: Ophiuroidea). An autoradiographic study was applied for analysis of the seasonal changes in the number of spermatogenic cells, the duration of spermatogenesis and the spermatocyte production rate. The annual testicular cycle can be divided into five phases; spermatogenesis occurs during phases 2 to 4 (November to June). Spermatogenesis during phases 3 and 4 (March to June) was sensitive to the sea water temperature as an external environment, while that during phase 2 (November to February) was insensitive to it. The water temperature directly controls the speed of the later phase of spermatogenesis (from spermatocyte to spermatozoon), but it is not able to control the spermatocyte production rate. It is therefore suggested that the water temperature plays an important role in spermatogenesis during phases 3 and 4 by controlling the speed of the later phase of spermatogenesis, and that other environmenta...

Techniques for the detection of leukocytospermia in human semen.

During the cytological evaluation of 1200 spermiocytograms, we used prestained microscope slides (Testsimplets) in 200 cases as a screening method. In 50 cases, a check was made to determine whether leukocytospermia could be detected with aid of Cytur-Test. The findings were compared with smears that were routinely stained by the Papanicolaou technique and subsequently differentiated. The cytological examination of human sperm with the aid of Testsimplets provides a technically simple and rapid procedure that has sufficient informative value to permit the diagnosis or exclusion of the leukocytospermia and the sperm morphology. Leukocytospermia could also be detected by the use of Cytur-Test. The presence of larger numbers of spermatogenic cells does not lead to a blue coloration of the Cytur-Test areas. In cases of leukocytospermia there is a clear blue coloration. These two rapid techniques are useful for the diagnosis of leukocytospermia.

Effects of Testicular and Ovarian Inhibin‐Like Activity, Using In Vitro and In Vivo Systems

Inhibin‐like activities in charcoal‐treated bovine follicular fluid (FF) and medium from cultured Sertoli cells (SCCM) were assayed in an in vitro bioassay system, using cultured pituitary cells. Addition of both fluids resulted in parallel dose‐dependent decreases of the concentration of follicle‐stimulating hormone (FSH) in the medium, both in the presence or absence of luteinizing hormone‐releasing hormone (LH‐RH).A single injection of FF into immature and adult male and female rats resulted in decreased peripheral levels of FSH, but not of LH, after 4 or 8 h. This decrease was larger and occurred faster in adult female rats than in prepubertal female animals. Injection of FF into adult female rats, immediately after unilateral ovariectomy (ULO) prevented the specific increase of FSH levels, occurring in control animals. This suppression could not be obtained after treatment with steroids.Daily treatment of adult female or immature male rats for periods longer than 5 days did not result in prolonged suppression of circulating FSH concentrations; LH levels were significantly increased. The female animals showed cyclic vaginal smear changes and normal ovulation; in the male rats testis weight and numbers of spermatogenic cells were reduced.It is concluded that testicular and ovarian inhibin‐like activities have similar properties. Injections of FF into male and female rats cause similar effects on FSH and LH. The effect of FF in ULO‐animals suggests that inhibin could play a role in the short‐term regulation of the number of developing follicles in the ovary. Injection of FF into male rats causes a probably transient impairment of spermatogenesis through an initial suppression of FSH.