A role for nocturnal serum testosterone surge in regulating spermatogenesis in the adult non-human primate

Adult male bonnet monkeys exhibit nychthemeral rhythms in testosterone (T) secretion but the precise role of this heightened level of T secretion in regulating spermatogenesis is not known. Intranasal administration of microdoses (500 mu g or 250 mu g/day) of Norethisterone (IN-NET) to adult monkeys (n = 6) at 1600 h each day selectively and completely suppressed the nocturnal surge levels of serum T. Concomitant with this was a significant reduction (P<0.01) in serum LH but not FSH levels. DNA flow cytometric analysis of testicular biopsy tissue showed by week 10 of IN-NET treatment an arrest in meiotic transformation of primary spermatocytes (4C) to round/elongate (1C/HC) spermatids and by week 20 there was a complete absence of 4C, 1C and HC cells (with a relative accumulation in 2C cells). The accumulated meiotic (4C) cells at week 10 showed an increase (>80%, P<0.01) in coefficient of variation and a decrease in intensity of DNA-bound ethidium bromide fluorescence, parameters characteristic of degenerating 'apoptotic' subpopulation of germ cells. While two monkeys exhibited acute oligozoospermia 4 became azoospermic by 20 weeks of IN-NET treatment. A complete, qualitative reversal in the regressive changes in spermatogenesis and near-normal sperm output were apparent at the end of a 20-week recovery phase. These data demonstrate that prolonged, selective suppression of nocturnal surge levels of serum T secretion exerts a primary effect on meiosis in spermatogenesis leading to oligo/azoospermic status in adult bonnet monkeys.

Chromosome complements in the spermatogenesis of two penaeid prawns, Penaeus merguiensis and P. esculentus.

Although the fisheries for, and mariculture of, penaeid prawns are of major commercial importance, there has been relatively little research undertaken on the chromosome number, structure and composition in the Penaeidae. One reason for this is due to the relatively small size and large number of chromosomes, which makes production of histological material difficult. In this paper, we report a simple and effective technique for determining chromosome complements during spermatogenesis in two species of penaeid prawns, Penaeus merguiensis and P. esculentus in Australia. The first estimates of the number of chromosomes in these species are given.

Identification of a novel nucleolin related protein (NRP) gene expressed during rat spermatogenesis

Nucleolin is a major nucleolar phosphoprotein involved in various steps of ribosome biogenesis in eukaryotic cells. As nucleolin plays a significant role in ribosomal RNA transcription we were interested in examining in detail the expression of nucleolin across different stages of spermatogenesis and correlate with the transcription status of ribosomal DNA in germ cells.

Hollow sperm syndrome during spermatogenesis in the giant tiger shrimp Penaeus monodon (Fabricius 1798) from eastern Australia

During spermatogenesis, giant tiger shrimp (Penaeus monodon) from Queensland, eastern Australia had a high proportion of testicular spermatids that appeared 'hollow' because their nuclei were not visible with the haematoxylin and eosin stain. When examined by transmission electron microscopy, the nuclei of hollow spermatids contained highly decondensed chromatin, with large areas missing fibrillar chromatin. Together with hollow spermatids, testicular pale enlarged (PE) spermatids with weakly staining and marginated chromatin were observed. Degenerate-eosinophilic-clumped (DEC) spermatids that appeared as aggregated clumps were also present in testes tubules. Among 171 sub-adult and adult P. monodon examined from several origins, 43% displayed evidence of hollow spermatids in the testes, 33% displayed PE spermatids and 15% displayed DEC spermatids. These abnormal sperm were also found at lower prevalence in the vas deferens and spermatophore. We propose 'Hollow Sperm Syndrome (HSS)' to describe this abnormal sperm condition as these morphological aberrations have yet to be described in penaeid shrimp. No specific cause of HSS was confirmed by examining either tank or pond cultured shrimp exposed to various stocking densities, temperatures, salinities, dietary and seasonal factors. Compared with wild broodstock, HSS occurred at higher prevalence and severity among sub-adults originating from farms, research ponds and tanks. Further studies are required to establish what physiological, hormonal or metabolic processes may cause HSS and whether it compromises the fertility of male P. monodon.

Gonadal cell surface receptor for plasma retinol-binding protein A method for its radioassay and studies on its level during spermatogenesis

A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps: direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme.The binding of retinol-binding protein to the receptor is saturable and reverible. The interaction shows a Kd value of 2.1 · 10−10 M. The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testoterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifically induced by testoterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome.

Impaired spermatogenesis in Finnish boars and bulls

Maintenance of breeding efficiency and high semen quality is essential for reproductive success in farm animals. Early recognition of possible inheritable factors causing infertility requires constant attention. This thesis focuses on describing different manifestations of impaired spermatogenesis, their impact on fertility and partly also their incidence in populations. The reasons for spermatogenic failure are various. An interruption of germ cell differentiation, spermatogenic arrest, can lead to infertility. The incidence of azoospermia was investigated in the 1996 2005 survey of Finnish AI and farm breeding boars. We focused on the diagnosis, testicular morphometry and the possible reasons for the condition. The incidence of azoospermia was significantly higher in Yorkshire boars than in the Landrace breed. The most common diagnosis in Yorkshire boars was germ cell arrest at the primary spermatocyte level. The second most frequent diagnosis in Yorkshire boars was segmental aplasia of the Wolffian ducts with idiopathic epididymal obstruction. Other reasons for azoospermia were infrequent. In the second study we investigated the incidence of two relatively well-defined specific sperm defects in Finnish Yorkshire and Landrace boars during the same survey, the immotile short-tail sperm (ISTS) defect and the knobbed acrosome (KA) defect. In the Finnish Yorkshire boars the inherited ISTS defect, and the probably inherited KA defect, were important causes of infertility during 1996 2005. The ISTS defect was found in 7.6% and the KA defect in 0.8% of the Yorkshire boars. No Landrace boars were diagnosed with either of these two defects. In the third study we described a new sterilizing sperm defect in an oligoasthenoterazoospermic bull. Because of its morphological characteristics this defect was termed the multinuclear-multiflagellar sperm (MNMFS) defect. The number of Sertoli cells in the seminiferous tubuli was highly increased in the MNMFS bull compared with the number in normal bulls. In the following two studies we used a combined approach of fluorescence in situ hybridization (FISH), flow cytometry and morphometric studies to provide information on the cytogenetic background of macrocephalic bull spermatozoa. We described cellular features of diploid spermatozoa and compared the failures in the first and second meiotic divisions. In the last study we describe how the transplantation of testicular cells was used to determine whether spermatogonia derived from donor animals are able to colonize and produce motile spermatozoa in immune-competent unrelated boars suffering the ISTS defect. Transplantation resulted in complete focal spermatogenesis, indicated by the appearance of motile spermatozoa and confirmed by genotyping.

Behaviour of the germ cell specific lamin through mammalian spermatogenesis as probed with monoclonal antibodies.

We had earlier identified a 60 kDa nuclear lamin protein (lamin(g)) unique to the germ cells of rat testis which was subsequently shown to be antigenically conserved in germ cells of grasshopper, rooster, frog and plants. We have now obtained eight monoclonal antibodies in mouse against this lamin(g) antigen. While all the eight Mabs reacted with lamin(g) antigen in an immunoblot analysis, only three Mabs (A(11)C(7), A(11)D(4), C1F7) showed strong reactivity in the immunofluorescence analysis of the germ cells. The Mabs A(11)C(7) and A(11)D(4) showed a slight cross-reactivity with rat liver lamin B. Indirect immunofluorescence analysis of pre-meiotic, meiotic and post-meiotic germ cells with Mabs have shown that while the lamin(g) is localized in the lamina structures of spermatogonia and round spermatids, it is localized to the phase dense regions of pachytene spermatocytes which is in conformity with our previous observations using rabbit polyclonal antibodies. The localization of the antigen in the germ cells was also confirmed by immunohistochemical staining of the thin sections of seminiferous tubules. By immunostaining the surface spread pachytene spermatocytes, the antigen was further localized to the telomeric ends of the paired homologous chromosomes. Using anti-somatic lamin B antibodies, we have also demonstrated the absence of somatic lamins in meiotic and post-meiotic germ cells. The lamina structure of pre-meiotic spermatogonial nucleus contains both somatic lamin B and lamin(g) as evidenced by immunofluorescence studies with two differently fluorochrome labelled anti-lamin B and anti-lamin(g) antibodies. The selective retention of lamin(g) in the pachytene spermatocytes is probably essential for anchoring the telomeric ends of the paired chromosomes to the inner nuclear membrane.

Quantitation of spermatogenesis by DNA flow cytometry: Comparative study among six species of mammals

Suspensions of testicular germ cells from six species of mammals were prepared and stained for the DNA content with a fluorochrome (ethidium bromide) adopting a common technique and subjected to DNA flow cytometry. While uniform staining of the germ cells of the mouse, hamster, rat and monkey could be obtained by treating with 0.5% pepsin for 60 min followed by staining with ethidium bromide for 30 min, that of the guinea pig and rabbit required for optimal staining pepsinization for 90 min and treatment with ethidium bromide for 60 min. The procedure adopted here provided a uniform recovery of over 80% of germ cells with each one of the species tested and the cell population distributed itself according to the DNA content (expressed as C values) into 5 major classes-spermatogonia (2C), cells in S-phase, primary spermatocytes (4C), round spermatids (1C), and elongating/elongated spermatids (HC). Comparison of the DNA distribution pattern of the germ cell populations between species revealed little variation in the relative quantities of cells with 2C (8-11%), S-phase (6-9%), and 4C (6-9%) amount of DNA. Though the spermatid cell populations exhibited variations (1C:31-46%, HCI:7-20% and and HC2:11-25%) they represented the bulk of germ cells (70-80%). The overall conversion of 2C to 1C (1C:2C ratio) and meiotic transformation of 4C cells to IC (1C:4C ratio) kinetics were relatively constant between the species studied. The present study clearly demonstrates that DNA flow cytometry can be adopted with ease and assurance to quantify germ cell transformation and as such spermatogenesis by analysing a large number of samples with consistency both within and across the species barrier. Any variation from the norms in germ cell proportions observed following treatment, for e.g. hormonal stimulation or deprivation can then be ascribed due to a specific effect of the hormone/drug on single/multiple steps in germ cell transformation

Expression and Localization of Opioid Receptors in Male Germ Cells and the Implication for Mouse Spermatogenesis

The presence of endogenous opioid peptides in different testicular cell types has been extensively characterized and provides evidence for the participation of the opioid system in the regulation of testicular function. However, the exact role of the opioid system during the spermatogenesis has remained controversial since the presence of the mu-, delta-and kappa-opioid receptors in spermatogenic cells was yet to be demonstrated. Through a combination of quantitative real-time PCR, immunofluorescence, immunohistochemistry and flow cytometry approaches, we report for the first time the presence of active mu-, deltaand kappa-opioid receptors in mouse male germ cells. They show an exposition time-dependent response to opioid agonist, hence suggesting their active involvement in spermatogenesis. Our results contribute to understanding the role of the opioid receptors in the spermatogenesis and could help to develop new strategies to employ the opioid system as a biochemical tool for the diagnosis and treatment of male infertility.

A histological study of the spermatogenesis in Ompok pabda (Hamilton-Buchanan 1822)

The present study deals with the histological analysis of testicular development in Ompok pabda. For the study, male gonads were collected month wise from January to September at Freshwater Station, BFRI, Mymensingh. From the analysis, 4 stages of sperm formation, namely, spermatogonia, spermatocytes, spermatids and spermatozoa, were distinguished. The percent distribution of spermatozoa was highest in July (about 92%). Maximum GSI value was 1.129±0.271 found in July. By analyzing the histology of spermatogenesis it was established that this species breeds once in a year.

Rapid evolution, genetic variations, and functional association of the human spermatogenesis-related gene NYD-SP12

NYD-SP12 is a recently identified spermatogenesis-related gene with a pivotal role in human testis development. In this study, we analyzed between-species divergence and within-species variation of NYD-SP12 in seven representative primate species, four wo

Effect of butachlor on spermatogenesis, testosterone amount and testes tissue Rutilus frisii kutum Kamenskii 1901

Morphological assessment of sexually mature Rutilus frisii kutum Kamenskii 1901 caught from the rivers (Shirud, Khoshkrud, Sepidrud and Chelavand Rivers) flowing in the southwest Caspian Sea region was conducted and sperm volume, total sperm count and sperm concentration of abnormal sperms were determined after exposing the spawners to 60% herbicide butachlor (machete). Spawners under study were maintained in tanks (1000 l) at the Shahid Ansari Teleost Fish Hatchery and exposed to two different concentrations (25% and 75% of its LC50 value) of butachlor. Results obtained indicate that exposure to high butachlor toxicity (75% of its LC50 value) decreased sperm volume to 0.61 ± 0.42 cc in 2-3 year old fishes and to 0.55 ± 0.42 cc in fishes above 3 years of age, while that in fish exposed to low butachlor toxicity (25% of its LC50 value) decreased to 1.55 ± 0.42 cc in 2-3 year old fishes and to 1.28 ± 0.42 cc in fishes above 3 years of age. The sperm volume under normal conditions in R. frisii kutum is 4.6 ± 0.42 cc in 2-3 year olds and 4.58 ± 0.42 cc in fishes above 3 years of age. The total sperm count in R. frisii kutum is 39.74 ± 2.5 billion spermatozoa/cc in 2-3 year olds and 42.99 ± 2.5 billion spermatozoa/cc in fishes above 3 years of age. When exposed to high butachlor toxicity, total sperm count dropped to 16.92 ± 2.5 billion spermatozoa/cc in 2-3 year olds and to 15.98 ± 2.5 billion spermatozoa/cc in fishes above 3 years of age. Similarly total sperm count in R. frisii kutum exposed to low butachlor toxicity was recorded as 23.6 ± 2.5 billion spermatozoa/cc in 2-3 year olds and 29.4 ± 2.5 billion spermatozoa/cc in fishes above 3 years of age. Under normal conditions, on the basis of morphology, spermatozoa showed only 10 ± 1.92% of abnormal sperms. The number of abnormal sperms increased by 28.6 ± 1.92% in fishes exposed to high butachlor toxicity, while that in fishes exposed to low butachlor toxicity increased by 19.7 ± 1.92% in 2-3 year olds and 16.6 ± 19.2% in fishes above 3 years of age. It is evident from the results obtained that increase in level of pollution caused a decrease in sperm volume but an increase in the percentage of abnormal sperms. Results obtained indicate that exposure to high butachlor toxicity (75% of its LC50 value) decreased testostron hormone to 0.31 ± 0.22 ng/ml in high butachlor toxicity, and to 0.45 ± 0.22 ng/ml in low butachlor toxicity (25% of its LC50 value). Testostron hormone dropped to 0.53 ± 0.22 ng/ml in 2-3 year olds and to 0.79 ± 0.22ng/ in fishes above 3 years of age. The testostron hormone under normal conditions in R. frisii kutum is 2.7 ± 0.22 ng/ml. It is evident from the results obtained that increase in level of pollution caused a decrease in testostron hormone