Changes in testicular function following specific deprivation of LH in the adult male rabbit

Sexually mature male rabbits actively immunized against highly purified ovine LH (oLH) were used as a model system to study the effects of endogenous LH deprivation (and therefore testosterone) on spermatogenesis as well as pituitary FSH secretion. Immunization against oLH generated antibody titres capable of cross-reacting and neutralizing rabbit LH and this resulted in a significant reduction (P<0.01) in serum testosterone levels by 2-4 weeks of immunization. A significant increase in circulating FSH concentration (from a basal level of similar to 1 ng to 60-100 ng/ml; P<0.01) was observed within 4-6 weeks of immunization, perhaps a consequence of the negative feedback effect of the lack of testosterone. The effect of LH deprivation on spermatogenesis assessed by DNA flow cytometry and histological analyses of testicular biopsy tissue revealed that lack of testosterone primarily results in a rapid reduction and complete absence of round (1C) and elongated (HC) spermatids. The immediate effect of LH/testosterone deprivation thus appears to be at the step of meiotic transformation of primary spermatocytes (4C) to 1C. A significant reduction (>80%; P<0.01) in the 4C population and a relative accumulation (>90%; P<0.01) in spermatogonia (2C) was also observed, suggesting a need for testosterone during the transformation of 2C to 1C. In all but one of the rabbits, both qualitative and quantitative recovery in spermatogenesis occurred during the recovery phase, even at a time when only a marginal increase in serum testosterone (compared with the preimmunization) levels was observed as a result of a rapid decline in the cross-reactive antibody titres. These results clearly show that LH/testosterone deprivation in addition to primarily affecting the meiotic step also regulates the conversion of 2C to 4C during spermatogenesis.

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.

Transformation of 16-dehydroprogesterone and 17?-hydroxyprogesterone by Mucor piriformis

Mucor piriformis was used to study the mode of transformation of 16-dehydroprogesterone (I, pregna-4, 16-diene-3, 20-dione) and 17 alpha-hydroxyprogesterone (II, 17 alpha-hydroxypregn-4-ene-3, 20-dione). Biotransformation products formed from I were 14 alpha-hydroxypregna-4, 16-diene-3, 20-dione (Ia), 7 alpha, 14 alpha-dihydroxypregna-4 16-diene-3, 20-dione (Ib), 3 beta, 7 alpha, 14 alpha-trihydroxy-5 alpha-pregn-16-en-20-one (Ic), and 3 alpha, 7 alpha, 14 alpha-trihydroxy-5 alpha-pregn-16-en-20-one (Id). Metabolites Ic and Id appear to be hitherto unknown. Timecourse studies suggested that the transformation is initiated by hydroxylation at the 14 alpha-position (Ia) followed by hydroxylation at the 7 alpha-position (Ib). Microsomes (105,000 g sediment) prepared from 16-dehydroprogesterone-induced cells hydroxylate I to its 14 alpha-hydroxy derivative (Ia) in the presence of NADPH. Incubation of Ia with the organism resulted in the formation of Ib, Ic and Id. Biotransformation products formed from compound II were 17 alpha, 20 alpha-dihydroxypregn-4-en-3-one (IIa), 7 alpha, 17 alpha-dihydroxypregn-4-ene-3, 20-dione (IIb), 6 beta, 17 alpha, 20 alpha-trihydroxypregn-4-en-3-one (IIc) and 11 alpha, 17 alpha, 20 alpha-trihydroxypregn-4-en-3-one (IId). Time-course studies indicated that IIa is the initial product formed, which is further hydroxylated either at the 6 beta or 11 alpha position. Incubation of IIa with the organism resulted in the formation of IIc and IId. Reduction of the 4-en-3-one system and 20-keto group has not been observed before in organisms of the order Mucorales. In addition, M. piriformis has been shown to carry out hydroxylation at the C-6, C-7, C-11 and C-14 positions in the steroid molecules tested.

Comparative studies on the effects of specific immunoneutralization of endogenous FSH or LH on testicular germ cell transformations in the adult bonnet monkey (Macaca radiata)

PROBLEM: It is yet to be determined clearly whether the two hormones FSH and T act synergistically in the same cell type-the Sertoli cells-to control overall spermatogenesis or influence independently the transformation of specific germ cell types during spermatogenesis in the adult mammal. METHOD: Adult male bonnet monkeys specifically deprived of either FSH or LH using immunoneutralization techniques were monitored for changes in testicular germ cell transformation by DNA flow cytometry. RESULTS: FSH deprivation caused a significant reduction (>40%; P < 0.05) in [H-3] thymidine incorporation into DNA of proliferating 2C (spermatogonial) cells, a marked inhibition (>50%) in the transformation of 2C to primary spermatocytes (4C) and a concomitant, belated reduction (50%) in the formation of round spermatids (1C). In contrast, specific LH/T deprivation led to an immediate arrest in the meiotic transformation of 4C to 1C/HC leading to an effective and significant block (<90%; P < 0.01) in sperm production. CONCLUSION: Thus, LH rather than FSH deprivation has a more pronounced and immediate effect as the former primarily blocks meiosis (4C --> 1C/HC) which controls production of spermatids. These data provide evidence for LH/T and FSH regulating spermatogenic process in the adult primate by primarily acting at specific germ cell transformation steps.

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

Processing of DNA double-stranded breaks and intermediates of recombination and repair by saccharomyces cerevisiae Mre11 and its stimulation by Rad50, Xrs2, and Sae2 proteins

Saccharomyces cerevisiae RAD50, MRE11, and XRS2 genes are essential for telomere length maintenance, cell cycle checkpoint signaling, meiotic recombination, and DNA double-stranded break (DSB) repair via nonhomologous end joining and homologous recombination. The DSB repair pathways that draw upon Mre11-Rad50-Xrs2 subunits are complex, so their mechanistic features remain poorly understood. Moreover, the molecular basis of DSB end resection in yeast mre11-nuclease deficient mutants and Mre11 nuclease-independent activation of ATM in mammals remains unknown and adds a new dimension to many unanswered questions about the mechanism of DSB repair. Here, we demonstrate that S. cerevisiae Mre11 (ScMre11) exhibits higher binding affinity for single-over double-stranded DNA and intermediates of recombination and repair and catalyzes robust unwinding of substrates possessing a 3' single-stranded DNA overhang but not of 5' overhangs or blunt-ended DNA fragments. Additional evidence disclosed that ScMre11 nuclease activity is dispensable for its DNA binding and unwinding activity, thus uncovering the molecular basis underlying DSB end processing in mre11 nuclease deficient mutants. Significantly, Rad50, Xrs2, and Sae2 potentiate the DNA unwinding activity of Mre11, thus underscoring functional interaction among the components of DSB end repair machinery. Our results also show that ScMre11 by itself binds to DSB ends, then promotes end bridging of duplex DNA, and directly interacts with Sae2. We discuss the implications of these results in the context of an alternative mechanism for DSB end processing and the generation of single-stranded DNA for DNA repair and homologous recombination.

N-Terminal disordered domain of saccharomyces cerevisiae Hop1 protein Is dispensable for DNA binding, bridging, and synapsis of double-stranded DNA molecules but is ecessary for spore formation

The cytological architecture of the synaptonemal complex (SC), a meiosis-specific proteinaceous structure, is evolutionarily conserved among eukaryotes. However, little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in interhomolog recombination. Previously, we showed that Hop1 is a structure-specific DNA-binding protein, exhibits higher binding affinity for the Holliday junction, and induces structural distortion at the core of the junction. Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular synapsis between duplex DNA molecules. Here, we show that Hop1 possesses a modular domain organization, consisting of an intrinsically disordered N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD). Furthermore, we found that Hop1CTD exhibits strong homotypic as well as heterotypic protein protein interactions, and its biochemical activities were similar to those of the full-length Hop1 protein. However, Hop1CTD failed to complement the meiotic recombination defects of the Delta hop1 strain, indicating that both N- and C-terminal domains of Hop1 are essential for meiosis and spore formation. Altogether, our findings reveal novel insights into the structure-function relationships of Hop1 and help to further our understanding of its role in meiotic chromosome synapsis and recombination.

Structural Insights into Saccharomyces cerevisiae Msh4-Msh5 Complex Function Using Homology Modeling

The Msh4-Msh5 protein complex in eukaryotes is involved in stabilizing Holliday junctions and its progenitors to facilitate crossing over during Meiosis I. These functions of the Msh4-Msh5 complex are essential for proper chromosomal segregation during the first meiotic division. The Msh4/5 proteins are homologous to the bacterial mismatch repair protein MutS and other MutS homologs (Msh2, Msh3, Msh6). Saccharomyces cerevisiae msh4/5 point mutants were identified recently that show two fold reduction in crossing over, compared to wild-type without affecting chromosome segregation. Three distinct classes of msh4/5 point mutations could be sorted based on their meiotic phenotypes. These include msh4/5 mutations that have a) crossover and viability defects similar to msh4/5 null mutants; b) intermediate defects in crossing over and viability and c) defects only in crossing over. The absence of a crystal structure for the Msh4-Msh5 complex has hindered an understanding of the structural aspects of Msh4-Msh5 function as well as molecular explanation for the meiotic defects observed in msh4/5 mutations. To address this problem, we generated a structural model of the S. cerevisiae Msh4-Msh5 complex using homology modeling. Further, structural analysis tailored with evolutionary information is used to predict sites with potentially critical roles in Msh4-Msh5 complex formation, DNA binding and to explain asymmetry within the Msh4-Msh5 complex. We also provide a structural rationale for the meiotic defects observed in the msh4/5 point mutations. The mutations are likely to affect stability of the Msh4/5 proteins and/or interactions with DNA. The Msh4-Msh5 model will facilitate the design and interpretation of new mutational data as well as structural studies of this important complex involved in meiotic chromosome segregation.

The HORMA domain: an evolutionarily conserved domain discovered in chromatin-associated proteins, has unanticipated diverse functions

The HORMA domain (for Hop1p, Rev7p and MAD2) was discovered in three chromatin-associated proteins in the budding yeast Saccharomyces cerevisiae. This domain has also been found in proteins with similar functions in organisms including plants, animals and nematodes. The HORMA domain containing proteins are thought to function as adaptors for meiotic checkpoint protein signaling and in the regulation of meiotic recombination. Surprisingly, new work has disclosed completely unanticipated and diverse functions for the HORMA domain containing proteins. A. M. Villeneuve and colleagues (Schvarzstein et al., 2013) show that meiosis-specific HORMA domain containing proteins plays a vital role in preventing centriole disengagement during Caenorhabditis elegans spermatocyte meiosis. Another recent study reveals that S. cerevisiae Atg13 HORMA domain acts as a phosphorylation-dependent conformational switch in the cellular autophagic process. (C) 2014 Elsevier B.V. All rights reserved.

N-Terminal Disordered Domain of Saccharomyces cerevisiae Hop1 Protein Is Dispensable for DNA Binding, Bridging, and Synapsis of Double-Stranded DNA Molecules But Is Necessary for Spore Formation

The cytological architecture of the synaptonemal complex (SC), a meiosis-specific proteinaceous structure, is evolutionarily conserved among eukaryotes. However, little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in interhomolog recombination. Previously, we showed that Hop1 is a structure-specific DNA-binding protein, exhibits higher binding affinity for the Holliday junction, and induces structural distortion at the core of the junction. Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular synapsis between duplex DNA molecules. Here, we show that Hop1 possesses a modular domain organization, consisting of an intrinsically disordered N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD). Furthermore, we found that Hop1CTD exhibits strong homotypic as well as heterotypic protein protein interactions, and its biochemical activities were similar to those of the full-length Hop1 protein. However, Hop1CTD failed to complement the meiotic recombination defects of the Delta hop1 strain, indicating that both N- and C-terminal domains of Hop1 are essential for meiosis and spore formation. Altogether, our findings reveal novel insights into the structure-function relationships of Hop1 and help to further our understanding of its role in meiotic chromosome synapsis and recombination.

Molecular phylogeny of Glyphochloa (Poaceae, Panicoideae), an endemic grass genus from the Western Ghats, India

The genus Glyphochloa (Poaceae: Panicoideae: Andropogoneae: Rottboellinae) is endemic to peninsular India and is distributed on lateritic plateaus of low and high altitude in and around Western Ghats and the Malabar Coast. The genus presumably originated and diversified in the Western Ghats. Species relationships in the genus Glyphochloa were deduced here based on molecular phylogenies inferred using nuclear ribosomal ITS sequences and plastid intergenic spacer regions (atpB-rbcL, trnT-trnL, trnL-trnF), and new observations were made of spikelet morphology, caryopsis morphology and meiotic chromosome counts. We observed two distinct clades of Glyphochloa s.l. One of these (group I') includes Ophiuros bombaiensis, and is characterized by a single-awned lower glume and a base chromosome number of 6; it grows in low elevation coastal areas. The other clade (group II') has a double-awned lower glume, a base chromosome number of 7, and is restricted to higher elevation lateritic plateaus; G. ratnagirica may belong to the group II clade, or may be a third distinct lineage in the genus. A sister-group relationship between group I and II taxa (with or without G. ratnagirica) is not well supported, although the genus is recovered as monophyletic in shortest trees inferred using ITS or concatenated plastid data. We present a key to species of Glyphochloa and make a new combination for O. bombaiensis.

Meiosis in flowering plants and other green organisms.

Sexual eukaryotes generate gametes using a specialized cell division called meiosis that serves both to halve the number of chromosomes and to reshuffle genetic variation present in the parent. The nature and mechanism of the meiotic cell division in plants and its effect on genetic variation are reviewed here. As flowers are the site of meiosis and fertilization in angiosperms, meiotic control will be considered within this developmental context. Finally, we review what is known about the control of meiosis in green algae and non-flowering land plants and discuss evolutionary transitions relating to meiosis that have occurred in the lineages giving rise to the angiosperms.

Induced diploid meiogynogenesis in the African clariid catfish Heterobranchus longifilis (Valenciennes, 1840) (Pisces: Clariidae)

Diploid meiotic gynogenesis was induced in African catfish, Heterobranchus longifilis by injection of 0.5ml/kg ovaprim on the breeders, followed by application of UV light irradiation on the spermatozoa and temperature shocking of activated eggs. Diploidy was restored by shocking haploid activated eggs at 5 degree C for 40 minutes. The normal control spermatozoa did not receive any UV irradiation nor temperature shock, while the haploid control spermatozoa were irradiated, but did not receive cold shock. The percentage hatchability in the treated group was 25%, while in the control it was 53%. Less than 15 fingerlings had morphological aberrations. After two weeks of indoor rearing, the survival percentage of the treated group was 45% in the control experiment. Cytogenetic analysis of chromosomes revealed 25 chromosomes in the haploid embryo and 50 chromosomes each in diploid gynogenesis and normal diploid control

Individual growth and seasonal cycle of Daphnia middendorffiana in an Alpine lake [Translation from: Memorie dell'Istituto Italiano di Idrobiologia Dott.Marco de Marchi 26 41-83, 1970]

The problem of the peculiar reproductive biology of the cladoceran Daphnia middendorffiana is investigated from a cytological viewpoint, and by direct observation the meiotic phenomena of the eggs both subitaneous and resting is studied. and during maturation, the true mechanism of the succession of reproductive phases of different ecological significance. Samples were collected in the Italian Alpine Lake of Campo 4°.