Chlorpyrifos impairs sperm parameters and number of Sertoli and Leydig cells in rats after exposure during the peripubertal period.

Chlorpyrifos is an organophosphate insecticide used to control pests in crops. Thus, humans are constantly exposed through ingestion of contaminated food or water, inhalation of contaminated air, and through the skin. The juvenile and peripubertal periods comprise a window of development of the reproductive system, sensitive to toxic agents. Considering the scarcity of data on exposure to the insecticide during these periods, the aim of this study was to evaluate the effects of chlorpyrifos on the testis during the juvenile and peripubertal periods. Thirty Wistar rats with an initial age of 25 days were distributed into 3 groups: control, which received corn oil (vehicle); CPS5, which received 5 mg/Kg b.w. of chlorpyrifos; and CPS15, which received 15 mg/Kg b.w. of chlorpyrifos. The groups were treated via gavage daily for 40 days and on the 41st experimental day, the animals were anesthetized and submitted to euthanasia to collect the organs. Blood was collected to obtain plasma and testosterone measurement. The testicles were removed, weighed and used for sperm count analyses, histopathological and morphometric analyzes and for oxidative stress analyses. Spermatozoa from the vas deferens were collected for analyzes of sperm morphology and acrosome integrity. The results showed that the two concentrations of chlorpyrifos caused a decrease in the number of Leydig and Sertoli cells and germ cells and increased the number of morphologically abnormal sperm and sperm with acrosomal damage. Furthermore, a decrease in lipid peroxidation was observed in the CPS5 and CPS15 groups, and a decrease in glutathione-S-transferase activity in the CPS5 group. We conclude that exposure to chlorpyrifos harms the daily production of sperm, as well as their quality, in addition to causing an imbalance in the oxidoreductive balance of the testicle.

Analysis of Plant-Specific ANTH Domain-Containing Protein in Marchantia polymorpha.

Membrane trafficking is a fundamental mechanism for protein and lipid transport in eukaryotic cells and exhibits marked diversity among eukaryotic lineages with distinctive body plans and lifestyles. Diversification of the membrane trafficking system is associated with the expansion and secondary loss of key machinery components, including RAB GTPases, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and adaptor proteins, during plant evolution. The number of AP180 N-terminal homology (ANTH) proteins, an adaptor family that regulates vesicle formation and cargo sorting during clathrin-mediated endocytosis, increases during plant evolution. In the genome of Arabidopsis thaliana, 18 genes for ANTH proteins have been identified, a higher number than that in yeast and animals, suggesting a distinctive diversification of ANTH proteins. Conversely, the liverwort Marchantia polymorpha possesses a simpler repertoire; only two genes encoding canonical ANTH proteins have been identified in its genome. Intriguingly, a non-canonical ANTH protein is encoded in the genome of M. polymorpha, which also harbors a putative kinase domain. Similar proteins have been detected in sporadic lineages of plants, suggesting their ancient origin and multiple secondary losses during evolution. We named this unique ANTH group phosphatidylinositol-binding clathrin assembly protein-K (PICALM-K) and characterized it in M. polymorpha using genetic, cell biology-based and artificial intelligence (AI)-based approaches. Our results indicate a flagella-related function of MpPICALM-K in spermatozoids, which is distinct from that of canonical ANTH proteins. Therefore, ANTH proteins have undergone significant functional diversification during evolution, and PICALM-K represents a plant-unique ANTH protein that is delivered by neofunctionalization through exon shuffling.

Autophagy regulates plastid reorganization during spermatogenesis in the liverwort Marchantia polymorpha.

Autophagy is a highly conserved system that delivers cytoplasmic components to lysosomes/vacuoles. Plastids are also degraded through autophagy for nutrient recycling and quality control; however, the involvement of autophagic degradation of plastids in plant cellular differentiation remains unclear. Here, we investigated whether spermiogenesis, the differentiation of spermatids into spermatozoids, in the liverwort Marchantia polymorpha involves autophagic degradation of plastids. Spermatozoids of M. polymorpha possess one cylindrical plastid at the posterior end of the cell body. By fluorescently labeling and visualizing plastids, we detected dynamic morphological changes during spermiogenesis. We found that a portion of the plastid was degraded in the vacuole in an autophagy-dependent manner during spermiogenesis, and impaired autophagy resulted in defective morphological transformation and starch accumulation in the plastid. Furthermore, we found that autophagy was dispensable for the reduction in plastid number and plastid DNA elimination. These results demonstrate a critical but selective role of autophagy in plastid reorganization during spermiogenesis in M. polymorpha.

A Physcomitrella PIN protein acts in spermatogenesis and sporophyte retention.

The auxin efflux PIN-FORMED (PIN) proteins are conserved in all land plants and important players in plant development. In the moss Physcomitrella (Physcomitrium patens), three canonical PINs (PpPINA-C) are expressed in the leafy shoot (gametophore). PpPINA and PpPINB show functional activity in vegetative growth and sporophyte development. Here, we examined the role of PpPINC in the life cycle of Physcomitrella. We established reporter and knockout lines for PpPINC and analysed vegetative and reproductive tissues using microscopy and transcriptomic sequencing of moss gametangia. PpPINC is expressed in immature leaves, mature gametangia and during sporophyte development. The sperm cells (spermatozoids) of pinC knockout mutants exhibit increased motility and an altered flagella phenotype. Furthermore, the pinC mutants have a higher portion of differentially expressed genes related to spermatogenesis, increased fertility and an increased abortion rate of premeiotic sporophytes. Here, we show that PpPINC is important for spermatogenesis and sporophyte retention. We propose an evolutionary conserved way of polar growth during early moss embryo development and sporophyte attachment to the gametophore while suggesting the mechanical function in sporophyte retention of a ring structure, the Lorch ring.

Autophagy regulates organelle reorganization during spermiogenesis in the liverwort <i>Marchantia polymorpha</i>.

Sperm mitochondria generally exhibit distinctive and diverse morphologies in animals and plants. Bryophytes, a plant group consisting of liverworts, mosses, and hornworts, produce motile male gametes, called spermatozoids, that possess a fixed number of two mitochondria in their cell bodies. Electron microscopy observations have revealed the detailed morphological aspects of plant spermatozoids, including mitochondrial morphology; however, the mechanism by which mitochondria are reorganized during spermiogenesis in bryophytes remains largely unknown. Our recent study using the liverwort, <i>Marchantia polymorpha</i>, revealed that the mitochondrial number is reduced to one via mitochondrial fission and macroautophagic/autophagic degradation, which subsequently becomes two via asymmetric division to form large anterior and small posterior mitochondria. Other cytoplasmic components, such as peroxisomes, are also degraded via autophagy; however, mitochondria are degraded at a time distinct from other cytoplasmic components. We also found that some cytoplasmic components were degraded in the vacuole independent of autophagy. Our study highlights the dynamic reorganization of organelles via multiple degradation pathways during spermiogenesis in <i>M. polymorpha</i>.

HAG1 and SWI3A/B control of male germ line development in P. patens suggests conservation of epigenetic reproductive control across land plants.

KEY MESSAGE: Bryophytes as models to study the male germ line: loss-of-function mutants of epigenetic regulators HAG1 and SWI3a/b demonstrate conserved function in sexual reproduction. With the water-to-land transition, land plants evolved a peculiar haplodiplontic life cycle in which both the haploid gametophyte and the diploid sporophyte are multicellular. The switch between these phases was coined alternation of generations. Several key regulators that control the bauplan of either generation are already known. Analyses of such regulators in flowering plants are difficult due to the highly reduced gametophytic generation, and the fact that loss of function of such genes often is embryo lethal in homozygous plants. Here we set out to determine gene function and conservation via studies in bryophytes. Bryophytes are sister to vascular plants and hence allow evolutionary inferences. Moreover, embryo lethal mutants can be grown and vegetatively propagated due to the dominance of the bryophyte gametophytic generation. We determined candidates by selecting single copy orthologs that are involved in transcriptional control, and of which flowering plant mutants show defects during sexual reproduction, with a focus on the under-studied male germ line. We selected two orthologs, SWI3a/b and HAG1, and analyzed loss-of-function mutants in the moss P. patens. In both mutants, due to lack of fertile spermatozoids, fertilization and hence the switch to the diploid generation do not occur. Pphag1 additionally shows arrested male and impaired female gametangia development. We analyzed HAG1 in the dioecious liverwort M. polymorpha and found that in Mphag1 the development of gametangiophores is impaired. Taken together, we find that involvement of both regulators in sexual reproduction is conserved since the earliest divergence of land plants.

Characterisation of evolutionarily conserved key players affecting eukaryotic flagellar motility and fertility using a moss model.

Defects in flagella/cilia are often associated with infertility and disease. Motile male gametes (sperm cells) are an ancestral eukaryotic trait that has been lost in several lineages like flowering plants. Here, we made use of a phenotypic male fertility difference between two moss (Physcomitrella patens) ecotypes to explore spermatozoid function. We compare genetic and epigenetic variation as well as expression profiles between the Gransden and Reute ecotype to identify a set of candidate genes associated with moss male infertility. We generated a loss-of-function mutant of a coiled-coil domain containing 39 (ccdc39) gene that is part of the flagellar hydin network. Defects in mammal and algal homologues of this gene coincide with a loss of fertility, demonstrating the evolutionary conservation of flagellar function related to male fertility across kingdoms. The Ppccdc39 mutant resembles the Gransden phenotype in terms of male fertility. Potentially, several somatic (epi-)mutations occurred during prolonged vegetative propagation of Gransden, causing regulatory differences of for example the homeodomain transcription factor BELL1. Probably these somatic changes are causative for the observed male fertility defect. We propose that moss spermatozoids might be employed as an easily accessible system to study male infertility of humans and animals in terms of flagellar structure and movement.

DEK1 displays a strong subcellular polarity during Physcomitrella patens 3D growth.

Defective Kernel 1 (DEK1) is genetically at the nexus of the 3D morphogenesis of land plants. We aimed to localize DEK1 in the moss Physcomitrella patens to decipher its function during this process. To detect DEK1 in vivo, we inserted the tdTomato fluorophore into PpDEK1 gene locus. Confocal microscopy coupled with the use of time-gating allowed the precise DEK1 subcellular localization during 3D morphogenesis. DEK1 localization displays a strong polarized signal, as it is restricted to the plasma membrane domain between recently divided cells during the early steps of 3D growth development as well as during the subsequent vegetative growth. The signal furthermore displays a clear developmental pattern because it is only detectable in recently divided and elongating cells. Additionally, DEK1 localization appears to be independent of its calpain domain proteolytic activity. The DEK1 polar subcellular distribution in 3D tissue developing cells defines a functional cellular framework to explain its role in this developmental phase. Also, the observation of DEK1 during spermatogenesis suggests another biological function for this protein in plants. Finally the DEK1-tagged strain generated here provides a biological platform upon which further investigations into 3D developmental processes can be performed.

Cryopreservation of Marchantia polymorpha spermatozoa.

The liverwort Marchantia polymorpha has become one of the model organisms, since it has less genetic redundancy, sexual and asexual modes of reproduction and a range of genomic and molecular genetic resources. Cryopreservation of fertile spermatozoa eliminates time, space and labor for growing and maintaining male plants in reproductive phase, and also provides an optional way to backup lines. Here we report a protocol to cryopreserve spermatozoa of M. polymorpha in liquid nitrogen. A cryoprotective solution containing sucrose, glycerol and egg yolk and controlled cooling and warming processes led to successful recovery of motile M. polymorpha spermatozoa after the cryogenic process. The survival rate and average motility of spermatozoa after cryopreservation were maintained at 71 and 54% of those before cryopreservation, respectively. Cryopreserved spermatozoa were capable of fertilization to form normal spores. The technique presented here confers more versatility to experiments using M. polymorpha and could be applied to preservation of plant spermatozoa in general.

Refrigeração do sêmen da garoupa-verdadeira Epinephelus marginatus / Refrigerated storage of dusky grouper Epinephelus marginatus sperm

Foram estudados diferentes diluentes no processo de refrigeração do sêmen da garoupa-verdadeira. Inicialmente, a taxa e a duração da motilidade e a concentração espermática foram avaliadas, caracterizando a qualidade do sêmen fresco. Para os testes de refrigeração a 4ºC, diferentes diluentes foram testados em atmosfera normal e modificada (100% oxigênio). O sêmen fresco apresentou concentração espermática de 3,1±0,2 x 109 células mL-1, motilidade média de 90%, permanecendo móvel, em média, por 3.060 segundos. No experimento de refrigeração, a taxa e a duração média da motilidade foram mantidas adequadas durante 144 horas para o diluente A (70%; 3100 segundos) em atmosfera normal. Na atmosfera modificada, a qualidade do sêmen caiu drasticamente durante as primeiras 24 horas, independentemente do diluente empregado, não propiciando vantagem em sua utilização. A refrigeração do sêmen da garoupa-verdadeira permite manter por até 144 horas uma apropriada qualidade espermática.(AU)

This study aimed to evaluate different extenders in refrigerated storage of dusky grouper sperm. Initially, motility rate, motility time, and sperm density were analyzed to characterize the fresh sperm quality. Different diluents were used in the refrigerated storage sperm at 4ºC in normal atmosphere and modified atmosphere (100% oxygen). The fresh sperm has a spermatozoa density of 3.1±0.2 x 109 spermatozoa mL-1, motility rate 90% and motility time of 3,060 seconds. In the experiment of refrigerated storage, the motility rate and the motility time were maintained appropriate for 144 hours for the extender A (70%; 3,100 seconds) in normal atmosphere. In the modified atmosphere, the sperm quality decreased drastically during the first 24 hours, independently of the diluent used, not propitiating advantages. The refrigerated sperm of dusky grouper is a viable alternative for the short-term storage, being possible, to maintain for up to 144 hours an appropriate sperm viability.(AU)

Scanning Electron Microscopy of Motile Male Gametes of Land Plants.

The only motile cells produced in land plants are male gametes (spermatozoids), which are reduced to non-flagellated cells in flowering plants and most gymnosperms. Although a coiled architecture is universal, the complexity of land plant flagellated cells varies from biflagellated in bryophytes to thousands of flagella per gametes in the seed plants Ginkgo and cycads. This wide diversity in number of flagella is associated with vast differences in cell size and shape. Scanning electron microscopy (SEM) has played an important role in characterizing the external form, including cell shape and arrangement of flagella, across the varied motile gametes of land plants. Because of the size and scarcity of released swimming sperm, it is difficult to concentrate them and prepare them for observation in the SEM. Here we detail an SEM preparation technique that yields good preservation of sperms cells across plant groups.

Effects of aqueous extract of celery (Apium graveolens L.) leaves on spermatogenesis in healthy male rats.

OBJECTIVES: Nowadays, a lot of attention has been paid to the therapeutic properties of herbs, including evaluation of the effects of these plants on fertility in laboratory animals. Apium graveolens L. (celery) has been widely used in traditional medicine for treatment of various disorders including impotency. Therefore, this study was designed to investigate the effects of aqueous extract of A. graveolens on testicular tissue and spermatogenesis in healthy male rats. MATERIALS AND METHODS: In this research, 24 apparently healthy male rats were divided into three groups, including eight rats in each. The first group as control received only distilled water 1 ml/animal/day. The second and third groups orally received 100 and 200 mg/kg b.w. of the extract, respectively, for 30 days. The day after the last administration of the extract, the rats were sacrificed, the testes were removed entirely, and the morphometric studies were carried out. Epididymal sperm count and histological studies of testicular tissue were conducted. RESULTS: The comparison between the treated and control groups revealed a remarkable increase in the seminiferous tubules diameter, testes volume (p≤0.001), and the number of spermatogonia, primary spermatocytes and spermatozoa. Furthermore, the increase in the number of spermatids and epididymal weight were only significant at high doses of the extract (p≤ 0.05). CONCLUSIONS: The results from this study indicated that administration of celery leaf extract may improve spermatogenesis process and also be useful for some sperm fertility parameters.

Importancia de la evaluación del estrés oxidativo en el semen humano / Importance of the assessment of oxidative stress in human semen

Utilizando bases de datos Medline, Science Direct, Endocrine Society y Sociedad Argentina de Andrología se realizó una revisión bibliográfica para estudiar e interpretar el efecto del estrés oxidativo (EO) en el proceso reproductivo. Los seres vivos que utilizan oxígeno para obtener energía son liberadores de especies reactivas de oxígeno (ERO). Existen situaciones andrológicas, factores medioambientales y compuestos químicos que incrementan las citoquinas proinflamatorias, generan EO alterando la regulación del proceso espermatogénico. Los espermatozoides son susceptibles al daño oxidativo, debido al elevado contenido de ácidos grasos poliinsaturados (AGPI) en su membrana y el escaso citoplasma que limita el contenido de enzimas antioxidantes. El EO induce daño peroxidativo en la membrana espermática y fragmentación del ADN en los genomas nucleares y mitocondriales. El test estrés espermático modificado (MOST) estima la resistencia espermática a la lipoperoxidación. El incremento de ERO en hombres con trastornos reproductivos y su correlación con alteraciones seminales revelan la importancia de la evaluación del EO en el estudio integral del hombre que consulta por infertilidad.

Data from Medline, Science Direct, Endocrine Society and organisms of information of Argentina Society of Andrology were used to study and interpret the effect of oxidative stress (OS) in the reproductive process. The living things use the oxygen for energy they release reactive oxygen species (ROS). There andrology situations enviromental factors, chemical agents that increase of proinflammatory cytokines, OS generated by altering the regulation of spermatogenesis process. The spermatozoids due to high concentration of polyunsaturated fatty acids (PFA) in its membrane are sensitive to the oxidative damage induced for ROS and it also contain low levels of antioxidant enzymes for their limited cytoplasm. OS induced peroxidative damage in sperm membrane and DNA fragmentation in the nuclear and mitochondrial genomes. The modified sperm stress test (MOST) estimates sperm resistance to lipoperoxidation. The ROS increased in men with reproductive disorders and its correlation with seminal alterations reveal the importance of the evaluation of OS in the study of man consulting for infertility.

Multiflagellated sperm cells of Ceratopteris richardii are bathed in arabinogalactan proteins throughout development.

UNLABELLED: • PREMISE OF THE STUDY: Sperm cell differentiation in ferns involves the origin of an elaborate locomotory apparatus, including 70+ flagella, and the structural modification of every cellular component. Because arabinogalactan proteins (AGPs) are implicated in molecular signaling and in regulation of plant development, we speculated that these glycoproteins would be present during spermiogenesis in ferns.• METHODS: Using ß-glucosyl Yariv reagents that specifically bind to and inhibit AGPs and immunogold localizations with monoclonal antibodies JIM13, JIM8, and LM6, we examined the specific expression patterns of AGPs and inhibited their function during sperm cell development in the model fern Ceratopteris richardii.• KEY RESULTS: Developing sperm cells stained intensely with Yariv phenylglycosides, demonstrating the presence of AGPs. JIM13-AGP epitopes were widespread throughout development in the expanding extraprotoplasmic matrix (EPM) in which flagella elongate, cytoplasm is eliminated, and spherical spermatids become coiled. JIM8 and LM6 epitopes localized to the plasmalemma on growing flagella and on the rapidly changing sperm cell body. Spermatids treated with ß-glucosyl lacked an EPM and formed fewer, randomly arranged flagella.• CONCLUSIONS: We demonstrated that AGPs are abundant in the EPM and along the plasmalemma and that the three AGP epitopes have specific expression patterns during development. Coupled with inhibition studies, these results identify AGPs as critical to the formation of an extraprotoplasmic matrix and the consequent origin and development of flagella in an orderly and precise fashion around the cell. We speculate that AGPs may play additional roles as signaling molecules involved in cell shaping, cytoskeletal development, vesicle trafficking, and cytoplasmic elimination.

Evaluation of the semen characteristics after induced spermiation in the bullfrog Lithobates catesbeianus / Características e avaliação do sêmen de rã-touro Lithobates catesbeianus induzidos à espermiação

Evaluation of semen characteristics after hormonal induction of the bullfrog could provide valuable information on the gametes of this species, which may be useful for projects related to artificial fertilization, animal improvement, and cryopreservation. Bullfrog males were induced to spermiate with buserelin acetate (GnRHa), and their semen was subsequently analyzed. GnRHa (0.4 µg) was administered to the bullfrog males with secondary sexual characteristics such as weight > 200 g, yellow chin, nuptial callus, and amplexus reflex, being the semen collected after 60 min. The semen volume was 5.76 mL, light-colored. The other characteristics of the semen were: vigor of 4.80, motility of 93%, concentration of 14.24 × 106 mL-1, and content of normal spermatozoa of 70%. The volume, color, vigor, motility, sperm concentration, and content of normal spermatozoa were adequate in these bullfrog semen samples. Evaluation of the bullfrog semen samples based on this set of parameters is essential for decision-making about the quality and destination of the semen.

Avaliação de que as características do sêmen, após a indução hormonal da rã-touro, pode fornecer informações valiosas sobre os gametas desta espécie, podendo ser útil para projetos relacionados à fertilização artificial, melhoramento animal, e criopreservação. Machos de rã-touro foram induzidos à espermiação com acetato de buserelina (GnRHa) e o sêmen foi posteriormente analisado. GnRHa (0,4 mg) foi administrado à rã- touro do sexo masculino com características sexuais secundárias, tais como peso superior a 200 g, papo amarelo, calo nupcial, e reflexo amplexo, e seu sêmen foi coletado após 60 min. O sêmen da rã-touro apresentou volume de 5,76 mL, coloração turva, vigor espermático de 4,80; motilidade espermática de 93%, concentração de 14,24 x 106 SPTZ mL-1 e 70% de espermatozoides normais. O volume, cor, vigor, motilidade, concentração espermática e o número de espermatozoides normais das amostras do sêmen de rã-touro são adequados. O conjunto dos parâmetros para avaliação das amostras de sêmen de rã-touro é indispensável para a tomada de decisão sobre a qualidade e destino do mesmo.

El efecto de los agroquímicos en la espermatogénesis / The effect of the agrochemicals in the spermatogenesis

Se relacionó la exposición ocupacional a agroquímicos con parámetros seminales vinculados con la espermatogénesis. En 104 muestras seminales de pacientes con infertilidad idiopática y 20 a 45 años de edad del Servicio de Reproducción Hospital Centenario (Rosario, Argentina), se efectuó espermograma y estudios funcionales según normas OMS (1999). Se agruparon en G1 (n=42) trabajadores expuestos a agroquímicos y G2 (n=62) hombres sin riesgo espermatogénico. La concentración espermática se determinó en cámara de Neubauer, con Papanicolaou se evaluó la concentración de células germinales y la morfología espermática. Se observó en concentración espermática (espermatozoides.10(6)/ml) G1: 21,1±7,1; G2: 41,6±9,2; morfología ( por ciento espermatozoides normales) G1: 5,2±1,3; G2: 8,2±3,6 y células germinales.10(6)/ml en G1: 0,91±0,51; G2: 0,32±0,21. La concentración espermática fue menor en G1, las alteraciones morfológicas y células germinales mayores en G1. Estos resultados indican que la exposición a agroquímicos altera la espermatogénesis y es un factor a considerar cuando se estudia infertilidad masculina.

The occupational exposition to agrochemicals was related with sperm parameters linked with spermatogenesis. In 104 semen samples of patients with idiopatic infertility of the Reproduction Service of Centenario Hospital (Rosario, Argentina), sperm study and functional tests according to WHO (1999) have been carried out. Two groups were formed: G1 (n=42) workers exposed to agrochemicals; G2 (n=62) men with no spermatogenic risk. The sperm concentration was determined in Neubauer camera, the concentration of germinal cells and the sperm morphology were evaluated with Papanicolaou. The results were: sperm concentration (spermatozoids 10(6)/ml) G1: 21.1 ± 7.1; G2: 41.6 ± 9.2; morphology ( percent normal spermatozoids) G1: 5.2 ± 1.3; G2: 8.2 ± 3.6; and germinal cells 10(6)/ml in G1: 0.91 ± 0.51; G2: 0.32 ± 0.21. The sperm concentration was lower in G1, major morphological changes and germinal cells in G1. This results show that the exposition to agrochemicals alters the spermatogenesis, it is a factor to consider in the male infertility.