Sperm Release Research Articles

HUBUNGAN PEMANFAATAN TUMBUHAN PASAK BUMI (Eurycoma longifolia Jack.) SEBAGAI OBAT TRADISIONAL DAN BIOAKTIVITASNYA

Pasak bumi (Eurycoma longifolia Jack.) is a species belonging Simarobuaceae has been long used as traditional medicine, which known very well as aphrosidiac. The studies have been conducted to reveal the benefits of E. longifolia through ethnobotany, phytochemical, and bioessay approaches, but the review articles on the its utilization and bioactivity limited. This article is based on scientific articles published on line or off-line, then synthesized so that to be information the relationship between use and bioactivity. Ethnobotany of E. longifolia have been used as a medicine for fever, malaria, improve stamina, antimalarial, antidiabetic, anticancer, aphrodisiac, overcoming erectile dysfunction. Bioactivity of E. longifolia is antiosteoporotic, antimicrobial, aphrodisiac, anticancer, angiogenesis, and hepatoprotective. Eurycomanone has activity as an antimalaria, antipyretic, aphrodisiac, and cytotoxic. The quassinoids, coumarin and glycosides of El have activity increase the production, quality, totality, synthesis and release of spermatozoa. Eurycoma longifolia is very potential to be developed as an antiosteoporotic and aprosidiac drug, but until now most of it is harvested directly from the forest, so to preserve it, we need to study the cultivation method.

Observations of Simultaneous Sperm Release and Larval Planulation Suggest Reproductive Assurance in the Coral Pocillopora acuta

Despite being an extensively studied group of corals, the reproductive biology of the scleractinian genus Pocillopora remains a mystery. Pocillopora acuta has been proposed to exhibit a mixed reproductive mode, sexually producing gametes (sperm and eggs) and asexually brooding larvae simultaneously within a single colony. Here, we report observations of night-time spawning of sperm during the peak monthly larval release period. We offer a new hypothesis for the regulation of sexual and asexual reproduction in the species and posit that sexual reproduction may occur more often than previously suggested. However, the success of internal oocyte fertilization and subsequent zygote development is dependent on sperm making contact with a fertile colony. We hypothesize that asexual development of larvae occurs when sperm is absent, but more extensive genetic, genomic, and histological data are required to determine the pathway by which unfertilized oocytes may develop. We also propose that this mixed mode of reproduction is an adaptation to mating failure, common in sessile marine invertebrates. The reproductive assurance enjoyed by the species may therefore be the key to its ecological and evolutionary persistence.

SUN-229 The Role of Testosterone in Spermatogenesis: Lessons from Proteome Profiling of Human Spermatozoa in Testosterone Deficiency

Testosterone is required during spermatogenesis to mantain the blood-testis barrier and for meiosis, Sertoli-spermatid adhesion and sperm release. Aim of the study was to better understand the effect of hypothalamic-pituitary-testicular axis on sperm protein composition and function.12 male patients with low testosterone and LH levels were consecutively enrolled for this study. Five normogonadic men were enrolled as control group in the protocol. If spermatozoa were detectable in seminal samples, the the ejaculates were washed and the sperm cells were selected using 50% PercollTM. The recovered sperm cells were then suspended in PBS and subjected to a residual leukocyte depletion using CD45 Dynabeads® magnetic cell sorting. The absence of leukocytes was further confirmed both by microscopic observation after staining and by performing a RT-PCR for CD45. Protein solubilization was independently performed on each sperm sample. A comparative proteomic analysis was performed comparing two pools: controls vs hypogonadic patients. The TMTduplexTM Isobaric Mass Tagging Kit was used for the peptide labeling. MS/MS analysis was performed using a LTQ Orbitrap Velos. Data were processed using Proteome Discoverer 1.4.1.14 software, using very stringent filtering criteria. Proteins identified by SEQUEST were analyzed using the PANTHER classification system. Proteomic results have been confirmed by Western Blot. The following primary antibody were used: anti-Lactoferrin polyclonal antibody and anti-PIP polyclonal antibodyWe included in our study 5 oligozoospermic hypogonadic patients with a sperm count >10 x 106/ml and without leukocyte contamination. 42 proteins were under-expressed in the pool of hypogonadic patients; 12 of these 42 increased proteins have been previously reported to be related with spermatogenesis, sperm motility, sperm-oocyte interaction and acrosomal reaction. The distribution of proteins for molecular function revealed that the protein pattern impaired by low blood testosterone is involved in protein binding and hydrolase. Twelve proteins have been over-expressed in the pool of hypogonadic patients, including markers of defective spermatogenesis. Western-blot analysis confirmed proteomic data. This is the first application of high resolution mass spectrometry -based proteomics aimed to reveal an array of proteins in spermatozoa reflecting an impairment of spermatogenesis and sperm function in male secundary hypogonadism.

Early varicocelectomy by percutaneous scleroembolization improves seminiferous tubules spermatozoa release in the adolescent phase of testicular growth.

Varicocele is a rather common andrological condition in adolescents, which can adversely affect testicular growth and seminal parameters, leading to infertility in about 20% of adults. The aim of this study was to investigate if treating varicocele before the age of 18 is a beneficial option to improve testicular hypotrophy and seminal parameters and if minimally invasive techniques could be an appropriate treatment choice for adolescent varicocele associated with spermatic vein reflux. A percutaneous scleroembolization was performed in sixty-four consecutive young patients (13-19years old) with left varicocele, preceded by a fluoroscopy. In thirty-four of them, semen samples were also collected. We examined semen samples and testicles dimensions before and after percutaneous varicocelectomy, compared to a nonoperated control group, with a six-month follow-up. Total sperm count and sperm morphology were significantly increased in the intervention group. Left testicular volume significantly increased in both groups, while only correction of varicocele improved spermatozoa release per unit of testis volume. We conclude that early varicocelectomy by percutaneous scleroembolization significantly ameliorates seminiferous tubules activity in the critical adolescent phase of testicular growth. It is suggested that adolescents should be; offered varicocele repair as soon as possible in order to improve reproductive potential.

Regulation of sperm storage and movement in the ruminant oviduct

Three regions of the ruminant oviduct play different roles in the progress of sperm: the uterotubal junction, isthmus, and ampulla. The uterotubal junction acts as a point of selection of sperm, requiring that sperm are progressively motile and express specific proteins in order to enter the oviduct. The isthmus stores sperm, preserving motility and viability until ovulation. Sperm are stored in the isthmus by binding to its mucosal epithelium. In bovine sperm, binding to the oviductal epithelium is promoted by proteins that are secreted by the seminal vesicles and coat the heads of sperm by associating with plasma membrane phospholipids. Putative oviductal receptors for the seminal vesicle proteins are members of the annexin protein family. Release of sperm from the storage site in the isthmus is gradual, which serves to ensure that sperm in the proper physiological state reach the oocytes at the appropriate moment and also to reduce incidence of polyspermic fertilization. The ampulla supports fertilization and may participate in guiding sperm toward the eggs. Further studies are needed to improve our understanding of the interactions between sperm and the female reproductive tract, in order to develop means to improve fertility in ruminants.

Novel concepts about normal sexual differentiation of reproductive neuroendocrine function and the developmental origins of female reproductive dysfunction: the sheep model

The neuroendocrine regulation of GnRH secretion plays a central role in timing gamete release in both sexes. This regulation is more complex in the female because the discontinuous release of ova is more complex than the continuous release of spermatozoa. This review provides an evolving understanding of the sex differences in reproductive neuroendocrine controls and how these differences arise. The rules for sexual differentiation of steroid feedback control of GnRH secretion conceptually parallel the well-established principles that underlie the sexual differentiation of the internal and external genitalia. In the context of the neuroendocrine regulation of the ovarian cycle, and using the sheep as a model, four steroid feedback controls for GnRH secretion are inherent (default). They require no ovarian developmental input to function appropriately during adulthood. Two steroid feedback controls regulate the preovulatory surge mode of GnRH secretion, and two regulate the pulsatile mode. If the individual is a male, three steroid feedback controls of GnRH secretion become unnecessary or irrelevant, and these are abolished or become functionally inoperative through programmed reductions in hypothalamic sensitivity. This central programming occurs through exposure of presynaptic GnRH neurons in the developing male brain to the androgenic and estrogenic actions of testicular steroids. In precocial species such as ruminants, this programming begins well before birth. Understanding how GnRH secretion normally becomes sexually differentiated is of practical importance to determining how inappropriate hormonal environments during development can variously malprogram the neuroendocrine system to produce a variety of reproductive dysfunctions relating to patterning of gonadotropin secretion.

Ubiquitin-proteasome system participates in the de-aggregation of spermadhesins and DQH protein during boar sperm capacitation.

We studied the participation of the ubiquitin-proteasome system (UPS) in spermadhesin release during in vitro capacitation (IVC) of domestic boar spermatozoa. At ejaculation, boar spermatozoa acquire low molecular weight (8-16 kDa) seminal plasma proteins, predominantly spermadhesins, aggregated on the sperm surface. Due to their arrangement, such aggregates are relatively inaccessible to antibody labeling. As a result of de-aggregation and release of the outer layers of spermadhesins from the sperm surface during IVC, antibody labeling becomes feasible in the capacitated spermatozoa. In vivo, the capacitation-induced shedding of spermadhesins from the sperm surface is associated with the release of spermatozoa from the oviductal sperm reservoir. We took advantage of this property to perform image-based flow cytometry to study de-aggregation and shedding of boar spermadhesins (AQN, AWN, PSP protein families) and boar DQH (BSP1) sperm surface protein which induces higher fluorescent intensity in capacitated vs ejaculated spermatozoa. Addition of a proteasomal inhibitor (100 µM MG132) during IVC significantly reduced fluorescence intensity of all studied proteins (P < 0.05) compared to vehicle control IVC. Western blot detection of spermadhesins did not support their retention during IVC with proteasomal inhibition (P > 0.99) but showed the accumulation of DQH (P = 0.03) during IVC, compared to vehicle control IVC. Our results thus demonstrate that UPS participates in the de-aggregation of spermadhesins and DQH protein from the sperm surface during capacitation, with a possible involvement in sperm detachment from the oviductal sperm reservoir and/or sperm-zona pellucida interactions. The activity of sperm UPS modulates de-aggregation of boar spermadhesins and DQH sperm surface protein/binder of sperm1 (BSP1) during the sperm capacitation.

Spermatogenesis, reproductive maturation, and spawning seasonality of male winter flounder, Pseudopleuronectes americanus: Comparisons among fishery stocks

Winter flounder, Pseudopleuronectes americanus, were collected from three fishery stocks in the United States (U.S.) during 2009–2013 to describe spermatogenesis. Spermatogenesis included rapid, synchronous transitions during autumn from mitotic spermatogonia to meiotic spermatocytes, then to haploid spermatids, and the release of spermatozoa into lobule lumen. As these transitions occurred several months prior to the spring spawning season, maturity of the testes are a poor indicator of spawning season, however, individuals can be readily evaluated prior to the spawning season for evidence of skipped spawning. Throughout this process, the gonad weight increased dramatically, with spawning-season maxima from 11 to 17% relative to individual somatic weight, suggesting considerable reproductive investment relative to other flatfishes. Fish condition also cycled seasonally, which indicated that males follow a capital breeding pattern that has been reported for female winter flounder. The timing of spermiogenesis was very similar in the fish among each U.S. stock (Southern New England [SNE], Georges Bank [GB], and the Gulf of Maine [GOM]) and published data from coastal Newfoundland, even though U.S. winter flounder spawned earlier in spring than fish in the Canadian stock. Male maturity varied in relation to both size and age, and over time, in patterns similar to those reported for female winter flounder, but at smaller and younger sizes than females. These intra-specific variations in reproductive seasonality, maturation, and skipped spawning suggest that winter flounder have the potential to adaptively respond to a dynamic environment in a region where ocean warming is occurring rapidly.

First person – Tushna Kapoor and Pankaj Dubey

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Pankaj Dubey and Tushna Kapoor are co-first authors on ‘Atypical septate junctions maintain the somatic enclosure around maturing spermatids and prevent premature sperm release in Drosophila testis’, published in BiO. Pankaj is a Postdoctoral fellow in the lab of Dr Subhojit Roy at Wisconsin Institute of Medical Research (WIMR) II, Madison, USA, investigating how to understand how cytoskeleton dynamics shapes neuronal physiology and transport. Tushna is a PhD student in the lab of Prof. Krishanu Ray at the Tata Institute of Fundamental Research, Mumbai, India, investigating the interplay between cell adhesion, the cytoskeleton and membrane dynamics.

Releasing small ejaculates slowly increases per-gamete fertilization success in an external fertilizer: Galeolaria caespitosa (Polychaeta: Serpulidae)

The idea that male reproductive strategies evolve primarily in response to sperm competition is almost axiomatic in evolutionary biology. However, externally fertilizing species, especially broadcast spawners, represent a large and taxonomically diverse group that have long challenged predictions from sperm competition theory-broadcast spawning males often release sperm slowly, with weak resource-dependent allocation to ejaculates despite massive investment in gonads. One possible explanation for these counter-intuitive patterns is that male broadcast spawners experience strong natural selection from the external environment during sperm dispersal. Using a manipulative experiment, we examine how male reproductive success in the absence of sperm competition varies with ejaculate size and rate of sperm release, in the broadcast spawning marine invertebrate Galeolaria caespitosa (Polychaeta: Serpulidae). We find that the benefits of Fast or Slow sperm release depend strongly on ejaculate size, but also that the per-gamete fertilization rate decreases precipitously with ejaculate size. Overall, these results suggest that, if males can facultatively adjust ejaculate size, they should slowly release small amounts of sperm. Recent theory for broadcast spawners predicts that sperm competition can also select for Slow release rates. Taken together, our results and theory suggest that selection often favours Slow ejaculate release rates whether males experience sperm competition or not.

Living on the edge: Gamete release and subsequent fertilisation in Fucus vesiculosus (Phaeophyceae) are weakened by climate change–forced hyposaline conditions

ABSTRACTPopulations at range margins of marine organisms are at the front line of climate-induced changes in abiotic factors and are thus particularly susceptible. Especially, macroalgae that are externally reproducing rely on optimal environmental conditions for gamete release and subsequent fertilisation. However, the effect of climate change–forced decrease in salinity on these critical life stages has been largely overlooked. We tested the impact of forecasted hyposaline conditions on a marginal population of the rockweed Fucus vesiculosus growing at 5.8 PSU on the Finnish coast of the Baltic Sea. We incubated individuals with receptacles for at least seven days at 2.5, 4.1, 5.8, and 7.2 PSU and determined their gamete release and subsequent fertilisation success. We further tested sperm performance at 3.5, 5.0, and 6.1 PSU. Salinity of 2.5 PSU, which is predicted to occur in the region by the end of this century, reduced egg release. In contrast, sperm and antheridia release were not consistently affected by the different salinities, but the size of sperm swells at 3.5 PSU. Because fertilisation success was drastically reduced at 2.5 and 4.1 PSU, we suggest that sperm performance was compromised such that sperm dysfunction hampered fertilisation success. Our results demonstrate that the forecasted hyposalinity negatively affects egg release and sexual reproduction in F. vesiculosus at its northern distribution limit. This macroalga can probably only withstand the future decrease in salinity when populations proliferate via asexual reproduction.

Pollen tube integrity regulation in flowering plants: insights from molecular assemblies on the pollen tube surface.

Contents Summary 687 I. Introduction 687 II. Pollen tube membrane-localized receptors coordinate cell integrity and sperm release 689 III. RALF peptides mediate autocrine and paracrine signaling 689 IV. ROS and ion channel signaling mediate intracellular response 690 V. Involvements from pollen tube cell wall components 690 VI. Concluding remarks 691 Acknowledgements 692 Author contributions 692 References 692 SUMMARY: Unlike in animals, sperm in flowering plants are immotile and they are embraced as passive cargoes by a pollen tube which embarks on a long journey in the pistil to deliver them to the female gametophyte for fertilization. How the pollen tube switches from a rapid polarized growth towards its target to an abrupt disintegration for sperm cell release inside the female gametophyte is puzzling. Recent studies have shown that members of the Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase family and their ligands, 5-kDa cysteine-rich peptide rapid alkalinization factors (RALFs), engage in an intricate balancing act involving autocrine and paracrine signaling to maintain pollen tube growth and induce timely tube rupture at the spatially confined pollen tube-female gametophyte interface. Here, we review recent progress related to pollen tube integrity control, mainly focusing on the molecular understanding of signaling as well as intracellular signaling nodes in Arabidopsis. Some missing links and future perspectives are also discussed.

Spermatogenesis and gonadal cycle in male Tamoya haplonema and Chiropsalmus quadrumanus (Cnidaria, Cubozoa)

Spermatogenesis has been little studied in cubozoans. This work describes spermatogenesis of two cubozoan species from Brazil, Tamoya haplonema and Chiropsalmus quadrumanus, using light microscopy and histochemical techniques to highlight and contrast stages of the gonadal cycle. Specimens were collected by hand at the water surface or with shrimp trawls in three areas in the state of São Paulo (SE Brazil): Cananéia lagoon estuarine system (south), Santos and São Vicente Bay (central), and São Sebastião and Ubatuba (north). Male box jellyfish gonads are located between the mesoglea and gastrodermis in a thin and independent layer, organized in elongated follicles, macroscopically resembling a fingerprint pattern. After subsampling, serial sections were performed on male gonad tissue. The resulting slides were stained with hematoxylin and eosin and methods for staining proteins, acidic materials and neutral polysaccharides. Light microscopy revealed that the follicle wall of the male gonads of both cubozoan species is composed of spermatogonia. Spermatocytes are found peripherally near the follicle wall, while spermatids and spermatozoa are located towards the center. Sperm release occurs through rupture of the follicular wall into the gastric pockets. Four stages of gonad development were documented for T. haplonema and three for C. quadrumanus. The sperm of C. quadrumanus has a rounded head while in T. haplonema the sperm head is conical with an elongated apical projection. In both species, there was no positive histochemical reaction for putative acrosomal vesicles precursors.

Salto/CG13164 is required for sperm head morphogenesis in Drosophila.

Producing mature spermatozoa is essential for sexual reproduction in metazoans. Spermiogenesis involves dramatic cell morphological changes going from sperm tail elongation and nuclear reshaping to cell membrane remodeling during sperm individualization and release. The sperm manchette plays a critical scaffolding function during nuclear remodeling by linking the nuclear lamina to the cytoskeleton. Here, we describe the role of an uncharacterized protein in Drosophila, salto/CG13164, involved in nuclear shaping and spermatid individualization. Salto has dynamic localization during spermatid differentiation, being progressively relocated from the sperm-nuclear dense body, which is equivalent to the mammalian sperm manchette, to the centriolar adjunct and acrosomal cap during spermiogenesis. salto-null male flies are sterile and exhibit complete spermatid individualization defects. salto-deficient spermatids show coiled spermatid nuclei at late maturation stages and stalled individualization complexes. Our work sheds light on a novel component involved in cytoskeleton-based cell-morphological changes during spermiogenesis.

Progesterone induces the release of bull spermatozoa from oviductal epithelial cells.

The mechanism that causes the detachment of spermatozoa from the oviductal reservoir around the time of ovulation remains to be elucidated. Because the cumulus cells of the bovine oocyte are known to secrete progesterone (P4), and P4 has been shown to act upon cation channels of spermatozoa (CatSper) in human spermatozoa, it was hypothesised that P4 could induce hyperactivation due to an influx of extracellular calcium, and this would facilitate detachment of spermatozoa from oviductal epithelial cells. Therefore, this study aimed to investigate the role and mechanism of action of P4 in the release of spermatozoa from bovine oviduct epithelial cells (BOEC). Initial dose-response assessments on sperm hyperactivation determined the optimum concentration of P4 (10 nM), mibefradil (a non-specific Ca2+ channel antagonist; 5µM), NNC 55-0396 dihydrochloride (NNC; a CatSper antagonist; 2µM), mifepristone (a classical and membrane P4 receptor antagonist; 400nM) and AG205 (a membrane P4 receptor antagonist; 10μM). BOEC explants were incubated with frozen-thawed bovine spermatozoa for 30min, following which loosely bound spermatozoa were removed. Two experiments were completed. In Experiment 1, BOECs were treated for 30min with either no treatment, P4, NNC, mibefradil, P4+mibefradil, P4+NNC, P4+mibefradil+NNC or P4+EGTA. In Experiment 2, BOECs were treated for 30min with either no treatment, P4, mifepristone, AG205, mifepristone+AG205, P4+mifepristone, P4+AG205 or P4+mifepristone+AG205. The number of spermatozoa remaining bound per millimetre squared of BOEC explant was determined. Progesterone stimulated the release of bound spermatozoa from BOEC explants, whereas NNC, mibefradil and EGTA inhibited this release. The release of spermatozoa by P4 was inhibited in the presence of both mifepristone and AG205, whereas the combination of both had the greatest inhibitory action on P4 release of spermatozoa. These findings suggest the presence of a P4 membrane receptor on bovine spermatozoa and that P4-induced release of spermatozoa from BOECs is likely mediated by extracellular Ca2+.

Hormone-induced sperm-release in the critically endangered Booroolong frog (Litoria booroolongensis): effects of gonadotropin-releasing hormone and human chorionic gonadotropin.

Research into the development of reproductive technologies for amphibians has increased in recent years due to the rapid decline of amphibian species globally. Reproductive technologies have great potential to overcome captive breeding failure and improve the propagation and genetic management of threatened species. However, the incorporation of these technologies into conservation breeding programs has been protracted, primarily as a result of trial-and-error approaches to the refinement of hormone therapies. The present study investigated the effects of: (1) GnRH-a dose (0, 0.5, 1, 2, 4, 8 or 16 μg g-1), and (2) hCG dose (0, 2.5, 5, 10, 20 or 40 IU g-1), on the sperm-release response of the critically endangered Booroolong frog. Administration of GnRH-a at a dose of 0.5 μg g-1 resulted in the greatest number of sperm released (mean total sperm = 3.5 ×106, n = 11). Overall, hCG was more effective at eliciting spermiation in Booroolong frogs, with peak sperm release (mean total sperm = 25.1 ×106, n = 10) occurring in response to a dose of 40 IU g-1. Sperm output in response to 40 IU g-1 hCG was greatest between 1 and 6 h and steadily declined between 8 and 24 h post-hormone administration. Percent sperm motility peaked between 4 and 10 h (58.1-62.7%), and sperm velocity between 4 and 12 h (24.3-27.2 μm s-1). Booroolong frogs join a small, but growing number of amphibian species that exhibit improved spermiation in response to hCG. Further research is required to identify optimal hormone-induction protocols for threatened amphibians and expedite the incorporation of reproductive technologies into CBPs.

Histological assessment of seasonal gonad maturation of red mullet (Mullus barbatus L., 1758) in Antalya Bay of Mediterranean in Turkey

ABSTRACT In this study, gonadal changes of red mullet (Mullus barbatus L., 1758) were investigated by histologically in Antalya Bay of Eastern Mediterranean in Turkey. A total of 890 specimens were collected monthly between May 2015 and October 2016 from fishing boats. The age of 890 red mullets was found to range from 0+ to VI+. Their gonads were dissected and fixed in 10% neutral buffered formaldehyde. They were embedded in paraffin blocks after histochemical processes. The sectioned tissue samples were stained with Haematoxylin-Eosin and Masson Trichrome. Two different stages in females and in males were observed according to gonadal development. Histologically, female individuals had in immature female (66 fish, 0+-IV+ ages), chromatin-nucleolus (67 fish, I+-VI+ ages), perinucleolus (63 fish, I+-IV+ ages), cortical alveoli (55 fish, I+-V+ ages), vitellogenesis (50 fish, I+-V+ ages), maturity (39 fish, I+-VI+ ages) and post-ovulation stages (59 fish, I+-VI+ ages). Male individuals had in spermatogenesis (96 fish, 0+-VI+ ages), spermiogenesis (97 fish, I+-IV+ ages), spermiation (85 fish, I+-VI+ ages), sperm release (84 fish, I+-VI+ ages) and resting stages (116 fish, I+-VI+ ages). Results show that the spawning season of M. barbatus is between May and September, and that egg diameters range from 31.00 ± 15 to 428.00 ± 40 µm.

Morphology of the spermatheca of Triatoma lecticularia (Hemiptera: Reduviidae) (Stal, 1859).

Triatoma lecticularia (Hemiptera: Reduviidae) (Stal, 1859) is a potential vector of Chagas's disease and the comprehension of its reproductive biology is an important tool to control this insect. In the reproductive tract of female insects, the spermatheca plays a crucial role storing male spermatozoa after mating. Whithin insects the spermatheca shows a wide morphological diversity and the analysis of this characteristic can contribute to understand the reproductive biology of the species. This study describes the histology and histochemistry of the spermatheca of T. lecticularia. Females have a pair of elongated spermathecal reservoirs without associated accessory gland. The reservoir opens into the common oviduct via a narrow muscular duct. The reservoir epithelium has single layer of columnar secretory cells. The control of the release of spermatozoa from the spermatheca occurs via the muscular duct. The anatomical features of the spermatheca of T. lecticularia resemble those described of other Reduviidae. However, the histological and histochemical features of spermatheca observed in T. lecticularia were important to explain the maintenance of the viability of the spermatozoa stored.

Friend or foe: Signaling mechanisms during double fertilization in flowering seed plants.

Since the first description of double fertilization 120 years ago, the processes of pollen tube growth and guidance, sperm cell release inside the receptive synergid cell, as well as fusion of two sperm cells to the female gametes (egg and central cell) have been well documented in many flowering plants. Especially microscopic techniques, including live cell imaging, were used to visualize these processes. Molecular as well as genetic methods were applied to identify key players involved. However, compared to the first 11 decades since its discovery, the past decade has seen a tremendous advancement in our understanding of the molecular mechanisms regulating angiosperm fertilization. Whole signaling networks were elucidated including secreted ligands, corresponding receptors, intracellular interaction partners, and further downstream signaling events involved in the cross-talk between pollen tubes and their cargo with female reproductive cells. Biochemical and structural biological approaches are now increasingly contributing to our understanding of the different signaling processes required to distinguish between compatible and incompatible interaction partners. Here, we review the current knowledge about signaling mechanisms during above processes with a focus on the model plants Arabidopsis thaliana and Zea mays (maize). The analogy that many of the identified "reproductive signaling mechanisms" also act partly or fully in defense responses and/or cell death is also discussed.

103 Hormonal stimulation and post-breeding sperm induction in the mountain yellow-legged frog, Rana muscosa

The first choice of any captive animal program should be to promote natural breeding through appropriate diet and environmental conditions. However, in cases of poor reproductive performance, the administration of exogenous hormones can induce breeding behaviours and gamete release. Part of the approach to the Mountain yellow-legged frog recovery program is the collection of sperm for cryobanking as a means of preserving genetic diversity in a cost- and space-effective manner. To develop a protocol for induction of sperm release from Mountain yellow-legged frogs, we tested the administration of gonadotropin-releasing hormone agonist (d-Ala6, des-Gly10 ethylamide LHRH derivative) and hCG singly or in combination. Once a month, animals were injected with a single dose of saline amphibian ringers (control) or 1 of 10 hormone treatments. Groups 1-4 received a single intraperitoneal injection of gonadotropin-releasing hormone agonist administered in 4 different doses: (1) 0.3 μg/g body weight (bw); (2) 0.4 μg/g bw; (3) 0.6 μg/g bw; and (4) 1 μg/g bw. Groups 5 and 6 were injected with 5 and 10IU hCG/g bw, respectively, and groups 7-10 received a combined injection of hCG and GnRH in four different doses: (7) 5 IU/g hCG with 0.3 μg/g GnRH; (8) 10 IU/g hCG with 0.3 μg/g GnRH; (9) 5 IU/g hCG and 0.6 μg/g GnRH; and (10) 10 IU/g hCG and 0.6 μg/g GnRH. Hormone treatments began in July 2015, 2 months after the end of the breeding season to allow males a post-breeding recovery time. Monthly administration continued from July until December 2015, ending before the brumation period. In 2016, treatments resumed again in July, after another breeding period. In 2017, hormone treatments began in April, during the reproductive season, and continued until July. From July 2015 through November 2016, sperm concentration and motility were examined in response to hormones treatment groups 1, 3, 5, 6, and 7-10. In April 2017, based on previous results, experiments were redesigned to include group 9 from 2015-16, and two new groups, 2 and 4. Spermiation was not easily hormonally induced when administered from August-October 2015, August-September 2016, and in July 2017. These low-response periods coincided with post-breeding months when only 3.7% (3/81) of males responded to either 10 IU/g hCG, 0.6 μg/g GnRH, or a combination of 5 IU/g hCG and 0.6 μg/g GnRH. Sperm production was significantly affected (P&amp;lt;0.05) by the month of hormone administration and hormone treatments during the responsive periods (P&amp;lt;0.0001) of April-June and October-November. However, hormone doses and treatments had no significant effect on the average or total sperm concentration per male (P&amp;lt;0.05). Motility and speed of forward progression were not significantly affected by treatment (P&amp;lt;0.05). This study demonstrates the seasonality of sperm production in this species, which may be attributed to testicular recrudescence during the summer months following breeding. Without hormonal stimulation, spermiation did not occur during the nonreproductive part of the year.