scholarly journals FACS-based isolation and RNA extraction of Secondary Cells from the Drosophila male Accessory Gland

2019 ◽  
Author(s):  
Clément Immarigeon ◽  
François Karch ◽  
Robert K. Maeda
Keyword(s):  
Cell Population ◽  
Seminal Fluid ◽  
Rna Extraction ◽  
Cell Types ◽  
Accessory Gland ◽  
Specific Cell ◽  
Male Accessory Gland ◽  
Accessory Gland Proteins ◽  
Accessory Glands ◽  
Study Gene Expression

ABSTRACTTo appreciate the function of an organ, it is often critical to understand the role of rare cell populations. Unfortunately, this rarity often makes it difficult to obtain material for study. This is the case for the Drosophila male accessory gland, the functional homolog of mammalian prostate and seminal vesicle. In Drosophila, this gland is made up of two morphologically distinct cell types: the polygonally-shaped main cells, which compose 96% of the organ, and the larger, vacuole-containing secondary cells (SCs), which represent the remaining 4% of cells (~40 cells per lobe). Both cell types are known to produce accessory gland proteins (Acps), which are important components of the seminal fluid and are responsible for triggering multiple physiological and behavioral processes in females, collectively called the post-mating response (PMR). While a few genes are known to be specific to the SCs, the relative rarity of SCs has hindered the study of their whole transcriptome. Here, a method allowing for the isolation of SCs is presented, enabling the extraction and sequencing of RNAs from this rare cell population. The protocol consists of dissection, protease digestion and mechanical dissociation of the glands to obtain individual cells. Then, the cells are sorted by FACS, and living GFP-expressing SC singulets are isolated for RNA extraction. This procedure is able to provide SC-specific RNAs from ~40 males per condition in the course of one day. Given the speed and low number of flies required, this method enables the use of downstream RT-qPCR and/or RNA sequencing to the study gene expression in the SCs from different genetic backgrounds, ages, mating statuses or environmental conditions.SUMMARYHere, we describe the dissociation and sorting of a specific cell population from the Drosophila male accessory glands (Secondary cells), followed by RNA extraction for sequencing and RT-qPCR. The dissociation consists of dissection, proteases digestion and mechanical dispersion, followed by FACS purification of GFP-expressing cells.

scholarly journals Rab-mediated trafficking in the secondary cells of Drosophila male accessory glands and its role in fecundity

2018 ◽  
Author(s):  
E. Prince ◽  
M. Brankatschk ◽  
B. Kroeger ◽  
D. Gligorov ◽  
C. Wilson ◽  
...  
Keyword(s):  
Mating Behavior ◽  
Seminal Fluid ◽  
Cell Types ◽  
Accessory Gland ◽  
Molecular Organization ◽  
Secretory Cells ◽  
Rab Proteins ◽  
Intracellular Membrane ◽  
Accessory Glands ◽  
Intracellular Membrane Transport

AbstractIt is known that the male seminal fluid contains factors that affect female post-mating behavior and physiology. In Drosophila, most of these factors are secreted by the two epithelial cell types that make up the male accessory gland: the main and secondary cells. Although secondary cells represent only 4% of the cells of the accessory gland, their contribution to the male seminal fluid is essential for sustaining the female post-mating response. To better understand the function of the secondary cells, here we investigate their molecular organization, particularly with respect to the intracellular membrane transport machinery. We determined that large vacuole-like structures found in the secondary cells are trafficking hubs labeled by Rab6, 7, 11 and 19. Furthermore, these cell-specific organelles are essential for the long-term post-mating behavior of females and that their formation is directly dependent upon Rab6. Our discovery adds to our understanding of Rab proteins function in secretory cells. We have created an online, open-access imaging resource as a valuable tool for the intracellular membrane and protein traffic community.

scholarly journals Comparative genomics identifies male accessory gland proteins in five Glossina species

2017 ◽  
Vol 2 ◽  
pp. 73 ◽  
Author(s):  
Muna F. Abry ◽  
Kelvin M. Kimenyi ◽  
Daniel K Masiga ◽  
Benard W. Kulohoma
Keyword(s):  
Comparative Genomics ◽  
Reproductive Cycle ◽  
Sequence Data ◽  
Control Strategies ◽  
Accessory Gland ◽  
Tsetse Fly ◽  
Whole Genome Sequence ◽  
Reproductive Proteins ◽  
Accessory Gland Proteins ◽  
Accessory Glands

Accessory gland proteins (ACPs) are important reproductive proteins produced by the male accessory glands (MAGs) of most insect species. These proteins are essential for male insect fertility, and are transferred alongside semen to females during copulation. ACPs are poorly characterized in Glossina species (tsetse fly), the principal vector of the parasite that causes life-threatening Human African Trypanosomiasis and Animal trypanosomiasis in endemic regions in Africa. The tsetse fly has a peculiar reproductive cycle because of the absence of oviposition. Females mate once and store sperm in a spermathecal, and produce a single fully developed larva at a time that pupates within minutes of exiting their uterus. This slow reproductive cycle, compared to other insects, significantly restricts reproduction to only 3 to 6 larvae per female lifespan. This unique reproductive cycle is an attractive vector control strategy entry point. We exploit comparative genomics approaches to explore the diversity of ACPs in the recently available whole genome sequence data from five tsetse fly species ( Glossina morsitans, G. austeni, G. brevipalpis, G. pallidipes and G. fuscipes). We used previously described ACPs in Drosophila melanogaster and Anopheles gambiae as reference sequences. We identified 36, 27, 31, 29 and 33 diverse ACP orthologous genes in G. austeni, G. brevipalpis, G. fuscipes, G. pallidipes and G. morsitans genomes respectively, which we classified into 21 functional classes. Our findings provide genetic evidence of MAG proteins in five recently sequenced Glossina genomes. It highlights new avenues for molecular studies that evaluate potential field control strategies of these important vectors of human and animal disease.

Male accessory gland substance: an egg-laying stimulant in Melanoplus sanguinipes (F.) (Orthoptera:Acrididae)

1969 ◽  
Vol 47 (6) ◽  
pp. 1199-1203 ◽  
Author(s):  
R. Pickford ◽  
Al B. Ewen ◽  
C. Gillott
Keyword(s):  
Seminal Vesicle ◽  
Chemical Substance ◽  
Accessory Gland ◽  
Egg Laying ◽  
Melanoplus Sanguinipes ◽  
Male Accessory Gland ◽  
Accessory Glands ◽  
Egg Deposition ◽  
Complete Set ◽  
Per Se

The rate of egg deposition by mature virgin females of the migratory grasshopper, Melanoplus sanguinipes (F.), increased markedly after implantation of accessory glands from mature males. Implants consisting of one-half of a complete set of glands, or of the 10 short hyaline glands alone, were about equally effective in increasing the rate of egg deposition. However, in each case the oviposition rate was less than that of normally mated females of the same age. Implanting only the white glands of the accessory gland complex had a lesser effect on egg-laying and implants of the long hyaline gland or of the seminal vesicle had little or no effect.Our results indicate that the physical act of mating per se or the presence of sperm probably do not provide the primary stimulus to egg-laying. The abdominal location of the implanted accessory gland does, however, suggest that some diffusible chemical substance is responsible for increasing egg deposition in these virgin females.

scholarly journals Seminal Plasma Proteomic Biomarkers of Oxidative Stress

2020 ◽  
Vol 21 (23) ◽  
pp. 9113
Author(s):  
Rossella Cannarella ◽  
Andrea Crafa ◽  
Federica Barbagallo ◽  
Laura M. Mongioì ◽  
Rosita A. Condorelli ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Male Infertility ◽  
Seminal Plasma ◽  
Molecular Mechanisms ◽  
Seminal Fluid ◽  
Accessory Gland ◽  
Male Accessory Gland ◽  
Abnormal Sperm ◽  
Clinical Conditions ◽  
Biomarkers Of Oxidative Stress

The prevalence of idiopathic male infertility is high, up to 75% of patients with abnormal sperm parameters. Hence, the research of its causes is mandatory. Oxidative stress (OS) can be responsible for male infertility in 30–80% of cases. In recent years, seminal plasma (SP) proteomics has developed as a useful tool to provide biomarkers of specific diseases. This systematic review aims to collect the available evidence on the changes of SP proteome in patients exposed to OS to provide possible SP biomarkers of sperm OS. To accomplish this, the following keyterms “seminal fluid proteome”, “seminal plasma proteome”, “oxidative stress”, and “sperm oxidative stress” were used and 137 records were found. Among these, 17 were finally included. Nine proteins involved with OS were found overexpressed in patients with OS. Twenty-three proteins were found differentially expressed in patients with clinical conditions associated with OS, such as varicocele, male accessory gland infection/inflammation, cigarette smoke, and obesity. These proteins do not seem to overlap among the clinical conditions taken into account. We speculate that specific SP proteins may mediate OS in different clinical conditions. Altogether, these results suggest that proteomics could help to better understand some of the molecular mechanisms involved in the pathogenesis of infertility. However, further studies are needed to identify potential biomarkers of male infertility with valuable clinical significance.

MALE ACCESSORY GLAND FACTORS ELICIT CHANGE FROM ‘VIRGIN’ TO ‘MATED’ BEHAVIOUR IN THE FEMALE CORN EARWORM MOTH HELICOVERPA ZEA

1993 ◽  
Vol 183 (1) ◽  
pp. 61-76 ◽  
Author(s):  
T. G. Kingan ◽  
P. A. Thomas-Laemont ◽  
A. K. Raina
Keyword(s):  
Sex Pheromone ◽  
Helicoverpa Zea ◽  
High Performance ◽  
Accessory Gland ◽  
Corn Earworm ◽  
Male Accessory Gland ◽  
Reverse Phase ◽  
Accessory Glands ◽  
Calling Behaviour ◽  
Chemical Messenger

After mating, the females of many species of moths become depleted of sex pheromone, calling behaviour is terminated, and they become transiently or permanently unreceptive to additional matings. In the corn earworm moth, Helicoverpa zea, we have found that the male accessory gland/duplex is required for evoking the post-mating depletion of sex pheromone but apparently not for the cessation of calling. The latter change requires the receipt of a spermatophore or a chemical messenger derived from non-accessory gland/duplex sources. Desalted extracts of combined accessory glands and duplexes caused a depletion of pheromone in injected females. Proteinaceous components in extracts purified by fractionation in cation-exchange cartridges and by reverse-phase high-performance liquid chromotography retain their pheromonostatic activity. In addition, this fractionated material shuts off calling behaviour and prevents mating in injected females, raising the possibility that redundant mechanisms exist in eliciting the different components of ‘mated’ behaviour.

scholarly journals Specificity and localization of lipolytic activity in adult Drosophila melanogaster

1994 ◽  
Vol 304 (3) ◽  
pp. 775-779 ◽  
Author(s):  
G M Smith ◽  
K Rothwell ◽  
S L Wood ◽  
S J Yeaman ◽  
M Bownes
Keyword(s):  
Drosophila Melanogaster ◽  
Lipase Activity ◽  
Specific Activity ◽  
Lipolytic Activity ◽  
Accessory Gland ◽  
Null Mutant ◽  
Male Accessory Gland ◽  
Accessory Glands ◽  
Esterase 6 ◽  
Adult Drosophila

The triacylglycerol lipases present in adult Drosophila melanogaster have been investigated. Different lipase activities are present in various tissues in the fly. In particular, an abundant lipase activity is present in the male accessory gland. An esterase null mutant was used to confirm that the enzyme activity was due to a distinct lipase and not non-specific activity from esterase 6 which is also abundant in accessory glands. The properties of the accessory-gland lipase were investigated, and pH optima and substrate utilization suggest that it has some similarities to vertebrate bile-salt-stimulated lipase. Lipase activity is significantly reduced in males and increased in females shortly after mating. This finding suggests that lipase activity is transferred to the female and may be important in mating and reproduction in Drosophila.

Male accessory gland proteins affect differentially female sexual receptivity and remating in closely related Drosophila species

2017 ◽  
Vol 99 ◽  
pp. 67-77 ◽  
Author(s):  
Béatrice Denis ◽  
Gaëlle Claisse ◽  
Arnaud Le Rouzic ◽  
Claude Wicker-Thomas ◽  
Gildas Lepennetier ◽  
...  
Keyword(s):  
Accessory Gland ◽  
Drosophila Species ◽  
Sexual Receptivity ◽  
Male Accessory Gland ◽  
Accessory Gland Proteins ◽  
Female Sexual Receptivity

scholarly journals HumanPapilloma VirusInfection in Patients with Male Accessory Gland Infection: Usefulness of the Ultrasound Evaluation

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Rosita A. Condorelli ◽  
Enzo Vicari ◽  
Laura M. Mongioi ◽  
Giorgio I. Russo ◽  
Giuseppe Morgia ◽  
...  
Keyword(s):  
Seminal Fluid ◽  
Hpv Infection ◽  
Accessory Gland ◽  
Seminal Vesicles ◽  
The Other ◽  
Male Accessory Gland ◽  
Papilloma Virus ◽  
Progressive Motility ◽  
Ultrasound Evaluation ◽  
Fluid Concentration

This study evaluated the ultrasound (US) features of 20 patients with MAGI and concomitantpapilloma virus(HPV) infection compared to 20 patients with microbial (presence ofChlamydia trachomatisalone) MAGI and 20 patients with amicrobial (inflammatory) MAGI. Patients with HPV infection showed significantly (p<0.05) higher total prostate, seminal vesicles, and epididymal US signs (18.0 ± 2.0) compared to the other 2 groups (12.0 ± 4.0 versus 10.0 ± 3.0, resp.). In addition, patients with MAGI and HPV had a higher prevalence of complicated forms of MAGI [prostatovesiculitis (PV) and prostate-vesiculo-epididymitis (PVE)] and a higher frequency of the fibrosclerotic variant compared to the other groups (70.0 ± 10.0% versus 48.0 ± 5.0% versus 15.0 ± 10.0%). Moreover, HPV infected patients had a higher number of US criteria suggestive for MAGI in the periurethral region of the prostate compared to the other groups. In particular, the patients showed a higher ratio between periurethral and lobar US criteria distribution (5.0 versus 0.5). Finally, the seminal fluid concentration ofCD45posleukocytes (2.0 ± 0.2 versus 1.3 ± 0.3 versus 1.0 ± 0.3 mil/mL) was significantly higher and sperm progressive motility was significantly lower in these patients compared to other groups.

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