Sperm Release Research Articles

Cathepsin D in prawn reproductive system: its localization and function in actin degradation.

Cathepsin D (CAT-D) is a well-known aspartic protease that serves a function as house-keeping lysosomal enzyme in all somatic cells. Its existence in reproductive tissues is highly variable, even in the somatic derived epithelial cells of reproductive tract. In Macrobrachium rosenbergii, existence of MrCAT-D and its translational product was detected in both somatic cells (Sertoli-like supporting cells) and developing spermatogenic cells as well as along accessory spermatic ducts. Specifically, MrCAT-D was localized onto the sperm surface rather than within the acrosomal matrix, as evident by similar staining pattern of anti-CAT-D on live and aldehyde fixed sperm. MrCAT-D in testicular extracts and sperm isolates showed active enzyme activities towards its specific fluorogenic substrate (MCA-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys (Dnp)-D-Arg-NH2). MrCAT-D also exerted its function towards hydrolyzing filamentous actin, the meshwork of which is shown to be localized at the junction between germ cells and supporting cells and spermatogonia in M. rosenbergii testicular epithelium. Together, we have localized MrCAT-D transcript and its translational product in both supporting and germ cells of testis and claimed its enzymatic function towards actin degradation, which may be related to sperm release from the epithelial cell interaction.

Two subgroups of receptor-like kinases promote early compatible pollen responses in the Arabidopsis thaliana pistil.

In flowering plants, cell-cell communication between the compatible pollen grain/growing pollen tube and the pistil is an essential component for successful sexual reproduction. In Arabidopsis thaliana, the later stages of this dialogue are mediated by several peptide ligands and receptors that guide pollen tubes to the ovules for the release of sperm cells. Despite a detailed understanding of these processes, a key gap remains regarding the nature of the regulators that function at the earlier stages which are essential steps leading to fertilization. Here, we report on new functions for A. thaliana Receptor-Like Kinase (RLK) genes belonging to the LRR-II and LRR-VIII-2 RLK subgroups in the female reproductive tract to regulate compatible pollen hydration and the early stages of pollen tube growth. Mutant pistils for the A. thaliana RKF1 gene cluster were observed to support reduced wild-type pollen hydration and, when combined with the SERK1 and SERK3/BAK1 mutations, reduced pollen tube travel distances occurred. As these mutant pistils displayed a wild-type morphology, we propose that the observed altered compatible pollen responses result from an impaired pollen-pistil dialogue at these early stages.

Evidence of sperm removal behaviour in an externally fertilizing species and compensatory behaviour for the risk of self-sperm removal.

The removal of rival sperm from a female's sperm storage organ acts as a strong sperm competition avoidance mechanism, which has been reported only in internally fertilizing species and not at all in externally fertilizing species. This study demonstrated for the first time that nest-holding males of Bathygobius fuscus, an externally fertilizing marine fish, remove the sperm of rival sneaker males from the spawning nest by exhibiting tail-fanning behaviour within the nest. Males showed tail-fanning behaviour when semen was artificially injected into the nest but not when seawater was injected, and in open nests this behaviour resulted in higher paternity rates for the focal male. The sperm removal behaviour entails the risk of removing their own sperm; therefore, additional sperm release behaviour is likely necessary to benefit from the sperm removal effect. Consistent with this, males increased post-fanning sperm release behaviour more in the semen than in the seawater injection treatment. Moreover, males who had removed sperm for a longer time spent more time releasing sperm after the removal, suggesting that the additional sperm release behaviour compensated for the loss of their own sperm. These results suggest that sperm removal behaviour is not restricted to internally fertilizing organisms and deserves further investigation in this and other species.

Identifying spawning events in fish by observing a spike in environmental DNA concentration after spawning

AbstractAn understanding of the reproductive biology of aquatic organisms is crucial for the efficient conservation and management of species and/or populations. Nevertheless, conventional spawning surveys such as visual‐ and capture‐based monitoring generally require laborious, time‐consuming work and are subject to monitoring biases such as observer bias, as well as miscounts due to false spawning. In addition, direct capture often damages eggs or individuals. Thus, an efficient noninvasive approach for monitoring spawning events on aquatic species would be a valuable tool to understand their reproductive biology and conserving biodiversity. Here, we proposed an environmental DNA (eDNA)‐based approach for monitoring and understanding spawning events by observing spikes in eDNA concentration after spawning events. We found in hybridization experiment using two medaka species (Oryzias latipes and Oryzias sakaizumii, 1:1 individual per tank) that a spike in eDNA concentration occurred in male species after spawning. Besides, the magnitude of the spike in eDNA concentration was dependent on the number of spawning activities with egg and sperm release. In the field survey during the reproductive season, eDNA concentrations after spawning were 3–25 times higher than before the expected time for spawning. Additionally, there was no spike in eDNA concentration during the non‐reproductive season. Therefore, our results demonstrated that spike in eDNA concentration is mainly caused by the released sperm during spawning events, and it can be used as evidence of spawning in the field survey. The presented approach could be a practical tool for studying reproductive biology and provides an opportunity to design effective conservation and environmental management actions.

ORP9 knockdown delays the maturation of junction-related endocytic structures in the testis and leads to impaired sperm release†.

The release of late spermatids from the seminiferous epithelium requires the internalization of intercellular junctions by Sertoli cell specific structures called "tubulobulbar complexes" (TBCs). These large, endocytic devices likely evolved from classic clathrin-mediated-endocytosis (CME) machinery, but have several important morphological differences to CME vesicles. Most notable among these differences is that extensive endoplasmic reticulum (ER) membrane contact sites (MCSs) occur with TBCs and not with clathrin-coated pits. One of the well-established functions of ER MCSs is lipid exchange. Previously, we have established that the ORP9 lipid exchange protein is localized to the TBC-ER MCS; however, the function of ORP9 and lipid exchange at the sites is not known. Here we use an in vivo knockdown approach to probe function. The testes of Sprague-Dawley rats were injected with ORP9 targeted siRNA or non-targeted reagents, and the tissues examined by bright field, super-resolution stimulated emission depletion, and electron microscopy. The knockdown of ORP9 was achieved and maintained with daily injections of siRNA for 2-3day intervals. Compared to controls, sections from ORP9 siRNA-injected testes had longer TBC tubes and fewer fused TBC bulbs. Late spermatids were also abnormally retained in the epithelium of knockdown tissue. These results suggest that ORP9 is necessary for normal TBC bulb vesiculation and fusion, most likely by changing the plasma membrane lipid profile of the TBC. These data also further support the conclusion that TBCs are part of the normal mechanism of sperm release.

NC1-peptide derived from collagen α3 (IV) chain is a blood-tissue barrier regulator

Collagen α3 (IV) chains are one of the major constituent components of the basement membrane in the mammalian testis. Studies have shown that biologically active fragments, such as noncollagenase domain (NC1)-peptide, can be released from the C-terminal region of collagen α3 (IV) chains, possibly through the proteolytic action of metalloproteinase 9 (MMP9). NC1-peptide was shown to promote blood–testis barrier (BTB) remodeling and fully developed spermatid (e.g., sperm) release from the seminiferous epithelium because this bioactive peptide was capable of perturbing the organization of both actin- and microtubule (MT)-based cytoskeletons at the Sertoli cell–cell and also Sertoli–spermatid interface, the ultrastructure known as the basal ectoplasmic specialization (ES) and apical ES, respectively. More importantly, recent studies have shown that this NC1-peptide-induced effects on cytoskeletal organization in the testis are mediated through an activation of mammalian target of rapamycin complex 1/ribosomal protein S6/transforming retrovirus Akt1/2 protein (mTORC1/rpS6/Akt1/2) signaling cascade, involving an activation of cell division control protein 42 homolog (Cdc42) GTPase, but not Ras homolog family member A GTPase (RhoA), and the participation of end-binding protein 1 (EB1), a microtubule plus (+) end tracking protein (+TIP), downstream. Herein, we critically evaluate these findings, providing a critical discussion by which the basement membrane modulates spermatogenesis through one of its locally generated regulatory peptides in the testis.

Transcriptome Analysis of Testis from HFD-Induced Obese Rats (Rattus norvigicus) Indicated Predisposition for Male Infertility.

Obesity is a worldwide life-threatening metabolic disorder, associated with various chronic diseases, including male infertility. Obesity was induced by high fat diet (HFD), and testis RNA was used for the transcriptome analysis using RNAseq via Illumina NovaSeq 6000 System and NovaSeq 6000 Kit. Gene expression level was estimated as FPKM (Fragments Per Kilobase of transcript per Million mapped reads). Differential expressed genes (DEGs) were annotated against gene ontology (GO) and KEGG databases. More than 63.66 million reads per sample were performed with 100 bp cutoff and 6 Gb sequencing depth. Results of this study revealed that 267 GO terms (245 biological processes (BP), 14 cellular components (CC), eight molecular functions (MF)), and 89 KEGG pathways were significantly enriched. Moreover, total numbers of 136 genes were differentially expressed (107 upregulated, 29 downregulated) with |FC| ≥ 2 and bh adjusted <0.05. Interesting DEGs were detected, including obesity and lipid metabolism-related genes, immune response-related genes, cytochrome P450 genes, including aromatase were upregulated, whereas genes related to male fertility and fertilization, cell adhesion, and olfactory receptors were downregulated. The combined expression pattern of the DEGs in obese animals indicated an increase in cholesterol metabolism. Furthermore, high aromatase activity enhances the testosterone turnover into estradiol and lowers the testosterone/estradiol (T/E) ratio, which ultimately reduces fertility. In addition, downregulation of cadherens junction components genes leads to the pre-mature release of sperm from Sertoli cells resulting in the reduction of fertility. Moreover, the downregulation of olfactory receptor genes reduces the chemotaxis capacity of sperms in tracking the oocyte for fertilization, which reduces male fertility. Furthermore, various obesity molecular markers were detected in our transcriptome. The results of this study will enhance our understanding of the molecular network of obesity development, development of obesity novel molecular diagnosis markers, molecular bases of obesity-induced infertility, and the development of anti-obesity drugs.

Extraordinary sperm to egg ratios in seabirds

Abstract Following copulation, females of many seabird species spend a prolonged period of time away from the colony, building up reserves for egg formation and incubation. Here, we report that the number of sperm associated with eggs of single-egg clutch seabirds was almost an order of magnitude greater than predicted from the relationship between ovum size and sperm numbers in multi-egg clutch non-seabirds. Sperm numbers were also several times greater than the estimated number necessary for maximal fertilization success. Our results are consistent with 3 unusual features of seabird reproduction: (1) single-egg clutches, (2) prolonged sperm storage, and (3) a lag period between the end of yolk formation and ovulation. We hypothesize that sperm release from storage is under precise temporal control in these species, with high sperm numbers acting as an insurance against infertility in single-egg clutches. If true, the lag period may have evolved to provide sufficient time for sperm to be released simultaneously from storage and accumulate at the site of fertilization prior to ovulation.

Disparities in Spawning Times Between in situ and ex situ Pillar Corals

Contrasts in spawning time between wild and onshore colonies of the pillar coral Dendrogyra cylindrus were conducted by comparing eight years of wild spawning observations with three years of observations on ex situ corals held in outdoor flow-through tanks. Wild colonies exhibited peak spawning 3 nights after the full moon and 90-96 minutes after sunset. Corals held ex situ peaked on night 4 after the full moon, with females continuing to spawn through night 8. Spawning occurred ~50 minutes later than in wild colonies, and the spawning window, particularly for females, was significantly greater. Fragments held ex situ for as few as 10 days exhibited this shift to later spawning, but corals held for greater than one year also exhibited significantly later spawning than those held less than one year. We propose that the wild colonies in Florida exhibit a historic natural spawning cycle. Lunar and solar cues determine date and time, but deviations in full moon from average dates affect time of sperm release. Comparisons with other spawning locations in the Caribbean suggest that sites closer to shore delay spawning until later in the evening. We propose that artificial light is altering the light spectrum that D. cylindrus colonies use to cue spawning time. Colonies held at onshore outdoor facilities, which are subject to high levels of artificial light, also have delayed and expanded spawning windows. The implications of widespread and increasing light pollution on spawning synchrony identify a potential stressor that could inhibit natural reef recovery.

Reactive Oxygen Species as Mediators of Gametophyte Development and Double Fertilization in Flowering Plants.

Reactive oxygen species (ROS) are toxic by-products of aerobic metabolism. In plants, they also function as important signaling molecules that regulate biotic and abiotic stress responses as well as plant growth and development. Recent studies have implicated ROS in various aspects of plant reproduction. In male gametophytes, ROS are associated with germline development as well as the developmentally associated programmed cell death of tapetal cells necessary for microspore development. ROS have a role in regulation of female gametophyte patterning and maintenance of embryo sac polarity. During pollination, ROS play roles in the generation of self-incompatibility response during pollen-pistil interaction, pollen tube growth, pollen tube burst for sperm release and fertilization. In this mini review, we provide an overview of ROS production and signaling in the context of plant reproductive development, from female and male gametophyte development to fertilization.

Spermcast mating with release of zygotes in the small dioecious bivalve Digitaria digitaria

Digitaria digitaria, a small astartid usually less than 10 mm in length, has a non-brooding behaviour in spite of its limited space for gonad development. This species lives in highly unstable environments with strong currents, which represent a challenge for fertilization and larval settlement. The studied population of D. digitaria from the Strait of Gibraltar area was dioecious, with significant predominance of females and sexual dimorphism, where females are larger than males. The reproductive cycle is asynchronous throughout the year, without a resting period, but with successive partial spawning events. The presence of stored sperm in the suprabranchial chamber and inside the gonad of some females, together with the release of eggs along the dorsal axis of both gills, points to internal oocyte fertilization. Bacteriocytes were found in the female and male follicle walls, but no bacteria were observed inside any of the gametes. Digitaria digitaria could represent a “missing link” between spermcast mating bivalves with brooded offspring and bivalves with broadcast release of eggs and sperm. The small size, limiting the oocyte production, together with the unstable environment could represent evolutionary pressures towards sperm uptake in D. digitaria.

Breeding and Larval Rearing of Asian Moon Scallop Amusium pleuronectes in Eastern Samar, Philippines

Asian moon scallop, Amusium pleuronectes, is among the most common commercially harvested scallop in the Philippines. This study investigated suitable conditioning methods, induced spawning, and documented scallop larval development at the Marine Fisheries Research and Development Center (MFRDC) Guiuan, Eastern Samar. Scallops held in tanks with flow-through water system without substrate yielded high survival rate of 48.06 ± 5.95%. Optimum water temperature was 28-29°C for maintaining scallops. Isochrysis galbana and Chaetoceros calcitrans were suitable microalgal feed for A. pleuronectes consumed at 7,388,888 cells min-1 per scallop. These significant results were applied in maintaining scallops at the hatchery. As a result, scallops with an average 19.8 mm shell length (SL) reached 37.5 mm SL within four months with a survival rate of 96.57 ± 2.04%, average daily growth rate (ADGR) of 0.13 ± 0.04 mm.day-1, and specific growth rate (SGR) of 3.92 ± 1.31%.d-1. Natural spawning was successful under controlled conditions while induced spawning trial through thermal stimulation, food shock, sexual stimulation, and serotonin injection resulted unsuccessful release of sperm and eggs. The estimated number of fertilized eggs per spawning ranged from 0.22-1.4 million. Fertilized eggs appeared spherical and dark in color with 54.2-62.57 µm in diameter. After nine hours, the larvae developed into trochophore stage with 59.08-84.4 µm in length. D-veliger with 120.37-157.07µm shell length developed after 24 hours. Development of the early umbone stage was reached on day 5; and on day 7, the umbo larvae become well-developed with shell length of 135.45-173.36 µm. On day 9, pedi-veligers were observed in the culture. Spat grew 312.41-509.48 µm on day 16 and survived until four months with final shell length of 4-10 mm. For the larval rearing, stocking density of A. pleuronectes larvae observed highest survival rate of 0.04 ± 0.03% at 200 larvae/L, while no larvae survived at 800 larvae/L due to contamination of protozoans in the culture medium.

A New Perspective on Female-to-Male Communication in Salamander Courtship.

Courtship behavior in salamanders is often complex and involves well-documented communication from males to females in multiple sensory modalities. Historically, behaviors exhibited during the major stages of courtship have been predominately framed as a male acting and signaling to "persuade" a passive female to participate in courtship and remain with him until sperm release is completed. In this review, we use courtship descriptions for lungless salamanders (Plethodontidae) as a case study to illustrate this historical bias of a male-centered perspective. We then re-examine the literature and summarize the many ways females are active participants during plethodontid courtships. We also relate female behaviors to the types of female-to-male communication that may occur. For example, females have been documented to approach a male and initiate courtship, participate in mutual head rubbing, and step astride the male's tail to begin the tail-straddling walk (a key courtship behavior observed in all plethodontids). Additionally, females have glands that may produce chemical signals that males respond to during courtship. We conclude that communication during courtship is more accurately described as a two-way interaction where each partner's behavior is coordinated with the other's via multi-modal signaling. Shifting the lens through which we view courtship and behavior provides insight into which female behaviors and anatomical features are most likely to be used for communication with males.

Fine-scale genital morphology affects male ejaculation success: an experimental test.

The evolution of male genital traits is usually ascribed to advantages that arise when there is sperm competition, cryptic female choice or sexual conflict. However, when male-female contact is brief and sperm production is costly, genital structures that ensure the appropriate timing of sperm release should also be under intense selection. Few studies have examined the role of individual structures in triggering ejaculation. We therefore conducted a series of anatomical manipulations of fine-scale features of the complex intromittent organ (gonopodium) of a freshwater fish with internal fertilization (Gambusia holbrooki) to determine their effects on sperm release. Mating in G. holbrooki is fleeting (less than 50 ms), so there should be strong selection for control over the timing of sperm release. We surgically removed three features at the tip of the gonopodium (claws, spines, awl-shape) to test for their potential role in triggering ejaculation. We show that the 'awl-shape' of the tip affects sperm release when a male makes contact with a female, but neither gonopodial claws nor spines had a detectable effect. We suggest that the claws and spines may instead function to increase the precision of sperm deposition (facilitating anchorage and contact time with the female's gonopore), rather than the initiation of ejaculation.

Tuberculosis testicular: presentación de un caso y revisión de la literatura

Descripción del caso clínico: Presentamos el caso de un paciente masculino de 33 años con historia familiar de tuberculosis pulmonar, quien acude a consulta por presentar fiebre de predominio nocturno y percepción de masa testicular derecha no dolorosa, sin otro síntoma asociado, marcadores tumorales en rangos normales.&#x0D; Relevancia: De acuerdo con cifras de la OMS (Organización Mundial de la Salud), la infección por Mycobacterium tuberculosis es considerada la infección oportunista más frecuente en el mundo.&#x0D; Conclusión: La presentación clínica característica de la tuberculosis testicular (TbT) es una masa testicular no dolorosa, difícil de diferenciar de una tumoración maligna. Los hallazgos inespecíficos en los exámenes de laboratorio e imagen acompañados de baja sospecha diagnóstica llevan a un alto porcentaje de diagnósticos tardíos. Hoy en día una cantidad considerable de pacientes se detectan por hallazgos de patología en la pieza quirúrgica. Si bien el pronóstico a corto y mediano plazo suele ser favorable, una de las complicaciones a largo plazo de la TbT es la infertilidad secundaria a la destrucción del parénquima testicular y/o la obstrucción del trayecto de salida de los espermatozoides.

Targeted mutation of secretogranin-2 disrupts sexual behavior and reproduction in zebrafish

The luteinizing hormone surge is essential for fertility as it triggers ovulation in females and sperm release in males. We previously reported that secretoneurin-a, a neuropeptide derived from the processing of secretogranin-2a (Scg2a), stimulates luteinizing hormone release, suggesting a role in reproduction. Here we provide evidence that mutation of the scg2a and scg2b genes using TALENs in zebrafish reduces sexual behavior, ovulation, oviposition, and fertility. Large-scale spawning within-line crossings (n = 82 to 101) were conducted. Wild-type (WT) males paired with WT females successfully spawned in 62% of the breeding trials. Spawning success was reduced to 37% (P = 0.006), 44% (P = 0.0169), and 6% (P < 0.0001) for scg2a-/- , scg2b-/- , and scg2a-/-;scg2b-/- mutants, respectively. Comprehensive video analysis indicates that scg2a-/-;scg2b-/- mutation reduces all male courtship behaviors. Spawning success was 47% in saline-injected WT controls compared to 11% in saline-injected scg2a-/-;scg2b-/- double mutants. For these mutants, spawning success increased 3-fold following a single intraperitoneal (i.p.) injection of synthetic secretoneurin-a (P = 0.0403) and increased 3.5-fold with injection of human chorionic gonadotropin (hCG). Embryonic survival at 24 h remained on average lower in scg2a-/-;scg2b-/- fish compared to WT injected with secretoneurin-a (P < 0.001). Significant reductions in the expression of gonadotropin-releasing hormone 3 in the hypothalamus, and luteinizing hormone beta and glycoprotein alpha subunits in the pituitary provide evidence for disrupted hypothalamo-pituitary function in scg2a and scg2b mutant fish. Our results indicate that secretogranin-2 is required for optimal reproductive function and support the hypothesis that secretoneurin is a reproductive hormone.

Sperm Allocation Strategies Depending on Female Quality in Medaka (Oryzias latipes).

Sperm production is costly, and males are expected to strategically allocate this potentially limiting resource to maximize their fitness. Sperm allocation theory predicts that males should adjust their sperm expenditure in relation to female quality. However, the available empirical evidence is limited. In this study, we assessed whether wild male medaka (Oryzias latipes) would allocate their sperm depending on female quality under controlled conditions. Behavioral observations revealed that spawning behavior could be classified into four stages: male quivering, female quivering, sperm release, and stay. Of these behaviors, only the duration of sperm release was positively correlated with the number of sperm ejaculated in the aquarium, suggesting that males may adjust the sperm number expended for each mating by controlling the duration of sperm release. The estimated amount of sperm released per spawning was positively and significantly correlated with the body depth and weight of paired females. This result is consistent with the prediction arising from the sperm allocation hypothesis, and suggests that male medaka may allocate their sperm depending on paired female body depth and weight. However, the number of released sperm did not correlate with the number of spawned eggs, which was positively correlated with body depth and weight of females. These results imply that medaka females may have counter-strategies against sperm allocation by males, and consequently, males might not be able to adjust the number of sperm efficiently. We disscuss our results from the perspective of sexual conflict over sperm as a limited resource.

Knockdown of IP3R1 disrupts tubulobulbar complex-ectoplasmic reticulum contact sites and the morphology of apical processes encapsulating late spermatids†.

Tubulobulbar complexes (TBCs) internalize intercellular junctions during sperm release. One of the characteristic features of TBCs is that they form "bulbs" or swollen regions that have well-defined membrane contact sites (MCS) with adjacent cisternae of endoplasmic reticulum. Previously, we have localized the IP3R calcium channel to the TBC bulb-ER contacts and have hypothesized that fluctuations in local calcium levels may facilitate the maturation of TBC bulbs into putative endosomes, or alter local actin networks that cuff adjacent tubular regions of the TBCs. To test this, we injected the testes of Sprague Dawley rats with small interfering RNAs (siRNAs) against IP3R1 and processed the tissues for either western blot, immunofluorescence, or electron microscopy. When compared to control testes injected with nontargeting siRNAs, Sertoli cells in knocked-down testes showed significant morphological alterations to the actin networks including a loss of TBC actin and the appearance of ectopic para-crystalline actin bundles in Sertoli cell stalks. There also was a change in the abundance and distribution of TBC-ER contact sites and large internalized endosomes. This disruption of TBCs resulted in delay of the withdrawal of apical processes away from spermatids and in spermiation. Together, these findings are consistent with the hypothesis that calcium exchange at TBC-ER contacts is involved both in regulating actin dynamics at TBCs and in the maturing of TBC bulbs into endosomes. The results are also consistent with the hypothesis that TBCs are part of the sperm release mechanism.

In‐Vivo Knockdown of Cortactin Disrupts Basal Tubulobulbar Complexes and Results in the Presence of Redundant Junction Related Ectoplasmic Specializations

In the mammalian testis, spermatogenic cells begin development in the germ cell niche or basal compartment of the seminiferous epithelium. They then translocate through massive junction complexes between neighboring Sertoli cells to enter the adluminal compartment and further differentiate. Removal of the junctions above and formation of new junctions below is necessary for the translocation of cells from one compartment to the other. Tubulobulbar complexes’ (TBCs) are Sertoli cell specific structures that are proposed to internalize intercellular junctions in the seminiferous epithelium. TBCs are actin cuffed endocytic structures that share many features in common with clathrin mediated endocytosis (CME). Both TBCs and CME vesicles begin with the association of a clathrin coated pit on the plasma membrane and the development of dendritic actin ‘cuffs’ on their neck regions. Rather than vesiculating, the necks of TBCs continue to lengthen into microns long structures, eventually developing an actin free ‘bulb’ region, which develops an ORP9 positive membrane contact site with an associated leaflet of endoplasmic reticulum. One of the proteins known to be highly specific for the dendritic actin around TBCs is the actin related protein, cortactin. It previously has been shown that knockdown of cortactin causes shortening and disorganization of those TBCs at the apex of the epithelium that are associated with late spermatids, and this is correlated with a delay in sperm release. However, it has not been shown if disruption of cortactin expression has similar effects on TBCs associated with the basal junction complexes and whether or not this has an effect on junction removal. Here we use electron microscopy (EM) and stimulated emission depletion (STED) super resolution microscopy to evaluate the phenotype of rat testes injected 5 times over a 9‐day period with siRNA targeting cortactin, and use western blots to confirm the knockdown. In vivo knockdown of cortactin in rat testes disrupts basal TBCs when sections labeled for ORP9 and cortactin/actin are evaluated by STED, and leads to the presence of redundant junction related material (ectoplasmic specializations) when evaluated by EM. These data are consistent with the conclusion that TBCs internalize basal junctions and are likely part of the mechanism of spermatocyte translocation from basal to adluminal compartments of the epithelium.Support or Funding InformationSupported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to A Wayne Vogl (RGPIN‐2018‐03727).

Cephalopod mating systems as models for the study of sexual selection and alternative reproductive tactics: a review

Cephalopods are a remarkable group for studies on sexual selection, due to their complex reproductive behavior, pronounced sexual dimorphism, polygamous behavior, as well as cumulative evidence of pre- and postcopulatory male-male competition and female choice in the group. is paper provides a review of sexual selection studies on cephalopods, with an emphasis on the loliginid squid mating system. In this system, males may adopt alternative reproductive tactics (ART) and display male intrasexual dimorphism, with each morph generally exhibiting distinct behaviors and ejaculate traits. Consort males typically transfer ejaculates to the female mantle cavity, while sneaker males typically transfer ejaculates to the female buccal region, where a sperm storage organ is located. Each site provides distinct fertilization environments for the ejaculates of each morph, differing in the availability of eggs, gamete protection, and timing between mating and fertilization. Accordingly, the ejaculates of each morph show putative adaptations for each site, differing in size, morphology, sperm release duration, and sperm behavior. Although solid evidence is lacking for most loliginids, cryptic female choice may occur in this system through, for example, spermatangia removal or egg string manipulation during fertilization. In addition, recent evidence for one loliginid species indicates that adoption of male ART is ontogenetic and may represent two alternative pathways: from immature males (i) directly to consort phenotype or (ii) going through a sneaker phase, then switching to consort morph. The second pathway would, therefore, imply striking change in behavior, physiology, and ejaculates. Moreover, data on the age of this species suggests that the adoption of sneaker tactic early in life may be associated with extending the male mating period by 25%. With such a peculiar system, loliginids should prove useful not only for comprehending the evolution of female promiscuity and ART in cephalopods, but also as an emerging nonvertebrate and nonarthropod model for testing sperm competition models.