Vaginal Plug Research Articles

Reproductive behavior: Decrypting cryptic female choice

Females in many species bias fertilization success and paternity after mating with multiple males. Two new studies uncover the mechanisms of this phenomenon, cryptic female choice, in Drosophila, linking it to pheromone-stimulated ejection of sperm and mating plug.

Male cuticular pheromones stimulate removal of the mating plug and promote re-mating through pC1 neurons in Drosophila females

In birds and insects, the female uptakes sperm for a specific duration post-copulation known as the ejaculate holding period (EHP) before expelling unused sperm and the mating plug through sperm ejection. In this study, we found that Drosophila melanogaster females shortens the EHP when incubated with males or mated females shortly after the first mating. This phenomenon, which we termed male-induced EHP shortening (MIES), requires Or47b+ olfactory and ppk23+ gustatory neurons, activated by 2-methyltetracosane and 7-tricosene, respectively. These odorants raise cAMP levels in pC1 neurons, responsible for processing male courtship cues and regulating female mating receptivity. Elevated cAMP levels in pC1 neurons reduce EHP and reinstate their responsiveness to male courtship cues, promoting re-mating with faster sperm ejection. This study established MIES as a genetically tractable model of sexual plasticity with a conserved neural mechanism.

Male cuticular pheromones stimulate removal of the mating plug and promote re-mating through pC1 neurons in Drosophila females.

In birds and insects, the female uptakes sperm for a specific duration post-copulation known as the ejaculate holding period (EHP) before expelling unused sperm and the mating plug through sperm ejection. In this study, we found that Drosophila melanogaster females shortens the EHP when incubated with males or mated females shortly after the first mating. This phenomenon, which we termed male-induced EHP shortening (MIES), requires Or47b+ olfactory and ppk23+ gustatory neurons, activated by 2-methyltetracosane and 7-tricosene, respectively. These odorants raise cAMP levels in pC1 neurons, responsible for processing male courtship cues and regulating female mating receptivity. Elevated cAMP levels in pC1 neurons reduce EHP and reinstate their responsiveness to male courtship cues, promoting re-mating with faster sperm ejection. This study established MIES as a genetically tractable model of sexual plasticity with a conserved neural mechanism.

Maternal exposure to phenanthrene induces testicular apoptosis and Sertoli cell dysfunction in F1 adult male mice: a histological and molecular study.

Phenanthrene, a polycyclic aromatic hydrocarbon, is found in abundance in environmental pollutants, food, and drinking water. This substance can accumulate in body tissues and exert harmful effects. Moreover, phenanthrene can cross the placental barrier, potentially impacting fetal development. We aimed to explore the impacts of maternal exposure to phenanthrene on testicular tissue and Sertoli cell function in F1 mice. Female rats with vaginal plugs were randomly assigned to one of three groups: control, sham, or phenanthrene. The control group received no intervention during pregnancy. In the sham and phenanthrene groups, corn oil and a phenanthrene solution, respectively, were administered via gavage once every 2 days. Offspring were separated by sex 21 days after birth. At 56 days postnatal, male F1 offspring were euthanized, and their testes were harvested for histological and molecular analyses. Phenanthrene exposure was associated with a lower testicular weight and volume, a smaller diameter of the seminiferous tubules, and a relative thinning of the germinal epithelium. These changes were associated with increased cellular apoptosis, as shown by the upregulation of caspase 3 expression. Additionally, we observed an increase in vacuolization and residual bodies within the tissue. Conversely, the number of Sertoli cells and expression levels of Sox9, as well as the Ocln and Itgb1 genes, were found to be lowered. Maternal exposure to phenanthrene impacts both germ cells and Sertoli cells, disrupting their function and leading to fertility disorders in male F1 offspring mice.

A simple method for repeated in vivo sperm collection from laboratory mice

PurposeMouse spermatozoa for archiving laboratory mice or for in vitro fertilization (IVF) are routinely obtained from the cauda epididymis of adult males sacrificed for this purpose. To avoid the death of the donor, we tested whether a precisely timed interruption of the mating act could be used for repeated sperm collection from laboratory mice.MethodsSperm donors (B6D2F1) were mated with a receptive female, and mating behavior was observed. The stud was separated from the female 1–2 s after the onset of the ejaculatory shudder. The ejected copulatory plug with the yellowish viscous ejaculate was carefully removed from the penile cup.ResultsA total of 80 ejaculates were successfully obtained from 100 ejaculations. The latency to first mount was 1.1 ± 1.1 min (mean ± SD) and to ejaculation 8.1 ± 4.7 min. The average number of mounts to ejaculation was 10.5 ± 5.8, and the mean number of spermatozoa per collected ejaculate was 1.86 ± 1.05 × 106. An average fertilization rate of 76% was observed after IVF.ConclusionsSeparating the stud from the female just before ejaculation is feasible, easy to learn, and requires no special equipment. The sperm count of collected ejaculates is lower than natural ejaculations, but higher than previous in vivo sperm collection methods achieved. We recommend this simple sperm collection method in mice, especially when the donor cannot be sacrificed and/or repeated sperm collection from the same animal is required for experimental purposes.

P-316 Fertility, pregnancy outcomes, and perinatal results related to experimental endometriosis in a murine model

Abstract Study question Does the transplantation of endometrial tissue fragments into the peritoneal wall of female mice reduce natural fertility and lead to compromised obstetric and perinatal outcomes? Summary answer Experimental murine endometriosis did not compromise fertility, but females with more ectopic endometria had litters with reduced birth weight and fewer offspring at weaning. What is known already It has been demonstrated that endometriosis is strongly associated with subfertility and is increasingly linked to late pregnancy complications, such as those caused by placental insufficiency. At the molecular level, the presence of ectopic endometrium may perturb the endometrial hormonal, cellular, and immunological milieu, negatively influencing the decidualization, placentation, and developmental programming of the embryo. However, it remains unclear if and how such early aberrant reproductive development relates to pregnancy outcomes in endometriosis. Thus, the use of animal models may be very useful in deepening our understanding of the pathogenic mechanisms of infertility and compromised pregnancy outcomes related to endometriosis. Study design, size, duration C57BL/6J female mice were divided into three groups: Control (42 animals underwent sham surgery), Endometriosis1 (19 animals underwent surgery for transplantation of one fragment of endometrial tissue on each side of the peritoneal wall), and Endometriosis2 (23 animals received two fragments on each side). The maximum experiment duration was 15 weeks, including a 4-week rest period post-surgery, up to 4 weeks for mating, 3 weeks for delivery, and 4 weeks until weaning of the offspring. Participants/materials, setting, methods Reproductive-age female mice from the three groups had a 4-week rest period post-surgery. Then, they were paired for mating with proven fertile males and checked for the presence of a vaginal plug daily. Pregnant females were euthanized after weaning the litter at 4 weeks. Tissue from the implants was collected for histological analysis. The occurrence of pregnancy, gestation duration, litter size, pup weight at birth and weaning, and mating return were compared among the groups. Main results and the role of chance All females in the experimental groups were confirmed for the presence of ectopic endometrium through histology. No statistically significant differences were observed in pregnancy rates, gestation duration, litter size at birth, and the number of live pups at birth when comparing the three groups (C, E1, and E2). However, the litter weight at birth was significantly lower in group E2 (Medial (Md): 8g; Percentile (P)25: 6; P75: 10) than in groups C (Md: 10g; P25: 8; P75: 10) and E1 (Md: 10g; P25: 10; P75: 12), and significantly higher in E1 compared to C. The number of live pups at weaning was significantly lower in E2 (Md: 5; P25: 3.5; P75: 7) compared to groups C (Md: 7; P25: 5; P75: 7) and E1 (Md: 8; P25: 7; P75: 8), and significantly higher in E1 than in C. The litter weight at weaning was significantly lower in E2 (Md: 96g; P25: 67; P75: 109) than in both E1 (Md: 116g; P25: 110; P75: 128) and C (Md: 104g; P25: 96; P75: 112). The litter weight at weaning was higher in E1 compared to group C. There was no statistically significant difference in mating return rates among the three groups. Limitations, reasons for caution This study is limited by the use of an animal model that does not have spontaneous endometriosis. Wider implications of the findings Litters from the E2 group may have had impaired development during gestation, as indicated by lower birth weight and higher perinatal mortality. Although this is not an ideal translational model for studying endometriosis-related infertility, it may serve as a suitable model for evaluating gestational and perinatal outcomes associated with endometriosis. Trial registration number Not applicable

Mating system variation and gene expression in the male reproductive tract of Peromyscus mice.

Genes involved in reproduction often evolve rapidly at the sequence level due to postcopulatory sexual selection (PCSS) driven by male-male competition and male-female sexual conflict, but the impact of PCSS on gene expression has been under-explored. Further, though multiple tissues contribute to male reproductive success, most studies have focused on the testes. To explore the influence of mating system variation on reproductive tract gene expression in natural populations, we captured adult males from monogamous Peromyscus californicus and polygynandrous P. boylii and P. maniculatus. We generated RNAseq libraries, quantified gene expression in the testis, seminal vesicle, epididymis, and liver, and identified 3627 mating system-associated differentially expressed genes (MS-DEGs), where expression shifted in the same direction in P. maniculatus and P. boylii relative to P. californicus. Gene expression variation was most strongly associated with mating behaviour in the seminal vesicles, where 89% of differentially expressed genes were MS-DEGs, including the key seminal fluid proteins Svs2 and Pate4. We also used published rodent genomes to test for positive and relaxed selection on Peromyscus-expressed genes. Though we did not observe more overlap than expected by chance between MS-DEGs and positively selected genes, 203 MS-DEGs showed evidence of positive selection. Fourteen reproductive genes were under tree-wide positive selection but convergent relaxed selection in P. californicus and Microtus ochrogaster, a distantly related monogamous species. Changes in transcript abundance and gene sequence evolution in association with mating behaviour suggest that male mice may respond to sexual selection intensity by altering aspects of sperm motility, sperm-egg binding and copulatory plug formation.

Alcohol intake during pregnancy reduces offspring bone epiphyseal growth plate chondrocyte proliferation through transforming growth factor β-1 inhibition in the Sprague Dawley rat humerus.

Intrauterine alcohol exposure delays bone maturation and intensifies osteoporosis and fracture risk. As most studies emphasize the neurological aspects of intrauterine alcohol exposure, there is a lack of research on the implications pertaining to osseous tissue. Previous studies investigated these effects in fetuses, with limited studies on postnatal life. Postnatal studies are crucial since peak bone growth occurs during adolescence. This study aimed at assessing the effects of prenatal alcohol exposure on the humerus proximal and distal growth plate chondrocytes in 3-week-old rats. Sprague Dawley rats (n=9) were assigned to either the ethanol group (n=3), saline (n=3), and untreated (n=3) group and time-mated. Once pregnant, as confirmed by the presence of a copulation plug, the former 2 groups were treated with 0.015 ml/g of 25.2% ethanol and 0.9% saline. The untreated group received no treatment. The left humeri belonging to 6 pups per group were used. Serial sections were cut with a microtome at 5 μm thickness. These sections were stained with haematoxylin and eosin for assessment of normal morphology or immunolabeled with anti-Ki-67 and transforming growth factor β-1 (TGFβ-1) antibody. Prenatal alcohol exposure adversely effected the growth plate sizes and the number of cells in the proliferative zone. Fewer TGFβ-1 immunopositive and proliferative chondrocytes were found using the anti-Ki-67 antibody. This may explain the growth retardation in offspring exposed to gestational alcohol, showing that gestational alcohol exposure inhibits cell proliferation, aiding the diminished stature.

Treatment of mice with maternal intermittent fasting to improve the fertilization rate and reproduction.

Maternal intermittent fasting (MIF) can have significant effects on several tissue and organ systems of the body, but there is a lack of research on the effects on the reproductive system. So, the aim of our study was to analyze the effects of MIF on fertility. B6C3F1Crl (C57BL/6N × C3H/HeN) male and female mice were selected for the first part of the experiments and were analyzed for body weight and fat weight after administration of the MIF intervention, followed by analysis of sperm counts and activation and embryo numbers. Subsequently, two strains of mice, C57BL/6NCrl and BALB/cJRj, were selected and administered MIF to observe the presence or absence of vaginal plugs for the purposes of mating success, sperm and oocyte quality, pregnancy outcome, fertility status and in vitro fertilization (IVF). Our results showed a significant reduction in body weight and fat content in mice receiving MIF intervention in B6C3F1Crl mice. Comparing the reproduction of the two strains of mice. However, the number of litters was increased in all MIF interventions in C57BL/6NCrl, but not statistically significant. In BALB/cJRj, there was a significant increase in the number of pregnant females as well as litter size in the MIF treatment group, as well as vaginal plugs, and IVF. There was also an increase in sperm activation and embryo number and the MIF intervention significantly increased sperm count and activation. Our results suggest that MIF interventions may be beneficial for reproduction in mice.

Xue Ping Tablets treat abnormal uterine bleeding via VEGF-ERK1/2 pathway

ObjectiveTo investigate the protective effects against abnormal uterine bleeding (AUB) and possible mechanisms of Xue Ping tablets (XPT) using a rat model. MethodsA total of 58 unmated female and 25 male SPF SD rats aged 8–9 weeks were selected. Eight unmated female rats were selected as the blank control group according to the complete random method. The other 50 rats were mated in a female/male ratio of 2:1. In the morning after mating, vaginal smears were collected. Presence of vaginal plug or sperm was regarded as the first day of pregnancy. All pregnant rats were given 8.3 mg/kg of mifepristone by gavage at 8:00 a.m. and 100 μg/kg misoprostol by gavage at 6:00 p.m. on the seventh day of pregnancy to induce incomplete abortion, thereby establishing a rat model of AUB. Forty rats were randomly divided into model, low- (220 mg/kg), medium- (441 mg/kg), high-dose (882 mg/kg) XPT, and positive control groups. The positive group was given 130 mg/kg Gong Xue Ning (GXN). The model group and the blank group were given an equal amount of distilled water. ResultsCompared with the model group, the volume of bleeding in the positive and middle- and high-dose XPT groups decreased (P < 0.05). Moreover, compared with the model group, the progesterone levels in the positive and XPT groups were significantly increased. Immunohistochemistry showed that XPT significantly decreased the expression levels of VEGF, p-ERK, NF-κB, SAA, MMP-2, MMP-9, TIMP-1, TIMP-2 and TIMP-3. WB results showed that XPT significantly decreased the expression levels of p-ERK, MMP-9, NF-κB, MMP-2 and VEGF. QRT-PCR results showed that XPT significantly decreased the expression levels of VEGF, NF-κB, SAA, MMP-2, TIMP-1, TIMP-2 and TIMP-3 (P < 0.05). ConclusionsXPT could reduce AUB by inhibiting the inflammatory factors involved in the VEGF-ERK1/2 pathway.

A spider mating plug functions to protect sperm.

Mating plugs in animals are ubiquitous and are commonly interpreted to be products of mating strategies. In spiders, however, mating plugs may also take on functions beyond female remating prevention. Due to the vagaries of female genital (spermathecal) anatomy, most spiders face the problem of having to secure additional, non-anatomical, protection for transferred sperm. Here, we test the hypothesis that mating plugs, rather than (or in addition to) being adaptations for mating strategies, may serve as sperm protection mechanism. Based on a comparative study on 411 epigyna sampled from 36 families, 187 genera, 330 species of entelegyne spiders, our results confirm the necessity of a sperm protection mechanism. We divided the entelegyne spermathecae into four types: SEG, SED, SCG and SCD. We also studied detailed morphology of epigynal tracts in the spider Diphya wulingensis having the SEG type spermathecae, using 3D-reconstruction based on semi thin histological series section. In this species, we hypothesize that two distinct types of mating plug, the sperm plug and the secretion plug, serve different functions. Morphological details support this: sperm plugs are formed on a modified spermathecal wall by the spilled sperm, and function as a temporary protection mechanism to prevent sperm from leaking and desiccating, while secretion plugs function in postcopulation both as a permanent protection mechanism, and to prevent additional mating. Furthermore, with the modified spermathecal wall of S2 stalk, the problem of shunt of sperm input and output, and the possibility of female multiple mating have been resolved. Variation in spermathecal morphology also suggests that the problem of sperm protection might be resolved in different ways in spiders. Considering mating plugs of varying shapes and origins in the vast morphospace of spiders, we conclude that mating plugs might serve different purposes that relate both to mating strategies, as well as to sperm protection.

Does morphological maturity anticipate physiological maturity? The pattern of sexual maturity in males of the seabob shrimp Xiphopenaeus kroyeri (Heller, 1862)

The seabob shrimp Xiphopenaeus kroyeri (Heller, 1862) is an important fishing resource and is intensely exploited in South America. Therefore, for the correct and sustainable use of this fishing resource, it is necessary to employ multiple tools to determine the sexual maturity of this species, a key condition for the maintenance of this important economic resource. In this work, we evaluated the morphological (development of secondary sexual characteristics) and physiological (maturation of the reproductive system) sexual maturities in males of X. kroyeri, through the macroscopic and microscopic description of the testes, vasa deferentia and petasma from the juvenile stage to the adult stage. The male reproductive system of seabob shrimp is a paired organ with testes connected to a vasa deferentia, which is anatomically differentiated into three regions: proximal (PVD) (subdivided into anterior, or PVDa, and posterior, or PVDp), medial (MVD) and distal (DVD), the latter region being associated with the terminal ampulla. The morphological maturity of males preceded physiological maturity, meaning the petasma must be formed for the production of seminal fluid and the formation of spermatozoa contained in spermatophores to occur. Petasma formation occurred in the size class 12 ┤13 mm CL, while the production of spermatozoa and spermatophores occurred from 14.1 mm CL. These spermatophores are completely formed in the PVDp and are glycoproteic. Spermatophores are stored in the simple tube of the MVD and DVD. In the DVD, there is an accessory gland forming the ampulla, which produces part of the seminal fluid. The deposition of spermatophores occurs internally in the female’s thelycum with the aid of the petasma, which deposits the seminal fluid in the seminal receptacles or spermathecae. At the end of this process, the thelycum is sealed by a secretion from the accessory gland in the male’s ampulla, forming a sperm plug. Based on the results obtained here, we recommend that only physiologically mature males of X. kroyeri (> 14.1 mm CL) should be exploited by artisanal fisheries and that macroscopic inspection of the ampulla to determine physiological maturity in this species should be avoided, as this region of the DVD has the accessory gland as its main part, a structure responsible for forming the sperm plug, which does not contain spermatozoa or participate in the formation of spermatophores.

The consumption of lard oil during pregnancy and postpartum periods has negative effects on cognitive function by altering the fatty acid profile and activating neuroinflammation via calcium signaling pathway in the maternal mice brain

It has been suggested that dietary intake of lipids and fatty acids may influence cognitive function, however, the effect of lard intake during pregnancy and postpartum periods on cognitive function of mother remains to be elucidated. We investigated the effect and mechanism of consuming soybean oil (SO), the mixed oil of lard and soybean oil at the ratio of 1:1 (LS) and lard oil (LO) during the pregnancy and postpartum periods on cognitive function of the maternal mice. All pregnant C57BL/6JNifdc mice were fed with soybean oil diet during day 0–10 (the day when vaginal plugs appeared in female mice was recorded as day 0), and then randomly assigned to SO, LS and LO groups (n = 10) from day 11 to day 44. The time in center zone and the number of times to enter in center zone were significantly higher in the SO group than in the LO group detected by the open-field test. The levels of neuroglial cells, NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex and pyroptosis related proteins in brain of the LO group were significantly higher than those in the SO group. RNA-sequencing results showed that the calcium signaling pathway related genes in brain, including Adcy8, Ntsr1, Trhr, Oxtr, Htr5b and Camk2d levels significantly higher in the LO group than in the SO group. Lipidomic analysis indicated that PG 18:2_18:2, PG 20:5_22:6, and CL 12:0_16:0_22:3_22:5 of glycerophospholipid metabolism in brain significantly connected with Htr5b of calcium signaling pathway. In conclusion, the intake of lard during the pregnancy and postpartum periods is detrimental to the cognitive function of maternal mice, which probably due to changes in the composition of fatty acid in the brain, thereby activating neuroinflammation via calcium signaling pathway in brain.

Treatment of spontaneously hypertensive rats during pregnancy and lactation with the antioxidant tempol lowers blood pressure and reduces oxidative stress.

Genetic and environmental factors interact in a complex manner in the pathogenesis of essential hypertension in humans. Oxidative stress is considered one of the more important environmental factors. We used the spontaneously hypertensive rat (SHR) model to test whether continuous feeding with the antioxidant tempol reduces maternal oxidative stress during pregnancy and potentially contributes to the prevention of cardiovascular disease onset. Pregnant female rats were divided into control and tempol-treated groups. Tempol was continuously administered in drinking water. The administration period lasted approximately 40 days, from the confirmation of a vaginal plug until birth of the pups and their subsequent weaning. The blood pressure (BP) of each adult female was measured three times during pregnancy and post parturition. Milk was collected three times from nursing mother rats in the immediate postpartum period. Markers of oxidative stress were measured: 8-hydroxyl-2'-deoxyguanosine (8-OHdG) levels in milk during the experimental period and 8-OHdG and corticosterone levels in urine of adult and neonatal rats. The urinary level of 8-OHdG in the tempol-treated group was significantly lower than that in the control group. Corticosterone levels were significantly lower in urine of neonatal rats from the tempol-treated group compared with the levels of the control group. The levels of total antioxidant in milk were significantly greater in the tempol-treated group than in the control group. This study demonstrated that continuous administration of tempol to pregnant SHRs reduced maternal oxidative stress and contributed to reduced oxidative stress in neonatal rats.

A model of pregnancy-associated malaria for inducing adverse pregnancy outcomes in ICR mouse

BackgroundBased on understanding of placental pathological features and safe medication in pregnancy-associated malaria (PAM), establishment of a stable pregnant mouse infection model with Plasmodium was urgently needed. MethodsICR mice with vaginal plugs detected were randomly divided into post-pregnancy infection (Malaria+) and uninfected pregnancy (Malaria−) cohorts. Age-matched mice that had not been mated were infected as pre-pregnancy infection group (Virgin control), which were subsequently mated with ICR males. All mice were inoculated with 1 × 106Plasmodium berghei ANKA-infected RBCs by intraperitoneal injection, and the same amount of saline was given to Malaria− group. We recorded the incidence of adverse pregnancy outcomes and the amounts of offspring in each group. ResultsThe Virgin group mice were unable to conceive normally, and vaginal bleeding, abortion, or stillbirth appeared in the Malaria+ group. The incidence of adverse pregnancy outcomes was extremely high and statistically significant compared with the control (Malaria−) group (P < 0.05), of which placenta exhibited pathological features associated with human gestational malaria. ConclusionsThe intraperitoneal injection of 1 × 106Plasmodium berghei ANKA-infected RBCs could establish a model of pregnancy-associated malaria in ICR mouse.

The seminal odorant binding protein Obp56g is required for mating plug formation and male fertility in Drosophila melanogaster.

In Drosophila melanogaster and other insects, the seminal fluid proteins (SFPs) and male sex pheromones that enter the female with sperm during mating are essential for fertility and induce profound post-mating effects on female physiology. The SFPs in D. melanogaster and other taxa include several members of the large gene family known as odorant binding proteins (Obps). Work in Drosophila has shown that some Obp genes are highly expressed in the antennae and can mediate behavioral responses to odorants, potentially by binding and carrying these molecules to odorant receptors. These observations have led to the hypothesis that the seminal Obps might act as molecular carriers for pheromones or other compounds important for male fertility, though functional evidence in any species is lacking. Here, we used functional genetics to test the role of the seven seminal Obps in D. melanogaster fertility and the post-mating response (PMR). We found that Obp56g is required for male fertility and the induction of the PMR, whereas the other six genes are dispensable. We found males lacking Obp56g fail to form a mating plug in the mated female's reproductive tract, leading to ejaculate loss and reduced sperm storage, likely due to its expression in the male ejaculatory bulb. We also examined the evolutionary history of these seminal Obp genes, as several studies have documented rapid evolution and turnover of SFP genes across taxa. We found extensive lability in gene copy number and evidence of positive selection acting on two genes, Obp22a and Obp51a. Comparative RNAseq data from the male reproductive tract of multiple Drosophila species revealed that Obp56g shows high male reproductive tract expression in a subset of taxa, though conserved head expression across the phylogeny. Together, these functional and expression data suggest that Obp56g may have been co-opted for a reproductive function over evolutionary time.

Sexual dimorphism in the proventriculus of the buff-tailed bumblebee Bombus terrestris (L. 1758) (Hymenoptera: Apidae).

Research on eusocial bee species like Bombus terrestris is primarily focused on the worker caste, which is why their morphology and anatomy are already well described. This includes the alimentary tract, which is adapted for feeding on nectar and pollen. Located at the transition between crop and ventriculus is a highly specialised compartment, the proventriculus. In female workers of B. terrestris, the proventriculus is surrounded by muscles and consists of four anterior lips. A detailed description, however, is only provided for B. terrestis worker bees while studies on the proventriculus of the male reproductive caste are absent. Here, we provide a detailed analysis of the differences between the proventriculus of the B. terrestris males and females through morphometrics, histology and scanning electron microscopy imaging, and unravel a distinct sexual dimorphism. The male proventriculus is wider resulting in a greater volume than the female proventriculus. Histological analysis revealed 4 distinctive chambers of the male proventriculus, which are completely covered with hairs on the inside. In contrast, those chambers in the proventriculus of female B. terrestris, are only rudimentarily present forming only small pouches with hairs in the junctions between the proventricular folds inside the proventriculus. The morphological differences in the proventriculus may be based on different modi vivendi, as males do not return to the colony and fly longer distances. This and the synthesis of sperm and mating plug might require higher energy reserves, leading to the necessity of higher food storage capacities.

Dynamic changes characteristics of the spermatozoon during the reproductive process of mud crab (Scylla paramamosain): From spermatophore formation, transportation to dispersion

To enhance comprehension of the destiny of the spermatozoon throughout the reproductive process of mud crab (Scylla paramamosain) is important for achieving large-scale artificial breeding of Scylla. The reproductive systems of sexually mature male and female mud crabs were studied, utilizing the histomorphological method. The spermatozoon traveled from testicular lobules through the seminiferous duct, into the anterior vas deferens (AVD) via the collecting duct. The spermatophore wall formed at the end of the AVD. During copulation, spermatophores stored in the median vas deferens (MVD) were transferred to the seminal receptacle through the posterior vas deferens (PVD), ejaculatory duct, and gonopod, with seminal plasma secreted by both MVD and PVD together. After mating, the sperm plug was formed within the seminal receptacle's ventral zone. At ovary stage Ⅲ, the spermatophore wall ruptured, and the sperm plug disappeared. Bundled sperm packets congregated into a dense mass, while a few spermatozoa dispersed along the intermediate chamber to the dorsal zone. At ovary stage Ⅳ, the dorsal zone also exhibited a dense sperm mass due to increased dispersal. At ovary stage Ⅴ, merely a modest portion of the dense sperm mass remained in the ventral zone, and the rest dissociated and dispersed within the seminal receptacle. Following ovulation, sperm mass remained in the ventral zone, re-dissociating and re-dispersing once ovarian development reached stage Ⅲ again. The phased dispersion supply mode of spermatozoa behind multiple ovipositions was unveiled. Furthermore, we observed the ultrastructural changes in the spermatozoon during the process of the capacitation, including operculum widening, actin-containing substance accumulation, and the density of chromatin in the nucleus decreased. As presented herein, the elucidation of the dynamic changes of the spermatozoon, as well as the pattern of spermatozoa supply, contributes significantly to our enhanced comprehension of the reproductive biology of Scylla.

Effect of carbofuran insecticide administration to fetal weight and length of mice (Mus musculus)

Introduction: Carbofuran could decrease cholinesterase levels are significantly associated with reduced gestation duration of and the risk of low birth weight and length. This study aims to determine a decrease in the fetal weight and length of pregnant mice exposed to carbofuran. Objective: This study used 18 female mice aged 10 weeks old with a weight of 25-30 grams. Female mice were mated with 18 male mice aged 12 weeks old which were synchronized using the hormones of Pregnant Mare Serum Gonadotrophin (PMSG) and Human Chorionic Gonadotrophin (HCG). This study was divided into three treatments, namely control (C), carbofuran at a dose of 0.0417 mg/mouse (T1), and carbofuran at a dose of 0.0208 mg/mouse (T2). Each pregnant mouse was treated with carbofuran for 10 days from day 6 up to day 15 of gestation when vaginal plugs were seen. Fetal collection was carried out on day 17 to measure the fetal weight and length. Data were analyzed using the ANOVA test and Duncan's test (significance 5%). Results: The results showed that the treatment groups (T1 and T2) had a decrease in the fetal weight and length compared to the control (C) (p&lt;0.05), nevertheless the treatment groups (T1 and T2) did not have significant difference (p&gt;0/05). Conclusion: It can be concluded that administration of carbofuran to pregnant mice could decrease the fetal weight and length.

The function of mating plugs in the spider Neriene emphana: mating strategy or sperm protection?

IntroductionIt is generally thought that mating plugs, where present, impede or reduce the possibilities of female subsequent mating. Behavioral studies on numerous spiders, where mating plugs are common, have generally supported this function. However, mating plugs in spiders could plausibly serve other functions as well. Namely, the structure of entelegyne spermathecae—the morphology of most spiders—could require a mechanism that would prevent sperm from leakage, desiccation, and backflow. Although the form and function of mating plugs in several spider species imply their potential adaptation for sperm protection, this function has never been empirically tested.MethodsHere, we test whether mating plugs in the sheet-web spider Neriene emphana serve as a sperm protective device by investigating its genital morphology, its copulation process, and the precise formation of its amorphous mating plugs. ResultsThis species constructs secretion plugs through male-female cooperation. Additionally, we found sperm plugs to be formed as a side product of sperm transfer, as well as an intermediate type of secretion plugs. These plug materials are transferred in different mating stages as documented by variations in the rhythm of male palpal application during copulation. We showed that complete copulations always resulted in formation of secretion plugs at spermathecal entrances via laborious deposition of male materials. DiscussionWhile our findings do not reject that secretion plugs in N. emphana prevent females from subsequent mating, we suggest that they must have evolved to provide sperm protection.