Sperm Penetration Research Articles

O-073 Delta-9-THC acts on the calcium channel CatSper and alters human sperm function

Abstract Study question Do the main psychoactive phytocannabinoid delta-9-tetrahydrocannabinol (THC) and its non-psychoactive analog cannabidiol (CBD) affect human sperm function? Summary answer THC reduces the ligand-dependent activation of sperm-specific Ca2+ channel CatSper and affects human sperm functions in vitro. What is known already Marijuana (Cannabis sativa) is one of the most commonly used recreational drugs worldwide. Although the impact of phytocannabinoids on reproductive health has been investigated, there is no evidence of a direct effect of THC on CatSper function. Study design, size, duration We studied the effects of the main psychoactive phytocannabinoid, THC, its non-psychoactive analog, CBD, as well as their major metabolites on Ca2+ influx via CatSper in human spermatozoa. THC and CBD were selected to further evaluate their action on progesterone-, prostaglandin-, and pH-induced activation of human CatSper. The effects of THC and CBD on hyperactivation, progressive motility in viscous media, and acrosomal exocytosis were also assessed. Participants/materials, setting, methods The impact of phytocannabinoids on CatSper activity was investigated on sperm samples from healthy volunteers using kinetic Ca2+ fluorimetry. Motility assessment was performed using Computer-Assisted Sperm Analysis (CASA). Sperm penetration into viscous media was assessed using a modified Kremer test. Acrosomal exocytosis was evaluated by flow cytometry using Pisum sativum agglutinin-stained spermatozoa. Main results and the role of chance Both THC and CBD suppress natural ligand-induced calcium influx via CatSper. In particular, THC inhibits progesterone-induced Ca2+ influx via CatSper at pharmacologically relevant concentrations in a non-competitive manner and reduces the pH-induced activation of CatSper. In addition, THC impaired sperm hyperactivation and penetration into viscous media and induced spontaneous acrosomal exocytosis in vitro. Limitations, reasons for caution This is an in vitro study. Future studies are needed to test the physiological relevance in vivo and whether THC can disrupt human sperm function. Wider implications of the findings The action of THC on CatSper in human sperm might impair the fertilization process. Healthcare providers, especially fertility clinicians, should be aware of the potentially negative effects of cannabis consumption on sperm physiology Trial registration number not applicable

Dissecting Sperm Mitochondrial G-Quadruplex Structures Using a Fluorescent Probe Biomarker to Monitor and Regulate Fertilization Capability.

To monitor the levels of mitochondrial DNA G-quadruplexes (mtDNA G4s) in spermatozoa and to explore the possibility using mtDNA G4s as a reliable marker in patients with multiple clinical insemination failures, a novel chemical TPE-mTO probe engineered in our previous work was used on both samples from the mice sperm and from patients with fertilization failure. Expression of valosin-containing protein and the zona-free hamster egg assay were used to evaluate mitophagy and human sperm penetration. RNA-sequencing was used to explore expression changes of key genes affected by mtDNA G4s. Results showed that the probe can track mtDNA G4s in spermatozoa easily and quickly with fewer backgrounds. Significantly increased mtDNA G4s were also found in patients with fertilization failure, using the flow-cytometry-based TPE-mTO probe detection method. A sperm-hamster egg penetration experiment showed that abnormal fertilization caused by increased mtDNA G4s can be effectively restored by a mitophagy inducer. This study provides a novel method for monitoring etiological biomarkers in patients with clinical infertility and treatment for patients with abnormal fertilization caused by mtDNA G4 dysfunction.

019 Sperm Toll-like receptor 2 regulates sperm penetration to uterine glands thereby triggering uterine inflammatory cascade in cattle

019 Sperm Toll-like receptor 2 regulates sperm penetration to uterine glands thereby triggering uterine inflammatory cascade in cattle

Antifreeze protein type III addition to freezing extender comprehensively improves post-thaw sperm properties in Okinawan native Agu pig

Sperm cryopreservation often leads to physical cell damage through ice crystal formation. This study evaluates the improvements to freezing extender cryoprotective activity due to antifreeze protein (AFP) addition, which primarily acts on ice crystal formation, through investigating the post-thaw sperm properties of Okinawan native Agu pig. Six individual boar sperm samples were diluted with the freezing extender supplemented with 1 μg/mL of AFP I or AFP III and then subjected to cryopreservation. Treatment with AFP I during the freezing procedure had no improvement for any characteristics after thawing compared to untreated sperm. In contrast, the addition of AFP III to the freezing extender strongly increased sperm motility, mitochondria and cell membrane integrity, and the acrosomal proteolytic activity of frozen-thawed sperm in 5 of 6 individuals (P < 0.05). Furthermore, cryoinjury prevention by AFP III significantly enhanced sperm viability (by ATP content), and maintained DNA quality and in vitro sperm penetrability compared with AFP I treatment (P < 0.05). These findings demonstrate that AFP III addition to the freezing extender of boar sperm is more effective in maintaining sperm characteristics than the extender without AFP III or AFP I, despite individual differences in response.

ACROSIN deficiency causes total fertilization failure in humans by preventing the sperm from penetrating the zona pellucida.

Does a homozygous nonsense mutation in ACR lead to total fertilization failure (TFF) resulting in male infertility in humans? A novel homozygous nonsense mutation of ACR (c.167G>A, p.Trp56X) was identified in two infertile brothers and shown to cause human TFF. ACROSIN, encoded by ACR, is a major acrosomal enzyme expressed only in the acrosome of the sperm head. Inhibition of acrosin prevents sperm penetration of the zona pellucida (ZP) in several species, including humans. Acr-knockout in hamsters causes male infertility with completely blocked fertilization. Of note, there are no reports of ACR mutations associated with TFF in humans. Whole-exome sequencing (WES) was used for the identification of pathogenic genes for male factor TFF in eight involved couples. Data from eight infertile couples who had experienced TFF during their IVF or ICSI attempts were collected. Functional assays were used to verify the pathogenicity of the potential genetic factors identified by WES. Subzonal insemination (SUZI) and IVF assays were performed to determine the exact pathogenesis of TFF caused by deficiencies in ACROSIN. A novel homozygous nonsense mutation in ACR, c.167G>A, p.Trp56X, was identified in two additional primary infertile brothers whose parents were first cousins. This rare mutation caused ACROSIN deficiency and acrosomal ultrastructural defects in the affected sperm. Spermatozoa lacking ACROSIN were unable to penetrate the ZP, rather than hampering sperm binding, disrupting gamete fusion, or preventing oocyte activation. These findings were supported by the fertilization success of SUZI and ICSI attempts, as well as the normal expression of ACTL7A and PLCζ in the mutant sperm, suggesting that ICSI without remedial assisted oocyte activation is an optimal treatment for ARCOSIN-deficient TFF. The absence of another independent pedigree to support our argument is a limitation of this study. The findings expand our understanding of the genes involved in human TFF, providing information for appropriate genetic counseling and fertility guidance for these patients. This study was supported by the National Natural Science Foundation of China (grant no. 82201803, 81901541, 82271639, and 32000584), University Synergy Innovation Program of Anhui Province (GXXT-2019-044), and the Nonprofit Central Research Institute Fund of the Chinese Academy of Medical Sciences (grant no. 2019PT310002). The authors declare no conflicts of interest. N/A.

Viscoelastic Properties of Zona Pellucida of Oocytes Characterized by Transient Electrical Impedance Spectroscopy.

The success rate in vitro fertilization is significantly linked to the quality of the oocytes. The oocyte's membrane is encapsulated by a shell of gelatinous extracellular matrix, called zona pellucida, which undergoes dynamic changes throughout the reproduction cycle. During the window of highest fertility, the zona pellucida exhibits a softening phase, while it remains rigid during oocyte maturation and again after fertilization. These variations in mechanical properties facilitate or inhibit sperm penetration. Since successful fertilization considerably depends on the state of the zona pellucida, monitoring of the hardening process of the zona pellucida is vital. In this study, we scrutinized two distinct genetic mouse models, namely, fetuin-B wild-type and fetuin-B/ovastacin double deficient with normal and super-soft zona pellucida, respectively. We evaluated the hardening with the help of a microfluidic aspiration-assisted electrical impedance spectroscopy system. An oocyte was trapped by a microhole connected to a microfluidic channel by applying suction pressure. Transient electrical impedance spectra were taken by microelectrodes surrounding the microhole. The time-depending recovery of zona pellucida deflections to equilibrium was used to calculate the Young's modulus and, for the first time, absolute viscosity values. The values were obtained by fitting the curves with an equivalent mechanical circuit consisting of a network of dashpots and springs. The observer-independent electrical readout in combination with a fitting algorithm for the calculation of the viscoelastic properties demonstrates a step toward a more user-friendly and easy-to-use tool for the characterizing and better understanding of the rheological properties of oocytes.

Shilajit improves sperm penetration on in vitro fertilization of pig oocytes

Shilajit improves sperm penetration on in vitro fertilization of pig oocytes

The Effect of Acidic and Alkaline Seawater on the F-Actin-Dependent Ca2+ Signals Following Insemination of Immature Starfish Oocytes and Mature Eggs.

In starfish, the addition of the hormone 1-methyladenine (1-MA) to immature oocytes (germinal vesicle, GV-stage) arrested at the prophase of the first meiotic division induces meiosis resumption (maturation), which makes the mature eggs able to respond to the sperm with a normal fertilization response. The optimal fertilizability achieved during the maturation process results from the exquisite structural reorganization of the actin cytoskeleton in the cortex and cytoplasm induced by the maturing hormone. In this report, we have investigated the influence of acidic and alkaline seawater on the structure of the cortical F-actin network of immature oocytes of the starfish (Astropecten aranciacus) and its dynamic changes upon insemination. The results have shown that the altered seawater pH strongly affected the sperm-induced Ca2+ response and the polyspermy rate. When immature starfish oocytes were stimulated with 1-MA in acidic or alkaline seawater, the maturation process displayed a strong dependency on pH in terms of the dynamic structural changes of the cortical F-actin. The resulting alteration of the actin cytoskeleton, in turn, affected the pattern of Ca2+ signals at fertilization and sperm penetration.

Effects of dietary supplementation of tomato pomace (Solanum lycopersicum L.) and L-Arg on reproductive performance of aged male broiler breeders

This study aimed to investigate the effects of different dietary supplementation of tomato pomace (TP) and L-arginine (L-Arg) supplementation on sperm characteristics, reproductive performance, and semen biochemical components of aged commercial male broiler breeders. Thirty Ross 308 male broiler breeders (58 wk old) were provided and assigned to 5 dietary treatment groups, including control (CON), 5% TP (TPS-5), 10% TP (TPS-10), 15% TP (TPS-15), and L-Arg supplemented (10% above the recommendation, LAS-10). The results indicated that the semen volume increased in the TPS-15 group compared to that of the LAS-10 (and CON on wk 9) throughout the study (P < 0.05). The sperm concentration significantly increased in TPS-10 and TPS-15 groups in comparison to the other experimental groups. On wk 5 and 7, the sperm viability increased in all TPS groups compared to the CON and LAS-10, while on wk 9, it only increased in the TPS-10 group in comparison to the LAS-10 group (P < 0.05). The hypo-osmotic swelling test decreased in the LAS-10 group compared to the other experimental groups on wk 5 and all TPS groups on wk 7 and 9 (P < 0.05). The sperm total motility and forward progressive motility decreased in the LAS-10 group compared to the other experimental groups (P < 0.05). In contrast, unprogressive motility and immotile sperms were increased in the LAS-10 group compared to the other experimental groups (P < 0.05). In addition, the sperm penetration and fertility rate increased in TPS-10 and TPS-15 groups in comparison to CON and LAS-10 groups (P < 0.05). However, hatchability was reduced in the LAS-10 group (P < 0.05). The semen adenosine triphosphate increased in TPS-10, TPS-15, and LAS-10 groups compared to the CON (P < 0.05). Finally, the semen TAC and superoxidase dismutase decreased in the LAS-10 group (P < 0.05), while the glutathione peroxidase increased in the TPS-15 group (P < 0.05). In conclusion, 15% dietary TPS is recommended to improve the reproductive performance of aged commercial male broiler breeders.

Inactivation of mammalian spermatozoa on the exposure of TiO2 nanorods deposited with noble metals

Titanium dioxide (TiO2) nanorods (NRs) are well-known semiconducting and catalytic material that has been widely applied, but their toxicities have also attracted recent interest. In this study, we investigated and compared the toxic effects of TiO2 NRs and TiO2 NRs loaded with Ag or Au NPs on boar spermatozoa. As a result, sperm incubated with Ag-TiO2 NRs showed lower motility than sperm incubated with controls (with or without TiO2 NRs) or Au-TiO2 NRs. In addition, sperm viability and acrosomal integrity were defective in the presence of Ag-TiO2 NRs, and the generation of intracellular reactive oxygen species (ROS) increased significantly when spermatozoa were incubated with 20 μg/ml Ag-TiO2 NRs. We discussed in depth the charge transfer mechanism between enzymatic NADPH and Ag-TiO2 NRs in the context of ROS generation in spermatozoa. The effects we observed reflected the fertilization competence of sperm incubated with Ag-TiO2 NRs; specifically sperm penetration and embryonic development rates by in vitro fertilization were reduced by Ag-TiO2 NRs. To summarize, our findings indicate that exposure to Ag-TiO2 NRs could affect male fertilization fecundity and caution that care be exercised when using these NRs.

Egg Development After In Vitro Insemination in Japanese Quail (Coturnix japonica).

In vitro fertilization has been widely used to produce offspring in several mammalian species. We previously successfully produced Japanese quail chicks using intracytoplasmic sperm injection (ICSI), whereas in vitro insemination was not successful. This may be due to the difficulties associated with mimicking the sperm-egg fusion process and subsequent events in physiological polyspermic fertilization in vitro. In the present study, we observed egg development after in vitro insemination and investigated the inactivation of metaphase-promoting factor (MPF) and cytostatic factor (CSF), which are downstream of the Ca2+ signaling pathway in the egg, due to fertilizing sperm. We found a sperm number-dependent increase in hole formation caused by sperm penetration of the perivitelline membrane, the extracellular coat surrounding the egg. Egg development was observed following in vitro insemination; however, the developmental rate and stages after 24-h culture were inferior to those of ICSI eggs, even when insemination was performed with a high number of sperm (2 × 104). We also noted the downregulation of inositol 1,4,5-trisphosphate receptor-1, ryanodine receptor-3, cyclin B1, and c-MOS, which are important regulatory components of MPF and CSF in the egg, which was dependent on the number of sperm used for insemination. However, the decreases observed in these components did not reach the levels observed in the ICSI eggs. Collectively, the present results suggest that a sperm number higher than 2 × 104 is required for the progression of the Ca2+ signaling pathway, which initiates subsequent egg development in Japanese quail.

Observation of the in vitro fertilization process in living oocytes using frozen–thawed sperm in rats

Previous studies have observed the fertilization process in rats using whole-mount preparation at different time-points after insemination. However, very few reports have described the various events during the fertilization process using an inverted microscope without whole-mount. Moreover, to the best of our knowledge, no reports have described the observation of changes in sperm motility associated with sperm penetration into oocytes. In this study, in vitro fertilization was performed using frozen–thawed sperm in various rat strains (SD, Wistar, LE, F344, and BN) and oocytes from the SD strain, and the process of sperm penetration into the oocytes and the subsequent development were observed. The sperm motility was assessed, and the correlation between the process of sperm penetration into the oocytes and sperm motility over time was examined. The motility of frozen sperm from the SD, Wistar, LE, and F344 increased at 2–3 h after thawing, at which time the sperm attached themselves to the zona pellucida. Sperm penetration into the zona pellucida occurred after 3–5 h, and pronuclei were formed in the cytoplasm of oocytes 5–9 h after insemination. The fertilities of frozen–thawed sperm from the SD, Wistar, LE, and F344 were 92.7%, 90.0%, 90.7%, and 68.7%, respectively. However, no increase in motility was observed after thawing of frozen sperm from the BN, and the fertility was only 21%. In addition, very few polyspermic oocytes were observed with use of frozen–thawed sperm of all strains. In summary, rats are suitable animals for the observation of sperm penetration into the oocytes, and we determined the timing of fertilization events in IVF using frozen–thawed rat sperm.

Polymyxin B neutralizes endotoxin and improves the quality of chicken semen during liquid storage

Despite its importance in gamete utilization for livestock production, poultry semen cryopreservation in a liquid state, is limited in the poultry industry due to a significant decline in sperm viability and functionality during liquid storage. Lipopolysaccharide (LPS) is released from gram-negative bacteria and impairs sperm function in mammals. Using exogeneous LPS, we show this endotoxin compromises sperm viability and function, including motility and penetrability to the inner peri-vitelline layer (IPVL) during liquid storage at 4 °C. This outcome was supported by LPS quantification showing an extreme increase in the first 24 h of storage. Polymyxin B (PMB) is an LPS neutralizer previously shown to improve fertility in boar semen, thus we explored the effect of PMB on chicken semen quality during liquid storage. Sperm viability and penetrability tests showed that PMB completely abolishes the deleterious effect by LPS. However, co-addition of PMB with penicillin G (PenG), an antibiotic against gram positive bacteria, reduces IPVL-penetrability while improving sperm viability post-storage. Furthermore, artificial insemination trials showed that PMB addition improves semen fertility at the post liquid storage. Our results show that chicken semen quality during liquid storage is impaired by bacterial LPS, but improved by PMB addition due to cancelled endotoxic effects, which offers a new approach for prolonged fertility of poultry semen storage in a liquid state.

Topical reinforcement of the cervical mucus barrier to sperm.

Close to half of the world's pregnancies are still unplanned, reflecting a clear unmet need in contraception. Ideally, a contraceptive would provide the high efficacy of hormonal treatments, without systemic side effects. Here, we studied topical reinforcement of the cervical mucus by chitosan mucoadhesive polymers as a form of female contraceptive. Chitosans larger than 7 kDa effectively cross-linked human ovulatory cervical mucus to prevent sperm penetration in vitro. We then demonstrated in vivo using the ewe as a model that vaginal gels containing chitosan could stop ram sperm at the entrance of the cervical canal and prevent them from reaching the uterus, whereas the same gels without chitosan did not substantially limit sperm migration. Chitosan did not affect sperm motility in vitro or in vivo, suggesting reinforcement of the mucus physical barrier as the primary mechanism of action. The chitosan formulations did not damage or irritate the ewe vaginal epithelium, in contrast to nonoxynol-9 spermicide. The demonstration that cervical mucus can be reinforced topically to create an effective barrier to sperm may therefore form the technological basis for muco-cervical barrier contraceptives with the potential to become an alternative to hormonal contraceptives.

Scutellarein stimulates human sperm function by increasing the levels of intracellular calcium and tyrosine phosphorylation.

As a kind of flavonoid, scutellarein is widely used to protect against various human diseases. Although the protective effects of scutellarein have been well studied, its influence on human reproduction remains unknown. In this research, we evaluated the effect of scutellarein on human sperm functions in vitro. Three different concentrations of scutellarein (1, 10, 100 μM) were applied to ejaculated human sperm. Fertilisation-essential functions, as well as the intracellular calcium concentration ([Ca2+ ]i ) and protein-tyrosine phosphorylation, two factors which are vital for sperm function regulation, were evaluated. The results demonstrated that all concentrations of scutellarein utilised in this study could significantly increase sperm spontaneous capacitation and acrosome reaction through the enhancement of [Ca2+ ]i . Besides, the level of tyrosine phosphorylation of sperm could also be increased by scutellarein. Meanwhile, the sperm motility could be improved by 10 and 100 μM scutellarein, which also make a significant enhancement in sperm penetration ability and hyperactivation. This is one of the limited studies showing the regulation of scutellarein on human spermatozoa functions and is helpful to enrich its application.

UTILIZATION OF INTRACYTOPLASMIC SPERM INJECTION VERSUS CONVENTIONAL IVF IN NON-MALE INFERTILITY PATIENT POPULATION: AGE BASED ANALYSIS

The primary indication for intracytoplasmic sperm injection (ICSI) is for treatment of couples with male factor infertility. However, ICSI has been proposed as a tool to overcome difficulties in sperm-oocyte interaction secondary to oocyte age and not necessarily sperm abnormalities. Our hypothesis is that ICSI might be helpful in assisting sperm penetration in oocytes from older women. Therefore, the objective of this study was to investigate if there is any advantage of performing ICSI over conventional insemination in different age groups.

Human sperm TMEM95 binds eggs and facilitates membrane fusion

Tmem95 encodes a sperm acrosomal membrane protein, whose knockout has a male-specific sterility phenotype in mice. Tmem95 knockout murine sperm can bind to, but do not fuse with, eggs. How TMEM95 plays a role in membrane fusion of sperm and eggs has remained elusive. Here, we utilize a sperm penetration assay as a model system to investigate the function of human TMEM95. We show that human TMEM95 binds to hamster egg membranes, providing evidence for a TMEM95 receptor on eggs. Using X-ray crystallography, we reveal an evolutionarily conserved, positively charged region of TMEM95 as a putative receptor-binding surface. Amino acid substitutions within this region of TMEM95 ablate egg-binding activity. We identify monoclonal antibodies against TMEM95 that reduce the number of human sperm fused with hamster eggs in sperm penetration assays. Strikingly, these antibodies do not block binding of sperm to eggs. Taken together, these results provide strong evidence for a specific, receptor-mediated interaction of sperm TMEM95 with eggs and suggest that this interaction may have a role in facilitating membrane fusion during fertilization.

Symposium 1-4 - Immunological infertility caused by gamete immunity

1. Infertility caused by anti-sperm antibodies (ASA) 1) Female infertility caused by sperm-immobilizing antibodies in the sera In some women, ASA are produced as allogenic immunity, which causes infertility. Various methods for the antibody detection have been developed, but sperm-immobilization antibodies (SI-Ab) detected by the sperm immobilization test (SIT) have the greatest influence on the development of infertility. Sperm-immobilizing antibodies are present in the female genital tract, and also in the follicle fluid.SI-Ab cause a post-coital test (PCT) negative or impaired sperm transport from the uterine cavity to the Fallopian tubes, and fertilization disorders. There is a method to quantitatively measure SI-Ab titer, SI50 unit, and treatment methods are decided based on this SI 50 value. In cases with a SI50 value of 10 or more, it is difficult to conceive with AIH, and IVF is effective after preventing antibody contamination into the culture medium. As a mechanism for producing sperm-immobilizing antibodies, it has been speculated that women with disease-specific genes may be exposed to enough amount of sperm through sexual intercourse and develop symptoms triggered by such as chlamydia infection. CD52 is considered to be a promising antigen against sperm-immobilization antibodies, and is expected to be applied for the development of contraceptive vaccines. 2) Male infertility caused by ASA that bind to ejaculated sperm In some men, ASA are produced as autoantibodies, causing infertility. In men, it is not useful for measuring serum antibodies, therefore antibodies that bind to ejaculated sperm (sperm-binding antibodies) should be detected with ImmunoSpheres○R. However, since there are antibodies that are not related to the development of infertility, the treatment strategy can be determined by confirming whether the antibody has a biological effect. Inhibitory effect on cervical sperm penetration is confirmed by PCT and fertilization disorder is examined by the hemizona assay (HZA). If HZA cannot be performed, ICSI is recommended because it is likely to be accompanied by fertilization disorder in cases where the ratio of ASA-bound sperm to total motile sperm is 80% or more. 2. Female infertility caused by anti-zona pellucida (ZP) antibodies The ZP plays an important role in terms of egg formation, fertilization, protection of the embryo. These include the recognition of sperm species of animals, the induction of acrosome reaction, and the prevention of polysperm fertilization. On the other hand, since there is a strong antibody production ability as an antigen, if an anti-ZP antibodies are produced, the function of the ZP is impaired by the antibody, and there is a possibility of developing infertility. It was reported that anti-ZP antibodies were detected at a high rate in the sera of infertile women of unknown cause by the anti-ZP antibody detection method using porcine ZP, followed by controversial reports by another investigators. Therefore, we developed a detection method using human ZP. Anti-ZP antibodies disturb the follicular development, inhibit sperm-ZP binding and hatching. For the purpose of appropriate treatments, it was shown that ICSI is effective for fertilization disorder and assisted hatching (AHA) is effective for hatching disorder, respectively.

Evaluation of sperm fertilization capacity of large Japanese field mice (Apodemus speciosus) exposed to chronic low dose-rate radiation after the Fukushima accident

Due to the risk of bias in early developmental processes of radio-sensitive germ cells, the effects of radiation on reproductive ability remain unclear. In this study, wild large Japanese field mice (Apodemus speciosus) within the ex-evacuation zone of the Fukushima Daiichi Nuclear Power Plant (FDNPP) exposed to chronic low dose-rate (LDR) radiation since 2011 were examined. We analyzed the in vitro fertilization (IVF) capacity of cryopreserved A. speciosus sperm from 2012 to 2021 using interspecific zona pellucida (ZP)-free and homogenous wild mice oocytes. The radioactivity concentrations of 134Cs and 137Cs were 3,460–37,630 Bq/kg in the Fukushima wild mice. Frozen-thawed sperm showed generally normal acrosomal and nuclear morphology. No significant differences in sperm penetration rate to that of ZP-free mouse oocytes were observed between the control and Fukushima sperm. When frozen-thawed A. speciosus sperm was used for IVF, 44%–90% homogenous wild mice oocytes fertilized and developed a 2-cell embryo. This study was the first to reveal that long-term chronic LDR radiation exposure (0.18–0.59 mGy/d) associated with the FDNPP accident showed no detrimental effect on the IVF capacity of cryopreserved sperm in wild A. speciosus. This study also provides an alternative framework to estimate radiation effects on wild mice reproduction.

A REVIEW ON ADVANCES IN THE DEVELOPMENT OF SPERMICIDES LOADED VAGINAL DRUG DELIVERY SYSTEM: STATE OF THE ART

Spermicides are bio-actives that might immobilize/kill the sperm in the vaginal tissues inducing contraception in the female genitalia. To inhibit sperm viability, the spermicidal drug might have to inhibit the penetration of sperm through the cervical tube of the uterus as well as attack sperm present in the vaginal walls. There are several classes of spermicidal agents, such as bactericides, sulfhydryl binding agents, natural compounds, and synthetic products. There are several classes of spermicidal agents that are widely reported. Spermicides could be available in different dosage forms as foams, gels, creams, films, sponges, and nanofibers. Available pharmaceutical spermicides showed particular importance for production on a large scale due to the continuous need for contraception. The upscaling of a process can be performed using a quality by design approach to ensure the achievement of the similarity principle between lab-scale and industrial scale. In addition, risk evaluation is performed to recognize all high-risk factors that can negatively affect the product to build the design space. Furthermore, the knowledge of the critical quality attributes enables the selection of the appropriate settings on a larger scale to establish a product of good quality and good packaging.