OBJECTIVE: Since the unexplained in vitro fertilization failure occurs frequently, it is of great importance and clinical value to identify potential underlying predictors. This study aimed to explore whether the percentage of sperm with a small acrosome was correlated with unexplained in vitro fertilization failure.
METHODS: A new acrosomal function evaluation index (the percentage of sperm with a small acrosome) was introduced into the analysis of sperm morphology. The association between the index and acrosome function by acrosin activity detection test and acrosome reaction test was investigated. In addition, the correlation with unexplained in vitro fertilization failure was further explored. Finally, the ROC curve was used to analyze the diagnostic efficacy on the failure of in vitro fertilization and the cutoff value was calculated.
RESULTS: As the increasing of the percentage of sperm with a small acrosome, the value of acrosin activity, acrosome reaction rate, and in vitro fertilization rate were reduced, with a statistically significant difference (P < 0.05). The index in the low fertilization rate group was significantly higher than that in the normal fertilization rate group (P < 0.05). Finally, the results of ROC curve found that when the index was 43.5%, the sensitivity and specificity were 74.2% and 95.3%, respectively.
CONCLUSIONS: The percentage of sperm with a small acrosome was positively correlated with unexplained in vitro fertilization failure, which could be potentially used as a prognostic index for the failure of in vitro fertilization.
BACKGROUND: [Ethics review acceptance No IIT20210339B].

Liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to the poultry industry. However, fertility rates from liquid-stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality remains unidentified. Therefore, the purpose of the present study was to monitor the dynamics of proteomic changes in spermatozoa during 48 h of liquid storage by 2-dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. A total of 57 protein spots were differentially expressed between fresh and stored spermatozoa; 42 spots were more and 15 were less abundant after 48 h of semen storage. Raw proteomic data are available via ProteomeXchange with identifier PXD043050. The selected differentially expressed proteins (DEPs) were validated by western blotting and localized in specific spermatozoa structures by immunofluorescence, such as the head (acrosin and tubulin α), midpiece (acrosin, aconitate hydratase 2, and glycerol-3-phosphate dehydrogenase) and tail (tubulin α). Most of the DEPs that changed in response to liquid storage were related to flagellum-dependent cell motility, energy derivation through oxidation of organic compounds and induction of fertilization, suggesting the complexity of the processes leading to the decrease in stored semen quality. The damaging effect of liquid storage on spermatozoa flagellum manifested as more microtubule proteins, such as tubulins and tektins, most likely formed by posttranslational modifications, tubulin α relocation from the tail to the sperm head, which appeared after 48 h of semen storage, and decreases in fibrous shelf proteins at the same time. Motility could be affected by dysregulation of Ca2+-binding proteins and disturbances in energy metabolism in spermatozoa flagellum. Regarding sperm mitochondria, DEPs involved in energy derivation through the oxidation of organic compounds indicated disturbances in fatty acid beta oxidation and the tricarboxylic acid cycle as possible reasons for energy deficiency during liquid storage. Disturbances in acrosin and 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase zeta may be involved in rapid declines in the fertility potential of stored turkey spermatozoa. These results showed the complexity of the processes leading to a decrease in stored semen quality and broadened knowledge of the detrimental effects of liquid storage on turkey spermatozoa physiology.

OBJECTIVE: To investigate the effect of pricking-reinforcing -reducing therapy (PRRT) on the semen quality and seminal plasma biochemical indexes of varicocele (VC) infertility patients.
METHODS: We randomly and equally assigned 160 patients with VC infertility into a PRRT and a control group, the former treated by PRRT and the latter with oral ShengjingCapsules. Before and after treatment, we obtained the semen parameters, sperm morphology, sperm survival rate, sperm acrosin activity, seminal plasma neutral α glucosidase and seminal plasma zinc in the patients and compared them between the two groups.
RESULTS: Before treatment, there were no statistically significant differences between the PRRT and control groups in sperm concentration (［16.81 ± 7.83］ vs ［16.80 ± 7.54］ ×106 /ml, P > 0.05), total sperm count (［42.01 ± 19.57］ vs ［41.99 ± 18.84］ ×106, P > 0.05), percentages of progressively motile sperm (PMS) (［15.37 ± 11.03］% vs ［14.68 ± 10.27］%, P > 0.05) and morphologically normal sperm ( MNS) (1.62 ± 1.51］% vs ［1.62 ± 1.13］%, P > 0.05), sperm survival rate (［28.11 ± 18.95］% vs ［28.23±18.38］%, P > 0.05) and sperm acrosin activity (［28.11 ± 14.64］ vs ［27.19 ± 14.07］ U/L, P > 0.05). After three months of treatment, all the patients showed evident increases in the above parameters (P < 0.05), even higher in the PRRT than in the control group, more significantly in sperm concentration (［38.88 ± 30.54］ vs ［25.60 ± 14.71］ ×106 /ml, P < 0.05), PMS (［32.60 ± 12.46］% vs ［27.67 ± 12.27］%, P < 0.05) and sperm acrosin activity (［65.74±31.81］ vs ［67.94±17.95］ U/L, P < 0.05), though not significantly in total sperm count (97.20 ± 76.35］ vs ［88.19 ± 39.56］ ×106, P > 0.05), MNS (［2.35 ± 1.83］% vs ［1.87 ± 1.20］%, P > 0.05) and sperm survival rate (［61.44 ± 20.02］% vs ［59.12 ± 22.48］%, P > 0.05). Compared with the baseline, after treatment, the patients in the PRRT group also exhibited elevated levels of neutral α-glucosidase (［14.42 ± 5.90］ vs ［28.43 ± 19.76］ U/L, P < 0.05) and seminal plasma zinc (［2.11 ± 1.22］ vs ［2.89 ± 1.23］ mmol/L, P < 0.05), and so did the controls (［14.44 ± 5.61］ vs ［26.66 ± 17.69］ U/L , P < 0.05) and (［2.09 ± 1.10］ vs ［2.82±1.08］ mmol/L, P < 0.05). No statistically significant difference, however, was observed between the two groups after treatment (P > 0.05).
CONCLUSIONS: PRRT can significantly improve semen quality in patients with VC infertility, even more effective than ShengjingCapsules in improving sperm concentration, PMS, sperm survival rate, and sperm acrosin activity, which may be related to its effect of elevating the levels of seminal plasma neutral-α glucosidase and zinc providing sufficient energy for basic sperm metabolism, maturation, energy acquisition and motility.

Low acrosin activity (LAA) is associated with sperm function anomaly and poor outcomes of in vitro fertilization. In this study, we confirm that 993 semen samples with LAA had a reduced sperm motility and low in vitro fertilization rate in comparison with 1332 normal controls (NC). Proteomic comparison between 11 LAA and 11 NC sperm samples identified 35 upregulated and 99 downregulated proteins in the LAA group. Indeed, proteomic data showed that acrosome enzymes Spam1 and Acrosin were among the downregulated proteins in the LAA group, which was validated by quantitative PCR and immunefluorescent staining of sperm cells. The KEEG pathway analysis revealed a deficiency of GSH and Gln biosynthesis in LAA sperm cells. Immunofluorescent staining of sperms and quantitative PCR verified downregulation of GLUL and GCLC, the key enzymes for GSH and Gln biosynthesis. Moreover, the results of ELISA assay confirmed low levels of GSH and Gln in LAA sperm cells. Mechanistic studies showed that addition of 10 mM H2O2 to semen samples led to a significant reduction of acrosin activity and sperm motility, most possibly by triggering premature acrosome release. In contrast, the presence of 20 mM GSH blocked the oxidative effects of H2O2. Since GSH counteracts the oxidative stress and Gln participates in TCA cycling, their deficiency may affect the redox balance as well as energy production of sperm cells. These findings shed new light on the pathological mechanisms of infertility associated with LAA. Male infertility patients could benefit from GSH supplement by improvement of acrosin activity and other sperm functions.

The clinical applications of acrosin activity are limited. We analyzed 61 578 male partners in infertile couples who visited the outpatient department of the Reproductive and Genetic Hospital of CITIC-Xiangya (Changsha, China) between August 2014 and December 2019 to determine the reference ranges and thresholds for acrosin activity in infertile Chinese men; to determine whether correlations exist between acrosin activity and age, sperm concentration, sperm morphology, or sperm motility; and to evaluate whether acrosin activity could serve as an effective prognostic indicator for choosing between in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in the clinic. The cut-off value for the normal reference range of acrosin activity for male partners in infertile couples was 24.78 µIU per 10 6 sperm. There was no significant association between acrosin activity and age, sperm concentration, semen volume, total sperm count, progressive motility, or total motile spermatozoa. A weak positive correlation was found between acrosin activity and normal sperm morphology. There was a statistically significant difference in abnormal acrosome morphology between the group with high acrosin activity (>24.78 µIU per 10 6 sperm) and the group with low acrosin activity (<24.78 µIU per 10 6 sperm). The group with a low IVF fertilization rate had a high index of abnormal acrosomal morphology at 21.2%, while the group with a high IVF fertilization rate had a low index of 0.2%. At an acrosin activity of <24.78 µIU per 10 6 sperm, in one cycle of the same patient, the fertilization rate, normal fertilization rate, and good-quality embryo rate for ICSI were significantly higher than those for IVF. Therefore, the most promising application of acrosin activity could be in the selection of ICSI over IVF for infertile male patients with complete fertilization failure or a low fertilization rate.

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.
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Previously, we reported that cysteine-rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine-rich secretory protein 2protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning.
This study aimed to identify cysteine-rich secretory protein 2 interacting partners. These binding partner interactions were investigated under different conditions, namely, non-capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187-induced acrosome reaction.
The incubated pig sperm samples were subjected to protein extraction. Extracted proteins were subjected to blue native gel electrophoresis and native immunoblots. Immunoreactive gel bands were excised and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC-MS for protein identification. The most prominent cystein-rich secretory protein 2 interacting proteins that appeared in both independent LC-MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with cystein-rich secretory protein 2 in pig sperm.
Blue native gel electrophoresis and native immunoblots revealed that cystein-rich secretory protein 2 was present within a ∼150 kDa protein complex under all three conditions. Interrogation of cystein-rich secretory-protein 2-immunoreactive bands from blue native gels as well as cystein-rich secretory protein 2 immunoprecipitated products using mass spectrometry consistently revealed that, beyond cystein-rich secretory protein 2, acrosin and acrosin binding protein were among the most abundant interacting proteins and did interact under all three conditions. Co-immunoprecipitation and immunoblotting indicated that cystein-rich secretory protein 2 interacted with pro-acrosin (∼53 kDa) and Aacrosin binding protein under all three conditions and additionally to acrosin (∼35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein-rich secretory protein 2 was assessed via in situ proximity ligation assays. The colocalization signal of cystein-rich secretory protein 2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of cystein-rich secretory protein 2 and acrsin binding protein colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post-acrosomal sheath region upon capacitation.
These results suggest that CRISP2 may act as a scaffold for protein complex formation and dissociation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.

The golden (Syrian) hamster (Mesocricetus auratus) is a small rodent belonging to the Cricetidae family. Golden hamsters have several unique characteristics that are advantageous in the study of reproductive and developmental biology: a highly stable 4-day estrous cycle, a high responsiveness to conventional superovulation methods, and a shortest gestation period (16 days) known among eutherian mammals. Besides these advantages, the technical ease of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in this species has contributed much to our understanding of the basic mechanisms of mammalian fertilization. However, the exceptionally strong in vitro developmental block of hamster embryos, especially at the two-cell stage, has hampered the production of genetically modified hamsters, which has resulted in limited use of this species for biomedical research. However, the recently developed in vivo genome editing method (improved genome editing via oviductal nucleic acid delivery, i-GONAD) has overcome this shortcoming and made production of gene-edited hamsters much easier than before. This method has the potential to provide a means of reexamining genes whose functions cannot be identified using mouse models, thus leading to the better understanding of gene functions in mammals. In this chapter, we present our procedure for editing the genome of the golden hamster using i-GONAD.

To obtain de novo male gametes capable of inducing full preimplantation blastocyst development using a novel three-dimensional (3D) culture system.
Mouse embryonic stem cells (mESCs) were spherified by plunging in sodium alginate followed by calcium chloride, delineating a 3D environment that simulates the seminiferous tubule. As a control, mESCs cultured on two-dimensional plates were used. Plates and spheres containing mESCs from both methods were exposed to Activin-A, bFGF, and KSR followed by exposure to BMP4, LIF, SCF, and EGF to promote differentiation into male germ-like cells.
Cells were assessed for VASA, DAZL, and BOULE on days 3 and 10. Cells were later injected into activated oocytes and monitored using time-lapse imaging on days 15, 22, 29, and 36. Control conceptuses generated using mature epididymal spermatozoa were also monitored via time-lapse imaging.
On day 3, cells differentiated on plates expressed VASA at 1% and DAZL at 29%. In spheres, VASA was expressed at a rate of 15% and DAZL at a rate of 45% (P<.001). On day 10, cells differentiated on plates had VASA expression of 7%, DAZL of 23%, and BOULE of only 0.5%. Cells differentiated into spheres expressed VASA at a rate of 20%, DAZL at 43%, and BOULE at 10% (P<.001). Subsequent differentiation in spheres on day 3 exhibited a DAZL (expressed in spermatogonia) expression of 43% and a VASA (further spermatogenesis progression) expression of 15%. On day 10, DAZL and VASA expressions were reassessed and increased to 45% and 18%, respectively. BOULE, a marker expressed solely in postmeiotic spermatocytes, was expressed at 8%, whereas acrosin was expressed in spermatids at 2%. On day 15, VASA expression plateaued at 17%, BOULE peaked at 10%, and acrosin reached 5%. On day 22, expression of VASA increased to 19%, BOULE decreased to 8%, and acrosin peaked at 7%. On day 29, VASA expression peaked at 20%, BOULE dropped to 2%, and acrosin remained stable at 7%. On day 36, VASA expression remained at 13%, whereas BOULE and acrosin expression decreased to 0% and 1%, respectively. The control cohort attained 88.4% fertilization and 76.9% blastocyst rates. De novo gametes achieved fertilization rates of 35.0%, 61.1%, 81.8%, and 50.0% on days 15, 22, 29, and 36, respectively. Neogametes-generated blastocyst rates were 5.0%, 16.7%, 36.4%, and 8.3% for days 15, 22, 29, and 36, respectively.
Our novel 3D differentiation model can generate functional gametes and is aimed at obviating the need for allogeneic/xenogeneic transplantation. The decreased overall marker expression and the reduced blastocyst development indicated that intrasphere germ cell differentiation correlated with the length of mouse spermatogenesis at approximately 30 days. Future experiments will be conducted to confirm the reproducibility of our findings and the eventual generation of offspring.

BACKGROUND: Xenotransplantation has been primarily performed using fresh donor tissue to study testicular development for about 20 years, and whether the cultured tissue would be a suitable donor is unclear. In this study, we combined testicular culture and xenotransplantation into an integrative model and explored whether immature testicular tissue would survive and continue to develop in this model.
METHODS: In the new integrative model group, the testes of neonatal rats on postnatal day 8 (PND 8) were cultured for 4 days ex vivo and then were transplanted under the dorsal skin of castrated nude mice. The xenografted testes were resected on the 57th day after xenotransplantation and the testes of rats in the control group were harvested on PND 69. The survival state of testicular tissue was evaluated from morphological and functional perspectives including H&E staining, immunohistochemical staining of 8-OH-dG, immunofluorescence staining, TUNEL assay, ultrastructural study, gene expression and protein analysis.
RESULTS: (a) We found that complete spermatogenesis was established in the testes in the new integrative model group. Compared with the control in the same stage, the seminiferous epithelium in some tubules was a bit thinner and there were vacuoles in part of the tubules. Immunofluorescence staining revealed some ACROSIN-positive spermatids were present in seminiferous tubule of xenografted testes. TUNEL detection showed apoptotic cells and most of them were germ cells in the new integrative model group. 8-OH-dG immunohistochemistry showed strongly positive-stained in the seminiferous epithelium after xenotransplantation in comparison with the control group; (b) Compared with the control group, the expressions of FOXA3, DAZL, GFRα1, BOLL, SYCP3, CDC25A, LDHC, CREM and MKI67 in the new integrative model group were significantly elevated (P < 0.05), indicating that the testicular tissue was in an active differentiated and proliferative state; (c) Antioxidant gene detection showed that the expression of Nrf2, Keap1, NQO1 and SOD1 in the new integrative model group was significantly higher than those in the control group (P < 0.05), and DNA methyltransferase gene detection showed that the expression of DNMT3B was significantly elevated as well (P < 0.05).
CONCLUSIONS: The new integrative model could maintain the viability of immature testicular tissue and sustain the long-term survival in vivo with complete spermatogenesis. However, testicular genes expression was altered, vacuolation and thin seminiferous epithelium were still apparent in this model, manifesting that oxidative damage may contribute to the testicular development lesion and it needs further study in order to optimize this model.