Perinuclear Theca Research Articles

P-067 A physiological approach for oocyte activation when using testicular spermatozoa

Abstract Study question Can poor fertilization with testicular spermatozoa be rescued by oocyte activation? We propose a more physiological approach to the standard method. Summary answer Couples who utilized testicular spermatozoa benefitted from assisted oocyte activation (AOA), particularly when a more physiological approach such as recombinant human phospholipase-C-zeta (rhPLCζ) was utilized. What is known already ICSI overcomes sperm acrosomal dysfunction and other inherent defects of the male gamete. However, partial or complete fertilization failure may still occur, often attributable to the absence of PLCζ. These cases are often treated by calcium ionophore, a very popular method that is considered quite safe, however it still raises concerns because it induces intracellular calcium oscillations in a very aspecific manner. Testicular spermatozoa are particularly at risk of suboptimal fertilization; therefore, the availability of a more physiological method would be welcome. Study design, size, duration Over a period of 12 months, 21 couples utilizing testicular spermatozoa for ICSI, that resulted in poor fertilization, were identified and treated in subsequent ICSI cycles with assisted oocyte activation (AOA), either by exposure to ionomycin or co-injection of rhPLCζ at the time of ICSI. Embryology and clinical outcomes were recorded and compared between the two AOA methods as well as the couples’ historical and subsequent cycles. Participants/materials, setting, methods Couples with a history of poor/absent fertilization after undergoing ICSI using testicular spermatozoa were counseled for AOA in subsequent cycles (IRB 0712009553). For consenting couples, AOA was performed either by exposing post-ICSI oocytes to 50mM of ionomycin (Ionomycin-AOA), or by co-injecting spermatozoa with 0.4pL of rhPLCζ (5mg/mL) during ICSI (rhPLCζ-AOA). Fertilization, embryo development, and clinical outcomes were compared between the couples’ historical and AOA cycles. Main results and the role of chance We included 21 couples (maternal age, 35.1±4 yrs; paternal age, 36.8±7 yrs). Female partners all had negative infertility workups that consisted of a comprehensive review of their medical history as well as a targeted physical examination and tests assessing ovarian reserve, ovulatory function, and absence of uterine and tubule structural abnormalities. The couples underwent 53 historical ICSI cycles using poor testicular spermatozoa (concentration, 0.04±0.1x106/ml, 0.5±1% motility) that yielded a 22.5% (94/417) fertilization and only an 11.5% (3/26) clinical pregnancy rate. All couples subsequently underwent ICSI-AOA cycles. Fourteen couples (maternal age, 35.5±4 yrs; paternal age, 37.6±6 yrs) who were treated with Ionomycin-AOA obtained a higher fertilization of 44.4% (99/223) (P<0.00001), which resulted in a clinical pregnancy rate of 26.3% (5/19). Thus far, four couples have delivered 1 boy and 3 girls, with no major or minor congenital malformations. The remaining 7 couples (maternal age, 35.8±4 yrs; paternal age, 37.9±7 yrs) underwent subsequent ICSI cycles with rhPLCζ-AOA, which yielded a remarkably higher fertilization of 50.0% (50/134) (P<0.00001). These couples went on to achieve a clinical pregnancy rate of 27.3% (3/11), resulting in the delivery of 1 girl and 2 boys. Initial follow-up has confirmed normal neonatal development. Limitations, reasons for caution The effectiveness of AOA in ICSI cycles with testicular spermatozoa is very encouraging, particularly when a more physiological method is utilized. Nonetheless, these findings need to be validated in a larger cohort, confirming successful outcome and children health. Wider implications of the findings ICSI utilizing surgically retrieved spermatozoa, particularly testicular, are plagued by suboptimal fertilization, and using AOA may represent a way to normalize the generation of conceptuses. Moreover, a broader adoption of AOA would certainly benefit from the use of a recombinant cytosolic factor normally present in the sperm perinuclear theca. Trial registration number n/a

P-225 Implementing a more physiological method to trigger oocyte activation in cases of extremely poor fertilization with ICSI

Abstract Study question Can we utilize a more physiological compound to induce oocyte activation in couples with history of complete/partial fertilization failure with ICSI? Summary answer Recombinant human phospholipase-C-zeta (rhPLCζ) successfully activated oocytes with same efficiency as ionomycin, consistently normalized fertilization and enhanced clinical outcomes. What is known already While ICSI overcomes most aspects of male gamete dysfunction, partial or complete fertilization failure may seldomly occur mostly due absence of sperm-specific protein, known as PLCζ, which is required to trigger oocyte activation. Assisted oocyte activation (AOA), most often performed with an antibiotic (ionomycin), activates oocytes by triggering calcium release from intracellular storage. This agent induces indiscriminate permeabilization of ooplasmic membrane-bound organelles. This action has been considered nonspecific and may have possible deleterious effects on subsequent embryo development. Therefore, we propose a physiological alternative that employs a recombinant form of PLCζ, the native protein residing in the sperm perinuclear theca. Study design, size, duration In the past 12 months, couples with poor ICSI fertilization were included. PLCζ were assessed to confirm sperm-related oocyte activation deficiency with a normal threshold of ≥ 30%. In a preliminary comparison, sibling oocytes were equally allocated to AOA by ionomycin or to rhPLCζ at time of ICSI. Fertilization, embryo development, and clinical outcomes between the two cohorts. Subsequently, we describe the clinical outcomes of couples treated solely by rhPLCζ. Participants/materials, setting, methods Thirty couples, confirmed with low level of PLCζ in the ejaculates, were offered AOA with their subsequent ICSI cycles (IRB#0712009553). Conventional AOA was performed by exposing post-ICSI oocytes to 50 µM ionomycin. The rhPLCζ-AOA method was performed by co-injecting spermatozoa with 0.4pL of a rhPLCζ (5 µg/µL) during ICSI. Chi-square tests were utilized to compare rhPLCζ-AOA versus ionomycin-AOA and rhPLCζ-AOA versus history cycles. Main results and the role of chance In the preliminary study, 9 patients were included (maternal age: 35.7 ± 6, paternal age: 36.7 ± 6). PLCζ positivity was 11.7±3.6% confirming a sperm-related oocyte activation deficiency. A total of 123 oocytes were retrieved in 9 cycles, with maturity of 78.0% (96/123). The 96 MII oocytes were allocated to be treated with ionomycin-AOA (n = 58) or rhPLCζ-AOA (n = 38). While conventional ionomycin-AOA yielded a fertilization rate at 55.2% (32/58), rhPLCζ-AOA resulted in 63.1% (24/38). Both compounds generated embryos at similar rates and one pregnancy was achieve in each cohort. On the bases of such comparable outcomes, 21 couples had 40 previous cycles that generated a fertilization rate of 18.3% (60/327), cleavage rate of 51.7% (31/60), Following the replacement of 23 embryos in 14 transfer cycles, these cycles yielded 2 clinical pregnancies, but all resulted in pregnancy loss. In the subsequent cycles with rhPLCζ-AOA (n = 32), an enhanced fertilization rate was achieved at 41.1% (116/280, P<0.00001). The treatment cycle yielded a higher cleavage rate at 80.2% (93/116, P<0.0001), and after replacing 25 embryos in 17 embryo transfer cycles, yielded 6 pregnancies and 5 of them resulted in delivered or are ongoing. Limitations, reasons for caution We demonstrated that physiological rhPLCζ-AOA achieved an excelled fertilization and clinical outcomes comparing to the traditional ionomycin method. Although further observation is needed to confirm its safety, this approach represents a more physiological method capable of achieving fertilization, higher embryo cleavage and ultimately better pregnancy outcomes. Wider implications of the findings While ICSI has gained large popularity, poor fertilization may occasionally occur. The availability of an assisted oocyte activation that utilize a native compound capable of restoring fertilization capability of the male gamete is appealing and holds great promises. Trial registration number N/A

Disruption in CYLC1 leads to acrosome detachment, sperm head deformity, and male in/subfertility in humans and mice

The perinuclear theca (PT) is a dense cytoplasmic web encapsulating the sperm nucleus. The physiological roles of PT in sperm biology and the clinical relevance of variants of PT proteins to male infertility are still largely unknown. We reveal that cylicin-1, a major constituent of the PT, is vital for male fertility in both mice and humans. Loss of cylicin-1 in mice leads to a high incidence of malformed sperm heads with acrosome detachment from the nucleus. Cylicin-1 interacts with itself, several other PT proteins, the inner acrosomal membrane (IAM) protein SPACA1, and the nuclear envelope (NE) protein FAM209 to form an ‘IAM–cylicins–NE’ sandwich structure, anchoring the acrosome to the nucleus. WES (whole exome sequencing) of more than 500 Chinese infertile men with sperm head deformities was performed and a CYLC1 variant was identified in 19 patients. Cylc1-mutant mice carrying this variant also exhibited sperm acrosome/head deformities and reduced fertility, indicating that this CYLC1 variant most likely affects human male reproduction. Furthermore, the outcomes of assisted reproduction were reported for patients harbouring the CYLC1 variant. Our findings demonstrate a critical role of cylicin-1 in the sperm acrosome–nucleus connection and suggest CYLC1 variants as potential risk factors for human male fertility.

Disruption in CYLC1 leads to acrosome detachment, sperm head deformity, and male in/subfertility in humans and mice

The perinuclear theca (PT) is a dense cytoplasmic web encapsulating the sperm nucleus. The physiological roles of PT in sperm biology and the clinical relevance of variants of PT proteins to male infertility are still largely unknown. We reveal that cylicin-1, a major constituent of the PT, is vital for male fertility in both mice and humans. Loss of cylicin-1 in mice leads to a high incidence of malformed sperm heads with acrosome detachment from the nucleus. Cylicin-1 interacts with itself, several other PT proteins, the inner acrosomal membrane (IAM) protein SPACA1, and the nuclear envelope (NE) protein FAM209 to form an ‘IAM–cylicins–NE’ sandwich structure, anchoring the acrosome to the nucleus. WES (whole exome sequencing) of more than 500 Chinese infertile men with sperm head deformities was performed and a CYLC1 variant was identified in 19 patients. Cylc1-mutant mice carrying this variant also exhibited sperm acrosome/head deformities and reduced fertility, indicating that this CYLC1 variant most likely affects human male reproduction. Furthermore, the outcomes of assisted reproduction were reported for patients harbouring the CYLC1 variant. Our findings demonstrate a critical role of cylicin-1 in the sperm acrosome–nucleus connection and suggest CYLC1 variants as potential risk factors for human male fertility.

Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human.

Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

Cryopreservation, in addition to protein tyrosine phosphorylation, alters the distribution of phosphatidyl inositol bisphosphate and the localization of cytoskeletal and signaling proteins (gelsolin, tyrosine kinase c-SRC and phospholipase C-ζ) in the perinuclear theca of boar sperm

Cryopreservation of boar spermatozoa affects the perinuclear theca (PT) and involves several proteins and molecules that play important roles during capacitation and the acrosomal reaction. The objective of the present study was to evaluate whether the deleterious effects of cryopreservation in addition to protein tyrosine phosphorylation are accompanied by changes in the distribution of phosphatidyl inositol bisphosphate (PIP2) and the localization of cytoskeletal and signaling proteins in the perinuclear theca of cryopreserved boar spermatozoa. For this purpose, by immunocytochemistry (IC) the changes in localization of phosphorylated proteins in tyrosine residues, gelsolin, c-SRC kinase and PLC-ζ, as well as in the distribution of phosphatidyl inositol bisphosphate were analyzed in thawed spermatozoa (T) non capacitated (NC), capacitated (C) and in those with acrosomal reaction (AR) and compared with fresh spermatozoa (F) under the same physiological status. Western blotting (WB) and co-immunoprecipitation were performed to confirm the presence of these proteins in PT and to determine the interaction between these molecules. IC showed that immunostaining for phosphorylated proteins significantly increased in the acrosomal region and flagellum in TNC spermatozoa (p < 0.05). The proportion of cells displaying immunolabeling for gelsolin in the acrosomal region decreased after capacitation in cryopreserved spermatozoa; the same change was found (p < 0.05) in the proportion of spermatozoa immunoreactive to PIP2 in the sperm head. c-SRC was observed in the equatorial segment and acrosomal region, subdomains that coincide with the site where phosphorylated proteins were detected. PLC-ζ immunolocalization in fresh spermatozoa underwent changes after capacitation and acrosomal reaction, with a significant increase in the equatorial segment and post-acrosomal region in cryopreserved spermatozoa (p < 0.05). WB analysis indicated the presence of gelsolin, c-SRC and PLC-ζ in PT; besides, we confirmed that gelsolin co-immunoprecipitated with c-SRC and PLC-ζ, which changes according to the physiological state of spermatozoa. As a conclusion, cryopreservation together with increased immunodetection of tyrosine phosphorylated proteins decreases the detection of PIP2 and alters the immunolocalization patterns of gelsolin, c-SRC and PLC-ζ in the PT in boar spermatozoa.

Rab2 and Rab6 are Implicated in Acrosome Formation during Spermatogenesis in Eriocheir sinensis: Based on Sperm Proteome.

Rab proteins are GTP-dependent small proteins that function as regulators of intracellular vesicle transport, fusion, and localization. However, few studies have investigated their function in Decapoda reproduction. The Eriocheir sinensis sperm has no tail and the nuclei are uncondensed. With the acrosome forming the majority of the sperm mass, it provides an ideal model for studying acrosome formation. We firstly analyzed the sperm proteome using LC-MS/MS. To study the functions of Rab2 and Rab6, related to the Golgi apparatus, in the acrosome formation during spermatogenesis, the genes of Rab2 and Rab6 were cloned based on the testis transcriptome of E.sinensis and poly-clonal antibodies were prepared. The presence of 2 Rab proteins was confirmed in the testis and sperm by western blot. We further observed the characteristics of target 2 Rab proteins using immunofluorescence (IF). A total of 1247 proteins including 7 Rab proteins, Rab1, Rab2, Rab5, Rab6, Rab11, Rab14, and Rab18 were identified in the sperm proteome. The IF results showed that Rab2 co-localizes with GM130, a cis-Golgi matrix protein, in the spermatagonia and spermatocytes. In the early spermatids, Rab2 and Rab6 participate in the formation of pre-acrosomal vesicles. In maturing spermatids, both Rab2 and Rab6 settle on the acrosomal membrane but present different characteristics wrapping the pre-acrosome. In the mature sperm, Rab2 localizes in the perinuclear theca surrounding the nuclei cup, while Rab6 remains on the acrosomal membrane. Our research found 7 Rab proteins based on the analysis of the sperm proteome in E.sinensis, and confirmed the involvement of Rab2 and Rab6 in acrosome formation. These findings provide a foundation for studying the functions of Rab proteins during spermatogenesis in Decapoda animals.

P-106 The inhibition of sperm SIRT1 enhances fertilization and modifies subsequent embryonic epigenetic quality

Abstract Study question Is SIRT1 present in sperm and thus involved in fertilization and/or sperm epigenetic code? Do the SIRT1-driven epigenetic marks be transmitted to the progeny? Summary answer SIRT1 localized at the sperm head and influences fertilization and sperm epigenetic code via its deacetylation activity. SIRT1-modulated epigenome is transmitted to zygotes and blastocysts. What is known already The addition or removal of acetyl group from lysine residua (i.e. acetylation) post-translationally modulates proteins through the action of acetylases and deacetylases, respectively. SIRT1 (also Sirtuin 1) is capable to deacetylate a number of proteins, including histones, with roles in male reproduction. Testicular SIRT1 deficit leads to subfertility and increases number of abnormal and immotile spermatozoa that fail to fertilize. However, neither sperm SIRT1 presence nor its function has been investigated despite the significance of acetylation in the events around fertilization. Moreover, the epigenetic mode of action of sperm SIRT1 highlights its relevance for the success of the pre-implantation embryo. Study design, size, duration Mouse sperm of 14-18-weeks-old males of CD-1 x C57Bl6 hybrid strain were isolated from the epididymis. Sperm were incubated under capacitating conditions in presence of sirtinol (5 µM), a selective SIRT1 inhibitor, or the vehicle (0.1% v/v DMSO) for 1 h in 37 °C and 5% CO2. Participants/materials, setting, methods Motility and acrosome reaction was evaluated as previously stated (Ritagliati et al., 2018). Moreover, sperm proteins were extracted into RIPA-buffer and acetylated proteins were depicted by western blot. Finally, in vitro fertilization and embryo culture were performed. Above parameters defining IVF outcomes (i.e., fertilization, cleavage, and blastocyst rate), immunocytochemistry of acetylated histone 4 in the 16th residua of lysine (H4K16) in zygotes and blastocyst was quantified. Main results and the role of chance SIRT1 was localized in the sperm head, apparently at the perinuclear theca and perforatorium. Inhibition of SIRT1 leads to the increase of lysine acetylation (2.28 vs 1, fold change values) and hyperactivation (16.55 ± 0.79 vs 10.84 ± 0.79 %sperm), the enhanced motility that facilitates sperm to penetrate the oocyte, as well as the acrosome reaction (28.69 ± 0.41 vs 17.98 ± 0.41 %sperm). Subsequently, these spermatozoa were able to better fertilize oocytes in vitro (90.5 ± 7.21 vs 66.25 ± 7.21, %). Interestingly, zygotes derived from sirtinol-pre-treated spermatozoa developed to 2-cell embryos at a higher rate than those fertilized by untreated spermatozoa (79.33 ± 7.21 vs 67.00 ± 7.21, %). Moreover, we observed significant hyperacetylation of H4K16 in the male pronuclei of zygotes and embryos derived from sirtinol-treated sperm (8.19 ± 1.02 vs 6.10 ± 2.09 and 45.55 ± 13.96 vs 27.62 ± 13.96, integrated density of the signals, respectively). Limitations, reasons for caution Although acetylation of H4K16 was accumulated in male pronuclei, we do not know which subset of genes was affected. Therefore, we only can assure that SIRT1-driven epigenetic marks are transmitted to the offspring, but their consequences should be further elucidated. For knowledge transfer to humans, a verification study is required. Wider implications of the findings the reduction in testicular SIRT1 expression is an age-related phenomenon. Therefore, our results support SIRT1 as the basis for new interventions against age-related male infertility. Moreover, due to the high reactivity of SIRT1 to its inactivation, SIRT1 becomes a target to pharmacologically enhance sperm fertilization ability in assisted reproductive techniques. Trial registration number Animal procedures were conducted in accordance with Act No. 246/1992 Coll. on the Protection of Animals Against Cruelty under the supervision of the Animal Welfare Committee of the Ministry of Education, Youth and Sports of the Czech Republic, approval ID MSMT-11925/2016-3.

P-027 Absence of perinuclear theca ACTRT1 protein induces sperm head deformation and primary male infertility in humans

Abstract Study question Given that Actrt1 knockout induces teratospermia, fertilization failure and severe male infertility in mice, will ACTRT1 deficiency affect sperm morphology and male fertility in humans? Summary answer The ∼110 kb microdeletion of X chromosome only including ACTRT1 gene was identified inducing sperm head deformation and fertilization failure in an infertile Chinese man. What is known already The actin-related proteins, ACTRT1, ACTRT2, ACTL7A, and ACTL9, interact to form a multimeric complex in the sperm subacrosomal region and is vital for the attachment of acrosome to nucleus. The disruption of ACTL7A or ACTL9 have been identified to cause acrosomal detachment, total fertilization failure and male infertility in humans and mice. Actrt1-knockout mice are severely subfertile also because of malformed sperm heads with detached acrosomes and a partial deficiency in fertilization. Loss/reduced expression and/or abnormal localization of PLCζ, a well-known sperm-borne activating factor, are identified in ACTL7A/ACTL9 mutant patients as well as Actrt1-, Actl7a-, and Actl9-deficient mice. Study design, size, duration We recruited a cohort of 85 infertile men with teratospermia which is characterized by deformed sperm heads in the center for reproductive medicine from August 2019 to June 2022. Genomic DNA (gDNA) of patients was extracted from peripheral blood, then whole-exome sequencing and in silico analyses were performed to identify gene mutations. Morphological analysis, functional assays, and assisted reproductive therapy were performed in 2022. Participants/materials, setting, methods The ACTRT1 deficiency was identified by whole-exome sequencing and confirmed by whole-genome sequencing, PCR and qPCR. Family members’ gDNA was collected to define the hereditary mode. Papanicolaou-staining, scanning and transmission electronic microscopy were performed to reveal sperm morphologies. Western blot and immunostaining of spermatozoa were conducted to explore the pathological mechanism of the ACTRT1 deficiency. Intracytoplasmic sperm injection (ICSI) combined with artificial oocyte activation (AOA) was applied for the assisted reproductive therapy of ACTRT1-deletion patient. Main results and the role of chance We identified a whole-gene deletion mutation of ACTRT1 on Chromosome X in an infertile male with teratospermia, which was inherited from his mother. Papanicolaou-staining, scanning electronic microscopy, and transmission electronic microscopy showed the sperm head deformation owing to acrosome detachment, which mimicked the previously reported phenotype of Actrt1-knockout mice. The results of western blot and immunostaining suggested that ACTRT1 deficiency induced a down-regulated expression of ACTL7A and PLCζ proteins, but not ACTRT2, in human sperm samples. The detached acrosomes induced fertilization failure in assisted reproductive therapy of both Actrt1-knockout mice and the ACTRT1-deletion patient, which could be effectively rescued by ICSI combined with AOA. Limitations, reasons for caution Additional cases are needed to confirm the genetic contribution of ACTRT1 mutations to male infertility with teratospermia and fertilization failure. In addition, the proband with ACTRT1 deletion was with severe oligozoospermia and mild asthenozoospermia. The effect of ACTRT1 mutations on sperm count and motility still need more investigation. Wider implications of the findings Our results reveal a gene–disease relationship between ACTRT1 deficiency and human male infertility owing to teratospermia and fertilization failure. This report also describe a good fertility outcome of assisted reproductive therapy with ICSI and AOA for the ACTRT1-deficient patient. Trial registration number not applicable

Human Sperm Head Vacuoles Are Related to Nuclear-Envelope Invaginations.

Nuclear vacuoles are specific structures present on the head of the human sperm of fertile and non-fertile men. Human sperm head vacuoles have been previously studied using motile sperm organelle morphology examination (MSOME) and their origin related to abnormal morphology, abnormal chromatin condensation and DNA fragmentation. However, other studies argued that human sperm vacuoles are physiological structures and consequently, to date, the nature and origin of the nuclear vacuoles remains to be elucidated. Here, we aim to define the incidence, position, morphology and molecular content of the human sperm vacuoles using transmission electron microscopy (TEM) and immunocytochemistry techniques. The results showed that ~50% of the analyzed human sperm cells (n = 1908; 17 normozoospermic human donors) contained vacuoles mainly located (80%) in the tip head region. A significant positive correlation was found between the sperm vacuole and nucleus areas. Furthermore, it was confirmed that nuclear vacuoles were invaginations of the nuclear envelope from the perinuclear theca and containing cytoskeletal proteins and cytoplasmic enzyme, discarding a nuclear or acrosomal origin. According to our findings, these human sperm head vacuoles are cellular structures originating from nuclear invaginations and contain perinuclear theca (PT) components, allowing us to define a new term of 'nuclear invaginations' rather than 'nuclear vacuoles'.

Testis-specific actin-like 7A (ACTL7A) is an indispensable protein for subacrosomal-associated F-actin formation, acrosomal anchoring, and male fertility.

Formation of the acrosome during spermiogenesis is an essential process for creating fertilization-competent sperm. Of the numerous aspects required for acrosome biogenesis, adherence of the acrosomal outer membrane to the nuclear surface is mediated by the subacrosomal perinuclear theca. However, the cellular dynamics and congruent functions pertaining to these acrosomal anchoring factors are not well understood despite many of them being implicated as potential causes for human male infertility. Actin-like 7A (ACTL7A) is one such factor for which deleterious polymorphisms have recently been shown to cause human male infertility. It is thought that acrosomal attachment is coordinated by cytoskeletal associations between the acrosome and nucleus via the acroplaxome. To further illuminate the mechanistic underpinnings of ACTL7A for essential acrosome associations, in this study, we investigated its dynamic localization in the developing germline, molecular associations with other cytoskeletal components, and the cellular consequences of ablation. Our intracellular localization data show ACTL7A to be dynamically present within the nucleus and subacrosomal space and later associated with postacrosomal regions of developing spermatids. Through the generation of an Actl7a knock-out mouse model, we consistently observed disruption of acrosomal biogenesis with abnormal migration of the acrosomal granule and peeling acrosomes during spermatid elongation. Significantly, we found a complete loss of subacrosomal filamentous actin (F-actin) structures in knock-out spermatids suggesting a regulatory role for subacrosomal F-actin. Considering our reported data together with existing literature, we propose a mechanistic model explaining the essential role of ACTL7A for acroplaxome-associated F-actin, acrosomal attachment integrity, and male fertility.

A novel homozygous mutation in ACTL7A leads to male infertility.

Maleinfertility,a global public health problem,exhibits complex pathogenic causes and genetic factors deserve further discovery and study. We identified a novel homozygous missense mutation c.224A > C (p.D75A) in ACTL7A gene in two infertile brothers with teratozoospermia by whole-exome sequencing (WES). In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) showed fertilization failure of the two affected couples. The three-dimensional (3D) models showed that a small section of α-helix transformed into random coil in the mutant ACTL7A protein and mutant amino acid lacked a hydrogen bond with Ser170 amino acid. Immunofluorescence revealed that ACTL7A protein was degraded in sperms of patients. Transmission electron microscopy (TEM) analysis of sperms from the infertile patients showed that the irregular perinuclear theca (PT) and acrosomal ultrastructural defects. Furthermore, ACTL7A mutation caused abnormal localization and reduced the expression of PLCZ1 in sperms of the patients, which may be the key reasons for the fertilization failure after ICSI. Our findings expand the spectrum of ACTL7A mutations and provide novel theoretical basis for genetic counseling.

The fate of porcine sperm CRISP2 from the perinuclear theca before and after in vitro fertilization†.

In a previous study, we reported that porcine sperm cysteine-rich secretory protein 2 (CRISP2) is localized in the post-acrosomal sheath-perinuclear theca (PT) as reduction-sensitive oligomers. In the current study, the decondensation and removal of CRISP2 was investigated during in vitro sperm capacitation, after both the induction of the acrosome reaction and in vitro fertilization. Confocal immunofluorescent imaging revealed that additional CRISP2 fluorescence appeared on the apical ridge and on the equatorial segment (EqS) of the sperm head following capacitation, likely due to cholesterol removal. After an ionophore A23187-induced acrosome reaction, CRISP2 immunofluorescence disappeared from the apical ridge and the EqS area partly not only owing to the removal of the acrosomal shroud vesicles, but to its presence in a subdomain of EqS. The fate of sperm head CRISP2 was further examined post-fertilization. In vitro matured porcine oocytes were co-incubated with boar sperm cells for 6-8h and the zygotes were processed for CRISP2 immunofluorescent staining. Notably, decondensation of CRISP2, and thus of the sperm PT, occurred while the sperm nucleus was still fully condensed. CRISP2 was no longer detectable in fertilized oocytes in which sperm nuclear decondensation and paternal pronucleus formation were apparent. This rapid dispersal of CRISP2 in the PT is likely regulated by redox reactions for which its cysteine-rich domain is sensitive. Reduction of disulfide bridges within CRISP2 oligomers may be instrumental for PT dispersal and elimination.

The perinuclear theca protein Calicin helps shape the sperm head and maintain the nuclear structure in mice.

The perinuclear theca (PT) is a cytoskeletal element encapsulating the sperm nucleus; however, our understanding of the physiological roles of PT in sperm is very limited. We show that Calicin interacts with itself and many other PT components, indicating it may serve as an organizing center of the PT assembly. Calicin is detectable first when surrounding the acrosome, then detected around the entire nucleus, and finally translocated to the postacrosomal region of spermatid heads. Intriguingly, loss of Calicin specifically causes surface subsidence of sperm heads in the nuclear condensation stage. Calicin interacts with inner acrosomal membrane (IAM) protein Spaca1 and nuclear envelope (NE) components to form an "IAM-PT-NE" structure. Intriguingly, Ccin-knockout sperm also exhibit DNA damage and failure of fertilization. Our study provides solid animal evidence to suggest that the PT encapsulating sperm nucleus helps shape the sperm head and maintain the nuclear structure.

SPERM FACTORS AND EGG ACTIVATION: ICSI and the discovery of the sperm factor and PLCZ1.

The discovery of PLCZ1 nearly 20 years ago as the primary Ca2+ oscillation-inducing factor in the sperm of mammals represented a significant breakthrough in our quest to elucidate the molecules and pathways that promote egg activation during fertilization. The advent of the intracytoplasmic sperm injection (ICSI) technique, which made fertilization possible without sperm capacitation, acrosome reaction, and gamete fusion, strengthened the research that led to the discovery of PLCZ1 and became an essential clinical tool for humans. The use of ICSI combined with the detection of PLCZ1 expression and mutations in infertile patients established the fundamental role of PLCZ1 in human fertility while leading to the discovery of novel components of the perinuclear theca, the site of the residence of PLCZ1 in sperm before fertilization. Remarkably, the more extensive use of ICSI in species other than humans and mice revealed poor success and exposed gaps in our understanding of PLCZ1 release and/or activation. Similarly, fertilization using sperm from mouse models lacking Plcz1 has produced striking results whose true implications are yet to be determined. Nevertheless, answers to these unresolved questions will produce a complete picture of the adaptations and molecular players that mammalian species employ to ensure the success of the triggering event of embryo development that has linked generations since the beginning of times.

Loss of perinuclear theca ACTRT1 causes acrosome detachment and severe male subfertility in mice.

The perinuclear theca (PT) is a cytoskeletal element encapsulating the sperm nucleus; however, the physiological roles of the PT in sperm are largely uncertain. Here, we reveal that ACTRT1, ACTRT2, ACTL7A and ACTL9 proteins interact to form a multimeric complex and localize to the subacrosomal region of spermatids. Furthermore, we engineered Actrt1-knockout (KO) mice to define the functions of ACTRT1. Despite normal sperm count and motility, Actrt1-KO males were severely subfertile owing to a deficiency in fertilization. Loss of ACTRT1 caused a high incidence of malformed heads and detachment of acrosomes from sperm nuclei, caused by loosened acroplaxome structure during spermiogenesis. Furthermore, Actrt1-KO sperm showed reduced ACTL7A and PLCζ protein content as a potential cause of fertilization defects. Moreover, we reveal that ACTRT1 anchors developing acrosomes to the nucleus, likely by interacting with the inner acrosomal membrane protein SPACA1 and the nuclear envelope proteins PARP11 and SPATA46. Loss of ACTRT1 weakened the interaction between ACTL7A and SPACA1. Our study and recent findings of ACTL7A/ACTL9-deficient sperm together reveal that the sperm PT-specific ARP complex mediates the acrosome-nucleus connection.

High Resolution Proteomic Analysis of Subcellular Fractionated Boar Spermatozoa Provides Comprehensive Insights Into Perinuclear Theca-Residing Proteins.

The perinuclear theca (PT) is a highly condensed, largely insoluble protein structure that surrounds the nucleus of eutherian spermatozoa. Recent reports have indicated that the PT unexpectedly houses several somatic proteins, such as core histones, which may be important post-fertilization during re-modelling of the male pronucleus, yet little is known regarding the overall proteomic composition of the PT. Here, we report the first in depth, label-free proteomic characterization of the PT of boar spermatozoa following the implementation of a long-established subcellular fractionation protocol designed to increase the detection of low abundance proteins. A total of 1,802 proteins were identified, a result that represents unparalleled depth of coverage for the boar sperm proteome and exceeds the entire annotated proteome of the Sus scrofa species so far. In the PT structure itself, we identified 813 proteins and confirmed the presence of previously characterized PT proteins including the core histones H2A, H2B, H3 and H4, as well as Ras-related protein Rab-2A (RAB2A) and Rab-2B (RAB2B) amongst other RAB proteins. In addition to these previously characterized PT proteins, our data revealed that the PT is replete in proteins critical for sperm-egg fusion and egg activation, including: Izumo family members 1–4 (IZUMO1-4) and phosphoinositide specific phospholipase ζ (PLCZ1). Through Ingenuity Pathway Analysis, we found surprising enrichment of endoplasmic reticulum (ER) proteins and the ER-stress response in the PT. This is particularly intriguing as it is currently held that the ER structure is lost during testicular sperm maturation. Using the String and Cytoscape tools to visualize protein-protein interactions revealed an intricate network of PT protein complexes, including numerous proteasome subunits. Collectively, these data suggest that the PT may be a unique site of cellular homeostasis that houses an abundance of protein degradation machinery. This fits with previous observations that the PT structure dissociates first within the oocyte post-fertilization. It remains to be explored whether proteasome subunits within the PT actively assist in the protein degradation of paternal cell structures post-fertilization and how aberrations in PT protein content may delay embryonic development.

Novel bi-allelic variants in ACTL7A are associated with male infertility and total fertilization failure.

What are the genetic causes of total fertilization failure (TFF) in a proband suffering from male infertility? Novel compound heterozygous variants (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) in actin-like protein 7A (ACTL7A) were identified as a causative genetic factor for human TFF. ACTL7A, an actin-related protein, is essential for spermatogenesis. ACTL7A variants have been reported to cause early embryonic arrest in humans but have not been studied in human TFF. We recruited a non-consanguineous family whose son was affected by infertility characterized by TFF after ICSI. Whole-exome sequencing was used to identify the potential pathogenic variants. Artificial oocyte activation (AOA) after ICSI was performed to overcome TFF and any resulting pregnancy was followed up. Sanger sequencing was performed to validate the variants. Pathogenicity of the identified variants was predicted by in silico tools. The ultrastructure of spermatozoa was studied by transmission electron microscopy (TEM). Immunofluorescence staining and western blotting were used to investigate the mechanism of the variants on the affected spermatozoa. Novel compound heterozygous variants in ACTL7A (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) were identified in a family with TFF after ICSI. In silico analysis predicted that the variants lead to a disease-causing protein. TEM showed that the ACTL7A variants caused ultrastructural defects in the acrosome and perinuclear theca. Protein expression of ACTL7A and phospholipase C zeta, a key sperm-borne oocyte activation factor, was significantly reduced in the affected sperm compared to healthy controls, suggesting that the ACLT7A variants lead to an oocyte activation deficiency and TFF. AOA by calcium ionophore (A23187) after ICSI successfully rescued the TFF and achieved a live birth for the patient with ACTL7A variants. Given the rarity of sperm-associated TFF, only one family with an only child carrying the ACTL7A variants was found. In addition, the TFF phenotype was not assessed in two or more ICSI cycles, due to the intervention in ICSI with AOA after one failed ICSI cycle. Further studies should validate the ACTL7A variants and its effect on male infertility in larger independent cohorts. : Our findings revealed a critical role of ACTL7A in male fertility and identified bi-allelic variants in ACTL7A associated with human TFF, which expands the genetic spectrum of TFF and supports the genetic diagnosis of TFF patients. We also rescued TFF by AOA and obtained a healthy live birth, which provides a potentially effective intervention for patients with ACTL7A pathogenic variants. This work was supported by the National Natural Science Foundation of China (81971374 and 81401267). No conflicts of interest were declared. N/A.

CONCURRENT ULTRASTRUCTURAL ANALYSIS AND GENOMIC ASSESSMENT OF SPERMATOZOA TO ACCURATELY DIAGNOSE RARE AND SEVERE FORMS OF MALE INFERTILITY

In men with ART failure, sperm DNA sequencing with ultrastructural analysis can identify specific sperm defects and guide ART treatment. In the past 2 years, 22 consenting men who failed IVF cycles due to severe astheno-/teratozoospermia were recruited; specimens from 3 fertile men were used as controls. To assess ultrastructure by transmission electron microscopy (TEM), pelleted spermatozoa were fixed, embedded, sectioned to 100-nm slides by ultramicrotome, and viewed with an electron microscope (JEOL-1400) at 300,000X magnification. Sperm head morphology, fibrous sheath intactness, and axonemal/periaxonemal structure were examined on at least 100 spermatozoa to diagnose specific sperm organelle defects. DNA was extracted and amplified from at least 500 spermatozoa for next-generation sequencing (NGS) to identify mutations contributing to the ultrastructural abnormalities. Four types of sperm ultrastructural defects were identified: globozoospermia, dysplasia of fibrous sheath (DFS), proximal centriole defect, and primary ciliary dyskinesia (PCD). One case of combined globozoospermia and DFS and one case of combined DFS and proximal centriole defect were identified. In 13 globozoospermic cases, 97–100% of the spermatozoa displayed complete spherical heads with the absence of acrosomes, perinuclear theca deformities, and dispersed chromatin. Microtubular arrangement of centrosome and flagellum was conserved. NGS identified deletions (DPY19L, PICK1, SPATA16) directly responsible for the globozoospermic phenotype. Based on the findings, assisted oocyte activation was performed for 7 couples with an oocyte-activating agent, boosting fertilization from 5.7% to 40.2% (p<0.00001), resulting in 5 pregnancies and 4 deliveries. In patients with DFS (n=6), flagellar deformities were observed in 90–100% of spermatozoa with mitochondria disorganization, disarranged microtubules, and cytoplasmic remnant enveloping coiled flagella, indicating incomplete late spermiogenesis. Normal acrosomes and nuclei were seen. NGS identified deletions (AKAP4, SPAG16, CATSPER1) involved in flagellar development and function. For patients with proximal centriole defects (n=2), the sperm nuclei and fibrous sheath appeared normal. Approximately 90% of proximal centrioles exhibited microtubular disorganization, confirmed by an ODF2 mutation identified by NGS. For a patient with PCD, chaotic flagellar microtubular arrangement and the absence of outer dynein arms prevailed in 90% of the axonemal cross-sections examined, confirmed by a DNAH5 gene deletion. Nonetheless, a few motile spermatozoa were identified and used for ICSI, yielding 80% fertilization and the delivery of a healthy twin. Concurrent ultrastructural and genomic analysis can pinpoint sperm organelle disruption and lead to successful ART cycles.

P–568 Homozygous Pathogenic Variants in ACTL9 Cause Fertilization Failure and Male Infertility in Human and Mouse

Abstract Study question What are the other male factors that cause total fertilization failure (TFF) excepting for variants in PLCZ1? Summary answer Homozygous variants in ACTL9 (actin like 9) cause abnormal localization of PLCζ in a loosened perinuclear theca (PT) structure and leads to TFF. What is known already In previous studies, investigators have reported that the female factors in TFF after intracytoplasmic sperm injection (ICSI) include pathogenic variants in WEE2, TLE6, and TUBB8, whereas for male factors, pathogenic variants in PLCZ1 were reported to be the primary cause of TFF, which account for approximately 30% of couples with male factors in TFF excluding globozoospermia. Most recently, it was reported that pathogenic variants in ACTL7A led to reduced expression and abnormal localization of PLCζ, thereby identifying this genetic variant as a potential cause of TFF. Study design, size, duration Fifty-four infertile couples with TFF or poor fertilization (fertilization rate of &amp;lt; 20%) at the Reproductive and Genetic Hospital of CITIC-Xiangya during January 2014 to June 2020 were recruited into this study. Participants/materials, setting, methods Male factors were identified in (MOAT). WES analysis was used to analyze the genetic factors of individuals with male factors. Sperm morphological study was conducted by H&amp;E staining and TEM. Immunostaining of PLCζ was used to analyze the status of sperm-borne activation factor. A knock-in mouse model was generated by CRISPER-Cas9 technology. Sperm from homozygous Actl9 variant mice were analyzed by TEM and ICSI. ICSI with AOA was performed in couples with ACTL9 variants. Main results and the role of chance A total of 54 couples with TFF or poor fertilization were screened, with 21 couples determined to have a male infertility factor by MOAT. Whole-exome sequencing of these 21 male individuals identified three homozygous pathogenic variants in ACTL9 in three individuals. ACTL9 variations led to abnormal ultrastructure of the PT, with PLCζ absent in the head and present in the neck of the mutant sperm, which contributed to failed normal calcium oscillations in oocytes and subsequent TFF. The key roles of ACTL9 in the PT structure and TFF after ICSI were further confirmed in Actl9-mutated mouse model. Furthermore, assisted oocyte activation by calcium ionophore exposure successfully overcame TFF and achieved live births in a couple with an ACTL9 variant. Limitations, reasons for caution The mechanism of how ACTL9 regulate PLCζ remains unknown. Wider implications of the findings: It provided a genetic marker and a therapeutic option for individuals who have undergone ICSI without successful fertilization. Trial registration number not applioable