Actl7b deficiency leads to mislocalization of LC8 type dynein light chains and disruption of murine spermatogenesis.

Actin-related proteins (Arps) are classified according to their similarity to actin and are involved in diverse cellular processes. ACTL7B is a testis-specific Arp, and is highly conserved in rodents and primates. ACTL7B is specifically expressed in round and elongating spermatids during spermiogenesis. Here, we have generated an Actl7b-null allele in mice to unravel the role of ACTL7B in sperm formation. Male mice homozygous for the Actl7b-null allele (Actl7b-/-) were infertile, whereas heterozygous males (Actl7b+/-) were fertile. Severe spermatid defects, such as detached acrosomes, disrupted membranes and flagella malformations start to appear after spermiogenesis step 9 in Actl7b-/- mice, finally resulting in spermatogenic arrest. Abnormal spermatids were degraded and levels of autophagy markers were increased. Co-immunoprecipitation with mass spectrometry experiments identified an interaction between ACTL7B and the LC8 dynein light chains DYNLL1 and DYNLL2, which are first detected in step 9 spermatids and mislocalized when ACTL7B is absent. Our data unequivocally establish that mutations in ACTL7B are directly related to male infertility, pressing for additional research in humans.

The activation of M2 muscarinic receptor inhibits cell growth and survival in human epithelial ovarian carcinoma.

Ovarian cancer is the fifth leading cause of cancer-related deaths in females. Many ovarian tumor cell lines express muscarinic receptors (mAChRs), and their expression is correlated with reduced survival of patients. We have characterized the expression of mAChRs in two human ovarian carcinoma cell lines (SKOV-3, TOV-21G) and two immortalized ovarian surface epithelium cell lines (iOSE-120, iOSE-398). Among the five subtypes of mAChRs (M1-M5 receptors), we focused our attention on the M2 receptor, which is involved in the inhibition of tumor cell proliferation. Western blot analysis and real-time PCR analyses indicated that the levels of M2 are statistically downregulated in cancer cells. Therefore, we investigated the effect of arecaidine propargyl ester hydrobromide (APE), a preferential M2 agonist, on cell growth and survival. APE treatment decreased cell number in a dose and time-dependent manner by decreasing cell proliferation and increasing cell death. FACS and immunocytochemistry analysis have also demonstrated the ability of APE to accumulate the cells in G2/M phase of the cell cycle and to increase the percentage of abnormal mitosis. The higher level of M2 receptors in the iOSE cells rendered these cells more sensitive to APE treatment than cancer cells. The data here reported suggest that M2 has a negative role in cell growth/survival of ovarian cell lines, and its downregulation may favor tumor progression.

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.

Male humans, mice and other animals produce sex cells known as sperm that seek out and fertilize egg cells from females. Sperm have a very distinctive shape with a head and a long tail that enables them to swim towards an egg. At the front of the sperm's head is a pointed structure known as the acrosome that helps the sperm to burrow into an egg cell. A structure known as the cytoskeleton is responsible for forming and maintaining the shape of acrosomes and other parts of cells. Two proteins, known as Cylicin 1 and Cylicin 2, are unique to the cytoskeleton of sperm, but their roles remain unclear. To investigate the role of the Cylicins during spermiogenesis, Schneider, Kovacevic et al. used an approach called CRISPR/Cas9-mediated gene-editing to generate mutant mice that were unable to produce either Cylicin 1 or Cylicin 2, or both proteins. The experiments found that healthy female mice were less likely to become pregnant when they mated with mutant males that lacked Cylicin 1 compared with males that had the protein. When they did become pregnant, the females had smaller litters of babies. Mutant male mice lacking Cylicin 2 or both Cylicin proteins (so-called "double" mutants), were infertile and mating with healthy female mice did not lead to any pregnancies. Further experiments found that the sperm of such mice had smaller heads than normal sperm, defective acrosomes, and curled tails that wrapped around the head. Schneider, Kovacevic et al. also examined the sperm of a human patient who had inherited genetic variants in the genes encoding both Cylicin proteins. Similar to the double mutant mice, the patient was infertile, and his sperm also had defective acrosomes and curled tails. These findings indicate that Cylicins are required to make the acrosome as sperm cells mature and help maintain the structure of the cytoskeleton of sperm. Further studies of Cylicins and other sperm proteins in mice may help us to understand some of the factors that contribute to male infertility in humans.