Nonobstructive azoospermia (NOA) and diminished ovarian reserve (DOR) are two disorders that can lead to infertility in males and females. Genetic factors have been identified to contribute to NOA and DOR. However, the same genetic factor that can cause both NOA and DOR remains largely unknown. To explore the candidate pathogenic gene that causes both NOA and DOR, we conducted whole-exome sequencing (WES) in a non-consanguineous family with two daughters with DOR and a son with NOA. We detected one pathogenic frameshift variant (NM_007068:c.28delG, p. Glu10Asnfs*31) following a recessive inheritance mode in a meiosis gene DMC1 (DNA meiotic recombinase 1). Clinical analysis showed reduced antral follicle number in both daughters with DOR, but metaphase II oocytes could be retrieved from one of them. For the son with NOA, no spermatozoa were found after microsurgical testicular sperm extraction. A further homozygous Dmc1 knockout mice study demonstrated total failure of follicle development and spermatogenesis. These results revealed a discrepancy of DMC1 action between mice and humans. In humans, DMC1 is required for spermatogenesis but is dispensable for oogenesis, although the loss of function of this gene may lead to DOR. To our knowledge, this is the first report on the homozygous frameshift mutation as causative for both NOA and DOR and demonstrating that DMC1 is dispensable in human oogenesis.

Caseinolytic protease proteolytic subunit (ClpP) and caseinolytic protease X (ClpX) are mitochondrial matrix peptidases that activate mitochondrial unfolded protein response to maintain protein homeostasis in the mitochondria. However, the role of ClpP and ClpX in spermatogenesis remains largely unknown. In this study, we demonstrated the importance of ClpP/ClpX for meiosis and spermatogenesis with two conditional knockout (cKO) mouse models. We found that ClpP/ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply during meiosis and attenuated zygotene-pachytene transformation of the male germ cells. The dysregulated spermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within the seminiferous tubules. We found mTORC1 pathway was over-activated after deletion of ClpP/ClpX in spermatocytes. Long-term inhibition of the mTORC1 signaling via rapamycin treatment in vivo partially rescue spermatogenesis. The data reveal the critical roles of ClpP and ClpX in regulating meiosis and spermatogenesis.

Oocyte vitrification technology is widely used for assisted reproduction and fertility preservation, which requires precise washing sequences and timings of cryoprotectant agents (CPAs) treatment to relieve the osmotic shock to cells. The gold standard Cryotop method is extensively used in oocyte vitrification and is currently the most commonly used method in reproductive centers. However, the Cryotop method requires precise and complex manual manipulation by an embryologist, whose proficiency directly determines the effect of vitrification. Therefore, in this study, an automatic microfluidic system consisting of a novel open microfluidic chip and a set of automatic devices was established as a standardized operating protocol to facilitate the conventional manual Cryotop method and minimize the osmotic shock applied to the oocyte. The proposed open microfluidic system could smoothly change the CPA concentration around the oocyte during vitrification pretreatment, and transferred the treated oocyte to the Cryotop with a tiny droplet. The system better conformed to the operating habits of embryologists, whereas the integration of commercialized Cryotop facilitates the subsequent freezing and thawing processes. With standardized operating procedures, our system provides consistent treatment effects for each operation, leading to comparable survival rate, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) level of oocytes to the manual Cryotop operations. The vitrification platform is the first reported microfluidic system integrating the function of cells transfer from the processing chip, which avoids the risk of cell loss or damage in a manual operation and ensures the sufficient cooling rate during liquid nitrogen (${\mathrm{LN}}_2$) freezing. Our study demonstrates significant potential of the automatic microfluidic approach to serve as a facile and universal solution for the vitrification of various precious cells.

Mitochondria are highly dynamic organelles and their activity is known to be regulated by changes in morphology via fusion and fission events. However, the role of mitochondrial dynamics on cellular differentiation remains largely unknown. Here, we explored the molecular mechanism of mitochondrial fusion during spermatogenesis by generating an Mfn2 (mitofusin 2) conditional knock-out (cKO) mouse model. We found that depletion of MFN2 in male germ cells led to disrupted spermatogenesis and meiosis during which the majority of Mfn2 cKO spermatocytes did not develop to the pachytene stage. We showed that in these Mfn2 cKO spermatocytes, oxidative phosphorylation in the mitochondria was affected. In addition, RNA-Seq analysis showed that there was a significantly altered transcriptome profile in the Mfn2 deficient pachytene (or pachytene-like) spermatocytes, with a total of 262 genes up-regulated and 728 genes down-regulated, compared with wild-type (control) mice. Pathway enrichment analysis indicated that the peroxisome proliferator-activated receptor (PPAR) pathway was altered, and subsequent more detailed analysis showed that the expression of PPAR α and PPAR γ was up-regulated and down-regulated, respectively, in the MFN2 deficient pachytene (or pachytene-like) spermatocytes. We also demonstrated that there were more lipid droplets in the Mfn2 cKO cells than in the control cells. In conclusion, our study demonstrates a novel finding that MFN2 deficiency negatively affects mitochondrial functions and alters PPAR pathway together with lipid metabolism during spermatogenesis and meiosis.

The role of two-pore channel type 2 (TPC2, encoded by tcpn2)-mediated Ca²⁺ release was recently characterized in zebrafish during establishment of the early spinal circuitry, one of the key events in the coordination of neuromuscular activity. Here, we extend our study to investigate the in vivo role of TPC2 in the regulation of caudal primary motor neuron (CaP) axon extension. We used a combination of TPC2 knockdown with a translation-blocking morpholino antisense oligonucleotide (MO), TPC2 knockout via the generation of a tpcn2^dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing and pharmacological inhibition of TPC2 via incubation with bafilomycin A1 (an H⁺-ATPase inhibitor) or trans-ned-19 (an NAADP receptor antagonist), and showed that these treatments attenuated CaP Ca²⁺ signaling and inhibited axon extension. We also characterized the expression of an arc1-like transcript in CaPs grown in primary culture. MO-mediated knockdown of ARC1-like in vivo led to attenuation of the Ca²⁺ transients in the CaP growth cones and an inhibition of axon extension. Together, our new data suggest a link between ARC1-like, TPC2 and Ca²⁺ signaling during axon extension in zebrafish.

Chemotherapy is extensively used to treat cancers and is often associated with ovarian damage and leads to premature ovarian insufficiency and infertility, while the role of mitochondria during ovarian damage with chemotherapy remains unknown. This study used a mouse model with oocyte-specific deletion of mitochondrial stress response gene Caseinolytic peptidase P (Clpp) to investigate mitochondrial homeostasis in oocytes from mice receiving a chemotherapeutic drug cyclophosphamide (CTX). We found that oocyte-specific deletion of Clpp reduced fecundity of the mice at advanced age. The deletion led to meiotic defects with elevated abnormal spindle rate and aneuploidy rate with impaired mitochondrial function in the MII oocytes from 8-week-old mice. Upon CTX treatment at 8-week-old, the oocyte competence and folliculogenesis from the oocyte-specific Clpp knockout mice was further deteriorated with dramatic impairment of mitochondrial distribution and function including elevated ROS level, decreased mitochondrial membrane potential, respiratory chain activity and ATP production. Taken together, the results indicate that that ClpP was required for oocyte competence during maturation and early folliculogenesis, and its deficiency deteriorate cyclophosphamide-induced ovarian damage.