During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse (Mlh3^DN/DN) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3^DN/DN males, like fully null Mlh3^-/- males, have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and synapsis events in early prophase I. Unlike Mlh3^-/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3^DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3^DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3^-/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3^DN/DN males. Double mutant mice homozygous for the Mlh3^DN/DN and Mus81^-/- mutations show losses in chiasmata close to those observed in Mlh3^-/- males, indicating that the MUS81-EME1-regulated crossover pathway can only partially account for the increased residual chiasmata in Mlh3^DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3^DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3^DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata. Meiosis is a specialized cell division whereby a diploid cell undergoes one round of DNA replication followed by two rounds of division, yielding up to four haploid gametes. This process depends on tethering of maternal and paternal homologous chromosomes, and by the formation of crossovers (COs) between homologs during prophase I. COs arise from programmed double-strand breaks (DSBs), and can form via one of at least two mechanisms (class I, class II). In mouse, class I represents the major CO pathway, with the MLH1-MLH3 (MutLγ) complex being critical. MLH3 contains a conserved metal binding motif, DQHA(X)₂E(X)₄E, required for its endonuclease function, and this activity is postulated to represent a “resolvase”activity for class I COs. We generated a point mutant (Mlh3^DN) in the endonuclease domain without altering the overall structure of MutLγ. Mlh3^DN/DN males have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and chromosome pairing. The MLH3^DN mutation permits normal loading of MutLγ, but key DSB repair factors persist in Mlh3^DN/DN males, indicating a temporal delay in repair and suggesting a mechanism by which alternative DSB repair pathways may be selected. Thus, the endonuclease domain of MLH3 is important for normal processing of DSB repair intermediates.

Is there an association between morphokinetic variables of meiotic maturation and the severity of aneuploidy following in vitro maturation (IVM) in the mouse? The severity of meiotic aneuploidy correlates with an extended time to first polar body extrusion (tPB1) and duration of meiosis I (dMI). Morphokinetic variables measured using time-lapse technology allow for the non-invasive evaluation of preimplantation embryo development within clinical assisted reproductive technology (ART). We recently applied this technology to monitor meiotic progression during IVM of mouse gametes. Whether there is a relationship between morphokinetic variables of meiotic progression and aneuploidy in the resulting egg has not been systematically examined at the resolution of specific chromosomes. Next-generation sequencing (NGS) is a robust clinical tool for determining aneuploidy status and has been reverse-translated in mouse blastocysts and oocytes. Therefore, we harnessed the technologies of time-lapse imaging and NGS to determine the relationship between the morphokinetics of meiotic progression and egg aneuploidy. Cumulus–oocyte complexes were collected from large antral follicles from hyperstimulated CD-1 mice. Cumulus cells were removed, and spontaneous IVM was performed in the absence or presence of two doses of Nocodazole (25 or 50 nM) to induce a spectrum of spindle abnormalities and chromosome segregation errors during oocyte meiosis. Comprehensive chromosome screening was then performed in the resulting eggs, and morphokinetic variables and ploidy status were compared across experimental groups (control, n = 11; 25 nM Nocodazole, n = 13; 50 nM Nocodazole, n = 23). We monitored IVM in mouse oocytes using time-lapse microscopy for 16 h, and time to germinal vesicle breakdown (tGVBD), tPB1, and dMI were analyzed. Following IVM, comprehensive chromosome screening was performed on the eggs and their matched first polar bodies via adaptation of an NGS-based preimplantation genetic testing for aneuploidy (PGT-A) assay. Bioinformatics analysis was performed to align reads to the mouse genome and determine copy number-based predictions of aneuploidy. The concordance of each polar body–egg pair (reciprocal errors) was used to validate the results. Ploidy status was categorized as euploid, 1–3 chromosomal segregation errors, or ≥4 chromosomal segregation errors. Additionally, aneuploidy due to premature separation of sister chromatids (PSSC) versus non-disjunction (NDJ) was distinguished. We applied and validated state-of-the-art NGS technology to screen aneuploidy in individual mouse eggs and matched polar bodies at the chromosome-specific level. By performing IVM in the presence of different doses of Nocodazole, we induced a range of aneuploidy. No aneuploidy was observed in the absence of Nocodazole (0/11), whereas IVM in the presence of 25 and 50 nM Nocodazole resulted in an aneuploidy incidence of 7.69% (1/13) and 82.61% (19/23), respectively. Of the aneuploid eggs, 5% (1/20) was due to PSSC, 65% (13/20) to NDJ, and the remainder to a combination of both. There was no relationship between ploidy status and tGVBD, but tPB1 and the dMI were both significantly prolonged in eggs with reciprocal aneuploidy events compared to the euploid eggs, and this scaled with the severity of aneuploidy. Eggs with ≥4 aneuploid chromosomes had the longest tPB1 and dMI (P < 0.0001), whereas eggs with one to three aneuploid chromosomes exhibited intermediate lengths of time (P < 0.0001). N/A. We used Nocodazole in this study to disrupt the meiotic spindle and induce aneuploidy in mouse oocytes. Whether the association between morphokinetic variables of meiotic progression and the severity of aneuploidy occurs with other compounds that induce chromosome segregation errors remain to be investigated. In addition, unlike mouse oocytes, human IVM requires the presence of cumulus cells, which precludes visualization of morphokinetic variables of meiotic progression. Thus, our study may have limited direct clinical translatability. We validated NGS in mouse eggs to detect aneuploidy at a chromosome-specific resolution which greatly improves the utility of the mouse model. With a tractable and validated model system for characterizing meiotic aneuploidy, investigations into the molecular mechanisms and factors which may influence aneuploidy can be further elaborated. Time-lapse analyses of morphokinetic variables of meiotic progression may be a useful non-invasive predictor of aneuploidy severity. This work was supported by the Bill & Melinda Gates Foundation (INV-003385). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. The authors have no conflict of interest to disclose.

Summary: Ovulation is a spatiotemporally coordinated process that involves several tightly controlled events, including oocyte meiotic maturation, cumulus expansion, follicle wall rupture and repair, and ovarian stroma remodeling. To date, no studies have detailed the precise window of ovulation at single-cell resolution. Here, we performed parallel single-cell RNA-seq and spatial transcriptomics on paired mouse ovaries across an ovulation time course to map the spatiotemporal profile of ovarian cell types. We show that major ovarian cell types exhibit time-dependent transcriptional states enriched for distinct functions and have specific localization profiles within the ovary. We also identified gene markers for ovulation-dependent cell states and validated these using orthogonal methods. Finally, we performed cell-cell interaction analyses to identify ligand-receptor pairs that may drive ovulation, revealing previously unappreciated interactions. Taken together, our data provides a rich and comprehensive resource of murine ovulation that can be mined for discovery by the scientific community.

During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse ( Mlh3 ^DN/DN ) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3 ^DN/DN males, like fully null Mlh3 ^-/- males, have no spermatozoa and are infertile, yet spermatocytes have normal DSBs and undergo normal synapsis events in early prophase I. Unlike Mlh3 ^-/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3 ^DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3 ^DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3 ^-/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3 ^DN/DN males. Double mutant mice homozygous for the Mlh3 ^DN/DN and Mus81 ^-/- mutations show losses in chiasmata that approach levels observed in Mlh3 ^-/- males, indicating that the MUS81-EME1-regulated crossover pathway accounts for some of the increased residual chiasmata observed in Mlh3 ^DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3 ^DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3 ^DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata. SUMMARY: The MLH1-MLH3 complex is essential for crossing over in mammalian meiosis. We generated a mutation in mouse MLH3 that alters its conserved endonuclease domain and show that it disrupts crossing over in a manner distinct from the full null Mlh3 mouse, but also results in male infertility.

Background/Objectives: Geographical differences exist in the clinical presentation of polycystic ovary syndrome (PCOS). The degree to which ovarian morphology contributes to this variability is unknown. Methods: This study compared ovarian ultrasound features between women with PCOS residing in two geographical regions (India and the United States) using stored de-identified ultrasound scans from 331 women with PCOS. Sonographic markers of interest included follicle number per ovary (FNPO), follicle number per cross-section (FNPS), ovarian volume (OV), ovarian area (OA), stromal area (SA), and stromal-to-ovarian area ratio (S/A). Results: Most participants in both regions met the accepted criteria for polycystic ovarian morphology (India 87% vs. U.S. 83%). The U.S.-based group had a higher prevalence of follicle excess (41% in U.S. vs. 29% in India; p = 0.037), whereas the prevalence of ovarian enlargement was similar across groups (India 37% vs. U.S. 31%, p = 0.252). FNPS was higher in the U.S.-based group (p = 0.046), while the India-based group had higher OV (p = 0.010). SA and S/A did not differ between groups, albeit OA was slightly larger in women with PCOS from India (p = 0.022). Associations between ovarian morphology and menstrual cycle length (ρ = 0.16–0.25), hirsutism score (ρ = 0.19–0.23), and total testosterone (ρ = −0.33–0.42) were noted in both groups (p < 0.05). Conclusions: Some variation in ovarian morphology may exist across geographic regions. However, the degree of variability is unlikely to warrant regional definitions for polycystic ovarian morphology at this time.