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There is a general agreement that granulosa cell apoptosis is the cause of antral follicle attrition. Less clear is whether this pathway is also activated in case of preantral follicle degeneration, as several reports mention that the incidence of granulosa cell apoptosis in preantral follicles is negligible. Our objective is therefore to determine which cell-death pathways are involved in preantral and antral follicular degeneration. Atretic preantal and antral follicles were investigated using immunohistochemistry and lasercapture microdissection followed by quantitative real-time reverse transcription polymerase chain reaction. Microtubule-associated light-chain protein 3 (LC3), sequestosome 1 (SQSTM1/P62), Beclin1, autophagy-related protein 7 (ATG7), and cleaved caspase 3 (cCASP3) were used as markers for autophagy and apoptosis, respectively. P62 immunostaining was far less intense in granulosa cells of atretic compared to healthy preantral follicles, while no difference in LC3 and BECLIN1 immunostaining intensity was observed. This difference in P62 immunostaining was not observed in atretic antral follicles. mRNA levels of LC3 and P62 were not different between healthy and atretic (pre)antral follicles. ATG7 immunostaining was observed in granulosa cells of preantral atretic follicles, not in granulosa cells of degenerating antral follicles. The number of cCASP3-positive cells was negligible in preantral atretic follicles, while numerous in atretic antral follicles. Taken together, we conclude that preantral and antral follicular atresia is the result of activation of different cell-death pathways as antral follicular degeneration is initiated by massive granulosa cell apoptosis, while preantral follicular atresia occurs mainly via enhanced granulosa cell autophagy. Summary Sentence Preantral and antral follicles make use of different cell-death pathways, as antral follicular degeneration is initiated by massive granulosa cell apoptosis, while preantral follicular atresia is mainly dependent on enhanced granulosa cell autophagy.

In eukaryotes, diploid cells give rise to haploid cells via meiosis, a program of two cell divisions preceded by one round of DNA replication. Although key molecular components of the meiotic apparatus are highly conserved among eukaryotes, the mechanisms responsible for initiating the meiotic program have diverged substantially among eukaryotes. This raises a related question in animals with two distinct sexes: Within a given species, are similar or different mechanisms of meiotic initiation used in the male and female germ lines? In mammals, this question is underscored by dramatic differences in the timing of meiotic initiation in males and females. Stra8 is a vertebrate-specific, cytoplasmic factor expressed by germ cells in response to retinoic acid. We previously demonstrated that Stra8 gene function is required for meiotic initiation in mouse embryonic ovaries. Here we report that, on an inbred C57BL/6 genetic background, the same factor is also required for meiotic initiation in germ cells of juvenile mouse testes. In juvenile C57BL/6 males lacking Stra8 gene function, the early mitotic development of germ cells appears to be undisturbed. However, these cells then fail to undergo the morphological changes that define meiotic prophase, and they do not display the molecular hallmarks of meiotic chromosome cohesion, synapsis and recombination. We conclude that, in mice, Stra8 regulates meiotic initiation in both spermatogenesis and oogenesis. Taken together with previous observations, our present findings indicate that, in both the male and female germ lines, meiosis is initiated through retinoic acid induction of Stra8.

STUDY QUESTION Twenty years after the inception of the first fertility preservation programme for pre-pubertal boys, what are the current international practices with regard to cryopreservation of immature testicular tissue? SUMMARY ANSWER Worldwide, testicular tissue has been cryopreserved from over 3000 boys under the age of 18 years for a variety of malignant and non-malignant indications; there is variability in practices related to eligibility, clinical assessment, storage, and funding. WHAT IS KNOWN ALREADY For male patients receiving gonadotoxic treatment prior to puberty, testicular tissue cryopreservation may provide a method of fertility preservation. While this technique remains experimental, an increasing number of centres worldwide are cryopreserving immature testicular tissue and are approaching clinical application of methods to use this stored tissue to restore fertility. As such, standards for quality assurance and clinical care in preserving immature testicular tissue should be established. STUDY DESIGN, SIZE, DURATION A detailed survey was sent to 17 centres within the recently established ORCHID-NET consortium, which offer testicular tissue cryopreservation to patients under the age of 18 years. The study encompassed 60 questions and remained open from 1 July to 1 November 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS Of the 17 invited centres, 16 completed the survey, with representation from Europe, Australia, and the USA. Collectively, these centres have cryopreserved testicular tissue from patients under the age of 18 years. Data are presented using descriptive analysis. MAIN RESULTS AND THE ROLE OF CHANCE Since the establishment of the first formal fertility preservation programme for pre-pubertal males in 2002, these 16 centres have cryopreserved tissue from 3118 patients under the age of 18 years, with both malignant (60.4%) and non-malignant (39.6%) diagnoses. All centres perform unilateral biopsies, while 6/16 sometimes perform bilateral biopsies. When cryopreserving tissue, 9/16 centres preserve fragments sized ≤5 mm3 with the remainder preserving fragments sized 6–20 mm3. Dimethylsulphoxide is commonly used as a cryoprotectant, with medium supplements varying across centres. There are variations in funding source, storage duration, and follow-up practice. Research, with consent, is conducted on stored tissue in 13/16 centres. LIMITATIONS, REASONS FOR CAUTION While this is a multi-national study, it will not encompass every centre worldwide that is cryopreserving testicular tissue from males under 18 years of age. As such, it is likely that the actual number of patients is even higher than we report. Whilst the study is likely to reflect global practice overall, it will not provide a complete picture of practices in every centre. WIDER IMPLICATIONS OF THE FINDINGS Given the research advances, it is reasonable to suggest that cryopreserved immature testicular tissue will in the future be used clinically to restore fertility. The growing number of patients undergoing this procedure necessitates collaboration between centres to better harmonize clinical and research protocols evaluating tissue function and clinical outcomes in these patients. STUDY FUNDING/COMPETING INTEREST(S) K.D. is supported by a CRUK grant (C157/A25193). R.T.M. is supported by an UK Research and Innovation (UKRI) Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health at the University of Edinburgh is supported by MRC (MR/N022556/1). C.L.M. is funded by Kika86 and ZonMW TAS 116003002. A.M.M.v.P. is supported by ZonMW TAS 116003002. E.G. was supported by the Research Program of the Research Foundation—Flanders (G.0109.18N), Kom op tegen Kanker, the Strategic Research Program (VUB_SRP89), and the Scientific Fund Willy Gepts. J.-B.S. is supported by the Swedish Childhood Cancer Foundation (TJ2020-0026). The work of NORDFERTIL is supported by the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115), the Swedish Research Council (2018-03094; 2021-02107), and the Birgitta and Carl-Axel Rydbeck’s Research Grant for Paediatric Research (2020-00348; 2021-00073; 2022-00317; 2023-00353). C.E is supported by the Health Department of the Basque Government (Grants 2019111068 and 2022111067) and Inocente Inocente Foundation (FII22/001). M.P.R. is funded by a Medical Research Council Centre for Reproductive Health Grant No: MR/N022556/1. A.F. and N.R. received support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. K.E.O. is funded by the University of Pittsburgh Medical Center and the US National Institutes of Health HD100197. V.B-L is supported by the French National Institute of Cancer (Grant Seq21-026). Y.J. is supported by the Royal Children’s Hospital Foundation and a Medical Research Future Fund MRFAR000308. E.G., N.N., S.S., C.L.M., A.M.M.v.P., C.E., R.T.M., K.D., M.P.R. are members of COST Action CA20119 (ANDRONET) supported by COST (European Cooperation in Science and Technology). The Danish Child Cancer Foundation is also thanked for financial support (C.Y.A.). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.

In principle, germline cells possess the capability to transmit a nearly unaltered set of genetic material to infinite future generations, whereas somatic cells are limited by strict growth constraints necessary to assure an organism’s physical structure and eventual mortality. As the potential to replicate indefinitely is a key feature of cancer, we hypothesized that the activation of a “germline program” in somatic cells can contribute to oncogenesis. Our group recently described over one thousand germline specific genes that can be ectopically expressed in cancer, yet how germline specific processes contribute to the malignant properties of cancer is poorly understood. We here show that the expression of germ cell/cancer (GC) genes correlates with malignancy in lung adenocarcinoma (LUAD). We found that LUAD cells expressing more GC genes can repair DNA double strand breaks more rapidly, show higher rates of proliferation and are more resistant to ionizing radiation, compared to LUAD cells that express fewer GC genes. In particular, we identified the HORMA domain protein regulator TRIP13 to be predominantly responsible for this malignant phenotype, and that TRIP13 inhibition or expression levels affect the response to ionizing radiation and subsequent DNA repair. Our results demonstrate that GC genes are viable targets in oncology, as they induce increased radiation resistance and increased propagation in cancer cells. Because their expression is normally restricted to germline cells, we anticipate that GC gene directed therapeutic options will effectively target cancer, with limited side effects besides (temporary) infertility.

Non-small cell lung cancer (NSCLC) continues to be a global health challenge, with limited treatment options and a high mortality rate. We recently found that lung cancer cells that express more genes that are typically restricted to the germline (germ cell cancer genes, GC genes) repair DNA double-strand breaks more rapidly, show higher rates of proliferation, and are more resistant to ionizing radiation, compared to cells that express fewer GC genes. Moreover, we found that the gene encoding TRIP13 (thyroid hormone receptor interactor 13) plays a significant role in this malignant phenotype. Here, we demonstrate that melatonin (MT), a hormone synthesized in the pineal gland, downregulates the expression of TRIP13, particularly in lung cancer cells with high expression of TRIP13. Moreover, this downregulation of TRIP13 by MT further inhibits the DNA repair proteins RAD51 and XRCC5, thereby impairing DNA repair via homologous recombination and non-homologous end joining. We further found that the melatonin receptor 1B (MTNR1B), rather than melatonin receptor 1 A (MTNR1A), is essential for MT mediated TRIP13 downregulation. Because we also found that treatment with MT still decreases cell proliferation of TRIP13-KO cells, combining MT with the TRIP13 inhibitor DCZ0415 would likely have an additive anti-proliferative therapeutic effect in the treatment of NSCLC.

Autologous transplantation of spermatogonial stem cells is a promising new avenue to restore fertility in infertile recipients. Expansion of the initial spermatogonial stem cell pool through cell culturing is a necessary step to obtain enough cells for effective repopulation of the testis after transplantation. Since in vitro propagation can lead to (epi-)genetic mutations and possibly malignant transformation of the starting cell population, we set out to investigate genome-wide DNA methylation status in uncultured and cultured primary testicular ITGA6+ sorted cells and compare them with germ cell tumor samples of the seminoma subtype. Seminomas displayed a severely global hypomethylated profile, including loss of genomic imprinting, which we did not detect in cultured primary testicular ITGA6+ cells. Differential methylation analysis revealed altered regulation of gamete formation and meiotic processes in cultured primary testicular ITGA6+ cells but not in seminomas. The pivotal POU5F1 marker was hypomethylated in seminomas but not in uncultured or cultured primary testicular ITGA6+ cells, which is reflected in the POU5F1 mRNA expression levels. Lastly, seminomas displayed a number of characteristic copy number variations that were not detectable in primary testicular ITGA6+ cells, either before or after culture. Together, the data show a distinct DNA methylation patterns in cultured primary testicular ITGA6+ cells that does not resemble the pattern found in seminomas, but also highlight the need for more sensitive methods to fully exclude the presence of malignant cells after culture and to further study the epigenetic events that take place during in vitro culture.

Background Spermatogonial stem cell transplantation (SSCT) is proposed as a fertility therapy for childhood cancer survivors. SSCT starts with cryopreserving a testicular biopsy prior to gonadotoxic treatments such as cancer treatments. When the childhood cancer survivor reaches adulthood and desires biological children, the biopsy is thawed and SSCs are propagated in vitro and subsequently auto-transplanted back into their testis. However, culturing stress during long-term propagation can result in epigenetic changes in the SSCs, such as DNA methylation alterations, and might be inherited by future generations born after SSCT. Therefore, SSCT requires a detailed preclinical epigenetic assessment of the derived offspring before this novel cell therapy is clinically implemented. With this aim, the DNA methylation status of sperm from SSCT-derived offspring, with in vitro propagated SSCs, was investigated in a multi-generational mouse model using reduced-representation bisulfite sequencing. Results Although there were some methylation differences, they represent less than 0.5% of the total CpGs and methylated regions, in all generations. Unsupervised clustering of all samples showed no distinct grouping based on their pattern of methylation differences. After selecting the few single genes that are significantly altered in multiple generations of SSCT offspring compared to control, we validated the results with quantitative Bisulfite Sanger sequencing and RT-qPCRin various organs. Differential methylation was confirmed only for Tal2 , being hypomethylated in sperm of SSCT offspring and presenting higher gene expression in ovaries of SSCT F1 offspring compared to control F1. Conclusions We found no major differences in DNA methylation between SSCT-derived offspring and control, both in F1 and F2 sperm. The reassuring outcomes from our study are a prerequisite for promising translation of SSCT to the human situation.

Radiation therapy is a common treatment modality for lung cancer, and resistance to radiation can significantly affect treatment outcomes. We recently described that lung cancer cells that express more germ cell cancer genes (GC genes, genes that are usually restricted to the germ line) can repair DNA double-strand breaks more rapidly, show higher rates of proliferation and are more resistant to ionizing radiation than cells that express fewer GC genes. The gene encoding TRIP13 appeared to play a large role in this malignant phenotype. However, the molecular regulatory mechanism of TRIP13 in radiation resistance remained largely unknown. Here, we show that TRIP13 is a key contributor to non-small cell lung cancer (NSCLC) treatment resistance, particularly in patients following radiation treatment, for whom levels of TRIP13 expression are correlated with a poor prognosis. Repeated irradiation of led to an increase of basal TRIP13 levels and radioresistance. This effect of radioresistance could be enhanced or abrogated by overexpressing or knocking out TRIP13. Elevated TRIP13 is also correlated with enhanced repair of radiation-induced DNA damage. We further showed the proteins NBS1 and RAD51 (homologous recombination. HR) and XRCC5 (non-homologous end-joining, NHEJ) to act downstream of TRIP13, although inhibition of TRIP13 mostly reduced the HR associated proteins in response to induced resistance to irradiation. This study elucidates a novel mechanism of treatment resistance in NSCLC cells, in which TRIP13 promotes HR mediated DNA repair and resistance to ionizing radiation.

Despite the importance of mutation in genetics, there are virtually no experimental data on the occurrence of specific nucleotide substitutions in human gametes. C>G transversions at position 755 of FGF receptor 2 (FGFR2) cause Apert syndrome; this mutation, encoding the gain-of-function substitution Ser252Trp, occurs with a birth rate elevated 200- to 800-fold above background and originates exclusively from the unaffected father. We previously demonstrated high levels of both 755C>G and 755C>T FGFR2 mutations in human sperm and proposed that these particular mutations are enriched because the encoded proteins confer a selective advantage to spermatogonial cells. Here, we examine three corollaries of this hypothesis. First, we show that mutation levels at the adjacent FGFR2 nucleotides 752-754 are low, excluding any general increase in local mutation rate. Second, we present three instances of double-nucleotide changes involving 755C, expected to be extremely rare as chance events. Two of these double-nucleotide substitutions are shown, either by assessment of the pedigree or by direct analysis of sperm, to have arisen in sequential steps; the third (encoding Ser252Tyr) was predicted from structural considerations. Finally, we demonstrate that both major alternative spliceforms of FGFR2 (Fgfr2b and Fgfr2c) are expressed in rat spermatogonial stem cell lines. Taken together, these observations show that specific FGFR2 mutations attain high levels in sperm because they encode proteins with gain-of-function properties, favoring clonal expansion of mutant spermatogonial cells. Among FGFR2 mutations, those causing Apert syndrome may be especially prevalent because they enhance signaling by FGF ligands specific for each of the major expressed isoforms.

Mesenchymal stem cells hold great promise for regenerative medicine as they can be easily isolated from different sources such as adipose tissue, bone marrow, and umbilical cord blood. Spontaneously arising pluripotent stem cells can be obtained in culture from murine spermatogonial stem cells (SSCs), while the pluripotency of the human counterpart remains a matter of debate. Recent gene expression profiling studies have demonstrated that embryonic stem cell- (ESC-) like cells obtained from the human testis are indeed closer to mesenchymal stem cells (MSCs) than to pluripotent stem cells. Here, we confirm that colonies derived from human testicular cultures, with our isolation protocol, are of mesenchymal origin and do not arise from spermatogonial stem cells (SSCs). The testis, thus, provides an important and accessible source of MSCs (tMSCs) that can be potentially used for nephrotoxicity testing in vitro. We further demonstrate, for the first time, that tMSCs are able to secrete microvesicles that could possibly be applied to the treatment of various chronic diseases, such as those affecting the kidney.