Semen parameters, including sperm counts, have rapidly declined in men across the globe over the last five decades. Although this decline remains unexplained, lifestyle factors may affect male fertility. Recently, several studies highlighted a potential link between non-steroidal anti-inflammatory drug (NSAID) usage, such as naproxen and ibuprofen, and declining male fertility. However, the mechanisms by which these common analgesics affect male fertility, including their effects on the blood-testis barrier (BTB), remain poorly characterized. Utilizing an in vitro rhesus macaque non-human primate (NHP) BTB model, we demonstrate that serum levels of naproxen and ibuprofen alter the function of BTB. Following short-term naproxen and ibuprofen treatment of NHP primary Sertoli cells, we show that these NSAIDs increase the transepithelial electrical resistance, indicating an overall strengthening of the Sertoli cell junctions. Furthermore, naproxen and ibuprofen treatment alter the expression of genes involved in maintaining the BTB. Specifically, the genes that were significantly expressed in response to ibuprofen exposure were enriched for human phenotypic abnormalities linked to male factor infertility. Together, these results suggest that short-term naproxen and ibuprofen treatment disrupt the function of the BTB by altering the integrity of the Sertoli cell junctions, proposing a potential role of NSAIDs in male factor infertility.

Mammalian spermatogenesis is a highly dynamic process involving male germ cell self-renewal and terminal differentiation into progenitor cells that produce testicular sperm in coordination with testicular somatic support cells. Multinuclear giant cells identified in mutant, toxin-exposed or genetically altered mice post-meiosis, reflect abnormal spermiogenesis. Here, analysis of fixed isolated testicular cells immunolabeled for microtubules, molecular motors, spindle binding proteins, and DNA from non-transgenic and GFP-CETN2-expressing CB6F1 or C57BL mice identify polynuclear primary spermatocytes within the normal testicular cell population. We hypothesize polynuclear primary spermatocytes arise from spermatogonia through aberrant cytokinesis or cell fusion of tethered spermatogonia. Polynuclear primary spermatocytes apparently progress in pre-prophase-I stages, completing first centriole duplication and homologous chromosome pairing/DNA cross-over events, forming multiple bipolar metaphase-I spindles of distinct phenotypes based on hypothesized spermatogonia derivation but without the microtubule clustering motor protein KIFC1. Remarkably, polynuclear spermatocytes are consistent with completing meiosis without undergoing first or second cytokinesis, seemingly generating polyploid spermatids post-meiosis. Polyploid spermatids perhaps undergo cytodifferentiation, forming sperm axonemes, acrosomes, centrosome nuclear binding, transient manchette microtubule assembly for nuclear shaping, associating with Sertoli cells and entering suspected spermiation. Collectively, these findings demonstrate remarkable testicular plasticity post-differentiation and the lax cell-cycle control of spermatogenesis, regardless of nuclear constituency.

Transforming acidic acid coiled-coil protein 3 (TACC3) and cytoskeleton associated protein 5 (cKAP5; or colonic hepatic tumor overexpressed gene, chTOG) are vital for spindle assembly and stabilization initiated through TACC3 Aurora-A kinase interaction. Here, TACC3 and cKAP5/chTOG localization with monospecific antibodies is investigated in eGFP-centrin-2- expressing mouse meiotic spermatocytes. Both proteins bind spermatocyte spindle poles but neither kinetochore nor interpolar microtubules, unlike in mitotic mouse fibroblasts or female meiotic oocyte spindles. Spermatocytes do not display a liquid-like spindle domain (LISD), although fusing them into maturing oocytes generates LISD-like TACC3 condensates around sperm chromatin but sparse microtubule assembly. Microtubule inhibitors do not reduce TACC3 and cKAP5/chTOG spindle pole binding. MLN 8237 Aurora-A kinase inhibitor removes TACC3, not cKAP5/chTOG, disrupting spindle organization, chromosome alignment, and impacting spindle pole γ-tubulin intensity. The LISD disruptor 1,6-hexanediol abolished TACC3 in spermatocytes, impacting spindle bipolarity and chromosome organization. Cold microtubule disassembly and rescue experiments in the presence of 1,6-hexanediol reinforce the concept that spermatocyte TACC3 spindle pole presence is not required for spindle pole microtubule assembly. Collectively, meiotic spermatocytes without a LISD localize TACC3 and cKAP5/chTOG exclusively at spindle poles to support meiotic spindle pole stabilization during male meiosis, different from either female meiosis or mitosis.

Objective: To demonstrate that functional spermatids can be derived in vitro from nonhuman primate pluripotent stem cells. Design: Green fl uorescent protein-labeled, rhesus macaque nonhuman primate embryonic stem cells (nhpESCs) were differentiated into advanced male germ cell lineages using a modified serum-free spermatogonial stem cell culture medium. In vitro-derived round spermatidlike cells (rSLCs) from differentiated nhpESCs were assessed for their ability to fertilize rhesus oocytes by intracytoplasmic sperm(atid) injection. Setting: Multiple academic laboratory settings. Patient(s): Not applicable. Intervention(s): Intracytoplasmic sperm(atid) injection of in vitro-derived spermatids from nhpESCs into rhesus macaque oocytes. Main Outcome Measure(s): Differentiation into spermatogenic cell lineages was measured through multiple assessments including ribonucleic acid sequencing and immunocytochemistry for various spermatogenic markers. In vitro spermatids were assessed for their ability to fertilize oocytes by intracytoplasmic sperm(atid) injection by assessing early fertilization events such as spermatid deoxyribonucleic acid decondensation and pronucleus formation/apposition. Preimplantation embryo development from the one-cell zygote stage to the blastocyst stage was also assessed. Result(s): Nonhuman primate embryonic stem cells can be differentiated into advanced germ cell lineages, including haploid rSLCs. These rSLCs undergo deoxyribonucleic acid decondensation and pronucleus formation/apposition when microinjected into rhesus macaque mature oocytes, which, after artificial activation and coinjection of ten-eleven translocation 3 protein, undergo embryonic divisions with approximately 12% developing successfully into expanded blastocysts. Conclusion(s): This work demonstrates that rSLCs, generated in vitro from primate pluripotent stem cells, mimic many of the capabilities of in vivo round spermatids and perform events essential for preimplantation development. To our knowledge, this work represents, for the fi rst time, that functional spermatid-like cells can be derived in vitro from primate pluripotent stem cells. (Fertil Steril Sci (R) 2021;2:365-75. (c) 2021 by American Society for Reproductive Medicine.)

With nearly ten million babies conceived globally, using assisted reproductive technologies, fundamental questions remain; e.g., How do the sperm and egg DNA unite? Does ICSI have consequences that IVF does not? Here, pronuclear and mitotic events in nonhuman primate zygotes leading to the establishment of polarity are investigated by multidimensional time-lapse video microscopy and immunocytochemistry. Multiplane videos after ICSI show atypical sperm head displacement beneath the oocyte cortex and eccentric para-tangential pronuclear alignment compared to IVF zygotes. Neither fertilization procedure generates incorporation cones. At first interphase, apposed pronuclei align obliquely to the animal-vegetal axis after ICSI, with asymmetric furrows assembling from the male pronucleus. Furrows form within 30° of the animal pole, but typically, not through the ICSI injection site. Membrane flow drives polar bodies and the ICSI site into the furrow. Mitotic spindle imaging suggests para-tangential pronuclear orientation, which initiates random spindle axes and minimal spindle:cortex interactions. Parthenogenetic pronuclei drift centripetally and assemble astral spindles lacking cortical interactions, leading to random furrows through the animal pole. Conversely, androgenotes display cortex-only pronuclear interactions mimicking ICSI. First cleavage axis determination in primates involves dynamic cortex-microtubule interactions among male pronuclei, centrosomal microtubules, and the animal pole, but not the ICSI site.

In the original version of this Article, the affiliation details for Jadranka Loncarek and Vito Mennella were incorrectly given as 'Cell Biology Program, The Hospital for Sick Children, Department of Biochemistry, University of Toronto, 555 University Avenue, Toronto, ON, M5G 1X8, Canada' and 'Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, 1050 Boyles Street, Frederick, MD, 21702, USA', respectively. This has now been corrected in both the PDF and HTML versions of the Article.

Oocytes, including from mammals, lack centrioles, but neither the mechanism by which mature eggs lose their centrioles nor the exact stage at which centrioles are destroyed during oogenesis is known. To answer questions raised by centriole disappearance during oogenesis, using a transgenic mouse expressing GFP-centrin-2 (GFP CETN2), we traced their presence from e11.5 primordial germ cells (PGCs) through oogenesis and their ultimate dissolution in mature oocytes. We show tightly coupled CETN2 doublets in PGCs, oogonia, and pre-pubertal oocytes. Beginning with follicular recruitment of incompetent germinal vesicle (GV) oocytes, through full oocyte maturation, the CETN2 doublets separate within the pericentriolar material (PCM) and a rise in single CETN2 pairs is identified, mostly at meiotic metaphase-I and -II spindle poles. Partial CETN2 foci dissolution occurs even as other centriole markers, like Cep135, a protein necessary for centriole duplication, are maintained at the PCM. Furthermore, live imaging demonstrates that the link between the two centrioles breaks as meiosis resumes and that centriole association with the PCM is progressively lost. Microtubule inhibition shows that centriole dissolution is uncoupled from microtubule dynamics. Thus, centriole doublets, present in early G2-arrested meiotic prophase oocytes, begin partial reduction during follicular recruitment and meiotic resumption, later than previously thought.

The forms and locations of centrosomes in mouse oocytes and in sea urchin eggs were followed through the whole course of fertilization and first cleavage by immu- nofluorescence microscopy. Centrosomes were identified with an autoimmune antiserum to centrosomal material. Staining of the same preparations with tubulin antibody and with the DNA dye Hoechst 33258 allowed the correlation of the forms of the centrosomes with the microtubule structures that they generate and with the stages of meiosis, syngamy, and mitosis. The results with sea urchin eggs conform to Boveri's view on the paternal origin of the functional centrosomes. Centrosomes are seen in spermatozoa and enter the egg at fertilization. Initially, the centrosomes are compact, but as the eggs enter the mitotic cycle the forms of the centrosomes go through a cycle in which they spread during interphase, apparently divide, and con- dense into two compact poles by metaphase. In anaphase, they spread to form flat poles. In telophase and during reconstitu- tion of the daughter nuclei, the centrosomal material is disposed as hemispherical caps around the poleward surfaces of the nuclei. Mouse sperm lack centrosomal antigen. In the unfertilized mouse oocyte, the meiotic spindle poles are dis- played as broad-beaded centrosomes. In addition, centrosomal material is detected in the cytoplasm as particles, about 16 in number, which are foci of small aster-like arrays of microtu- bules. The length and number of astral microtubules correlate with the size of the centrosomal foci. After sperm incorpo- ration, as the pronuclei develop and more cytoplasmic micro- tubules assemble, a few of the foci associate with the peripheries of the nuclei. The number of foci multiplies during the first cell cycle. At the end of interphase, all of the centrosomal foci have concentrated on the nuclear peripheries and the cytoplasmic microtubules have disappeared. At prophase, the centrosomes are seen as two irregular clusters, marking the poles which, at metaphase and anaphase, appear as rough bands with foci, and the spindle is typically barrel-shaped. At telophase, the centrosomes are seen as arcs that lie on the nuclear peripheries after cleavage. The ordering of microtubules in all the stages reflects the shapes of the centrosomes. The findings on the sea urchin confirm the classical theory of the paternal origin of centrosomes and contrast with observations tracing the mitotic poles of the mouse egg to maternal centrosomal material. This evidence strengthens the conclusion that mouse centrosomes derive from the oocyte.

The fertilizing sperm’s lengthiest unchartered voyage is through the longest, least-investigated organ in a man’s body – the Epididymis. Over six meters long in men, ~80 meters in stallions and over one-hundred times a mouse’s body length, there are few functions known aside from sperm storage and nutrition. While spermatogenesis is completed in the testes, here we demonstrate sperm centriole reduction occurs within the epididymis. Investigations of GFP-CENTR mice and controls demonstrate both the presence of centriole pairs in the upper caput region of the epididymis and, the destruction, first, of the distal and, then, of the proximal centriole as the sperm transits to the cauda and vas deferens in preparation for its climactic release. These centrioles can neither recruit γ-tubulin nor nucleate microtubules when eggs are inseminated or microinjected, yet numerous maternally-nucleated cytasters are found. These sperm centrioles appear as vestigial basal bodies, destroyed in the mid-to-lower corpus. Post-testicular sperm maturation, in which sperm centrioles found in the caput are destroyed prior to ejaculation, is a newly discovered function for the epididymis.

The ability of microtubules of the mitotic apparatus to control the positioning and initiation of the cleavage furrow during cytokinesis was first established from studies on early echinoderm embryos. However, the identity of the microtubule population that imparts cytokinetic signaling is unclear. The two main-and not necessarily mutually exclusive-candidates are the central spindle and the astral rays. In the present study, we examined cytokinesis in ammonia-activated sea urchin eggs, which lack paternally derived centrosomes and undergo mitosis mediated by unusual anastral, bipolar mini-spindles. Live cell imaging and immunolabeling for microtubules and the centralspindlin constituent and kinesin-related protein, MKLP1, demonstrated that furrowing in ammonia-activated eggs was associated with aligned arrays of centralspindlin-linked, opposed bundles of antiparallel microtubules. These autonomous, zipper-like arrays were not associated with a mitotic apparatus, but did possess characteristics similar to the central spindle region of control, fertilized embryos. Our results highlight the self-organizing nature of the central spindle region and its ability to induce cytokinesis-like furrowing, even in the absence of a complete mitotic apparatus.

SummaryAmong transgenic mice with ubiquitous Cre recombinase activity, all strains to date excise loxP‐flanked (floxed) alleles either at or before the zygote stage or at nondescript stages of development. This manuscript describes a new mouse strain, in which Cre recombinase, integrated into the Esrrb locus, efficiently excises floxed alleles in pre‐implantation embryos at the onset of the four‐cell stage. By enabling inactivation of genes only after the embryo has undergone two cleavages, this strain should facilitate in vivo studies of genes with essential gametic or zygotic functions. In addition, this study describes a new, highly pluripotent hybrid C57BL/6J x 129S1/SvImJ mouse embryonic stem cell line, HYB12, in which this knockin and additional targeted alleles have been generated. genesis 54:53–61, 2016. © 2015 Wiley Periodicals, Inc.

Centriole construction, now revealed by crystallography, proteomics, and imaging to be a sophisticated assembly of interlocking bricks, resembles LEGOs—albeit centrioles have remarkable dynamic capabilities, including self‐assembly and dis‐assembly, kinases and post‐translational modifications, self‐replication, and still mysterious mechanisms for transmission through each cell cycle and via the gametes during development. Centrioles are created by core proteins that aggregate to form unique ninefold‐symmetrical paracrystalline cylinders. The centrosome then coalesces as a cloud of pericentriolar material (PCM) around the centriole. Together they comprise the cell's microtubule organizing center (MTOC), which governs the shape, functions, and dynamics of the cell's microtubule (MT) arrays. This includes the meiotic and mitotic spindle apparatus for chromosome segregation, the accuracy of which is crucial for avoiding aneuploidies and resulting cancer, birth defects, or infertility. Centrioles’ replication and transmission mechanisms—and reduplication blocks—across cell cycles and generations, are only now becoming tractable to molecular analysis, which allows research to address questions about spindle assembly with neither centrioles nor centrosomes or de novo centriole formation. Here we discuss the latest insights into centriole and centrosome assembly and function and their transgenerational inheritance.

With advances in cancer therapies, survival rates in prepubescent patients have steadily increased. However, a number of these surviving patients have been rendered sterile owing to their rigorous oncologic treatment regimens. In addition to cancer treatments, men and women, who are genetically fertile, can become infertile owing to immune suppression treatments, exposure to environmental and industrial toxicants, and injury. Notwithstanding the great emotional burden from an inability to conceive a child with their partner, the financial burdens for testing and treatment are high, and successful treatment of these patients' sterility is rare. Recent advances in pluripotent stem cell differentiation and the generation of patient-specific, induced pluripotent stem cells indicate that stem cell replacement therapies or in vitro differentiation followed by IVF may be on the horizon. Here we discuss these recent advances, their relevance to treating male-factor and female-factor infertility, and what experimental procedures must be carried out in animal models before these exciting new treatments can be used in a clinical setting. The goal of this research is to generate functional gametes from no greater starting material than a mere skin biopsy.

Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and several biomedical rationales. Here, we consider several strategies for deriving gametes from PSCs from mice and primates (human and non-human) and their anticipated strengths, challenges and limitations. Although the ‘Weismann barrier’, which separates the mortal somatic cell lineages from the potentially immortal germline, has long existed, breakthroughs first in mice and now in humans are artificially creating germ cells from somatic cells. Spermatozoa with full reproductive viability establishing multiple generations of seemingly normal offspring have been reported in mice and, in humans, haploid spermatids with correct parent-of-origin imprints have been obtained. Similar progress with making oocytes has been published using mouse PSCs differentiated in vitro into primordial germ cells, which are then cultured after xenografting reconstructed artificial ovaries. Progress in making human oocytes artificially is proving challenging. The usefulness of these artificial gametes, from assessing environmental exposure toxicity to optimising medical treatments to prevent negative off-target effects on fertility, may prove invaluable, as may basic discoveries on the fundamental mechanisms of gametogenesis.

The promise of nuclear transfer (NT) in producing patient-specific embryonic stem cells for regenerative medicine holds great interest for the treatment of human diseases, whether as tools for "disease in a dish" discoveries or as replacement cells in patient therapies. With the discovery of induced pluripotent stem cells, current debates flourish over whether human NT is necessary, given the limitations of burdensome technical, ethical, and legal issues. Non-human primates (NHP) are superior models for understanding the stepwise events of successful primate NT and could provide easier extrapolations to events in humans than rodent cloning model. Early challenges to producing cloned NHP blastocysts using traditional NT technologies successful in rodent and cows were overcome by modifications to enucleation, artificial activation, and embryo culture in monkeys. Regardless, adoption of these technical advances in human NT has yet to produce stable human pluripotent embryonic stem cells, and recent reports suggest that human meiotic spindle removal as the first step in the cloning process remains detrimental to producing viable human ESC lines. This chapter provides a review on challenges in nuclear transfer in primates – non-human and human alike.

Embryonic stem (ES) cells are characterized by pluripotency, defined as the developmental potential to generate cell lineages derived from all three primary germ layers. In the past decade, great progress has been made on the cell culture conditions, transcription factor programs and intracellular signaling pathways that control both murine and human ES cell fates. ES cells of mouse vs. human origin have distinct culture conditions, responding to some tyrosine kinase signaling pathways in opposite ways. Previous work has implicated the Src family of non-receptor protein–tyrosine kinases in mouse ES cell self-renewal and differentiation. Seven members of the Src kinase family are expressed in mouse ES cells, and individual family members appear to play distinct roles in regulating their developmental fate. Both Hck and c-Yes are important in self-renewal, while c-Src activity alone is sufficient to induce differentiation. While these findings implicate Src-family kinase signaling in mouse ES cell renewal and differentiation, the role of this kinase family in human ES cells is largely unknown. Here, we explored Src-family kinase expression patterns and signaling in human ES cells during self-renewal and differentiation. Of the eleven Src-related kinases in the human genome, Fyn, c-Yes, c-Src, Lyn, Lck and Hck were expressed in H1, H7 and H9 hES cells, while Fgr, Blk, Srm, Brk, and Frk transcripts were not detected. Of these, c-Yes, Lyn, and Hck transcript levels remained constant in self-renewing human ES cells vs. differentiated EBs, while c-Src and Fyn showed a modest increase in expression as a function of differentiation. In contrast, Lck expression levels dropped dramatically as a function of EB differentiation. To assess the role of overall Src-family kinase activity in human ES cell differentiation, cultures were treated with inhibitors specific for the Src kinase family. Remarkably, human ES cells maintained in the presence of the potent Src-family kinase inhibitor A-419259 retained the morphology of domed, pluripotent colonies and continued to express the self-renewal marker TRA-1-60 despite culture under differentiation conditions. Taken together, these observations support a role for Src-family kinase signaling in the regulation of human ES cell fate, and suggest that the activities of individual Src-family members are required for the initiation of the differentiation program.

Differentiation of stem cells into spermatogenic lineages in vitro provides a unique window into the biological mechanisms responsible for driving pluripotent stem cells into essential progeny—haploid spermatids and viable sperm—as well as provides an innovative approach for determining novel root causes for male infertility. Our recent work outlined a novel approach for differentiating human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) into advanced spermatogenic lineages including haploid spermatids with correct parent-of-origin genomic imprints on two loci. The work described here in this chapter provides a foundation for building a true in vitro model for human spermatogenesis with which to model, diagnose and potentially treat male factor infertility.

In this study, we used a large non‐human primate model, the baboon, to establish a step‐wise protocol to generate CD34+ endothelial progenitor cells (EPCs) from embryonic stem cells (ESCs) and to demonstrate their reparative effects. Baboon ESCs were sequentially differentiated from embryoid body cultures for 9 days and then were specified into EPCs by culturing them in monolayer for 12 days. The resulting EPCs expressed CD34, CXCR4 and UEA‐1, but neither CD31 nor CD117. The EPCs were able to form intact lumen structures when seeded on Matrigel, took up Dil‐LDL, and responded to TNF‐α. Angioblasts specified in EGM‐2 medium and ECGS medium had 6.41 ± 1.16% (n = 3) and 9.32 ± 3.73% CD34+ cells (n = 3). The efficiency of generating CD34+ EPCs did not differ significantly from ECGS to EGM‐2 culture media, however, angioblasts specified in ECGS medium expressed a higher percentage of CD34+/CXCR4+ cells (3.49 ± 1.32%, n = 3) than those specified in EGM‐2 medium (0.49 ± 0.52%, n = 3). To observe their reparative capacity, we purified CD34+ progenitors after specification by EGM‐2 medium; inoculated fluorescently labelled CD34+ EPCs into an arterial segment denuded of endothelium in an ex vivo system. After 14 days of ex vivo culture, the grafted cells had attached and integrated to the denuded surface; in addition, they had matured further and expressed terminally differentiated endothelial markers including CD31 and CD146. In conclusion, we have proved that specified CD34+ EPCs are promising therapeutic agents for repairing damaged vasculature.

There is an increasing need for an animal model that can be used to translate basic research into clinical therapy. We documented the differentiation and functional competence of embryonic stem cell (ESC)-derived endothelial cells in baboons. Baboon angioblasts were sequentially differentiated from embryoid body cultures for 9 days in an angioblast differentiation medium with varying concentrations of BMP-4, FLT-3 ligand, stem cell factor, thrombopoietin, basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and knockout serum replacement. Real-time polymerase chain reaction results showed that ESC-derived angioblasts down-regulated NANOG and OCT3/4, upregulated T-brachyury and GATA2, and moderately expressed CD34; they did not express CD144, TEK, or VWF, and varied in levels of CD31 expression. Several populations of putative angioblasts appeared 3 days and 9 days after differentiation, as identified by flow cytometry. Angioblasts at this stage exhibited dual paths of differentiation toward hematopoietic and vascular fates. To examine whether derived angioblasts could reconstitute the endothelium, we built an ex vivo culture system and seeded fluorescently labeled angioblast cultures onto a denuded segment of the femoral artery. We found that the seeded cells were able to grow into the endothelium on the interior surface of denuded artery segments within 5 days after seeding. After 14 days of ex vivo culture, the transplanted cells expressed CD31, an endothelial marker. The control arteries, seeded with vehicle only, did not harbor cells with endothelial markers. We conclude that ESC-derived angioblasts are promising therapeutic agents for repairing damaged vasculature, and that the baboon model will be vital for optimizing therapies for human clinical studies.