Author: Mingyao Yang
Publisher:
Published: 2023
Total Pages: 0
ISBN-13:
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Spermatozoa serves as the vehicle for transmitting the male's genetic contribution to the succeeding generation. Producing substantial and functional spermatozoa is essential for male fertility. Mammalian spermatogenesis is a highly coordinated and continuous process in which spermatogonial stem cells (SSCs) undergo differentiation to produce functional spermatozoa. Precise gene regulation in germ cells directs their development, guaranteeing the ongoing production of a significant quantity of spermatozoa throughout the reproductive lifespan. Many mysteries surrounding gene regulation mechanisms during spermatogenesis remain unresolved. Spermatogenesis encompasses three pivotal transitions in germ cells: the shift from undifferentiated to differentiating state among spermatogonia, meiosis involving spermatocytes, and the transformation of spermatids through spermiogenesis. Retinoic acid (RA) signaling is vital in governing all three transitions of germ cells during spermatogenesis. RA is periodically synthesized by Sertoli cells and germ cells and regulates genes associated with germ cell differentiation, meiosis, proliferation, and apoptosis. Members of the PRAME (Preferentially expressed antigen in melanoma, also known as PRAME nuclear receptor transcriptional regulator) family are identified as repressors of RA signaling. This family constitutes a significant group that exhibits broad expression during germline development. PRAME members have conserved leucine-rich repeat (LRR) domains, which are folded into a horseshoe shape in their tertiary structure, facilitating protein--protein interactions in various molecular recognition processes, including signal transduction. The LRR domains of PRAME have the capability to engage with RA receptors (RARs), thus suppressing RAR signaling transduction in both cancer and embryonic stem cells (ESCs). However, the functional roles of the PRAME family during spermatogenesis are poorly understood. Furthermore, the interaction between these members of the PRAME family during spermatogenesis has not been investigated. To address these questions, we have directed our attention towards two members of the mouse Prame gene family, Prame like, X-linked 1 (Pramex1) (ID: 75829) and Prame like 1 (Pramel1) (ID:83491), and their involvement in spermatogenesis and oogenesis. To investigate their functional roles, we generated and characterized six different lines of Prame transgenic mice, including conditional Pramex1 knockout (cKO), Pramex1 global KO (gKO), Pramel1 cKO, Pramel1 gKO, Id4-eGfp+Pramel1 gKO and Pramex1/ Pramel1 double KO (dKO) mice. The study of these models provided valuable insights into the functions of the PRAME family during gametogenesis. Our hypothesis was that these two genes were involved in gametogenesis through repressing RA signaling pathway, and collectively contribute to the process of germ cell formation. The primary objectives of this project were to unravel the cellular and molecular mechanisms underlying the roles of these two members during gametogenesis, as well as to explore their collaborative interactions. We found that ablation of Pramex1 in the mutant mice causes apoptosis of pachytene spermatocytes during spermatogenesis, having negative effects on 5~7% of seminiferous tubules, with a Sertoli cell-only (SCO) phenotype during the first round of spermatogenesis in young testes. We speculated that these cellular defects resulted from the disruption of the RA signaling due to the depletion of Pramex1. Due to these cellular defects, the Pramex1 cKO mice had a 12% reduction in testis size and sperm count. However, the Pramex1 cKO mice were fertile even with these reproductive phenotypes observed in the young and mature males. The Pramex1 cKO females appeared to be normal with no observed abnormal phenotypes. Unlike the Pramex1 conditional deletion, which did not affect male fecundity, Pramel1 deficiency led to a 43% increase in fecundity of juvenile males and an 18% decrease in fecundity of mature Pramel1 gKO males. The enhanced fecundity in young Pramel1 gKO males resulted from a 32% increase in sperm production during the first round of spermatogenesis. Conversely, the reduction in fecundity in mature mice resulted from fewer germ cells being processed in subsequent rounds of spermatogenesis. For the second round of spermatogenesis, we discovered that Pramel1 global deficiency led to apoptosis of the initial progenitor cells, characterized as ID4-eGFPMid cells by using the Id4-eGfp+Pramel1 gKO line. Apoptosis of progenitor induced the formation of the SCO phenotype in about 7% of seminiferous tubules in the Pramel1 gKO mice. The SCO phenotype was rescued through the administration of the RA inhibitor, WIN18,446, indicating that PRAMEL1 serves as a repressor of RA signaling during spermatogonia development. Furthermore, our data indicated that Pramel1 not only affects progenitors in young males, but also contributed to the maintenance of undifferentiated spermatogonial populations in mature mice, as demonstrated by the heat-stress experiment. Overall, our results revealed that PRAMEL1 acts as a fine tuner in RA signaling to ensure the proper establishment of the first, second and subsequent rounds of spermatogenesis. Results obtained from the Pramex1 and Pramel1 mutant mice indicated that both genes play minor roles during spermatogenesis. We hypothesize that the individual members of the Prame family act as fine-tuners of RA signaling, and collectively, the family represses RA signaling to regulate gametogenesis. Interestingly, when either Pramex1 or Pramel1 was knocked out in their single knockout (sKO) mice, we observed a compensatory upregulation of the other gene. To confirm the compensatory ability of the two members, we further determined the impact of the double deletion of Pramex1/Pramel1 on gametogenesis by using a Pramex1/Pramel1 dKO mice model. In the case of sKO mice, the male mice had a relatively mild phenotypes except for the 7% SCO tubules, while the female mice remained unaffected. When we examined the dKO mice, we discovered more severe defects, suggesting that the two genes aligned with a genetic model exhibiting synergistic genetic enhancement. The double deletion similarly led to a reduction in fecundity by approximately 50% in both male and female gametogenesis. The reduced fecundity of male dKO mice was attributed to their (12-50%) smaller testis size, a (12-58%) decrease in sperm production, and a 47% reduction in litter size compared to WT mice. Moreover, the dKO juvenile and mature males exhibited a larger SCO region, an increased number of apoptotic cells, but fewer undifferentiated spermatogonia compared to sKOs and WT mice. Similarly, the diminished fecundity observed in dKO females was evident in a 57% smaller litter size, which could be attributed to a 51% decrease in the total oocyte count in the ovaries compared to WT females. We believed that the synergistic genetic enhancement of these two genes was a result of their mutual genetic compensatory ability. We also revealed that their compensatory ability was driven by their shared function of finely repressing RA signaling during gametogenesis. Our RA treatment experiment determined that the RA inhibitor effectively rescued the defects observed in sKOs and dKO mice. Besides, we confirmed that the PRAMEX1 also interacted with RAR[alpha], similar to PRAMEL1. In summary, our study sheds light on the compensatory ability played by Pramex1 and Pramel1 under the regulation of RA signaling, suggesting a collective contribution among the Prame members for gametogenesis. In conclusion, this study delved into the functional roles and genetic interactions of the mouse Pramex1 and Pramel1 during gametogenesis. These investigations revealed the important roles of Pramex1 and Pramel1 in germ cell development. Furthermore, we elucidated the mechanisms by which the two genes (Pramex1 and Pramel1) both suppress RA signaling. Remarkably, these two genes demonstrated synthetic genetic enhancement during gametogenesis through their complementary ability in their shared pathway, repressing RA signaling. The discoveries from this project offer valuable insights into the Prame gene family, indicating that these genes collectively play crucial roles during gametogenesis.
