Evolution Of The Mammalian Testis Location And Molecular Evolutionary Mechanisms Of Healthy 'cryptorchidism'

The healthy male reproductive system and normal reproductive ability are the important guarantee for individual reproduction,population reproduction and expansion,gene exchange of mammals.The testis is the core reproductive organ in the male reproductive system,which is temperature dependent.Therefore,a cooler environment than in the abdomen is indeed required for the normal spermatogenesis and sperm storage.Local high temperature is recognized by the World Health Organization as one of the factors that have a strong damaging effect on male reproductive ability,and it has been found that there is a certain relationship between the temperature of the testis and the fertility decline.In most mammals,including humans,the testis descents into the scrotum,which is posited outside the body core,to achieve the lower temperature.In fact,mammals have a rich diversity of testicular positions,not all mammalian testes are located in the scrotum naturally.It is more interesting that mammals with natural “cryptorchid testis” have reproductive and life health similar to those of species with scrotal testicles.In spite of the interesting observations,it is still controversial of the evolutionary history of the testicular positions and molecular studies on the mechanisms under the evolution of diverse testicular positions in mammals are scarce.Therefore,the present study aims to,first,investigate the mammalian evolutionary history of testicular positions(i.e.,the completely descended scrotal testis,the incompletely descended ascrotal testis and the undescended ascrotal testis)in mammals by reconstructing the ancestral state.Next,to reveal the potential molecular mechanisms under the evolution of the diverse testicular positions as well as under the normal reproductive and life health,the present study intends to explore the evolutionary pattern of the cryptorchidism-related genes and genes in the Desert Hedgehog(DHH)signaling pathway in mammals.Although most mammals develop testes which fall completely into a pair of scrotums,testes of species such as the platypus,elephant,sloth,armadillo,and hedgehog naked mole rat do not fall into scrotums and remain in the abdominal or inguinal region.Because it is not easy for soft tissues such as the testis and the scrotum to be preserved in fossils,the evolutionary history of the position of the testis is not clear till now.Thus,to infer the evolutionary history of the testicular position in mammals,the present study chose 63 mammals from 17 orders and reconstructed the ancestral state of the testicular position by maximum likelihood(ML)method.The results showed that ancestors of mammals have the completely descended scrotal testis.Moreover,species in different lineages(i.e.,Monotremata,Afrotheria,Cingulata,Rodentia,Chiroptera,Cetartiodactyla,Carnivora,Perissodactyla,Eulipotyphla)developed the incompletely descended ascrotal testis and the undescended ascrotal testis independently.The difference in testicular position among different species may be affected by environmental changes and the unique evolutionary history of mammals,which reflects the association between phenotypes and adaptive evolution.The process of the testicular descent is complex which involves multiple genes and pathways,and a variety of delicate endocrine factors and mechanical regulators.Anti-Müllerian hormone(AMH),Insulin-like 3(INSL3)and Relaxin family peptide 2(RXFP2)are involved in the first stage of the testicular descent.Meanwhile,the prominent feature of this stage is the enlargement of the gubernaculum.Then the gubernaculum develops into the gubernaculum bulb with mesenchymal core and muscular periphery.The traction of the gubernaculum is the primary force leading the testicular descent in both the two descent stages.In the second stage of the testicular descent,androgen(consists largely of testosterone)is the main hormonal regulator.Testosterone interacts with the genital femoral nerve(GFN)to promote the release of the calcitonin gene-related peptide(CGRP).CGRP induces the rhythmic contraction and motility in the second stage of testicular descent,thereby promoting testicular descent.In mammals with scrotal testes,the impeded or the abnormal testicular descent can lead to cryptorchidism.The cryptorchid testis stays in the abdominal cavity or groin,where the temperature is higher than that of the scrotum,resulting in chronic heat stress damage to the testis.This is associated with sperm abnormalities and male infertility.In addition,patients with cryptorchidism often suffer abnormal development of Sertoli cells and Leydig cells,and endocrine disorders.However,it is interesting that in normal circumstances,natural ascrotal testes have never shown such pathological changes.To further investigate genes underlying the mechanisms of the reproductive and life health in ascrotal testis mammals,here we studied 380 cryptorchidism-related genes in 49 representative mammalian species at the molecular evolutionary level.The results showed that signals of positive selection and rapid evolution focused on Gene Ontology terms associated with muscle development(muscle tissue development,striated muscle tissue development and muscle organ development),reproductive development(reproductive structure development,reproductive system development,male gonad development,development of primary male sexual characteristics,male sex differentiation),as well as Hedgehog signaling pathway,suggesting their important contribution in the evolution of testicular descent.Additionally,combined with the exploration of genes coevolved with the testicular position trait,and the specific amino acid substitutions in the ascrotal testis mammals,the results found that 1)spermatogenesis regulation,2)genomic stability and accuracy of DNA replication maintain,3)autophagy regulation were the potential mechanisms involved in the reproductive and life health in the natural cryptorchid mammals.Moreover,convergence of GTPase function may play a vital role in the convergence of the ascrotal testis in mammals.The testis contains not only germ cells in different stages,but also a variety of somatic cells with important functions.Leydig cells locate in the intercellular substance of seminiferous tubules and secrete hormones important for the testicular descent.Leydig cells are the target cells of the protein DHH which is produced and secreted from Sertoli cells.Given that DHH and DHH signaling pathway regulate the proliferation and differentiation of Leydig cells,it has been suggested that paracrine of Sertoli cells is the precondition of Leydig cells differentiation.Dysregulation or inhibition of the DHH signaling pathway can hinder the normal testicular descent,gonad development and normal degeneration of male Mullerian duct.To further explore the evolutionary pattern of the DHH signaling pathway in between ascrotal and scrotal species,we analyzed 43 genes in 28 laurasiatherian species,which possess different testicular locations.It was shown that the negative regulation-related genes in the DHH signaling pathway was positively selected and rapidly evolved in ascrotal species(e.g.,MEGF8 and CUL1),suggesting the enhancement of the negative regulation might drive the evolution of ascrotal testis in Laurasiatheria.A series of in silico functional prediction showed a number of ascrotal species specific amino acid substitutions may result in changes of physiochemical properties and structure of proteins.Consequently,these specific substitutions likely affect the function of DHH signaling pathway proteins,and contribute to the evolution of the descent of the testis.In conclusion,based on the analyses of reconstructing the ancestral state of the testicular positions in mammals,and studying the evolutionary pattern of the cryptorchidism-related genes as well as the DHH signaling pathway genes,results and conclusions in the present study help us understand the evolution history of the testicular positions in mammals;on the other hand,it provides more insights into the genetic mechanisms of the diverse testicular positions in mammals and further understanding the factors influencing testicular descent.In the future,these theoretical reference and insights for the occurrence,and early prevention and diagnosis of cryptorchidism in humans and other mammals.