Supporting Sertoli Cells Research Articles

Isolation and In Vitro Culture of Germ Cells and Sertoli Cells from Human Fetal Testis.

In the human fetal testis, fetal germ cells (FGCs) are progressively surrounded by supporting Sertoli cells inside seminiferous cords. During the second trimester, the FGCs develop asynchronously and can be observed in several stages of development. However, the mechanism that regulates the transition between the different developmental stages as well as the formation of spermatogonia is currently not well understood. For this, it is necessary to develop suitable isolation protocols and a platform for in vitro culture of FGCs of different stages. Here, we report a method to isolate distinct populations of FGCs and Sertoli cells from second trimester human testis using a panel of conjugated antibodies for THY1, PDPN, ALPL, KIT, and SUSD2 for fluorescence-activated cell sorting (FACS) followed by in vitro culture up to 7days. This platform provides the base for cellular and molecular characterization of the different testicular cell populations to investigate the transition between FGCs and spermatogonia and shed some light on crucial processes of early human gametogenesis unknown until now.

A comparative analysis of genes differentially expressed between rete testis cells and Sertoli cells of the mouse testis

The rete testis (RT) is a region of the mammalian testis that plays an important role in testicular physiology. The RT epithelium consists of cells sharing some well-known gene markers with supporting Sertoli cells (SCs). However, little is known about the differences in gene expression between these two cell populations. Here, we used fluorescence-activated cell sorting (FACS) to obtain pure cultures of neonatal RT cells and SCs and identified differentially expressed genes (DEGs) between these cell types using RNA sequencing (RNA-seq). We then compared our data with the RNA-seq data of other studies that examined RT cells and SCs of mice of different ages and generated a list of DEGs permanently upregulated in RT cells throughout testis development and in culture, which included 86 genes, and a list of 79 DEGs permanently upregulated in SCs. The analysis of studies on DMRT1 function revealed that nearly half of the permanent DEGs could be regulated by this SC upregulated transcription factor. We suggest that useful cell lineage markers and candidate genes for the specification of both RT cells and SCs may be present among these permanent DEGs.

Expression of Protein Markers in Spermatogenic and Supporting Sertoli Cells Affected by High Abdominal Temperature in Cryptorchidism Model Mice.

Cryptorchidism is a congenital abnormality resulting in increased rates of infertility and testicular cancer. We used cryptorchidism model mice that presented with the translocation of the left testis from the scrotum to the abdominal cavity. Mice underwent the surgical procedure of the left testis at day 0 and were sacrificed at days 3, 5, 7, 14, 21, and 28 post-operatively. The weight of the left cryptorchid testis decreased significantly at days 21 and 28. The morphological changes were observed after 5 days and showed detached spermatogenic cells and abnormal formation of acrosome at day 5, multinucleated giant cells at day 7, and atrophy of seminiferous tubules at days 21 and 28. The high abdominal temperature disrupted the normal expression of cell adhesion molecule-1, Nectin-2, and Nectin-3 which are essential for spermatogenesis. In addition, the pattern and alignment of acetylated tubulin in cryptorchid testes were also changed at days 5, 7, 14, 21, and 28. Ultrastructure of cryptorchid testes revealed giant cells that had been formed by spermatogonia, spermatocytes, and round and elongating spermatids. The study's findings reveal that cryptorchidism's duration is linked to abnormal changes in the testis, impacting protein marker expression in spermatogenic and Sertoli cells. These changes stem from the induction of high abdominal temperature.

Deep SED-Net with interactive learning for multiple testicular cell types segmentation and cell composition analysis in mouse seminiferous tubules.

The development of mouse spermatozoa is a continuous process from spermatogonia, spermatocytes, spermatids to mature sperm. Those developing germ cells (spermatogonia, spermatocyte, and spermatids) together with supporting sertoli cells are all enclosed inside seminiferous tubules of the testis, their identification is key to testis histology and pathology analysis. Automated segmentation of all these cells is a challenging task because of their dynamical changes in different stages. The accurate segmentation of testicular cells is critical in developing computerized spermatogenesis staging. In this paper, we present a novel segmentation model, SED-Net, which incorporates a squeeze-and-excitation (SE) module and a dense unit. The SE module optimizes and obtains features from different channels, whereas the dense unit uses fewer parameters to enhance the use of features. A human-in-the-loop strategy, named deep interactive learning, is developed to achieve better segmentation performance while reducing the workload of manual annotation and time consumption. Across a cohort of 274 seminiferous tubules from stages VI to VIII, the SED-Net achieved a pixel accuracy of 0.930, a mean pixel accuracy of 0.866, a mean intersection over union of 0.710, and a frequency weighted intersection over union of 0.878, respectively, in terms of four types of testicular cell segmentation. There is no significant difference between manual annotated tubules and segmentation results by SED-Net in cell composition analysis for tubules from stages VI to VIII. In addition, we performed cell composition analysis on 2346 segmented seminiferous tubule images from 12 segmented testicular section results. The results provided quantitation of cells of various testicular cell types across 12 stages. The rule reflects the cell variation tendency across 12 stages during development of mouse spermatozoa. The method could enable us to not only analyze cell morphology and staging during the development of mouse spermatozoa but also potentially could be applied to the study of reproductive diseases such as infertility.

Дисліпідемія та репродуктивні розлади у самців щурів внаслідок тривалого споживання гідрогенізованої пальмової олії

The paper focuses on the problem of unbalanced nutrition. The effects of hydrogenated palm oil (solid form) consumption at a dose of 3 g / kg body weight, which exceeded the daily norm by 1,5 times, for 75 days in adult male rats have been experimentally shown. The simulated experimental conditions led to the development of dyslipidemia in the serum and the liver (hypertriglyceridemia, hypercholesterolemia). Signs of androgen deficiency were also found: the content of total and free testosterone in the serum was significantly less than the physiological norm. Also, defects in spermatogenesis were recorded in the experimental animals: total gametes concentration and proportion of morphologically normal gametes decreased, the pathological forms increased. Disorders of androgen-synthetic and spermatogenic functions in the testes were accompanied by histological changes. In testicular tissues, structural rearrangements of a number of seminal tubules have been observed, possibly due to an increase in inactive Leydig cell forms. A decrease in the index of spermatogenesis was found, this reflects the number of generations of spermatogenic cells in the wall of the seminal tubules. An increased number of tortuous seminal tubules with exfoliated germ cells in the lumen have been reported. The reason may be the loss of communication between the germ cells and the supporting Sertoli cells. When the link between the germ cell and the supporting cells is lost, the effects of testosterone are impossible, which leads to a violation of spermatogenesis.

Role of Follicle-Stimulating Hormone in Spermatogenesis.

Spermatogenesis is a concerted sequence of events during maturation of spermatogonia into spermatozoa. The process involves differential gene-expression and cell-cell interplay regulated by the key endocrine stimuli, i.e., follicle-stimulating hormone (FSH) and luteinizing hormone (LH)-stimulated testosterone. FSH affects independently and in concert with testosterone, the proliferation, maturation and function of the supporting Sertoli cells that produce regulatory signals and nutrients for the maintenance of developing germ cells. Rodents are able to complete spermatogenesis without FSH stimulus, but its deficiency significantly decreases sperm quantity. Men carrying loss-of-function mutation in the gene encoding the ligand (FSHB) or its receptor (FSHR) present, respectively, with azoospermia or suppressed spermatogenesis. Recently, the importance of high intratesticular testosterone concentration for spermatogenesis has been questioned. It was established that it can be completed at minimal intratesticular concentration of the hormone. Furthermore, we recently demonstrated that very robust constitutive FSHR action can rescue spermatogenesis and fertility of mice even when the testosterone stimulus is completely blocked. The clinical relevance of these findings concerns a new strategy of high-dose FSH in treatment of spermatogenic failure.

Roles of Reactive Oxygen Species in the Spermatogenesis Regulation

Spermatogenesis is a complex process of male germ cells proliferation and maturation from diploid spermatogonia, through meiosis, to mature haploid spermatozoa. The process involves dynamic interactions between the developing germ cells and their supporting Sertoli cells. The gonadal tissue, with abundance of highly unsaturated fatty acids, high rates of cell division, and variety of testis enzymes results very vulnerable to the overexpression of reactive oxygen species (ROS). In order to address this risk, testis has developed a sophisticated array of antioxidant systems comprising both enzymes and free radical scavengers. This chapter sets out the major pathways of testis generation, the metabolism of ROS, and highlights the transcriptional regulation by steroid receptors of antioxidant stress enzymes and their functional implications. It also deals with of the advantages of the system biology for an antioxidant under steroid control, the major selenoprotein expressed by germ cells in the testis, the phospholipid hydroperoxide glutathione peroxidase (PHGPx/GPx4) having multiple functions and representing the pivotal link between selenium, sperm quality, and species preservation.

MEF2 Is Restricted to the Male Gonad and Regulates Expression of the Orphan Nuclear Receptor NR4A1

Leydig cell steroidogenesis is controlled by the pituitary gonadotropin LH that activates several signaling pathways, including the Ca(2+)/calmodulin kinase I (CAMKI) pathway. In other tissues, CAMKI regulates the activity of the myocyte enhancer factor 2 (MEF2) transcription factors. MEF2 factors are essential regulators of cell differentiation and organogenesis in numerous tissues but their expression and role in the mammalian gonad had not been explored. Here we show that MEF2 factors are expressed in a sexually dimorphic pattern in the mouse gonad. MEF2 factors are present in the testis throughout development and into adulthood but absent from the ovary. In the testis, MEF2 was localized mainly in the nucleus of both somatic lineages, the supporting Sertoli cells and the steroidogenic Leydig cells. In Leydig cells, MEF2 was found to activate the expression of Nr4a1, a nuclear receptor important for hormone-induced steroidogenesis. In these cells MEF2 also cooperates with forskolin and CAMKI to enhance Nr4a1 promoter activity via two MEF2 elements (-318 and -284 bp). EMSA confirmed direct binding of MEF2 to these elements whereas chromatin immunoprecipitation revealed that MEF2 recruitment to the proximal Nr4a1 promoter was increased following hormonal stimulation. Modulation of endogenous MEF2 protein level (small interfering RNA-mediated knockdown) or MEF2 activity (MEF2-Engrailed active dominant negative) led to a significant decrease in Nr4a1 mRNA levels in Leydig cells. All together, our results identify MEF2 as a novel testis-specific transcription factor, supporting a role for this factor in male sex differentiation and function. MEF2 was also positioned upstream of NR4A1 in a regulatory cascade controlling Leydig cell gene expression.

Germ-line activation of the luteinizing hormone receptor directly drives spermiogenesis in a nonmammalian vertebrate.

In both mammals and teleosts, the differentiation of postmeiotic spermatids to spermatozoa (spermiogenesis) is thought to be indirectly controlled by the luteinizing hormone (LH) acting through the LH/choriogonadotropin receptor (LHCGR) to stimulate androgen secretion in the interstitial Leydig cells. However, a more direct, nonsteroidal role of LH mediating the spermiogenic pathway remains unclear. Using a flatfish with semicystic spermatogenesis, in which spermatids are released into the seminiferous lobule lumen (SLL), where they develop into spermatozoa without direct contact with the supporting Sertoli cells, we show that haploid spermatids express the homolog of the tetrapod LHCGR (Lhcgrba). Both native Lh and intramuscularly injected His-tagged recombinant Lh (rLh) are immunodetected bound to the Lhcgrba of free spermatids in the SLL, showing that circulating gonadotropin can reach the intratubular compartment. In vitro incubation of flatfish spermatids isolated from the SLL with rLh specifically promotes their differentiation into spermatozoa, whereas recombinant follicle-stimulating hormone and steroid hormones are ineffective. Using a repertoire of molecular markers and inhibitors, we find that the Lh-Lhcgrba induction of spermiogenesis is mediated through a cAMP/PKA signaling pathway that initiates the transcription of genes potentially involved in the function of spermatozoa. We further show that Lhcgrba expression in germ cells also occurs in distantly related fishes, suggesting this feature is likely conserved in teleosts regardless of the type of germ cell development. These data reveal a role of LH in vertebrate germ cells, whereby a Lhcgrba-activated signaling cascade in haploid spermatids directs gene expression and the progression of spermiogenesis.

The Dynamic of the Apical Ectoplasmic Specialization between Spermatids and Sertoli Cells: The Case of the Small GTPase Rap1

Despite advances in assisted reproductive technologies, infertility remains a consistent health problem worldwide. Spermiation is the process through which mature spermatids detach from the supporting Sertoli cells and are released into the tubule lumen. Spermiation failure leads to lack of mature spermatozoa and, if not occasional, could result into azoospermia, major cause of male infertility in human population. Spermatids are led through their differentiation into spermatozoa by the apical ectoplasmic specialization (aES), a testis-specific, actin-based anchoring junction restricted to the Sertoli-spermatid interface. The aES helps spermatid movement across the seminiferous epithelium, promotes spermatid positioning, and prevents the release of immature spermatozoa. To accomplish its functions, aES needs to undergo tightly and timely regulated restructuring. Even if components of aES are partly known, the mechanism/s through which aES is regulated remains still elusive. In this review, we propose a model by which the small GTPase Rap1 could regulate aES assembly/remodelling. The characterization of key players in the dynamic of aES, such as Rap1, could open new possibility to develop prognostic, diagnostic, and therapeutic approaches for male patients under treatment for infertility as well as it could lead to the identification of new target for male contraception.

Zmynd15 Encodes a Histone Deacetylase-dependent Transcriptional Repressor Essential for Spermiogenesis and Male Fertility

Spermatogenesis is a complex process through which male germ line stem cells undergo a multi-step differentiation program and sequentially become spermatogonia, spermatocytes, spermatids, and eventually spermatozoa. In this process, transcription factors act as switches that precisely regulate the expression of genes that in turn control the developmental program of male germ cells. Transcription factors identified to be essential for normal haploid gene expression all display transcription-activating effects and thus serve as the "on" switch for haploid gene expression. Here, we report that ZMYND15 acts as a histone deacetylase-dependent transcriptional repressor and controls normal temporal expression of haploid cell genes during spermiogenesis. Inactivation of Zmynd15 results in early activation of transcription of numerous important haploid genes including Prm1, Tnp1, Spem1, and Catpser3; depletion of late spermatids; and male infertility. ZMYND15 represents the first transcriptional repressor identified to be essential for sperm production and male fertility.

Genome-wide analysis of DNA binding and transcriptional regulation by the mammalian Doublesex homolog DMRT1 in the juvenile testis

The DM domain proteins Doublesex- and MAB-3-related transcription factors (DMRTs) are widely conserved in metazoan sex determination and sexual differentiation. One of these proteins, DMRT1, plays diverse and essential roles in development of the vertebrate testis. In mammals DMRT1 is expressed and required in both germ cells and their supporting Sertoli cells. Despite its critical role in testicular development, little is known about how DMRT1 functions as a transcription factor or what genes it binds and regulates. We combined ChIP methods with conditional gene targeting and mRNA expression analysis and identified almost 1,400 promoter-proximal regions bound by DMRT1 in the juvenile mouse testis and determined how expression of the associated mRNAs is affected when Dmrt1 is selectively mutated in germ cells or Sertoli cells. These analyses revealed that DMRT1 is a bifunctional transcriptional regulator, activating some genes and repressing others. ChIP analysis using conditional mutant testes showed that DNA binding and transcriptional regulation of individual target genes can differ between germ cells and Sertoli cells. Genes bound by DMRT1 in vivo were enriched for a motif closely resembling the sequence DMRT1 prefers in vitro. Differential response of genes to loss of DMRT1 corresponded to differences in the enriched motif, suggesting that other transacting factors may modulate DMRT1 activity. DMRT1 bound its own promoter and those of six other Dmrt genes, indicating auto- and cross-regulation of these genes. Many of the DMRT1 target genes identified here are known to be important for a variety of functions in testicular development; the others are candidates for further investigation.

Non-classical actions of testosterone and spermatogenesis

Testosterone is essential to maintain spermatogenesis and male fertility. In the absence of testosterone stimulation, spermatogenesis does not proceed beyond the meiosis stage. After withdrawal of testosterone, germ cells that have progressed beyond meiosis detach from supporting Sertoli cells and die, whereas mature sperm cannot be released from Sertoli cells resulting in infertility. The classical mechanism of testosterone action in which testosterone activates gene transcription by causing the androgen receptor to translocate to and bind specific DNA regulatory elements does not appear to fully explain testosterone regulation of spermatogenesis. This review discusses two non-classical testosterone signalling pathways in Sertoli cells and their potential effects on spermatogenesis. Specifically, testosterone-mediated activation of phospholipase C and calcium influx into Sertoli cells is described. Also, testosterone activation of Src, EGF receptor and ERK kinases as well as the activation of the CREB transcription factor and CREB-mediated transcription is reviewed. Regulation of germ cell adhesion to Sertoli cells and release of mature sperm from Sertoli cells by kinases regulated by the non-classical testosterone pathway is discussed. The evidence accumulated suggests that classical and non-classical testosterone signalling contribute to the maintenance of spermatogenesis and male fertility.

HP1γ function is required for male germ cell survival and spermatogenesis

BackgroundHP1 proteins are conserved components of eukaryotic constitutive heterochromatin. In mammals, there are three genes that encode HP1-like proteins, termed HP1α, HP1β and HP1γ, which have a high degree of homology This paper describes for the first time, to our knowledge, the physiological function of HP1γ using a gene-targeted mouse.ResultsWhile targeting the Cbx3 gene (encoding the HP1γ protein) with a conditional targeting vector, we generated a hypomorphic allele (Cbx3hypo), which resulted in much reduced (barely detectable) levels of HP1γ protein. Homozygotes for the hypomorphic allele (Cbx3hypo/hypo) are rare, with only 1% of Cbx3hypo/hypo animals reaching adulthood. Adult males exhibit a severe hypogonadism that is associated with a loss of germ cells, with some seminiferous tubules retaining only the supporting Sertoli cells (Sertoli cell-only phenotype). The percentage of seminiferous tubules that are positive for L1 ORF1 protein (ORF1p) in Cbx3hypo/hypo testes is greater than that for wild-type testes, indicating that L1 retrotransposon silencing is reversed, leading to ectopic expression of ORF1p in Cbx3hypo/hypo germ cells.ConclusionsThe Cbx3 gene product (the HP1γ protein) has a non-redundant function during spermatogenesis that cannot be compensated for by the other two HP1 isotypes. The Cbx3hypo/hypo spermatogenesis defect is similar to that found in Miwi2 and Dnmt3L mutants. The Cbx3 gene-targeted mice generated in this study provide an appropriate model for the study of HP1γ in transposon silencing and parental imprinting.

Changes in the Peritubular Tissue of Rat Testis after Cadmium Treatment

The present study demonstrates histological and immunohistochemical changes in the peritubular testicular tissue of rat testis after application of cadmium chloride. After 5-day cadmium exposure, advanced deterioration of the boundary testicular tissue, mainly oedema, disarrangement of collagen fibres and peritubular cells, dilatation and thrombosis of blood vessels were observed. Changes in the boundary tissue were accompanied with desquamation of the germinal epithelium. Immunohistochemically, positive reaction for alpha-smooth muscle actin and desmin in tunica media of large testicular blood vessels basically was not affected. No reaction for vimentin was seen in endothelial cells of blood capillaries, whereas positive reaction presented only these cells in large blood vessels. The myofibroblasts positively reacting for desmin and alpha-smooth muscle actin form a single incomplete layer in the lamina propria of seminiferous tubules. Vimentin reactivity in the myofibroblasts and in the supporting Sertoli cells as well as Leydig cells in damaged testicular tissue was not observed. An increase in fibroblasts and free inflammatory cells positive for vimentin in the peritubular space on the peripheric area of the testis was observed.

Mammalian testis: A target of in vivo electroporation

Mammalian spermatogenesis consists of three biologically significant processes: stem cell self-renewal and differentiation, meiosis, and haploid cell morphogenesis. Understanding the molecular mechanisms behind these processes might provide clues to the puzzle of species preservation and evolution, and to treatments for male infertility. However, few useful in vitro systems exist to investigate these processes at present. To elucidate these mechanisms, in vivo electroporation of the testis might be a convenient option. Since DNA solution can be injected into the seminiferous tubule via the rete testis, similar to germ cell transplantation, it is easy to transfect expression vectors into various differentiated germ cells and supporting Sertoli cells with adequate electric shock. Unfortunately, it is difficult to create transgenic animals using this method because of its low efficiency. However, gain- and loss-of-function assays, promoter assays, and tagged-protein behavior assays can be conducted with this technique, as in in vitro culture systems.

Molecular mechanisms underlying sex change in hermaphroditic groupers

Groupers are widely distributed throughout the tropical and subtropical waters of the world and are regarded as a favourite marine food fish. However, their large-scale aquaculture has been hindered by the rarity of natural males. Being protogynous hermaphrodites, groupers have been considered as study model for development and reproduction, especially for sex determination or sex differentiation, owing to the advantage that grouper gonad development undergoes transition from ovary to intersexual gonad and then to testis, and primordial germ cells and different stages of gametic cells during oogenesis and spermatogenesis are synchronously observed in the transitional gonads. Recently, a series of genes related to the reproduction regulation or sex differentiation have been identified in the groupers, mainly by researchers in China. One important finding was that the grouper gene, doublesex/male abnormal 3-related transcription factor 1 (DMRT1), is not only differentially expressed in gonads at different stages, but that it is also restricted to specific stages and specific cells of spermatogenesis. Grouper DMRT1 protein exists only in spermatogonia, primary spermatocytes and secondary spermatocytes, but not in the supporting Sertoli cells. Moreover, no introns were found in the grouper DMRT1, and no duplicated DMRT1 genes were detected. The finding implies that the intronless DMRT1 that is able to undergo rapid transcriptional turnover might be a significant gene for stimulating spermatogenesis in the protogynous hermaphroditic gonad. Additionally, we have found that grouper expression of sex-determining region Y-related high-mobility group-box gene 3 (SOX3) is a significant time point for enterable gametogenesis of primordial germ cells, because SOX3 is obviously expressed and localized in primordial germ cells. As SOX3 continues to express, the SOX3-positive primordial germ cells develop toward oogonia and then oocytes, whereas, when SOX3 expression is ceased, the SOX3-positive primordial germ cells develop toward spermatogonia. Therefore, we suggest that SOX3, as a transcription factor, might have more important roles in oogenesis than in spermatogenesis. Based on the findings, a hypothetic molecular mechanism underlying sex change is proposed in the hermaphroditic groupers, and some candidate genes related to the grouper sex change are also suggested for further research.

Regulation of Early Wave of Germ Cell Apoptosis and Spermatogenesis by Deubiquitinating Enzyme CYLD

Spermatogenesis involves an early wave of germ cell apoptosis, which is required for maintaining the balance between germ cells and supporting Sertoli cells. However, the signaling mechanism regulating this apoptotic event is poorly defined. Here we show that genetic deficiency of Cyld, a recently identified deubiquitinating enzyme, attenuates the early wave of germ cell apoptosis and causes impaired spermatogenesis in mice. Interestingly, the loss of CYLD in testicular cells leads to activation of the transcription factor NF-kappaB and aberrant expression of antiapoptotic genes. We further show that CYLD negatively regulates a ubiquitin-dependent NF-kappaB activator, RIP1. CYLD binds to RIP1 and inhibits its ubiquitination and signaling function. These findings establish CYLD as a pivotal deubiquitinating enzyme (DUB) that regulates germ cell apoptosis and spermatogenesis and suggest an essential role for CYLD in controlling the RIP1/NF-kappaB signaling axis in testis.

Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers

DMRT1 has been suggested to play different roles in sex determination and gonad differentiation, because different expression patterns have been reported among different vertebrates. The groupers, since their gonads first develop as ovary and then reverse into testis, have been thought as good models to study sex differentiation and determination. In this study, we cloned the full-length cDNAs of DMRT1 gene from orange-spotted grouper ( Epinephelus coioides), and prepared corresponding anti- EcDMRT1 antiserum to study the relationship of DMRT1 to sex reversal. One important finding is that the grouper DMRT1 is not only differentially expressed in different stage gonads, but also restricted to specific stages and specific cells of spermatogenesis. Grouper DMRT1 protein exists only in spermatogonia, primary spermatocytes and secondary spermatocytes, but not in the supporting Sertoli cells. Moreover, we confirmed that EcSox3 is expressed not only in oogonia and different stage oocytes, but also in Sertoli cells and spermatogonia, and EcSox9 is expressed only in Sertoli cells. The data suggested that grouper DMRT1 might be a more specific sex differentiation gene for spermatogenesis, and play its role at the specific stages from spermatogonia to spermatocytes. In addition, no introns were found in the grouper DMRT1, and no duplicated DMRT1 genes were detected. The finding implicates that the intronless DMRT1 that is able to undergo rapid transcriptional turnover might be a significant gene for stimulating spermatogenesis in the protogynous hermaphroditic gonad.

CIB1 Is Essential for Mouse Spermatogenesis

CIB1 is a 22-kDa calcium binding, regulatory protein with approximately 50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of effectors, such as integrin alphaIIb, PAK1, and polo-like kinases, in different tissues. However, the in vivo functions of CIB1 are not well understood. To elucidate the function of CIB1 in whole animals, we used homologous recombination in embryonic stem cells to generate Cib1(-/-) mice. Although Cib1(-/-) mice grow normally, the males are sterile due to disruption of the haploid phase of spermatogenesis. This is associated with reduced testis size and numbers of germ cells in seminiferous tubules, increased germ cell apoptosis, and the loss of elongated spermatids and sperm. Cib1(-/-) testes also show increased mRNA and protein expression of the cell cycle regulator Cdc2/Cdk1. In addition, mouse embryonic fibroblasts (MEFs) derived from Cib1(-/-) mice exhibit a much slower growth rate compared to Cib1(+/+) MEFs, suggesting that CIB1 regulates the cell cycle, differentiation of spermatogenic germ cells, and/or differentiation of supporting Sertoli cells.