Impaired Blastocyst Formation in Lnx2-Knockdown Mouse Embryos.

Ligand of Numb-protein X 2 (LNX2) is an E3 ubiquitin ligase that is known to regulate Notch signaling by participating in NUMB protein degradation. Notch signaling is important for differentiation and proliferation in mammals, and plays a significant role in blastocyst formation during early embryonic development. In this study, we investigated Lnx2 in mouse preimplantation embryos. Expression analysis showed that Lnx2 is expressed in oocytes and preimplantation embryos. Lnx2-knockdown embryos normally progress to the morula stage, but the majority of them do not develop into normal blastocysts. Transcript analysis revealed that the expression levels of genes critical for cell lineage specification, including octamer-binding transcription factor 4 (Oct4), are increased in Lnx2 knockdown embryos. Furthermore, the expression levels of Notch and Hippo signaling-related genes are also increased by Lnx2 knockdown. Collectively, our results show that Lnx2 is important for blastocyst formation in mice, suggest that this may act via lineage specification of inner cell mass, and further show that Lnx2 may be involved in transcriptionally regulating various genes implicated in early embryonic development.

Analysis of mouse male germ cell-specific or -predominant Tex13 family genes encoding proteins with transcriptional repressor activity.

Mammalian spermatogenesis is a highly organized process with successive mitotic, meiotic, and postmeiotic phases. This unique developmental process is characterized by the involvement of spermatogenic cell-specific genes. In this study, we identified and investigated testis expressed gene 13 (Tex13) family genes, consisting of Tex13a, Tex13b, Tex13c1, and Tex13d, in mice. All of these genes were transcribed specifically or predominantly in male germ cells, and their transcription was developmentally regulated. Proteins encoded by the Tex13 genes were predicted to have a conserved domain of ~ 145 amino acids. Tex13a, Tex13c1, and Tex13d encode additional C-terminal regions containing a short conserved sequence termed a zinc finger-RAN binding protein 2 (zf-RanBP2) or zf-RanBP2-like domain. As TEX13B reportedly has transcriptional repressor activity, we examined the effect of the TEX13 proteins on transcriptional regulation using a reporter assay. All of the TEX13 proteins exhibited transcriptional repressor activity. This activity was revealed to reside in the TEX13B-corresponding regions of TEX13A, TEX13C1, and TEX13D. Further, we found that the C-terminal regions of TEX13A, TEX13C1, and TEX13D also have inhibitory activities. These results suggest that male germ cell-specific or -predominant TEX13 proteins commonly function in transcriptional repression as transcription cofactors and/or RNA binding proteins.

Identification of a novel embryo-prevalent gene, Gm11545, involved in preimplantation embryogenesis in mice.

In mammals, the early embryo travels down the oviduct to the uterus and prepares for implantation. The unique features of preimplantation development include compaction followed by blastocyst formation. This first cell lineage specification involves various proteins including cell polarity regulators, kinases, and transcription factors. In this study, a novel gene named predicted gene 11545 (Gm11545) expressed predominantly in mouse early embryos was identified and characterized at the transcript, protein, cellular, and functional levels. The Gm11545 protein localized to both cytoplasmic and membrane regions of preimplantation embryos. Remarkably, knockdown of Gm11545 led to arrest of mouse embryos at the morula stage and consequent impairment of blastocyst formation. Expression patterns of the key transcription factors critical for early lineage specification, octamer-binding transcription factor 4 and caudal type homeobox 2, were affected by Gm11545 depletion. Based on the collective findings, we propose that the novel protein identified in this study, Gm11545, is implicated in cell proliferation and cell lineage specification critical for blastocyst formation.-Kim, J., Kim, J., Jeong, J., Hong, S. H., Kim, D., Choi, S., Choi, I., Oh, J. S., Cho, C. Identification of a novel embryo-prevalent gene, Gm11545, involved in preimplantation embryogenesis in mice.

SPATC1L maintains the integrity of the sperm head-tail junction.

Spermatogenesis is a tightly regulated process involving germ cell-specific and germ cell-predominant genes. Here we investigate a novel germ cell-specific gene, Spatc1l (spermatogenesis and centriole associated 1 like). Expression analyses show that SPATC1L is expressed in mouse and human testes. We find that mouse SPATC1L localizes to the neck region in testicular sperm. Moreover, SPATC1L associates with the regulatory subunit of protein kinase A (PKA). Using CRISPR/Cas9-mediated genome engineering, we generate mice lacking SPATC1L. Disruption of Spatc1l in mice leads to male sterility owing to separation of sperm heads from tails. The lack of SPATC1L is associated with a reduction in PKA activity in testicular sperm, and we identify capping protein muscle Z-line beta as a candidate target of phosphorylation by PKA in testis. Taken together, our results implicate the SPATC1L-PKA complex in maintaining the stability of the sperm head-tail junction, thereby revealing a new molecular basis for sperm head-tail integrity.

The sperm-associated antigen 6 interactome and its role in spermatogenesis.

Mammalian SPAG6, the orthologue of Chlamydomonas reinhardtii PF16, is a component of the central apparatus of the '9 + 2' axoneme that controls ciliary/flagellar motility, including sperm motility. Recent studies revealed that SPAG6 has functions beyond its role in the central apparatus. Hence, we reexamined the role of SPAG6 in male fertility. In wild-type mice, SPAG6 was present in cytoplasmic vesicles in spermatocytes, the acrosome of round and elongating spermatids and the manchette of elongating spermatids. Spag6-deficient testes showed abnormal spermatogenesis, with abnormalities in male germ cell morphology consistent with the multi-compartment pattern of SPAG6 localization. The armadillo repeat domain of mouse SPAG6 was used as a bait in a yeast two-hybrid screen, and several proteins with diverse functions appeared multiple times, including Snapin, SPINK2 and COPS5. Snapin has a similar localization to SPAG6 in male germ cells, and SPINK2, a key protein in acrosome biogenesis, was dramatically reduced in Spag6-deficient mice which have defective acrosomes. SPAG16L, another SPAG6-binding partner, lost its localization to the manchette in Spag6-deficient mice. Our findings demonstrate that SPAG6 is a multi-functional protein that not only regulates sperm motility, but also plays roles in spermatogenesis in multiple cellular compartments involving multiple protein partners.

Profiling of testis-specific long noncoding RNAs in mice.

BACKGROUND: Spermatogenesis, which is the complex and highly regulated process of producing haploid spermatozoa, involves testis-specific transcripts. Recent studies have discovered that long noncoding RNAs (lncRNAs) are novel regulatory molecules that play important roles in various biological processes. However, there has been no report on the comprehensive identification of testis-specific lncRNAs in mice. RESULTS: We performed microarray analysis of transcripts from mouse brain, heart, kidney, liver and testis. We found that testis harbored the highest proportion of tissue-specific lncRNAs (11%; 1607 of 14,256). Testis also harbored the largest number of tissue-specific mRNAs among the examined tissues, but the proportion was lower than that of lncRNAs (7%; 1090 of 16,587). We categorized the testis-specific lncRNAs and found that a large portion corresponded to long intergenic ncRNAs (lincRNAs). Genomic analysis identified 250 protein-coding genes located near (≤ 10 kb) 194 of the loci encoding testis-specific lincRNAs. Gene ontology (GO) analysis showed that these protein-coding genes were enriched for transcriptional regulation-related terms. Analysis of male germ cell-related cell lines (F9, GC-1 and GC-2) revealed that some of the testis-specific lncRNAs were expressed in each of these cell lines. Finally, we arbitrarily selected 26 testis-specific lncRNAs and performed in vitro expression analysis. Our results revealed that all of them were expressed exclusively in the testis, and 23 of the 26 showed germ cell-specific expression. CONCLUSION: This study provides a catalog of testis-specific lncRNAs and a basis for future investigation of the lncRNAs involved in spermatogenesis and testicular functions.

Reduced Fertility and Altered Epididymal and Sperm Integrity in Mice Lacking ADAM7.

The mammalian epididymis is a highly convoluted tubule that connects the testis to the vas deferens. Its proper functions in sperm transport, storage, and maturation are essential for male reproduction. One of the genes predominantly expressed in the epididymis is ADAM7 (a disintegrin and metalloprotease 7). Previous studies have shown that ADAM7 synthesized in the epididymis is secreted into the epididymal lumen and is then transferred to sperm membranes, where it forms a chaperone complex that is potentially involved in sperm fertility. In this study, we generated and analyzed mice with a targeted disruption in the Adam7 gene. We found that the fertility of male mice was modestly but significantly reduced by knockout of Adam7. Histological analyses revealed that the cell heights of the epithelium were dramatically decreased in the caput of the epididymis of Adam7-null mice, suggesting a requirement for ADAM7 in maintaining the integrity of the epididymal epithelium. We found that sperm from Adam7-null mice exhibit decreased motility, tail deformation, and altered tyrosine phosphorylation, indicating that the absence of ADAM7 leads to abnormal sperm functions and morphology. Western blot analyses revealed reduced levels of integral membrane protein 2B (ITM2B) and ADAM2 in sperm from Adam7-null mice, suggesting a requirement for ADAM7 in normal expression of sperm membrane proteins involved in sperm functions. Collectively, our study demonstrates for the first time that ADAM7 is required for normal fertility and is important for the maintenance of epididymal integrity and for sperm morphology, motility, and membrane proteins.

Profiling of testis-specific or testis-predominant genes expressed in mouse male germ cell lines GC-1 and GC-2.

BACKGROUND: Spermatogenesis is a tightly organized process that utilizes an intrinsic genetic program composed of germ cell-specific genes. Although mouse germ cell-related cell lines are available, few germ cell-specific genes have been comprehensively identified in such cell lines. OBJECTIVE: We aimed to profile gene expression in the male mouse germ cell-related cell lines, GC-1 and GC-2, characterize their transcriptomic nature, and identify potential testis- or germ cell-specific or -predominant genes expressed in these cell lines. METHODS: We performed profiling analysis of genes transcribed in the mouse germ cell-related cell lines, GC-1 and GC-2, using our previous microarray data together with public transcriptome information. We analyzed the expression of a number of the cell line genes predicted to be preferentially expressed in testis by RT-PCR. RESULTS: We found that most testis-specific or -predominant mRNAs are not expressed in GC-1 and GC-2 cells, implying that these cell lines have lost their testis- or germ cell-specific genetic characteristics. RT-PCR analysis of genes predicted to be expressed in the cell lines with preferential testicular expression showed the testis-specific or -predominant expression of nine genes and verified four of them as being expressed in the germ cell lines. Among them, only cyclin-dependent kinase inhibitor 3 genes (Cdkn3) showed testis and germ cell specificity. CONCLUSION: Our study provides extensive transcriptomic information to shed light on the limited testicular characteristics of the mouse male germ cell-derived cell lines, GC-1 and GC-2, and offers a list of germ cell line genes with testicular preference.

Targeted ablation of the histidine-rich Ca(2+)-binding protein (HRC) gene is associated with abnormal SR Ca(2+)-cycling and severe pathology under pressure-overload stress.

The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild-type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca(2+) transients, and maximal SR Ca(2+) uptake rates in the heart. However, after-contractions were developed in 50 % of HRC-KO cardiomyocytes, compared to 11 % in WT mice under stress conditions of high-frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca(2+) spontaneous SR Ca(2+) release and delayed afterdepolarizations with ISO in HRC-KO, compared to WT cells. The frequency of Ca(2+) sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca(2+) load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca(2+)-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC-null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca(2+)-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.

Identification and characterization of promoter and regulatory regions for mouse Adam2 gene expression.

ADAM2, a member of the 'a disintegrin and metalloprotease' (ADAM) family, is a key protein in mammalian fertilization that is specifically expressed in testicular germ cells. Here, we investigated the transcriptional regulation of the mouse Adam2 gene. An in silico analysis identified two conserved non-coding sequences located upstream of the mouse and human ADAM2 genes. The upstream region of the mouse Adam2 gene was found to lack typical TATA and CAAT boxes, and to have a high GC content. Our in vitro transient transfection-reporter analysis identified a promoter in this region of the mouse Adam2 gene, along with regulatory regions that inhibit the activity of this promoter in somatic cells. Site-directed mutagenesis revealed that the caudal-type homeobox 1 and CCTC-binding factor motifs are responsible for the inhibitory activities of the repressor regions. Finally, electrophoretic mobility shift assays showed putative transcription factor-promoter DNA complexes, and DNA-affinity chromatography and proteomic analyses identified myelin gene regulatory factor as a binding partner of the Adam2 promoter. This provides the first identification and characterization of promoter and repressor regions that regulate the transcription of the mouse Adam2 gene, and offers insights into the regulation of this germ-cell-specific gene.

Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes.

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.

Myosin regulatory light chains are required to maintain the stability of myosin II and cellular integrity.

Myosin II is an actin-binding protein composed of MHC (myosin heavy chain) IIs, RLCs (regulatory light chains) and ELCs (essential light chains). Myosin II expressed in non-muscle tissues plays a central role in cell adhesion, migration and division. The regulation of myosin II activity is known to involve the phosphorylation of RLCs, which increases the Mg2+-ATPase activity of MHC IIs. However, less is known about the details of RLC-MHC II interaction or the loss-of-function phenotypes of non-muscle RLCs in mammalian cells. In the present paper, we investigate three highly conserved non-muscle RLCs of the mouse: MYL (myosin light chain) 12A (referred to as MYL12A), MYL12B and MYL9 (MYL12A/12B/9). Proteomic analysis showed that all three are associated with the MHCs MYH9 (NMHC IIA) and MYH10 (NMHC IIB), as well as the ELC MYL6, in NIH 3T3 fibroblasts. We found that knockdown of MYL12A/12B in NIH 3T3 cells results in striking changes in cell morphology and dynamics. Remarkably, the levels of MYH9, MYH10 and MYL6 were reduced significantly in knockdown fibroblasts. Comprehensive interaction analysis disclosed that MYL12A, MYL12B and MYL9 can all interact with a variety of MHC IIs in diverse cell and tissue types, but do so optimally with non-muscle types of MHC II. Taken together, our study provides direct evidence that normal levels of non-muscle RLCs are essential for maintaining the integrity of myosin II, and indicates that the RLCs are critical for cell structure and dynamics.

Anti-Ischemic Effects of PIK3IP1 Are Mediated through Its Interactions with the ETA-PI3Kγ-AKT Axis.

Oxidative stress, caused by the accumulation of reactive oxygen species (ROS) during acute myocardial infarction (AMI), is one of the main factors leading to myocardial cell damage and programmed cell death. Phosphatidylinositol-3-kinase-AKT (PI3K-AKT) signaling is essential for regulating cell proliferation, differentiation, and apoptosis. Phosphoinositide-3-kinase (PI3K)-interacting protein 1 (PIK3IP1) is an intrinsic inhibitor of PI3K in various tissues, but its functional role during AMI remains unknown. In this study, the anti-ischemic role of PIK3IP1 in an in vitro AMI setting was evaluated using H9c2 cells. The MTT assay demonstrated that cell viability decreased significantly via treatment with H2O2 (200-500 µM). The TUNEL assay results revealed substantial cellular apoptosis following treatment with 200 µM H2O2. Under the same conditions, the expression levels of hypoxia-inducible factor (HIF-1α), endothelin-1 (ET-1), bcl-2-like protein 4 (BAX), and cleaved caspase-3 were elevated, whereas those of PIK3IP1, LC3II, p53, and Bcl-2 decreased significantly. PIK3IP1 overexpression inhibited H2O2-induced and PI3K-mediated apoptosis; however, PIK3IP1 knockdown reversed this effect, suggesting that PIK3IP1 functions as an anti-apoptotic molecule. To identify both the upstream and downstream molecules associated with PIK3IP1, ET-1 receptor type-specific antagonists (BQ-123 and BQ-788) and PI3K subtype-specific antagonists (LY294002 and IPI-549) were used to determine the participating isoforms. Co-immunoprecipitation was performed to identify the binding partners of PIK3IP1. Our results demonstrated that ROS-induced cardiac cell death may occur through the ETA-PI3Kγ-AKT axis, and that PIK3IP1 inhibits binding with both ETA and PI3Kγ. Taken together, these findings reveal that PIK3IP1 plays an anti-ischemic role by reducing the likelihood of programmed cell death via interaction with the ETA-PI3Kr-AKT axis.

Identification of heat shock protein 5, calnexin and integral membrane protein 2B as Adam7-interacting membrane proteins in mouse sperm.

In mammals, sperm acquire their motility and ability to fertilize eggs in the epididymis. This maturation process involves the acquisition of particular proteins from the epididymis. One such secretory protein specifically expressed in the epididymis is Adam7 (a disintegrin and metalloprotease 7). Previous studies have shown that Adam7 that resides in an intracellular compartment of epididymal cells is transferred to sperm membranes, where its levels are dependent on the expression of Adam2 and Adam3, which have critical roles in fertilization. Here, using a proteomics approach based on mass spectrometry, we identified proteins that interact with Adam7 in sperm membranes. This analysis revealed that Adam7 forms complexes with calnexin (Canx), heat shock protein 5 (Hspa5), and integral membrane protein 2B (Itm2b). Canx and Hspa5 are molecular chaperones, and Itm2b is a type II integral membrane protein implicated in neurodegeneration. The interaction of Adam7 with these proteins was confirmed by immunoprecipitation-Western blot analysis. We found that Adam7 and Itm2b are located in detergent-resistant regions known to be highly correlated with membrane lipid rafts. We further found that the association of Adam7 with Itm2b is remarkably promoted during sperm capacitation owing to a conformational change of Adam7 that occurs in concert with the capacitation process. Thus, our results suggest that Adam7 functions in fertilization through the formation of a chaperone complex and enhanced association with Itm2b during capacitation in sperm.

Testicular germ cell-specific lncRNA, Teshl, is required for complete expression of Y chromosome genes and a normal offspring sex ratio.

Heat shock factor 2 (HSF2) regulates the transcription of the male-specific region of the mouse Y chromosome long arm (MSYq) multicopy genes only in testes, but the molecular mechanism underlying this tissue specificity remains largely unknown. Here, we report that the testicular germ cell-specific long noncoding RNA (lncRNA), NR_038002, displays a characteristic spatiotemporal expression pattern in the nuclei of round and elongating spermatids. NR_038002-knockout male mice produced sperm with abnormal head morphology and exhibited reduced fertility accompanied by a female-biased sex ratio in offspring. Molecular analyses revealed that NR_038002 interacts with HSF2 and thereby activates expression of the MSYq genes. We designate NR_038002 as testicular germ cell-specific HSF2-interacting lncRNA (Teshl). Together, our study is the first to demonstrate that the testis specificity of HSF2 activity is regulated by the lncRNA Teshl and establishes a Teshl-HSF2-MSYq molecular axis for normal Y-bearing sperm qualities and consequent balanced offspring sex ratio.

Transcriptome Analysis of Testicular Aging in Mice.

Male reproductive aging, or andropause, is associated with gradual age-related changes in testicular properties, sperm production, and erectile function. The testis, which is the primary male reproductive organ, produces sperm and androgens. To understand the transcriptional changes underlying male reproductive aging, we performed transcriptome analysis of aging testes in mice. A total of 31,386 mRNAs and 9387 long non-coding RNAs (lncRNAs) were identified in the mouse testes of diverse age groups (3, 6, 12, and 18 months old) by total RNA sequencing. Of them, 1571 mRNAs and 715 lncRNAs exhibited changes in their levels during testicular aging. Most of these aging-related transcripts exhibited slight and continuous expression changes during aging, whereas some (9.6%) showed larger expression changes. The aging-related transcripts could be classified into diverse expression patterns, in which the transcripts changed mainly at 3-6 months or at 12-18 months. Our subsequent in silico analysis provided insight into the potential features of testicular aging-related mRNAs and lncRNAs. We identified testis-specific aging-related transcripts (121 mRNAs and 25 lncRNAs) by comparison with a known testis-specific transcript profile, and then predicted the potential reproduction-related functions of the mRNAs. By selecting transcripts that are altered only between 3 and 18 months, we identified 46 mRNAs and 34 lncRNAs that are stringently related to the terminal stage of male reproductive aging. Some of these mRNAs were related to hormonal regulation. Finally, our in silico analysis of the 34 aging-related lncRNAs revealed that they co-localized with 19 testis-expressed protein-coding genes, 13 of which are considered to show testis-specific or -predominant expression. These nearby genes could be potential targets of cis-regulation by the aging-related lncRNAs. Collectively, our results identify a number of testicular aging-related mRNAs and lncRNAs in mice and provide a basis for the future investigation of these transcripts in the context of aging-associated testicular dysfunction.

Normal fertility in male mice lacking ADAM32 with testis-specific expression.

The a disintegrin and metalloprotease (ADAM) family proteins comprise a group of membrane-anchored proteins. ADAM32 is expressed specifically in testis and is closely related phylogenetically to ADAM2 and ADAM3, which are known to be critical for fertilization in mice. To assess the biological role of ADAM32, we analyzed Adam32-mutant mice. We found that male mice lacking ADAM32 have normal fertility, testicular integrity, and sperm characteristics. ADAM32 was found to exist at lower levels than ADAM2 and ADAM3 in wild-type testis and sperm, respectively. The present study demonstrates that ADAM32 is dispensable for fertility and appears to be functionally unrelated to ADAM2 and ADAM3 in mice.

Differential activity of 16K rat prolactin in different organic systems.

The 16K isoform of rat prolactin (16K rPRL) performs multiple functions in various systems including angiogenesis, tumorigenesis, and reproduction. Recently, 16K rPRL has attained prominence as a possible therapeutic target in pathophysiological conditions. However, the integral function and mechanism of 16K rPRL in various systems has not been elucidated. To this end, a transient gain-of-function animal model was adopted. An expression DNA plasmid containing 16K rPRL or rPRL gene was introduced into the muscle of adult mice by direct injection. The mRNA and protein expression levels of 16K rPRL were detected by initial RT-PCR and subsequent Southern blot and western blot, respectively. When the expression vector was introduced, the results were as follows: First, 16K rPRL combined with rPRL reduced angiogenesis in the testis whereas rPRL alone induced angiogenesis. Second, 16K rPRL combined with rPRL reduced WBC proliferation, whereas rPRL alone increased WBC proliferation. Third, 16K rPRL combined with rPRL reduced diestrus, whereas rPRL alone extended diestrus. Fourth, 16K rPRL combined with rPRL unexpectedly increased testosterone (T) levels, whereas rPRL alone did not increase T levels. Taken together, our data suggest that the 16K rPRL isoform performs integral functions in angiogenesis in the testis, WBC proliferation, and reproduction, although the action of 16K rPRL is not always antagonistic.

Expressional and functional analyses of epididymal SPINKs in mice.

Epididymal maturation is critical for acquisition of motility and fertilizing capacity by sperm. During epididymal transit, the surface of sperm undergoes prominent sequential changes through interactions with secreted proteins, including protease inhibitors. In the present study, we characterized three epididymis-specific SPINKs (serine protease inhibitors, Kazal-type): SPINK8, SPINK11, and SPINK12. We found that these epididymal SPINKs are expressed in an epididymal region-specific manner and their expression is developmentally regulated. Remarkably, cellular analyses revealed that SPINK8 and SPINK12 are transferred to the sperm. To investigate the in vivo properties of SPINK12, we analyzed knockout mice generated by CRISPR/Cas9-mediated genome editing. Loss of SPINK12 did not alter epididymal tubule structure or sperm phenotypes. Spink12 mutant mice exhibited normal fertility, suggesting that SPINK12 is functionally redundant in the epididymis.

ADAM2 promotes migration of neuroblasts in the rostral migratory stream to the olfactory bulb.

Neuroblasts migrate from the subventricular zone along the rostral migratory stream (RMS) to the olfactory bulb (OB). While the migration occurs by movement over other cells, the molecular mechanisms are poorly understood. We have found that ADAM2 (a disintegrin and metalloprotease 2) is expressed in migrating RMS neuroblasts and functions in their migration. The brains from ADAM2 knockout (KO) mice showed a smaller OB than that seen in wild-type (WT) mice at postnatal day 0. In addition, the RMS in ADAM2 KO mice appeared thinner and less voluminous in its rostral part and thicker in its caudal part. Estimates of migration in vivo using bromodeoxyuridine labeling revealed that neuroblasts from KO mice show a decreased migration rate compared with those from WT mice. Direct assays of migration by imaging living slices also showed a decreased migration speed and loss of directionality in the KO mice. This phenotype was similar to that seen in RMS containing slices from WT mice exposed to a peptide that mimicked the disintegrin loop of ADAM2. Finally, RMS explants from KO or WT mice that were cultured in Matrigel also revealed striking differences. The cells migrating out of explants from WT mice showed robust cell-cell interactions. In contrast, fewer cells migrated out of explants from ADAM2 KO mice, and those that did were largely dispersed and their migration inhibited. These experiments suggest that ADAM2 contributes to RMS migration, possibly through cell-cell interactions that mediate the rapid migration of the neuroblasts to their endpoint.