Similar high molecular weight forms of growth hormone-releasing hormone are found in rat brain and testis

We have utilized a new radioimmunoassay for rat growth hormone-releasing hormone (GHRH) to investigate the presence of GHRH in different organ systems of adult rat, and specifically the rat central nervous system (CNS). The highest concentration of GHRH was found, as expected, in the hypothalamus, but significant amounts were also located in the brain cortex, predominantly the frontal cortex, as well as in the testis. Smaller amounts were identified in the cerebellum and brain stem. Sephadex and reversed phase high performance liquid chromatography demonstrated that while hypothalamic GHRH exclusively eluted at the position of rat GHRH (1-43), in testis and brain the major form was predominantly (testis) or wholly (brain) of a higher molecular weight. While this molecular species has yet to be further characterized, the data suggest the similar GHRH-like species exist in the CNS as well as the testis.     

Immunohistochemical Localization of Mitochondrial Fatty Acid ��-Oxidation Enzymes in Rat Testis

The testis consists of two types of tissues, the interstitial tissue and the seminiferous tubule, which have different functions and are assumed to have different nutritional metabolism. The localization of enzymes of the mitochondrial fatty acid β-oxidation system in the testis was investigated to obtain a better understanding of nutrient metabolism in the testis. Adult rat testis tissues were subjected to immunoblot analysis for quantitation of the amounts of enzyme proteins, to DNA microarray analysis for gene expression, and to immunofluorescence and immunoelectron microscopy for localization. Quantitative analysis by immunoblot and DNA microarray revealed that enzymes occur abundantly in Leydig cells in the interstitial tissue but much less so in the seminiferous tubules. Immunohistochemistry revealed that Leydig cells in the interstitial tissue and Sertoli cells in the seminiferous tubules contain a full set of mitochondrial fatty acid β-oxidation enzymes in relatively plentiful amounts among the cells in the testis, but that this is not so in spermatogenic cells. This characteristic localization of the mitochondrial fatty acid β-oxidation system in the testis needs further elucidation in terms of a possible role for it in the nutritional metabolism of spermatogenesis. (J Histochem Cytochem 58:195–206, 2010)     

Role of neurotropins in rat embryonic testis morphogenesis (cord formation)

The process of seminiferous cord formation is the first morphological event that differentiates a testis from an ovary and indicates male sex determination. Cord formation occurs by embryonic Day 14 (Day 0 = plug date; E14) in the rat. A series of experiments were conducted to determine if neurotropins and their receptors are important for the process of rat embryonic cord formation. The expression of low affinity neurotropin receptor (p75/LNGFR) was determined by immunohistochemistry on sections of both testis and ovary from E13 through birth (Day 0, P0) with an antibody to p75/LNGFR. The staining for p75/LNGFR was present in the mesonephros of E13 gonads and in a sex-specific manner appeared around developing cords at E14 in the embryonic testis. At birth, staining for p75/LNGFR was localized to a single layer of cells (i.e., peritubular cells) that surrounded the seminiferous cords. The genes for both neurotropin 3 (NT3) and for corresponding high affinity neurotropin trkC receptor were found to be expressed in the E14 rat testis, as well as other neurotropins and receptors. Immunocytochemical analysis of E14 rat testis demonstrated that NT3 was localized to the Sertoli cells and trkC was present in individual cells of the interstitium at E16 and in selected preperitubular cells at E18. Previously, the peritubular cells adjacent to the cords were demonstrated to be derived from migrating mesonephros cells around the time of cord formation. To determine if neurotropins were involved in cord formation, the actions of neurotropins were inhibited. A high affinity neurotropin receptor (trk)-specific kinase inhibitor, K252a, was used to treat organ cultures of testes from E13 rats prior to cord formation. Treatment of E13 testis organ cultures with K252a completely inhibited cord formation. K252a-treated organ cultures of E14 testis that contained cords did not alter cord morphology. A second experiment to inhibit neurotropin actions utilized a specific antagonist trk-IgG chimeric fusion protein and E13 testis organ cultures. The trk-IgG molecules dimerize with endogenous trk receptors and inhibit receptor signaling and activation of ligand function. Forty percent of E13 testis organ cultures treated with trkC-IgG had significantly reduced cord formation. TrkA-IgG had no effect on initiation of cords; however, in fifty percent of the treated organs, a "swollen" appearance of the cord structures was observed. Experiments using trkB-IgG chimeric protein on E13 organ cultures had no effect on cord formation or cord morphology. The testes from trkC and NT3 knockout mice were examined to determine if there were any morphological differences in the testis. NT3 knockouts appeared to have normal cord morphology in E15 and E17 testis. TrkC knockout mice also had normal cord morphology in E14 and P0 testis. Both NT3 and trkC knockout-mice testis had less interstitial area than wild-type controls. In addition, the trkC knockout mice have an increased number of cells expressing p75LNGFR within the cords when compared to controls or NT3 knockout mice. Combined observations suggest compensation between the different neurotropin ligands, receptors, and/or possibly different growth factors for this critical biological process. In summary, results suggest a novel nonneuronal role for neurotropins in the process of cord formation during embryonic rat testis development. The hypothesis developed is that neurotropins are involved in the progression of male sex differentiation and are critical for the induction of embryonic testis cord formation.     

Role of transforming growth factor-alpha and the epidermal growth factor receptor in embryonic rat testis development

Embryonic testis development requires the morphogenesis of cords and growth of all cell populations to allow organ formation. It is anticipated that coordination of the growth and differentiation of various cell types involves locally produced growth factors. The current study was an investigation of the hypothesis that transforming growth factor-alpha (TGF-alpha) is involved in regulating embryonic testis growth. TGF-alpha has previously been shown to function in the postnatal testis. TGF-alpha and other members of the epidermal growth factor (EGF) family act through the epidermal growth factor receptor (EGFR) to stimulate cell proliferation and tissue morphogenesis. To understand the potential actions of TGF-alpha in the embryonic testis, general cell proliferation was investigated. Characterization of cell proliferation in the rat testis throughout embryonic and postnatal development indicated that each cell type has a distinct pattern of proliferation. Germ cell growth was transiently suppressed around birth. Interstitial cell growth was high embryonically and decreased to low levels around birth. A low level of Sertoli cell proliferation was observed at the onset of testis cord formation. Sertoli cell proliferation in early embryonic development was low; the levels were high later in embryonic development and remained high until the onset of puberty. Both TGF-alpha and the EGFR were shown to be expressed in the embryonic and postnatal rat and mouse testis. Perturbation of TGF-alpha function using neutralizing antibodies to TGF-alpha on testis organ cultures dramatically inhibited the growth of both embryonic and neonatal testis. TGF-alpha antibodies had no effect on cord formation. The TGF-alpha antibody was found to be specific for TGF-alpha in Western blots when compared to EGF and heregulin. Testis growth was also inhibited by perturbation of EGFR signaling using an EGFR kinase inhibitor. Therefore, TGF-alpha appears to influence embryonic testis growth but not morphogenesis (i.e., cord formation). Treatment of embryonic testis organ cultures with exogenous TGF-alpha also perturbed development, leading to an increased proliferation of unorganized cells. Testis from EGFR and TGF-alpha knockout mice were analyzed for testis morphology. TGF-alpha knockout mice had no alterations in testis phenotype, while EGFR knockout mice had a transient decrease in the relative amount of interstitial cells before birth. Observations suggest that there may be alternate or compensatory factors that allow testis growth to occur in the apparent absence of TGF-alpha actions in the mutant mice. In summary, the results obtained suggest that TGF-alpha is an important factor in the regulation of embryonic testis growth, but other factors will also be involved in the process.     

Expression and cellular localization of the mRNA for the 25-kDa heat-shock protein in the mouse

The 25-kDa heat-shock protein (Hsp25) is a member of the small heat-shock protein family but its function remains largely unknown. In the present study we examined the expression and cellular localization of Hsp25 mRNA in mice under physiological, unstressed conditions using Northern blot and in situ hybridization analyses with specific oligonucleotide probes. At the organ level, high amounts of Hsp25 mRNA were detected in the oesophagus, skin,eye, stomach, lung and urinary bladder, with moderate amounts in the heart, skeletal muscle, aorta, adrenal gland, ovary, testis, uterus, large intestine, and thymus. At the cellular level, intense to moderate signals for Hsp25 mRNA were localized in the muscle cells of smooth, heart and skeletal types, in the epithelial cells of stratified squamous and transitional types and of the oviduct, in the steroid endocrine cells of the adrenal cortex and corpus luteum, as well as in the spermatocytes of the testis. In contrast, the signal was scarcely detectable in the nervous tissues, lymphatic tissues, the columnar epithelial cells of the digestive tract, or the parenchymal cells of the liver, pancreas and kidney. These results suggest some significant role for Hsp25 in distinct populations of mouse cells under physiological conditions.     

A Gas Chromatographic Method for the Determination of N-Acetyl-l-Aspartic Acid, N-Acetyl-α- Aspartylglutamic Acid and β-Citryl-l-Glutamic Acid and Their Distributions in the Brain and Other Organs of Various Species of Animals

Abstract: A simple and sensitive gas-chromatographic method for the determination of N-acetyl- l -aspartic acid (NA-Asp), N-acetyl-α-aspartylglutamic acid (NA-Asp-Glu) and β-citryl- l -glutamic acid (β-CG) was developed. The organ, regional and phylogenetic distributions of these compounds were studied. NA-Asp and NA-Asp-Glu were highly concentrated in nervous tissue, and less than 1% of the amounts in the nervous tissues were found in nonnervous organs. These two compounds showed a reciprocal relationship in their regional distribution in mature brains, but such a relationship was not evident or was even reversed in immature brains. The two compounds also showed different developmental changes in different regions of the brain. Fish brain contained a relatively high concentration of NA-Asp, but only a trace amount of NA-Asp-Glu. By contrast, a 10 times higher concentration of NA-Asp-Glu than NA-Asp was found in frog brain. Reptilian brain contained similar amounts of each compound. Avian and mammalian brain had NA-Asp at a roughly 10 times higher concentration than NA-Asp-Glu. β-CG occurred at the highest concentration in the immature brain of rat and guinea pig, but disappeared in the mature brains. The adult frog brain, however, contained a large amount of β-CG. In the adult rat, testis contained the highest concentration of β-CG.     

Absence asymmetry: the evolution of monorchid beetles (Insecta: Coleoptera: Carabidae)

Asymmetrical monorchy, or the complete absence of one testis coupled with the presence of its bilateral counterpart, is reported for 174 species of the carabid beetle tribes Abacetini, Harpalini, and Platynini (Insecta: Coleoptera: Carabidae) based on a survey of over 820 species from throughout the family. This condition was not found in examined individuals of any other carabid beetle tribes, or of other adephagan beetle families. One monorchid taxon within Platynini exhibits symmetrical vasa deferentia at the beginning of the pupal stadium, suggesting that developmental arrest of the underdeveloped vas deferens takes place in pupation. The point at which development of the testis is interrupted is unknown. Complete absence of one organ of a bilateral pair--absence asymmetry--is rarely found in any animal clade and among insects is otherwise only known for testes in the minute-sized beetles of the family Ptiliidae, ovaries in Scarabaeinae dung beetles, and ovaries of some aphids. Based on current phylogenetic hypotheses for Carabidae, testis loss has occurred independently at least three times, and up to five origins are possible, given the variation within Abacetini. Clear phylogenetic evidence for multiple independent origins suggests an adaptive or functional cause for this asymmetry. A previously posited taxon-specific hypothesis wherein herbivory in the tribe Harpalini led to testis loss is rejected. Optimal visceral packing of the beetle abdomen is suggested as a general explanation. Specifically, based on the function of various organ systems, we hypothesize that interaction of internal organs and pressure to optimize organ size and space usage in each system led to the multiple origins and maintenance of the monorchid condition. Testes are the only redundant and symmetrically paired structures not thought to be developmentally linked to other symmetrical structures in the abdomen. Among all possible organs, they are the most likely--although the observed frequency is very small--to bypass constraints that maintain bilateral symmetry, resulting in absence asymmetry. However, based solely on our observations of gross morphology of internal organs, no function conclusively explains the ontogenetic loss of one testis in these taxa. Unlike the analogous absence asymmetry of organs in other animal groups, no dramatic body-form constraint--e.g., snakes and lung loss, ptiliid beetles' small body-size and relatively giant sperm--or adaptive scenario of improved locomotory performance--e.g., birds and ovary loss due to flight constraints-applies to these carabid beetles. We tentatively suggest that testis loss is driven wholly by an interaction among the internal organs of these beetles, possibly due to selective pressure to maximize the comparatively large accessory glands found in these taxa. However, as the ordering of these evolutionary events of testis loss and accessory gland size increase is not known, large accessory glands might have secondarily evolved to compensate for a decreased testicular output.     

The effect of alpha-tocopherol on lipid peroxidation of microsomes and mitochondria from rat testis

The testis is a remarkably active metabolic organ; hence it is suitable not only for studies of lipid metabolism in the organ itself but also for the study of lipid peroxidation processes in general. The content of fatty acids in testis is high with a prevalence of polyunsaturated fatty acids (PUFA) which renders this tissue very susceptible to lipid peroxidation. Studies were carried out to evaluate the effect of alpha-tocopherol in vitro on ascorbate-Fe(++) lipid peroxidation of rat testis microsomes and mitochondria. Chemiluminescence and fatty acid composition were used as an index of the oxidative destruction of lipids. Special attention was paid to the changes produced on the highly PUFA [C20:4 n6] and [C22:5 n6]. Lipid peroxidation of testis microsomes or mitochondria induced a significant decrease of both fatty acids. Total chemiluminescence was similar in both kinds of organelles when the peroxidized without (control) and with ascorbate-Fe(++) (peroxidized) groups were compared. Arachidonic acid was protected more efficiently than docosapentaenoic acid at all alpha-tocopherol concentrations tested when rat testis microsomes or mitochondria were incubated with ascorbate-Fe(++). The maximal percentage of inhibition in both organelles was approximately 70%; corresponding to an alpha-tocopherol concentration between 1 and 0.25 mM. IC50 values from the inhibition of alpha-tocopherol on the chemiluminescence were higher in microsomes (0.144 mM) than mitochondria (0.078 mM). The protective effect observed by alpha-tocopherol in rat testis mitochondria was higher compared with microsomes, associated with the higher amount of [C20:4 n6]+[C22:5 n6] in microsomes that in mitochondria. It is proposed that the vulnerability to lipid peroxidation of rat testis microsomes and mitochondria is different because of the different proportion of PUFA in these organelles The peroxidizability index (PI) was positively correlated with the level of long chain fatty acids. The results demonstrated the protective effect of alpha-tocopherol on lipid peroxidation in microsomes and mitochondria from rat testis.     

Partial characterization of a new basic nuclear protein from rat testis elongated spermatids.

A basic protein designated TP2 has been isolated from rat testis elongated spermatids. This new protein contains basic and acidic amino acids in relative amounts similar to those in histone F2al but is unusually rich in serine and proline. Techniques which were developed for preparing relatively homogeneous populations of spermatid nuclei were used to demonstrate that TP2 is most abundant and most actively synthesized in spermatids representing steps 12 through 15 of spermiogenesis.     

Quantitative investigations on the topography of twelve structural elements of the male gonad

For elucidating the problem whether the tissue composition of the male gonad is identical or different in the equatorial zone and polar zones of the organ, 104 left testes were studied obtaining them from the cadaver of patients dying from various acute and chronic diseases not affecting the endocrine system. In the histological preparations of the polar and equatorial zones of the testis, the proportion [%] were calculated of 6 types of convoluted tubules designated with numbers from 1 to 6 in the sequence of decreasing spermatogenic activity, and by the morphometric method, the proportions [%] were determined of the tubular lumina, spermatogenic epithelium, tubular membrane, stroma, blood vessels, and LEYDIG's cells. In the whole analysed group, only tubules type 2 were more numerous in the equatorial part, while tubules type 6 prevailed in the polar parts. The lumina of the tubules occupied a greater % of the testicular structure in the polar parts as compared with the central part. Other tissue elements were present in similar amounts or the differences between their amounts in these parts were only nearly significant statistically. The study showed also that the number of type 1 tubules decreased significantly with progressing age, while the number of type 6 tubules increased. A statistically significant fall of the proportion [%] of the spermatogenic epithelium and increased proportion of tubular membranes and stroma were another age-related changes. It is worth stressing that these age-related processes progressed parallelly in the equatorial part and in the polar parts of the testis which suggest that ageing of the organ occurs simultaneously in the whole gonad.     

Localization of lysosomal acid phosphatase mRNA in mouse tissues.

We studied the expression of lysosomal acid phosphatase (LAP) in mouse by hybridizing Northern blots and tissue sections with the mouse LAP cDNA. Three mRNA species of 2.3, 3.2 and 5.2 KB were identified, which differ in the length of their 3' untranslated region (UTR). The 3.2 KB mRNA is expressed in equal amounts in all tissues and represents the major species in most tissues, whereas the amounts of the 2.3 and 5.2 KB species differ. In situ hybridization of different tissues of adult mice showed a uniform expression of LAP, as expected for a housekeeping gene, except in testis and brain. In testis we found an increase in the LAP mRNA level in spermatocytes. By Northern blot analysis of young mouse testis, this increase could be attributed to late pachytene primary spermatocytes or secondary spermatocytes. In brain tissue the neurons were predominantly labeled, especially the Purkinje and pyramidal cells, whereas glial cells expressed only low amounts of LAP mRNA. Very high LAP expression was also found in the epithelial cells of the choroid plexus. Analysis of LAP expression during mouse embryonic development between Days 9.5 and 17.5 revealed a prominent expression relative to other tissues in the neural tube from Day 9.5 to Day 13.5.     

Subcellular fractionation of rainbow trout gonads with emphasis on microsomal enzymes involved in steroid metabolism

Summary Rainbow trout gonads were subfractionated by differential centrifugation with emphasis on obtaining preparations suitable for the study of steroid-metabolizing enzymes. A fractionation scheme was evaluated for the mature testis and for 3 ovarian developmental stages. The distribution of cell organelles among the fractions was determined using enzyme-markers and electron microscopy. The fractionation scheme was found to be suitable for separating mitochondria and microsomes which were recovered at similar yields to those that had been reported for other extraheptic fish tissues. Fractionation of the mature ovary was fraught with problems probably because a large yolk protein cytosole fraction interfered with the recovery of microsomes. However, no difference in the specific activity of microsomal NADPH-cytochrome c-reductase between the various organ preparations was evident. The testis microsomes contained detectable amounts of cytochrome P450, whereas its content in the various ovary microsomes was too low to be detected. Progesterone 17-hydroxylase was detected in microsomes from testes and early developing ovaries, and microsomal aromatase activity was present in microsomes from early developing, mature and postovulatory ovaries. Furthermore, the testis microsomes contained a highly active UDP glucuronosyltransferase with testosterone used as a substrate.     

灰仓鼠重要内脏器官生长指数及其变化

对室内培育的灰仓鼠进行同一季节不同年龄段和同一年龄段不同季节某些器官的测量。结果显示, 在初生至25 d 的高速生长期, 体重与肝脏、心脏、肺、脾脏和肾的生长非常接近Huxley 的相对生长公式Y = b x^k , 其回归方程分别为: 肝脏Y = 0.013 47 x^{1.515 9} ; 心脏Y = 0.000 18 x^2.163 ; 肺Y = 0.028 48 x^{0.798 2} ; 脾脏Y = 0.000 24 x^{1.583 6} ; 肾脏Y = 0.000 2418 x^{2.310 4} , 并高度相关。睾丸的回归方程在60 日内与Y = b x^k公式非常拟合, Y = 0.000 0108 x^{3.049 4} , r = 0.989 9。除性腺外, 肝脏、心脏、肺、脾脏和肾脏的比值最高值均在25 d 之前。在性成熟期不同年龄段, 性腺比值较稳定, 其它器官比值在性别和年龄段有显著性差异, 雌性普遍高于雄性。在10 月龄不同季节, 只有体重和睾丸比值有显著性差异(7 月最高) , 表示在不同季节利用相近体重比较器官指数差异应将年龄因素作为重要参考依据。     

Rat ATP citrate-lyase. Molecular cloning and sequence analysis of a full-length cDNA and mRNA abundance as a function of diet, organ, and age

ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. We have isolated a full-length cDNA copy of 4.3 kilobase pairs encoding the ATP-citrate lyase mRNA by screening rat liver cDNA library using oligonucleotide probes designed from peptide sequences obtained from the purified rat enzyme. Expression of this cDNA in bacteria, followed by immunoblotting with antibody directed against the ATP citrate-lyase, further demonstrated the identity of this clone. Nucleic acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1100 amino acids in length with a calculated molecular weight of 121,293. RNA blot analysis indicated an mRNA species of about 4.3 kilobase pairs in livers of chow-fed rats. Rats maintained on low fat, high carbohydrate diets exhibited a striking increase (50-fold) in the level of liver ATP citrate-lyase mRNA as compared with the control animals maintained on a normal diet. The tissue distribution of this mRNA in chow-fed animals revealed a relatively high abundance of the message in liver and adrenal, moderate levels were found in lung, brain, and large intestine with only trace amounts of the message in small intestine, stomach, testis, spleen, pancreas, kidney, and heart. During rat development, the ATP citrate-lyase mRNA was relatively high in the liver at parturition, followed by a reduction in its level during suckling. Higher amounts of the mRNA were detected again in adult animals. The isolation and characterization of the mRNA for ATP citrate-lyase will allow further studies on the reaction mechanism and metabolic regulation of this key enzyme in lipogenesis and cholesterogenesis.     

Steroid deficiency syndromes in mice with targeted disruption of Cyp11a1

Steroid deficiencies are diseases affecting salt levels, sugar levels, and sexual differentiation. To study steroid deficiency in more detail, we used a gene-targeting technique to insert a neo gene into the first exon to disrupt Cyp11a1, the first gene in steroid biosynthetic pathways. Cyp11a1 null mice do not synthesize steroids. They die shortly after birth, but can be rescued by steroid injection. Due to the lack of feedback inhibition by glucocorticoid, their circulating ACTH levels are exceedingly high; this results in ectopic Cyp21 gene expression in the testis. Male Cyp11a1 null mice are feminized with female external genitalia and underdeveloped male accessory sex organs. Their testis, epididymis, and vas deferens are present, but undersized. In addition, their adrenals and gonads accumulate excessive amounts of lipid. The lack of steroid production, abnormal gene expression, and aberrant reproductive organ development resemble various steroid deficiency syndromes, making these mice good models for studies of steroid function and regulation.     

Localization of angiotensin converting enzyme (dipeptidyl carboxypeptidase) in swine sperm by immufluorescence

Testis and epididymis are known to have high amounts of angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1). We investigated the localization of the enzyme in these tissues by an immunofluorescent technique and found that the enzyme was localized in the spermatids and residual bodies in the Sertoli cells of the testis. Furthermore, the enzyme was shown to be present in the cytoplasmic droplet of epididymal sperm and also in detached cytoplasmic droplets in semen. The enzyme was not detected in the interstitium of testis and epididymis except for the endothelial cells of the vessel.     

Ribonuclease-RNAase inhibitor complex from rat testis. Purification of the RNAase inhibitor

The RNAase inhibitor from rat testis has been purified to homogeneity. The purified protein appeared as a single spot after two-dimensional electrophoresis. The calculated Mr value is 48,000 which coincides with that obtained for the native protein on gel filtration chromatography, thus indicating a single polypeptide chain. The amino acid composition and the characteristics of the inhibitor activity are reported and compared to those of other RNAase inhibitors from mammalian tissues. The naturally occurring ribonuclease-RNAase inhibitor complex from rat testis has also been studied and compared with the rat testis inhibitor-RNAase A as model complex. The ribonuclease released from the natural rat testis complex showed heterogeneity of size. The significance of the rat testis ribonuclease/RNAase inhibitor system is discussed in terms of the important functionality of this organ.     

Intraspecific variation in testis asymmetry in birds: evidence for naturally occurring compensation

In many taxa, the left and right testes often differ in size. The compensation hypothesis states that one testis of the pair serves as a ‘back-up’ for any reduced function in the other and provides a mechanism to explain intraspecific variation in degree and direction of gonad asymmetry. Although testis asymmetry is common in birds, evidence for natural testis compensation is unknown. Using a novel quantitative approach that can be applied to any bilateral organ or structure, we show that testis compensation occurs naturally in birds and can be complete when one testis fails to develop. Owing to a recurrent risk of testis impairment and an evolutionary trade-off between natural and sexual selections acting on the arrangement of internal organs in species with abdominal and/or seasonal testes, compensation adds an important, but neglected, dimension to measures of male reproductive investment.