The testicular cycle of Gambusia affinis holbrooki (Teleostei: Poeciliidae)

Fifteen male mosquito fish ( Gambusia affinis holbrooki ) were collected in 1989 on the 15th of each month to perform a quantitative histologic study of the annual testicular cycle including a calculation of the gonadosomatic index, testicular volume, and the total volume per testis occupied by each germ cell type. The cycle comprises two periods: spermatogenesis and quiescence. The spermatogenic period begins in April with the development of primary spermatogonia into secondary spermatogonia, spermatocytes and round spermatids. In May, the first spermatogenic wave is completed and the testicular volume begins to increase up to June when the maximum testicular volume and gonadosomatic index are reached. Germ cell proliferation with successive spermatogenetic waves continues until August. In September germ cell proliferation ceases and neither secondary spermatogonia nor spermatocytes are observed. However, spermiogenesis continues until October. In November, spermiogenesis has stopped and the testis enters the quiescent period up to April. During this period only primary spermatogonia and spermatozoa are present in the testis. In addition, a few spermatids whose spermiogenesis was arrested in November are observed. Testicular release of spermatozoa is continuous during the entire spermatogenesis period. The spermatozoa formed at the end of this period (September-October) remain in the testis during the quiescent period and are released at the beginning of the next spermatogenesis period in April. Developed Leydig cells appear all year long in the testicular interstitium, mainly around both efferent ducts and the testicular tubule sections showing S₄ spermatids.     

Seasonal changes in the seminiferous epithelium of rhesus and bonnet monkeys

With a view to elucidate seasonal variations in testicular spermatogenesis, quantitative analysis of spermatogenic cells was carried out in non-human primate species viz. rhesus (Macaca mulatta) and bonnet (M. radiata) monkeys during breeding (October-December) and non-breeding (May-June) seasons. The results revealed significant inhibition of testicular germ cell population during non-breeding compared with the breeding period in both the species. Quantitative determination of Sertoli cell-germ cell ratio showed a marked decrease in the number of type A-spermatogonia, spermatocytes (non-pachytene and pachytene) and spermatids (in steps 1-12 of spermiogenesis) in rhesus monkey during the non-breeding period. Bonnet monkeys exhibited the significant decline in the number of primary spermatocytes and spermatids during the non-breeding phase. In addition, average diameter of round seminiferous tubules and nuclear diameter of Leydig cells also decreased significantly in rhesus monkeys. However, bonnet monkeys did not show any significant change in nuclear diameter/morphology of Leydig cells, testicular tubular diameter and number of type A-spermatogoniae. Sertoli cell number did not show any significant change during both breeding and non-breeding periods in both the species. The results of this study indicate a prominent seasonal variation in testicular spermatogenic/Leydig cells in rhesus monkeys than those observed in bonnet monkeys.     

Long day photoperiods and temperature of 20 degrees C induce spermatogenesis in blinded and non-blinded marbled newts during the period of testicular quiescence

Adult male marbled newts (Triturus marmoratus) were collected at the end of the spermatogenesis period and exposed to different photoperiods (natural-daylength-simulated photoperiod, total darkness, 8L:16D, 12L:12D, 16L:8D, and continuous light) for 3 mo. Temperature was maintained at 20 degrees C. Two additional groups of newts were blinded and exposed to either the natural-simulated photoperiod and to 16 h of light per day respectively. Quantitative histologic studies on testicular development and germ cell volume per testis were performed. The newts captured in the field at the beginning (initial controls) or at the end of the experiments (final controls) were in the period of testicular quiescence. Newts kept in total darkness or exposed to a short photoperiod (8L:16D) showed germ cell development up to primary spermatocytes, whereas germ cell development in the newts exposed to long photoperiods (12L:12D or 16L:8D) progressed to elongated spermatids. The newts exposed either to intermediate photoperiods (natural-simulated photoperiod) or to constant light showed an intermediate degree of germ cell development (up to round spermatids). No significant differences between non-blinded and blinded animals were found. These results suggest that (1) mild temperature initiates testicular development in the period of testicular quiescence, (2) long photoperiods associated with mild temperatures produce spermatogenesis in this period, (3) complete darkness or constant light are less effective than some intermediate photoperiod, and (4) the effect of photoperiod on testicular function in newts is not related to ocular photoreception.     

Role of environmental temperature and photoperiod in regulation of seasonal testicular activity in the frog, Rana perezi.

To analyze the role of environmental temperature and photoperiod in the regulation of the annual testicular cycle in Rana perezi, we performed experiments combining high (25 +/- 1 degrees C) or low (6 +/- 1 degrees C) temperature and different photoperiod regimens (18L:6D, 12L:12D, and 6L:18D (hours light:hours dark)) during three phases of the reproductive cycle: winter stage (December) and prebreeding (February) and postbreeding (May, June) periods. Low temperature and short photoperiod in winter induced the arrest of the maturation phase of spermatogenesis and the activation of primary spermatogonia proliferation and spermiohistogenesis. Rana perezi testis responded to long days stimulus in winter, even at low temperature, with induction of the maturation phase of the cycle. Exposure of male frogs to either high temperature or long photoperiod induced a decrease in testosterone levels in winter. During the prebreeding period, an increase in environmental temperature caused a reduction in testosterone, and a lengthening in photoperiod produced the opposite effect. Photoperiod had no effect on testosterone levels during the postbreeding period, but low temperature increased testosterone plasma levels. These results suggest that both temperature and photoperiod effects can vary seasonally, depending on the phase of the annual reproductive cycle in R. perezi.     

Annual reproductive cycle and rate of the spermatogenic process in male mosquitofish <Emphasis Type="Italic">Gambusia affinis</Emphasis>

The present study investigates the relationship between the annual cycle of testicular development and external environment and the rate of spermatogenesis in the mosquitofish Gambusia affinis based on histological observations of testes. The annual reproductive cycle of the mosquitofish was divided into two periods, i.e., the spermatogenic period (May–October) and resting period (October–April). In the spermatogenic period, the transition from spermatogonia to spermatocytes begins and meiosis actively progresses. In the resting period, the transition from spermatogonia to spermatocytes ceases, meiosis of spermatocytes that already shifted by this period gradually progresses, and a considerable number of sperm balls are produced. Onset of spermatogenesis seems to be related to both a rise in water temperature and a prolonged photoperiod. 5-bromo-2-deoxyuridine (BrdU) was a useful in vivo marker of DNA synthesizing spermatogenic cells. The results of immunohistochemical detection of injected BrdU indicated that 5 days are needed for the conversion of spermatocytes to spermatids, 5 days for spermatids to spermatozoa, and 10 days for spermatozoa to sperm balls.     

Immunohistochemical analysis of cAMP response element-binding protein in mouse testis during postnatal development and spermatogenesis

Basal activity and cellular localization of cAMP response element-binding protein (CREB) was examined in mouse testis during postnatal development and spermatogenesis. Testes of ICR mice sampled on postnatal day (PND) 3, 7, 14, 21, 28, 35, 42, and 49 were analyzed using Western blotting. Basal CREB activity was significantly higher in early phase (PND 3–7) developing testes than in intermediate- and late-phase developing (PND 14–42) and adult testes (PND 49). Furthermore, immunohistochemical analysis demonstrated the change of CREB phosphorylation in various testicular cell types during postnatal development. In particular, CREB phosphorylation in seminiferous tubules of the adult testis varied according to the spermatogenic cycle, while phosphorylation was evident in spermatogonia during all stages. Phosphorylation was moderate in pachytene spermatocytes of stages I–III and intense in round and elongate spermatids of spermiogenesis in stages XII–IX. These results suggest that CREB plays an important role in cell proliferation and differentiation in the early phase of postnatal development and spermatogenesis of mouse testis.     

Immunohistochemical detection of the retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) in the marbled newt along the annual cycle

Retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) expression in the testis of the marbled newt were investigated with special attention to the changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. The annual testicular cycle of the marbled newt (Triturus marmoratus marmoratus) comprises three periods: (a) proliferative period (germ cell proliferation from primordial germ cells to round spermatids, April-June); (b) spermiogenesis period (July-September); and (c) quiescence period (interstitial and follicular cells form the glandular tissue, October-April). In the proliferative period, primordial germ cells and primary spermatogonia immunostained intensely to the three types of RXRs and also to FXR. In the other periods, immunostaining to these antibodies was weak or absent. Secondary spermatogonia stained weakly to the four antibodies in the proliferative period, and only to FXR, also weakly, in the spermiogenesis period. Immunoreactive primary spermatocytes were weakly labeled with the RXR antibodies in the proliferative period. Spermatids and spermatozoa did not stain to any antibody in any period. Follicular cells only immunostained to RXR-gamma and only in the quiescence period when they are forming the glandular tissue, together with the interstitial cells. As follicular cells, interstitial cells only immunostained in the quiescence period; however, they immunoreacted to the three types of RXRs. These findings suggest that in the newt, RXRs and FXR are involved in spermatogenesis control by regulating the proliferation of primordial germ cells and spermatogonia. In addition, RXR-gamma seems to be also involved in the development of the glandular (steroidogenic) tissue.     

Flow cytometric quantification of rat spermatogenic cells after hypophysectomy and gonadotropin treatment

DNA flow cytometry was evaluated as a tool to analyze stage-specific changes that occur in absolute cell numbers in the testes. Hypophysectomy was selected as a model system for perturbing testicular cell types, since the cytological sequelae of this treatment post-hypophysectomy in the rat are well documented in the literature. Rat spermatogenic cells in stages II-V, VII, and IX-XIII of the seminiferous epithelial cycle (as defined by Leblond and Clermont, 1952) were quantified in numbers per standard length of seminiferous tubule by DNA flow cytometry after hypophysectomy and subsequent gonadotropin treatment. In agreement with previous histological studies, we found that acrosome- and maturation-phase spermatids disappeared from the seminiferous epithelium after 17 days post-hypophysectomy, whereas meiosis and early spermiogenesis continued at least 164 days. The number of meiotic cells and round spermatids gradually decreased after hypophysectomy. Changes were observed as early as Day 6 post-hypophysectomy. Treatment with human chorionic gonadotropin (hCG) alone maintained most cell numbers within normal limits, and follicle-stimulating hormone (FSH) was needed in addition to hCG to maintain the normal number of cells with the amount of DNA contained in primary spermatocytes and spermatogonia in G2/M-phase (4C) in stages IX-XIII and elongated spermatids (1C') in stages II-V of the epithelial cycle. The absolute numbers of spermatogenic cells at different phases of maturation provide a useful reference for quantitative studies of spermatogenesis. Pathological changes in the seminiferous epithelium can be detected and quantified by DNA flow cytometry.     

Cell population indexes of spermatogenic yield and testicular sperm reserves in adult jaguars (Panthera onca)

The intrinsic yield of spermatogenesis and supporting capacity of Sertoli cells are the desirable indicators of sperm production in a species. The objective of the present study was to quantify intrinsic yield and the Sertoli cell index in the spermatogenic process and estimate testicular sperm reserves by histological assessment of fragments obtained by testicular biopsy of five adult jaguars in captivity. The testicular fragments were fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 μm thickness. In the seminiferous epithelium of the jaguar, 9.2 primary spermatocytes in pre-leptotene were produced by “A” spermatogonia. During the meiotic divisions only 3.2 spermatids were produced by a primary spermatocyte. The general spermatogenic yield of the jaguar was about 23.4 cells and each Sertoli cell was able to maintain about 19.2 germ cells, 11 of them were round spermatids. In each seminiferous epithelium cycle about 166 million spermatozoa were produced by each gram of testicular tissue. In adult jaguars, the general spermatogenic yield was similar to the yield observed in pumas, greater than that observed for the domestic cat, but less compared to most domestic animals.     

Spermatogenic cycle in seals (Callorhinus ursinus L.)

In the fur-seal germ cells at various stages of development are situated in the spermatic canaliculi as concentric layers in accordance with the stages of the spermatogenic cycle. By means of PAS-reaction 18 stages have been revealed in spermatogenesis of the fur-seal and basing on the first 15 of them, 15 stages of the spermatogenic cycles have been presented. Transformations of the nucleus during the process of spermatids development proceed similar to other animals studied, formation of acrosomes is accompanied with a specific for the given species development of temporal formation--tubulo-bulbar complexes, reduced at terminal stages of spermiogenesis.     

Involvement of sex steroid hormones in the early stages of spermatogenesis in Japanese huchen (Hucho perryi )

In higher vertebrates, considerable progress has been made in understanding the endocrine regulation of puberty; however, in teleosts, the regulatory mechanisms of spermatogenesis during the first annual cycle remain unclear. The present study was conducted to understand the regulatory mechanisms of spermatogenesis throughout the different stages of the first spermatogenic cycle and to check the ability of various steroids and hormones to induce in vitro spermatogonial proliferation in Japanese huchen (Hucho perryi ). The results indicate that the serum level of 11-ketotestosterone (11-KT) was positively associated with germ cell type; the level first began to rise with the appearance of late-type B spermatogonia and continued to increase gradually throughout the active spermatogenic stages and spermiogenesis, reaching a peak value 2 wk before spawning, and then declined. During the spermatogenic stages, the serum concentration of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP) was undetectable. Only a small peak was detected with the appearance of spermatocytes and spermatids, and at the time of spawning, the level increased dramatically, reaching its maximum value with the onset of milt production. Despite the high variation in serum levels of 17beta-estradiol (E2) both between months and among the individuals, E2 was found during the whole reproductive cycle. From these results, we concluded that 1) 11-KT is necessary for the initiation of spermatogenesis and sperm production, and it probably plays a role in spermiation, 2) 17alpha,20beta-DP is essential for the final maturation stage, could play a significant role in the mitosis phase and meiosis process, and probably participates in the regulation of spawning behavior, and 3) estrogen is an indispensable male hormone that plays a physiological role in some aspects of testicular functions, especially during the mitotic phase. The three steroids were also able to induce DNA synthesis, spermatogonial renewal, and/or spermatogonial proliferation in vitro.     

精子发生过程中的相关基因

在哺乳动物精子发生过程中, 原生殖细胞发育成为精原细胞, 再发育为精母细胞, 精母细胞经过两次减数分裂成为圆形精细胞, 这些圆形精细胞经过细胞变态形成精子。精子发生过程经历了复杂的细胞分化阶段, 这一阶段受许多因素的调控作用, 其中生精细胞内的基因调节起着决定作用。精子发生中的重要基因与一系列精子发生过程中阶段性的细胞事件密切相关, 例如减数分裂重组、联会丝复合物的形成、姊妹染色体的结合、减数分裂后精子的变态以及减数分裂周期中的关键点和必需因子等。生精细胞许多特异基因的阶段特异性表达, 参与了精子发生这一特殊的细胞分化过程。近年来随着基因克隆、表达和功能研究技术的发展和应用, 发现了许多与精子发生相关的基因, 而且有的被证明在精子发生过程中具有重要作用。文章较全面综述了这一研究领域的一些进展, 着重讨论了与精子发生相关的周期蛋白基因、原癌基因、无精子因子基因、细胞骨架基因、热休克基因、核蛋白转型基因、中心体蛋白基因和细胞凋亡相关基因等。     

La fécondation par injection d'une spermatide dans l'ovule

There was a recent large spreading of intracytoplasmic sperm injection (ICSI) to treat male infertility in most of in vitro fertilization (IVF) laboratories. The recent data confirm the efficacy of ICSI even by using testicular sperm or sperm with grossly abnormal phenotype (round head, absence of motility). Moreover it appears that ICSI could pass beyond the last events of spermatogenesis (i.e. spermiogenesis), since normal development follows fertilization with the male gamete, spermatid, recovered just after completion of meiosis. It is obvious that the natural properties of a mature spermatozoon (motility, ADN compaction, oocyte recognition and penetration) are only necessary to reach the site of fertilization (into the female tube) and to pass through the protective enveloppes around the oocyte (cumulus oophorus, zona pellucida, plasma membrane). The current view that spermatids lack genetic maturation comparing to eggs is not valid since eggs are only secondary oocytes at a meiotic stage equivalent to that of secondary spermatocytes. Moreover genetic imprinting occurs before meiosis, and cytoplasmic structures which seem necessary for embryo development are already present in spermatids. ICSI using spermatid cells is relevant to men suffering non obstructive azzospermia if spermatids are recovered from either the ejaculate or the testicular tubes. Several normal babies were born after injection of round spermatids. Since these spermatogenic cells are present in the ejaculate of most of the patients with non obstructive azoospermia (76% in our lab), one can estimate to 5–10% the proportion of sterile men potentially concerned by conception with spermatids. However certain of these men may have occasional sperm found with testicular sperm extraction and it is to early to know if such iatrogenic extraction is always preferable to ejaculate spermatid collection.     

The germ cell development strategy and seasonal changes in spermatogenesis and Leydig cell morphologies of the spiny lizard Sceloporus mucronatus (Squamata: Phrynosomatidae)

The viviparous lizards of the Sceloporus genus exhibit both seasonal and continuous spermatogenesis. The viviparous lizard Sceloporus mucronatus from Tecocomulco, Hidalgo, México, exhibits seasonal spermatogenesis. This study demonstrates the relationship between changes in testis volume, spermatogenesis activity, and Leydig cells during the male reproductive cycle of S. mucronatus. A recrudescence period is evident, which starts in the winter when testicular volume is reduced and climaxes in February, when the greatest mitotic activity of spermatogonia occurs. The testicular volume and Leydig cell index increase gradually during the spring with primary spermatocytes being the most abundant cell type observed within the germinal epithelium. In the summer, the secondary spermatocytes and undifferentiated round spermatids are the most abundant germinal cells. The breeding season coincides with spermiogenesis and spermiation; testicular volume also increases significantly as does the Leydig cell index where these cells increase in both cytoplasmic and nuclear volume. During fall, testicular regression begins with a significant decrease in testicular volume and germinal epithelium height, although there are remnant spermatozoa left within the lumen of the seminiferous tubules. During this time, the Leydig cell index is also reduced, and there is a decrease in cellular and nuclear volumes within these interstitial cells. Finally, during quiescence in late fall, there is reduced testicular volume smaller than during regression, and only spermatogonia and Sertoli cells are present within the seminiferous tubules. Leydig cells exhibit a low index number, their cellular and nuclear volumes are reduced, and there is a depletion in lipid inclusion cytoplasmically.     

The ability of whale haploid spermatogenic cells to induce calcium oscillations and its relevance to oocyte activation

Interspecies microinsemination assay was applied to examine the ability of minke whale haploid spermatogenic cells to induce Ca2+ oscillations and oocyte activation. Populations of round spermatids (RS), early-stage elongating spermatids (e-ES), late-stage elongating spermatids (1-ES) and testicular spermatozoa (TS) were cryopreserved in the presence of 7.5% glycerol on board ship in the Antarctic Ocean. Repetitive increases of intracellular Ca2+ concentration occurred in 0, 65, 81 and 96% of BDF1 mouse oocytes injected with the postthaw RS, e-ES, 1-ES and TS, respectively. A normal pattern of the Ca2+ oscillations was observed in 26-47% of the responding oocytes. Most oocytes that exhibited Ca2+ oscillations, regardless of the oscillation pattern, resumed meiosis (83-94%). These results indicate that whale spermatogenic cells acquire SOAF activity, which is closely related to their Ca2+ oscillation-inducing ability at the relatively early stage of spermiogenesis.     

Characterization and immunolocalization of beta-D-glucuronidase in mouse testicular germ cells and spermatozoa

Spermatozoa released from the seminiferous tubules are terminally differentiated cells with no known synthetic activity. Their components are synthesized in the spermatogenic cells during spermatogenesis. In this study, we report the characterization and immunolocalization of beta-glucuronidase in mouse testicular germ cells and spermatozoa. The enzyme is an exoglycohydrolase with dual localization, being present in lysosomes and endoplasmic reticulum of several mouse and rat tissues. The purified germ cell preparations (spermatocytes, round spermatids, and condensed/elongated spermatids) when assayed for beta-glucuronidase activity showed that the spermatocytes contained five times more enzyme activity per cell than the spermatids. Polyacrylamide gel electrophoresis, carried out under native and denaturing conditions, demonstrated that the germ cells express only the lysosomal form of the enzyme (pI 5.5-6.0) with a subunit molecular mass of 74 kDa. Immunocytochemical studies revealed a positive reaction in the Golgi membranes, Golgi-associated vesicles, and lysosomes of late spermatocytes (pachytene spermatocytes) and a stage-specific localization during spermiogenesis. The forming or formed acrosome of the elongated spermatids (stages 9-16) and epididymal spermatozoa was highly immunopositive. Comparison of immunoprecipitation curves and kinetic properties of the enzyme present in spermatocytes and spermatozoa revealed no major differences. Taken together, our results demonstrate that beta-glucuronidase activities present in the lysosomes of spermatocytes and the sperm acrosome are kinetically and immunologically similar.     

Seasonal spermatogenic cycle and morphology of germ cells in the viviparous lizard Mabuya brachypoda (Squamata,Scincidae)

We describe seasonal variations of the histology of the seminiferous tubules and efferent ducts of the tropical, viviparous skink, Mabuya brachypoda, throughout the year. The specimens were collected monthly, in Nacajuca, Tabasco state, Mexico. The results revealed strong annual variations in testicular volume, stages of the germ cells, and diameter and height of the epithelia of seminiferous tubules and efferent ducts. Recrudescence was detected from November to December, when initial mitotic activity of spermatogonia in the seminiferous tubules were observed, coinciding with the decrease of temperature, photoperiod and rainy season. From January to February, early spermatogenesis continued and early primary and secondary spermatocytes were developing within the seminiferous epithelium. From March through April, numerous spermatids in metamorphosis were observed. Spermiogenesis was completed from May through July, which coincided with an increase in temperature, photoperiod, and rainfall. Regression occurred from August through September when testicular volume and spermatogenic activity decreased. During this time, the seminiferous epithelium decreased in thickness, and germ cell recruitment ceased, only Sertoli cells and spermatogonia were present in the epithelium. Throughout testicular regression spermatocytes and spermatids disappeared and the presence of cellular debris, and scattered spermatozoa were observed in the lumen. The regressed testes presented the total suspension of spermatogenesis. During October, the seminiferous tubules contained only spermatogonia and Sertoli cells, and the size of the lumen was reduced, giving the appearance that it was occluded. In concert with testis development, the efferent ducts were packed with spermatozoa from May through August. The epididymis was devoid of spermatozoa by September. M. brachypoda exhibited a prenuptial pattern, in which spermatogenesis preceded the mating season. The seasonal cycle variations of spermatogenesis in M. brachypoda are the result of a single extended spermiation event, which is characteristic of reptilian species. J. Morphol. © 2012 Wiley Periodicals, Inc.     

Inhibition of Spermiogenesis of Eupyrene Spermatozoa by Elevated,Sublethal Temperatures During Pupal-Adult Development of the Bertha Armyworm,Mamestra con figurata Wlk. (Lepidoptera,Noctuidae)

Summary

Eupyrene spermiogenesis and spermatozoa production in Mamestra configurata Walker were inhibited by temperatures >24° C during a “critical period” of about 7 days during the first half of post-diapause pupal-adult development. The critical period coincided with the appearance of early-stage eupyrene spermatids. If temperatures >24° C occurred during the critical period, eupyrene spermatozoa production was reduced, the result of irreversible autolysis of the early- and mid-stage eupyrene spermatids. Each day of exposure to 25 or 27.5° C during the critical period reduced the number of eupyrene cysts with spermatozoa on average by 12 to 14%. There were no observable effects on eupyrene spermiogenesis and spermatozoa production of exposing post-diapausing pupae to 25 or 27.5° C before or after the critical period. Apyrene spermatogenesis was not affected by 25 or 27.5° C.

Exposure of pupae to 25°or 27.5° C during diapause had no apparent effect on eupyrene spermiogenesis and spermatozoa production. Meiosis usually was not detected among the eupyrene cysts with lalje primary spermatocytes until after diapause, indicating that meiosis is suppressed during diapause. By providing a meiotic block, diapause governs the time of onset of the temperature-sensitive critical period of spermiogenesis. In nature, male pupae of M. configurata in diapause are protected from sterilizing temperatures in the soil during August and post-diapause developing pupae pass through the critical period of spermiogenesis in spring (April to June) when soil temperatures throughout the range of M. configurata in Canada are well below 25° C. The distribution of M. configurata in the northern region of the temperate zone may in part be attributable to the sensitivity of spermatogenesis to relatively mild temperatures.