Period Of Sperm Storage Research Articles

Evaluation of antioxidant enzymes in the female genital fluid of the bat Corynorhinus mexicanus during sperm storage

Abstract Female Corynorhinus mexicanus bats store sperm for three months. One cause of the death of sperm is the action of reactive oxygen species (ROS). Three antioxidant enzymes – superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) – regulate the REDOX state of the environment in which gametes are stored. The aim of this study was to evaluate antioxidant enzymatic activity in female genital fluid during the period of sperm storage (October-December). The activity of the enzymes in the female bats’ genital fluid was determined by spectrophotometry. SOD activity was 20 USOD/ml/min in each month. Total GPX activity was 70 UGPX/ml/min in October, but increased in November and December to 145 UGPX/ml/min. Specific CAT activity was 2000 UK/ml/min in October, but in November it increased to 16 000 UK/ml/min, then to 32 000 UK/ml/min in December. The ROS fluorescence indices in the sperm ranged from 3000 to 8000 with no significant differences. These data show that antioxidant enzymes participate in the maintenance of sperm viability in the female reproductive tract of C. mexicanus.

Transcriptome sequencing reveals ovarian immune response and development during female sperm storage in viviparous black rockfish (Sebastes schlegelii)

Black rockfish (Sebastes schlegelii) is a viviparous teleost fish whose spermatozoa were transferred into the female ovary cavity and stored for up to five months and then fertilized with the matured eggs. There is no clarity about the molecular characteristics of ovarian follicles during female sperm storage in Sebastes schlegelii. In this study, histological observation, transcriptomic analysis and hormone level detection were performed in ovaries at stages of pre-mating (PRM), post-mating (POM) and pre-fertilization (PRF). Histological observation displayed that oocytes developed from the primary growth (PG) stage to the mature stage during the three stages. Furthermore, somatic cells around the oocyte were proliferated and spermatozoa were found near the layer of epithelial cells. Transcriptomic analysis showed that there were 437 and 747 differentially expressed genes (DEGs) in ovarian comparison of PRM-vs-POM and POM-vs-PRF, respectively. GO enrichment and KEGG analysis revealed that lots of DEGs from PRM-vs-POM were linked to immune-related pathways, such as antigen processing and presentation, immune response, and complement and coagulation cascade. Meanwhile, seven DEGs associated with immune response were differentially expressed after spermatozoa treatment in ovarian tissue in vitro. While the DEGs from POM-vs-PRF were mostly enriched in the pathways related to homeostasis maintenance and cellular junction and metabolism. In addition, we found increased estrogen (E2) and 11-ketotestosterone (11-KT) level and decreased testosterone level in ovarian follicles during the sperm storage period by ELISA, suggesting that sex hormones are involved in the dynamic change of ovarian follicles. In total, this study could provide new hints for understanding the immune adaption and developmental signatures of ovarian follicles post copulation in black rockfish and other viviparous fish.

Differential gene expression analysis related to sperm storage in spermathecas of Amphioctopus fangsiao

Sperm storage in the female body is an important strategy in animal reproductive behavior. Amphioctopus fangsiao is an economically important cephalopod that has a sperm storage period of up to seven months. There are few studies concerning the mechanism of sperm storage in A. fangsiao. In this study, we performed transcriptome gene expression profiling of the oviductal glands at different phases (presence and absence of sperm storage). In total, 7943 differentially expressed genes (DEGs) comprising 4737 upregulated and 3206 downregulated genes were identified. GO and KEGG enrichment analyses were used to search for sperm storage-related genes. A protein interaction network was constructed to examine the interactions between genes. Nineteen genes associated with immunity, apoptosis, and autophagy were obtained and verified by qRT-PCR. This is the first comprehensive analysis of sperm storage-related genes in A. fangsiao. The results provide basic insights into the complex sperm storage mechanism of A. fangsiao.

Female sperm storage in the bonnethead Sphyrna tiburo oviducal gland: Immunolocalization of steroid hormone receptors in sperm storage tubules

Female sperm storage (FSS) has been demonstrated to occur in representatives from all major vertebrate groups and has been hypothesized to have several possible adaptive benefits that may maximize reproductive success of its practitioners. However, while the range of taxa that exhibit FSS and its possible evolutionary benefits have received significant attention in past years, the physiological mechanisms by which FSS occurs in vertebrates have only recently been explored. In this study, we examined the potential role of gonadal steroid hormones in regulating FSS in the bonnethead Sphyrna tiburo, a small hammerhead species in which females have been shown to be capable of storing male spermatozoa for up to 6 – 7 months following copulation. Like past studies on this species, we observed associations between plasma concentrations of the gonadal steroids 17β-estradiol, testosterone, and progesterone with FSS in female bonnetheads, suggesting roles for these hormones in regulating this process. Using immunohistochemistry, we also observed presence of androgen receptor, estrogen receptor alpha (ERα), and progesterone receptor in epithelial cells of sperm storage tubules in the bonnethead oviducal gland, as well as occurrence of ERα in stored spermatozoa, specifically during the sperm storage period. These results suggest that E2, T, and P4 may regulate certain aspects of FSS in bonnethead indirectly through actions on the female reproductive tract, whereas E2 may also have direct effects on sperm function. This is the first study on the regulation of FSS in sharks and has formed a basis for future work geared towards improving our understanding of this process in chondrichthyans.

Interaction of Epididymal Epithelia and their Secretions with Spermatozoa Supports Functional and Morphological Changes During Long-Term Storage in the Chinese Soft-Shelled Turtle (Pelodiscus sinensis).

Post-testicular maturation of spermatozoa is crucial for attaining the morphological and functional capabilities needed for successful fertilization. Epididymal epithelia offer a favorable environment for spermatozoa that are stored long term in the turtle epididymis; however, sperm-epithelial interactions during storage, which are enormously important for sperm functional and morphological maturation, are still largely unknown in turtles. The present study examined the epididymis during the sperm-storage period (November-April) in the Chinese soft-shelled turtle (Pelodiscus sinensis). Light and transmission electron microscopy were used to determine the cellular features of each epididymal segment (caput, corpus, and cauda) and their epithelial interactions with the spermatozoa. Spermatozoa were mainly located in the lumena of caput, corpus, and cauda epididymides. Numerous spermatozoa were bound to apical surfaces of the epithelia, and several were even embedded in the epithelial cytoplasm of the caput and corpus epididymides. No embedded spermatozoa were found in the cauda epididymis. In all epididymal segments, principal and clear cells showed the synthetic activity, evidenced by a well-developed endoplasmic reticulum network and high and low electron-dense secretory materials, respectively. Principal and clear cells in the caput and corpus segments showed embedded spermatozoa in electron-dense secretions and in the lipid droplets within the cytoplasm. No lysosomes were observed around the embedded spermatozoa. The lumena of the caput and corpus segments showed few apocrine and low electron density secretions. In the lumen of the cauda epididymidis, different secretions, such as holocrine with low and high electron density and their fragmentation, apocrine, and dictyosome, were found and are summarized. Altogether, sperm physical interactions with secretions either in the cytoplasm of epithelium or in the lumen may support the viability, morphological maintenance, and transfer of various proteins involved in long-term sperm storage in the turtle. This interaction could help us to understand the mechanisms of long-term sperm storage and provide more insights into the reproductive strategies of turtle sperm preservation.

Changes in membrane carbohydrates distribution associated to epididymal sperm maturation during the prolonged sperm storage period of Corynorhinus mexicanus bat (Chiroptera: Vespertilionidae)

The Corynorhinus mexicanus bat provides an interesting experimental model for the study of epididymal sperm maturation because after testicular regression, this bat stores sperm in epididymal cauda for several months. Earlier research conducted by our group suggested that sperm maturation in this specie must be completed in the caudal region of the epididymis, although the precise factor linked with this phenomenon is unknown. Therefore, the aim of this work was to analyze changes in the distribution of N-acetylglucosamine and/or sialic acid, Fucose and Mannose carbohydrates in different membrane domains of sperm cells as they change from the caput to the cauda of the epididymis, as well as, their changes in different dates of capture. The sperm cells present a redistribution of N-acetylglucosamine and/or sialic when they arrived in the caudal region (September 11), but after storage until October 22 the distribution of N-acetylglucosamine and/or sialic acid changed. Mannose residues were found predominantly towards the acrosome during their entry into and transit through the three regions of the epididymis. The flow cytometry assay indicated that fluorescence intensity due to the presence of of N-acetylglucosamine and/or sialic acid on the sperm decreases as the sperm pass through the epididymal duct and as storage time in the cauda goes on. The Mannose fluorescence intensity, decreased in corpus and cauda from September 24 to October 8, though no differences appeared on the latter date. The presence of Fucuse was corroborated only by flow cytometry. In conclusion, the carbohydrate distribution on sperm membrane can be considered as part of the process of epididymal sperm maturation and is associated with the phenomenon of prolonged sperm storage that is characteristic of this specie. This adaptation allows the males to synchronize with the period of receptivity of the females, and then, carry out the matings.

Reproductive biology, multiple paternity and polyandry of the bull shark Carcharhinus leucas.

To improve understanding of bull shark Carcharhinus leucas reproductive biology, we analysed reproductive traits from 118 bull sharks caught along Reunion Island coasts (Western Indian Ocean), including 16 gravid females. Specific microsatellite loci were used to investigate the frequency of multiple paternity. Males and females reached maturity at c. 234 cm and 257 cm total length (LT ), respectively, and litter sizes ranged from 5 to 14 embryos. Analysis of the 16 litters collected in various months of the year indicated that parturition occurs between October and December, with a size at birth c. 60-80 cm LT and that the gestation period is probably c. 12 months. Assuming a 1 year resting period and a period of sperm storage (4-5 months) between mating (in June-September) and fertilisation, the reproductive cycle of bull sharks at Reunion Island would be biennial. At least 56.25% of the litters investigated were polyandrous, sired by 2-5 males. Several males that each sired several litters conceived during the same or distinct mating seasons were detected, suggesting both a seasonal aggregation of sharks to mate and some male fidelity to mating site. Altogether, these findings provide valuable information for both shark risk management and conservation of the species in the Western Indian Ocean.

Efektivitas Waktu Penyimpanan Sperma Ikan Komet (Carassium auratus auratus) dalam Larutan NaCl terhadap Derajat Pembuahan

The research was conducted to determine the effect of storage time in 0.9% NaCl solution on sperm quality and the degree of fertilization goldfish, using a completely randomized design with 7 treatments consisting of sperm storage duration 0 hours, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, and 12 hours with 3 replications. The result showed that the goldfish sperm storage duration 2 hours still has a higher value of motility and the degree of fertilization. While the value of motility and the degree of fertilization lowest found in sperm storage duration of 12 hours. The deadlinefor sperm storage period goldfish 6 hours still have good motility value of 60% so it still possible for the process of fertilization and hatching.

Transcriptome profiling of the spermatheca identifies genes potentially involved in the long-term sperm storage of ant queens

Females of social Hymenoptera only mate at the beginning of their adult lives and produce offspring until their death. In most ant species, queens live for over a decade, indicating that ant queens can store large numbers of spermatozoa throughout their long lives. To reveal the prolonged sperm storage mechanisms, we identified enriched genes in the sperm-storage organ (spermatheca) relative to those in body samples in Crematogaster osakensis queens using the RNA-sequencing method. The genes encoding antioxidant enzymes, proteases, and extracellular matrix-related genes, and novel genes that have no similar sequences in the public databases were identified. We also performed differential expression analyses between the virgin and mated spermathecae or between the spermathecae at 1-week and 1-year after mating, to identify genes altered by the mating status or by the sperm storage period, respectively. Gene Ontology enrichment analyses suggested that antioxidant function is enhanced in the spermatheca at 1-week after mating compared with the virgin spermatheca and the spermatheca at 1-year after mating. In situ hybridization analyses of 128 selected contigs revealed that 12 contigs were particular to the spermatheca. These genes have never been reported in the reproductive organs of insect females, suggesting specialized roles in ant spermatheca.

146 SPERM STORAGE: EXPRESSION OF PROGESTERONE RECEPTORS, STRUCTURAL PROTEINS, AND HEAT SHOCK PROTEINS IN THE AVIAN OVIDUCT

In avian species, sperm is stored for a long period in sperm storage tubules (SST) located in the utero-vaginal junction. It has been shown that progesterone (P4) regulates the release of sperm from SST for fertilization [Ito et al. 2011 Endocrinology 152, 3952–3621] in quails. In the same context, heat shock proteins (HSP) such as the HSP70 family activate sperm motility in quails [Hiyama et al. 2013 Reproduction 147, 167–178] and may participate in sperm release. In addition, we can hypothesise that at least some structural proteins from SST cells could play a role during sperm storage and release by interacting with sperm. Our objective was to study the potential effect of P4, HSP, and structural proteins on sperm storage potential, by analysing the expression of P4 receptors (PR and mPRα), HSPA8 and HSPB1, CNN1, TAGLN and DES genes in the avian genital tract. Uterus, vagina, and utero-vaginal junctions were collected from 2 divergent lines of hens. One lines displays a long period of sperm storage (21 days, line F+, n = 5), whereas the second displays a shorter period of sperm storage (10 days, line F–, n = 6). Total RNA was extracted and reverse transcribed, and then mRNA coding for PR, mPRα, HSPA8, HSPB1, CNN1, TAGLN and DES were assessed by qPCR (CFX96 Touch Real-Time PCR Detection System, Bio-Rad Laboratories, Hercules, CA, USA). Quantification was performed by using the relative standard curve method. Results were normalized by the geometric mean value of 2 reference genes, GAPDH and S17. Relative amounts of mRNA in each tissue were compared between lines by a Mann-Whitney test. Differences were considered significant when P < 0.05. No significant difference was observed between lines for both P4 receptors, as well as for HSPA8, regardless of the tissue. Expression of CNN1, TAGLN, and DES was significantly higher in the utero-vaginal junction of line F– than line F+ (P < 0.01). Expression of HSPB1 was significantly higher in utero-vaginal junction of line F– than of line F+ (P < 0.05). In conclusion, our data suggest that HSPB1 and structural proteins CNN1, TAGLN, and DES are involved in the regulation of sperm storage in the utero-vaginal junction in hens.

Tyrosine phosphorylation as evidence of epididymal cauda participation in the sperm maturation process ofCorynorhinus mexicanusbat

The bat Corynorhinus mexicanus provides an interesting experimental model for the study of epididymal sperm maturation because after spermatogenesis and the regression of the testes, this bat stores sperm in the epididymal cauda for several months. Earlier research conducted by our group suggested that sperm maturation in this species must be completed in the caudal region of the epididymis. One of the major signal transduction events during sperm maturation is the tyrosine phosphorylation of sperm proteins. The aim of the present study was to comparatively evaluate tyrosine phosphorylation in spermatozoa obtained from the caput, corpus and cauda of the epididymis during the sperm storage period. The maturation status of the sperm was determined by the percentage of capacitation and tyrosine phosphorylation in sperm obtained from the epididymis. The highest proportion of tyrosine phosphorylation was registered after the sperm had reached the cauda epididymis during the middle of the storage period. In conclusion, in Corynorhinus mexicanus and most likely in other chiropteran species with an asynchronous male reproductive pattern, epididymal sperm maturation ends in the caudal region of the epididymis and is related to the time that the sperm remains in the epididymis before mating activity.

15 SPERM STORAGE IN FEMALE REPRODUCTIVE TRACT: STUDY OF MOLECULES INVOLVED

Because of prolonged sperm storage in their oviduct, domestic hens can produce fertile eggs for up to 3 weeks following a single AI. The oviduct secretions may have an effect on sperm survival, but its composition during fertilization is unknown. In the present study, we compared the proteomic content of uterine fluid collected from two lines of hens divergent by their duration of fertility period (DFP), which defined sperm-storage duration. The first line displays a shorter period of sperm storage (10 days, line DFP–), whereas the second displays a longer period of sperm storage (21 days, DFP+). The aim was to identify proteins or peptides that may be involved in spermatozoa survival. Uterine fluid was collected 10 h after oviposition either before (n = 5/line) or 24 h after (n = 5/line) AI. Samples were pooled by condition: DFP+ before AI, DFP+ after AI, DFP– before AI, and DFP– after AI. Bottom-up approach using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano LC-MS/MS was performed (3 replicates). Data were matched against the NCBInr database (2014) using Mascot, and identifications were validated by the peptide and protein Prophet algorithm using Scaffold software. To determine the differences in protein expression, spectral counting and XIC quantitative methods were employed using Scaffold Q+ (P < 0.05, ratio > 2). Two proteins were up-regulated and one was down-regulated in oviductal secretion of both lines in response to AI. However, AI induced a significantly different abundance between protein content of DFP– and DFP+ fluids. A panel of 8 proteins, included one DFP+-specific protein, was more abundant in DFP+ line than in DFP–. Only one protein was less abundant in DFP+ line than in DFP–. In conclusion, the presence of sperm in the genital tract induced quantitative differences of the protein content of the uterine fluid in DFP– and DFP+ hen lines. These differences imply proteins that are known as male proteins (sperm, seminal plasma, testis). Analysis of sperm protein modifications after storage will help us to understand the functional implication of these candidates.

Changes in glucose and carnitine levels and their transporters in utero‐tubal junction in relation to sperm storage in the vespertilionid bat, Scotophilus heathi

Prolonged sperm storage over winter is a common feature of reproduction in some bats. In order to understand how sperm storage in the female genital tract of the vespertilionid bat, Scotophilus heathi (Greater yellow bat), is controlled, we compared concentrations of glucose and the fatty-acid carrier carnitine in the blood, and carnitine concentrations and levels of expression of the glucose transporters (GLUTs) and the carnitine transporter OCTN2 in the utero-tubal junction of females during non-storage (early winter) and sperm-storage periods (late winter-early spring). During the sperm-storage period (December-January) blood glucose concentrations declined, as did the expression of GLUT3 and GLUT5 in the utero-tubal junction. At the same time there were increases in the concentration of carnitine, and expression of OCTN2 and hormone-sensitive lipase (HSL) in the utero-tubal junction. These results suggest that prolonged sperm storage is enhanced by decreased glucose availability but increased free fatty acid availability at the site of sperm storage. Increases in expression of GLUT4 and GLUT8 in late winter suggest a role for these GLUTs in increasing sperm motility prior to fertilization.

Changes in the expression of HSL and OCTN2 in the female reproductive tract of the bat, Scotophilus heathii in relation to sperm storage

The aim of this study was to compare the changes in the expression of hormone sensitive lipase (HSL) and organic cation transporter 2 (OCTN2) in different regions of the female reproductive tract of bats (Scotophilus heathii) during the period of sperm storage. Western blot analysis revealed that both HSL and OCTN2 showed elevated expression in the utero-tubal junction, the site of sperm storage, compared to the vagina, cervix and uterus. An immunohistochemical study showed localization of HSL and OCTN2 in the cytoplasm of luminal epithelial cells in the utero-tubal junction of S. heathii, which suggests a role of fatty acids metabolism at the site of sperm storage. Furthermore, this study showed the presence OCTN2 immunostaining in the tail of stored sperm, which suggests a direct role of carnitine in sperm physiology. However, the role of carnitine in sperm storage needs further investigation. It is hypothesized that the utero-tubal junction may be finely tuned with fat depletion to support sperm storage in the female genital tract of S. heathii.

Sperm storage in the female reproductive tract of Scotophilus heathii: Role of androgen

The aim of the present study was to investigate the mechanism of androgen-mediated, prolonged sperm-storage in the female reproductive tract of the bat, Scotophilus heathii. The bat treated in vivo with flutamide, an androgen antagonist, showed loss of spermatozoa at the storage site, utero-tubal junction. Immunocytochemistry and Western blot analysis revealed the presence of increased expression of Bcl2 in the epithelial cell lining of the utero-tubal junction during the period of sperm-storage. Treatment with testosterone in vitro caused a significant dose-dependent increase in expression of the survival factor Bcl2, whereas treatment with flutamide together with testosterone caused a significant decline in Bcl2 in the utero-tubal junction of S. heathii. Together with the expression of Bcl2, the utero-tubal junction also expresses the death signal, caspase3. Expression of caspase3 decreased during January, but increased in February during the late stage of sperm storage. Androgen stimulated Bcl2 synthesis in the utero-tubal junction via the non-genomic MAP kinase signaling pathway. In conclusion, this study suggests that androgen promotes sperm storage in S. heathii by stimulating the survival factor Bcl2 in the utero-tubal junction. It is further hypothesized that a balance between the survival factor, Bcl2, and the death signal, caspase3, determines the duration of sperm storage in S. heathii.

Dynamics of sperm DNA fragmentation in patients carrying structurally rearranged chromosomes

This investigation was conducted to assess the baseline level of sperm DNA fragmentation (SDF) in a cohort of patients presenting chromosomal rearrangements (nine reciprocal translocations and two inversions). In a separate experiment, a dynamic analysis to calculate the rate of SDF (rSDF), after a varying period of sperm storage (0 h, 1 h, 4 h, 8 h and 24 h) at 37 °C, was performed. Results were compared with eight fertile donors. Different experimental approaches to assess SDF, such as terminal transferase dUTP nick-end labelling (TUNEL), sperm chromatin structure assay (SCSA) and sperm chromatin dispersion test (SCDt), were used. No differences for the baseline level of SDF were found. Carriers of reorganized genomes showed statistically higher levels of SDF than did control donors (p = 0.025 for TUNEL; p = 0.022 for SCSA; p = 0.014 for SCDt). However, 54.5% (6/11) of the patients presented values similar to those of control donors. There was no significant difference in rSDF (p = 0.34). Nevertheless, the results suggest that a high variability for SDF and rSDF exists in these patients. Routine analysis of SDF and rSDF should be considered in patients presenting rearranged genomes to determine fertility status for assisted reproductive techniques (ART) purposes.

In vitro adenine nucleotide catabolism in African catfish spermatozoa

It has been shown recently that African catfish ( Clarias gariepinus) spermatozoa possess relatively low ATP content and low adenylate energy charge (AEC). One of the possible explanations for this phenomenon is that the spermatozoa actively catabolize adenine nucleotides. A relatively high rate of such catabolism could then contribute to the low ATP concentration and low adenylate energy charge observed in the spermatozoa in vitro. To check this hypothesis, we investigated ATP content and adenine nucleotide catabolism in African catfish spermatozoa stored at 4 °C in the presence of glycine as an energetic substrate. Our results indicate that the storage of African catfish sperm at 4 °C in the presence of glycine causes time-dependent ATP depletion. In contrast to ATP, the AMP content increases significantly during the same period of sperm storage, while the ADP increases only slightly. Moreover, a significant increase of inosine and hypoxanthine content was also found. Hypoxanthine was accumulated in the storage medium, but xanthine was found neither in spermatozoa nor in the storage medium. It indicates that hypoxanthine is not converted to xanthine, probably due to lack of xanthine oxidase activity in catfish spermatozoa. Present results suggest that adenine nucleotides may be converted to hypoxanthine according to the following pathway: ATP→ADP→AMP (adenosine/IMP)→inosine→hypoxanthine. Moreover, hypoxanthine seems to be the end product of adenine nucleotide catabolism in African catfish spermatozoa. In conclusion, our results suggest that a relatively high rate of adenine nucleotide catabolism contributes to the low ATP concentration and low adenylate energy charge observed in African catfish spermatozoa in vitro.

Reproductive strategies of a warm temperate vespertilionid, the large-footed myotis, Myotis moluccarum (Microchiroptera:Vespertilionidae)

A population of M. moluccarum, which is known to be polyoestrous and monotocous, giving birth to two consecutive offspring annually, was investigated to determine the female reproductive cycle. The population was visited, and observations recorded, at approximately 5-week intervals from July 1994 to February 1995. At each visit, 2–4 females were collected and their reproductive tracts were removed, fixed and processed for light microscopy. During visits conducted after parturition periods, the age of the offspring was estimated in order to determine the duration of parturition. The two gestation periods of the breeding season were of approximately equal duration (11.5–12 weeks) despite the initial pregnancy occurring over much cooler months. While some copulations take place prior to ovulation, sperm storage is not a necessity, and copulation can occur until the time of ovulation. First ovulations of the season take place in early August and although several follicles may mature only one is ovulated. Analysis of climatic variables indicates that females undergo the first ovulation and pregnancy of the season during the cooler part of the year whereas lactation occurs during the warmer, wetter months. A post-partum oestrus occurs soon after parturition and adult males leave the population before the birth of the second offspring. Individual variation in the time before the post-partum oestrus may account for the reduced synchrony noted in the second parturition period. Monthly changes in the corpus luteum and ovaries are documented. The reproductive cycle of M. moluccarum resembles most closely that of tropical and sub-tropical vespertilionids; however, pre-ovulatory copulation, a short period of sperm storage and the development of typical over-wintering follicles (aspects of reproduction seen in species from cooler climates) remain inherent in the large-footed myotis.

Sperm storage in a seasonally reproducing African vespertilionid, the banana bat (Pipistrellus nanus) from Malawi

Spermatogenesis in the banana bat (Pipistrellus nanus) from southern Malawi began in the hot, wet season (February to April) and spermatozoa were released to the cauda epididymides at the beginning of the cool, dry season (May). Mating occurred between mid‐June and early July, in the middle of the cool, dry season. After mating, spermatozoa were stored by the female until ovulation in August and by the male until at least September. The period of female sperm storage effectively lengthened the reproductive cycle so that early spermatogenesis occurred during the second half of one hot, wet season and births at the beginning of the following hot, wet season. During the period of sperm storage, males and females roosted together and group membership was labile, suggesting that the mating system may be promiscuous (both males and females mating with more than one partner) and that sperm competition may occur. The similarity between the chronology of reproduction in Malawi, Kenya, and South Africa leads us to propose that sperm storage occurs at all three localities.

Spermathecae of Salamandrina terdigitata (Amphibia: Salamandridae): Patterns of sperm storage and degradation.

The spermathecae of female Salamandrina terdigitata were observed using light and transmission electron microscopy during the fallspring period of sperm storage and secretory activity and during the summer stasis. When sperm are stored inside the spermathecae, the product synthesized by spermathecal epithelial cells is exported into the lumen, where it bathes the sperm. During sperm storage some spermatozoa undergo degradation by the spermathecal epithelium. This process, which includes sperm capture by the apical microvilli, formation of endocytic vacuoles and production of lysosomes, becomes prominent shortly after oviposition. In many instances, cells filled with vacuolized spermatozoa and/or residual bodies undergo desquamation from the spermathecal epithelium and enter the lumen together with residual sperm. Desquamated cells, together with residual sperm, are a common feature in the spermathecal lumina at the end of the egg-laying season. Concomitant to the activity of the spermathecal epithelium, macrophages move into the spermathecae from the stroma and contribute to the degradation of both the residual sperm and desquamated epithelial cells. As a result of this degradation activity, spermathecae observed during the short summer stasis appear devoid of secretory product and sperm. By late summer, however, the spermathecae already show early signs of an imminent resumption of biosynthetic activity. © 1995 Wiley-Liss, Inc.