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Measurements of extremely high osmolalities in cauda epididymidal fluids of hibernating bat species led to an investigation of the junctional complex morphology of the epithelium of this sperm storage site. Freeze fracture replicas revealed the presence, at certain times of the year, of a tight junction architecture that resembled that traditionally thought to be exclusive to the blood-testis barrier, the strongest permeability barrier in the body. It is hypothesized that seasonal establishment of these specialized Sertoli cell-like tight junctions is necessary to the maintenance of the high osmotic state of the luminal environment, allowing for the prevention of dilution of its contents by paracellular routes and its protection from bursting under the osmotic pressure contained within.

Previous experiments have established that the long-lived spermatozoa of hibernating bats are resistant to the acrosome reaction and fertilization in vitro conventional techniques. We tested the hypothesis that the membranes of these spermatozoa are more resistant to perturbation than those of other mammals. We exposed them to non-specific bilayer destabilizing agents and abrupt changes in incubation temperature and tested their response by observing their status (motility and viability) after a time interval compared with other mammals (golden hamster, rabbit, human). The results did not support the hypothesis. The inherent longevity of bat spermatozoa may thus be a function of some component other than unique resilience of their plasma membrane.

Descriptions are provided of the histology and ultrastructure of the male internal reproductive tracts from three species of Camponotus, representing three subgenera. This study is the first to provide ultrastructural information on the testes (including spermatogenesis and spermiogenesis), seminal vesicles, and accessory glands in ants. Testes contain about ten follicles each, and each follicle is capable of producing hundreds of cysts in which spermatozoa develop. Structural evidence of meiosis in late pupal testes includes cytoplasmic bridges between spermatocytes, centriole elimination, and fusion of mitochondria. Developing spermatids are in close contact with cyst cells in the region of the acrosome. Mature spermatozoa are similar in ultrastructure to those described previously for two other subfamilies of ants (Myrmicinae and Dolichoderinae). The ultrastructure of the seminal vesicle suggests that it is not merely a passive organ for sperm storage. Large numbers of both mitochondria and membranous whorls suggest a pH-regulating and/or hormonal function. The accessory gland is made up of secretory cells that contain a diversity of secretory granules. SDS-PAGE reveals several proteins found in the accessory glands but absent in the adjacent genitalia. Preliminary analyses indicate that carbohydrate is an important component of accessory gland secretions.

Mature spermatozoa from spermathecae of founding queens were obtained from 5 species of ants, representing the major subfamilies Myrmicinae (Acromyrmex versicolor, Crematogaster sp.) and Dolichoderinae (Tapinoma sessile, Conomyrma insana, Conomyrma wheeleri). The ultrastructure of ant spermatozoa has many features in common with that of higher insects and is similar to that of other Hymenoptera. Structural similarities to spermatozoa of other Hymenoptera include an acrosome containing an internal rod that extends into the nucleus, two elongate mitochondrial derivatives, a centriolar adjunct, and an axonemal arrangement of 9 + 9 + 2 that includes well-developed coarse, or accessory, tubules. Spermatozoa obtained from A. versicolor, a species that is known to store and utilize viable sperm from this supply for over 10 years, show greater development of the mitochondrial derivatives than do the other species. The most distinctive feature of ant spermatozoa in comparison to other Hymenoptera is the large size of the centriolar adjunct relative to the other organelles. The centriolar adjunct is located posterior to the nucleus, anterior to the mitochondrial derivatives, and opposite the axoneme.

In the leaf-nosed bat, Macrotus californicus, a 4.5-month period of delayed early embryogenesis (October-March) precedes a 3.5-month period of normal embryogenesis (March-June). This prolonged gestation provides a unique opportunity to correlate ovarian changes with the events following implantation. The present study investigated luteal cell development and follicular biology during gestation. Circulating progesterone (P) levels following implantation were unchanged before transition to normal development, and were maximal at the start of active gestation. Luteal cell diameters increased during this period. Serum P levels declined prior to parturition, when cells staining positive for 3 beta-hydroxy-5-steroid dehydrogenase-5,4-isomerase (3 beta-HSD) activity were reduced in number and diameter, and enzyme staining was less intense in tissue slices. Subcellular steroidogenic organelles were present during delayed development, but smooth endoplasmic reticulum (SER) was markedly increased after the resumption of normal development at which time also luteal cells reacted positively to staining for 17 beta-HSD. Before parturition, lipid droplet accumulation and reduced SER suggested a reduction in steroid secretion. Large multilaminar follicles stained positive for 3 beta-HSD activity throughout gestation and for 17 beta-HSD except in late delayed development. Thus, the delay in embryogenesis may be due to an inadequately developed corpus luteum or to the steroidogenic activity of the multilaminar follicles.

The seasonal chronology of the events of the reproductive cycle, and changes in the structure and function of the primary and accessory organs of the male bent-winged bat, Miniopterus schreibersii, were studied at latitude 37 degrees S in temperate southeastern Australia. The testicular cycle commenced in late spring (November), and sperm appeared in the seminiferous tubules and epididymides in early fall (March). The cycle of the accessory sex gland complex generally paralleled the testicular cycle, reaching maximum hypertrophy at the time of insemination in late fall (April/May). Thereafter, the primary and secondary sex glands (except the ampullary gland) involuted as the animals entered winter torpor. However, a cauda epididymal store of sperm persisted until late spring, and sperm were often observed, as well, in the ampullary gland duct and alveoli throughout winter. This study has confirmed that male Miniopterus differs from other vespertilionids in that accessory gland activity declines following the fall breeding in keeping with the fact that, unlike in other vespertilionids, insemination, ovulation and conception are concurrent events in the fall in this species. The reduced secretory status of the Leydig cells and exceptionally low levels of circulating androgens throughout the year, in combination with the presence of viable epididymidal sperm for most of gestation, are all interesting features of this reproductive cycle.

The reproductive biology of the bat Pipistrellus hesperus was investigated histologically using animals obtained from nature at monthly intervals throughout the year. The female shows proestrous morphological changes in late summer and autumn, which continue until early spring. Insemination is probably effected several times during proestrous, since both sexes are intermittently active throughout this period (August--April). Permanent arousal from torpor and return to normal metabolism in the spring results in ovulation. Both ovaries are functional and both uterine horns may be utilized. P. hesperus is monestrous; two young per bat is usual. The male spermatogenic cycle is initiated in late June and the first sperm are available for ejaculation in September. Some sperm remain in the seminiferous tubules until early March. The epididymides also serve in sperm storage with the caput emptied by late February and the cauda in April. Leydig cells are largely nonsecretory from March through July, a few are secretory in August and most are secretory in September and October. Active cells gradually decline in number until few are secretory in late March. Accessory sex glands are functionally cyclic: they are small from April through August, hypertrophy in September, and gradually involute to the resting stage through March, to achieve total involution in April. The penis, similar in structure to that of other vespertilionids, is thought specialized to insure effective insemination.

The anatomy, biology, and chronology of reproduction in the male of the long penile form of Mormopterus planiceps was studied in southeast South Australia and Victoria. In the morphology of its primary and accessory reproductive organs, M. planiceps was generally reminiscent of other Molossidae; however, in the specialized (sebaceous) nature of the Cowper's gland ducts, in the presence of para-anal glands, and in the unusual, horizontally bifid glans penis and the greatly elongated os penis, it was distinct from other Molossidae studied to date. Young of the year were not reproductively active. Adults displayed a single annual spermatogenic cycle that commenced in spring (September/October) and culminated in spermiogenesis in autumn (February-May), during which period plasma levels of testosterone overtook androstenedione. Thereafter, spermatogenesis appeared to cease (though scattered sperm were seen in the seminiferous tubules until August), but abundant epididymal sperm reserves persisted until September/(October). The accessory glands were hypertrophied during this period, becoming involuted by October. Although the numbers of animals available for study were small, these observations, together with the appearance of spermatozoa in the ductus deferens in August/September suggested that mating could occur during the interval from autumn to spring. Late winter/spring insemination is normal for molossids from temperate environments. However, protracted spermatogenesis commencing in spring that is not accompanied by the availability of spermatozoa until autumn, and a subsequent apparent extension of fertility (epididymal sperm storage, accessory gland hypertrophy) beyond the testicular gametogenic phase, are aspects of the male reproductive cycle in M. planiceps that have not heretofore been described in another molossid bat.

The renal morphology of three species of desert dwelling Perognathus rodents were compared to Dipodomys and two species of sympatric cricetid rodents. Perognathus has a highly adapted unipolar kidney capable of urine concentration up to 7,500 mOsm/KgH2O. Two major modifications were observed in these kidneys. (1) There is elongation of both the inner and outer medulla. When the thickness of the regions in P. penicillatus are factored by kidney weight it is found that the outer medulla is 3.8 and the inner medulla is 2.2 times greater in length than the extensively studied Dipodomys merriami. (2) There is great variance in glomerular size with approximately 20% of the glomeruli being very large and located in the juxta medullary region. These glomeruli are 1.5--1.6 times greater in diameter than the more numerous superficial glomeruli. These structural modifications are quantitated and the functional implications are discussed particularly in relation to urine concentrating ability in these desert inhabitants.

In Macrotus californicus (Phyllostomatidae), normal embryogenesis(March-June) is preceded by a period of delayed development (October- March) characterized by implantation and slow growth of the embryo to the primitive streak stage. The events of the annual reproductive cycle can be correlated with ovarian dynamics. Waves of follicular growth appear to be initiated in January and June. Increased multilaminar follicles resulting from the second wave of recruitment appear from August to October. Vesiculation of these follicles is seen in both ovaries from July to January; however, the single Graafian follicle forms only in the right ovary just prior to ovulation in late October-early November. Left ovarian ovulation can be induced by right ovariectomy. High atresia from July to December may retard embryo genesis by failing to provide an optimal hormone milieu for the conceptus. In addition, luteal cells are small during the initial months of embryonic development.The first wave of follicular growth results in the appearance of an increased percentage of growing follicles in April; resultant enhanced estrogen levels may influence the resumption of normal development, an event which also coincides with luteal cell hypertrophy. It would appear possible, therefore,that delayed development in Macrotus is an expression of luteal cell insufficiency and uterine nutritional incompetence resulting from depressed steroid levels. Termination of delay may be brought about by the action of increased levels of estrogen and/or progesterone on the endometrium, perhaps by influencing the activity of mast cells whose products are known in some species to enhance vascularity which in turn could account for added substances essential to normal fetal growth.

The major reproductive events in the male eastern pipistrelle, are similar to those of other hibernating vespertilionids. The eastern pipistrelle stores epididymal spermatozoa throughout hibernation, a time when the testes are involuted but accessory gland activity is maintained. However, this species differs from others in that epididymal and testicular spermatozoa persist longer and the weights of the accessory glands are not strongly differentiated between winter and spring/summer. It is suggested that the reproductive period is extended in this species as a function of a more prolonged period of hibernation, resulting in only a brief period of sexual quiescence in mid-summer. The eastern pipistrelle (Pipistrellus subflavus) resembles the canyon bat (P. hesperus) in that some testicular spermatozoa persist during winter. Many aspects of the reproductive anatomy and chronology of these two species are similar; however, eastern pipistrelles apparently lack a seminal vesicle and possess a distinctly different baculum.

The reproductive biology of the bat Macrotus waterhousii was investigated anatomically and physiologically several times monthly throughout the year. The male spermatogenic cycle is initiated in June; sperm are available in August. Testicular hypertrophy is accompanied by increased levels of testicular ascorbic acid and circulating plasma testosterone. Sperm are present in the seminiferous tubules and later the epididymides from August to early December. Regression in testicular size begins in late September and testes are involuted by early December. Levels of testicular ascorbic acid and plasma testosterone decline at this time, although detectable amounts of both are present year-round. Mature sperm are absent from the male tract after late November or early December. The accessory sex gland complex and Cowper's glands undergo an annual cycle in synchrony with the testicular cycle. The accessory complex contains fructose, the concentration of which varies seasonally in direct proportion to glandular hypertrophy. Only trace levels of fructose are elaborated by Cowper's glands undergo an annual cycle in synchrony with the testicular cycle. The accessory complex contains fructose, the concentration of which varies seasonally in direct proportion to glandular hypertrophy. Only trace levels of fructose are elaborated by Cowper's glands. In contrast to vespertilionid bats, where the accessory glands remain enlarged and active when testes involute, those in Macrotus regress. The penis differs structurally from that of other temperate North American bats, primarily by lacking a bony baculum and specialized erectile accessory corpus cavernosus bodies. Males are not reproductively active in their first autumn.

Developmental delay is correlated with torpor in the temperate zone bent-winged bat, Miniopterus schreibersii (latitude 37 degrees S) as a period of pre-implantation delay (delayed implantation) followed by a short post-implantation delay (delayed development). During this time, the number of steroidogenic organelles in luteal cytoplasm is greatly reduced compared with normal embryogenesis, and granular endoplasmic reticulum is prominent. Nidation, which occurs while the animals are hibernating, is not accompanied by marked changes in luteal ultrastructure, although the number of lipid droplets decreases somewhat. Progesterone rises slightly but not significantly; however, a pre-nidation decrease in high 17 beta-estradiol levels may play a role in implantation. Following implantation, the conceptus remains delayed at the blastocyst stage for several weeks. During this time the bats remain torpid and the only change in luteal cell ultrastructure is an increase in smooth endoplasmic reticulum as differentiation begins toward the trilaminar stage. At the end of developmental delay hypertrophy of the luteal cell begins and mitochondria and lipid droplets increase, markedly. By this time arousal from hibernation has occurred, placentation takes place and normal development is underway. At placentation, smooth endoplasmic reticulum reaches its maximum in luteal cytoplasm; estrogen and progesterone levels peak about 6 weeks later. For the remaining 2 months of gestation, signs of luteolysis appear. These observations suggest that the corpus luteum of developmental delay, though sub-optimally functional, is prolonged in its luteinization by the arrival of winter when the bats enter torpor. The capacity for maximal steroidogenesis is acquired at the end of winter, some weeks after implantation, when arousal occurs and normal development ensues.

The cauda epididymidis, uterine corpus, and cornua and uterotubal junction of Myotis function to retain and preserve normal spermatozoa throughout hibernation. In none of the sites do spermatozoa show features that might account for their extended viability. Spermatozoa stored in the uterus and epididymis show no special orientation toward the epithelium lining these sites, whereas an intimate relationship is established between some sperm and the epithelial cells of the uterotubal junction which might either account for extended postcoital sperm survival or forecast their removal from further participation. Transmission and scanning electron microscopic observations do not disclose any morphological changes in stored luminal spermatozoa. A low rate of phagocytosis of sperm is evident in the female tract during hibernation. However, spermatozoa are evidently not vulnerable to being removed from the storage sites until spring arousal when ovulation occurs. Both uterotubal epithelial cells and phagocytes appear to be involved in the disposal of spermatozoa in the female, whereas epididymal spermatozoa apparently are primarily voided during urination. A mechanism that delays capacitation must underlie the ability of spermatozoa to survive in the female reproductive tract of the hibernating bat.

Corpora lutea were surgically collected from superovulated ewes 36 h post-injection of human chorionic gonadotropin (hCG) (Day 2), dissociated (0.2% collagenase), plated, and maintained in culture Days 2-10 in Medium 199 supplemented with 5% calf serum. Accumulation of progesterone in the cultures did not decrease (p greater than 0.05) from Day 3 (17.5 +/- 5.1 nmol/10(6) cells) to Day 10 (4.8 +/- 1.7 nmol/10(6) cells). Calf serum (5%) in the medium supported greater (p less than 0.05) progesterone production than fetal calf serum (5%) or medium without added serum. Steroidogenic cells did not increase (Days 2-10) in numbers, but increased (p less than 0.01) in mean cell diameter (Day 2, 11.7 +/- 0.4 micron; Day 10, 24.5 +/- 1.6 micron). Steroidogenic capacity on Day 10 of cells cultured Days 2-10 (in vitro) was not different (p greater than 0.05) from that of cells collected from the ovary on Day 10 (in vivo); however, steroidogenic cells recovered from plates had greater (p less than 0.01) mean cell diameters (24.5 +/- 1.6 micron, in vitro, compared to 15.2 +/- 1.0 micron, in vivo). Transmission electron microscopy revealed that cultured cells (Days 5, 10) possessed less smooth endoplasmic reticulum but more lipid droplet inclusions, ribosomes, and rough endoplasmic reticulum than cells obtained in situ (Day 10). Electron-dense secretory granules were rarely seen. Although subcellular morphology of ovine luteal cells in culture was altered, these changes did not appear to significantly affect the ability of these cells to produce progesterone.

The reproductive biology of the female little mastiff bat (Mormopterus planiceps) was studied from specimens obtained throughout the year in southeast Australia, within the region occupied only by the long penile form of this species. Mormopterus planiceps appeared to undergo a single pregnancy each year and was monotocous. Conception occurred during late winter/early spring after a protracted proestrus, during which the uterine/vaginal epithelia attained an extraordinary thickness; spermatozoa were present in the uterine corpus, vagina, and intramural oviduct for at least 2 months prior to ovulation, although only those present in the oviducts were entire and thus appeared to be viable. Following ovulation, a massive postovulatory infiltration of phagocytes occurred; and the thickness of the uterine corpus epithelium was dramatically reduced. As in other molossids, the tract was bicornuate and dextrally functional. The length of gestation was difficult to determine because early embryonic stages, up to implantation, appeared to span several months (late July/August/September) as did parturition (December/January). Growth of the young was slow; nevertheless, females attained sexual maturity in their first year. Several unusual features included the presence of a long os clitoridis, and tubuloalveolar sudoriferous and associated lobulated, sebaceous, paravaginal glands, which surrounded and emptied into the lower vagina. A deep fornix anterior and lateral to the cervix probably serves to receive the secondary glans penis. The epithelium of the uterine corpus was stratified and indistinguishable, in its cytology and cyclicity, from that of the vagina; furthermore, it lacked a glandular endometrium. This portion of the female tract likely receives the elongated primary glans. These findings are discussed in relation to other Molossidae and to the reproductive biology of male M. planiceps. Although the number of animals sampled was relatively small, the data suggest that this species does not exhibit the usual temperate molossid pattern of late winter/spring coincidence of spermatogenesis and ovulation. It would seem that pregnancy may begin, at least in some individuals, during the inhospitable winter months (when epididymal and uterine spermatozoa are abundant but spermatogenesis has largely terminated) and that additional conceptions continue into the early spring. The occurrence of sperm storage in both sexes of this species is unique among Molossidae studied to date.

Knowledge relative to the reproduction of nonhibernating bats is reviewed. Events in the male, as they are now understood, are summarized for all families for which data exist. Attention is given to the wide species diversity of male accessory sex oragns in respect to gross structure and glandular complement. Stability or variability or organization of the male reproductive system is noted. Functional relationships between primary and accessory sex organs are considered and any synchrony between these organs and their functional cyclicity identified. Various frequencies of male reproductive cycles are examined for each species within families. Factors influencing male reproductive evens in nonhibernating Chiroptera are briefly considered. It has long been speculation that reproductive cycles under natural conditions are strikingly related to the environment, with such features as temperature, moisture, length of day and available food all implicated. These cues, however, appear to be filtered through varied genetic potential and physiological patterns, since not all species respond similarly, even though residing together in a homogeneous environment. Variation between certain species in frequency and length of the oestrous cycle seem to be accommodated to by parallel adjustments in the male, including extended sperm production or storage and continued secretory activity of accessory sex glands.

Studies on the pathogenesis of rabies in two species of experimentally infected insectivorous Chiroptera, the Mexican free-tailed bat (Tadarida mexicana), a quasi hibernator, and the little brown bat (Myotis lucifugus), a deep hibernator, provided evidence that brown adipose tissue may serve as an extraneural site for storage and multiplication of rabies virus. Although the Mexican free-tailed bat proved to be relatively insusceptible to experimental rabies infection, virus was demonstrated in the brown fat of 22 per cent of those animals shown to be infected by viral assay in white Swiss mice. Rabies infection in this species was most evident 20 to 40 days after intramuscular inoculation of virus. Rabies virus was found to be widely distributed in the little brown myotis 9 to 26 days following inoculation and virus concentrations in some of the tissues approached the level of the stock mouse brain virus suspension used in inoculating these bats. The shorter incubation period and higher virus titers in the tissues assayed reflect the increased susceptibility of Myotis lucifugus as compared with the Mexican free-tailed bat. Virus was demonstrated in the brown fat of 30 per cent of the experimentally infected Myotis. In the experimentally infected Myotis lucifugus and in the Syrian hamster which is highly susceptible to rabies infection, rabies virus was isolated more frequently from the brown fat than from the salivary gland indicating that in a susceptible host brown adipose tissue may be as frequent a site of viral proliferation as salivary gland. Since rabies virus was found to persist for long periods of time in the brown fat of experimentally infected bats and was occasionally demonstrated in this tissue alone, it is suggested that brown adipose tissue provides a mechanism by which these animals may serve as reservoirs for this agent in nature. The possibility that similar mechanisms may be involved in the maintenance of other viral agents during interepidemic periods is discussed.

Studies on the influence of environmental temperature on the pathogenesis of rabies in two species of experimentally infected Chiroptera, the Mexican free-tailed bat (Tadarida mexicana) and the little brown bat (Myotis lucifugus), provided evidence that little or no viral multiplication occurs in the inactive host during experimentally induced hibernation. When inoculated animals are wakened from hibernation by transfer to a warm room, virus previously in "cold storage" multiplies, reaching detectable levels in various tissues. Similar results were obtained with two strains of rabies virus, a canine rabies street virus which produced a fatal infection in man and a strain isolated from the pooled brown fat of naturally infected little brown bats. However, certain differences in the characteristics of these virus strains were observed. The canine rabies virus strain produced an encephalitic disease in mice and overt symptoms in bats; the bat rabies virus producing an encephalomyelitic disease in mice and infrequent symptoms in bats. The bat rabies virus had a greater predilection for brown adipose tissue than the canine strain. Results obtained with the bat rabies virus in hibernating animals indicate that after a period of latency in a dormant animal activated virus may reach the salivary gland more rapidly, with greater frequency, and attain higher concentrations than in animals which have not experienced a period of hibernation. The significance of these results as they relate to the natural history of bat rabies is discussed.