Histology and functional morphology of the female reproductive tract of the tortoise Gopherus polyphemus

The oviducts of 25 tortoises (Gopherus polyphemus) were examined by using histology and scanning electron microscopy to determine oviductal functional morphology. Oviductal formation of albumen and eggshell was of particular interest. The oviduct is composed of 5 morphologically distinct regions; infundibulum, uterine tube, isthmus, uterus, and vagina. The epithelium consists of ciliated cells and microvillous secretory cells throughout the oviduct, whereas bleb secretory cells are unique to the infundibulum. The epithelium and endometrial glands of the uterine tube histologically resemble those of the avian magnum which produce egg albumen and may be functionally homologous. The isthmus is a short, nonglandular region of the oviduct and appears to contribute little to either albumen or eggshell formation. The uterus retains the eggs until oviposition and may form both the fibrous and calcareous eggshell. The endometrial glands are histologically similar to the endometrial glands of the isthmus of birds, which are known to secrete the fibers of the eggshell. These glands hypertrophy during vitellogenesis but become depleted during gravidity. The uterine epithelium may supply "plumping water" to the egg albumen as well as transport calcium ions for eggshell formation. The vagina is extremely muscular and serves as a sphincter to retain the eggs until oviposition. Sperm are found within the oviductal lumen and endometrial glands from the posterior tube to the anterior uterus throughout the reproductive cycle. This indicates sperm storage within the female tract, although the viability and reproductive significance of these sperm are unknown.     

Seasonal variation of the oviduct of the American alligator,Alligator mississippiensis (Reptilia: Crocodylia)

The annual oviductal cycle of the American alligator, Alligator mississippiensis, is described using light and electron microscopy. Previous work done by Palmer and Guillette ([ 1992 ] Biol Reprod 46:39–47) shed some light on the reproductive morphology of the female alligator oviduct; however, their study was limited and did not report details relating to variation across the reproductive season. We recognize six variable regions of the oviduct: infundibulum, tube, isthmus, anterior uterus, posterior uterus, and vagina. Each area shows variation, to some degree, in the histochemistry and ultrastructure of oviductal secretions. Peak secretory activity occurs during the months of May and June, with the greatest variation occurring in the tube and anterior uterus. During the month of May, high densities of neutral carbohydrates and proteins are found within the tubal and anterior uterine glands. The epithelium of the entire oviduct secretes neutral carbohydrates throughout the year, but many regions lack protein secretions, and the posterior uterine glands show little secretory activity of any type throughout the year. After oviposition, secretory activity decreases drastically, andthe oviduct resembles that of the premating season. This study also provides evidence to support the homology between alligator and bird oviducts. Sperm were observed in glands at the tubal‐isthmus and utero‐vaginal junctions in preovulatory, postovulatory and postovipository females. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Synthesis, distribution, and levels of an egg lipoprotein from the apple snail Pomacea canaliculata (Mollusca: Gastropoda)

The site of synthesis of mollusc lipoproteins is hitherto unknown and was investigated for perivitellin 2 (PV2), an egg lipoprotein found in the freshwater snail Pomacea canaliculata. Tissues (albumen gland, gonad-digestive gland complex, and muscle) from vitellogenic females were incubated in vitro with 14C-leucine at 25 degrees C for 12 hr. At the end of incubation, soluble proteins from tissue homogenates and medium were analyzed for de novo protein synthesis by electrophoresis and HPLC, and radiolabeled proteins were quantified by liquid scintillation. Two albumen gland radiolabeled proteins (67 and 31 kDa) co-migrated with the subunits of PV2, and they represented 6.0% of the total labeled protein in that tissue. Western blot analysis confirmed the presence of PV2 only in the albumen gland. In vivo experiments where adult females were injected with 3H-leucine revealed that PV2 was not present in hemolymph. ELISA analysis in all tissues of the snail confirmed the PV2 presence only in the albumen gland and developing eggs with levels of 26 and 98 mg/g protein, respectively. Therefore, the albumen gland is the only site for PV2 synthesis, and no extra-gland synthesis, circulation, or accumulation could be found. PV2 subunits were further characterized analyzing N-terminal sequences which showed no homology with other proteins.     

Secretory proteins in the reproductive tract of the snapping turtle, Chelhydra serpentina

SDS-polyacrylamide gel electrophoresis was used to separate the secretory proteins produced by the epithelial and endometrial glands of the uterine tube and uterus in the snapping turtle Chelydra serpentina. The proteins were analyzed throughout the phases of the reproductive cycle from May to August, including preovulatory, ovulatory, postovulatory or luteal, and vitellogenic phases. The pattern of secretory proteins is quite uniform along the length of the uterine tube, and the same is true of the uterus, but the patterns for uterine tube and uterus are clearly different. We identify 13 major proteins in C. serpentina egg albumen. Bands co-migrating with 11 of these are found in the uterine tube, but at most 4 are found in the uterus, suggesting that the majority of the albumen proteins are most likely secreted in the uterine tube, not in the uterus. Although some of the egg albumen proteins are present in the uterine tube only at the time of ovulation, most of the bands corresponding to albumen proteins are present throughout the breeding season even though the snapping turtle is a monoclutch species. These results suggest that the glandular secretory phase in the uterine tube is active and quite homogeneous in function regardless of location or phase of the reproductive cycle.     

Sperm storage in caecilian amphibians

Background

Female sperm storage has evolved independently multiple times among vertebrates to control reproduction in response to the environment. In internally fertilising amphibians, female salamanders store sperm in cloacal spermathecae, whereas among anurans sperm storage in oviducts is known only in tailed frogs. Facilitated through extensive field sampling following historical observations we tested for sperm storing structures in the female urogenital tract of fossorial, tropical caecilian amphibians.

Findings

In the oviparous Ichthyophis cf. kohtaoensis, aggregated sperm were present in a distinct region of the posterior oviduct but not in the cloaca in six out of seven vitellogenic females prior to oviposition. Spermatozoa were found most abundantly between the mucosal folds. In relation to the reproductive status decreased amounts of sperm were present in gravid females compared to pre-ovulatory females. Sperm were absent in females past oviposition.

Conclusions

Our findings indicate short-term oviductal sperm storage in the oviparous Ichthyophis cf. kohtaoensis. We assume that in female caecilians exhibiting high levels of parental investment sperm storage has evolved in order to optimally coordinate reproductive events and to increase fitness.     

Sperm storage in the oviduct of the American alligator

Oviducts of the American alligator (Alligator mississippiensis) were examined histologically for the presence of stored sperm. Two regions containing sperm were identified, one at the junction of the posterior uterus and the vagina (UVJ) and the other at the junction of the tube and isthmus (TIJ). In these areas, sperm were found in the lumina of oviductal glands. The glands in these areas of the oviduct are diffuse and shallow and appear to allow better access to sperm than glands located elsewhere. Histochemically, the glands of the UVJ reacted weakly for carbohydrates and proteins, whereas those of the TIJ reacted strongly for these same two components, secretions of which are associated with sperm storage structures in other reptiles. Sperm were not in contact with the glandular epithelium, and glands at the UVJ contained more sperm than those at the TIJ. Oviductal sperm storage was observed not only in recently mated females but in all females possessing uterine eggs as well as all females known to be associated with a nest. We conclude that female alligators are capable of storing sperm in their oviductal glands, but not from one year to the next.     

Changes in oviductal morphology of the skink, Lampropholis guichenoti, associated with egg production

We describe changes in the morphology of the oviductal epithelium of an oviparous skink, Lampropholis guichenoti, during the course of egg production and oviposition: to characterize the luminal epithelial changes; to provide a baseline for understanding uterine changes in viviparous species; and to establish whether the plasma membrane transformation of uterine epithelial cells is indeed a feature restricted to viviparous species. Oviducts from vitellogenic, gravid, and postgravid females were observed using scanning electron microscopy. Cellular characteristics of the oviductal epithelium previously used to determine the plasma membrane transformation were assessed morphologically. Three anatomically different areas were defined within the oviduct, but no plasma membrane transformation was observed in the oviparous skink, suggesting that this is a phenomenon particular to viviparity.     

A histological investigation of the occurrence of non-reproductive female bluefin tuna Thunnus thynnus in the Mediterranean Sea

The presence of non-reproductive Atlantic bluefin tuna Thunnus thynnus females in the Mediterranean Sea was investigated through histological analysis of the gonads. Three hundred and twenty-six ovary samples were collected from adults captured at different locations in the Mediterranean Sea during the reproductive seasons between 1998 and 2008. Only three specimens were considered to be in a non-reproductive state: two of them were in a reabsorbing state showing ovaries with early vitellogenic oocytes and extensive α and β atresia of vitellogenic follicles; the third showed gonads with perinucleolar oocytes and was considered to be in a resting state. The low occurrence of non-reproductive individuals found in this study makes it unlikely that non-reproductive individuals aggregate with reproductive ones during their migration towards spawning grounds. Further research is suggested in order to investigate the potential presence of non-reproductive individuals on non-spawning grounds during the reproductive season.     

Metabolism of ovorubin,the major egg lipoprotein from the apple snail

The site of synthesis of molluscs lipoproteins is little known and was investigated for the egg lipoprotein perivitellin 1 (PV1) or ovorubin in the freshwater snail Pomacea canaliculata. Tissues (albumen gland, gonad–digestive gland complex and muscle) of vitellogenic females were incubated in vitro at 25°C for 12 h with ¹⁴C Leucine. After that, soluble proteins from tissue homogenates and medium samples were analysed for de novo protein synthesis by electrophoresis and HPLC, and radiolabelled proteins quantified by liquid scintillation. Gonad–digestive gland complex did not synthesise ovorubin, in spite its high protein synthesis levels. Three albumen gland radiolabelled proteins (35, 32 and 28 kDa) comigrated with the subunits of ovorubin and represented 1.3% of the total labelled protein of that tissue. Western blot analysis with polyclonal antibodies confirmed that these were ovorubin subunits. In vivo experiments where vitellogenic females were injected with ³H Leucine, revealed that ovorubin was not present in hemolymph. ELISA analysis confirmed ovorubin presence only in albumen gland and developing eggs with levels of 800 and 582 mg/g protein, which represent 30.3 and 28.4 mg ovorubin/g of tissue, respectively. Therefore, albumen gland is the single site of ovorubin synthesis as no extragland synthesis, circulation or accumulation could be detected in the apple snail.     

Microscopic structures of the ovary and female genital ducts of Supachai's caecilian,Ichthyophis supachaii Taylor, 1960 (Amphibia: Gymnophiona)

The structures of the female reproductive system (ovary, oviduct and cloaca) of Ichthyophis supachaii were investigated by dissection, histology and light microscopy. Paired, elongated, sac‐like ovaries are parallel to the gut and fat bodies. Follicle stages include germinal nests of oogonia and primary oocytes, early and late previtellogenic follicles, early and late vitellogenic follicles and atretic follicles. Germinal nests of oogonia comprise oogonia and prefollicular cells. Nests of primary oocytes contain clusters of synchronously developing primary oocytes enclosed by connective tissue. Primary oocytes are associated with follicular cells. Previtellogenic follicles initially form the vitelline envelope, theca cell layers and patches of ooplasmic glycoproteins. Vitellogenic follicles contain heterogeneously sized spherical yolk granules. Atresia is present in several stages of developing follicles. The oviduct is divided into the anterior, middle and posterior parts. All oviductal parts are lined by non‐ciliated epithelium. A small number of mucous cells are present in the middle part. The cloaca of female I. supachaii is divided into the anterior and posterior chambers. The anterior chamber is lined by glandular stratified columnar epithelium, while the posterior chamber has stratified cuboidal epithelium with less mucus production. Our results contribute to useful information on the reproductive biology of caecilians.     

Low mitochondrial DNA variation among American alligators and a novel non-coding region in crocodilians

We analyzed 1317-1823 base pairs (bp) of mitochondrial DNA sequence beginning in the 5' end of cytochrome b (cyt b) and ending in the central domain of the control region for 25 American alligators (Alligator mississippiensis) and compared these to a homologous sequence from a Chinese alligator (A. sinensis). Both species share a non-coding spacer between cyt b and tRNA(Thr). Chinese alligator cyt b differs from that of the American alligator by 17.5% at the nucleotide level and 13.8% for inferred amino acids, which is consistent with their presumed ancient divergence. Only two cyt b haplotypes were detected among the 25 American alligators (693-1199 bp surveyed), with one haplotype shared among 24 individuals. One alligator from Mississippi differed from all other alligators by a single silent substitution. The control region contained only slightly more variation among the 25 American alligators, with two variable positions (624 bp surveyed), yielding three haplotypes with 22, two, and one individuals in each of these groups. Previous genetic studies examining allozymes and the proportion of variable microsatellite DNA loci also found low levels of genetic diversity in American alligators. However, in contrast with allozymes, microsatellites, and morphology, the mtDNA data shows no evidence of differentiation among populations from the extremes of the species range. These results suggest that American alligators underwent a severe population bottleneck in the late Pleistocene, resulting in nearly homogenous mtDNA among all American alligators today.     

Developmental and age-related changes in proteins in the female reproductive tract of Heligmosomoides polygyrus (Nematoda)

Proteins in the female reproductive tract of Heligmosomoides polygyrus at days 8, 16, 35, 90, and 140 postinfection (PI) were examined using polyacrylamide gel electrophoresis. Sixteen-day-old and 140-day-old worms also were examined histochemically. Egg production of these worms was assessed for each age group. In analyzing proteins using electrophoresis, the reproductive tracts were separated into 3 sections: the tip, or anteriormost part of tract, containing oogonia; the middle region, containing developing oocytes; and the posterior region, containing the uterus with fertilized eggs. Three major reproductive tract proteins were identified as having molecular weights of greater than 140 kDa, 115 kDa, and 82 kDa. These were found in all parts of the reproductive tract from worms of all ages except those at 8 days PI (which are too young to produce eggs) and are believed to be yolk proteins. Four low molecular weight proteins (L1-4) are believed to be nucleoproteins. L4 was absent from the posterior section of the reproductive tracts and L3 was limited to the posterior sections and may be associated with sperm stored in the uterus. Of 5 high molecular weight proteins the second heaviest, designated H2, appeared to be relatively more concentrated in the posterior sections of the reproductive tract. An 85-kDa protein was limited to the tip and middle sections of reproductive tracts. Histochemical tests on sectioned H. polygyrus showed strong positive reactions for protein in cytoplasmic granules in developing oocytes and in eggs of younger worms (16 days) but a reduced reaction in older worms (140 days). Strains for collagen showed a slight positive reaction in and between developing oocytes and a strong reaction in the egg shells. Stains for nucleoproteins particularly reacted with sperm stored in the uterus, and slightly reacted with fertilized eggs and the nucleoli of the intestinal and ovarian epithelium. Egg production by H. polygyrus increased to 123 eggs/female/day by 16 days PI but declined from 121 eggs/female/day at 35 days PI to 64 eggs/female/day in worms 140 days old. Electrophoresis indicated no loss in the different types of proteins in the reproductive tract of older worms, but histochemistry and protein content assays suggest that older worms that produce fewer eggs contain a relatively smaller amount of protein in the female reproductive tract.     

Detection by microsatellite analysis of early embryonic mortality in an alligator population in Florida

In the 1980s, alligators (Alligator mississippiensis) of Lake Apopka (Florida, USA) underwent a population decline associated with decreased egg viability, effects that have been associated with endocrine-disrupting, persistent organochlorine pesticides. It is currently unknown whether the decreased egg viability is due to fertilization failure or early embryonic death. Therefore, we conducted a preliminary study to evaluate the use of microsatellite DNA loci to determine the fertilization status of nonviable eggs. Using microsatellite analysis, we compared genotypes from blasto-disks and embryos with the genotypes from females trapped at the nest. Four of five nonviable egg samples tested yielded evidence of fertilization. No evidence of unfertilized eggs was obtained, but amplifiable DNA could not be obtained from one entirely nonviable clutch. Thus, we demonstrate that early embryonic mortality in alligators can be detected by microsatellite analysis, but also suggest substantial effort is needed to improve the recovery of DNA and amplification of alligator microsatellite loci.     

HoxA10-like proteins in the reproductive tract of the viviparous lizard Eulamprus tympanum and the oviparous lizard Lampropholis guichenoti

The gene HoxA10 and its protein product are essential for the formation of the extensions of the plasma membrane called uterodomes or pinopods in mammalian uterine epithelia. In mice, the presence of the HoxA10 protein and uterodomes is needed for uterine receptivity to blastocyst implantation. The viviparous lizard Eulamprus tympanum displays uterodomes whereas the oviparous lizard Lampropholis guichenoti does not. To explore the theory that HoxA10 is involved in the formation of uterodomes we investigated whether HoxA10 immunoreactive proteins were present in both species during their reproductive cycles. Oviduct proteins from vitellogenic, gravid or non-reproductive L. guichenoti (n=19) and E. tympanum (n=28) were separated by electrophoresis and analysed by Western blot and specific antibodies to HoxA10. E. tympanum displayed HoxA10 immunoreactive bands at 59 and 63 kDa in 20 out of the 28 samples. All of the L. guichenoti samples displayed HoxA10 immunoreactive bands, 18 had bands at 59 and 64 kDa and 1 animal had a single band at 59 kDa. There were no significant differences in the level of HoxA10 immunoreactivity between the different stages of reproductive cycle in either species. The different molecular mass of the larger band in L. guichenoti (64 kDa) compared to E. tympanum (63 kDa) indicates that the two lizards express different isoforms of the HoxA10-like proteins and it will be interesting in future studies to determine whether there are differences in the biological activity of the proteins that regulate different physiological functions in the uterus of viviparous and oviparous lizards.     

A histochemical study of the secretions of reproductive glands and of the egg envelopes of Achatina fulica (Pulmonata: Stylommatophora)

Histochemical investigations of the secretions of reproductive glands—albumen gland, apical uterus, basal uterus and prostate gland—indicate the presence of galactogen in the albumen gland, acid mucopolysaccharide in the apical uterus, and lipoprotein in the basal uterus and prostate gland of A. fulica. The proteinaceous secretions produced by the glands do differ in their terminal reactive sites. Intense alkaline phosphatase reaction is found in albumen gland and apical uterus; carbonic anhydrase activity could be detected mainly in the uterine glands. The cyclical secretory activity of the reproductive glands has been studied preparatory to egg-laying and in the spent phase. Histochemical characteristics of the egg envelopes—albumen, shell membrane and egg-shell—suggest a possible sequential deposition of glandular products during the descent of eggs through the repv. ductive tract. The factors contributing to the stability and resistant nature of the egg envelopes, and the possible role of nutritive materials contained in reproductive gland secretions in the development of the embryo (even while the eggs are inside the uterus), are discussed.     

Changes in femoral robusticity and porosity during the reproductive cycle of the female alligator (Alligator mississippiensis)

To help understand the nature of skeletal changes during the reproductive cycle of the female alligator, we compared femoral robusticity (density) and porosity of cross-sections from the midshafts of femora from the following groups of female alligators: (1) immature; (2) pre-ovulatory; (3) postovulatory with soft-shelled oviducal eggs; (4) post-ovulatory with hard-shelled oviducal eggs; (5) post-ovulatory with eggs in the nest; (6) post-ovulatory with hatched eggs; and (7) mature, quiescent. Femora from alligators with eggs in the nest were significantly less robust (dense) than those of the other groups except those with hard-shelled oviducal eggs. Cross sections from the midshaft of femora from alligators with eggs in the nest were significantly more porous than those from all the other groups. The results indicated that calcium was mobilized from the femoral shaft shortly before eggs were laid and that femoral density returned to normal levels for mature alligators 1–2 months after egg-laying.     

扬子鳄的现状与保护

扬子鳄为我国特有的珍稀爬行动物,被我国政府列为国家一级保护,在我国有关它的记载已有三千多年的历史。古典文献把它记作“鼍’,民间称为“土龙”、“猪婆龙”,拉丁学名叫Alligatorsinensis。据史料记载,约五百多年前,它在长江中下游种群分布稠密,分布区范围约在东经11     

Are non-reproductive male highveld mole-rats, Cryptomys hottentotus pretoriae physiologically suppressed while in the confines of the natal colony?

The highveld mole-rat, Cryptomys hottentous pretoriae is a social, subterranean, co-operative breeder with a high reproductive skew. The relationship between reproductive status and reproductive physiology in male highveld mole-rats was investigated. Testicular morphometrics, histological parameters, plasma testosterone concentrations and sperm motility parameters were studied in 31 males (14 reproductive and 17 non-reproductive males). Reproductive males were significantly larger than non-reproductive males, with testicular mass and volume corrected for body mass considerably larger for the reproductive males. Circulating plasma testosterone concentrations of reproductive males were not significantly higher than non-reproductive males (reproductive males 10.3±1.8 nmol l⁻¹ vs non-reproductive males 7.3±1.74 nmol l⁻¹). Sperm motility parameters were measured, but no significant differences were found between reproductive and non-reproductive males. All 14 reproductive males had motile sperm, whereas 13 of the 17 (76%) of non-reproductive males possessed motile sperm. A typical ejaculate of a reproductive male contains 48.3% motile sperm characterized by a high percentage of flagellar defects, whereas the non-reproductive male has an ejaculate containing 45.6% motile sperm with a high percentage of head defects. It is apparent that non-reproductive males are not physiologically suppressed from reproducing. Moreover, the non-reproductive males are excluded from incestuous matings as a result of being the offspring of the reproductive female.     

Fundamentals of viviparity: comparison of seasonal changes in the uterine epithelium of oviparous and viviparous Lerista bougainvillii (Squamata: Scincidae)

Distinct differences in epithelial response between oviparous and viviparous species of skinks led us to investigate morphological differences in the uterus of a species that exhibits bi-modal reproduction and that may indicate specialities for the different requirements of viviparity and oviparity. The uteri of females from oviparous and viviparous populations of the Australian scincid lizard, Lerista bougainvillii, are described in detail to determine whether the occurrence of uterodomes and the plasma membrane transformation, found in other viviparous species but not oviparous species, are indeed features characteristic of viviparity. Oviductal tissue was dissected at three different stages of reproduction from lizards from both populations: 1) vitellogenic, 2) gravid or pregnant, and 3) non-reproductive or quiescent. Tissue was observed using both scanning and transmission electron microscopy. Lerista bougainvillii has a simple placental morphology with simple squamous epithelium. In contrast to mammals and other viviparous skinks, L. bougainvillii does not undergo a plasma membrane transformation, but early signs of placentation in viviparous individuals are indicated by changes in the uterine surface that occur largely after embryonic stage 30. There are no obvious cellular differences between the uteri of oviparous and viviparous L. bougainvillii at the non-reproductive and vitellogenic phase of the reproductive cycle but throughout gestation/gravidity, the cellular differences that could be related to the changing functional requirements with the retention of the viviparous embryo, became apparent. A plasma membrane transformation with ensuing uterodome formation does not occur, which suggests that these more sophisticated changes are a feature of advanced placental development in reptiles.     

Egg Turning During Incubation has no Effect Upon the Growth of Embryos of Alligator mississippiensis

Alligator eggs are not turned during incubation, instead the embryo adheres to the top inside of the shell. Turning is alleged to shear off the embryo and kill it. Avian egg turning allegedly facilitates embryonic development by stimulating growth of the area vasculosa and minimizing the effects of unstirred yolk and albumen layers. From day 10 to day 45 of incubation, alligator eggs were experimentally turned, gently, through ± 60° in an hourly cycle. This turning regime killed only 6 out of 25 embryos. Compared with unturned controls, no significant effects were observed on the growth, production of extraembryonic fluids or utilization of albumen and yolk for those embryos that survived turning. The protein concentration of amniotic fluid at various stages of alligator development was examined in eggs incubated at 30 and 33°C. The fluid contained very little protein (max <8 mg) at any time: the protein concentration did not change consistently as development progressed. Differences in response to egg turning in birds and reptiles may be associated with the length of the incubation period, the protein content of the albumen and the mechanism of albumen utilization.