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Evidence of increased endometrial vascular permeability at the time of implantation in the short-nosed fruit bat, Cyanopterus sphinx
Animal Reproduction Science 101 (2007) 179–185
Short communication
Evidence of increased endometrial vascular permeability at the time of implantation in the short-nosed fruit bat, Cyanopterus sphinx Pranab Lal Pakrasi ∗ , Anjana Tiwari Embryo Physiology Laboratory, Center of Advanced Study, Department of Zoology, Banaras Hindu University, Varanasi 221005, India Received 21 November 2005; received in revised form 11 November 2006; accepted 20 November 2006 Available online 1 December 2006
Abstract Early embryonic development and implantation were studied in tropical short-nosed fruit bat Cyanopterus sphinx. We report preimplantation development and embryo implantation. Different stages of cleavage were observed in embryo by direct microscopic examination of fresh embryos after retrieving them either from the oviduct or the uterus at different days, up to the day of implantation. Generally, the embryos enter the uterus at the 8-cell stage. Embryonic development continued without any delay and blastocyst were formed showing attachment to the uterine epithelium at the mesometrial side of the uterus. A distinct blue band was formed in the uterus. The site of blastocyst attachment was visualized as a blue band following intravenous injection of pontamine blue. Implantation occurred 9 ± 0.7 days after mating. This study reports that bat embryonic development can be studied like other laboratory animals and that this bat shows blue dye reaction, indicating the site and exact time of implantation. This blue dye reaction can be used to accurately find post-implantational delay. We prove conclusively that this species of tropical bat does not have any type of embryonic diapause. © 2006 Elsevier B.V. All rights reserved. Keywords: Bat; Preimplantation development; Implantation; Blue dye reaction
1. Introduction Delayed implantation occurs when development of the conceptus is suspended at the blastocyst stage (Renfree, 2000). Embryonic diapause is a widespread adaptation and is found in ∗
Corresponding author. Tel.: +91 542 2575437; fax: +91 542 2368174. E-mail address: [email protected] (P.L. Pakrasi).
0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.11.017
180
P.L. Pakrasi, A. Tiwari / Animal Reproduction Science 101 (2007) 179–185
97 species from nine genera of the mammalia. Chiropterans have also adapted it as a reproductive strategy to achieve successful pregnancy (Oxberry, 1979). Temperate zone bats normally adopt embryonic diapause for successful implantation (Kimura and Uchida, 1983; Kawamoto, 2003). Cyanopterus sphinx (Suborder: Megachiroptera, Family: Pteropodidae) shows bimodal polyestry, i.e. breeds twice in quick succession, producing one offspring in March and June/July (Krishna, 1978). Krishna and Dominic (1983) reported that the first pregnancy lasts for a period of ∼150 days, whereas the second pregnancy begins in early April and has a gestation length of 120 days. So, the time period of the first pregnancy is selected as that having delayed implantation. It is also interesting to note that this Pteropid ovulates alternatively from two ovaries and both uterine horns are equally developed (Krishna and Dominic, 1983). On the basis of the available field data and gross, visible observations, it is of considerable interest to study the implantation phenomenon, as well as preimplantation embryo development, in this bat. However, the exact chronology of embryo development, time of implantation or the occurrence of delay, especially in the early post-implantational stages, is not yet known. This report attempts to fill these gaps in our knowledge. We report the blue dye reaction in the uterus, which is supposed to be the first discernible event in the process of mammalian implantation, as established in mice and rat (Ljungkvist and Nilsson, 1974). The objectives of this paper are: (1) to demonstrate the occurrence of any delay before implantation, (2) to visualize the implantation site via the blue dye reaction and (3) to delineate the exact time of blastocyst attachment in the short-nosed fruit bat C. sphinx (Suborder: Megachiroptera, Family: Pteropodidae), which is widely distributed in India (Ellerman and Morrison-Scot, 1951). 2. Materials and methods Bats were captured live daily from four to five sites within a 6-km radius of Banaras Hindu University campus from November 10 till the last date of finding live sperm in vaginal smears. All females weighed between 40 and 45 g and their wingspan exceeded 46 cm. The pelage was dark in colour. Bats were kept in our Departmental animal-house for approximately 1 month and provided with ripe fruits and water ad libitum. As soon as they were brought to the laboratory, vaginal smear was taken from each female to check mating. Females showing a live sperm positive (sp+) smear were assigned Day 1 (D1) of pregnancy. Bats showing no sperm in their smears were rejected from the study. Daily vaginal smear checks were performed from November 10 to observe the presence of live (motile) sperm. Sperm positive animals were then kept in separate cages according to their localities and assigned to different groups (according to day of pregnancy). The blue dye injection was started from D7 when the females showed blastocysts in their uterine flushing. Then, all the bats received 2% pontamine blue dye (Sigma, St. Louis, MO, USA) in 0.2 ml of 0.15 M NaCl solution intravenously through the cephalic vein and the dye was allowed to circulate for 5 min. The animals were then sacrificed under anesthesia. Uterine horns and ovaries along with oviducts were dissected out. The organs were freed from adherent fat and the oviducts and uterine horn were flushed separately with Medium 199 (Himedia Laboratories, Mumbai, India). The flushings were examined under a stereomicroscope for the presence of embryos. Eight to 12 bats were killed at every observation point. Uteri and ovaries exhibiting a blue dye reaction were saved. Routine histology was performed on these tissues with haematoxylin and eosin stain after fixation in Bouin’s fluid and proper dehydration in alcohol.
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2.1. Statistics The data were analyzed by one-way analysis of variance (ANOVA), followed by the Newman–Keul’s multiple range test (Bruning and Kintz, 1977). A probability of 0.01 was taken as the level of significant difference. Data were expressed as means ± S.E.M. 3. Results 3.1. Preimplantation embryo development Our results showed that sperm started appearing in the vaginal smear around November 10 and showed a gradual increase in the smear, as well as in uterine flushings. However, ovulation was not observed. This gradual increase of the number of sperm continued up to November 24. Suddenly, the count of fresh live sperm showed a decrease in vaginal smears. Our observations also revealed that sp+ bats showed no sperm when killed 24 h after the first appearance of sperm in the vaginal smear. From November 17 onwards, a single fertilized ovum with polar bodies was observed in the flushings of oviducts of bats killed from sperm positive bats. The fertilized zygote took 38–42 h to initiate cleavage and 2-cell embryos were observed in the oviduct on D3. Second cleavage took 24 h, and were observed under the microscope after sacrificing the bat on D4. The embryos at the 6–8-cell stage were the earliest embryos retrieved from uteri. In some cases, the embryos could be retrieved from the uterus from D4, showing normal cleavage. Embryos of 8-cell and later stages were present in uterine flushings of bats killed on D5 onwards. Subsequently, they formed morulae on D6 and underwent compaction. First appearance of a blastocoel was observed on D7 and an expanded blastocyst was observed on D8, irrespective of the location of capture. 3.2. Blue dye reaction Injection of macromolecular pontamine blue dye resulted in a distinct blue band in the uterus. We observed only one blue band in any one of the uterine horns (Fig. 1) on D9. After D10, blue bands were always associated with uterine swelling, which increased gradually (data not shown). By accumulating all the data from different locations, the Table 1 was formulated, showing that Table 1 Preimplantation embryonic development, ovarian weight and corpus luteal diameter from D1 to implantation. Days (pc) (no. of animals, n)
Embryonic stages retrieved
Location
Ovarian weighta (mg)
D1 (12) D2 (12) D3 (10) D4 (12) D5 (12) D6 (10) D7 (11) D8 (12) D9 (12)
Sperm attached ovum, zygote Zygote 2-Cell stage 4–8-Cell stage 8–16-Cell stage Compaction First appearance of blastocoel Blastocyst Blue spot in one uterine horn
Oviduct Oviduct Oviduct Uterus Uterus Uterus Uterus Uterus
1.15 1.17 1.23 1.49 1.89 2.41 2.58 2.94 3.09
a
± ± ± ± ± ± ± ± ±
0.03 0.01 0.02 0.05 0.04 0.03 0.08 0.03 0.13
Corpus luteum sizea (m) 206.81 208.18 215.66 410.50 475.84 591.37 599 634.8 639.84
± ± ± ± ± ± ± ± ±
33.40 31.79 75 67.80 64.10 62.01 79.30 94.31 66.84
Five animals were taken for each group. Values are mean ± S.E.M. All groups are significantly different at P < 0.01.
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Fig. 1. Blue dye reaction in the short-nosed fruit bat C. sphinx. Clear blue band around one uterine horn towards the uterotubal junction indicating cellular manifestation of implantation in this species. The ipsilateral ovary shows very significant size differences in comparison to the contralateral ovary. Original magnification ×20.48. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
this bat displayed 9 ± 0.7 days of preimplantation embryo development. The bats captured from four different locations showed that the phenomenon of mating spans a period of 7–8 days in ∼80% of the bat captured (data not shown). However, we did not find any appreciable variations within the same locations. 3.3. Delayed implantation In all the sperm positive bats, preimplantation embryonic development was observed without any interruption or retardation of cell division before implantation. This clearly indicates the absence of embryonic delay at the blastocyst stage. As soon as the embryos attained blastocyst stage, they underwent implantation and, subsequently, post-implantation development continued as the implantation swellings showed continuous growth (unpublished observation); thus, any possibility of delayed development can be ruled out. 3.4. Histological observations Histological observations showed that the ovulation occurred from the ipsilateral ovary having only one corpus luteum (Fig. 2a) along with two to four growing follicles. There is ovulation in the contralateral ovary. Histology showed different stages of maturing follicles (Fig. 2b). A significant increase in ovarian weight was observed after ovulation, which reached to maximum at implantation (3.09 ± 0.13 versus 1.15 ± 0.03; Table 1). Similarly, the size of the corpus luteum (Fig. 2a) also increased significantly and reached to a maximum of 639.84 ± 66.84 m (Table 1) in diameter at the time of implantation. None of the ovaries showed any old corpus lutea. The histology of the blue spot area of the uterus showed a bilaminar blastocyst attached superficially to the epithelium at the mesometrial side of the endometrium (Fig. 3). Histology of the implantation also showed least decidual cell formation.
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Fig. 2. Microscopy of ipsilateral and contralateral ovary bearing blue spot. (a) Ipsilateral ovary showing huge corpus luteum and only a few early follicles, indicating single ovulation in the ovary. (b) Transverse section of contralateral ovary showing many follicles but no corpus luteum, indicating unilateral ovulation in this species. Bar = 100 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 3. Transverse section of the blue band area of the uterus showing mesometrial implantation in C. sphinx. Bar = 500 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
4. Discussion The results clearly show that it is possible to identify pregnancy in bats by sperm positive vaginal smear, followed by retrieval of polar body stage embryo, as in other laboratory animals. In C. sphinx, copulation, fertilization and ovulation occur simultaneously. In almost all the animals, sp+ smears show ovulation and fertilization. There is no incidence of delayed ovulation, which is reported in another tropical species Scotophilus heathi (Abhilasha and Krishna, 1996). Our results differ completely from earlier reports on preimplantation embryo development in other bats (Heideman, 1989; Rasweiler, 1979; Rasweiler and Badwaik, 1997). There is no report on the physiological basis of survival of embryos in the uterus in bats, including C. sphinx. In all other Pteropids studied, only embryos of Pteropus giganteus enter the uterus as early as the morula stage (Marshall, 1949). In two other species of Pteropids Rousettus amplexicaudatus (Kohlbrugge, 1913) and Rousettus leschenaulti (Rasweiler, 1979), the embryo enters the uterus
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as a zona-free blastocyst. These experiments are mainly histological studies; so, our observations are more relevant in comparison to earlier studies. Visualization of a single distinct blue band around the uterine horn shows that only one implantation occurs in C. sphinx. This corroborates the single ovulation from the ipsilateral ovary (Krishna and Dominic, 1983). The corpus luteum takes care of the pregnancy, as it showed a progressive increase in diameter up to the stage of implantation of the blastocyst (Table 1), which agrees with reports in S. heathi (Krishna and Dominic, 1988), Taphozus georgiana (Kitchener, 1973), T. longimanus (Krishna and Dominic, 1982) and Rhinolophus hipposideros (Mathews, 1937). Several species of Insectivora (Harrison, 1948), such as Elephantulus (Van Der Horst and Guillman, 1946), hedgehog (Deanesly, 1934), Blarina (Pearson, 1944) and Ericulus (Strauss, 1939), also show rapid development of the corpus luteum. The blue dye reaction reflects increased vascular permeability at the blastocyst attachment site (Psychoyos, 1960). The mesometrial attachment of the blastocyst confirms earlier observations in other Pteropid bats (Rasweiler, 1979). However, we have not found any appreciable decidual tissue in the blue spot area where vascular permeability occurs. This needs further study and contrasts to observations in mice (Nilsson, 1967), rats (Psychoyos, 1973), rabbits (Hoffman et al., 1978) and hamsters (Evans and Kennedy, 1978). It should be noted that the blue band found in the uterine horns were not sharp, i.e. they were diffuse, in comparison to mice and rats. It is well established that prostaglandins and histamines are involved in the localized increase in vascular permeability at the time of implantation. We have also observed that 500 g of indomethacin could inhibit the blue dye reaction in C. sphinx (P.L. Pakrasi and Anjana Tiwari, unpublished data). A detailed study is in progress. The present findings establish the approximate time and chronology of preimplantation embryo development and the documentation of implantation in this bat. There is no delayed implantation in this species; blastocyst become attached normally. Rasweiler commented that “by histological studies of tracts obtained during early pregnancy, it is difficult to identify exactly when actually the delays begins” (Rasweiler and Badwaik, 1997). The blue dye reaction shows the precise timing of implantation and also provides an accurate method to assess the pace of post-implantation development. The blue dye technique, therefore, is a useful tool to study delayed development in bats and other animals. Acknowledgements We are grateful to the Rockefeller Foundation, USA, for providing basic infrastructure and the University Grants Commission, New Delhi, India, for financial support. A. Tiwari thanks the Indian Council of Medical Research, New Delhi, India, for financial assistance in the form of a Senior Research Fellowship. The authors are grateful to Professor Sinha of Department of English for his input in the manuscript. References Abhilasha, A., Krishna, A., 1996. High androgen production by ovarian thecal interstitial cells: a mechanism for delayed ovulation in a tropical vespertilionid bat, Scotofilus heathi. J. Reprod. Fertil. 106, 207–211. Bruning, J.L., Kintz, B.L., 1977. Newman’s Keul’s multiple range test. In: Computational Handbook of Statistics, second ed. Scott Foresman, Glenview, IL. Deanesly, R., 1934. The reproductive processes of certain mammals. Part IV. The reproductive cycle of the female hedgehog. Philos. Trans. R. Soc. Lond. B 223, 239–276.
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Ellerman, J.R., Morrison-Scot, T.C.S., 1951. In checklist of Palaeoretic and Indian mammals. British Museum (Natural History), London, pp. 1758–1946. Evans, C.A., Kennedy, T.G., 1978. The importance of prostaglandin synthesis for the initiation of blastocyst implantation in the hamster. J. Reprod. Fertil. 54, 255–261. Harrison, R.J., 1948. The development and fate of the corpus luteum in the vertebrate series. Biol. Rev. 23, 224–255. Heideman, P.D., 1989. Delayed development in Fisher’s pygmy fruit bat Haplonycteris fischeri, in the Philipines. J. Reprod. Fertil. 85, 363–382. Hoffman, L.H., Dipietro, D.L., McKenna, T.J., 1978. Effects of indomethacin on uterine capillary permeability and blastocyst development in rabbits. Prostaglandins 15, 823–828. Kawamoto, K., 2003. Endocrine control of the reproductive activity in hibernating bats. Zool. Sci. 20, 1057–1069. Kimura, K., Uchida, T.A., 1983. Ultrastructural observation of delayed implantation in Japanese long fingered bat Miniopterus schreibersii fuliginosus. J. Reprod. Fertil. 69, 187–193. Kitchener, D.J., 1973. Reproduction in the common sheath-tailed bat Taphozus Georgians (Thomas) (Microchiroptera; Emballonuridae) in Western Australia. Aust. J. Zool. 21, 378–389. Kohlbrugge, J.F.H., 1913. Befruchtung und Keimbildung bei der Fledermaus “Xantharpya amplexicaudata”. Verh. K. Akad. Wet. Amst. 2 (17), 1–37. Krishna, A., 1978. Aspect of reproduction in some Indian bats. Ph.D. thesis. Banaras Hindu University, Varanasi, India. Krishna, A., Dominic, C.J., 1982. Reproduction in the Indian sheath-tailed bat. Acta Theriol. 27, 97–106. Krishna, A., Dominic, C.J., 1983. Reproduction in the female short nosed fruit bat, Cyanopterus sphinx. Period. Biol. 85, 23–30. Krishna, A., Dominic, C.J., 1988. Histological and histochemical observations on the corpus luteum of the Indian Vespertillionid bat, Scotophilus heathi Horsefield. Zool. Anz. 220, 8–16. Ljungkvist, I., Nilsson, O., 1974. Blastocyst-endometrial contact and pontamine blue reaction during normal implantation in the rat. J. Endocr. 60, 149–154. Marshall, A.J., 1949. Pre-gestational changes in the giant fruit bat (Pteropus giganteus), with special reference to an assymetric endometrial reaction. Proc. Linn. Soc. Lond. 161, 26–36. Mathews, L.H., 1937. The female sex cycle in the British horseshoe bat Rhinolophus ferrum equine insulanus. Trans. Zool. Soc. Lond. 23, 224–255. Nilsson, O., 1967. Attachment of rat and mouse blastocysts onto the uterine epithelium. Int. J. Fertil. 12, 5–13. Oxberry, B.A., 1979. Female reproductive patterns in hibernating bats. J. Reprod. Fertil. 56, 359–367. Pearson, O.P., 1944. Reproduction in the shrew (Blarina brevicauda Say). Am. J. Anat. 84, 143–174. Psychoyos, A., 1960. Nouvelle contribution a l’etude de la nidation de l’oeuf chez la ratte. C. R. Acad. Sci. III 251, 3073–3075. Psychoyos, A., 1973. Endocrine control of egg implantation. In: Greep, R.O., Astwood, E.G., Geiger, S.R. (Eds.), Handbook of Physiology. American Physiological Society, Washington, D.C., pp. 187–215. Rasweiler IV, J.J., 1979. Early embryonic development and implantation in bats. J. Reprod. Fertil. 56, 403–416. Rasweiler IV, J.J., Badwaik, N.K., 1997. Delayed development in the short-tailed fruit bat, Carollia perspicillata. J. Reprod. Fertil. 109, 7–20. Renfree, M.B., 2000. Diapause. Annu. Rev. Physiol. 62, 353–375. Strauss, F., 1939. Die bildung der corpus luteum bei. Centtetiden Biomorphosis 1, 489–544. Van Der Horst, C.J., Guillman, 1946. The corpus luteum of Elephantulus during pregnancy, its form and function. S. Afr. J. Med. Sci. 11, 87–102.
Short communication
Evidence of increased endometrial vascular permeability at the time of implantation in the short-nosed fruit bat, Cyanopterus sphinx Pranab Lal Pakrasi ∗ , Anjana Tiwari Embryo Physiology Laboratory, Center of Advanced Study, Department of Zoology, Banaras Hindu University, Varanasi 221005, India Received 21 November 2005; received in revised form 11 November 2006; accepted 20 November 2006 Available online 1 December 2006
Abstract Early embryonic development and implantation were studied in tropical short-nosed fruit bat Cyanopterus sphinx. We report preimplantation development and embryo implantation. Different stages of cleavage were observed in embryo by direct microscopic examination of fresh embryos after retrieving them either from the oviduct or the uterus at different days, up to the day of implantation. Generally, the embryos enter the uterus at the 8-cell stage. Embryonic development continued without any delay and blastocyst were formed showing attachment to the uterine epithelium at the mesometrial side of the uterus. A distinct blue band was formed in the uterus. The site of blastocyst attachment was visualized as a blue band following intravenous injection of pontamine blue. Implantation occurred 9 ± 0.7 days after mating. This study reports that bat embryonic development can be studied like other laboratory animals and that this bat shows blue dye reaction, indicating the site and exact time of implantation. This blue dye reaction can be used to accurately find post-implantational delay. We prove conclusively that this species of tropical bat does not have any type of embryonic diapause. © 2006 Elsevier B.V. All rights reserved. Keywords: Bat; Preimplantation development; Implantation; Blue dye reaction
1. Introduction Delayed implantation occurs when development of the conceptus is suspended at the blastocyst stage (Renfree, 2000). Embryonic diapause is a widespread adaptation and is found in ∗
Corresponding author. Tel.: +91 542 2575437; fax: +91 542 2368174. E-mail address: [email protected] (P.L. Pakrasi).
0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.11.017
180
P.L. Pakrasi, A. Tiwari / Animal Reproduction Science 101 (2007) 179–185
97 species from nine genera of the mammalia. Chiropterans have also adapted it as a reproductive strategy to achieve successful pregnancy (Oxberry, 1979). Temperate zone bats normally adopt embryonic diapause for successful implantation (Kimura and Uchida, 1983; Kawamoto, 2003). Cyanopterus sphinx (Suborder: Megachiroptera, Family: Pteropodidae) shows bimodal polyestry, i.e. breeds twice in quick succession, producing one offspring in March and June/July (Krishna, 1978). Krishna and Dominic (1983) reported that the first pregnancy lasts for a period of ∼150 days, whereas the second pregnancy begins in early April and has a gestation length of 120 days. So, the time period of the first pregnancy is selected as that having delayed implantation. It is also interesting to note that this Pteropid ovulates alternatively from two ovaries and both uterine horns are equally developed (Krishna and Dominic, 1983). On the basis of the available field data and gross, visible observations, it is of considerable interest to study the implantation phenomenon, as well as preimplantation embryo development, in this bat. However, the exact chronology of embryo development, time of implantation or the occurrence of delay, especially in the early post-implantational stages, is not yet known. This report attempts to fill these gaps in our knowledge. We report the blue dye reaction in the uterus, which is supposed to be the first discernible event in the process of mammalian implantation, as established in mice and rat (Ljungkvist and Nilsson, 1974). The objectives of this paper are: (1) to demonstrate the occurrence of any delay before implantation, (2) to visualize the implantation site via the blue dye reaction and (3) to delineate the exact time of blastocyst attachment in the short-nosed fruit bat C. sphinx (Suborder: Megachiroptera, Family: Pteropodidae), which is widely distributed in India (Ellerman and Morrison-Scot, 1951). 2. Materials and methods Bats were captured live daily from four to five sites within a 6-km radius of Banaras Hindu University campus from November 10 till the last date of finding live sperm in vaginal smears. All females weighed between 40 and 45 g and their wingspan exceeded 46 cm. The pelage was dark in colour. Bats were kept in our Departmental animal-house for approximately 1 month and provided with ripe fruits and water ad libitum. As soon as they were brought to the laboratory, vaginal smear was taken from each female to check mating. Females showing a live sperm positive (sp+) smear were assigned Day 1 (D1) of pregnancy. Bats showing no sperm in their smears were rejected from the study. Daily vaginal smear checks were performed from November 10 to observe the presence of live (motile) sperm. Sperm positive animals were then kept in separate cages according to their localities and assigned to different groups (according to day of pregnancy). The blue dye injection was started from D7 when the females showed blastocysts in their uterine flushing. Then, all the bats received 2% pontamine blue dye (Sigma, St. Louis, MO, USA) in 0.2 ml of 0.15 M NaCl solution intravenously through the cephalic vein and the dye was allowed to circulate for 5 min. The animals were then sacrificed under anesthesia. Uterine horns and ovaries along with oviducts were dissected out. The organs were freed from adherent fat and the oviducts and uterine horn were flushed separately with Medium 199 (Himedia Laboratories, Mumbai, India). The flushings were examined under a stereomicroscope for the presence of embryos. Eight to 12 bats were killed at every observation point. Uteri and ovaries exhibiting a blue dye reaction were saved. Routine histology was performed on these tissues with haematoxylin and eosin stain after fixation in Bouin’s fluid and proper dehydration in alcohol.
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2.1. Statistics The data were analyzed by one-way analysis of variance (ANOVA), followed by the Newman–Keul’s multiple range test (Bruning and Kintz, 1977). A probability of 0.01 was taken as the level of significant difference. Data were expressed as means ± S.E.M. 3. Results 3.1. Preimplantation embryo development Our results showed that sperm started appearing in the vaginal smear around November 10 and showed a gradual increase in the smear, as well as in uterine flushings. However, ovulation was not observed. This gradual increase of the number of sperm continued up to November 24. Suddenly, the count of fresh live sperm showed a decrease in vaginal smears. Our observations also revealed that sp+ bats showed no sperm when killed 24 h after the first appearance of sperm in the vaginal smear. From November 17 onwards, a single fertilized ovum with polar bodies was observed in the flushings of oviducts of bats killed from sperm positive bats. The fertilized zygote took 38–42 h to initiate cleavage and 2-cell embryos were observed in the oviduct on D3. Second cleavage took 24 h, and were observed under the microscope after sacrificing the bat on D4. The embryos at the 6–8-cell stage were the earliest embryos retrieved from uteri. In some cases, the embryos could be retrieved from the uterus from D4, showing normal cleavage. Embryos of 8-cell and later stages were present in uterine flushings of bats killed on D5 onwards. Subsequently, they formed morulae on D6 and underwent compaction. First appearance of a blastocoel was observed on D7 and an expanded blastocyst was observed on D8, irrespective of the location of capture. 3.2. Blue dye reaction Injection of macromolecular pontamine blue dye resulted in a distinct blue band in the uterus. We observed only one blue band in any one of the uterine horns (Fig. 1) on D9. After D10, blue bands were always associated with uterine swelling, which increased gradually (data not shown). By accumulating all the data from different locations, the Table 1 was formulated, showing that Table 1 Preimplantation embryonic development, ovarian weight and corpus luteal diameter from D1 to implantation. Days (pc) (no. of animals, n)
Embryonic stages retrieved
Location
Ovarian weighta (mg)
D1 (12) D2 (12) D3 (10) D4 (12) D5 (12) D6 (10) D7 (11) D8 (12) D9 (12)
Sperm attached ovum, zygote Zygote 2-Cell stage 4–8-Cell stage 8–16-Cell stage Compaction First appearance of blastocoel Blastocyst Blue spot in one uterine horn
Oviduct Oviduct Oviduct Uterus Uterus Uterus Uterus Uterus
1.15 1.17 1.23 1.49 1.89 2.41 2.58 2.94 3.09
a
± ± ± ± ± ± ± ± ±
0.03 0.01 0.02 0.05 0.04 0.03 0.08 0.03 0.13
Corpus luteum sizea (m) 206.81 208.18 215.66 410.50 475.84 591.37 599 634.8 639.84
± ± ± ± ± ± ± ± ±
33.40 31.79 75 67.80 64.10 62.01 79.30 94.31 66.84
Five animals were taken for each group. Values are mean ± S.E.M. All groups are significantly different at P < 0.01.
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Fig. 1. Blue dye reaction in the short-nosed fruit bat C. sphinx. Clear blue band around one uterine horn towards the uterotubal junction indicating cellular manifestation of implantation in this species. The ipsilateral ovary shows very significant size differences in comparison to the contralateral ovary. Original magnification ×20.48. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
this bat displayed 9 ± 0.7 days of preimplantation embryo development. The bats captured from four different locations showed that the phenomenon of mating spans a period of 7–8 days in ∼80% of the bat captured (data not shown). However, we did not find any appreciable variations within the same locations. 3.3. Delayed implantation In all the sperm positive bats, preimplantation embryonic development was observed without any interruption or retardation of cell division before implantation. This clearly indicates the absence of embryonic delay at the blastocyst stage. As soon as the embryos attained blastocyst stage, they underwent implantation and, subsequently, post-implantation development continued as the implantation swellings showed continuous growth (unpublished observation); thus, any possibility of delayed development can be ruled out. 3.4. Histological observations Histological observations showed that the ovulation occurred from the ipsilateral ovary having only one corpus luteum (Fig. 2a) along with two to four growing follicles. There is ovulation in the contralateral ovary. Histology showed different stages of maturing follicles (Fig. 2b). A significant increase in ovarian weight was observed after ovulation, which reached to maximum at implantation (3.09 ± 0.13 versus 1.15 ± 0.03; Table 1). Similarly, the size of the corpus luteum (Fig. 2a) also increased significantly and reached to a maximum of 639.84 ± 66.84 m (Table 1) in diameter at the time of implantation. None of the ovaries showed any old corpus lutea. The histology of the blue spot area of the uterus showed a bilaminar blastocyst attached superficially to the epithelium at the mesometrial side of the endometrium (Fig. 3). Histology of the implantation also showed least decidual cell formation.
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Fig. 2. Microscopy of ipsilateral and contralateral ovary bearing blue spot. (a) Ipsilateral ovary showing huge corpus luteum and only a few early follicles, indicating single ovulation in the ovary. (b) Transverse section of contralateral ovary showing many follicles but no corpus luteum, indicating unilateral ovulation in this species. Bar = 100 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 3. Transverse section of the blue band area of the uterus showing mesometrial implantation in C. sphinx. Bar = 500 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
4. Discussion The results clearly show that it is possible to identify pregnancy in bats by sperm positive vaginal smear, followed by retrieval of polar body stage embryo, as in other laboratory animals. In C. sphinx, copulation, fertilization and ovulation occur simultaneously. In almost all the animals, sp+ smears show ovulation and fertilization. There is no incidence of delayed ovulation, which is reported in another tropical species Scotophilus heathi (Abhilasha and Krishna, 1996). Our results differ completely from earlier reports on preimplantation embryo development in other bats (Heideman, 1989; Rasweiler, 1979; Rasweiler and Badwaik, 1997). There is no report on the physiological basis of survival of embryos in the uterus in bats, including C. sphinx. In all other Pteropids studied, only embryos of Pteropus giganteus enter the uterus as early as the morula stage (Marshall, 1949). In two other species of Pteropids Rousettus amplexicaudatus (Kohlbrugge, 1913) and Rousettus leschenaulti (Rasweiler, 1979), the embryo enters the uterus
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as a zona-free blastocyst. These experiments are mainly histological studies; so, our observations are more relevant in comparison to earlier studies. Visualization of a single distinct blue band around the uterine horn shows that only one implantation occurs in C. sphinx. This corroborates the single ovulation from the ipsilateral ovary (Krishna and Dominic, 1983). The corpus luteum takes care of the pregnancy, as it showed a progressive increase in diameter up to the stage of implantation of the blastocyst (Table 1), which agrees with reports in S. heathi (Krishna and Dominic, 1988), Taphozus georgiana (Kitchener, 1973), T. longimanus (Krishna and Dominic, 1982) and Rhinolophus hipposideros (Mathews, 1937). Several species of Insectivora (Harrison, 1948), such as Elephantulus (Van Der Horst and Guillman, 1946), hedgehog (Deanesly, 1934), Blarina (Pearson, 1944) and Ericulus (Strauss, 1939), also show rapid development of the corpus luteum. The blue dye reaction reflects increased vascular permeability at the blastocyst attachment site (Psychoyos, 1960). The mesometrial attachment of the blastocyst confirms earlier observations in other Pteropid bats (Rasweiler, 1979). However, we have not found any appreciable decidual tissue in the blue spot area where vascular permeability occurs. This needs further study and contrasts to observations in mice (Nilsson, 1967), rats (Psychoyos, 1973), rabbits (Hoffman et al., 1978) and hamsters (Evans and Kennedy, 1978). It should be noted that the blue band found in the uterine horns were not sharp, i.e. they were diffuse, in comparison to mice and rats. It is well established that prostaglandins and histamines are involved in the localized increase in vascular permeability at the time of implantation. We have also observed that 500 g of indomethacin could inhibit the blue dye reaction in C. sphinx (P.L. Pakrasi and Anjana Tiwari, unpublished data). A detailed study is in progress. The present findings establish the approximate time and chronology of preimplantation embryo development and the documentation of implantation in this bat. There is no delayed implantation in this species; blastocyst become attached normally. Rasweiler commented that “by histological studies of tracts obtained during early pregnancy, it is difficult to identify exactly when actually the delays begins” (Rasweiler and Badwaik, 1997). The blue dye reaction shows the precise timing of implantation and also provides an accurate method to assess the pace of post-implantation development. The blue dye technique, therefore, is a useful tool to study delayed development in bats and other animals. Acknowledgements We are grateful to the Rockefeller Foundation, USA, for providing basic infrastructure and the University Grants Commission, New Delhi, India, for financial support. A. Tiwari thanks the Indian Council of Medical Research, New Delhi, India, for financial assistance in the form of a Senior Research Fellowship. The authors are grateful to Professor Sinha of Department of English for his input in the manuscript. References Abhilasha, A., Krishna, A., 1996. High androgen production by ovarian thecal interstitial cells: a mechanism for delayed ovulation in a tropical vespertilionid bat, Scotofilus heathi. J. Reprod. Fertil. 106, 207–211. Bruning, J.L., Kintz, B.L., 1977. Newman’s Keul’s multiple range test. In: Computational Handbook of Statistics, second ed. Scott Foresman, Glenview, IL. Deanesly, R., 1934. The reproductive processes of certain mammals. Part IV. The reproductive cycle of the female hedgehog. Philos. Trans. R. Soc. Lond. B 223, 239–276.
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