STUDIES OF THE HUMAN UNFERTILIZED TUBAL OVUM*t

Transcription

1 FERTILITY AND STERILITY 1973 by The Williams & Wilkins Co. Vol. 24, No.8, August 1973 Printed in U.S.A. STUDIES OF THE HUMAN UNFERTILIZED TUBAL OVUM*t C. NORIEGA, M.D., AND C. OBERTI, M.D. Department of Gynecology, Hospital San Borja, and Department of Morphology, University of Chile School of Medicine, Santiago, Chile The ultrastructure of the human ovum in different stages of development has been studied from ovarian wedges or oocytes removed from prominent follices. l - a Also, oocytes matured in vitro have been described. 4 The relationship of the ovum, the zona pellucida, and surrounding follicular cells has been presented. Cytoplasmic organelles and the nuclear characteristics and changes throughout the maturation processes have also been well described. 5 Isolated ova recovered from the oviduct in the pronuclear stage of fertilization have been documented with the aid of phase-contrast microscopy and electron microscopy.6, 7 The search for ova in the female tract has been clinically reported, and some morphologic characteristics of such ova were defined. 8- lo The present study describes the structure and ultrastructure of unfertilized human tubal ova from specimens obtained during elective laparotomies. MATERIALS AND METHODS High fertility patients (8-20 pregnancies) selected for tubal ligation were studied within a given normal cycle with basal body temperature, vaginal smear, fern test, and endometrial biopsies. The occurrence of ovulation was detected by culdoscopy and, once confirmed, a laparotomy was immediately performed. Observation of the corpus luteum (Fig. 1) and its biopsy documented the phenomenon. Received April 9, * Supported by a grant from The Population Council. t Presented at the Twenty-Ninth Annual Meeting of The American Fertility Society, April 5-7, 1973, San Francisco, California. 595 Eight tubal ova were recovered in 14 laparotomies between the 12th and 19th days of the cycle using sterile culture medium. After being identified by stereomicroscopy, they were photographed and immediately fixed with glutaraldehyde, postfixed with osmium tetroxide, and embedded in Epon. Control and serial sections were performed. The more recent ova were observed with a Siemens-Halske 100- kv. electron microscope. During the processing two specimens were lost. RESULTS Unfertilized human ova, when recovered from the oviduct at laparotomy, are surrounded by a loose layer of corona cells outside the zona pellucida (Figs. 2 and 3). The perivitelline space is well formed and contains the first polar body. On higher magnification, the zona pellucida appears homogeneous around the perivitelline space. Although no structures are identified between the zona and the vitelline membrane, the perivitelline space contains fine granular material. The oocyte surface shows scattered projections into the perivitelline space, and round electron-dense cortical granules are under the membrane (Fig. 4). Mitochondria with transverse crystae concentrate in the periphery of the ovum; some of these are associated with an agranular double membrane of the endoplasmic reticulum (Fig. 5). The endoplasmic reticulum surrounds less dense electron vacuolar formations. Large lysosomes are also observed; few compound aggregates and multivesicular bodies are seen in the cytoplasm. The chromosomes are electron-dense, fine

2 596 NORIEGA AND OBERTI Vol. 24 FIG. 1. An early corpus luteum at time of laparotomy. FIG. 2. Human tubal ovum recovered with cumulus and corona cells. Phase-contrast microscopy. x 100. granular irregular structures with no limiting membranes (Fig. 6). In two specimens, abundant vesicles were observed in the cytoplasm, and also two and three nuclear structures containing nucleoli (Fig. 7). In the periphery of the zona, follicular cells have lost most of their cytoplasmic projections, and abundant vacuoles are distributed on the periphery of these cells (Fig. 8). DISCUSSION In our studies, human ovulation has been accurately determined with the aid of

3 August 1973 HUMAN UNFERTILIZED TUBAL OVUM 597 FIG. 3. Human tubal ovum. Corona cells, homogeneous zona pellucida, and oocyte surface with microvilli. x 3000.

4 FIG. 4. Electron micrograph of fine granular material within perivitelline space. Few polymorphous microvilli and cortical granules. x 13, FIG. 5. Mitochondria and associated agranular endopll}smic reticulum. x 20,

5 August 1973 HUMAN UNFERTILIZED TUBAL OVUM 599 FIG. 6. Chromosomes of unfertilized human tubal ovum with dense fine granular material. x FIG. 7. Abundant vesicles and three nuclei. One contains a well-defined nucleolus. x 6000.

6 600 NORIEGA AND OBERTI Vol. 24 FIG. 8. Follicular cells with multiple cytoplasmic vacuoles and few retracted cytoplasmic projections. x pelvic endoscopy. The results describe some of the ultrastructural characteristics of the human unfertilized tubal ovum. The absence of cytoplasmic processes of the corona cells and the homogeneous appearance of the zona pellucida could be defined. A well-developed perivitelline space was also evident. The oocyte surface presents few microvilli projections and dense cortical granules which are abundant near the cell surface, results which are similar to those reported by Zamboni 11 for mammalian ova. Organelles such as mitochondria and endoplasmic reticulum are spread throughout the cytoplasm. Irregular, fine, dense chromosome and scarce Golgi complexes are present. In two specimens, more than two nuclear-like structures were observed with nucleoli and surrounded by a membrane. These ova presented abundant vacuoles in the cytoplasm, which suggests a delayed or prolonged period of time in the oviduct. These observations are in accordance with those obtained with the aging of mouse eggs in the fallopian tube, as indicated by Szollosi.1 2 It was reported that a nucleus reconstitutes without formation of the second polar body. In 2 of our cases, the ova demonstrated more than two nuclei. In both well-vascularized corpus luteum biopsies, it was indicated that ovulation had occurred more than 72 hr. prior. These ova also presented multiple vacuoles in the cytoplasm as evidence of degenerative changes in the oviduct. The timing of the clinical events that documented ovulation in the human and the morphologic changes of the ova recovered are in close agreement with those commented on by Clewe et ai.,8 where the aging of the ova is difficult to relate to the ovulatory phenomenon.

7 August 1973 HUMAN UNFERTILIZED TUBAL OVUM 601 SUMMARY High fertility patients accepted for tubal sterilization were followed during a normal cycle with four clinical parameters prior to an elective laparotomy. Culdoscopy was also performed to document ovulation. Ova were collected in the postovulatory phase by retrograde flushing of the fallopian tube. Eight tubal ova were identified and photographed. Some were processed for electron microscopy. Two were lost during fixation. Our results describe the morphology of 6 unfertilized tubal ova obtained in 14 laparotomies. In 4, the ovum appears surrounded by a homogeneous zona pellucida that shows no typical structure and, beyond it, by a layer of follicular cells (corona radiata) with noncytoplasmic processes. The ovum exhibits few smooth, round microvilli in its perivitelline space, and the cytoplasm contains electron-dense granules below the vitelline membrane and numerous organelles scattered throughout it: viz., mitochondria, endoplasmic reticulum, Golgi's apparatus, and nonmembrane-bound chromosomes. Two ova with advanced degenerative changes in the cytoplasm presented two and three nuclei with a clear nuclear membrane and nucleoli, although fertilization had not taken place. REFERENCES 1. BAKER, T. G., AND FRANCHI, L. L. The fine structure of oogohia and oocytes in human ovaries. J Cell Sci 2:213, HERTIG, A. T., AND ADAMS, E. C. Studies on the human oocyte and its follicle. J Cell Bioi 34:647, HERTIG, A. T. The primary human oocyte. Some observations on the fine structure of Balbiani's vitelline body and the origin of the annulate lamellae. AmerJ Anat 122:107, ZAMBONI, L., AND THOMPSON, R. S. Fine morphology of human oocyte maturation in vitro. Bioi Reprod 7:425, BACA, M., AND ZAMBONI, L. The fine structure of human follicular oocytes. J Ultrastuct Res 19:354, DICKMANN, Z., CLEWE, T. H., BONNEY, W. A., AND NOYES, R. W. The human egg in the pronuclear stage. Anat Rec 152:293, ZAMBONI, L., MIS HELL, D. R., BELL, J. H., AND BACA, M. Fine structure of the human ovum in the pronuclear stage. J Cell Bioi 30:579, CLEWE, T. H., MORGENSTERN, L. L., NOYES, R. H. BONNEY, W. A., BURRUS, S. B., AND DEFOE, V. J. Searches for ova in the human uterus and tubes. Amer J Obstet Gynec 109:313, CROXATTO, H. B., FUENTEALBA, B., DIAZ, S., PASTENE, L., AND TATUM, H. J. A single nonsurgical technique to obtain unimplanted eggs from human uteri. AmerJ Obstet Gynec 112:662, CROXATTO, B., DIAZ, S., FUENTEALBA, B., CROXATTO, D., CARILLO, D., AND FABRES, C. Studies on the duration of egg transport in the human oviduct. 1. The time interval between ovulation and egg recovery from the uterus in normal women. Fertil Steril 23:447, ZAMBONI, L. Ultrastructure of mammalian oocytes and ova. Bioi Reprod 2:44, SZOLLOSI, D. Morphological changes in mouse eggs due to aging in the Fallopian tube. Amer J Anat 130:209, 1971.