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Enzyme histochemical analysis of the process of atresia in mouse ovulatory follicles
152
153
STRU~ CRI~IA FOR D E V E I D P M ~ A L STAGES IN P I K E - P ~ C H (Stizostedion lucioperca L.) OVARIAN POLLI~.F-q. H . S t e n ~ c k and E.Antila. Department of Medical Biology , University of Helsinki, SF-00170 Helsinki, Finland.
DNA FLOW CYTOMETRIC QUANTITATION OF RAT SPE~TOGENIC CELLS AFTER HYPOPHYSECTOMY. J. T6Doari. I. Tsutsumi and G.S. diZere~a. Livingston Reproductive Biology Laboratory, University of Southern California School of Medicine, Los Angeles, California 90033.
Follicle development during annual maturationcycle was studied by light and electron microscpy in the pike-perch. Conspicuous features were the alteration in the location of the ovum nucleus and reduction in the num/~er of nucleoli, increase in tu~ bular steroidogenic type mitochondria, in~ crease in sa,Doth endoplasmatic reticulum, and in particular structural changes in the fol~ licular layer (fig.). The ultrastructural results correlated with age, gonado-scn~tic-index and length of fishes. Based on the above findings five developmental stages could be established.
Spermatogenesis is regulated by pituitary gonadotropins FSH and LH, and is impaired in the absence of these hoznnone s. This disruption was studied by quantitating spermatogenic cells in defined stages of the seminif erous epithelial cycle (II-V, VII, IX-XIII) at 3, 6, i0, 17, 27, 41, and 59 days after hypophysectomy using DNA flow cytometry. First signs of spermatogenio failure were found on day 6, when numbers of pachytene spermatocytes (4C) and round spez-matids (IC) in stage VII had decreaaed to 72% and 79% of control, respectively. Stages IX-XII also showed reduced cell numbers (4C: 77%; IC: 8S%), whereas stages II-V had 87~ 4C cells and 103% IC cells. At i0 days, step 9-12 spermatids had declined to 51 ~, and the amount of 4C cells in stages VII and IX-XII was 62%. Between I0 and 27 days, elongated spermatids disappeared. Round spermatids declined to under 10% in 59 days, but still spe~nat ids up %o step 8 of spez~niogenesi s could be seen. Meiosis continued despite hypophysectomy, while the amount of meiotic cells (4C) in stages I-VIII decreased to 27% at 41 days and 31% at 59 days; in stages IX-XIII, respective values were 23% and 22%. The gonadotropin dependent stages of developing germ cells can be identified and quantitatively characterized by DNA flow cytometry using stage specific sections of rat seminiferous epithelium.
I
~
~_____--Folltclecells~ ] Tunica p r o p r i a ~ o .
radlat~externa
IV
"
] ]
= 1 WIm~A - - ~
154
155
ENZYME HISTOCHEMICAL ANALYSIS OF THE PROCESS OF ATRESIA IN MOUSE OVULATORY FOLLICLES C.J.F. Van Noorden, W. Spllet, I.M.C. vogeis, ~. van ~chle, i . ~ruln ana u.G. De ~cfiepper. Academmc meamcai ten=re, unmverslty of Amsterdam, The Netherlands
DIFFERENTATION ANTIGENS OF GAMETOGENESIS IN CARP (CYPRINUS CARPIO L., TELEOSTEI) A. van Winkoop and L.P.M. Timmermans. Department ol Experimental Animal Morphology and Cell Biology, Agricultural University, Wageningen, the Netherlands.
During each menstrual cycle 15 to 20 follicles may grow but most of these degenerate in a process called atresia except the few that will ovulate. It is not known whether the selection procedure which oocyte will ovulate and which will become atretic is a randomized process or whether it is a survival of the ~ittest. However, this question may be of importance for in vitro fertilization because after hormon--/l stlmulatlpn far more graafian follicles develop than during a natural cycle. It may be assumed that at least some of the collected oocytes normally would have become atretic. Therefore, we have studied some parameters with histochemlcal means in order to determine early metabolic changes in atretic follicles. It will be reported that the earliest changes we have found were an increased activity of lactate and succlnate dehydrogenase locally at the periphery of the follicle. Serial sections stained for collagen with plkrosirius red revealed some Infiltration of connective tissue in those areas. In later stages of atresia, these areas with increased enzyme activity were extended to the interior of the follicle. These findings suggest that atresia is a process initiated at the peripheral granulosa cells and there are no indications yet that the metabolism of the oocyte itself is involved in the onset of atresla.
Gametogenesis in carp, a commercially important flesh water fish, is marked by respectively the onset ol primordial germ cell proliferation (at six weeks after fertilization), gonadal sex diflerentiation (week 10), meiosis and oocyte development in the Iemale gonad (from week 16 onwards), and sperm.%togenesis in males (starting at week 18) . Monoclonal antibodies have been prepared against membrane antigens of carp spermatozoa and recently against isolated carp spermatogonia. Several o l these monoclona] antibodies deline germ cell d i f l e r e n t i a t i o n antigens representative for the above mentioned stages ol gametogenesis. We are currently investigating the functional importance, as well as the molecular nature ol the identilied antigenic determinants. . Parmentier H.K. et al., Cell Tiss. Res. 236: 99-105, 1984. ¢* Parmentier H.K. & Timmermans L.P.M., 3. Exp. Embryol. Morphol. 90: 13-32, 1985. 55S
153
STRU~ CRI~IA FOR D E V E I D P M ~ A L STAGES IN P I K E - P ~ C H (Stizostedion lucioperca L.) OVARIAN POLLI~.F-q. H . S t e n ~ c k and E.Antila. Department of Medical Biology , University of Helsinki, SF-00170 Helsinki, Finland.
DNA FLOW CYTOMETRIC QUANTITATION OF RAT SPE~TOGENIC CELLS AFTER HYPOPHYSECTOMY. J. T6Doari. I. Tsutsumi and G.S. diZere~a. Livingston Reproductive Biology Laboratory, University of Southern California School of Medicine, Los Angeles, California 90033.
Follicle development during annual maturationcycle was studied by light and electron microscpy in the pike-perch. Conspicuous features were the alteration in the location of the ovum nucleus and reduction in the num/~er of nucleoli, increase in tu~ bular steroidogenic type mitochondria, in~ crease in sa,Doth endoplasmatic reticulum, and in particular structural changes in the fol~ licular layer (fig.). The ultrastructural results correlated with age, gonado-scn~tic-index and length of fishes. Based on the above findings five developmental stages could be established.
Spermatogenesis is regulated by pituitary gonadotropins FSH and LH, and is impaired in the absence of these hoznnone s. This disruption was studied by quantitating spermatogenic cells in defined stages of the seminif erous epithelial cycle (II-V, VII, IX-XIII) at 3, 6, i0, 17, 27, 41, and 59 days after hypophysectomy using DNA flow cytometry. First signs of spermatogenio failure were found on day 6, when numbers of pachytene spermatocytes (4C) and round spez-matids (IC) in stage VII had decreaaed to 72% and 79% of control, respectively. Stages IX-XII also showed reduced cell numbers (4C: 77%; IC: 8S%), whereas stages II-V had 87~ 4C cells and 103% IC cells. At i0 days, step 9-12 spermatids had declined to 51 ~, and the amount of 4C cells in stages VII and IX-XII was 62%. Between I0 and 27 days, elongated spermatids disappeared. Round spermatids declined to under 10% in 59 days, but still spe~nat ids up %o step 8 of spez~niogenesi s could be seen. Meiosis continued despite hypophysectomy, while the amount of meiotic cells (4C) in stages I-VIII decreased to 27% at 41 days and 31% at 59 days; in stages IX-XIII, respective values were 23% and 22%. The gonadotropin dependent stages of developing germ cells can be identified and quantitatively characterized by DNA flow cytometry using stage specific sections of rat seminiferous epithelium.
I
~
~_____--Folltclecells~ ] Tunica p r o p r i a ~ o .
radlat~externa
IV
"
] ]
= 1 WIm~A - - ~
154
155
ENZYME HISTOCHEMICAL ANALYSIS OF THE PROCESS OF ATRESIA IN MOUSE OVULATORY FOLLICLES C.J.F. Van Noorden, W. Spllet, I.M.C. vogeis, ~. van ~chle, i . ~ruln ana u.G. De ~cfiepper. Academmc meamcai ten=re, unmverslty of Amsterdam, The Netherlands
DIFFERENTATION ANTIGENS OF GAMETOGENESIS IN CARP (CYPRINUS CARPIO L., TELEOSTEI) A. van Winkoop and L.P.M. Timmermans. Department ol Experimental Animal Morphology and Cell Biology, Agricultural University, Wageningen, the Netherlands.
During each menstrual cycle 15 to 20 follicles may grow but most of these degenerate in a process called atresia except the few that will ovulate. It is not known whether the selection procedure which oocyte will ovulate and which will become atretic is a randomized process or whether it is a survival of the ~ittest. However, this question may be of importance for in vitro fertilization because after hormon--/l stlmulatlpn far more graafian follicles develop than during a natural cycle. It may be assumed that at least some of the collected oocytes normally would have become atretic. Therefore, we have studied some parameters with histochemlcal means in order to determine early metabolic changes in atretic follicles. It will be reported that the earliest changes we have found were an increased activity of lactate and succlnate dehydrogenase locally at the periphery of the follicle. Serial sections stained for collagen with plkrosirius red revealed some Infiltration of connective tissue in those areas. In later stages of atresia, these areas with increased enzyme activity were extended to the interior of the follicle. These findings suggest that atresia is a process initiated at the peripheral granulosa cells and there are no indications yet that the metabolism of the oocyte itself is involved in the onset of atresla.
Gametogenesis in carp, a commercially important flesh water fish, is marked by respectively the onset ol primordial germ cell proliferation (at six weeks after fertilization), gonadal sex diflerentiation (week 10), meiosis and oocyte development in the Iemale gonad (from week 16 onwards), and sperm.%togenesis in males (starting at week 18) . Monoclonal antibodies have been prepared against membrane antigens of carp spermatozoa and recently against isolated carp spermatogonia. Several o l these monoclona] antibodies deline germ cell d i f l e r e n t i a t i o n antigens representative for the above mentioned stages ol gametogenesis. We are currently investigating the functional importance, as well as the molecular nature ol the identilied antigenic determinants. . Parmentier H.K. et al., Cell Tiss. Res. 236: 99-105, 1984. ¢* Parmentier H.K. & Timmermans L.P.M., 3. Exp. Embryol. Morphol. 90: 13-32, 1985. 55S