15. 1. 1975
Specialia
a) Tr~gergebundene AminosXuren. Als fester Tr~iger dient vernetztes Polystyrol. Die daran gebundene Aminoschutzgruppe ist ein BenzhydryloxycarbonylAnalogon, bei dem ein Phenylring durch den des Polystyrols ersetzt ist. Vernetztes Polystyrol wird benzoyliert 7, mit Natriumborhydrid zum Carbinol reduziertT, s und zum entsprechenden gemischten Kohlensh.urephenylester umgesetzt 9. Dutch Reaktion mit der entsprechenden Aminos~ture erh~tlt man das Derivat% Der Reaktionsverlauf wird auf jeder Stufe durch Elementaranalyse und I R - S p e k t r n m verfolg~. Die Beladung liegt bei 1,8-2,5 Aminos~iurereste pro 10 Monomereinheiten. b) Kupplungsreaktion. ]tquimolare Mengen yon Prolinpicolylester a0 und tr~tgergebundenes, an der Aminogruppe geschtitztes Alanin werden in D M F mit DCCI und Hydroxybenzotriazol ~t gekuppelt. Der Umsatz wird m i t der Durchflussanalyse verfolgt s. Wenn die Aminokomponente nicht vollst~indig verbraucht ist, wird nochmals eine entsprechende Menge des Alaninderivates zugesetzt. Nach beendeter Reaktion (fiber Nacht bei 0 ~ wird gewaschen, bis das Filtrat beim Eindampfen keine Rfickst~tnde mehr enth~lt und ninhydrin-negativ ist. c) Freisetzung des Pepfids. Sie erfolgt durch Zugabe yon 10%iger Trifluoressigs/iure ill Methylenchlorid. Nach 10 Min wird abfiltriert und mehrfach mit DMF gewaschen, his das Filtrat ninhydrin-negativ ist. Darauf wird das Filtrat im H o c h v a k n u m eingedampft und mit D MF aufgenommen. Die Abtrennung der Aminos~iure erfolgt durch Ionenaustausch an SE-Sephadex oder Gelpermeations-Chromatographie all Sephadex G-10 mit DNIF als Elutionsmittel. Hierauf kann die Kupplung mit der n~Lchsten trXgergebundenen Aminos~ure erfolgen. Nach vollendeter Synthese wird die Carboxylschutzgruppe dutch katalytische Hydrogenolyse abgespalten. Das Peptid ist chromatographisch rein ~2
133
Das Peptid wurde such unter Verwendung yon Poly~thylenglykolmonostearyl/~ther als Carboxylschutzgruppe ~ snythetisiert. Der einzige Unterschied zum geschilderten Verfahren ist die Abtrennung der freien Aminos~ture; sic erfolgt hier durch Ausf~llen des Peptidesters mit Athanol oder durch Ultrafiltration2.
Summary. A new strategy for peptide synthesis on polymeric supports is described. This alternating liquidsolid phase peptide synthesis procedure facilitates the ready separation of reacted and unreacted amino component at the end of each coupling reaction. Incomplete coupling reactions and incomplete deprotection reactions, the most serious problems in previously described methods of peptide synthesis on polymeric supports, have in this procedure no influence on the success of a synthesis. The principle is exemplified by the synthesis of the pentapeptide Gly-Val-Gly-Ala-Pro. H. FRANK und H, HAGENMAIER
Chemisches Institut der Universitiit, Au/ tier 1VIorgenstelle, D-74 Ti~bingen 7 (Bundesrepublik Deutschland, BRD), 6. September 1974.
7 p. RIVAILLE, A. ROBINSON, M. KAMEN und G. MILHAUD, Helv. chim. A c t a 5d, 2772 (1971). 8 G. L. SOUTHARD, G. S. BROOKE, J. ~V[. PETTEE, T e t r a h e d r o n Lett. 7969, 3505. - H. FRA~CK, D i s s e r t a t i o n Tiibingen (1973). 9 p. SIEBER und 13. IsELnr Helv. chim. A c t a 57, 622 (1968). ~0 R. CAMBLE, R. GARNER und G. T. YOUNG, J. chem. Soc. (C) 7969, 1911. 11 W. K6NIG und R. GEIGER, chem. Ber. 103, 788 (1970). 12 H. HAGEN~AIER und H. FRANK, J. Chromat. Sci. 70, 663 (1972).
S t u d i e s on t h e M a i n t e n a n c e of P o r c i n e G r a a f i a n F o l l i c l e s in O r g a n C u l t u r e I t is now well established for a variety of mammalian species t h a t the changes characteristic of pre-ovulatory maturation in the Graafian follicle are dependent upon an adequate supply of gonadotrophic hormones, and especially on the so called 'LH-surge' which occurs in the pig about 40 h before ovulation. It is conceivable, however, t h a t two of the processes involved (resumption of meiosis by tile oocyte and synthesis of steroid hormones by the somatic tissues of the follicle), require different endocrine control mechanisms (see NEAL and BAKERS). The precise r61e played by F S H and Lift remains unknown and is difficult to study in vivo. The purpose of the present investigation was therefore to develop a suitable technique for maintaining porcine Graafian follicles in organ culture so t h a t the effects of gonadotrophic hormones administered during the phases of pre-ovulatory maturation could be assessed. If successful, these techniques would also provide a means of studying the interrelationships between the germinal and somatic components of the follicle. Materials and methods. 16 sexually mature pigs, either purebred Large Whites or Large White crosses maintained at the pig unit of the Edinburgh School of Agriculture, were used in this study. Their ovaries were removed during mid-ventral laparotomy at known stages of the oestrous cycle (on days 17 to 21). Approximately 80 follicles with antra, each follicle measuring 4 to 10 mm ill diameter, were cleanly dissected from the ovarian
tissue within 1 to 2 h of surgery. Some of the follicles were fixed for histology to serve as controls, while the remainder were set up for organ culture using one of the following procedures: a) the follicles were placed 'free floating' in culture medium within plastic Petri dishes containing Eagle's minimal essential medium supplemented with calf serum (20% v/v) and glutamine. Racks of 4 of these dishes (each with 2 to 5 follicles) were placed in modified 'Kilner' preserving jars (see BAKER and NEAL~), which were gassed at 0 to 0.703 kg/cm 2 with 5% CO 2 in either air or 55% oxygen; b) as for system (a), except t h a t the follicles were supported by lens tissue on a stainless steel grid, such that the follicle only received nutrients which diffused through the tissue ; c) method (b) was modified by the addition of a second piece of lens tissue which was draped over the follicle to act as a 'wick' which wetted the entire surface of the follicle with medium; d) the follicles, together with 2 ml of nutrient medium, were placed in roller culture tubes which were rotated at 4 rpm within the incubator. The same gas mixtures were used as in (a) above, but the tubes were not gassed at the high pressures.
1 p. NEALand T. G. BAKER, J. Endocr., in press (1974). 2 T. G. BAKERand P. NEAL,Biophysik 6, 39 (1969).
134
Specialia
Some of the cultures were s u p p l e m e n t e d w i t h insulin (50 ~g/ml), while others received gonadotrophins. The latter consisted of h u m a n chorionic g o n a d o t r o p h i n (HCG: ' P r e g n y l ' ; Organon Laboratories, London, at a dose of 1-10 IU/ml), N I H - F S H - S 9 (0.01-1.0 mg/ml), or N I H - L H $18 (0.01-1.0 mg/ml), either singly or in combination. The culture m e d i u m was c o m p l e t e l y replaced w i t h fresh n u t r i e n t on the 4th d a y of culture and the e x p l a n t e d follicles were fixed w i t h Bouin's aqueous fluid for histology
Fig. 1. 4 mm Graafian follicle cultured for 4 days using method (c) and with insulin in the nutritive medium. Ovary recovered on day 17 of the oestrous cycle. • 70.
Fig. 2. 8 m m follicle cultured 'free-floating' in m e d i u m c o n t a i n i n g HCG. The ooeyte, granulosa and theca h a v e cultured fairly well, b u t the cumulus cells are undergoing pyknosis. • 400.
EXPERIENTIA31/1
on t h e 8th day. The follicles were serially sectioned at 5-8 ~m and stained w i t h h a e m a t o x y l i n and eosin. Comparisons were m a d e of the histological appearance of the control (non-cultured) follicles and of those cultured according to the various procedures listed above. Results. Culture of Graafian follicles 'free floating' in the m e d i u m w i t h i n plastic P e t r i dishes or in t h e roller tubes (methods a and d) g a v e poor results: t h e s t r a t u m granulosum became d e t a c h e d f r o m the t h e c a i n t e r n s of the follicle, and b o t h layers showed v a r y i n g degrees of pyknosis. The oocyte and its surrounding cumulus cells consistently showed d e g e n e r a t i v e changes (Figure 2). B y the 8th d a y of organ culture, degeneration was adv a n c e d and t h e histology of the c u l t u r e d follicles was m a r k e d l y different from t h a t of t h e controls. The m o s t consistently good results were o b t a i n e d w h e n follicles were c u l t u r e d on stainless steel grids, especially w h e n lens tissue was draped over the follicle as in m e t h o d (c) above (Figure 3). F u r t h e r m o r e , gassing at 0.703 k g / c m 2 w i t h 5% C02 in 55% o x y g e n was more effective t h a n w i t h air at the same pressure (Figure 3). Follicles cultured under these conditions possessed few p y k n o t i c granulosa ceils and the s t r a t u m granulosum rarely showed m a r k e d signs of b e c o m i n g detached from the theca interns. H o w ever, a small localized area of damage, which seemed to occur in t h a t p a r t of the follicle wall which was in c o n t a c t w i t h t h e grid, was found in m o s t of t h e follicles: it was characterized by a t h i n n i n g of the granulosa layer and a mild degree of pyknosis. The oocytes in these follicles generally responded well to culture, and s o m e t i m e s progressed from the d i c t y a t e stages of meiotic prophase to m e t a p h a s e I or I I : by the 8th day m a n y had become mildly eosinophi!ic (Figure 4). The a d d i t i o n of insulin to t h e culture m e d i u m gave v a r i a b l e results, a l t h o u g h in general t e r m s it was considered beneficial. N o n e of t h e g o n a d o t r o p h i c hormones i m p r o v e d t h e histological appearance of the cultured follicles: t h e luteinization which was d e t e c t e d in some of t h e follicles seemed to be d e p e n d e n t u p o n the h o r m o n a l status of t h e animal p r o v i d i n g t h e follicles, r a t h e r t h a n on t h e a d d i t i o n of HCG, L H or F S H to t h e medium. Discussion. Most of t h e m e t h o d s of organ culture e m p l o y e d in the present s t u d y were unsuccessful, and the t r e a t e d follicles showed extensive signs of degeneration. H o w e v e r , those cultured at h i g h pressure w i t h 5% CO~ and 55% o x y g e n were well m a i n t a i n e d if supported b y stainless steel rafts and draped w i t h lens tissue. P y k n o t i c changes were largely confined to a small area of the wall of t h e follicle which had been in c o n t a c t w i t h the grid. The r e m a i n d e r of t h e granulosa and t h e c a layers, t o g e t h e r w i t h the oocyte within its cumulus, generally cultured well. The m e t h o d s used and the results obtained were t h u s strikingly similar to observations for sheep (MOOR et al. 3) and h u m a n ( B A K E R and N ~ A L a) follicles. The a p p a r e n t lack of a n y beneficial effect in v i t r o due to a d d i t i o n of g o n a d o t r o p h i e hormones was also recorded in these studies. H o w e v e r , M o o r et al. a, o b t a i n e d their best results w i t h sheep follicles r e m o v e d from animals at oestrus, or from those which had been p r e t r e a t e d w i t h P M S G prior to r e m o v a l of the ovaries for organ culture. W e h a v e n o t so far assayed the m e d i u m recovered at t h e end of t h e culture period for progesterone and t h u s are not able to confirm the observation t h a t levels of this h o r m o n e increase w h e n the follicle shows early signs of luteinization (see M o o r et al. 3). 3 R. M. ~/[OOR, M. F. HAY, J. E. A. )/~CINTOSH a n d t3. V. CALDWELL, J. Endocr. 58, 599 (1973).
4, T. G. t3AKERand P. N~AL, J. Anat. 117, in press (1974).
15. 1. 1975
Specialia
135
Our o b s e r v a t i o n t h a t t h e m o s t successful c u l t u r e s were o b t a i n e d w h e n t h e levels of o x y g e n were h i g h e s t is in k e e p i n g w i t h t h e results of THIBAULT a n d Gs ~. These a u t h o r s c u l t u r e d r a b b i t follicles w i t h a gas p h a s e of 5% CO~ in air a n d f o u n d t h a t t h e b e s t results were o b t a i n e d w i t h a p r e s s u r e of 5 to 10 b a r s (ca. 5.1 or 10.2 kg/cm2). Similar results h a v e b e e n o b t a i n e d b y BAKER a n d NEAL 6 for G r a a f i a n follicles o b t a i n e d f r o m m o u s e ovaries. The i n c r e a s e d gas p r e s s u r e m a y result in a h i g h p a r t i a l p r e s s u r e of dissolved o x y g e n while k e e p i n g t h e c o n c e n t r a t i o n in t h e gas p h a s e b e l o w t h e t o x i c level. If t h i s s u g g e s t i o n is true, it is p r o b a b l e t h a t we will n e e d t o use e l e v a t e d gas p r e s s u r e s if t h e p r e s e n t f i n d i n g s are t o be i m p r o v e d upon. N e v e r t h e l e s s , t h e results of t h e p r e s e n t s t u d y h a v e p r o v i d e d a t e c h n i q u e for m a i n t a i n i n g p o r c i n e G r a a f i a n follicles in o r g a n culture, a n d s t u d i e s c u r r e n t l y u n d e r w a y involve i n v e s t i g a t i o n s on t h e c o n t r o l of preo v u l a t o r y m a t u r a t i o n in b o t h t h e fluid a n d cellular c o m p o n e n t s of t h e follicle ~.
Fig. 3. Follicle recovered on day 18 and cultured for 4 days using method (e). Insulin and HCG were added to the medium. • 400.
Rdsumd. N o u s a v o n s 6tudi6 les follicules de G r a a f d a n s diff6rents m i l i e u x de c u l t u r e o r g a n i q u e classique, le b u t 6 v e n t u e l 6 r a n t de voir c o m m e n t le L H p r o v o q u e la reprise de la m6iose. Les follicules o n t 6t6 pr61ev6s de l ' o v a i r e de 16 t r u i e s se t r o u v a n t ~ diff6rents s t a d e s du cycle o e s t r i e n e t mis en c u l t u r e sous p r e s s i o n a u g m e n t 6 e (0.703 kg/cm~). E n g6n6ral, les couches de cellules et m ~ m e l ' o v o c y t e s u p p o r t 6 r e n t b i e n la m o d e de c u l t u r e d6crit. T. G. BAKER, 1~. H. F. HUNTER a n d P. ~EAL Department o/Obstetrics and Gynaecology, University of Edinburgh, Hormone Laboratory, Royal Infirmaryl Edinburgh EH3 9ER (Scotland) ; and School of Agriculture, University of Edinburgh (Scotland), 2d June 1974.
5 C. THIBAULT a n d M. GI~RARD, A n n l s Biol. a n i m . B i o c h i m , B i o p h y s . 73, 145 {1973). 6 T. G. BAKER a n d P. NEAL, in Oogenesis (Eds. J . D. BIGGERS a n d
A. W. SCHUETZ; University Park Press, Baltimore 1972), p. 377. The expenses incurred in this study were defrayed from a grant to T.G.B. and R.H.F.H. by the Wellcome Trust. We are also grateful to Messrs. J. P. HALL and M. PELT for assistance in the surgery.
Specialia
a) Tr~gergebundene AminosXuren. Als fester Tr~iger dient vernetztes Polystyrol. Die daran gebundene Aminoschutzgruppe ist ein BenzhydryloxycarbonylAnalogon, bei dem ein Phenylring durch den des Polystyrols ersetzt ist. Vernetztes Polystyrol wird benzoyliert 7, mit Natriumborhydrid zum Carbinol reduziertT, s und zum entsprechenden gemischten Kohlensh.urephenylester umgesetzt 9. Dutch Reaktion mit der entsprechenden Aminos~ture erh~tlt man das Derivat% Der Reaktionsverlauf wird auf jeder Stufe durch Elementaranalyse und I R - S p e k t r n m verfolg~. Die Beladung liegt bei 1,8-2,5 Aminos~iurereste pro 10 Monomereinheiten. b) Kupplungsreaktion. ]tquimolare Mengen yon Prolinpicolylester a0 und tr~tgergebundenes, an der Aminogruppe geschtitztes Alanin werden in D M F mit DCCI und Hydroxybenzotriazol ~t gekuppelt. Der Umsatz wird m i t der Durchflussanalyse verfolgt s. Wenn die Aminokomponente nicht vollst~indig verbraucht ist, wird nochmals eine entsprechende Menge des Alaninderivates zugesetzt. Nach beendeter Reaktion (fiber Nacht bei 0 ~ wird gewaschen, bis das Filtrat beim Eindampfen keine Rfickst~tnde mehr enth~lt und ninhydrin-negativ ist. c) Freisetzung des Pepfids. Sie erfolgt durch Zugabe yon 10%iger Trifluoressigs/iure ill Methylenchlorid. Nach 10 Min wird abfiltriert und mehrfach mit DMF gewaschen, his das Filtrat ninhydrin-negativ ist. Darauf wird das Filtrat im H o c h v a k n u m eingedampft und mit D MF aufgenommen. Die Abtrennung der Aminos~iure erfolgt durch Ionenaustausch an SE-Sephadex oder Gelpermeations-Chromatographie all Sephadex G-10 mit DNIF als Elutionsmittel. Hierauf kann die Kupplung mit der n~Lchsten trXgergebundenen Aminos~ure erfolgen. Nach vollendeter Synthese wird die Carboxylschutzgruppe dutch katalytische Hydrogenolyse abgespalten. Das Peptid ist chromatographisch rein ~2
133
Das Peptid wurde such unter Verwendung yon Poly~thylenglykolmonostearyl/~ther als Carboxylschutzgruppe ~ snythetisiert. Der einzige Unterschied zum geschilderten Verfahren ist die Abtrennung der freien Aminos~ture; sic erfolgt hier durch Ausf~llen des Peptidesters mit Athanol oder durch Ultrafiltration2.
Summary. A new strategy for peptide synthesis on polymeric supports is described. This alternating liquidsolid phase peptide synthesis procedure facilitates the ready separation of reacted and unreacted amino component at the end of each coupling reaction. Incomplete coupling reactions and incomplete deprotection reactions, the most serious problems in previously described methods of peptide synthesis on polymeric supports, have in this procedure no influence on the success of a synthesis. The principle is exemplified by the synthesis of the pentapeptide Gly-Val-Gly-Ala-Pro. H. FRANK und H, HAGENMAIER
Chemisches Institut der Universitiit, Au/ tier 1VIorgenstelle, D-74 Ti~bingen 7 (Bundesrepublik Deutschland, BRD), 6. September 1974.
7 p. RIVAILLE, A. ROBINSON, M. KAMEN und G. MILHAUD, Helv. chim. A c t a 5d, 2772 (1971). 8 G. L. SOUTHARD, G. S. BROOKE, J. ~V[. PETTEE, T e t r a h e d r o n Lett. 7969, 3505. - H. FRA~CK, D i s s e r t a t i o n Tiibingen (1973). 9 p. SIEBER und 13. IsELnr Helv. chim. A c t a 57, 622 (1968). ~0 R. CAMBLE, R. GARNER und G. T. YOUNG, J. chem. Soc. (C) 7969, 1911. 11 W. K6NIG und R. GEIGER, chem. Ber. 103, 788 (1970). 12 H. HAGEN~AIER und H. FRANK, J. Chromat. Sci. 70, 663 (1972).
S t u d i e s on t h e M a i n t e n a n c e of P o r c i n e G r a a f i a n F o l l i c l e s in O r g a n C u l t u r e I t is now well established for a variety of mammalian species t h a t the changes characteristic of pre-ovulatory maturation in the Graafian follicle are dependent upon an adequate supply of gonadotrophic hormones, and especially on the so called 'LH-surge' which occurs in the pig about 40 h before ovulation. It is conceivable, however, t h a t two of the processes involved (resumption of meiosis by tile oocyte and synthesis of steroid hormones by the somatic tissues of the follicle), require different endocrine control mechanisms (see NEAL and BAKERS). The precise r61e played by F S H and Lift remains unknown and is difficult to study in vivo. The purpose of the present investigation was therefore to develop a suitable technique for maintaining porcine Graafian follicles in organ culture so t h a t the effects of gonadotrophic hormones administered during the phases of pre-ovulatory maturation could be assessed. If successful, these techniques would also provide a means of studying the interrelationships between the germinal and somatic components of the follicle. Materials and methods. 16 sexually mature pigs, either purebred Large Whites or Large White crosses maintained at the pig unit of the Edinburgh School of Agriculture, were used in this study. Their ovaries were removed during mid-ventral laparotomy at known stages of the oestrous cycle (on days 17 to 21). Approximately 80 follicles with antra, each follicle measuring 4 to 10 mm ill diameter, were cleanly dissected from the ovarian
tissue within 1 to 2 h of surgery. Some of the follicles were fixed for histology to serve as controls, while the remainder were set up for organ culture using one of the following procedures: a) the follicles were placed 'free floating' in culture medium within plastic Petri dishes containing Eagle's minimal essential medium supplemented with calf serum (20% v/v) and glutamine. Racks of 4 of these dishes (each with 2 to 5 follicles) were placed in modified 'Kilner' preserving jars (see BAKER and NEAL~), which were gassed at 0 to 0.703 kg/cm 2 with 5% CO 2 in either air or 55% oxygen; b) as for system (a), except t h a t the follicles were supported by lens tissue on a stainless steel grid, such that the follicle only received nutrients which diffused through the tissue ; c) method (b) was modified by the addition of a second piece of lens tissue which was draped over the follicle to act as a 'wick' which wetted the entire surface of the follicle with medium; d) the follicles, together with 2 ml of nutrient medium, were placed in roller culture tubes which were rotated at 4 rpm within the incubator. The same gas mixtures were used as in (a) above, but the tubes were not gassed at the high pressures.
1 p. NEALand T. G. BAKER, J. Endocr., in press (1974). 2 T. G. BAKERand P. NEAL,Biophysik 6, 39 (1969).
134
Specialia
Some of the cultures were s u p p l e m e n t e d w i t h insulin (50 ~g/ml), while others received gonadotrophins. The latter consisted of h u m a n chorionic g o n a d o t r o p h i n (HCG: ' P r e g n y l ' ; Organon Laboratories, London, at a dose of 1-10 IU/ml), N I H - F S H - S 9 (0.01-1.0 mg/ml), or N I H - L H $18 (0.01-1.0 mg/ml), either singly or in combination. The culture m e d i u m was c o m p l e t e l y replaced w i t h fresh n u t r i e n t on the 4th d a y of culture and the e x p l a n t e d follicles were fixed w i t h Bouin's aqueous fluid for histology
Fig. 1. 4 mm Graafian follicle cultured for 4 days using method (c) and with insulin in the nutritive medium. Ovary recovered on day 17 of the oestrous cycle. • 70.
Fig. 2. 8 m m follicle cultured 'free-floating' in m e d i u m c o n t a i n i n g HCG. The ooeyte, granulosa and theca h a v e cultured fairly well, b u t the cumulus cells are undergoing pyknosis. • 400.
EXPERIENTIA31/1
on t h e 8th day. The follicles were serially sectioned at 5-8 ~m and stained w i t h h a e m a t o x y l i n and eosin. Comparisons were m a d e of the histological appearance of the control (non-cultured) follicles and of those cultured according to the various procedures listed above. Results. Culture of Graafian follicles 'free floating' in the m e d i u m w i t h i n plastic P e t r i dishes or in t h e roller tubes (methods a and d) g a v e poor results: t h e s t r a t u m granulosum became d e t a c h e d f r o m the t h e c a i n t e r n s of the follicle, and b o t h layers showed v a r y i n g degrees of pyknosis. The oocyte and its surrounding cumulus cells consistently showed d e g e n e r a t i v e changes (Figure 2). B y the 8th d a y of organ culture, degeneration was adv a n c e d and t h e histology of the c u l t u r e d follicles was m a r k e d l y different from t h a t of t h e controls. The m o s t consistently good results were o b t a i n e d w h e n follicles were c u l t u r e d on stainless steel grids, especially w h e n lens tissue was draped over the follicle as in m e t h o d (c) above (Figure 3). F u r t h e r m o r e , gassing at 0.703 k g / c m 2 w i t h 5% C02 in 55% o x y g e n was more effective t h a n w i t h air at the same pressure (Figure 3). Follicles cultured under these conditions possessed few p y k n o t i c granulosa ceils and the s t r a t u m granulosum rarely showed m a r k e d signs of b e c o m i n g detached from the theca interns. H o w ever, a small localized area of damage, which seemed to occur in t h a t p a r t of the follicle wall which was in c o n t a c t w i t h t h e grid, was found in m o s t of t h e follicles: it was characterized by a t h i n n i n g of the granulosa layer and a mild degree of pyknosis. The oocytes in these follicles generally responded well to culture, and s o m e t i m e s progressed from the d i c t y a t e stages of meiotic prophase to m e t a p h a s e I or I I : by the 8th day m a n y had become mildly eosinophi!ic (Figure 4). The a d d i t i o n of insulin to t h e culture m e d i u m gave v a r i a b l e results, a l t h o u g h in general t e r m s it was considered beneficial. N o n e of t h e g o n a d o t r o p h i c hormones i m p r o v e d t h e histological appearance of the cultured follicles: t h e luteinization which was d e t e c t e d in some of t h e follicles seemed to be d e p e n d e n t u p o n the h o r m o n a l status of t h e animal p r o v i d i n g t h e follicles, r a t h e r t h a n on t h e a d d i t i o n of HCG, L H or F S H to t h e medium. Discussion. Most of t h e m e t h o d s of organ culture e m p l o y e d in the present s t u d y were unsuccessful, and the t r e a t e d follicles showed extensive signs of degeneration. H o w e v e r , those cultured at h i g h pressure w i t h 5% CO~ and 55% o x y g e n were well m a i n t a i n e d if supported b y stainless steel rafts and draped w i t h lens tissue. P y k n o t i c changes were largely confined to a small area of the wall of t h e follicle which had been in c o n t a c t w i t h the grid. The r e m a i n d e r of t h e granulosa and t h e c a layers, t o g e t h e r w i t h the oocyte within its cumulus, generally cultured well. The m e t h o d s used and the results obtained were t h u s strikingly similar to observations for sheep (MOOR et al. 3) and h u m a n ( B A K E R and N ~ A L a) follicles. The a p p a r e n t lack of a n y beneficial effect in v i t r o due to a d d i t i o n of g o n a d o t r o p h i e hormones was also recorded in these studies. H o w e v e r , M o o r et al. a, o b t a i n e d their best results w i t h sheep follicles r e m o v e d from animals at oestrus, or from those which had been p r e t r e a t e d w i t h P M S G prior to r e m o v a l of the ovaries for organ culture. W e h a v e n o t so far assayed the m e d i u m recovered at t h e end of t h e culture period for progesterone and t h u s are not able to confirm the observation t h a t levels of this h o r m o n e increase w h e n the follicle shows early signs of luteinization (see M o o r et al. 3). 3 R. M. ~/[OOR, M. F. HAY, J. E. A. )/~CINTOSH a n d t3. V. CALDWELL, J. Endocr. 58, 599 (1973).
4, T. G. t3AKERand P. N~AL, J. Anat. 117, in press (1974).
15. 1. 1975
Specialia
135
Our o b s e r v a t i o n t h a t t h e m o s t successful c u l t u r e s were o b t a i n e d w h e n t h e levels of o x y g e n were h i g h e s t is in k e e p i n g w i t h t h e results of THIBAULT a n d Gs ~. These a u t h o r s c u l t u r e d r a b b i t follicles w i t h a gas p h a s e of 5% CO~ in air a n d f o u n d t h a t t h e b e s t results were o b t a i n e d w i t h a p r e s s u r e of 5 to 10 b a r s (ca. 5.1 or 10.2 kg/cm2). Similar results h a v e b e e n o b t a i n e d b y BAKER a n d NEAL 6 for G r a a f i a n follicles o b t a i n e d f r o m m o u s e ovaries. The i n c r e a s e d gas p r e s s u r e m a y result in a h i g h p a r t i a l p r e s s u r e of dissolved o x y g e n while k e e p i n g t h e c o n c e n t r a t i o n in t h e gas p h a s e b e l o w t h e t o x i c level. If t h i s s u g g e s t i o n is true, it is p r o b a b l e t h a t we will n e e d t o use e l e v a t e d gas p r e s s u r e s if t h e p r e s e n t f i n d i n g s are t o be i m p r o v e d upon. N e v e r t h e l e s s , t h e results of t h e p r e s e n t s t u d y h a v e p r o v i d e d a t e c h n i q u e for m a i n t a i n i n g p o r c i n e G r a a f i a n follicles in o r g a n culture, a n d s t u d i e s c u r r e n t l y u n d e r w a y involve i n v e s t i g a t i o n s on t h e c o n t r o l of preo v u l a t o r y m a t u r a t i o n in b o t h t h e fluid a n d cellular c o m p o n e n t s of t h e follicle ~.
Fig. 3. Follicle recovered on day 18 and cultured for 4 days using method (e). Insulin and HCG were added to the medium. • 400.
Rdsumd. N o u s a v o n s 6tudi6 les follicules de G r a a f d a n s diff6rents m i l i e u x de c u l t u r e o r g a n i q u e classique, le b u t 6 v e n t u e l 6 r a n t de voir c o m m e n t le L H p r o v o q u e la reprise de la m6iose. Les follicules o n t 6t6 pr61ev6s de l ' o v a i r e de 16 t r u i e s se t r o u v a n t ~ diff6rents s t a d e s du cycle o e s t r i e n e t mis en c u l t u r e sous p r e s s i o n a u g m e n t 6 e (0.703 kg/cm~). E n g6n6ral, les couches de cellules et m ~ m e l ' o v o c y t e s u p p o r t 6 r e n t b i e n la m o d e de c u l t u r e d6crit. T. G. BAKER, 1~. H. F. HUNTER a n d P. ~EAL Department o/Obstetrics and Gynaecology, University of Edinburgh, Hormone Laboratory, Royal Infirmaryl Edinburgh EH3 9ER (Scotland) ; and School of Agriculture, University of Edinburgh (Scotland), 2d June 1974.
5 C. THIBAULT a n d M. GI~RARD, A n n l s Biol. a n i m . B i o c h i m , B i o p h y s . 73, 145 {1973). 6 T. G. BAKER a n d P. NEAL, in Oogenesis (Eds. J . D. BIGGERS a n d
A. W. SCHUETZ; University Park Press, Baltimore 1972), p. 377. The expenses incurred in this study were defrayed from a grant to T.G.B. and R.H.F.H. by the Wellcome Trust. We are also grateful to Messrs. J. P. HALL and M. PELT for assistance in the surgery.
