Cytotechnology 8: 215-217, 1992. 9 1992KluwerAcademicPublishers. Printedin the Netherlands.

Evaluation of the physiological value of porcine luteal cells isolated in various stages of the luteai phase: Tissue culture approach E.L. Gregoraszczuk and A. Wojtusiak Laboratory of Endocrinology and Tissue Culture, Department of Physiology, Institute of Zoology, Jagiellonian University, 30-060 Krak6w, lngardena 6, Poland Received 6 May 1992; acceptedin original form 2 June 1992

Key words: porcine luteal cells, progesterone, in vitro model Abstract Porcine luteal ceils were collected from corpora lutea in four different stages of the luteal phase and cultured as monolayers. Progesterone (P4) secretion was assayed using radioimmunoassays (Gregoraszczuk, 1991). Luteal cells cultured from porcine corpora lutea collected in the early luteal phase maintained steroidogenic capacity for 6 days in culture until the time comparable with midluteal corpora lutea. Luteal ceils collected from mature and regressing corpora lutea did not dedifferentiate during 2 days of culture. After this time secretion of progesterone decreased to undetectable amounts characteristic of old corpora lutea. The regression in the culture progressed. The results demonstrate that the degree of the decline of progesterone depends on the type of corpus luteum, which is connected to particular time intervals of the luteal phase. Before starting experiments it is necessary to take into consideration the stage of the luteal phase from which the material is collected for culture. This study provides evidence that long term culture is useful for investigating a variety of aspects of luteal function only if cells are collected in the early luteal phase. Short term culture is suitable for investigation of cells collected from mid and late luteal phase. Regulation of luteal function is dependent on stage of the luteal phase.

Introduction Steroid secretion is the main feature of ovarian cells. It, however, reveals characteristic fluctuation during the specific time called the "life span". Corpus luteum is the compartment of the ovary whose life span is very strictly limited by numerous factors. The regular sexual cycle is dependent on the precision of the life span of corpus luteum. Thus the results obtained in vitro can be compared with in vivo situation only when luteal ceils in culture function in accordance with then physiological life span. Previous experi-

ments (unpublished) showed that luteal cells isolated from midluteal phase corpora lutea of pig do not dedifferentiate during 2 days in culture. The present investigation was undertaken to show: firstly to what extent luteal ceils harvested from 4 consecutive stages of the luteal phase of porcine corpus luteum maintain their secretory pattern typical for that particular time interval of the corpus luteum life span; secondly, whether the life span of luteal cells continues in culture similarly to in vivo and if so, for how long. In instances where the results obtained in vio'o appear to be comparable to that in vivo, then cul-

216 tured luteal cells collected at a strictly defined stage of the life span of a corpus luteum might be r e c o m m e n d e d as an in vitro model for studying reproduction.

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Materials and methods The ovaries were collected from pigs in a local slaughterhouse. Ovaries in 4 stages of the luteal phase were used: 1) developing corpora lutea early ( 0 - 3 days after ovulation); 2) developing corpora lutea - midway ( 5 - 8 days after ovulation); 3) mature corpora lutea ( 8 - 1 0 days after ovulation); and 4) regressing corpora lutea ( 12-14 days after ovulation). The corpora lutea from ovaries in particular stage of luteal phase were excised, decapsulated, minced with scissors and then pooled. The tissue was enzymatically dissociated according to the technique described earlier (Gregoraszczuk, 1983). The cells were grown as monolayers in Leighton tubes. The cultures were grown in quadruplicate in each of 3 experiments. After 0, 2, 4 and 6 days of culture, the medium was changed and frozen for further steroid analysis. Progesterone secretion was assayed using radioimmunoassay. For details see Gregoraszczuk, 1991. Statistical analyses Statistical differences were determinated by Student t-test.

Results a n d discussion The difference in contents of progesterone in suspension prior to culture (0 days) and after 2, 4 and 6 days of culture is shown in Fig. 1. The statistically significant increase of progesterone secretion after 2, 4 and 6 days of culture of luteal cells from corpora lutea of type 1 (CL 1) was observed (p < 0.001, p < 0.01, p < 0.05, respectively). Corpora lutea of type 2 secreted higher amounts of progesterone during 2 and 4 days of culture (p < 0.001 and p < 0.01, respec-

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Fig. I. Progesteronesecretion by culture of luteal cells isolated from corpora lutea of different stages of luteal phase. Developing corpus luteum - early CL 1, developing corpus luteum midway CL 2, mature corpus luteum CL 3, and regressing corpus luteum CL 4. * p < 0.05; ** p < 0.01, *** p < 0.001. tively). Corpora lutea of type 3 secreted a higher amount of progesterone only during first 2 days of culture (p < 0.05). After 4 and 6 days, progesterone secretion was lower than in suspension prior to culture. Corpora lutea o f type 4 secreted a very low amount of progesterone and no increase of progesterone secretion during the culture period was observed (Fig. 1). The data show that, in the case of corpora lutea ovarian component with strictly determined life span, it is necessary to take into consideration the part of the luteal phase in which the cells were obtained. Our previous data (Gregoraszczuk and Stoklosowa, 1978) showed that the cells harvested from the postovulatory corpora lutea exhibited the best growth in culture. The cells collected from old corpora lutea did not proliferate in culture as energetically as those from younger types of corpora lutea. Those observations and the present data are an agreement with H o y e r et al., (1988) who described steroidogenic capacity, cellular composition and ultrastructure of cells cultured from ovine corpora lutea collected early in the luteal phase (day 2 after ovulation), and maintained in culture until a time comparable with day 10 of midluteal phase. Kong et al., 1989 showed that ovine luteal cells collected shortly after ovulation and maintained in culture for a time period analogous to the luteal phase pos-

217 s e s s e d an a b i l i t y to r e s p o n d to l u t e o t r o p i c as w e l l as l u t e o l y t i c factors. In s u m m a r y , the e x p e r i m e n t s s h o w e d that p o r c i n e luteal c e l l s c o l l e c t e d in the e a r l y luteal p h a s e are g o o d m a t e r i a l for l o n g t e r m c u l t u r e , w h i l e c e l l s f r o m m i d a n d late luteal p h a s e are s u i t a b l e for short term culture.

Acknowledgements I arn g r a t e f u l to Prof. S. S t o k l o s o w a for h e r valuable comments, critical remarks and constant i n t e r e s t d u r i n g the c o u r s e o f this s t u d y . T h i s w o r k w a s s p o n s o r e d b y the S m a l l S u p p l y P r o g r a m m e f o u n d o f the W o r l d H e a l t h O r g a n i s a tion S p e c i a l P r o g r a m m e o f R e s e a r c h , D e v e l o p m e n t a n d R e s e a r c h T r a i n i n g in H u m a n R e p r o d u c tion, a n d Z - 12/91 K B N W a r s z a w a .

References Gregoraszczuk E and Stoklosowa S (1978) Growth pattern of the cultured porcine corpus luteum cells. Bulletin de L'academie Polonaise des Sciences XXVI, 8: 567-569. Gregoraszczuk E (1983) Steroid hormone release in cultures of pig corpus luteum and granulosa cells: Effect of LH, hCG, PRL and estradiol. Endocr. Exp. 17: 59-68. Gregoraszczuk E (1991) The interaction of testosterone and gonadotropins in stimulating estradiol and progesterone secretion by cultures of corpus luteum cells isolated in early and midluteal phase. Endocrinol. Japonica 38: 229-237. Hoyer PB, Kong W, Crichton EG, Bevan L and Krutzsch PM (1988) Steroidogenic capacity and ultrastructural morphology of cultured ovine luteal cells. Biol. Reprod. 38: 909-920. Kong W, Marion SL and Hoyer PB (1989) Luteotropic and luteolytic responsiveness of ovine luteal cells in long-term culture. Biol. Reprod. 41: 707-714.

Address for offprints: E.L. Gregoraszczuk, Laboratory of Animal Endocrinology and Tissue Culture, Department of Animal Physiology, Institute of Zoology, Jagiellonian University, 30-060 Krak6w, Ingardena 6, Poland.

Evaluation of the physiological value of porcine luteal cells isolated in various stages of the luteal phase: tissue culture approach.

Porcine luteal cells were collected from corpora lutea in four different stages of the luteal phase and cultured as monolayers. Progesterone (P4) secr...
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