ENDOCRINE REGULATIONS, VOL. 31, 41–46, 1997 PROGESTERONE, ANDROGEN AND ESTRADIOL PRODUCTION BY PORCINE LUTEAL CELL SUBPOPULATIONS: DEPENDENCE ON CELL COMPOSITION AND PERIODS OF LUTEAL PHASE E.L. GREGORASZCZUK Laboratory of Animal Endocrinology and Tissue Culture, Department of Animal Physiology, Institute of Zoology, Jagiellonian University, 30-060 Krakow, Ingardena 6, Poland The size and number small (Sc) and large (Lc) luteal cells and the steroids they secrete were determined in porcine corpora lutea (CL) collected on days 1-3, 8-10 and 14-16 of the estrus cycle.
The cells were separated with the aid of Ficoll linear gradient. The size of Sc increased in middle- luteal phase (MLP) in comparison with early luteal phase (ELP) and retained consistent value until the end of the luteal phase (LLP). Lc increased in size with advancing luteal phase, their number per CL being greater in MLP than in ELP but decreased in LLP. In contrast, the number of Sc decreased in MLP in comparison with ELP and did not change in LLP. At the initial luteal phase the majority of small cells did not show any histochemical reactivity for delta5-3ß-hydroxysteroid dehydrogenase which then increased with luteal phase progress. Lc collected during ELP showed 5.7 times higher androgen secretion than Sc. A decrease of androgen secretion by Lc was observed as the luteal phase progressed but it was three times higher in Sc than in Lc collected during LLP.
Both types of luteal cells produced estradiol even without the addition of substrates. Endogenous estradiol content was always higher in Lc than in Sc. Moreover, estradiol secretion by both cell types was higher in LLP than in MLP and ELP. These findings suggest that the view on granulosa origin of Lc and theca origin of Sc as raised by others may be oversimplified. In appears that Sc most likely undergo hypertrophy, but retain their prior phenotype and also that some Lc gradually dedifferentiate to Sc as the corpus luteum ages.
Key words: Porcine Corpus Luteum – Small and Large Luteal Cells – Steroid Secretion – Progesterone – Androgens – Estradiol – Tissue Culture As in a variety of species, pig corpora lutea con- Lc (CRAN 1983; ALILA and HANSEL 1984; FARIN et al.
sist of at least two morphologically distinguishable 1988) or that Lc gradually “dedifferentiate” to Sc luteal cell types, which are assumed to be transformed (FISCH et al. 1989; GREGORASZCZUK 1996) as the cor- from follicular granulosa and theca cells (LEMON and pus luteum ages. However, it remains unclear whether LOIR 1977; LEMON and MAULEON 1982; BUHR 1987; the conversion of Sc to Lc involves the differentia- GREGORASZCZUK 1990; GREGORASZCZUK 1995). Early tion into other cell types or merely the hypertrophy histologists concluded that large luteal cells (Lc) are of a cell which retains its prior phenotype.
of granulosa origin whereas small cells (Sc) arise In porcine preovulatory follicles the theca cells from the theca of the preovulatory follicles (HANSEL possess not only the ability to synthesize androgens, et al. 1973; O’SHEA et al. 1979). However, such view which are then aromatized by granulosa cells to appears to be oversimplified. There are several lines estradiol (BJERSING 1967), but also to secrete estradiol of evidence suggesting that either Sc grow to became in the absence of additional androgen substrate DIURNAL RHYTHM OF HORMONES IN ASTHMA (STOKLOSOWA et al. 1982), and they express P450 (c17) Steroid analysis: Progesterone, androgen and during follicular development (CONLEY et al. 1995).
estradiol were detected in the medium by radioim- Like the follicle from which it originated, the por- munoassay described elsewhere (STOKLOSOWA et al.
cine corpus luteum produces significant quantities of androgens, in addition to large amounts of both Progesterone assay: A highly specific antibody estrogens and progesterone (GREGORASZCZUK 1992).
raised in sheep against 11á-hydroxy-progesterone However, it remains to be clarified which specific hemisuccinate coupled to bovine serum albumin was cell population produces which steroids, and whether used. The cross-reaction with pregnenolone was 2.9 these cell populations functionally stay the same dur- %. All other tested steroids showed less than 1 % ing the entire luteal phase.
cross-reaction. [1,2,6,7-3 H] progesterone (Radio- chemical, Amersham, England, spec. act. 80 Ci/ Material and Methods mmol) was used as the tracer. The limit of assay sen- sitivity was 50 pg. The coefficients of variation within Animals: Pig ovaries were collected from a local and between assays were 15 % and 2.5 %, respec- abattoir. The luteal cells were isolated from the cor- pus luteum and assessed according to previously de- Androgen assay. Antiserum was induced in rab- scribed criteria (GREGORASZCZUK 1992).
Cell separation: Dissected corpora lutea were BSA as the antigen. The antisera exhibited 100 % enzymatically dissociated according to our own tech- binding of testosterone and cross-reacted 100 % with nique (GRAGORASZCZUK 1983). The cells were sus- 5α-androsterone, 20 % with dihydrotestosterone, 15.7 pended in M199 medium. Cell viability by the trypan % with delta -androstenedione, 3 % with dehy- blue exclusion test was 85 %. To separate the luteal droepiandrosterone and 7.4 % with androsterone. The cells into the populations based on size, dispersed cells tracer used was [1,2,6,7/3H] testosterone (Radio- were separated at unit gravity (CELSEP; Du Pont, chemical Centre, Amersham, England). The limit of France). The CELSEP system uses a continuous shal- assay sensitivity was 5 pg. The coefficients of varia- low density gradient. Solutions of 2 % and 4 % Ficoll tion within and between assays were 7.5 % and 9.5 in tissue culture medium containing 1 % serum were %, respectively.
used to prepare a linear gradient (GREGORASZCZUK Estradiol assay. A highly specific antibody 1996). After 2 h sedimentation, 25 ml fractions were against estradiol 17ß-6-oxime-BSA antigen was collected. Fractions with the same type of cells were raised in rabbits. It bound estradiol 100 % and gave pooled, washed four times with fresh medium and negligible cross-reactions with estrone (0.8 %), resuspended in culture medium (M199). After sort- estriol (0.8 %) and 16-keto-estradiol 17ß (1 %).
ing, the cells in each fraction were recounted with a All the other twelve tested steroids showed a cross- hemocytometer and cell viability was determined. The reaction level lower than 0.001 %. The tracer em- cells were plated (24-well plate, Nunc) in 1 ml per ployed was [2,4,6,7,16,17-3H] estradiol (Radio- well of fresh M199 medium containing 10 % calf se- chemical Centre, Amersham, England, spec. act rum and incubated at 37 °C (95 % air/5 % CO , 100 % 140 Ci/mmol). The limit of assay sensitivity was 5 humidity) overnight (16 h). Cell culture densities and pg. The coefficients of variation within and be- normalized steroid concentrations were based on the tween assays were less than 7.5 % and 8 %, re- number of viable cells. The suspensions of the iso- lated cells were submitted to the histochemical test Statistical evaluation: All data points are ex- for the activity of 3ß-hydroxysteroid dehydrogenase pressed as means ± S.E. from at least three different (delta5, 3ß-HSD), (FISCHER and KHAN 1972) using experiments (n=3) each in triplicate, resulting in at dehydroepiandrosterone (DHA) as substrate, NAD as least nine observations. Significant differences be- cofactor, and nitrotetrazolium blue (NBT) as hydro- tween the concentrations of steroids in large and small gen acceptor. The cells which showed the presence of luteal cells and the time of the luteal phase were evalu- formazan granules after the reaction were identified ated by analysis of variance followed by Duncan’s as steroidogenic ones.
new multiple range test.
DIURNAL RHYTHM OF HORMONES IN ASTHMA The percentage representation of individual cell types after the sedimentation has been described else- where (GREGORASZCZUK 1996). The data was based on the counts of large and small luteal cells, as cat- egorized by cell diameter, after enzymatic dispersion of luteal tissue on various days of the luteal phase.
The small steroidogenic luteal cells increased in size in middle luteal phase and remain of consistent size to the end of the luteal phase, i.e. 10-20 µm; 20- 25 µm; 20-25 µm in early (ELP), middle (MLP) and late (LLP), luteal phase, respectively (Fig. 1b), while large cells increased in size with advancing luteal age (20-30 µm, 30-40 µm and 40-45 µm in ELP, MLP and LLP, respectively (Fig. 1a).
The number of large steroidogenic cells per CL was greater in the MLP (4.5 x 105) than in the ELP (3.5 x 105), and decreased in LLP (2.9 x 105) (Fig.
1a).However, the number of small steroidogenic luteal cells decreased in MLP in comparison with ELP and did not change in LLP (6.0 x 106 in ELP; 3.0 x 106 in MLP and 2.35 x 106 LLP) (Fig. 1b). At ELP, a major- ity of small cells showed no enzymatic activity and the reactivity for the delta5, 3ß-HSD activity remark- ably increased with luteal phase progresses (0.5 x 106, 2.5 x 106 and 4.7 x 106 cells in ELP, MLP and LLP, respectively).
Progesterone secretion: No significant changes in basal progesterone production by cells collected from the early and late luteal phases by either small or large cells were observed, but large cells secreted 10 times more progesterone than small cells. The highest secretion of progesterone was noted at MLP.
Large cells collected from this type of corpus luteum secreted 20 times more progesterone than small cells collected at the same luteal phase (Fig. 2a).
Androgen secretion: Androgen secretion ex- pressed per 105 cells was 5.7 times higher in the large cells collected during ELP than in the small cells. A decrease of androgen secretion by large cells was observed as the luteal phase progressed (83.1, 28.4 Fig. 1 Cell diameter (µm) and cells numbers expressed as cells/CL of (A) large, (B) small and (C) nonsteroidogenic cells and 19.5 pg/105 cells in ELP, MLP and LLP, respec- (NS) obtained from porcine CL at 1-3; 8-10 and 14-16 days tively). On the other hand, androgen secretion by of the luteal phase. Values are means ± SEM of total cells at small cells collected during LLP was 3 times higher each stages for 3 experiments. Means significantly different than that by large cells (Fig. 2b).
(P < 0.05) from each other are designated by different letters.
DIURNAL RHYTHM OF HORMONES IN ASTHMA Estradiol secretion: Both types of cells produced estradiol without any addition of substrates. Endog- enous estradiol-17ß content expressed per 105 cells was always higher in the large cells than in small ones.
Moreover, estradiol secretion by large and small cells was higher in LLP than in MLP and ELP (Fig. 2c).
Basal estradiol secretion by large cells isolated at LLP was 1.1 times and 1.3 times higher than that from large cells at MLP and ELP, respectively. Basal estradiol secretion by small cells collected at LLP was 1.5 times and 1.6 times higher than that from the same type of cells at MLP and ELP, respectively (Fig.
Although the origin of the two luteal cell populations is uncertain, a well known hypothesis is that bovine large luteal cells are derived from granu- losa cells, while small cells are of theca interna ori- gin (ALILA and HANSEL 1984). However, steroid syn- thesis (androgen and estradiol) during the entire luteal phase by the two cell types does not support this theory of their follicular origin. Actually, the finding that both small and large luteal cells secrete andro- gen and estrogen does not support the above hypoth- esis as suggested for bovine luteal cells.
Pig corpora lutea differ from ovine and bovine ones in that they produce, in addition to progesterone, also androgen (GREGORASZCZUK 1992) and estradiol (GREGORASZCZUK 1983, 1992; PRZALA et al. 1984). It has been suggested earlier (13,14) that the theca-de- rived cells secrete androgen, whereas granulosa-de- rived cells aromatize androgen to estradiol. However, our present results indicate that, whereas both types of cells secreted androgens, large cells collected from early luteal cells secreted 5.5 times more androgen than small cells did. Thus, these data suggest that in corpora lutea hemorrhagica (1-3 days after ovulation), large cells are probably theca-origin cells or undif- ferentiated theca cells which retain their original phe- notype. STOKLOSOWA et al.(1978) showed that theca interna cells isolated from preovulatory follicles are larger (17-23 µm in diameter) than granulosa cells Fig. 2 (A) progesterone, (B) androgen, (C) estradiol secretion isolated from the same follicles (10-14 µm in diam- by small and large luteal cell subpopulations obtained at the eter). The ability of large cells to secrete androgen early (ELP) mid – (MLP) and late (LLP) luteal phases decreased as the luteal phase progressed. In the late expressed per 105 cells. Means ± SEM for 3 experiments (in triplicate) for each stage. Means significantly different (P < luteal phase, small cells are the source of androgen 0.05) from each other are designated by different letters.
secretion. It is also possible that large cells “dedif- DIURNAL RHYTHM OF HORMONES IN ASTHMA ferentiate” to small luteal cells with advancing luteal plain the differences in experimental findings. Moreo- age. In the mid-luteal phase, when androgen secre- ver, the stages of the luteal phases used to by BUHR tion by large cells decline, an increase of secretion of (1987) differ from those used in the present study. In estradiol by them was noted. GREGORASZCZUK (1996) this experiment we used corpora lutea hemorrhagica showed that both cell types were capable of estradiol (1-3 days after ovulation). In such a short time after production without the addition of substrates. The ovulation, GREGORASZCZUK and WOJTUSIAK (1982) content of endogenous estradiol was always higher identified four types of luteal cells based on two pa- in large cells than in small ones and was higher in rameters, e.g. the size and 3ß-HSD activity. TAYLOR LLP than in MLP.
et al. (1987) also identified four types of luteal cells Detailed morphometric analysis of the porcine in the porcine corpus luteum based on steroid and corpus luteum showed that small cells increased in relaxin synthesis capacity. It is most likely that small size but decreased in number in MLP and remain of cells undergo hypertrophy but retain their prior phe- consistent size and number until the end of the luteal notype, and that some large cells gradually differen- phase. The number of large cells expressed as cell/ tiate to small cells as the corpus luteum ages. The CL was greater in MLP then in ELP, so it is possible observed decrease in androgen secretion by large cells that some small cells grow to become large cells as and the increase of androgen secretion by small cells the luteal phase progresses. This would accord with in LLP strongly support this hypothesis which, how- the data by LEI et al. (1991) in cows and by O’SHEA ever, should be subjected to further confirmation.
et al. (1986) and FARIN et al. (1986) in sheep. The number of large cells decreased at the end of the luteal phase resulting in a progressive increase in the small nonsteroidogenic cell. Consistent with the hypoth- The author would like to thank Prof. Stanislawa esis that small cells become large cells, the treatment Stoklosowa, M.D., for her constant interest and Mgr.
of sheep with LH from day 5 to 10 of the cycle re- M. Duda for RIA. This work was supported by the sulted in the increase of the number of large cells as grant KBN 6PO4C 11208/95 as well as by the SMA compared with untreated controls (FARIN et al. 1988).
fund of the World Health Organization, Special Pro- However, SWALL et al. (1986) showed in sheep that gramme of Research Development and Research the ratio of the number of large/small cells increased Training in Human Reproduction.
as the luteal phase progressed, suggesting that luteolysis is associated with a preferential loss of small cells. On the other hand, (BRANNIAN and STOUFFER 1991) working with monkey luteal cell ALILA HW, HANSEL W: Origin of different cell types in the subpopulations at different stages of the menstrual bovine corpus luteum as characterized by specific cycle, showed that the ratio of large/small cells tended monoclonal antibodies. Biol Reprod 31, 1015– to decline as the cycle progressed.
So far morphometric studies of cell populations BJERSING L: On the morphology and endocrine function during the entire luteal phase in porcine corpus lu- of granulosa cells in ovarian follicles and corpora lutea: Biochemical, histochemical and ultrastruc- teum have not been reported. The only data (BUHR tural studies on porcine ovary with special refer- 1987) showed that the number of large cells per gram ence to steroid hormone synthesis. Acta of CL did not differ between days 10,16 and 18, but Endocrinol 125, 1–23, 1967 the number of small cell per gram of CL declined BRANNIAN JD, STOUFFER RL: Progesterone production by after day 15. Presented results showed that during monkey luteal cell subpopulations at different the luteal phase the number of luteal cells recovered stages of the menstrual cycle: Changes in ago- from porcine corpus luteum varied.
nist responsiveness. Biol Reprod 44, 141–149, Actually, there are marked differences between the laboratories in the techniques used to disperse and BUHR MM: Effect of lipoproteins and luteinizing hormone isolate cell subpopulations and also in the classifica- on progesterone production by large and small luteal cells throughout the porcine estrus cycle. J tions of small and large luteal cells which could ex- Anim Sci 65, 1027–1032, 1987 DIURNAL RHYTHM OF HORMONES IN ASTHMA CONLEY AJ, KAMINSKI MA, DUBOWSKI SA, JABLONKA–SHARIFF LEI ZM, CHEGINI N, RAO CV: Quantitative cell composi- A, REDMER DA, REYNOLDS LP:Immunohistochemical tion of human and bovine corpora lutea from vari- localization of 3ß-hydroxysteroid dehydrogenase and ous reproductive states. Biol Reprod 44, 1148– P450 hydroxylase during follicular and lutea de- velopment in pigs, sheep and cows. Biol Reprod 52, LEMON M, LOIR M: Steroid release in vitro by two luteal cell types in the corpus luteum of the pregnant CRAN DG: Follicular development in the sheep after prim- sow. J Endocrinol 72, 351–359, 1977 ing with PMSG. J Reprod Fertil 67, 415–423, LEMON M, MAULEON P: Interaction between luteal cell types from the corpus luteum of the sow in progester- FARIN CE, MOELLER CL, SAWYER HR, GAMBONI E, one synthesis in vitro. J Reprod Fertil 64, 315– NISWENDER GD: Morphometric analysis of cell types in the ovine corpus luteum throughout the O’SHEA JD, CRAN DG, HAY MF: The small luteal cell of estrus cycle. Biol Reprod 35, 1299–1308, 1986 the sheep. J Anat 128, 239–243, 1979 FARIN CE, MOELLER CL, SAWYER HR, GAMBONI E, O’SHEA JD, RODGERS RJ, WRIGHT PJ: Cellular composi- NISWENDER GD: Effect of luteinizing hormone and tion of the sheep corpus luteum in the mid- and human chorionic gonadotropin on cells popula- late luteal phase of the estrus cycle. J Reprod Fertil tion in the ovine corpus luteum. Biol Reprod 38, 76, 685-691, 1986 PRZALA J, WIESAK T, GRAZUL A, CIEPLINSKA E: The effect FISCH B, MARGARA RA, WINSTON MR, HILLIER SG: Cellu- of prolactin on estradiol 17ß- and testosterone lar basis of luteal steroidogenesis in the human plus 5-dihydrotestosterone secretion by porcine ovary. J Endocrinol 122, 303–309, 1989 luteal cells in vitro. Clin Endocrinol 83, 334- FISCHER TV, KHAN RH: Histochemical studies of rat ovarian follicular cells in vitro. In Vitro 7, 201–205, 1972 STOKLOSOWA S, BAHR J, GREGORASZCZUK EL: Some mor- GREGORASZCZUK EL, WOJTUSIAK A: Histochemical evalua- phological and functional characteristic of cells tion of delta5, 3ß-OHSD activity in two types of of the porcine theca interna in tissue culture. Biol porcine corpora lutea and granulosa cells in tis- Reprod 19, 712–719, 1978 sue culture. Acta Histochemica 70, 22–30, 1982 STOKLOSOWA S, GREGORASZCZUK EL, CHANNING CP: GREGORASZCZUK EL: Steroid hormone release in cultures Estrogen and progesterone secretion by isolated of pig corpus luteum and granulosa cells. Effect cultured porcine thecal and granulosa cells. Biol of LH, hCG, PRL and estradiol. Endocrin Reprod 26, 943–952, 1982 Experimentalis 17, 59–63, 1983 SWALL RH, GAMBONI F, MAYAN MH, NISWENDER GD: GREGORASZCZUK EL: Different response of porcine large Changes in the distribution of sizes of ovine luteal and small luteal cells to PRL in terms of proges- cells during the estrus cycle. Biol Reprod 34, 911– terone and estradiol secretion in vitro. Exp Clin Endocrinol 96, 234–237, 1990 TAYLOR MJ, CLARK CL, FRAWLEY LS: Evidence for the GREGORASZCZUK EL: Interrelations between steroid hor- existence of a luteal cell types that is steroidog- mone secretion and morphological changes of enic and release relaxin. Proc Soc Exp Biol Med porcine corpora lutea at various periods of luteal 185, 469–473, 1987 phase. Endocrine Regulations 26, 189–194, 1992 GREGORASZCZUK EL: Effect of FSH on progesterone se- Corresponding author: cretion by porcine larger and small luteal cells E.L. Gregoraszczuk isolated from early–developing corpora lutea.
Laboratory of Animal Endocrinology Exp. Clin Endocrinol 103, 272–274, 1995 and Tissue Culture GREGORASZCZUK EL: Large and small cells of the porcine corpus luteum: Different capacity to secrete Department of Animal Physiology estradiol and aromatize exogenous androgen dur- Institute of Zoology ing mid- and late luteal phase. Exp Clin Jagiellonian University Endocrinol 104, 278–283, 1996 Ingardena 6, 30-060 Krakow, Poland HANSEL W, CONCANNON PW, LUKASZEWSKA JH: Corpora lutea of the large domestic animals. Biol Reprod Accepted: December 15, 1996 8, 222–228, 1973

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Martin howe qc: curriculum vitae and practice summary

Martin Howe QC: Curriculum Vitae and Practice Summary MARTIN HOWE Q.C. 8 New Square, Lincoln's Inn London WC2A 3QP Tel: 020 7405 4321 Fax: 020 7405 9955 Chambers website: SUMMARY OF PROFESSIONAL PRACTICE Chambers of Mark Platts-Mills QC Called to the Bar: 1978. Appointed Queen's Counsel: 1996.