Research Centre “Sigra” Faculty of Veterinary Medicine

Latvian University of Agriculture

Institute of Anatomy and Antropology Rīga Stradiņa University

Regīna Tamane

Investigation of the morphofunctional changes of the corpus luteum of ovaries in physiological

processes in the cow and of morphofunctionality of cow’s ovarian cysts

Summary of the promotion work

Rīga-Jelgava-Sigulda 2003

2

The promotion work was carried out in the Research Centre “Sigra” of the Faculty of Veterinary Medicine the Latvian University of Agriculture and at the institute of Anatomy and Anthropology of the Rīga Stradiņa University. Scientific supervisors of the promotion work: Aleksandrs Jemeļjanovs, Dr.med.vet., Dr.habil.agr., Full Member of Latvian Academy of Sciences, Vice-President of Latvian Agricultural and Forest Academy of Sciences, Director of Research Centre “Sigra” of Latvian University of Agriculture, professor; Māra Pilmane, Dr.med., Dr.habil.med., Corresponding Member of Latvian Academy of Sciences, Director of the Institute of Anatomy and Anthropology of Rīga Stradiņa University, professor. Reviewers of the promotion work: Dr. habil. vet med. Zigmunds Brūveris Dr. med., dr. habil. biol. Velta Ose-Klinklāva Dr. vet. med. Inese Zītare The defence of the thesis will take place in the Faculty of Veterinary Medicine at Latvian University of Agriculture on 30 January 2004 at 11.00 o’clok. Address – Nr. 8 Helmaņa Str., Jelgava.

3

INTRODUCTION The reproductive problems in the productive animals are still a current issue throughout the world as they cause economic losses. It is important to investigate morphology and functionality of ever organ of the reproductive system to be able to find out the losses associated with the reproduction problems. It would give an opportunity to understand the mechanism of the possible disturbances and to control them more effectively improving the animal reproductive ability. In the regulation of the reproductive system a great role is played by ovaries and their functional structures, the most important of which is the ovary corpus luteum. It should be considered as a biological clock that regulates the reproductive cycle, but in early stages of pregnancy it protects the foetus providing Favourable conditions in the uterus. The structure and physiology of the corpus luteum of ovaries in the cow has been of interest of scientists since early time. Evidences are show by investigations in early last century (McNutt, 1924; 1926). Morphology of the corpus luteum both macroscopic and microscopic and even electronmicroscopic has been investigated a lot applying different methods, however, every time scientists find something new about this unusual endocrine organ. So, Okuda (1982) in his investigation initially described at least 6 types of cells in the corpus luteum. Later Okuda (1988) mentioned large and small luteal cells, which were described in details some times later by Fields (1996) using the electronmicroscopy. Macroscopic indices as well – the size and volume of the corpus luteum – initially were determined in slaughtered animals (Edvarts, 1965; Grunert, 1982), but in the latest investigations by using ultrasonography (Kähn, 1991; Singh, 1997). However, it is not enough to have only morphological investigations to be able to understand completely the corpus luteum function. It is also necessary to investigate the corpus luteum morphofunctionality. Not only that functionality is important which the pituitary hormones determine, but also by the morphofunctional activity and interaction of the corpus luteum cells. A hypothesis that hormones produced by the corpus luteum cells have only a local effect affecting development, functions and life cycle of corpus luteum, was advanced already in the 1970’s (Knobil, 1973). This hypothesis was proved in the 1990’s when a succession of investigations demonstrated that any hormonal activity is associated with its receptors in the target cells. The corpus luteum produced hormone progesterone activity is also associated with its receptors. Thus, the presence of the progesterone receptors in the corpus luteum cells shows the morphofunctional activity of its cells. Not only the life cycle of the corpus luteum, but also the further procedure of normal reproductive cycle and successful pregnancy depend on the morphofunctional activity of exactly these luteal cells

4

Statement of the problem.

Many investigations have been carried out on the activity of the corpus luteum, mainly in primates (Duffy et al., 1994; 1995; 1997; Hild-Petito et al., 1997; Misao et al, 1998; Ottander et al., 2000) and rats (Park et al., 1991; 1995; Rothchild, 1996; Teleria et al., 1999). The expression of progesterone receptor and mRNS expression of progesterone receptors in the corpus luteum cells has been studied in these investigations. However, equivalent investigations on the corpus luteum in productive animals are few (Pate et al., 1996; Rae et al., 1998 a, b; Rueda et al., 2000). Thus, summarising the carried out work, it becomes evident that:

1) It is necessary to find out the changes of morphofunctional activities of the cyclical and pregnancy corpus luteum of the ovary in cows during the reproductive cycle and pregnancy; unfortunately, there is lack of such investigations;

2) There are few investigations dealing with the changes of macroscopic parameters and histological structure of the pregnancy corpus luteum of the ovary in cows during the course of pregnancy;

3) There are no investigations of the association of the changes of macroscopic parameters and histological structure of the pregnancy corpora lutea with their morphofunctional activity in cows, but such data could correlate;

4) There are no investigations of morphofynctional activity of the luteal cyst cells.

Goal of the work. To investigate morphafunctional changes of the ovary corpus luteum during the reproductive cycle and pregnancy in cows and to investigate morphofunctionality of the ovary cysts in the cow.

Task of the work. 1. Determine the developmental stages of the cow ovary pregnancy corpus

luteum and cyclical corpus luteum; 2. Describe macroscopic and microscopic morphology of the cow ovary

pregnancy corpus luteum, cyclical corpus luteum and cysts tissues; 3. Analyse the expression of luteal cells of the ovary corpora lutea and luteal

cysts in the cow by using histochemical and immunohistochemical reactions;

5

4. Try to find correlation between the expressions of progesterone receptors and mRNS, i. e., determine the cell morphofunctional activity;

5. Perform a comparative characterisation of the cyclical and pregnancy corpus luteum morphofunctionality;

6. Characterise the dynamics of the expression of progesterone receptors and mRNS during pregnancy, reproductive cycle and cystic degeneration.

It was essential to make out the morphofunctional activity changes of the cow ovary corpus luteum during the reproductive cycle and pregnancy. For better understanding of these changes, it was necessary to investigate the changes of the corpora lutea macroscopic parameters and histological structures during the course of reproductive cycle and pregnancy. This investigation includes a histological evaluation of the cow’s ovary corpus luteum tissue in pregnancy and in the cycle as well as a comparison of macroscopic indices and morphofunctional activity of the both types of the corpora lutea.

Novelty of the results.

1) In Latvia an extensive investigation of the ovary corpus luteum and ovary cysts in the Latvian brown cows has been carried out for the first time, comprising material of 24 slaughtered non-pregnant cows, 37 pregnant cows and 7 cows with ovary cystic degeneration.

2) Steroid hormone receptors have been determined in veterinary medicine for the first time in Latvia using the immunhistochemical method of biotin- streptavidin complex.

3) The investigation contains characterisation of the morphofunctionality both of the cyclic corpus luteum and pregnancy corpus luteum throughout the length of the life cycle of the corpus luteum that ensures an opportunity to evaluate differences between these glands.

4) Investigation of regression of the cyclic corpus luteum and pregnancy corpus luteum in cows is important that demonstrates essential differences of these corpora lutea morphofunctional activity.

5) Important is also the discovery that morphofunctional activity of the luteal cyst cells may not lag behind in morphofunctional activity of the cyclic corpus luteum cells.

6) The novelty of this research is correlation of macroscopic indices of bovine ovary corpora lutea with morphofunctional activity of these corpora lutea as well as the evaluation of the cystic tissue morphofunctional activity.

6

Approbation of the results of investigation.

The results of investigation have been presented in the following conferences: 1. International scientific conference “Current Issues in Veterinary

Medicine”, Jelgava, 2000, 29 September. Oral report “ Morphofunctional characterisation of the ovary corpus luteum in cattle during the reproductive cycle”;

2. 4th Congress, of the World Latvian Physicians, Rīga, 2001, 20-22 June. Poster report “Morphofunctional characterisation of the ovary corpus luteum during the pregnancy and cystic degeneration”;

3. 2nd Congress of the World Latvian Scientists, Rīga, 2001,14 - 15 August. Poster report “Morphofunctional characterisation of the ovary corpus luteum during the pregnancy”;

4. EAAP 52nd Congress in Budapest, Hungary, 2001, 26. – 29. August. Poster report “Morphofunctional Characterisation of bovine corpus luteum (CL) graviditationis”;

5. 4th International Conference on Farm Animal Endocrinology, Salsomaggiore, Parma, Italy, 2001, 7. – 10. October. Poster report “Expression of progesterone receptors and mRNA in cows corpus luteum during oestrus cycle”;

6. Scientific conference of residents organised by the Medical Continuing Education Institute of the University of Latvia, Riga, Skolas 1a, 2002, 28. February. Oral report “Expression of progesterone receptors in the corpus luteum of cows”;

7. 97 Versamlung der Anatomischen Geseltschaft, Halle, Deuchland, 22 Bis 25 Marz 2002. Poster report “Correlation between morphofunctional activity and morphofunctional parameters in bovine corpus luteum during oestrus cycle”;

8. Meeting of the Clinical Integrated Morphology Association, Rīga, Kronvalda bulv. 9, 2002, 6. June. Oral Report “’Morphofunctional characterisation of the ovary cyclic corpus luteum and pregnancy corpus luteum in cattle”;

9. World Veterinary congress 27th, Tunisia, 2002, 25. – 29. September. Poster report “Expression of progesterone receptors in tissue of cows ovarian cysts”;

10. Medical field scientific conference at Rīga Stradiņš University, Rīga, Dzirciema 16., 2003, 7. Mart. Poster report: “ Morphofunctionality of cows ovaries cysts”

11. The 6th Baltic-Nordic workshop in Veterinary Anatomy, Kaunas, June 19.-20., 2003. Poster report: “Regression of cow’s corpus luteum graviditationis”;

12. The Profesor Gaston Victor Backman scientific conference, Rīga, Oktober 3. – 4., 2003. Poster report: “The bovine corpus luteum during pregnancy”.

7

The research has been carried out in cooperation with the Research Centre “Sigra” of the Latvian University of Agriculture (director-prof. A. Jemeļjanovs), Department of Histology of the Faculty of Medicine of the University of Latvia (from 1999 to 2001; head- prof. M. Pilmane) and the Institute of Anatomy and Anthropology of the Rīga Stradiņa University (from 2001 to 2003; director- prof. M. Pilmane). The thesis comprises 115 pages with an annotation in Latvian, annotation in English, introduction, literature description, material and methods, results, discussion, novelty of the results, conclusions, references, annex.

MATERIAL AND METHODS

Material

The material for investigation was obtained from ovaries of cows as soon they where slaughtered. In all, 68 ovaries of the slaughtered cows and heifers were used. Determining the uterus content and structure within the ovaries of the slaughtered animals, all the obtained material was divided as follows:

1) 37 ovaries of pregnant cows and their pregnancy corpora lutea; 2) 24 ovaries of non-pregnant morphologically healthy cows and their

cyclical corpora lutea; 3) 7 cow ovaries with pathological lesions- ovary cystic degeneration.

There not include in the investigation cows with the macroscopically established changes in the endometrium, with the exudation in the cavity of uterus, with the mummification of the foetus. Pregnancy corpora lutea In order to determine precisely the month of pregnancy the following schemes of detection of the foetus age were used: Afanasjevs (1983) based on morphological signs, and Lommetz (1986) based on the skeleton development of the foetus. Determining the foetus age, the obtained pregnancy corpora lutea were arranged in the following way: • from the 1st to the 2nd month of pregnancy-4 pregnancy corpora lutea; • from the 2nd to the 3rd month of pregnancy-2 pregnancy corpora lutea; • from the 3rd to the 4th month of pregnancy-14 pregnancy corpora lutea; • from the 4th to the 5th month of pregnancy-9 pregnancy corpora lutea; • from the 5th to the 6th month of pregnancy-6 pregnancy corpora lutea; • from the 6th the 7th month of pregnancy-1 pregnancy corpus luteum; • from the 7th to the 8th month of pregnancy-1 pregnancy corpus luteum.

8

Cyclical corpora lutea The obtained material of cyclical corpora lutea was grouped according to the stages of luteal phases. It was made using both macroscopic (Stock,1984; Okuda et al., 1984; 1988) and microscopic (Horstmann et al., 1973; Peukert- Adam, 1981; Okuda, 1984) evaluation criteria. The decision is shown in Table 1. Table 1. Stages of cyclical corpora lutea

N of CL

Macroscopical classification

N of CL

Microscopical classification

N of CL Final stage

3 AF 3 AF 3 AF

2 VF 2 VF 5 VF

3 VF 13 N 6 N 4 AR

6 N

2 AR 6 AR

6 PR 4 PR 4 PR N of CL –number of cyclical corpora lutea; AF –early formation; VF - late formation; N –mature CL; AR – early regression; PR – total regression. Ovary cysts For the classification of bovine ovary solitary cysts the thickness of the cyst wall was measured in the thinnest place of its cross-section. According to these measurements, 5 of the obtained cysts were luteal cysts but 2 of them were follicular cysts.

Methods For the characterisation of corpora lutea macroscopic parameters (cm) – linear dimensions were used: 1) the widest place perpendicularly to the ovary – length of the corpus

luteum; 2) the widest part parallel to the ovary – width of the corpus luteum; 3) as well as the body volume which was determined using the immersion

method (ml).

1. Methods of histological examination From each sample 9 histological sections were taken and haemotoxylin – eosin stained, as well as used for 2 histological examinations.

9

Totally 420 histological preparations were prepared. The corpora lutea obtained from the slaughtered animals were placed into a fixative consisting of 2% formaldehyde and 0.2 % picric acid in 0.1 M phosphate-buffered saline (pH 7.2) to cover the tissue in the volume ratio 5:1. Tissue specimens were kept in the fixative for 5 days. For rinsing the fixative solution out of the tissue a thyroid buffer solution (phosphate buffer + sacharose, pH 7) was used and every 24 h was changed. During the period of rinsing tissue specimens were kept in the temperature of 0◦ – +5◦ C. After that the tissue specimens were imbedded into paraffin. The blocks of paraffin imbedded bovine corpora lutea were cut with a microtome 6 – 7 μm thick.

1.1. Staining of tissue preparations To make a qualitative evaluation of morphofunctional condition of ovary corpora lutea in the cow preparation were stained with haemotoxilyn and eosin (Пирс, 1962; Меркулов, 1969). A quantitative Unna – Brashe method with methylene green pironin was applied to determine the activity of nucleic acids (Меркулов, 1969; Саркисов и др., 1996). For determination of progesterone receptors a streptavidin – biotin immunohistochemical method with marked enzymes was used (Traish and Wotiz, 1990).

1.1.1. Staining of preparations with haemotoxilyn and eosin After removal of paraffin preparations were stained with haemotoxilyn for 7 minutes. Then, they were rinsing in water for 10 minutes. After that, they were stained with eosin and then the preparations were rinsed with water for a short duration. The staining was followed by the dehydration in alcohol solutions of increasing concentrations and xylol. The stained preparations were spread with polysterol and covered with a coverglass. Basophilic parts of the stained cells appeared bluish violet but those of the acidophilic ones were pink to dark red.

1.1.2. Unna-Brashe method of staining DNA and RNA with methylene green pironin

After removal of paraffin, preparations were dried with a filter paper and stained with methylene green pironin for 2 hours: 0.15 g methylene green + 0.25 g pironin G or Y + 2.5 ml of 96% alcohol + 20 ml glycerine + 100ml of 0.5% solution of carbonic acid. After staining preparations were dried with a

10

filter paper and dehydrated in acetone for 5 minutes, then in acetone-xylol (1:1) solution. The nuclei were stained green, bluish green or dark blue depending on the activity of DNA. Granular structures of the cytoplasm were stained pink or red of various shades depending on the activity of RNA in the cells. The level of RNA concentration was evaluated comparing preparation by a semiquantitative method of multistage system (Меркулов, 1969; Саркисов и др., 1996). There was examined RNA (mRNA) expression in nuclei of luteal cells in our investigation. As reactive was considered granular structures in the nucleus stained dark pink or red. Preparations were not rinsed in water but in acetone, as nucleic acids are water-soluble. Control with enzyme ribonuclease (1h 37˚ C)

1.1.3. Immunohistochemical reaction for the detection of progesterone receptors

Removal of paraffin was carried out according to routine scheme but more thoroughly using two rinsing solutions both for xylol and alcohol. At the end of the removal of paraffin preparations were rinsed in two portions of distilled water, each for 5 minutes. After that, they were rinsed in TRIS (Tris-Buffered saline, pH 7.4) or PBS (Phosphate-Buffered saline, pH 7.4) for 5 minutes. Next preparations were put into solution Natrii Citric 4% (pH 10), in a microwave oven not less than 750 W for 2 minutes. Cooled preparations were rinsed in TRIS for 5 minutes (rinsing with TRIS or PBS followed after each stage of the reaction). Then, every section was spread with 100 μl of hydrogen peroxide for 5 minutes. After that, 30 μl of primary antibodies – Rabbit Anti-Human Progesterone Receptor in dilution 1:25 (DAKO, Denmark) were spread on each section for 2 hours. Both the optimum dilution of antibodies and the optimum time of exposition for the bovine ovary corpus luteum tissue were determined experimentally. After the primary antibodies the preparations were spread with LSAB + LINK – biotin bound secondary antibodies for 30 minutes. Then preparations were spread with LASB + KIT – streptavidin which is bound with the enzyme peroxidase. For visualisation of the obtained results the preparations were stained with haemotoxilyn for a short period of time. Control: 1) without primary antibodies; 2) without secondary antibodies. In case of a positive reaction, the relevant places are stained brown. The above-described reaction corresponds to the scheme described in literature only in general outline. During the course of work it was modified: 1) the optimum dilution of the primary antibodies 1:25 was developed; 2) the optimum time of exposition 2 hours was determined.

11

1.2. Visualisation of results and data processing The preparations were analysed under the LEICA and KARL ZEIS microscopes with 400 times of magnification. A KARL ZEIS microscope with camera was used to obtain microphotographs. Microphotographs were taken with 400 times of magnification and FUJICOLOR SUPERIA films of light sensitivity 400. For the cell counting the Автандилов (1990) semiquantitative method was applied: the positive cells were counted in 6 visual fields and then the average number of positive cells per section was calculated. Two histological sections were used for the cell counting. For mathematical processing of the obtained data calculation of these mean value, standard deviation and standard error as well as correlation between data – Microsoft Excel computer software was used. For evaluation of significance of differences and comparison of the mean values of progesterone receptors and mRNA in various stages of development of cyclical corpus luteum and pregnancy corpus luteum Stjudent T-test was applied.

RESULTS AND DISCUSSION

Pregnancy corpus luteum

The data of this study revealed that in 75% of cases a typical protuberance of the ovary pregnancy corpus luteum in cows was loss than 1.0 cm, while the largest part of the corpus luteum was situated in the ovary tissue. Okuda (1982) also obtained similar research data. In 24.3% of the examined pregnancy corpora lutea there were central cavities. These data correspond with Arthur’s (1996) data about the presence of central cavities in 25% of cases but contradict with Okuda’s et al (1988) data about the presence of central cavities in the pregnancy corpora lutea saying that only in 5.1 % of cases they are present. Some typical morphological peculiarities of the pregnancy corpus luteum have been stated. During the course of pregnancy parameters of the corpus luteum do not change statistically significantly (see Figure 1). The increase of width in the 2nd month of pregnancy and its decrease in the 5th month takes place with probability P = 90% (p < 0.1). Volume, in its turn, decreases statistically significantly already in the 3rd month of pregnancy (p < 0.05). If in the 2nd month of pregnancy it reaches 8.0 ml (these data correspond with Jähn’s (1998) data), then in the 7th month of pregnancy it occupies only 4 ml of the volume.

A

AB

B

0

0,5

1

1,5

2

2,5

3

3,5

I mo II mo III mo IV mo V mo VI mo VII mo

Corpora lutea graviditationis

Len

gth

and

wid

th (c

m)

012345678

Vol

ume

(ml)

Length Width Volume

Figure 1. Morphological characterisation of corpus luteum graviditationis I mo., II mo., III mo., IV mo., V mo., VI mo., VII mo. –corpora lutea of respective months of pregnancy; A – statistically significant increase of parameter (p < 0,05); B – statistically significant decrease of parameter (p < 0,05). There is a poor agreement between morphological parameters of the pregnancy corpus luteum that provides evidence for the fact that parameters are changing independently of each other (see Tab. 2). A negative medium high agreement (r = - 0.51) is between length and width indicating that in the beginning of pregnancy when the corpora lutea are shorter their volume is larger but in the second part of pregnancy their length is a little increased while the volume is considerably decreased. Table 2. Correlation between parameters of corpus luteum graviditationis

PR Rec mRNA Length Width Volume PR Rec 1 mRNA 0.88 1 Length -0.56 -0.43 1 Width -0.12 -0.12 0.33 1 Volume 0.98 -0.51 -0.51 0.06 1

The consistency of the corpus luteum becomes looser and darker in colour during the course of pregnancy; thus, the corpus luteum has a tendency to darken, become looser, with less volume, but retaining its parameters (size). This interesting tendency can be explained with structural changes within the corpus luteum tissue during pregnancy.

12

The studies show that in the first and second month of pregnancy the large luteal cells prevail in the corpora lutea tissue parenchyma. In the first month of pregnancy the cells of the corpus luteum are very active cells. It is indicated both by progesterone receptor expression in cells and mRNA expression that shows evidence that cells produce progesterone intensively. Also in the second month of pregnancy the corpus luteum cells show great morphofunctional activity (see Figure 2.). The cell activity of the pregnancy corpus luteum in the early stage of pregnancy from the 8th to 20th day is supported by τ interferon secreted by the embryo trophoblast binuclear cells. τ Interferon blocks arachidonic acid transfer into PGF2α and stimulates progesterone synthesis in the large luteal cells (Peters, 1996). About on the 21st day the formation of the placenta starts and the effect of τ interferon on the pregnancy corpus luteum cells gradually diminishes. That explains the decrease of the luteal cell activity in the 3rd month of pregnancy. Undoubtedly, the pituitary hormones LH and FSH (gonadotropic hormones – luteinizing hormone and follicle-stimulating hormone) play an important role because exactly in the first months of pregnancy LH concentration in blood is high but afterwards it rapidly decreases (Schallenberger et al., 1985).

BIB

-2

0

2

4

6

8

10

12

I mo II mo III mo IV mo V mo VI mo VII mo

Corpora lutea graviditationis

Num

ber o

f rea

ctiv

e ce

lls

PR Rec

mRNA

Figure 2. Morphofunctional activity of corpus luteum graviditationis I mo., II mo., III mo., IV mo., V mo., VI mo., VII mo. – corpora lutea of respective months of pregnancy; PR Rec – expression of progesterone receptors, mRNA – expression of matrix ribonucleic acid; B – statistically significant decrease of parameter (p < 0,05). It was found that in the third month of pregnancy proportion between the large and small luteal cells is equalised, and the small luteal cells become more active. Davis et al. (1996) study gives evidence that the formation of the large luteal cells in the pregnancy corpus luteum occurs from the small luteal cells –

13

that is why they are not affected by the small luteal cell produced PGF2α. Luteal cells themselves produce several growing factors, including also basic fibroblast growing factor (bFGF) which is responsible not only for proliferation of fibroblasts but also for the luteal cell growing and proliferation (Niswender et al., 2000). As the luteal cell growing process seems to stop at about the third month of pregnancy when the placenta is completely formed (King, 1991; Peters, 1996), then possibly placental hormones play their role which hinder the luteal cell growth. These processes, however, can also explained by the decrease of LH concentration (Niswender et al., 2000). The growth of luteal cells was hindered in the corpus luteum and there were few stroma cells in its tissue. While the corpus luteum was regressing the proliferation of connective tissue was not observed (see Figure 3.).

A B Figure 3. Structural changes of pregnancy corpus luteum

A –Tissue of 2.5 months pregnancy corpus luteum; B –Tissue of 7 months pregnancy corpus luteum. Investigations carried out by Niswender et al. (1985) un Hild-Petito et al. (1989) provide with the information that the large luteal cells produce progesterone 4 – 20 times more than the small luteal cells. The cell ratio changes stand for the decrease of the concentration of progesterone produced by the corpus luteum (Okuda, 1982). Nevertheless, it does not cause statistically significant decrease of the cell morphofunctional activity (see Figure 2.). The small luteal cells also are able to produce progesterone, although, they need the LH stimulating effect (Fitz et al., 1983). The role of LH in the maintenance of the corpus luteum activity decreases significantly after completion of the placenta (Schallenberger et al., 1985). In most animals the placenta formation is complete and starts its functions on about the 45th day of pregnancy (King, 1991; Peters, 1996). Nevertheless, the function of the corpus luteum is maintained even until the 200th day of pregnancy (Niswender et al., 2000). The general increase of progesterone concentration cause the decrease of sensitivity of the hypothalamic-pituitary tract hindering the LH release, while 14

15

the decrease of progesterone produced by ovaries cause the increase of FSH concentration. (Schallenberger et al., 1985). As a result it causes a statistically significant decrease (p < 0,05) of morphofunctional activity of corpus luteum in the fourth months of pregnancy (see Figure 2.). The obtained data show, the small luteal cells start prevailing in the parenchyma of the corpus luteum tissue in the fourth month of pregnancy the activity of which depends on LH. The mean number of progesterone receptor and mRNA reactive cells per visual field of a slide in the corpus luteum tissue in the 5th and 6th months of pregnancy show evidence of the decrease of number of the active cells (see Figure 2.). In the 4th month of pregnancy lymphocytes appeared in great quantity in tissues because the progesterone produced by luteal cells decreases. Progesterone has a peculiarity of suppressing functions of immune cells and the local immunity response (Davis et al., 1996; Parkinson et al., 1996; Pate, 1996). Infiltration of immune cells introduces the intensification of regressive changes in luteal cells. Cytokines produced by the immune cells decrease even more the synthesis of progesterone stimulated by LH and stimulate the cytokines synthesis in the immune cells (Davis et al., 1996; Parkinson et al., 1996; Pate, 1996). Cytokines causes the cytotoxicity affect in luteal cells (Pate, 1996), which was determined in the 4th month of pregnancy as a karyopyknosis and cytolysis. These processes continue to increase with progressing of pregnancy and in the 7th month of pregnancy they include all the luteal cells (see Figure 3. B). The corpus luteum cells in the 7th month of pregnancy are non-active due to their destruction. Despite of a few connective tissues in the pregnancy corpus luteum stroma, a possible intensive proliferation of myocytes and pericytes takes place in the blood capillaries. As a result of this process the decrease of the capillary lumen was observed in the 5th month of pregnancy, but in the 7th month of pregnancy even a partial obliteration (see Figure 3. B). These processes are stimulated by the growing factors produced by the apoptotic luteal cells – heparin binding growing factor (HBGF) and tissue inhibitor of metalloproteinases (TIMPs) (Davis et al., 1996; Pate, 1996). The obtained data give evidence that by decreasing the expression of progesterone receptors in cells of the pregnancy corpus luteum, mRNA expression also decreases in luteal cells. These parameters are closely associated with each other (see Table 2.) although in the pregnancy corpus luteum the question is not only about the saturation of progesterone receptors and decrease of progesterone synthesis caused by it. The ability to bind progesterone and also to synthesise progesterone is determined to a large event by the process of pregnancy and biologically active substances associated with it - τ interferon produced by the trophoblast binuclear cells and hormones produced by the placenta including progesterone as well as gonadotropic hormones LH and FSH. The luteal tissue regression also plays a great role in the decline of morphofunctional activity. Statistical data of this

research give evidence of a clear decrease of the corpus luteum activity during the time of pregnancy. Nevertheless, the corpora lutea tissues are variable. It means that it is not possible to determine precisely when exactly the process of regression will start and when it will end in the pregnancy corpus luteum because it can be individual in every animal. Gase et al. (1987) in their study pointed that they had succeeded in finding morphofunctionally active cell clusters even in the last pregnancy month corpus luteum, although, an opinion exists that the corpus luteum is able to function only until the seventh month of pregnancy (Niswender et al. 2000).

Cyclical corpus luteum In this research a typical protuberance > 0.1 cm of cyclical corpora lutea, except the early stage of their formation and total regression stage, or even their placement an the surface of the ovary was observed in 60% of cases. Theissen (1970), who described the corpus luteum protrusion outside the ovary in 73% of cases also, obtained similar results. In 17% of the cyclical corpora lutea examined in this investigation there was a typical central cavity, in addition, the central cavity was most typical in mature corpora lutea. Kito et al. (1986) had established central cavities in 37% of cases of the cyclical corpora lutea, and that investigation also approves that the central cavities are typical most often in mature corpora lutea. Statistically significant (p < 0,05) increases of the morphological parameters of the cyclical corpus luteum both length, width and volume takes place during the early to the late formation stage (see Figure 4.).

A

B

A

B

A

B

0

0,5

1

1,5

2

2,5

3

3,5

AF VF N AR PR

Developmental stages of corpus luteum

Leng

th a

nd w

idth

(cm

)

0

1

2

3

4

5

Vol

ume

(ml)

Length Width Volume

Figure 4. Morphological characterisation of cyclical corpus luteum

16

AF – early formation, VF – late formation, N – mature CL, AR – early regression, PR - – total regression. A – statistically significant increase of

parameter (p < 0,05); B – statistically significant decrease of parameter (p < 0,05). It can be explained by examining the structure of the cyclical corpus luteum tissue. The corpus luteum is characterised by very rapid growth and development. Its cells possess a high metabolic activity and ability to multiply (Reynolds et al., 1999). If in the early stage of formation of the cyclical corpus luteum there were little, often-binuclear cells in the tissue structure, then in the late stage of formation there already were large luteal cells. Yigit un Arikan (2001) confirmed that the corpus luteum tissue, obtained in the early formation stage of the luteal phase, contained the most amount of small luteal cells. Okuda (1982) in his studies pointed out the appearance of large luteal cells in the cyclical corpus luteum tissue already on the 4th day after ovulation, i.e., at the end of the early formation stage. In the late formation stage there was observed that the distances among the cells were smaller. Luteal cells participate not only in the processes of proliferation and growing during the formation stage of the cyclical corpus luteum. The investigation shows that the parameters of morphofunctional activity, expression of progesterone receptors and expression of mRNA in luteal cells, reach their maximum value already in the late formation stage of luteal cells. That shows evidence – that cells not only bind progesterone with their receptors in the cell nuclei but also synthesise actively mRNA in order to produce progesterone (see Figure 5.).

B B0

2

4

6

8

10

12

14

AF VF N AR PR

Developmental stages of cyclical corpus luteum

Num

ber o

f rea

ctiv

e ce

lls

PR Rec mRNA

Figure 5 Morphofunctional activity of cyclical corpus luteum AF – early formation, VF – late formation, N – mature CL, AR – early regression, PR - total regression; PR Rec – expression of progesterone receptors, mRNA – expression of matrix ribonucleic acid;

17

18

B – statistically significant decrease of parameter (p < 0,05). Expression of progesterone receptors in the follicle theca interna cells occurs just after the increase of LH concentration, short before the ovulation (Hild-Petito et al., 1988). FSH ensures the appearance of progesterone receptors in the granular cells (Schallenberger et al., 1985), which explains the high morphofunctional activity of luteal cells in the formation stage of the cyclical corpus luteum which was determined in our study. Okuda et al. (1999) investigation showed that the expression of LH receptors increased rapidly in the luteal cells from the 2nd to 6th day of the reproduction cycle, remained up to the 15th day of the cycle that ensured the functional activity of the small luteal cells during that time. In the mature stage the large luteal cells we were prevailing in the tissue structure of the corpus luteum, which were closed to each other. On about the 9th to 12th day after the ovulation luteinization of the granular and theca cells were finished and hypertrophy of the granular cells reached its maximum (Priedkalns, 1993). The corpus luteum tissue has obtained more tight consistency, although, its size and volume does not have statistically significant difference from the late formation stage (see Figure 4.). The morphofunctional activity of cells also has not changed statistically significantly (see Figure 5.) though, exactly this stage is said to have the highest progesterone concentration in the blood (Young et al., 2000). Possibly, there has been some importance of the so-called distant effect when the cell morphofunctional activity, which started in the previous stages, did not decrease at once but gradually. The most intensive capillary network was observed in the corpora lutea tissue of the mature stage. These data correspond with Young et al. (2000) opinion – that the corpora lutea tissues of the middle luteal phase are characterised by a remarkable capillary network. The high progesterone concentration and saturation of progesterone receptors result in the changes, which takes place in the corpus luteum during the early regression stage. Saturation progesterone receptors and decreasing progesterone concentration increase FSH secretion. FSH stimulates the growth of new follicles. Oxytocin and oestrogen receptors begin to appear in the mucous of the uterus that facilitates PGF2α synthesis in the endometrium. PGF2α induces the corpus luteum regression (Edqvist and Forsberg, 1997). Statistically significant changes in the cyclical corpus luteum of the early regression stage were observed neither in the morphological parameters – size and volume (see Figure 4.) nor in the functional activity – expression of progesterone receptors and mRNA in luteal cells (see Figure 5.). Nevertheless, exactly in this stage the first signs of luteolysis – cytolysis, karyolysis, karyopyknosis were found. The appearance of these signs is determined by PGF2α, synthesised by the endometrium, which introduces luteolysis decreasing the synthesis of progesterone in the large luteal cells and provoking oxytocin synthesis in them

(Edqvist and Forsberg, 1997). This, in its turn, suppresses progesterone synthesis even more in the small luteal cells, which start producing PGF2α. Endothelin–1 produced by the endotheliocytes activates PGF2α receptors in the large luteal cells. The synthesis of endothelin-1 is facilitated by PGF2α vasoconstrictive effect (Pate, 1996; Niswender et al., 2000). Immune cells also participate in the luteolysis. If the immune cells activity is suppressed during the period of high concentration of progesterone (Davis et al., 1996; Parkinson et al., 1996; Pate, 1996), then these cells start an active function causing cytotoxicity effect with their cytokines in the luteal cells while the progesterone concentration decrease. Despite of the destructive changes in tissues, many luteal cells are able to bind progesterone and synthesise it, which indicated by the rather high level of mRNA expression in luteal cells. Duffy et al. (1994; 1995; 1996) also obtained similar results. A statistically significant decrease of the size and volume in observed of the cyclical corpus luteum in the total regression stage (see Figure 4.). It can be explained by observing the corpus luteum tissue in this stage. In the cyclical corpus luteum connective tissues are prevailing adding the light colour and tough consistency. A few luteal cells are characterised by vacuolised cytoplasm and pyknotic nuclei (see Figure 6.).

A B Figure 6. Structural changes of cyclical corpus luteum

A – tissue of cyclical corpus luteum in mature stage; B – tissue of cyclical corpus luteum in total regression stage. The great structural changes in the cyclical corpus luteum tissue also explain a statistically significant decrease of morphofunctional activity of luteal cells (see Figure 5.).

19

20

The decrease of morphofunctional activity of luteal cells of the cyclical corpus luteum was already found in the mature stage. It can be explained by the fact that progesterone receptors cannot bind progesterone unlimitedly because saturated and their amount decreases in the cells (Edqvist and Forsberg, 1997). Nevertheless, for some time after the saturation of receptors the synthesis of mRNA continues which explains the fact that mRNA expression in the luteal cells remains for a longer time. Nevertheless, there is a great positive agreement between the expression of progesterone receptors and expression of mRNA in the cells of the corpus luteum. It indicates that expression of progesterone receptors and mRNA in cells increases and decreases simultaneously in all the developmental stages of the cyclical corpus luteum (see Table 3.).

Table 3. Correlation between parameters of cyclical corpus luteum PR Rec mRNS Length Width Volume PR Rec 1 mRNA 0.92 1 Length 0.64 0.36 1 Width 0.57 0.23 0.95 1 Volume 0.55 0.55 0.95 0.98 1

A medium high agreement between the expression of progesterone receptors and size of the corpus luteum (see Table 3.) provides evidence that not in all the developmental stages of the corpus luteum morphofunctional activity increases or decreases together with the increase or decrease of the size. The volume and size of the cyclical corpus luteum increase statistically significant (p < 0.05) from the early stage of formation to the late stage of formation. Nevertheless, the morphofunctional activity of luteal cells between those stages does not increase statistically significant. Contrary, in the total regression stage both size, volume and morphofunctional activity of the cyclical corpus luteum decreases statistically significant (p < 0.05) (see Figure 4.). There is a medium high agreement between the expression of mRNA and the length as well as volume of the cyclical corpus luteum (see Table 3.). It shows that not in all the developmental stages of the cyclical corpus luteum the expression of mRNA expression changes together with the length and volume. There is a statistically significant increase of the volume and length (p < 0.05) of the corpus luteum during the transfer from the early formation to the late formation stage (see Figure 4.). Then there is not statistically significant increase of mRNA expression of luteal cells between these stages (see Figure 5). Nevertheless, in the total regression stage the sizes, volume (see Figure 4.) and mRNA expression of the cyclical corpus luteum decrease statistically significant (p < 0.05). (see Figure 5.). Between mRNA expression and width of the cyclical corpus luteum a poor agreement occurs (see Table 3.), that shows evidence that the intensity of

21

mRNA expression does not change together with the width of the corpus luteum in no one of the stages. Between the parameters of the cyclical corpus luteum - length and width there is a high positive agreement shoring that the cyclical corpus luteum grows at the same time both in the length and width. Afterwards these parameters decrease simultaneously. Between the size and volume of the cyclical corpus luteum there also is a high positive agreement giving evidence that the volume of the cyclical corpus luteum changes together with its size (see Table 3.).

Comparison of the cyclical and pregnancy corpus luteum The pregnancy corpus luteum is functioning as a cyclical corpus luteum until the appearance of τ interferon (in cows it is the 14th – 16th day of pregnancy). Thus, possibly tissues of the pregnancy corpus luteum of the first month of pregnancy will be similar to those of the cyclical corpus luteum. Most of investigations (Okuda, 1982; Lei et al., 1991; Fields et al., 1992; Singh et al., 1997) suggest that so it really is because τ interferon affects the development of the pregnancy corpus luteum only on about the 14th to 16th day which prevents luteolysis. Therefore, only at this moment differences in the structures and functional activities of the cyclical and pregnancy corpus luteum appear. In this investigation it was found that the structure of the pregnancy corpus luteum tissues of the 1st and 2nd month of pregnancy is similar to that of the tissue structure of the cyclical corpus luteum. The large luteal cells are prevailing, the capillary network is clearly formed, but the stroma cells – fibroblasts, macrophages, immune cells are comparatively few. Possibly, it is the effect of hormones of the placenta under formation on the pregnancy corpus luteum. τ Interferon not only inhibits luteolysis of the corpus luteum but also ensures a little its growing and great functional activity. The size of the pregnancy corpus luteum, especially the length, is larger then the length of the cyclical corpus luteum in the late formation stage and mature stage. The volume of the pregnancy corpus luteum during the early pregnancy is statistically significantly (p < 0.05) larger than that of the cyclical corpus luteum in the mature stage. Similar data have been also obtained by Lukaszewska et al. (1980); Wheater et al. (1987); Jaskowski et al. (1993); Aslan et al. (1998). The luteal cells of the pregnancy corpus luteum have a statistically higher significant potential ability to bind progesterone in the first three moths of pregnancy. Their ability to synthesise progesterone is statistically significant lower (p < 0.05) than that of luteal cells of the cyclical corpus luteum in its most active stage. If in luteal cells of the cyclical corpus luteum progesterone receptors are located in nuclei, then in luteal cells of the pregnancy corpus luteum progesterone receptors have tendency to be located in the cell cytoplasm, around the nucleus (see Figure 7.).

A B Figure 7. Expression of progesterone receptors in luteal cells of pregnancy corpus

luteum (A) and in luteal cells of cyclical corpus luteum (B). It means that only a great amount of progesterone receptors does not ensure an intensive synthesis of progesterone. Maybe the location of progesterone receptors in the cytoplasm does not ensure such an intensive synthesis of progesterone as it is in case of their location in the nucleus. Initially intensive expression of progesterone receptors in luteal cells of the pregnancy corpus luteum is created both by the luteotropic influence of τ interferon and production of luteotropic substances stimulated by LH in luteal cells themselves - PGI2, PGE2 (Niswender et al., 2000). During the course of pregnancy the progesterone is produced not only by the corpus luteum as it is during the reproductive cycle but also by the placenta. Alongside its formation, the concentration of progesterone increases, and progesterone quickly saturates its receptors in luteal cells. The secretion of progesterone in luteal cells of the cyclical corpus luteum also decreases together with the saturation of progesterone receptors. However, it is not assisted by the progesterone produced by the placenta. Furthermore, the early pregnancy corpus luteum is “older”, than the cyclical corpus luteum in its mature stage. Consequently, progesterone own decreases its production via autocrine mechanism both in cells of the pregnancy corpus luteum and luteal cells of the cyclical corpus luteum. The results of these processes are different. If the cyclical corpus luteum is subject to very rapid regression, then the pregnancy corpus luteum not only does not retain its structure but also even retains its certain morphofunctional activity for a comparatively long time. The main progesterone producers in the cyclical corpus luteum are the large luteal cells which are formed from the granular cells of follicles and progesterone receptors of which are formed under the influence of FSH in the preovulatory follicle (Fields, 1996). The number of these cells does not change in the corpus luteum. They only become hypertrophied contrary to the small luteal cells which are dependent on LH. The small luteal cells are able to proliferate (Milvae et al.,

22

23

1996). The great luteal cells initially form the parenchyma of the pregnancy corpus luteum but contrary to the great luteal cells of the cyclical corpus luteum them are able to be formed from the small luteal cells (Davis et al., 1996). This explains the different procedures of many factors in the pregnancy and cyclical corpus luteum. The regression of the pregnancy corpus luteum starts already in the second month of pregnancy. It differs significantly from regression of the cyclical corpus luteum, caused by a programmed saturation of progesterone receptors and a following decrease of progesterone concentration and secretion of PGF2α (Pate, 1996; Edqvist and Forsberg, 1997; Niswender et al., 2000). The formation of the placenta and decrease of hypothalamic pituitary system’s sensitivity cause the regression of the pregnancy corpus luteum. As a result is rapidly falling of LH level in the 3rd month of pregnancy (Schallenberger et al., 1985). Regression of cyclical corpus luteum occurs very fast because there acts a strong luteolytic factor PGF2α. It is associated with an intensive proliferation of the stroma tissues, which gradually substitute the parenchyma. Regression of pregnancy corpus luteum is slow because the activity of immune cells is inhibited by the high general level of progesterone, which is ensured by the placenta (Pate, 1996; Davis et al., 1996; Parkinson et al., 1996). Hormones of the placenta, possibly, also inhibit hypertrophy of luteal cells and proliferation of stroma cells. It explains the fact that the small luteal cells prevail in the regressive pregnancy corpus luteum and there are very few connective tissue cells contrary to the cyclical corpus luteum. Contrary to the cyclical corpus luteum parameters of the pregnancy corpus luteum have no connection with its volume. Its tissues in the 7th month are formed solely of cells of a destructive parenchyma, which are unable to ensure the tissue density that in why the volume decreases but the size remains the same. Much smaller cells of connective tissues, which ensure both the decreases of size and volume, form the regressive cyclical corpus luteum.

Cystic degeneration of ovaries in the cow In our investigation ovary cysts tissues were very variable both in regard to histological structure and functional activity. Cysts with thinner walls were with a thinner demarcation layer of connective tissues, which marked tissues of the cyst from the side of the lumen. The cysts, which were sill in the process of formation there were some groups of granular cells in the lumen. These data correspond with those of Foley’s investigations (1996). In the cysts with thicker walls, a thicker layer of connective tissues between the lumen and the cyst tissue was found (see Figure 8.).

A B Figure 8.Walls of ovary cysts

A – wall of follicular cyst; B – wall of luteal cyst That suggests that the thickness of the wall of the cyst could be associated with the age of the cyst. Older cysts possibly have thicker walls. Caroll (1990) has come to a similar conclusion. Possibly the thick fibber layer is formed to protect the cyst tissues from maceration. The tissues of luteal cysts did not differ much from the tissues of the cyclical corpus luteum – they were also formed by luteal cells and stroma cells, only the proportions of the cells in tissues of different cysts varied. However, one common characteristic feature of most of the cysts investigated. A lot of cysts tissue had characteristic immune cells in great quantity both macrophages and lymphocytes. These cells showed a high activity of mRNA. The presence and activity of immune cells is explained in great extent by morphofunctional activity of the cysts themselves. Morphofunctional activity of cells concerning the production of progesterone is variable and it has a positive agreement with the thickness of walls. These data of ours are the same as Douthwaite et al. (2000) that there is a positive correlation between the thickness of the wall of the cyst and progesterone concentration in the blood. The expression of progesterone receptors and mRNA of the most active cyst in our investigation is similar to that of the expression of progesterone receptors and mRNA of the cyclical corpus luteum in mature stage. These data correspond with Odore et al. (1999) obtained data (see Table 4.).

24

25

Table 4 Thickness of cyst wall and functional activity

Cysts Wall thickness (mm)

Expression of progesterone receptors expression of mRNA

1 3.0 3.33 ± 1.75 6.50 ± 1.87 2 2.5 2.17 ± 1.72 2.50 ± 1.38 3 2.5 2.00 ± 1.41 1.50 ± 1.05 4 4.0 2.17 ± 1.94 2.50 ± 1.87 5 1.5 0.33 ± 0.52 0.33 ± 0.52 6 1.5 0.67 ± 0.82 2.83 ± 1.47 7 4.5 3.67 ± 1.03 9.00 ± 2.00

Correlation

r = 0.84 r = 0.70

In the cyst tissue, however, there is a rapid regression as it is observed in the cyclical corpus luteum because the cyst maintains a stable concentration of progesterone maintaining progesterone receptors also in endometrium and not allowing it to synthesise PGF2α. In normal condition progesterone receptors of luteal cells saturate and synthesis of progesterone decreases. That also occurs in luteal cyst tissue that is suggested by an irregular activity of the tissues of the cysts found in this research. The cyst tissues are characterised by groups of active cells surrounded by groups of non-active cells. In the tissues of the one cyst both very active progesterone bonding and producing cells and fully non-active cells were found. Possibly, the cyst activity is maintained and regression inhibited by progesterone, which is in the cavity of the cyst. Therefore, the started regression occurs slowly. The great activity of macrophages and lymphocytes in the tissue of the cysts as well as the destructive changes of luteal cells of the cysts themselves suggest about regressive changes. It is possible that progesterone concentration and also existence of the cyst is maintained by the hormones which are present in the liquid of the cyst cavity because they are of much more higher concentration in the cyst cavity then in the blood (Odore et al., 2000).

CONCLUSIONS I Characterisation of the pregnancy corpus luteum

1) In 75,5% of cases protuberance is less than 1 cm and there is not statistically significant changes of its parameters during the course of pregnancy while there is a statistically significant decrease of the volume already in the 3rd month of pregnancy;

2) There is a tendency of the expression of progesterone receptors to decrease in luteal cells during the time of pregnancy reaching a

26

statistically significant fall (p < 0.05) in the 4th moth of pregnancy. The expression of mRNA in luteal cells also has a tendency to decrease reaching a statistically significant fall (p < 0.05) in the 7th month of pregnancy;

3) Changes of its volume correlate with those of the morphofunctional activity of luteal cells though, there is no correlation between the volume of the corpus luteum changes of luteal cell activity during pregnancy and parameters of the pregnancy corpus luteum;

4) Its regression starts in the 2nd - 3rd months of pregnancy and it is extended.

II Characterisation of the cyclical corpus luteum:

1) Its location in the ovary and protuberance depended on the stage of luteal phase of reproductive cycle;

2) Parameters of the cyclical corpus luteum during the luteal phase change together with its volume, but they correlate with the functional activity of the cells not in all the stages of the luteal phase;

3) There are no statistically significant changes of the expression of progesterone receptors and also mRNA in luteal cells of the cyclical corpus luteum during the course of the cycle reaching a statistically significant (p < 0.05) fall only in the stage of total regression;

4) Life cycle of the cyclical corpus luteum is characterised by rapid growth , intensive production of progesterone and as much as rapid regression;

III Comparison of the pregnancy and cyclical corpus luteum:

1) In regard to the size and tissue structure, the cyclical corpus luteum in the late formation and mature stage goes not differ from pregnancy corpus luteum in the 1st – 2nd month of pregnancy. There is a statistically significant lag behind only in volume (p < 0.05);

2) Changes of mRNS expression in luteal cells of both the pregnancy corpus luteum and the cyclical corpus luteum are closely associated with changes of progesterone receptor’s expression in luteal cells

3) Ability to bind progesterone is more pronounced in luteal cells of the early pregnancy corpus luteum while luteal cells of the cyclical corpus luteum in the late formation stage are more active in producing progesterone;

4) Regression of the pregnancy corpus luteum differs significantly from regression of the cyclical corpus luteum both in regard to the time and speed of its process and facilitating factors of regression and also structural change of tissue;

IV Cystic degeneration of ovaries:

27

1) Cystic tissues of ovaries are variable, with a great number of immune cells, thinner or thicker border layer of connective tissues in the side of the lumen;

2) The thickness of the cyst wall correlates positively with morphofunktional activity of luteal cells of cystic tissues indicating to the luteinisation of the cyst wall during the course of time;

3) Morphofunctional activity of the most active cyst luteal cells is similar to that of luteal cells in the cyclical corpus luteum in the mature stage;

28

ANNOTATION

In this research, morphofunctional activity of the pregnancy corpora lutea of ovaries in 37 cows, the cyclical corpora lutea of ovaries in 24 cows and solitary cysts of corpora lutea of ovaries in 7 cows were characterised by applying linear dimensions – length, width, volume, and microscopic morphology as well as immunohistochemical method with biotin and streptavidin complex to determine expression of progesterone receptors in luteal cells, Unna-Brashe method to determine expression of mRNA in luteal cells. Invariability of the linear dimensions – lengths and widths was characteristic with the pregnancy corpora lutea of ovaries in the cow investigated during all the seven month of pregnancy. There is a statistically significant (p < 0.05) decrease of the volume of the pregnancy corpora lutea already in the third month of pregnancy. Exactly in this month of pregnancy the ratio of the large to the small luteal cells the parenchyma of the corpus luteum tissues begin to equalise but in the fourth month of pregnancy a massive infiltration of immune cells and signs of degeneration of luteal cells occur in the tissue of the corpus luteum. Exactly, in this month of pregnancy there is a statistically significant (p < 0.05) decrease of expression of progesterone receptors in luteal cells. The expression of mRNA in luteal cells also decreases reaching a statistically significant (p < 0.05) fall in the seventh month of pregnancy. Parameters of the pregnancy corpus luteum do not correlate between themselves and do not also make correlative regularities with indices of morphofunctional activities. Nevertheless between the indices of volume and morphofunctional activity of the pregnancy corpus luteum there is a great positive agreement (r = 98; r = 86). There is a great agreement between indices of morphofunctional activities (r = 88). There is a statistically significant increase (p < 0.05) of all the linear dimensions of the cyclical corpus luteum in the late stage of formation, but in the stage of total regression there is a statistically significant decrease. A great agreement exists between these parameters (r = 0.95; r = 0.95; r = 0.98). Changes of linear dimensions are also reflected in the structure off tissues of the cyclical corpus luteum. If the small luteal cells prevail in the early stage of formation, then the large luteal cells prevail in the late stage of formation. Exactly, in this stage there is the most intensive expression of progesterone receptors and mRNA in luteal cells. It is characteristic with the mature stage of the cyclical corpus luteum that tissues are dense and vascularisation is maximal. The first signs of regression occur in the stage of early regression. Then there is a massive infiltration of immune cells, destructive signs of luteal cells and an intensive proliferation of the stromal cells. These processes continue during the stage of total regression. There are insignificant changes of morphofunctional activity of luteal cells during all the luteal phase reaching a statistically significant (p < 0.05) fall only in the stage of total regression. There

29

is a great agreement between the two indices of morphofunctional activity (r = 92). The main difference between the cyclical and pregnancy corpus luteum is in their regression. The regression of the pregnancy corpus luteum lasts for months and is characterised with a gradual destruction of luteal cells, loss of activity, small amount of the stromal cells in tissues. The rapid destruction of luteal cells and loss of activity as well as an intensive proliferation of connective tissues instead of luteal cells characterise the regression of the cyclical corpus luteum. Tissues of ovary cysts in the cow are of different ratios of cells, however, the thicker are the cyst walls the thicker in the layer of connective tissue fibres between the cyst tissues and its lumen. Morphofunctional activity of the cyst tissues is variable. However, the expression of mRNA and progesterone receptors correlate greatly with the thickness of the cyst wall (r = 0.84; r = 70), That indicates to the possible formation of luteal cysts when the process of luteinization of the follicular cyst walls takes place.

30

PUBLICATIONS

1. Tamane R., Pilmane M., Jemeļjanovs A. “Morphofunctional characterisation of bovine corpus luteum during oestrus cycle”, Current Issues in Veterinary Medicine 2000, 213 – 218.

2. Tamane R., Pilmane M., Jemeļjanovs A. “Morphofunctional characterisation of the ovary corpus luteum during the pregnancy and cystic degeneration”, 4th Congress, of the World Latvian Physicians Theses, 2001, 187 – 188.

3. Tamane R., Pilmane M., Jemeļjanovs A. “Morphofunctional characterisation of bovine corpus luteum (CL) graviditationis”, Book of Abstracts of the 52nd Annual Meeting of the European Association for Animal Production, 2001, 203.

4. Tamane R., Pilmane M., Jemeļjanovs A. “Expression of progesterone receptors and mRNA in cows corpus luteum during estrus cycle”, BASE, 5 (special issue), 2001, 92 – 93.

5. Tamane R., Pilmane M., Jemeļjanovs A. “Morphofunctional characterisation of the ovary corpus luteum during the pregnancy and cystic degeneration”, Acta Universitatis Latviensis, IV, Medicine, 2001, 214 – 230.

6. Tamane R., Pilmane M., Jemeljanovs A. “Correlations between morphofunctional activity and morphological parameters in bovine corpus luteum during estrus cycle”, Verhandlungen der Anatomischen Gesellschaft, Supplement zum 184. Band des Anatomischen Anzeigers – Annals of Anatomy, 2002, 143.

7. Tamane R., Jemeljanovs A., Pilmane M. “Comparison between bovine cyclical corpus luteum and pregnancy corpus luteum ”, Current Issues in Veterinary Medicine 2002, 277 – 281.

8. Tamane R., Pilmane M., Jemeljanovs A. “Expression of progesterone receptors in tissue of cows ovarian cysts”, World Veterinary congress 27th, Book of abstracts, 2002, 211 – 212.

9. Tamane R., Pilmane M., Jemeļjanovs A. “Morphofunctionality of cows ovaries cysts”, Medical field scientific conferencies theses, 2003, 66.

10. Tamane R., Pilmane M., Jemeljanovs A. “Morphofunctional characterization of bovine corpus luteum during oestrous cycle”, Rīgas Stradiņa universitātes Zinātniskie raksti 2002, 2003, 236 – 238.

11. Tamane R., Pilmane M., Jemeļjanovs A. “Morphofunctional characterisation of bovine ovarian cysts”, accepted for publishing in LLU Zinātniskajos rakstos.

31

12. Tamane R., Pilmane M., Jemeljanovs A. “Regression of cow’s corpus luteum graviditationis”, Proceedings the 6th Baltic-Nordic workshop in Veterinary Anatomy, 2003, 43 – 44;

13. Tamane R., Pilmane M., Jemeljanovs A. “The bovine corpus luteum during pregnancy”, Proceedings the Profesor Gaston Victor Backman scientific conference, 2003, 75 – 78;

14. Tamane R., Pilmane M., Jemeljanovs A., Dabužinskiene A. “Expression of progesterone receptors in bovine corpus luteum during pregnancy”, accepted for publishing in “Medicina”, the scientific proceedings of Kaunas Medical University;

15. Tamane R., Pilmane M., Jemeljanovs A., Ļvovs D. “Relationship between apoptosis and morphofunctional activity in luteal cells of bovine corpus luteum during pregnancy”, accepted for publishing in Rīgas Stradiņa universitātes Zinātniskajos rakstos.