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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Luteinization
  5. Luteal Regression
  6. Histologic and Hormonal Annual Cycle of CLs from Eurasian Lynx
  7. Summary
  8. Acknowledgement
  9. Funding
  10. Conflicts of interest
  11. References

The corpus luteum (CL) is a transient hormone gland on the ovary that produces progesterone (P4) for the maintenance of pregnancy. It develops from residual follicular granulosa and theca cells after ovulation. Very little is known about the cellular and hormonal processes within CLs obtained from pregnant and pseudopregnant felids. Therefore, our aim was to review the luteal function in feline CLs of different reproductive stages in conjunction with our data obtained in domestic cats and Eurasian lynxes. Corpus luteum function in lynxes is of particular interest, as a post-partum luteal activity was suggested based on repeated ultrasonography and endocrine examinations. Histology of CL from pregnant and pseudopregnant domestic cats clearly reflects the luteal function. The formation of the CL after ovulation is characterized by transforming of theca and granulosa cells into steroidogenic luteal cells and is accompanied by increased intraluteal and circulating P4 levels. Luteal regression is steadily progressive; the first signs (coarsed vacuolization, increased proportion of non-steroidogenic cells) are visible already in CL from the second trimester of pregnancy.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Luteinization
  5. Luteal Regression
  6. Histologic and Hormonal Annual Cycle of CLs from Eurasian Lynx
  7. Summary
  8. Acknowledgement
  9. Funding
  10. Conflicts of interest
  11. References

The corpus luteum (CL) is a hormone-regulated, transient reproductive gland and develops from residual follicular granulosa and theca cells after ovulation (Niswender et al. 2000). The main function of CLs, the synthesis of progesterone, is essential for the establishment and maintenance of early pregnancy. The life span of the CL varies between and within species; it is influenced by reproductive events such as mating or pregnancy. Eventually, the corpus luteum enters a dynamic regression process, during which it loses the capacity to produce progesterone and undergoes structural involution (Bowen-Shauver and Telleria 2003). The life cycle of CLs can be divided into three major events: luteinization (i.e. conversion of an ovulatory follicle), pregnancy-induced luteal maintenance/rescue and functional/structural luteal regression. While factors (luteotrophic hormones, extra- and intraluteal growth factors) that regulate the luteal function widely vary among different species, the composition of the CL (luteinized theca and granulosa cells) and the enzymes and proteins involved in the steroidogenic pathway are relatively similar among species (Christenson and Devoto 2003). Progesterone is the major and essential luteal steroid, but luteal tissue can also produce other steroids such as androgens, estrogens and 5α-reduced progestins.

Very little is known about the cellular and hormonal processes within CLs obtained from pregnant and pseudopregnant felids. Therefore, our aim was to review the luteal function in feline CLs of different reproductive stages and to introduce our own data obtained in domestic cats and Eurasian lynxes. The Eurasian lynx was chosen, in addition to the domestic cat, because of their unique non-cat like ovarian cycle (Göritz et al. 2009). During pregnancy, serum P4 levels of Eurasian lynxes are significantly increased. After parturition and weaning, they never return to baseline levels of non-breeding females. Based on repeated ultrasonography and endocrine examinations, a post-partum luteal activity was suggested (Göritz et al. 2009). Also in pseudopregnant lynxes, persistent CLs and elevated P4 values indicate luteal activity throughout the year. The lynx luteal cell function was assessed on samples obtained from hunted and road-killed animals in Sweden (Carnaby et al. 2012).

Luteinization

  1. Top of page
  2. Contents
  3. Introduction
  4. Luteinization
  5. Luteal Regression
  6. Histologic and Hormonal Annual Cycle of CLs from Eurasian Lynx
  7. Summary
  8. Acknowledgement
  9. Funding
  10. Conflicts of interest
  11. References

The pre-ovulatory LH surge results in luteinization of granulosa and theca cells. Furthermore, it alters the steroidogenic pathway; after luteinization, progesterone is the primary produced steroid hormone. Theca- and granulosa-derived luteal cells give rise to two distinct types of luteal cells that differ morphologically and physiologically. The cells derived predominantly from granulosa cells have been designated as large steroidogenic luteal cells (LLC), and those from theca cells have been designated as small steroidogenic luteal cells (SLC) (Murphy 2000). In addition to steroidogenic cells, the corpus luteum contains endothelial cells, fibroblasts, pericytes and cells originating from the bloodstream [for review: (Niswender et al. 2000)].

As in other mammalian species, the life cycle of the CL in the domestic cat is characterized by marked changes in morphology and endocrine regulation patterns. In the domestic cat, the CL formation starts immediately after ovulation, when ovarian follicular cells become compactly grouped and begin to hypertrophy, reaching the polyhedral form by day 7, which is typical for active luteal cells (Dawson 1941). Immediately after ovulation, the wall of the follicle is deeply plicated (Dawson 1941), and the foldings are composed of theca interna and granulosa cells (Fig. 1a) that transform to luteal cells. The theca cells lose their fibroblastic form and become rounded or polyhedral (Fig. 1b). The hypotrophy of the granulosa cells proceeds slowly, their reticulated pattern is gradually lost, and the cells become more and more compactly arranged. Their hypertrophy is accompanied by lipid accumulation within the cytoplasm (Fig. 1a), an indication of storage of progesterone precursors rather than active steroid secretion (Niswender et al. 2000). By the 7th day, the luteal cells have attained almost maximum size and adopted the typical rounded (polyhedral) form (Fig. 2a). The small lipid inclusions are located mainly in the cell periphery. The developing CL is widely infiltrated by theca cells and blood vessels causing the further increase in the gland (Dawson 1941). Both luteal cells and whole CL reach maximum size at days 12–16; during this period, the luteal cells remain typically vacuolated. In addition, the relation between SLC and LLC is shifted towards bigger luteal cells (Arikan et al. 2009); LLC produce >80% of total P4 during mid-luteal phase (Niswender et al. 2000). Young CLs obtained from pseudopregnant animals (Fig. 2e,f) are characterized by a mixture of different sized vacuolated steroidogenic luteal cells and are partly composed by fusiform luteal cells (Fig. 2f).

image

Figure 1. Histological picture of different developmental stages of corpora lutea (CL) obtained from domestic cats undergoing ovariohysterectomia. Freshly ovulated CLs were characterized by the bloody ovulation scar. (a) Formation process of the CL right after ovulation. Theca interna cells invade the inner space and mix with granulosa cells, both of them undergoing luteinization. (b) Cellular composition of freshly ovulated CL [weight: 33 mg; P4: 99.1 μg/g] at a more advanced stage, characterized by irregular polyhedral luteal cells, which contain lipid droplets of different size. Large luteal cells (black arrows: granulosa origin) can be differentiated from small luteal cells (white arrows: theca origin) and stroma cells (arrow heads). Scale bar = 50 μm

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image

Figure 2. Histological pictures of corpora lutea (CL) obtained from pregnant and pseudopregnant domestic cats. (a–c) CL of pregnancy; the approximate day of pregnancy was assessed according to the crown-rump-length of foetuses. (d–f) CL of pseudo-pregnancy. (a) The whole CL at day 21 of pregnancy is composed of equal sized, round, steroidogenic luteal cells, which contain small lipid inclusion in the cell periphery [weight: 30 mg; P4: 63.8 μg/g]. (b) The presented CL was obtained on days 39–40 [weight: 23 mg; P4: 15.7 μg/g]. At the end of second trimester, the cellular structure of luteal tissue becomes irregular with unclear cell borders and increasing portion of stroma cells; steroidogenic luteal cells show a coarse vacuolization with tendency for bigger and rarer lipid droplets within one cell. This process is even more evident towards the end of pregnancy. (c) The luteal tissue of a CL from day 47 to 48 is presented [weight: 12 mg; P4: 13.4 μg/g]. (d and e) depict a quite early stage of pseudo-pregnancy characterized by higher weight of the CL, clear luteal cell structure (no yet to differentiate from pregnancy) and different cell composition within different parts of the CL [d and e are from the same CL (weight: 31 mg)]. (f) depicts the appearance of a pseudopregnant CL [weight: 5 mg], which is already characterized by a progressive stage of luteal regression (coarse vacuolization, unclear borders between luteal cells, high portion of fibrotic cells). Scale bar = 50 μm

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Serum progesterone is known to depend on the amount of steroidogenic tissue and ovarian blood flow (Niswender et al. 2000); thus, the progesterone excretion follows the morphological changes. In the domestic cat, already 1–2 days after ovulation, serum progesterone exceeds 2.0 ng/ml, which was set as the threshold level, and then rises steadily for 10–12 days to concentrations above 15 ng/ml. In pseudopregnant cats (infertile mating), P4 levels stay elevated at that level till 25–40 days post-mating (Paape et al. 1975; Shille and Stabenfeldt 1979), whereas in pregnant cats, progesterone continues to increase throughout days 25–30 post-mating to values up to 40 ng/ml, followed by a steadily decline towards parturition (Verhage et al. 1976). The differences in the life span and P4 secretion of pseudopregnant vs. pregnant CLs are caused by luteotrophic and luteolytic factors that partly originate from feto-placental complexes. Most of them are still unknown, although there is an association between pregnancy (and placental function) in cats and the hormones relaxin (Stewart and Stabenfeldt 1985), prolactin (Banks et al. 1983; Addiego et al. 1987) and prostaglandin F2α (Tsutsui and Stabenfeldt 1993). In addition, local oxytocin synthesis is suggested to be involved in the ovarian–uterine interaction (Siemieniuch et al. 2011).

Luteal Regression

  1. Top of page
  2. Contents
  3. Introduction
  4. Luteinization
  5. Luteal Regression
  6. Histologic and Hormonal Annual Cycle of CLs from Eurasian Lynx
  7. Summary
  8. Acknowledgement
  9. Funding
  10. Conflicts of interest
  11. References

Luteal regression is defined as lysis or structural demise of the CL. During normal luteal regression, two closely related events occur: firstly, there is loss of the capacity to synthesize and secrete P4; secondly, the cells that comprise the CL break down (Niswender et al. 2000; Bowen-Shauver and Telleria 2003). The appearance of lipid vacuoles in the cytoplasm of steroidogenic cells expresses characteristic patterns of luteal function and luteal regression. Steroid cells show abundant smooth-surfaced endoplasmatic reticulum, but after reaching a maximum size, some luteal cells change markedly. The fine peripheral vacuolization disappears (Guraya 1969), and the luteal cells retain coarse lipid inclusions (Fig. 2b).

In a late corpus luteum, the cytoplasm of luteal cells apparently undergoes some drastic physicochemical changes as indicated by the decrease in the amount of diffuse lipoproteins and the accumulation of coarse lipid inclusion bodies of variable sizes and cytochemical nature (Guraya 1969). It is suggested that luteal cells during later stages of cat pregnancy begin to store hormone precursors rather than secrete hormones (Fig. 2b,c).

In pseudopregnant domestic cats, luteal regression starts from day 21 onwards and lasts approximately 40 days (Paape et al. 1975). Also in pregnant females, serum progesterone starts to decline after 25–30 days reaching 12.6 ng/ml by day 50 and 4–5 ng/ml just before parturition (Verhage et al. 1976). Coincidently, from day 28 onwards, first regressive luteal changes are detectable in feline CLs. Immediately after parturition, serum P4 values dropped to <1 ng/ml, but further fate of the CL seems to depend on lactation, probably, a relation to the luteotrophic functions of prolactin. The morphology of persistent CLs in lactating queens resembles the general appearance of CLs within the second week of pregnancy. These post-partum CLs stay until the end of 4th week of lactation (Dawson 1946). In non-lactating queens, the degree and type of vacuolization remain almost unchanged, but there is a gradual reduction in cell size. As long as the polyhedral appearance is maintained, there is no evidence of necrosis of luteal cells (Dawson 1946).

Histologic and Hormonal Annual Cycle of CLs from Eurasian Lynx

  1. Top of page
  2. Contents
  3. Introduction
  4. Luteinization
  5. Luteal Regression
  6. Histologic and Hormonal Annual Cycle of CLs from Eurasian Lynx
  7. Summary
  8. Acknowledgement
  9. Funding
  10. Conflicts of interest
  11. References

The life cycle of lynx CLs is characterized by the presence of steroidogenic luteal cells throughout the entire year (Fig. 3). The breeding season of lynxes in Scandinavia is dependent on latitude and lasts from February until early April, with mating activity peaking in March (Kvam 1990) and birthing approximately 68 days later. The image from March is considered as a typical example of functional luteal tissue if compared with CLs from pregnant domestic cats (Fig. 2a). The sample from December presented functional luteal cells with an increased vacuolization and higher proportion of connective tissue. Smaller sized luteal cells and a prevalence of connective tissue are clearly visible in the September samples (Fig. 3). This image is comparable to histological pattern of luteal regression depicted for pregnant and pseudopregnant domestic cats (Fig. 2). Analysing ovarian samples obtained throughout the year did not produce a clear picture of annual CL life cycle in lynx. At each time of the year, CLs were found on the ovaries (mean number per animal = 6.1) and both, fully functional and luteolytic, histological appearences were present (Carnaby et al. 2012) all year round.

image

Figure 3. Histological picture of corpora lutea from Eurasian lynxes obtained from hunted or road-killed animals. (a) Sample from March; (b) sample from July [weight: 220 mg; P4: 2.2 μg/g, E2: 18.5 ng/g]; (c) sample from September [weight: 210 mg; P4: 2.5 μg/g, E2: 10.2 ng/g] and (d) sample from December [weight: 310 mg; P4: 2.2 μg/g, E2: 9.9 ng/g]. CLs from all seasons contain steroidogenic luteal cells, with different degrees of luteal regression (b and c). CLs from March were collected immediately after death of the animal, whereas samples from other months were obtain from tissue-bank facility of National Veterinary Institute (SVA), Uppsala, Sweden, where samples from autopsied hunted or road-killed lynxes are stored at −20°C. These samples showed signs of decay. Scale bar = 20 μm

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The intraluteal steroid hormones follow the same pattern as described for serum (Göritz et al. 2009), with elevated P4 (72.3 μg/g) and E2 (450 ng/g) levels determined in CL from pregnant animals, but constant levels through the rest of the year (February–April 4.0 ± 0.7 μg/g P4, n = 19 and 39.3 ± 19.1 ng/g E2, n = 23; May–January 2.6 ± 0.7 μg/g P4, n = 32 and 15.0 ± 5.2 ng/g E2, n = 28). Interestingly, intraluteal prostaglandins method described in (Finkenwirth et al. 2010) were not elevated in CLs during pregnancy, but just prior to breeding season in ovaries obtained in January and February.

PGE was the predominant intraluteal prostaglandin with highest concentrations in January (3.1 ± 0.5 μg/g, n = 8), gradually decreasing thereafter towards the end of the year (November/December = 1.2 ± 0.1 μg/g, n = 16). Concurrently, significantly higher amounts of PGF2α (0.6 ± 0.1 μg/g) and PGFM (0.9 ± 0.1 μg/g) were found in CLs from January to February (n = 22) in comparison with the rest of the year (0.19 ± 0.11 μg/g and 0.5 ± 0.04 μg/g, respectively; n = 67). The seasonal elevation of intraluteal prostaglandins was neither related to significant changes in peripheral serum concentrations nor to their faecal metabolite compositions. Therefore, we suggest that intraluteal prostaglandins are related to pre-oestrus intraovarian signals as prerequisites for final luteal regression of persistent CLs and for follicular growth induction before ovulation. This hypothesis is supported by histological and hormonal findings on CLs of pregnant lynxes from April (Carnaby et al. 2012). Ovaries from pregnant animals contained two types of CLs. The first type CLs were bigger in size with large luteal cells (P4: 72.3 ± 46.3 μg/g; E2: 454.0 ± 37.1 ng/g), whereas the second type consisted of very small CLs with advanced luteal regression and lower steroid concentrations (P4: 8.3 ± 2.1 μg/g E2: 31.5 ± 14.4 ng/g). Previously, we already suggested that lynxes are able to onset oestrous, ovulate and become pregnant while being under the influence of CLs from preceding seasons (Painer et al. 2011).

Summary

  1. Top of page
  2. Contents
  3. Introduction
  4. Luteinization
  5. Luteal Regression
  6. Histologic and Hormonal Annual Cycle of CLs from Eurasian Lynx
  7. Summary
  8. Acknowledgement
  9. Funding
  10. Conflicts of interest
  11. References

Histology of corpora lutea from pregnant and pseudopregnant domestic cats clearly reflects the luteal function. The formation of the CL after ovulation is characterized by transforming of theca and granulosa cells into steroidogenic luteal cells and is accompanied by increasing intraluteal and circulating P4 levels. Luteal regression occurs progressively; first signs (coarse vacuolization, increased proportion of non-steroidogenic cells) are already visible in CLs from the second trimester; the time period when pregnancy is not exclusively depended on CLs (Tsutsui et al. 2009) and increasing levels of PGFM (PGF2α metabolite) are detected in urine and faecal samples of cat species (Dehnhard et al. 2012).

In addition to histology, luteal function is indicated by intraluteal hormone levels. In this respect, intraluteal prostaglandin analysis provides more insights than their peripheral concentrations, because the unique structure of the vascular utero-ovarian plexus allows transport of luteolytic PGF2α directly from the uterus to ovaries bypassing the systemic circulation. Thus, the intraluteal levels of steroids and prostaglandins provide more information about hormone driven processes during luteogenesis and luteal regression.

Acknowledgement

  1. Top of page
  2. Contents
  3. Introduction
  4. Luteinization
  5. Luteal Regression
  6. Histologic and Hormonal Annual Cycle of CLs from Eurasian Lynx
  7. Summary
  8. Acknowledgement
  9. Funding
  10. Conflicts of interest
  11. References

The material from Eurasian lynx was provided by the Division of Wildlife Diseases at SVA, Sweden. Ovarian extracts were prepared by Kim Carnaby as a part of a cooperative study (Carnaby et al. 2012). We thank Marlies Rohleder, Katrin Paschmionka and Sigrid Holz for their technical assistance and Kim Carnaby for English correction.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Luteinization
  5. Luteal Regression
  6. Histologic and Hormonal Annual Cycle of CLs from Eurasian Lynx
  7. Summary
  8. Acknowledgement
  9. Funding
  10. Conflicts of interest
  11. References