Olga Amelkina

Life Cycle of Feline Corpora lutea: Histological and Intraluteal Hormone Analysis

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.

Corpora lutea of pregnant and pseudopregnant domestic cats reveal similar steroidogenic capacities during the luteal life span

In domestic cats, luteal phases of pregnancy and pseudopregnancy (non-pregnant luteal phase) differ in the course and level of plasma progesterone (P4). Therefore, we assumed differences in luteal steroidogenic capacities. Here we present a comprehensive analysis of intraluteal steroid biogenesis in the domestic cat. We quantitatively measured relative mRNA levels of steroidogenic acute regulatory protein (STAR), cytochrome P450 oxidases (CYP), hydroxysteroid dehydrogenases (HSD), steroid reductase (SRD) and enzymes involved in sulfoconjugation of steroids, i.e. sulfotransferase (SULT) and sulfatase (STS). Protein expression was analysed by Western Blot for HSD3B. Additionally, intraluteal steroid contents were determined. During the pseudopregnant luteal phase, expression of STAR (p=0.005), HSD3B1 (p<0.0001), CYP19A1 (p<0.0001) and HSD17B7 (p=0.008) decreased from formation of the corpus luteum (CL) onwards. HSD3B protein expression was highest in the development/maintenance stage of CL and declined during the subsequent luteal phase of pregnancy and pseudopregnancy. This was in accordance with decreasing intraluteal levels of P4, oestrogens and androgens. In contrast, expression of SRD5A1 (p<0.001) increased with progression through stages of the pseudopregnant CL, being indicative of P4 metabolism via an alternate pathway to dihydrotestosterone (DHT). Compared to the formation stage, expression of SULT1E1 was higher in all other luteal stages of pseudopregnancy (p=0.004), implying a potential sulfoconjugation of oestrogens. Expression of CYP11A1 and CYP17A1 was unaffected by the luteal stage (p>0.05), suggesting a permanent capacity of cat CL to convert progestogens via androgen and oestrogen pathways. In general, mRNA expression profiles of steroidogenic enzymes during the pregnant luteal phase reflected the pseudopregnancy profiles. Intraluteal oestrogen (p<0.0001) and androgen (p=0.008) levels were higher in the formation stage compared to the following luteal stages of pseudopregnancy. Concentrations of P4 were higher in the development/maintenance compared to the regression stages (p=0.01). We conclude that cat CL of the same histomorphological stage are characterised by identical steroidogenic capacities independently of an on-going pregnancy.

Synthesis and reception of prostaglandins in corpora lutea of domestic cat and lynx

Felids show different reproductive strategies related to the luteal phase. Domestic cats exhibit a seasonal polyoestrus and ovulation is followed by formation of corpora lutea (CL). Pregnant and non-pregnant cycles are reflected by diverging plasma progesterone (P4) profiles. Eurasian and Iberian lynxes show a seasonal monooestrus, in which physiologically persistent CL (perCL) support constantly elevated plasma P4 levels. Prostaglandins (PGs) represent key regulators of reproduction, and we aimed to characterise PG synthesis in feline CL to identify their contribution to the luteal lifespan. We assessed mRNA and protein expression of PG synthases (PTGS2/COX2, PTGES, PGFS/AKR1C3) and PG receptors (PTGER2, PTGER4, PTGFR), and intra-luteal levels of PGE2 and PGF2α Therefore, CL of pregnant (pre-implantation, post-implantation, regression stages) and non-pregnant (formation, development/maintenance, early regression, late regression stages) domestic cats, and prooestrous Eurasian (perCL, pre-mating) and metoestrous Iberian (perCL, freshCL, post-mating) lynxes were investigated. Expression of PTGS2/COX2, PTGES and PTGER4 was independent of the luteal stage in the investigated species. High levels of luteotrophic PGE2 in perCL might be associated with persistence of luteal function in lynxes. Signals for PGFS/AKR1C3 expression were weak in mid and late luteal stages of cats but were absent in lynxes, concomitant with low PGF2α levels in these species. Thus, regulation of CL regression by luteal PGF2α seems negligible. In contrast, expression of PTGFR was evident in nearly all investigated CL of cat and lynxes, implying that luteal regression, e.g. at the end of pregnancy, is triggered by extra-luteal PGF2α.

Comparative analysis of intraluteal steroidogenic enzymes emphasises the functionality of fresh and persistent corpora lutea during pro--and metoestrus in the lynx.

European lynx species demonstrate an atypical ovarian cycle compared to other felids. The physiological persistence of corpora lutea (CLs), reflected in constantly elevated progesterone (P4) concentrations in serum, is thought to ensure a seasonal monooestrus. Moreover, the coexistence of CLs from a recent ovulation (freshCLs) and persistent CLs from previous years (perCLs) on the same ovary has been proven. We assume that perCLs in lynxes occur due to fundamentally different mechanisms of luteal regression. Our study presents a detailed analysis of steroidogenic enzymes and steroids in fresh and perCLs obtained from Iberian lynxes during metoestrus, and in perCLs obtained from Eurasian lynxes during prooestrus. By quantitative PCR we measured relative mRNA amounts of steroidogenic acute regulatory protein (STAR), cytochrome P450 oxidases (CYPs), hydroxysteroid dehydrogenases (HSDs) and a steroid reductase (SRD). Protein expression in CLs was investigated for CYP11A1, CYP17A1, CYP19A1 and HSD3B. Additionally, the intraluteal and serum steroid content was determined. During metoestrus, mRNA amounts of STAR, CYP11A1, CYP19A1, HSD17B7 and SRD5A1 were significantly higher in perCLs compared to freshCLs. Protein of CYP11A1 was detected independently of the CL age in metoestrus, but expression was less evident in prooestrous perCLs. The protein signal of CYP17A1 was strong in freshCLs and perCLs of metoestrus, but weak at prooestrus. The presence of CYP19A1 protein was confirmed in each stage of the CL. These findings contribute to the hypothesis that CLs from previous years might support freshly developed CLs for pregnancy maintenance. However, initiation of ovulation might require a functional down-regulation of perCLs prior to breeding. It is noteworthy that the HSD3B1 mRNA amount was significantly elevated in fresh compared to perCLs (metoestrus). Accordingly, HSD3B protein was substantially present in freshCLs, whereas signals were literally absent in all perCLs. Elevated expression of HSD3B coincided with high intraluteal oestrogen concentrations in freshCLs; however, the enzyme pattern was less concordant with intraluteal P4 and androgen concentrations. Serum P4 concentrations of Iberian lynxes were constant between prooestrus and prolonged dioestrus. Moreover, constantly high serum oestrogen concentrations were measured during pro-, met- and prolonged dioestrus. The physiology of exceptionally high serum oestrogen concentrations outside the breeding season of lynxes merits further investigation. In conclusion our study supports the concept that the unique reproductive strategy of lynxes is directly linked to sustained intraluteal steroid biogenesis in persistent CLs.

Apoptosis-Related Factors in the Luteal Phase of the Domestic Cat and Their Involvement in the Persistence of Corpora Lutea in Lynx

The corpus luteum (CL) is a transient gland formed in the ovary after ovulation and is the major source of progesterone. In the Iberian and Eurasian lynx, CL physiologically persist after parturition and retain their capacity to produce progesterone, thus suppressing the ovarian activity. This unique reproductive characteristic has a big impact on the success of assisted reproduction techniques in the endangered Iberian lynx. The mechanisms behind CL persistence are not yet understood and require extensive studies on potential luteotropic and luteolytic factors in felids. Because the apoptosis system has been shown to be involved in structural regression of CL in many species, we aimed to investigate the capacity of perCL to undergo apoptosis. In addition, we performed initial studies on the apoptosis system in the luteal phase of the domestic cat. No previous research on this system has been made in this species. Our factors of interest included agents of the intrinsic apoptosis pathway, i.e., pro-survival B-cell CLL/lymphoma 2 (BCL2) and pro-apoptotic BCL2-associated X protein (BAX), the executioner caspase-3 (CASP3), as well as of the extrinsic pathway, i.e., pro-apoptotic receptor FAS, and tumor necrosis factor (TNF) and its receptors (pro-apoptotic TNFRSF1A and pro-survival TNFRSF1B). We analyzed the relative mRNA levels of these factors, as well as protein localization of CASP3 and TNF during stages of pregnancy and the non-pregnant luteal phase in CL of domestic cats. The same factors were investigated in freshly ovulated CL (frCL) and perCL of Iberian and Eurasian lynx, which were histologically analyzed. All factors were present in the CL tissue of both domestic cat and lynx throughout all analyzed stages. The presence of pro-apoptotic factors BAX, CASP3, FAS and TNFRSF1A in perCL of the Eurasian and Iberian lynx might indicate the potential sensitivity of perCL to apoptotic signals. The expression of pro-survival factors BCL2 and TNFRSF1B was significantly higher in perCL compared to frCL of studied Iberian lynx, suggesting the potential involvement of these factors in the structural integrity of perCL. In both Iberian lynx and pregnant and non-pregnant domestic cats, the expression of TNFRSF1A was significantly higher in forming CL compared to other stages, suggesting the conserved involvement of this factor in the tissue reorganization during formation of the feline CL. The mRNA levels of CASP3 and TNFRSF1B were highest during regression stages of domestic cat CL. The current study provides initial results on the possible involvement of the apoptosis system in the structure and function of the feline CL and in its physiological persistence.