Localization of prolyl endopeptidase mRNA in small growing follicles of porcine ovary

Prolyl endopeptidase (EC3.4.21.26) has been considered a unique intracellular enzyme catalyzing internal peptide bond hydrolysis of Pro-X. In this study, the distribution of prolyl endopeptidase activity and its mRNA was investigated in the follicles of porcine ovary. Both follicular fluid and granulosa cell fractions from small follicles showed higher activity than those from large follicles. Molecular cloning and Northern blot analysis suggested that only one species of prolyl endopeptidase gene was expressed in the ovary. In addition, in situ hybridization study revealed that the prolyl endopeptidase mRNA expresssion was more noticeable in the granulosa cell layers of small ovarian follicles than in those of large follicles, suggesting its importance in the early stage of follicular development. Mol. Reprod. Dev. 50:121–127, 1998. © 1998 Wiley-Liss, Inc.     

On the ultrastructure of follicles and isolated follicular granulosa cells of porcine ovary

Summary The endoplasmic reticulum in granulosa cells of primary, secondary, and small tertiary follicles of the porcine ovary is sparse and largely of the granular type.In granulosa cells of large tertiary follicles the endoplasmic reticulum shows distinct signs of proliferation. Some cells even contain whorls of endoplasmic reticulum membranes, essentially of the agranular variety.Direct continuity between endoplasmic reticulum membranes of the granular and agranular type as well as the continuous increase in agranular membranes suggest that these membranes may originate from the granular membranes.Granulosa cells isolated from large tertiary follicles by microdissection and keptin vitro show essentially the same ultrastructure as granulosa cells of intact large tertiary follicles.Some lipid droplets appear to be localized in cavities of the endoplasmic reticulum. It is suggested that the droplets contain precursor material for steroid hormone synthesis.Finally, the development of the agranular endoplasmic reticulum including the appearance of whorls in some granulosa cells of large tertiary follicles indicates that steroid synthesis may occur in such follicular granulosa cells.Read at the Meeting of the Swedish Society for Pathology in Umeå, September 25, 1965 (Bjersing, 1966).This investigation was supported by grants from the Swedish Medical Research Council (Projects No. 13 X-78-01, 12 X-78-02, and 12 X-78-03).     

Estrogen receptor alpha and beta expression in the porcine ovary

In order to investigate the expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the porcine ovary, in situ hybridization was applied. Specific ovine ERalpha and bovine ERbeta cDNA probes were labeled with [-32S]dCTP. In the porcine ovary, positive signals for ERbeta were found in both granulosa and theca cells of all types of antral follicles as well as in the corpora lutea at all stages of regression. ERalpha mRNA was limited exclusively to the granulosa cells of preovulatory follicles and was present in a few cells of early corpora lutea. Thus, we showed differential expression of ERalpha and ERbeta at the mRNA level. Large antral follicles and early corpora lutea are the site, where both forms of estrogen receptor are expressed.     

Androgen actions and the ovary

Although androgens and the androgen receptor (AR) have defining roles in male reproductive development and function, previously no role in female reproductive physiology beyond testosterone (T) as the precursor in estradiol (E(2)) biosynthesis was firmly established. Understanding the role and specific mechanisms of androgen action via the AR in the ovary has been limited by confusion on how to interpret results from pharmacological studies, because many androgens can be metabolized in vivo and in vitro to steroids that can also exert actions via the estrogen receptor (ESR). Recent genetic studies using mouse models with specific disruption of the Ar gene have highlighted the role that AR-mediated actions play in maintaining female fertility through key roles in the regulation of follicle health, development, and ovulation. Furthermore, these genetic studies have revealed that AR-mediated effects influence age-related female fertility, possibly via mechanisms acting predominantly at the hypothalamic-pituitary axis in a dose-dependent manner. This review focuses on combining the findings from pharmacological studies and novel genetic mouse models to unravel the roles of ovarian androgen actions in relation to female fertility and ovarian aging, as well as creating new insights into the role of androgens in androgen-associated reproductive disorders such as polycystic ovarian syndrome.     

Autofluorescence of the porcine endometrium during early pregnancy

Uteri recovered from cyclic gilts (n = 5) on Days 15-19 and pregnant animals (n = 34) on Days 13-40 were opened and examined under UV light. A line of greenish fluorescence was present in the mesometrial region in contact with embryonic membranes at Day 13. Small patches of reddish fluorescence subsequently appeared on the uterine mucosa near the embryonic disc, and these increased in intensity and size until they encompassed the entire area of contact between each conceptus and the endometrium, for lengths of about 20 cm, by Day 29. Fluorescence then diminished gradually and was almost totally absent by Day 40. Three additional gilts were unilaterally hysterectomized on Day 15 and treated with Evans blue dye 10 min before removal of the second uterine horn. Both horns were opened and compared under UV light, but no difference in the pattern of fluorescence could be detected. All fluorescence was associated with uterine rather than conceptus tissues. The occurrence of autofluorescence in uteri of pregnant pigs precludes use of Evans blue dye as an indicator of vascular permeability.     

PGE receptor characteristics on porcine luteal cells during the estrous cycle and early pregnancy.

This study examined the affinities and concentrations of prostaglandin E (PGE) receptors on porcine luteal cells during the estrous cycle and early pregnancy. Corpora lutea (CL) were obtained from nonpregnant gilts at days 9 (n = 4), 12 (n = 3), and 14 (n = 6); three gilts possessed red, vascular CL and three gilts had white nonvascular CL) of the estrous cycle, and days 9 (n = 4), 12 (n = 3), 14 (n = 5), and 30 (n = 5) of pregnancy. The CL were dissociated enzymatically to disperse single cells and the red blood cells were removed by elutriation. The luteal cells were assayed for specific PGE binding by displacement analysis with use of [3H] PGE2 and varying concentrations of unlabeled PGE2. The specific binding of [3H] PGE2 to luteal cells decreased (p < 0.05) from days 9 to 14 of the estrous cycle, but only decreased (p < 0.05) from days 9 to 12 of pregnancy. Specific binding was higher (p < 0.05) on day 14 of pregnancy than the comparable stage of the estrous cycle. The affinities of PGE receptors decreased (p < 0.05) only on the luteal cells dissociated from red, vascular CL of day 14 nonpregnant gilts compared with those of other days of the estrous cycle and pregnancy. The number of PGE receptors on porcine luteal cells was similar (p > 0.05) in pregnant and nonpregnant gilts, but decreased (p < 0.05) on days 12-14 postestrus. During early pregnancy, it was evident that high affinity PGE receptors are sustained on porcine luteal cells; however, the role of the PGE receptors in maternal recognition of pregnancy remains speculative.     

Cloning of the porcine interferon-γ receptor and its foeto-endometrial expression in early pregnancy

In early gestation, trophoblastic cells of porcine preimplanting conceptuses transiently and massively secrete two distinct interferons (IFNs), one of which is IFN-γ. In order to localize possible cellular target(s) for this IFN-γ, the expression of the porcine IFN-γ receptor and its developmental regulation have been investigated on the maternal endometrium and on the embryonic tissues. A cDNA encoding the porcine IFN-γ binding-chain (pIFNGR1) was isolated. When expressed in COS-7 cells, it displayed a specific binding to radiolabelled pIFN-γ and was shown to be a glycosylated membrane protein with an apparent molecular mass of 92 kDa. Porcine IFNGR1 mRNA was detected by RT-PCR not only in uterine epithelial cells but also in embryonic tissues from at least as early as day 10 of gestation. Moreover, membrane expression of the pIFN-γ receptor quantified by binding and crosslinking of ³²P-pIFN-γ was demonstrated in uterine epithelium and in the trophoblast. In the trophoblast, expression of the receptor was found to be developmentally regulated: although expression was weak on days 12 and 15 of gestation, it reached a level similar to that found on some IFN-γ–sensitive cells on day 16. This study shows that maternal endometrium is not the only possible target for trophoblastic IFN-γ: the induction of pIFN-γ receptor expression in the trophoblast around day 16 of gestation could suggest the appearance of responsiveness to pIFN-γ in this implanted tissue and therefore a possible delayed autocrine effect of trophoblastic pIFN-γ. Mol. Reprod. Dev. 51:225–234, 1998.© 1998 Wiley-Liss, Inc.     

Synthesis of steroid hormones in the porcine oviduct during early pregnancy

Past studies of the oviducts have documented oviductal steroid production during the oestrous cycle in pigs. The present study examined whether the pig oviducts are the source of steroid hormones during early pregnancy. In the ampulla and isthmus, the expression of 3β-hydroxysteroid dehydrogenase (3βHSD) and aromatase cytochrome P450 (CYP19) mRNA by real-time PCR, cellular localization and quantities of the studied proteins by immunofluorescence and Western blot analysis, and concentration of steroid hormones in oviductal flushings by radioimmunoassay, were studied. The expression of 3βHSD in the ampulla and isthmus was correlated (r?=?0.89) and higher on Days 2–3 and 15–16 than on Days 10–11 and 12–13. CYP19 expression was elevated in the ampulla on Days 2–3, 10–11 and 15–16 and in the isthmus on Days 2–3 vs. the other days studied. The studied proteins were localized in oviductal epithelial cells. In the ampulla, the quantity of 3βHSD protein did not change, and was greater in the isthmus on Days 2–3 vs. Days 12–13 of pregnancy. The P450arom protein quantity increased in the ampulla on Days 2–3 vs. Days 10–11 and 15–16 and vs. Days 10–11 and 12–13 in the isthmus. The concentrations of progesterone and androstenedione in oviductal flushings were lowest on Days 12–13 and on Days 2–3 and 15–16, respectively, while oestradiol-17β and oestrone levels did not change. Porcine oviducts are the sources of steroid hormones during early pregnancy. The expression of steroidogenic enzymes primarily increases during the embryos presence in the oviduct, i.e., on Days 2–3 of pregnancy.     

Androgens and FSH affect androgen receptor and aromatase distribution in the porcine ovary

In the present study the authors investigated whether androgens could interact with FSH to induce aromatase and androgen receptor expression in porcine granulosa cells. Dissected whole porcine follicles (small, medium, and large) were incubated for 8 hours in M199 medium supplemented with testosterone (10(-7) M), FSH (100 ng/ml) or both those hormones. After incubation, the follicles were fixed and immunostained to visualise androgen receptor and aromatase. In cultures of granulosa cells isolated from small and large follicles, oestrogen secretion was measured by appropriate RIA. Incubation of follicles with testosterone and FSH increased aromatase immunoreactivity in preantral and early antral (i.e. small) follicles. The immunostaining for androgen receptor was slightly higher in medium follicles, while such hormonal stimulation had no effect on small and large follicles. Moreover, granulosa cells isolated from small follicles cultured with both testosterone and FSH produced more estradiol than control cultures (40 pg vs. 100 pg/10(5) cells). The level was relatively close to that obtained in the culture of control granulosa cells isolated from large preovulatory follicles (105 pg/10(5) cells). These results indicate that testosterone acts synergistically with FSH to increase aromatase expression in the small porcine follicles.     

Localization of an activin/activin receptor system in the porcine ovary.

The aim of this study was to locate a possible activin/activin receptor system within porcine ovaries containing functional corpora lutea. In situ hybridization was used to assess the gene expression of beta(A)- and beta(B)-activin subunits, and immunohistochemical studies were done to detect activin-A protein and activin receptor type II. mRNA expression of the beta(A)- and beta(B)-activin subunits was found in the granulosa from the unilaminar follicle stage onward, in the developing thecal layer of multilaminar and small antral follicles, in the theca interna of mid-sized antral follicles, in corpora lutea, and in the ovarian surface epithelium. Immunoreactive activin A protein could be detected at the same ovarian sites, but in thecal tissue of small antral follicles only. This protein was also demonstrated at the peripheral zone of oocytes from multilaminar and antral follicles. A positive immunoreaction for activin receptor was found in granulosa cells from multilaminar and older follicles and in oocytes from the earliest stages of follicular development onward. In late multilaminar follicles and in antral follicles, the oolemma was stained. Except for small antral follicles, a positive activin receptor immunoreaction was absent in the follicular theca. Activin receptor immunoreaction was furthermore present in corpora lutea and in the ovarian surface epithelium. It is concluded that, within porcine ovaries containing functional corpora lutea, an activin/activin receptor system is present in all intact follicles, the corpora lutea and the surface epithelium. Within follicles, granulosa and theca cells are the main sites of activin synthesis, while oocytes and granulosa cells are the main activin binding sites.     

Follicular microvasculature in the porcine ovary

The microvasculature of porcine ovaries, with special regard to the follicles in the interstitial-stromal tissue, was studied by scanning electron microscopy (SEM) of vascular corrosion casts. Porcine ovaries displayed several coiled arteries in the hilus and many branches with small diameters and a tightly spiraling configuration in the cortical areas. However, small arterioles became straight before entering vascular complexes of follicles and finally divided into capillaries. Vascular baskets of various sizes (150-9,900 micro m in diameter) and architecture related to follicles in various developmental stages were observed in the ovarian cortex. Small follicles (150-300 micro m in diameter) began with a polygonal meshwork of a few large capillary meshes and developed to an obvious spherical microvascular network with a thin single layer of capillaries when reaching 500-700 micro m in diameter. The microvascular architecture of follicles 1,000-2,000 micro m in diameter developed further and had a three-layer vascular plexus. With a diameter of more than 2,000 micro m, the microvasculature of antral follicles was arranged as an inner vascular plexus of about 25 micro m, a middle plexus of about 100 micro m, and an outer capillary plexus of about 30 micro m in thickness. The present observations indicate that follicular vascular baskets of diverse sizes and architecture in various developmental stages support the gradual increase of follicular blood flow during follicle growth in the pig.     

Oxytocin receptors in the porcine endometrium during the estrous cycle and early pregnancy

Oxytocin (OT) receptors in the porcine endometrium were investigated at four stages of the estrous cycle (Days (D) 0, 5, 10 and 15, n = 3), and at two stages of early pregnancy (D5 and D15 after mating, n = 3) by a radioreceptor assay using ¹²⁵I-labeled OT antagonist [d(CH₂)₅,Tyr(Me)²,Thr⁴,Tyr-NH⁹₂]-vasotocin. Binding specificity was demonstrated by displacement with four peptides related to oxytocin ([Arg⁷]-vasopressin, [Thr⁴,Gly⁷]-OT, OVT, OT) and two peptides unrelated to oxytocin (luteinizing hormone-releasing hormone, [Ile³]-pressinoic acid (tocinoic acid)). The dissociation constant (K_d) of endometrial OT receptors on D0 (0.59 ± 0.10 nM) was similar to those on D10 and D15 (D10, 0.75 ± 0.21; D15, 0.60 ± 0.14 nM; mean ± SEM). In the early luteal stage (D5), K_d (2.41 ± 0.24 nM) was higher than on D0, D10 and D15 (P < 0.01). In early pregnancy, K_d values were 3.25 ± 0.29 nM on D5 and 2.44 ± 0.44 nM on D15. Binding site concentration (B_max) on D0 (910.0 ± 25.1 fmol mg⁻¹ protein) was significantly higher than on D5 and D10 (D5, 322.5 ± 71.7; D10, 147.5 ± 25.8 fmol mg⁻¹ protein; P < 0.01) of the estrous cycle and D5 and D15 (D5, 302.5 ± 82.6; D15, 315.0 ± 20.1 fmol mg⁻¹ protein; P < 0.01) of early pregnancy. In the two stages of early pregnancy, B_max values were constant and similar to that on D5 of the early luteal stage.Our results reveal the existence of specific OT binding sites in the porcine endometrium during the estrous cycle and early pregnancy. Furthermore, the fluctuation in the binding of OT to the endometrium during the different stages of the estrous cycle suggests that OT plays an important role in regulating the estrous cycle of the pig as seen in other animals.     

Conceptus influences the distribution of uterine leukocytes during early porcine pregnancy

Pregnancy in humans and rodents is associated with dramatic changes in leukocyte populations within the uterus. In these species, recruitment of leukocytes, mostly natural killer (NK) lymphocytes, accompanies decidualization of endometrial stroma even in the absence of pregnancy. In the pig, a nondecidualizing species, the predominant lymphocytes in the pregnant uterus are T and/or NK cells, but their distribution relative to embryonic attachment sites has not been reported. The objective of this study was to compare the abundance of leukocytes in porcine endometrium in contact with trophoblast with that between attachment sites during the early postattachment period. Uteri were recovered on Days 15-17 (n = 4), 18 and 19 (n = 4), 21 and 22 (n = 5), and 25-27 (n = 2) of gestation and from cycling pigs during the luteal phase (Day 15; n = 3). Leukocytes were identified in uterus obtained at versus between attachment sites using an antibody reactive with all leukocytes (CD44). In all pregnant animals, leukocytes were diffusely scattered throughout the endometrial stroma but were rare or absent in the luminal epithelium. Leukocyte density was approximately 3-fold greater in endometrium in contact with conceptuses than in endometrium between attachment sites throughout the early postattachment period. Leukocyte density during the luteal phase was similar to that between attachment sites, suggesting that leukocyte recruitment was a localized response to the embryo. The ability of an individual porcine conceptus to recruit maternal leukocytes to the adjacent stroma may be a vital step in early placental development and embryo survival.     

HTRA3 expression in non-pregnant rhesus monkey ovary and endometrium,and at the maternal-fetal interface during early pregnancy

Background  

HTRA3 is a recently identified member of the mammalian serine protease family HTRA (high temperature requirement factor A). In both the rodent and the human HTRA3 is transcribed into two mRNA species (long and short) through alternative splicing. We have previously shown that HTRA3 is expressed in the mature rat ovary and may be involved in folliculogenesis and luteinisation. HTRA3 is also upregulated during mouse and human placental development. The current study investigated whether HTRA3 is also localised in the primate ovary (rhesus monkey n = 7). In addition, we examined the non-pregnant rhesus monkey endometrium (n = 4) and maternal-fetal interface during early pregnancy (n = 5) to further investigate expression of HTRA3 in primate endometrium and placentation.     

Growth rates of follicles in the ovary of the cow

Follicular growth rates were studied in 5 Hereford-Holstein cross heifers on Day 14 of the oestrous cycle. The granulosa cell mitotic index (MI) was measured in non-atretic antral follicles of various diameters (0.13-8.57 mm) from Bouin-fixed ovaries collected before (199, control) and 2 h after colchicine treatment (189, treated). In control ovaries, follicles of 0.68-1.52 mm had a higher MI than those of other size classes (P less than 0.05). In colchicine-treated ovaries, the MI of follicles ranging from 0.68 to 8.57 mm increased more than that of other sized follicles, so that the mitotic time was shorter (0.78 h vs 1.32 h) in medium and large sized follicles (0.68-8.57 mm) than in smaller follicles (0.13-0.67 mm). Calculations based on the number of granulosa cells in follicles of various classes and from the time required to double the number of cells within a follicle indicate that a follicle takes 27 days to grow from 0.13 to 0.67 mm, 6.8 days from 0.68 to 3.67 mm and 7.8 days from 3.68 to 8.56 mm, indicating that growth rates varied with the size of the follicle. A period equivalent to 2 oestrous cycles would therefore be required for a follicle to grow through the antral phase, i.e. from 0.13 mm to preovulatory size. Increased MI, decreased mitotic time and increased atresia found in follicles larger than 0.68 mm could indicate a change in the follicular metabolism during its maturation.