The role of hypoxia-induced genes in ovarian angiogenesis.

The hypoxic microenvironment that occurs in fast-growing tissue such as the corpus luteum (CL) is a major contributor to its ability to survive via the induction of an intricate vascular network. Cellular responses to hypoxia are mediated by hypoxia-inducible factor-1 (HIF-1), an oxygen-regulated transcriptional activator. HIF-1, a heterodimer consisting of a constitutively-expressed ß subunit and an oxygen-regulated α subunit, binds to the hypoxia responsive element (HRE) present in the promoter regions of responsive genes. This review summarises evidence for the involvement of hypoxia and HIF-1α in CL development and function. Special emphasis is given to hypoxia-induced, luteal cell-specific expression of multiple genes (vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF-2), prokineticin receptor 2 (PK-R2), stanniocalcin 1 (STC-1) and endothelin 2 (EDN-2) that participate in the angiogenic process during CL formation.

Emerging roles of immune cells in luteal angiogenesis.

In the mammalian ovary, the corpus luteum (CL) is a unique transient endocrine organ displaying rapid angiogenesis and time-dependent accumulation of immune cells. The CL closely resembles 'transitory tumours', and the rate of luteal growth equals that of the fastest growing tumours. Recently, attention has focused on multiple roles of immune cells in luteal function, not only in luteolysis (CL disruption by immune responses involving T lymphocytes and macrophages), but also in CL development (CL remodelling by different immune responses involving neutrophils and macrophages). Neutrophils and macrophages regulate angiogenesis, lymphangiogenesis, and steroidogenesis by releasing cytokines in the CL. In addition, functional polarisation of neutrophils (proinflammatory N1 vs anti-inflammatory N2) and macrophages (proinflammatory M1 vs anti-inflammatory M2) has been demonstrated. This new concept concurs with the phenomenon of immune function within the luteal microenvironment: active development of the CL infiltrating anti-inflammatory N2 and M2 versus luteal regression together with proinflammatory N1 and M1. Conversely, excessive angiogenic factors and leucocyte infiltration result in indefinite disordered tumour development. However, the negative feedback regulator vasohibin-1 in the CL prevents excessive tumour-like vasculogenesis, suggesting that CL development has well coordinated time-dependent mechanisms. In this review, we discuss the physiological roles of immune cells involved in innate immunity (e.g. neutrophils and macrophages) in the local regulation of CL development with a primary focus on the cow.

Association among calving season and measures of energy status, resumption of ovulation and subclinical endometritis in early lactating dairy cows

A total of 65 multiparous cows that calved during the hot season (n = 21), temperate season (n = 23) or cold season (n = 21) were used to investigate the relationships between heat stress, seasonal changes, metabolic traits, body condition score (BCS), resumption of ovulation and occurrence of subclinical endometritis (SE). Cows were monitored for the first 7 weeks of lactation by ultrasonographic examination. Blood sampling and BCS evaluations were performed on a weekly basis. Plasma progesterone (P4) and blood metabolites related to energy status including  -hydroxybutyrate, nonesterified fatty acids, total cholesterol, blood glucose and blood urea nitrogen were analyzed. Resumption of ovulation postpartum was confirmed by the first detection of a corpus luteum or a rise in P4 ≥ 1 ng/ml. Moreover, subclinical endometritis was diagnosed by brush cytology on day 40 ± 2 of lactation and cows having polymorphonuclear cell percentages (PMN%) ≥ 5 were considered SE positive. As expected, mean temperatur e humidity index differed (P < 0.001) among hot (75-85), temperate (50-65) and cold seasons (35-45). The BCS in cows that calved during the hot season was lower (P < 0.001) when compared with those that calved during the cold and temperate seasons. The percentage of cows that resumed ovulation in the first 45 days of lactation was greater (P < 0.05) for those that calved during the hot season (90%) than the temperate season (60%). The concentration of NEFA was greater (P < 0.05) for cows that calved during th e cold season than the hot season during different weeks postpartum. The concentration of glucose in weeks 4 to 7 postpartum was greater (P < 0.01) for cows that calved during the temperate season when compared to cows that calved during the hot season. Ther e was no difference in the prevalence of SE according to calving season. The percentage of PMN tended (P < 0.1) to be greater in cows with SE during the te mperate season than during the hot season. In conclusion, calving season may influence BCS, blood metabolites and resumption of ovulation whereas no association with prevalence of subclinical endometritis was observed.

Expression of KIT in the ovary, and the role of somatic precursor cells.

KIT is a type III receptor protein tyrosine kinase, and KITL its cognate ligand. KIT can mediate its effects via several intracellular signalling pathways, or by formation of a cell-cell anchor with its ligand. Through these mechanisms, KIT controls fundamental cellular processes, including migration, proliferation, differentiation and survival. These cellular processes are modulated by soluble KIT, a cleavage product of KIT, generated at the cell membrane. A cell-retained KIT cleavage fragment also arises from this cleavage event. This cleavage fragment must be distinguished from truncated KIT (trKIT), which originates through cryptic promoter usage. The expression of trKIT is highly restricted to postmeiotic germ cells in the testis. In contrast, KIT, together with its cleavage products, is present in somatic cells and germ cells in the gonads of both sexes. A functional KITL/KIT system is mandatory for normal population of the gonads by germ cells. Signalling via the KITL/KIT system promotes the growth, maturation, and survival of germ cells within the gonads, and prevents meiotic entry and progression. In addition to its importance in germ cell biology, the KITL/KIT system is crucial for gonadal stromal differentiation. During foetal life, KIT is expressed by testicular stromal precursor cells, which develop into Leydig cells. In the ovary, stromal cell KIT expression accompanies theca layer development around advanced follicles. After ovulation, KIT-immunopositive cells translocate from the theca layer to the luteal ganulosa where they contribute to a delicate cellular network that extends between the fully luteinised large luteal cells. In the outer regions of the developing corpus luteum, a highly conspicuous subpopulation of KIT/CD14-double-immunopositive cells can be observed. KIT/CD14-double-immunopositive cells are also seen in the haematopoietic-like colonies of long-term granulosa cultures established from late antral follicles. These cultures demonstrate expression of pluripotency marker genes such as octamer binding transcription factor-3/4 and sex determining region Y-box 2. The KIT/CD14-double-immunopositive cells can be purified and enriched by KIT-immunopositive magnetic cell sorting. Subsequent exposure of the KIT-expressing cells to the hanging drop culture method, combined with haematopoietic differentiation medium, provides the signals necessary for their differentiation into endothelial and steroidogenic cells. This suggests that monocyte-derived multipotent cells are involved in ovarian tissue remodelling. In summary, multicelluar KITL/KIT signalling organizes the stroma in the ovary and testis; monocyte-derived multipotent cells may be involved.

Efectos de la selección y de la superovulación en las pérdidas gestacionales de ratonas CF1 / Effects of selection and superovulation on gestational losses in CF1 mice

En un par de líneas de ratones seleccionadas para alto (s') y bajo peso (s), originadas a partir de una población no seleccionada de la cepa CF1 (t), se modificó la estructura ovárica. El diámetro de los folículos ováricos y el número de folículos y de cuerpos lúteos se incrementaron en las hembras de la línea s', sin expresarse en un mayor tamaño de camada al nacimiento, posiblemente, por un aumento de las pérdidas gestacionales. Se probó si los efectos conjuntos de la selección de peso a largo plazo y de la estimulación ovárica incrementaban las pérdidas gestacionales. Se utilizaron dos grupos de hembras por línea: sin y con estimulación ovárica (5UI de eCG y 5UI de hCG). Las hembras se sacrificaron a las 56-72 hs y a los 7 días postservicio y después de la primera parición. Se observaron los números de cuerpos lúteos (CL), embriones (E) y sitios de implantación (SI) y el tamaño de camada al nacimiento (TC). Se estimaron las pérdidas totales (PT) y las pérdidas de cuerpos lúteos (PCL), de embriones (PE) y de fetos (PF). Los promedios de CL, E, SI y TC variaron en el mismo sentido de la selección practicada y fueron significativamente mayores (P<0,05) para las hembras estimuladas, a excepción de TC. La línea s' tuvo un potencial reproductivo superior pero un mayor costo biológico (mayor PT y más tardía) cuando se la comparó con las otras líneas. La estimulación ovárica produjo menores eficiencias reproductivas totales para las tres líneas y pérdidas gestacionales mayores y más tardías, principalmente de SI. Las hembras de la línea no seleccionada (t), no estimuladas, con pesos intermedios, parieron un mayor número de crías, partiendo de un número intermedio de CL, E y SI, con una menor y más temprana mortalidad embrionaria, demostrando ser las más eficientes desde el punto de vista reproductivo y productivo.

The ovarian structure was modified as a consequence of weight selection in a pair of mouse lines selected for high (s') and low weight (s). Lines were founded from an unselected population of CF1 strain (t). The follicle diameter and the number of the ovarian follicles and the corpora lutea were higher in s' females, but they did not reach a larger litter size at birth, may be, by an increase in the gestational losses. In these lines, the co-effects of long-term weight selection and ovarian stimulation were tested to evaluate if they increased gestational losses. Two groups of females per line were employed: without and with ovarian stimulation (5UI of eCG and 5UI of hCG). Females were slaughtered at 56-72hs and at 7 days post-breeding and after first parturition. The number of corpora lutea (CL), embryos (E) and implantation sites (SI), and litter size at birth (TC) were observed. Total losses (PT) and corpora lutea (PCL), embryo (PE) and fetus (PF) losses were estimated. Mean CL, E, SI and TC varied in the same direction of the selection made and they were significantly higher (P<0.05) in stimulated females, though not for TC. Line s' had a higher reproductive potential but a greater biological cost (higher and later gestational mortality) when compared with the other lines. Ovarian stimulation produced lower total reproductive efficiencies for the three lines and higher and later gestational losses, mainly for implantation sites. Females from unselected line (t), without ovarian stimulation, with intermediate weights, bore larger litters, starting from an intermediate number of CL, E and SI, with a lower and earlier embryo mortality, showing to be the most efficient from a reproductive and productive point of view.

Ovulation, fertilization, and early embryonic development in the menstruating fruit bat, Carollia perspicillata.

To characterize periovulatory events, reproductive tracts were collected at 12 hr intervals from captive-bred, short-tailed fruit bats, Carollia perspicillata, on days 1-3 post coitum and examined histologically. Most bats bred readily. Graafian follicles developed large antra and exhibited preovulatory expansion of the cumulus oophorus. Ovulation had occurred in some on the morning, and in most by the evening, of day 1. The single ovum was released as a secondary oocyte and fertilized in the oviductal ampulla. Ovulated secondary oocytes were loosely associated with their cumulus cells, which were lost around the initiation of fertilization. Supernumerary spermatozoa were occasionally noted attached to the zonae pellucidae of oviductal ova, but never within the perivitelline space. By day 2, most ova had reached the pronuclear stage and by day 3, early cleavage stages. Several lines of evidence indicate that C. perspicillata is a spontaneous ovulator with a functional luteal phase. Most newly mated females had recently formed, but regressing corpora lutea, and thickened (albeit menstrual) uteri despite having been housed with males only for brief periods (<23 days). Menstruation is usually periovulatory in this species. Furthermore, the interval between successive estrus periods in most mated females that failed to establish ongoing pregnancies at the first was 21-27 days. Menstruation involved substantial endometrial desquamation, plus associated bleeding, and generally extended to the evening of day 3, the last time point studied. In nearly all females with a recent corpus luteum (n = 24 of 25; 96%), the preovulatory or newly ruptured follicle was in the opposite ovary.

Studies on morphological features of foetal and adult ovaries in Kano brown goats.

In this work we studied the structures of 51 foetal and 14 adult ovaries obtained from slaughtered Kano brown does in Nsukka abattoir. The ages of the adult does were determined by dentition and foetuses by crown rump length method. The foetal and adult ovaries were divided into five different groups using specific age intervals as Gestation day (GD) 50-65, 66-95, 96-125 and 126-145 and adults. For histological studies the ovaries were fixed, processed and routinely stained with H&E. The ovarian follicles were classified into 5 types according to granulosa cell layers surrounding the oocytes. The number of ovarian follicles per microscopic field, number of granulosa cells surrounding type 1 and 1A follicles and diameter of the ovarian follicles were determined for each group at 400x magnification. Grossly the foetal ovaries were like pin head, oval in shape, uniformly smooth and creamy in colour. The adult ovaries had follicles with different sizes. The adult mean ovarian weights were significantly higher (P<0.01) than those of the foetuses. Microscopically, the GD 50-65 ovaries had no distinct cortex and medulla. Oogonia were numerous among other stromal cells toward the periphery of the ovary. By GD 66-95 the ovaries contained types 1, 1a, 2 and 3 follicles. GD 96-125 ovaries contained type 4 follicles with early antrum formation and those of GD 126-145 comprised type 5 among other follicles. The adult ovaries comprised all the ovarian follicle types. The number of type 1 follicles increased significantly (P<0.01) with foetal age. It was least in the adults. The diameter of adult follicles was significantly higher (P<0.01) than those of the foetuses. This result provides baseline information on the morphological development of ovaries in Kano brown goats.

Unique patterns of Notch1, Notch4 and Jagged1 expression in ovarian vessels during folliculogenesis and corpus luteum formation.

Notch signaling functions to regulate cell-fate decisions by modulating differentiation, proliferation, and survival of cells. Notch receptors and ligands are expressed in embryonic vasculature and are required for the remodeling of the primary embryonic vasculature of mice. Here, we characterize the expression patterns of Notch1, Notch4, and Jagged1 proteins during the process of folliculogenesis and corpus luteum formation in the mouse ovary, an organ with dynamic physiological angiogenic growth. These Notch proteins and ligand are expressed in a subset of ovarian vessels, including both mature ovarian vasculature as well as angiogenic neovessels. Their expression in the ovary was found in both endothelial and vascular associated mural cells. Our data suggest a complex regulatory role for the Notch signaling pathway during mouse oogenesis and ovarian neovascularization.

Mutant mouse models and their contribution to our knowledge of corpus luteum development, function and regression.

The corpus luteum is a unique organ, which is transitory in nature. The development, maintenance and regression of the corpus luteum are regulated by endocrine, paracrine and autocrine signaling events. Defining the specific mediators of luteal development, maintenance and regression has been difficult and often perplexing due to the complexity that stems from the variety of cell types that make up the luteal tissue. Moreover, some regulators may serve dual functions as a luteotropic and luteolytic agent depending on the temporal and spatial environment in which they are expressed. As a result, some confusion is present in the interpretation of in vitro and in vivo studies. More recently investigators have utilized mutant mouse models to define the functional significance of specific gene products. The goal of this mini-review is to identify and discuss mutant mouse models that have luteal anomalies, which may provide some clues as to the significance of specific regulators of corpus luteum function.

Morphodynamics of the follicular-luteal complex during early ovarian development and reproductive life.

Female reproductive activity depends upon cyclic morphofunctional changes of the ovarian tissue during the female's fertile period, but the primum movens of an active gonadal rearrangement can be found from early phases of embryo development. To offer a basic account of the main steps of ovarian dynamics, we review the morphofunctional behavior of the follicular-luteal complex in an integrated study using light microscopy and transmission and scanning electron microscopy as well as through the use of numerous drawings. Particular emphasis is given to some reproductive aspects including (1) germ-somatic cell relationships and onset of folliculogenesis during early gonadal development; (2) follicular development and oocyte-follicle cell associations through adult folliculogenesis, finally leading to ovulation; (3) morphodynamics of corpus luteum formation, development, and regression, and (4) degenerative processes involving germ and somatic cells. The results reported, many of which originated in our laboratory, arise from some experiments on laboratory mammals but mostly from a large selection of human specimens. The data obtained are integrated and correlated with classic reports as well as with current views. Crucial biochemical, histophysiological, and clinical aspects are also emphasized.

Microvascular endothelial cells differ in basal and hypoxia-regulated expression of angiogenic factors and their receptors.

Phenotypically and functionally different types of microvascular endothelial cells (MVECs) derived from the developing corpus luteum were isolated and characterized by our group. We investigated whether these cytokeratin-positive (CK+) and cytokeratin-negative (CK-) MVECs differed in the expression of angiogenic factors and their regulation under hypoxia. Using quantitative RT-PCR, VEGF and its receptors, Flk-1 and Flt-1, were detected in CK- MVECs. The mRNA expression of Flk-1 mRNA was 100 times as high as that of Flt-1 mRNA. CK+ MVECs expressed VEGF and Flt-1 mRNA, but were devoid of Flk-1 transcripts. No Ang-1 mRNA was demonstrated in either cell type, and Ang-2 mRNA was found only in CK- MVECs. Tie-2 mRNA was detected in both MVEC types, but levels were 150 times as high in CK- MVECs as in CK+ MVECs. mRNA of hypoxia-inducible factors Hif-1alpha and Hif-1beta was expressed in both MVEC types. After hypoxia, neither VEGF, nor Flk-1, nor Tie-2 mRNA expression was altered in either MVEC type. Flt-1 expression and Ang-2 mRNA expression were significantly increased at about 2.5-fold (P < 0.05) in CK- MVECs, but not in CK+ MVECs. Our study demonstrates the varying expression and regulation of angiogenesis-related factors and receptors in phenotypically different MVEC types.

Coordinated expression of splice variants for luteinizing hormone receptor messenger RNA during the development of bovine corpora lutea.

We characterized splice variants for LH receptor mRNA in the bovine corpus luteum and examined the levels of expression of all the splice variants during the development of corpus luteum. Total RNA was extracted from bovine corpora lutea, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to amplify a part of the extracellular and transmembrane domains. As a result, four distinct bands were observed on analyses with polyacrylamide gel electrophoresis, and sequencing of the products revealed that the largest form (519 bp) was the full-length A form and that the three shorter forms (438, 253, and 172 bp) were its splicing variants. Splicing patterns of the 438-, 253-, and 172-bp forms are identical to the F, B, and G forms, respectively, found in the ovine ovary. In order to examine the level of expression of all these splice variants simultaneously, semiquantitative RT-PCR was set up and performed. The relative intensities for all four forms of LH receptor mRNA significantly increased (P < 0.05) from Stage I (Days 1-4; Day 1 = day of ovulation) to IIS (Days 5-10, CL weight < 4 g), and from Stage IIS to IIL (Days 5-10, CL weight > or = 4 g). The intensities of all forms for LH receptor mRNA decreased slightly, but not significantly, from Stage IIL to III (Days 11-17). The ratios of the levels for all the forms did not change significantly during the development of the corpus luteum. Determination of the steady-state levels of LH receptor mRNA by Northern blotting showed similar changes during the development of the corpus luteum to changes of the splice variants examined by the semiquantitative RT-PCR. These results suggest that at least three splicing variants of LH receptor mRNA (i.e., F, B, and G forms) exist in the bovine corpus luteum and that the levels of all the forms of LH receptor mRNA increased in a coordinated manner during the development of bovine corpus luteum.

Correlación entre embriocardia, saco vitelino y cuerpo lúteo en embarazo menor de diez semanas para eveluar su viabilidad y evolución

Existen reportes en la literatura mundial que, considerando algunos parámetros de ecografía temprana, hacen predicciones sobre le evolución del embarazo. Se pretendió demostrar estos hallazgos reportados universalmente para su aplicabilidad a la población que consulta al Hospital Militar Central, para la cual se realizaron ecografías transvaginales a 220 pacientes con embarazos menores de 10 semanas, en la Unidad de Ecografía Ginecoobstétrica del Hospital Militar Central o en la Unidad de Diagnóstico obstétrico y Ginecológico, (Obgyn), midiendo el diámetro del saco vitelino, cuantificando la embriocardia y valorando la presencia de cuerpo lúteo. Se realizó el seguimiento de las pacientes al final del período de estudio. Fueron excluidas 56 pacientes y con las 164 restantes no fue posible corroborar lo reportado en la bibliografía. Por tal motivo no podemos hacer ninguna predicción sobre el pronóstico del embarazo basados en las mediciones de ciertos parámetros por ecografía transvaginal en embarazos tempranos

Inherent variability among measures of fertility of rats and its implications in the design of mating trials.

Mating trials are used extensively to assess the effects of experimental treatments on the fertility of male or female subjects. Such trials are often insensitive because many measures of fertility are associated with large inherent variability, which makes it difficult to confirm, by statistical significance, that observed differences among treatment means are actually due to treatment rather than due to chance. Unfortunately, and despite this insensitivity, most researchers choose replication arbitrarily on the basis of cost, convenience, or conventional practice and interpret results without assessing the actual power and sensitivity of their experiments. This study was undertaken to characterize the relationship between the number of rats used in fertility trials and experimental power and sensitivity. The variables examined included pregnancy rates, numbers of fetuses per mated or per pregnant female, numbers of fetuses per corpus luteum, and numbers of fetal resorptions. The relationship between replication and experimental power and sensitivity was estimated via statistical approaches utilizing data from Sprague-Dawley rats used in a mating trial at 105-109 days of age. Tabular data are presented showing the numbers of rats needed per treatment group as a function of the minimal treatment differences to be detected and type II error probability selected. The application of these data in the planning of future studies and in interpreting their outcome is discussed.

Desenvolvimento de pré-embriöes de ratas Wistar da Colônia do Biotério do Centro de Biologia da Reproduçäo - UFJF / Pre-embryo development of Wistar rats from the Colony of Centro de Biologia da Reproduçäo Vivarium - UFJF

Durante a fase pré-embrionária, o concepto fica exposto às secreçöes tubárias que podem conter produtos tóxicos aos quais a mäe pode ter sido exposta no início do período gestacional. A determinaçäo dos padröes referentes ao número de pré-embriöes e suas diferentes fases do desenvolvimento é importante para a pesquisa de agentes com potencial tóxico. Neste trabalho estabeleceu-se a padronizaçäo do desenvolvimento dos pré-embriöes na fase de 2, 3 e 4 células, mórula e blastocisto, de ratas Wistar da colônia do Biotério do Centro de Biologia da Reproduçäo - UFJF.

Differential distribution of laminin chains in the development and sex differentiation of mouse internal genitalia.

The distribution of laminin chains and basement membranes (BMs) in the ontogenesis and sex differentiation of male and female mouse gonads and mesonephros was studied by conventional and immunocytochemical light and electron microscopy. The alpha 1 (synonymous to A) chain was recognized with MAbs against fragment E3, and three chains of laminin with PAbs raised against EHS-laminin. BMs, which formed around the mesonephric duct, the mesonephric tubules, and the paramesonephric duct, contained the laminin alpha 1 chain. The alpha 1 chain appeared with epithelial differentiation in the developing gonads in both sexes. The alpha 1 chain was first evident around the embryonic gonadal cords and remained, after development, in the BMs of the testicular cords and ovarian follicles. The laminin alpha 1 chain was also detected in BMs of the myoid cells around the epithelial rete cords, and transiently in the surface epithelium and in the corpus luteum. Laminin beta-gamma chains were found in many locations where the alpha 1 chain was not detected. These included the mesenchyme of the early mesonephros, the BMs of blood vessels and surface epithelium in the differentiated testis and ovary, between the theca cells in the ovary, and in some corpora lutea. The morphological differentiation of the BMs of the embryonic testicular cords proceeded rapidly. In contrast, the BM of the ovarian cords remained relatively poorly differentiated during the prenatal phases, and developed concomitantly with the differentiation of the follicles. The results show that BMs in the differentiating internal genitalia are heterogeneous with respect to their laminin chains, and suggest that all known laminin chains must be analyzed in the differentiation of gonadal epithelia for a complete role of the BMs in gonadal sex differentiation.

Prevalence of atretic corpora lutea in ovaries of bovine fetuses.

Both ovaries from 88 bovine fetuses in the fifth month or later of gestation were studied histologically to determine the prevalence, origin, and time of appearance of atretic corpora lutea (ACL). Ovaries from 36 (41%) fetuses had ACL; fetuses < 6 months of gestation did not have ACL. Six fetuses had more than 25 ACL, but there was no apparent relationship between fetal age and number of ACL. Formation of ACL involved disintegration of the stratum granulosum of secondary follicles, concomitant with proliferation and invasion by vascularized elements of the theca. Fully developed ACL consisted of a large primary oocyte surrounded by a prominent zona pellucida and encased in a well-vascularized, largely thecal, fibrocellular wall. They measured approximately 0.5 to 1.0 mm in diameter. Empty, collapsed zona pellucidas were seen in many of the degenerating ACL.

[Respective roles of thecal and granulosa cells in the morphogenesis of the corpus luteum]. / Rôles respectifs des cellules de la thèque et de la granulosa dans la morphogenèse du corps jaune.

Studies on human ovary embryogenesis and adult post-ovulatory surgically resected ovaries allowed the authors to propose a new conception on the genesis of corpus luteum: large luteal cells would be derived from theca interna cells and small luteal cells would come from externa theca cells. So there would be a structural unity of luteal tissue which might explain steroidogenesis potential of these two types of cells, derived both from the same ovarian stroma. On the other hand if granulosa cells played an important function in bio-conversion of androgens to oestradiol by aromatase activity before ovulation, their role is over after the expulsion of the oocyte out of the follicle; derived from primitive sex cords of coelomic mesothelium origin, granulosa cells act to support and surround the oocyte; they disappeared at the disappearance of the oocyte from the follicle.