Oocytic Tumor Necrosis Factor Alpha: Localization in the Neonatal Ovary and throughout Follicular Development in the Adult Rat1

Abstract

Tumor necrosis factor alpha (TNF alpha) has previously been immunolocalized within mouse oocytes. Our first objective was to examine TNF alpha immunolocalization in ovaries of adult, fetal, and neonatal rats. Our second objective was to examine TNF alpha mRNA in ovaries by Northern blot analysis and in oocytes by reverse-transcriptase polymerase chain reaction (RT-PCR). Our final objective was to determine whether oocytes contained bioactive TNF alpha. Ovaries and oviducts were collected throughout the estrous cycle in adult rats, fetal ovaries were obtained 1 day before expected delivery, and neonatal ovaries were collected 2 days after birth. TNF alpha was localized in tissues by a biotin-avidin immunocytochemical procedure. Immunocytochemistry in adult ovaries showed that the ooplasm of the oocyte was the primary site of TNF alpha localization within the follicle. Immunostaining was present in all oocytes in the adult, including ovulated oocytes within the oviduct. Oocytic TNF alpha immunostaining was also present within oocytes in the neonate; however, fetal oocytes did not contain immunoreactive TNF alpha. Northern blots showed that ovaries in the adult, neonate, and fetus all contained TNF alpha mRNA. RT-PCR analysis of oocytes collected from preovulatory follicles generated a cDNA band of 500 bp, corresponding to the predicted size for amplified TNF alpha cDNA. Subsequent Southern blot analysis showed that the 500-bp band hybridized to the TNF alpha probe, indicating that preovulatory oocytes contain TNF alpha mRNA. Preovulatory oocytes were used in TNF alpha cytotoxicity assays with L929 cells. Oocytes contained TNF alpha bioactivity that was similar to that of recombinant murine TNF alpha in the bioassay. Our results provide evidence for the identification of immunoreactive and bioactive TNF alpha within oocytes in the rat, which is further supported by the presence of TNF alpha mRNA within the oocyte. These studies also indicate that TNF alpha may appear in the oocyte around the time of birth.

Interactions between progesterone and tumor necrosis factor-α in the regulation of primordial follicle assembly

“…Previous literature suggests that estrogen is another steroid hormone important for follicle assembly in the mouse (Jefferson & Newbold 2006, Pepe & Billiar 2006, rat (Kezele & Skinner 2003), and baboon (Pepe & Billiar 2006), but estrogen likely plays a greater role in arresting the primordial follicle in the primordial stage and not allowing the primordial to primary follicle transition (Kezele & Skinner 2003). TNFa has been implicated as a growth factor that promotes the oocyte apoptosis thought to be necessary for follicle assembly to occur (Chen & Marcinkiewicz 1993, Marcinkiewicz & Krishna 1994, Kondo & Maruo 1995, Marcinkiewicz & Balchak 2002. In the present study, TNFa treatment of cultured ovaries does not change the rate or percentage of assembled follicles compared with controls.…”

“…TNFa mRNA levels in whole ovary samples were too low to detect differences in expression between control and P 4 -treated ovaries. At this developmental stage, TNFa expression has been demonstrated to be limited to oocytes in ovaries (Marcinkiewicz & Krishna 1994, Marcinkiewicz & Balchak 2002. It is possible that TNFa mRNA from oocytes has been so diluted by somatic cell RNA as to interfere with TNFa microarray detection.…”

“…The surviving oocytes become surrounded by pregranulosa cells to form primordial follicles. Studies have shown that ovaries and oocytes express tumor necrosis factor-a (TNFa) at the time of follicular assembly in rodents, and TNFa treatment can increase oocyte apoptosis (Chen & Marcinkiewicz 1993, Marcinkiewicz & Krishna 1994, Kondo & Maruo 1995, Marcinkiewicz & Balchak 2002. Observations suggest that TNFa is a stimulator of oocyte apoptosis that leads to oocyte nest breakdown and promotes assembly of primordial follicles.…”

Identification of a C1q family member associated with cortical granules and follicular cell apoptosis in Carassius auratus gibelio

“…1995; Marcinkiewicz et al, 1994;Naz et al, 1997). and Morrison and Marcinkiewicz (2002) demonstrated that TNF could decrease oocyte and primordial follicle number through stimulation of oocyte apoptosis in rat ovary.…”

Involvement of Meiotic Maturation of Oocytes in Promoting Granulosa Cell Proliferation.

“…Both TGF-β and TNF-α are known to be present in oocytes in heterologous and homologous coculture systems [8,16,23], and they are also present in oocytes to modulate rat granulosa cell DNA synthesis [10,21]. Moreover, TGF-β1 has been known to reproduce the effect of the soluble factor(s) elicited by the oocyte in the synthesis of hyaluronic acid stimulated by FSH in cumulus cells [27,32].…”