Normal Female Reproduction Research Articles

Conditional Ablation of Progesterone Receptor Membrane Component 2 Causes Female Premature Reproductive Senescence.

The nonclassical progesterone receptors progesterone receptor membrane component (PGRMC) 1 and PGRMC2 have been implicated in regulating cell survival of endometrial and ovarian cells in vitro and are abundantly expressed in these cell types. The objective of this study was to determine if Pgrmc1 and Pgrmc2 are essential for normal female reproduction. To accomplish this objective, Pgrmc1 and/or Pgrmc2 floxed mice (Pgrmc2fl/fl and Pgrmc1/2fl/fl) were crossed with Pgr-cre mice, which resulted in the conditional ablation of Pgrmc1 and/or Pgrmc2 from female reproductive tissues (i.e.,Pgrmc2d/d and Pgrmc1/2d/d mice). A breeding trial revealed that conditional ablation of Pgrmc2 initially led to subfertility, with Pgrmc2d/d female mice producing 47% fewer pups/litter than Pgrmc2fl/fl mice (P = 0.001). Pgrmc2d/d mice subsequently underwent premature reproductive senescence by parities 2 to 5, producing 37.8% fewer litters overall during the trial compared with Pgrmc2fl/fl mice (P = 0.020). Similar results were observed with Pgrmc1/2d/d mice. Based on ovarian morphology and serum P4, the subfertility/infertility was not due to faulty ovulation or luteal insufficiency. Rather an analysis of midgestation implantation sites revealed that postimplantation embryonic death was the major cause of the subfertility/infertility. As with our previous report of Pgrmc1d/d mice, Pgrmc2d/d and Pgrmc1/2d/d mice developed endometrial cysts consistent with accelerated aging of this tissue. Given the timing of postimplantation embryonic demise, uterine decidualization may be disrupted in mice deficient in PGRMC2 or PGRMC1/2. Overall, this study revealed that Pgrmc1 and/or Pgrmc2 are required for the maintenance of uterine histoarchitecture and normal female reproductive lifespan.

Loss of BMAL1 in ovarian steroidogenic cells results in implantation failure in female mice.

The circadian clock plays a significant role in many aspects of female reproductive biology, including estrous cycling, ovulation, embryonic implantation, onset of puberty, and parturition. In an effort to link cell-specific circadian clocks to their specific roles in female reproduction, we used the promoter that controls expression of Steroidogenic Factor-1 (SF1) to drive Cre-recombinase-mediated deletion of the brain muscle arnt-like 1 (Bmal1) gene, known to encode an essential component of the circadian clock (SF1-Bmal1(-/-)). The resultant SF1-Bmal1(-/-) females display embryonic implantation failure, which is rescued by progesterone supplementation, or bilateral or unilateral transplantation of wild-type ovaries into SF1-Bmal1(-/-) dams. The observation that the central clock, and many other peripheral clocks, are fully functional in this model allows the assignment of the implantation phenotype to the clock in ovarian steroidogenic cells and distinguishes it from more general circadian related systemic pathology (e.g., early onset arthropathy, premature aging, ovulation, late onset of puberty, and abnormal estrous cycle). Our ovarian transcriptome analysis reveals that deletion of ovarian Bmal1 disrupts expression of transcripts associated with the circadian machinery and also genes critical for regulation of progesterone production, such as steroidogenic acute regulatory factor (Star). Overall, these data provide a powerful model to probe the interlocking and synergistic network of the circadian clock and reproductive systems.

Prostaglandin E and F receptors in the uterus

Prostaglandins (PGs) are necessary for normal female reproduction. PGs act via their respective receptors and execute various functions in their target tissues depending on which type of receptor being activated. PG receptors are G-protein coupled receptors mediating various vital processes in the uterus throughout the reproductive cycle and pregnancy. They are essential for the normal functioning of uterine endometrium, myometrium and cervix. In this mini review, we explore the expression, functions and regulations of EP1-4 and FP in uterus. Recent reports show that PG receptors are regulated spatio-temporally in endometrium throughout the reproductive cycle. In myometrium, EPs and FP are differentially expressed and have prominent roles in the contraction and relaxation of the smooth muscle. In the cervix, PG receptors are essential for normal cervical ripening. PG receptors are important in several reproductive functions including reproductive cyclicity, embryo implantation, embryo spacing, uterine contraction or relaxation, and cervical ripening. Flawed regulation or signaling by PG receptors lead to many pathological conditions of the female reproductive tract such as dysmenorrhea, menorrhagia, endometriosis, cancer and pre-term or post-term pregnancies. Although several studies have shown the expression of PG receptors in different cell types of the uterus, we still do not fully understand their functions in different cell types, how they are regulated and their implications in normal health and diseases. Better understanding of the PG receptor signaling mechanism would offer valuable insight that could be used for diagnosis and therapy.

Androgen receptor functions in male and female reproduction.

The androgen receptor (AR) is a ligand-dependent transcription factor involved in the regulation of many different physiological processes. Dysfunction of AR causes diverse clinical conditions, such as testicular feminization mutation (Tfm) syndrome and prostate cancer. However, the molecular basis of the AR in these disorders largely remains unknown, as a result of a lack of genetic models. Using conditional targeting technique with Cre-loxP system, we successfully generated null AR mutant (ARKO) mice. The ARKO males grew healthily, but they showed typical Tfm abnormalities. The ARKO males exhibited late onset of obesity with impaired bone metabolism and sexual behaviors. No overt abnormality was found in female ARKO mice, but a premature ovarian failure-like phenotype was found with impaired folliculogenesis. Thus, andorogen/AR system supports normal reproduction as well as normal female reproduction. (Reprod Med Biol 2007; 6: 11-17).

258 EXPRESSION PATTERN OF THE MATERNAL FACTOR ZYGOTE ARREST 1 (ZAR1) GENE IN OVINE OOCYTES AND PRE-IMPLANTATION EMBRYOS

Zygote arrest 1 (ZAR1) is an ovary-specific maternal factor that plays essential roles during the oocyte-to-embryo transition. It is evolutionary conserved in vertebrates, and the protein is characterized by the presence of a homeobox zing finger domain, suggesting its role in transcription regulation. Discovered in mice (Wu et al. 2003 Nat. Genet. 33, 187-191), its expression pattern has been analyzed in oocytes and pre-implantation embryos in mice, cattle, and pig (Pennetier et al. 2004 Biol. Reprod. 71, 1359-1366; Brevini et al. 2004 Mol. Reprod. Dev. 69, 375-380; Reprod. Biol. Endocrinol. 21, 4-12). Because no information on Zar1 ortholog in the ovine species is available, the aim of this study was to assess its presence and to analyze its expression pattern in oocytes and pre-implantation embryos. Reverse transcription-polymerase chain reaction (RT-PCR) was performed in germinal vesicle (GV) and IVM metaphase II (MII) oocytes, and, following IVFC, in 2- (2C), 4- (4C), 8- (8C), and 12-16- (12C-16C) cell embryos, morulae, and blastocysts. Primers were designed on the basis of bovine and swine conserved sequences and contained an intron-spanning region. The PCR product was sequenced and the alignment, performed with BLAST (www.ncbi.nlm.nih.gov/BLAST/), confirmed its homology with the Zar1 orthologous genes present in public databases, sharing 98%, 92%, and 89% nucleotide identity with the bovine, porcine, and human sequences, respectively. Real-time PCR revealed that Zar1 is present at its highest level in the GV oocyte, has a major decrease at the MII stage (around 10-fold), remains constant in the embryo first cleavage steps (2C-4C), drops again at the 8C stage (around 30-fold), and disappears in morulae and blastocysts. Our data demonstrate that a Zar1 ortholog is present in the ovine species. Results are also in accordance with the expression patterns characterized in other mammalian species and suggest that Zar1 transcript in the ovine oocyte may be necessary for normal female reproduction in the transition from oocyte to embryonic life.

Premature ovarian failure in androgen receptor-deficient mice

Premature ovarian failure (POF) syndrome, an early decline of ovarian function in women, is frequently associated with X chromosome abnormalities ranging from various Xq deletions to complete loss of one of the X chromosomes. However, the genetic locus responsible for the POF remains unknown, and no candidate gene has been identified. Using the Cre/LoxP system, we have disrupted the mouse X chromosome androgen receptor (Ar) gene. Female AR(-/-) mice appeared normal but developed the POF phenotype with aberrant ovarian gene expression. Eight-week-old female AR(-/-) mice are fertile, but they have lower follicle numbers and impaired mammary development, and they produce only half of the normal number of pups per litter. Forty-week-old AR(-/-) mice are infertile because of complete loss of follicles. Genome-wide microarray analysis of mRNA from AR(-/-) ovaries revealed that a number of major regulators of folliculogenesis were under transcriptional control by AR. Our findings suggest that AR function is required for normal female reproduction, particularly folliculogenesis, and that AR is a potential therapeutic target in POF syndrome.

Haploinsufficiency of Chicken Ovalbumin Upstream Promoter Transcription Factor II in Female Reproduction

The chicken ovalbumin upstream promoter transcription factor II, COUP-TFII, is a member of the orphan nuclear receptor transcription factor family. Genetic ablation of COUP-TFII results in early embryonic lethality and demonstrates that this gene is required for cardiac and vascular development. Expression of COUP-TFII persists throughout postnatal life in various tissues including the female reproductive tract. However, the physiological function of COUP-TFII in female reproduction has not been extensively analyzed. Here, we provide phenotypic evidences that haploinsufficiency of COUP-TFII in mice demonstrates an important role of COUP-TFII for normal female reproduction. COUP-TFII +/- females show significantly reduced fecundity, irregular estrus cycles, delayed puberty, and retarded postnatal growth. Analysis of the reduced fertility revealed that although ovarian function was normal with respect to ovulation, the ovaries have reduced ability to synthesize progesterone in response to exogenous gonadotropins. This reduction is due to the reduction of the expression of steroidogenic enzymes important for progesterone synthesis and the reduction of vascularization in COUP-TFII heterozygotes. Analysis of uterine function demonstrated a reduced response to an experimentally induced decidual cell reaction indicating that the ability of the uterus to support embryo implantation was reduced. Taken together, our data show global impact of gene dosage effects of COUP-TFII on female postnatal life and indicates requirement of COUP-TFII in normal female reproduction, in particular for uterine endometrial functions during the peri-implantation period.

220.TGFβ1 deficient mice exhibit impaired follicle growth and luteal maintenance

Transforming Growth Factor β1 (TGFβ1) is essential for normal female reproduction. Mice with a targeted deletion in the TGFβ1 gene (TGFβ1–/–) have severely impaired fertility with pregnancy occurring in <25% of mated females. TGFβ1 is implicated in several aspects of ovarian function, including potentiation of granulosa cell proliferation and suppression of luteal cell apoptosis. Our initial observations indicate that estrous cycling is disrupted in TGFβ1–/– mice and that ovulation rate is reduced. To further investigate how impaired ovarian function contributes to the infertility of TGFβ1–/– mice, ovaries were isolated from TGFβ1+/+ and TGFβ1–/– littermates at proestrus and fixed and sectioned for examination of follicle morphology and growth. BrdU labelling was performed to detect granulosa cell proliferation and blood samples were obtained for analysis of gonadotrophins and ovarian steroid hormones. Histological examination showed that ovaries from TGFβ1–/– mice were smaller than those of TGF–1+/+ mice, however large antral follicles were observed, indicating that TGFβ1 is not essential for granulosa cell proliferation. Compared to TGFβ1+/+ ovaries however, there were fewer antral follicles and only rare corpora lutea. Interestingly, in some cases there were large numbers of macrophages surrounding small follicles suggesting increased follicular atresia and/or altered macrophage activity in the TGFβ1–/– ovaries. Ovaries and serum were also isolated from females at d4 post-coital for assessment of corpora lutea morphology. TGFβ1–/– ovaries weighed less and had fewer corpora lutea than TGFβ1+/+ ovaries. TGFβ1–/– corpora lutea also contained increased numbers of apoptotic cells and infiltrating macrophages indicative of premature luteal regression. Circulating progesterone levels were reduced in TGFβ1–/– females, as was progesterone production per corpus luteum further indicating a functional defect in luteal maintenance. Cumulatively these observations show that TGFβ1 has essential roles in regulation of ovarian macrophage populations, in normal follicular development and in the generation, maintenance and steroidogenic function of corpora lutea.